**Section 5**

**Systemic, Neuronal and Hormonal Pathologies** 

310 Oxidative Stress and Diseases

[45] Hodosy J, Celec P. Daytime of sampling, tooth-brushing and ascorbic acid influence

[46] Reznick AZ, Shehadeh N, Shafir Y, Nagler RM. Freeradicals related effects and

[47] Santini SA, Marra G, Giardina B, Cotroneo P, Mordente A, Martorana GE, Manto A,

[49] Natarajan Sulochana K, Lakshmi S, Punitham R, Arokiasamy T, Sukumar B,

[52] Beckman JA, Goldfine AB, Gordon MB, Garrett LA, Keaney JF, Creager MA. Oral

diabetic patients. A pilot clinical trial. Med Sci Monit 2002; 8:CR131-137. [50] Celik S, Baydas G, Yilmaz O. Influence of vitamin E on the levels of fatty acids and MDA in some tissues of diabetic rats. Cell biochem Funct 2002;20:67-71. [51] Peponis V, Papathanasiou M, Kapranou A, Magkou C, Tyligada A, Melidonis A, Droses

of diabetic patients. Br J Ophthalmol. 2002;86:1369-73.

mellitus. Am J Heart Circ Physiol 2003;285:H2392-98.

lipid peroxidation in uncomplicated IDDM. Diabetes 1997;46:1853-58. [48] Paolisso G, D'Amore A, Balbi V, Volpe C, Galzerano D, Giugliano D, Sgambato S,

gingival status. Dis Markers 2005; 21(4): 203-207.

mellitus. Arch Oral Biol 2006; 51(8): 640-648.

1994;266:E261-8.

salivary thiobarbituric acid reacting substances—a potential clinical marker of

antioxidants in saliva and serum of adolescents free radicals Type 1 diabetes

Chirlanda G. Defective plasma antioxidant defenses and enhanced susceptibility to

Verricchio M, D'Onofrio F. Plasma vitamin C affects glucose homeostasis in healthy subjects and in non-insulin-dependent diabetics. Am J Physiol

Ramakrishnan S. Effects of oral supplementation of free aminoacids in type 2

T, Sitaras NM. Protective role of oral antioxidant supplementation in ocular surface

antioxidant therapy improves endothelial function in type 1 and not type 2 diabetes

**14** 

Levente Lázár

*Hungary* 

**The Role of Oxidative Stress in Female** 

In a healthy body, reactive oxygen species (ROS) and antioxidants remain in balance. Oxidative stress occurs when the generation of reactive oxygen species and other radical species exceeds the scavenging capacity by antioxidants of antioxidative agents in organism, due to excessive production of reactive oxidagen species and/or inadequate intake or increased utilization of antioxidants. Most ROS are formed at operation of electrone transport chains in mitochondria, endoplasmatic reticulum, plasmatic and nuclear membranes. Minor ROS amounts are generated by some enzymes through autooxidation of different molecules. Reactive oxygen specieses can also be formed by exogenous exposures such as alcohol, tobacco smoke, and environmental pollutants. Elimination of reactive oxygen species is catalysed by certain enzymes such as superoxide dismutses (SOD), catalases and peroxidases. Antioxidants (including vitamins C and E) and antioxidant cofactors (such as selenium, zinc, and copper) are capable to dispose, scavenge, or suppress ROS formation. "Oxidative stress" rises when due to some reasons the steady-state ROS concentration is increased, leading to oxidative modification of cellular constituents, resulting disturbance of cellular metabolism and regulatory pathways (Lushchak. 2011). Cellular ROS and their control by antioxidants are involved in the physiology of the female reproductive system. Physiological ROS levels play an important regulatory role through various signalling and transduction pathways in folliculogenesis, oocyte maturation, corpus luteum, uterine function, embryogenesis, embryonic implantation and fetoplacental development. Imbalances between antioxidants and ROS production are considered to be responsible for the initiation or development of pathological processes affecting female

The establishment of pregnancy requires a receptive uterus able to respond to a variety of biochemical and molecular signals produced by the developing conceptus, as well as specific interactions between the uterine endometrium and the extra-embryonic membranes. Therefore, placental development and function are prerequisites for an adequate supply of nutrients and oxygen to the fetus and successful establishment of pregnancy. Oxidative stress has been proposed as the causative agent of female sterility, recurrent pregnancy loss and several pregnancy-related disorders as preeclampsia, intra-uterine growth restriction

**1. Introduction** 

reproductive processes.

(IUGR) and gestational diabetes.

**Reproduction and Pregnancy** 

*1st Departmet of Obstetrics and Gynecology,* 

*Semmelweis University Budapest,* 

### **The Role of Oxidative Stress in Female Reproduction and Pregnancy**

#### Levente Lázár

*1st Departmet of Obstetrics and Gynecology, Semmelweis University Budapest, Hungary* 

#### **1. Introduction**

In a healthy body, reactive oxygen species (ROS) and antioxidants remain in balance. Oxidative stress occurs when the generation of reactive oxygen species and other radical species exceeds the scavenging capacity by antioxidants of antioxidative agents in organism, due to excessive production of reactive oxidagen species and/or inadequate intake or increased utilization of antioxidants. Most ROS are formed at operation of electrone transport chains in mitochondria, endoplasmatic reticulum, plasmatic and nuclear membranes. Minor ROS amounts are generated by some enzymes through autooxidation of different molecules. Reactive oxygen specieses can also be formed by exogenous exposures such as alcohol, tobacco smoke, and environmental pollutants. Elimination of reactive oxygen species is catalysed by certain enzymes such as superoxide dismutses (SOD), catalases and peroxidases. Antioxidants (including vitamins C and E) and antioxidant cofactors (such as selenium, zinc, and copper) are capable to dispose, scavenge, or suppress ROS formation. "Oxidative stress" rises when due to some reasons the steady-state ROS concentration is increased, leading to oxidative modification of cellular constituents, resulting disturbance of cellular metabolism and regulatory pathways (Lushchak. 2011). Cellular ROS and their control by antioxidants are involved in the physiology of the female reproductive system. Physiological ROS levels play an important regulatory role through various signalling and transduction pathways in folliculogenesis, oocyte maturation, corpus luteum, uterine function, embryogenesis, embryonic implantation and fetoplacental development. Imbalances between antioxidants and ROS production are considered to be responsible for the initiation or development of pathological processes affecting female reproductive processes.

The establishment of pregnancy requires a receptive uterus able to respond to a variety of biochemical and molecular signals produced by the developing conceptus, as well as specific interactions between the uterine endometrium and the extra-embryonic membranes. Therefore, placental development and function are prerequisites for an adequate supply of nutrients and oxygen to the fetus and successful establishment of pregnancy. Oxidative stress has been proposed as the causative agent of female sterility, recurrent pregnancy loss and several pregnancy-related disorders as preeclampsia, intra-uterine growth restriction (IUGR) and gestational diabetes.

The Role of Oxidative Stress in Female Reproduction and Pregnancy 315

various biomarkers of oxidative stress has been investigated in normal cycling human ovaries (Maruyama et al. 1997, Matsui et al., 1996), justifiing the regulatory role of ROS and antioxidants in oocyte maturation, folliculogenesis, ovarian steroidogenesis and luteolysis (Shiotani et al., 1991; Behrman et al., 2001; Sugino et al., 2004). Studies demonstrate intensified lipid peroxidation in the preovulatory Graafian follicle (Paszkovski et al., 1995). The significance of reactive oxygen specieses and antioxidant enzymes as copper zinc superoxide dismutase (Cu, Zn-SOD), manganese superoxide dismutase (Mn-SOD), and glutathione peroxidase, in oocyte maturation was provided by Riley et al. (1991) using immmunohistochemical localization and mRNA expression (Tamate et al., 1995). The antioxidant enzymes neutralize reactive oxygen specieses and protect the oocyte. In corpora lutea collected from pregnant and nonpregnant patients, it was observed that during normal situations Cu–Zn SOD expression rise from early luteal to midluteal phase and decrease during regression of the corpus luteum. Studies investigating the correlation between adrenal-4 binding protein (Ad4BP) and superoxide dismutase expression also suggest an association between oxidative stress and ovarian steroidogeneis (Matsui et al.,1996). Antibody to adrenal 4-binding protein (Ad4BP) was utilized to localize Ad4BP in the nuclei of theca and granulosa cells. Ad4BP is a steroidogenic transcription factor that induces transcription of the steroidogenic P450 enzyme. Both human granulosa and luteal cells respond to hydrogen peroxide with an extirpation of gonadotropin action and inhibition of progesterone secretion (Sabuncu et al., 2001). The production of both progesterone and estradiol hormones is reduced when hydrogen peroxide is added to a culture of human

chorionic gonadotropin-stimulated luteal cells (Agrawal et al., 2006).

**2.2 Changes in endometrium** 

a mechanism for menstruation (Sugino et al., 2004).

for cytoplasmic maturation in metaphase II oocytes (El Mouattasim et al., 1999).

Levels of three oxidative stress biomarkers, conjugated dienes, lipid hydroperoxide and thiobarabituric acid reactive substances were found significantly lower in the follicular fluid compared with serum levels (Jozwik et al., 1999). The preovulatory follicle has a potent antioxidant defense, which is depleted by the intense peroxidation (Jozwik et al., 1999). Also the antioxidant enzymes glutathione peroxidase and Mn-SOD are considered to be markers

Cyclical changes in the endometrium are accompanied by changes in the expression of antioxidants. Enzymes, such as thioredoxin, have a higher expression in the early secretory phase (Murayama et al, 1997). There is also a cyclical variation in the expression of superoxide dismutase in the endometrium. Superoxide dismutase activity decreased in the late secretory phase while ROS levels increased and ROS triggered the release of prostaglandin F2 α (Sugino et al., 1996). Estrogen or progesterone withdrawal led to increased expression of cyclooxygenase-2 (COX-2). Stimulation of the cyclooxygenase enzyme is brought about by ROS via activation of the transcription factor NF-κB, suggesting

Nitrogen monoxide (.NO) has also important role in decidualisation and preparation of the endometrium for implantation by regulatation of the endometrial, myometrial and microvascular functions. Expression of endothelial and inducible NO synthase (NOS) have been demonstrated in the human endometrium (Tseng et al., 1996), and the endometrial vessels (Taguchi et al,. 2000). Highest levels of transcripts of endothelial NOS mRNA have

#### **2. Effect of oxidative stress on female reproductive system**

The female reproductive system is a complex multiorgan system which require an optimal biological environment. Aerobic metabolism utilizing oxygen is essential for reproductive homeostazis. Aerobic metabolism is associated with the generation of prooxidant molecules called ROS including hydroxyl radical, superoxide anion, hydrogen peroxide, and nitric oxide. The balance between the prooxidants and antioxidants maintain the cellular homeostasis, whenever there is an imbalance in this equilibrum leading to enhanced steadystate level a state of oxidative stress is initiated. Free radicals are key signal molecules modulating reproductive functions by the influence of the endometrial and fallopian tube function, maturation of oocytes, sperm, implantation of the preembryo and early embryo development (Figure 1.)

Fig. 1. Oxidative stress in female reproduction

#### **2.1 Ovarian function**

Aerobic metabolism utilizing oxygen is essential for developement of the gametes, also free radicals play a significant role in physiological processes within the ovary. The expression of

The female reproductive system is a complex multiorgan system which require an optimal biological environment. Aerobic metabolism utilizing oxygen is essential for reproductive homeostazis. Aerobic metabolism is associated with the generation of prooxidant molecules called ROS including hydroxyl radical, superoxide anion, hydrogen peroxide, and nitric oxide. The balance between the prooxidants and antioxidants maintain the cellular homeostasis, whenever there is an imbalance in this equilibrum leading to enhanced steadystate level a state of oxidative stress is initiated. Free radicals are key signal molecules modulating reproductive functions by the influence of the endometrial and fallopian tube function, maturation of oocytes, sperm, implantation of the preembryo and early embryo

> **Embryo Developement**

**Embryopathy** 

Aerobic metabolism utilizing oxygen is essential for developement of the gametes, also free radicals play a significant role in physiological processes within the ovary. The expression of

**2. Effect of oxidative stress on female reproductive system** 

development (Figure 1.)

Fig. 1. Oxidative stress in female reproduction

**2.1 Ovarian function** 

various biomarkers of oxidative stress has been investigated in normal cycling human ovaries (Maruyama et al. 1997, Matsui et al., 1996), justifiing the regulatory role of ROS and antioxidants in oocyte maturation, folliculogenesis, ovarian steroidogenesis and luteolysis (Shiotani et al., 1991; Behrman et al., 2001; Sugino et al., 2004). Studies demonstrate intensified lipid peroxidation in the preovulatory Graafian follicle (Paszkovski et al., 1995). The significance of reactive oxygen specieses and antioxidant enzymes as copper zinc superoxide dismutase (Cu, Zn-SOD), manganese superoxide dismutase (Mn-SOD), and glutathione peroxidase, in oocyte maturation was provided by Riley et al. (1991) using immmunohistochemical localization and mRNA expression (Tamate et al., 1995). The antioxidant enzymes neutralize reactive oxygen specieses and protect the oocyte. In corpora lutea collected from pregnant and nonpregnant patients, it was observed that during normal situations Cu–Zn SOD expression rise from early luteal to midluteal phase and decrease during regression of the corpus luteum. Studies investigating the correlation between adrenal-4 binding protein (Ad4BP) and superoxide dismutase expression also suggest an association between oxidative stress and ovarian steroidogeneis (Matsui et al.,1996). Antibody to adrenal 4-binding protein (Ad4BP) was utilized to localize Ad4BP in the nuclei of theca and granulosa cells. Ad4BP is a steroidogenic transcription factor that induces transcription of the steroidogenic P450 enzyme. Both human granulosa and luteal cells respond to hydrogen peroxide with an extirpation of gonadotropin action and inhibition of progesterone secretion (Sabuncu et al., 2001). The production of both progesterone and estradiol hormones is reduced when hydrogen peroxide is added to a culture of human chorionic gonadotropin-stimulated luteal cells (Agrawal et al., 2006).

Levels of three oxidative stress biomarkers, conjugated dienes, lipid hydroperoxide and thiobarabituric acid reactive substances were found significantly lower in the follicular fluid compared with serum levels (Jozwik et al., 1999). The preovulatory follicle has a potent antioxidant defense, which is depleted by the intense peroxidation (Jozwik et al., 1999). Also the antioxidant enzymes glutathione peroxidase and Mn-SOD are considered to be markers for cytoplasmic maturation in metaphase II oocytes (El Mouattasim et al., 1999).

#### **2.2 Changes in endometrium**

Cyclical changes in the endometrium are accompanied by changes in the expression of antioxidants. Enzymes, such as thioredoxin, have a higher expression in the early secretory phase (Murayama et al, 1997). There is also a cyclical variation in the expression of superoxide dismutase in the endometrium. Superoxide dismutase activity decreased in the late secretory phase while ROS levels increased and ROS triggered the release of prostaglandin F2 α (Sugino et al., 1996). Estrogen or progesterone withdrawal led to increased expression of cyclooxygenase-2 (COX-2). Stimulation of the cyclooxygenase enzyme is brought about by ROS via activation of the transcription factor NF-κB, suggesting a mechanism for menstruation (Sugino et al., 2004).

Nitrogen monoxide (.NO) has also important role in decidualisation and preparation of the endometrium for implantation by regulatation of the endometrial, myometrial and microvascular functions. Expression of endothelial and inducible NO synthase (NOS) have been demonstrated in the human endometrium (Tseng et al., 1996), and the endometrial vessels (Taguchi et al,. 2000). Highest levels of transcripts of endothelial NOS mRNA have

The Role of Oxidative Stress in Female Reproduction and Pregnancy 317

The partial oxygen tension in the intervillous space declines from the second to the third trimester, reaching about 40 mm Hg in the third trimester (Soothill et al., 1986). The exact mechanism by which trophoblasts sense oxygen tension is currently unclear; however, several potential pathways have been identified. Many of these pathways utilize the ROS formation, but it is currently unclear whether hypoxia results an increase or decrease in their cellular levels (DeMarco & Caniggia, 2002). In hypoxic conditions, trophoblast oxygen sensing mechanisms utilize several different pathways to control gene expression. These pathways often utilize redox-sensitive transcription factors, of which the hypoxia inducible factor (HIF) family are the best characterized. HIF-1 is a transcription factor and master regulator of the cellular response to low oxygen levels (Majmundar et al., 2010), showing prominent expression in first trimester villi (Wang&Semenza, 1993). HIF-1 regulates the expression of genes such as p53, p21, and Bcl-2 required for cells to adapt to a low oxygen environment and apoptosis. HIF-1 is able to be stabilized under normoxic conditions by a variety of growth factors and cytokines including epidermal growth factor (EGF), insulin, heregulin, insulin-like growth factors 1 and 2, transforming growth factor, and interleukin-1 (Zelzer et al., 1998; Feldser et al., 1999; Hellwig-Burgel et al., 1999; Laughner et al., 2001;

Several other transcription factors involved in trophoblast differentiation are responsive to hypoxia. The transcription factors Id1 stream stimulatory factor-1 and -2 (USF1 and USF2) mediate the effects of Mash2 are all up-regulated in 2% oxygen in comparison to 20% oxygen (Jiang et al., 2000; Jiang & Mendelson, 2003). The up-regulation of Mash2, USF1 and USF2 may inhibit cytotrophoblast fusion into syncytiotrophoblast (Jiang et al., 2000; Jiang& Mendelson, 2003). The elevation of intracellular Ca2+ is believed to activate an HIF-1 independent signalling pathway that involves the transcription factor activator protein-1 (AP-1), with cooperation between the HIF-1 and AP-1 pathways allowing fine regulation of gene expression under hypoxia (Laderoute et al., 2002; Salnikow et al., 2002). AP-1 is a dimeric transcription factor composed from the products of the Jun and Fos protooncogenes (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) (Dakour et al., 1999). AP-1 transcription factors are believed to play an important role in trophoblast differentiation. In the villus, AP-1 transcription factor expression is limited; however, extravillous trophoblasts express c-Jun, JunB, c-Fos, FosB and Fra-2 both in the first trimester and later in gestation

The other protective system is formed by antioxidant enzymes, playing a key role in the response of trophoblast to the burst of perfusion by maternal blood. With the increase of oxygen saturation and oxidative stress the activity in intervillous space the placenta employs a number of physiologic adaptations (Burton, 2009). Levels and activity of antioxidant enzymes: catalase, glutathione peroxidase, manganese and cooper, zinc superoxide dismutase are increased within placental tissues. This response is evolved as a defense mechanism to reduce harm to placental tissues exposed to this burst of oxidative

We can summarize that the trophoblast differentiation is essential for the success of human pregnancy, and despite some conflicting experimental evidence, hypoxia appears to play a vital role in regulating trophoblast differentiation in the first trimester. The regulation of trophoblast differentiation by hypoxia is a result of complex interactions between factors associated with oxidative stress, oxygen sensing mechanisms and the release of

Fukuda et al., 2002; Stiehl et al., 2002).

(Bamberger et al., 2004.)

stress (Jauniaux et al., 2000).

been reported in the late secretory phase of the endometrium (Tseng et al., 1996). These changes have been hypothesized to be important in the genesis of menstruation and endometrial shedding.

#### **2.3 Fallopian tube function**

Several studies demonstrated the presence of cytokines, prostaglandins, metabolites of lipid peroxidation and ROS in fluid samples of fallopain tube (Tamate et al., 1985). The eqilibrum of these components serves as an optimal milieu for fertilization and the transport of the preembryo. An endogenous nitrogen monoxide system exists in the fallopian tubes. Nitric oxide has a relaxing effect on smooth muscle and it has similar effects on tubular contractility. Deficiency of NO may lead to tubal motility dysfunction, resulting in retention of the ovum, delayed sperm transport and infertility. Increased NO levels in the fallopian tubes are cytotoxic to the invading microbes and also may be toxic to spermatozoa (Rosselli et al., 1995), leading to infetility.

#### **2.4 Embryo implantation and placenta**

The human embryo undergoes interstitial implantation by invading the maternal decidua at the blastocyst stage (Riley& Behrman, 1991). Although placental villi are bathed in maternal blood in the hemochorial placenta (Tamate et al., 1995), prior to 10 weeks of gestation maternal blood flow to the placenta is blocked by extravillous trophoblasts. Placentation is initiated when the blastocyst makes contact with the epithelial lining of the uterus shortly after implantation. Placental villi which consist of a mesenchymal core surrounded by a monolayer of mononuclear villous cytotrophoblast stem cells which either fuse to form the overlying multinucleated syncytiotrophoblast or, in anchoring villi, differentiate into extravillous trophoblasts which grow out from the villous and spread laterally around the placenta (Irving et al., 1995).

Invasive extravillous trophoblasts play an important role in adapting the decidua to sustain pregnancy. Extravillous trophoblasts invade the walls of the uterine spiral arteries and adapt these vessels into large bore conduits capable of delivering the increased blood supply required in the second and third trimesters (Robertson et al., 1967; Zhou et al., 1997). As the extravillous trophoblasts invade the spiral arteries early in pregnancy they form plugs which occlude the spiral arteries and prevent maternal blood from entering the intervillous space, creating a physiologic hypoxic environment (Hustin &nd Schaaps,1987; Jaffe et al., 1997; Burton et al., 1999).

Early placental and embryonic development occurs in a state of low oxygen in histiotroph manner (Evans et al., 2004). The early gestation placenta is poorly protected against oxidative damage, as the antioxidant enzymes Cu,Zn-SOD and Mn-SOD are not expressed by the syncytiotrophoblast until approximately 8–9 weeks of gestation (Watson et al., 1997). Premature perfusion of this space during this first 10 weeks of development increases the risk of pregnancy loss (Jauniaux et al., 2000). The low oxygen environment during early placental development is essential for normal placental angiogenesis, and this angiogenesis is promoted by hypoxia-induced transcriptional and post-transcriptional regulation of angiogenic factors, as vascular endothelial growth factor and placental growth factor (Charnock-Jones&Burton, 2000).

been reported in the late secretory phase of the endometrium (Tseng et al., 1996). These changes have been hypothesized to be important in the genesis of menstruation and

Several studies demonstrated the presence of cytokines, prostaglandins, metabolites of lipid peroxidation and ROS in fluid samples of fallopain tube (Tamate et al., 1985). The eqilibrum of these components serves as an optimal milieu for fertilization and the transport of the preembryo. An endogenous nitrogen monoxide system exists in the fallopian tubes. Nitric oxide has a relaxing effect on smooth muscle and it has similar effects on tubular contractility. Deficiency of NO may lead to tubal motility dysfunction, resulting in retention of the ovum, delayed sperm transport and infertility. Increased NO levels in the fallopian tubes are cytotoxic to the invading microbes and also may be toxic to spermatozoa (Rosselli

The human embryo undergoes interstitial implantation by invading the maternal decidua at the blastocyst stage (Riley& Behrman, 1991). Although placental villi are bathed in maternal blood in the hemochorial placenta (Tamate et al., 1995), prior to 10 weeks of gestation maternal blood flow to the placenta is blocked by extravillous trophoblasts. Placentation is initiated when the blastocyst makes contact with the epithelial lining of the uterus shortly after implantation. Placental villi which consist of a mesenchymal core surrounded by a monolayer of mononuclear villous cytotrophoblast stem cells which either fuse to form the overlying multinucleated syncytiotrophoblast or, in anchoring villi, differentiate into extravillous trophoblasts which grow out from the villous and spread laterally around the

Invasive extravillous trophoblasts play an important role in adapting the decidua to sustain pregnancy. Extravillous trophoblasts invade the walls of the uterine spiral arteries and adapt these vessels into large bore conduits capable of delivering the increased blood supply required in the second and third trimesters (Robertson et al., 1967; Zhou et al., 1997). As the extravillous trophoblasts invade the spiral arteries early in pregnancy they form plugs which occlude the spiral arteries and prevent maternal blood from entering the intervillous space, creating a physiologic hypoxic environment (Hustin &nd Schaaps,1987; Jaffe et al.,

Early placental and embryonic development occurs in a state of low oxygen in histiotroph manner (Evans et al., 2004). The early gestation placenta is poorly protected against oxidative damage, as the antioxidant enzymes Cu,Zn-SOD and Mn-SOD are not expressed by the syncytiotrophoblast until approximately 8–9 weeks of gestation (Watson et al., 1997). Premature perfusion of this space during this first 10 weeks of development increases the risk of pregnancy loss (Jauniaux et al., 2000). The low oxygen environment during early placental development is essential for normal placental angiogenesis, and this angiogenesis is promoted by hypoxia-induced transcriptional and post-transcriptional regulation of angiogenic factors, as vascular endothelial growth factor and placental growth factor

endometrial shedding.

**2.3 Fallopian tube function** 

et al., 1995), leading to infetility.

placenta (Irving et al., 1995).

1997; Burton et al., 1999).

(Charnock-Jones&Burton, 2000).

**2.4 Embryo implantation and placenta** 

The partial oxygen tension in the intervillous space declines from the second to the third trimester, reaching about 40 mm Hg in the third trimester (Soothill et al., 1986). The exact mechanism by which trophoblasts sense oxygen tension is currently unclear; however, several potential pathways have been identified. Many of these pathways utilize the ROS formation, but it is currently unclear whether hypoxia results an increase or decrease in their cellular levels (DeMarco & Caniggia, 2002). In hypoxic conditions, trophoblast oxygen sensing mechanisms utilize several different pathways to control gene expression. These pathways often utilize redox-sensitive transcription factors, of which the hypoxia inducible factor (HIF) family are the best characterized. HIF-1 is a transcription factor and master regulator of the cellular response to low oxygen levels (Majmundar et al., 2010), showing prominent expression in first trimester villi (Wang&Semenza, 1993). HIF-1 regulates the expression of genes such as p53, p21, and Bcl-2 required for cells to adapt to a low oxygen environment and apoptosis. HIF-1 is able to be stabilized under normoxic conditions by a variety of growth factors and cytokines including epidermal growth factor (EGF), insulin, heregulin, insulin-like growth factors 1 and 2, transforming growth factor, and interleukin-1 (Zelzer et al., 1998; Feldser et al., 1999; Hellwig-Burgel et al., 1999; Laughner et al., 2001; Fukuda et al., 2002; Stiehl et al., 2002).

Several other transcription factors involved in trophoblast differentiation are responsive to hypoxia. The transcription factors Id1 stream stimulatory factor-1 and -2 (USF1 and USF2) mediate the effects of Mash2 are all up-regulated in 2% oxygen in comparison to 20% oxygen (Jiang et al., 2000; Jiang & Mendelson, 2003). The up-regulation of Mash2, USF1 and USF2 may inhibit cytotrophoblast fusion into syncytiotrophoblast (Jiang et al., 2000; Jiang& Mendelson, 2003). The elevation of intracellular Ca2+ is believed to activate an HIF-1 independent signalling pathway that involves the transcription factor activator protein-1 (AP-1), with cooperation between the HIF-1 and AP-1 pathways allowing fine regulation of gene expression under hypoxia (Laderoute et al., 2002; Salnikow et al., 2002). AP-1 is a dimeric transcription factor composed from the products of the Jun and Fos protooncogenes (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) (Dakour et al., 1999). AP-1 transcription factors are believed to play an important role in trophoblast differentiation. In the villus, AP-1 transcription factor expression is limited; however, extravillous trophoblasts express c-Jun, JunB, c-Fos, FosB and Fra-2 both in the first trimester and later in gestation (Bamberger et al., 2004.)

The other protective system is formed by antioxidant enzymes, playing a key role in the response of trophoblast to the burst of perfusion by maternal blood. With the increase of oxygen saturation and oxidative stress the activity in intervillous space the placenta employs a number of physiologic adaptations (Burton, 2009). Levels and activity of antioxidant enzymes: catalase, glutathione peroxidase, manganese and cooper, zinc superoxide dismutase are increased within placental tissues. This response is evolved as a defense mechanism to reduce harm to placental tissues exposed to this burst of oxidative stress (Jauniaux et al., 2000).

We can summarize that the trophoblast differentiation is essential for the success of human pregnancy, and despite some conflicting experimental evidence, hypoxia appears to play a vital role in regulating trophoblast differentiation in the first trimester. The regulation of trophoblast differentiation by hypoxia is a result of complex interactions between factors associated with oxidative stress, oxygen sensing mechanisms and the release of

The Role of Oxidative Stress in Female Reproduction and Pregnancy 319

that various transcription factors are affected by thalidomide through redox regulation

Exposure to the anticonvulsant valproic acid during the first trimester of pregnancy is associated with an increased risk of congenital malformations including heart defects, craniofacial abnormalities, skeletal and limb defects, and most frequently, neural tube defects (NTDs). The mechanisms by which valproic acid induces teratogenic effects are not fully understood, although previous studies support a role for oxidative stress. Valproic acid can alter cell signaling through gene expression changes mediated through histone deacetylase inhibition (Phiel et al., 2001), and is a direct inhibitor of class I and II histone deacetylases. Several laboratories have shown that embryonic histone acetylation levels are increased following exposure to valproic acid (Menegola et al., 2005; Tung and Winn, 2010). Furthermore, studies have supported a role for histone deacetylase inhibition as a mechanism of teratogenesis as analogs of valproic acid that lack histone deacetylase inhibitory activity are less teratogenic (Gurvich et al., 2005). Gene microarray studies have also demonstrated that valproic acid targets genes regulated by histone deacetylase, including *Mt1* and *Mt2*, both of which are ROS-sensitive (Jergil et al., 2009). In addition, histone deacetylase inhibitors have also been shown to increase ROS production and induce apoptosis in several cancer cell lines (Carew et al., 2008). Therefore, alterations in gene expression and/or increases in ROS formation mediated by histone deacetylase inhibition

The widely used anticonvulsant, phenytoin, can double the incidence of structural and functional birth defects when used in pregnancy (Kaneko et al., 1991). It can induce vascular disruption, which leads to hypoxia and hypoperfusion (Danielsson et al.,1995). In addition, phenytoin results in oxidative DNA damage and dysmorphogenesis, which can be eliminated by antioxidants (Winn and Wells, 1995). Phenytoin also selectively increased NF-kB activity in targeted tissues. Blocking of these signaling events with p65 antisense oligonucleotides eliminated the associated embryopathies (Kennedy et al., 2004). Further evidence that oxidative stress is important in phenytoin mediated toxicity is exemplified by the fact that treatment with polyethylene-modified superoxide dismutase enhances embryo toxicity whereas antioxidant levels were modulated with

Chronic ethanol consumption can lead to the generation of ROS and, as a consequence, teratogenicity. Prolonged ethanol exposure lead to increased production of lipid peroxides and decreased expression of antioxidant enzymes. Ascorbic acid can prevent against ethanol toxicity through inhibition of ROS formation and NF-kB activation (Peng et al.,2005). Zebrafish embryos exposed to ethanol with lipoic acid and/or only partially attenuated ethanol embryo

Preeclampsia is a complex multisystem disorder that occurs during the pregnancy. The disease affects 5-8% of all pregnancies and is one of the leading couses of maternal and fetal

toxicity, suggesting that other mechanisms are also involved (Reimers et al., 2006).

**4. Oxidative stress in pathological pregnancies** 

(Hansen et al., 2001, 2002).

during development may induce teratogenesis.

phenytoin (Winn and Wells, 1999).

**4.1 Preeclampsia** 

inflammatory cytokines. Therefore, aberrations in any one of these factors, along with the temporal and spatial regulation of blood flow in the intervillous space has the potential to result in altered gene expression and trophoblast phenotype leading to fail of implantation.

#### **3. The role of oxidative stress in embryo and fetal malformation**

Basic principles of teratogenesis state that a teratogen must cause a specific malformation through a specific mechanism during a period in which the conceptus is susceptible to said mechanism (Karnofsky, 1965). Different mechanisms are responsible for malformations that are in agreement with these basic scientific principles.

A mechanism that has not been well described in teratology is oxidant induced or redox misregulation of developmental signals en route to dysmorphogenesis. The paucity of teratogenic study of redox misregulation is partially due to oxidative stress (Sies, 1985). Oxidizing and reducing equivalent imbalance in turn, leads to macromolecule damage, namely protein modification, lipid peroxidation, and DNA oxidation, and can lead to cell death. Unspecific oxidation of cellular components does not apply to basic principles of teratology or adequately explain the manifestation of teratogenic effects. If oxidative stress is the random, unspecific oxidation of cellular molecules, it does not adequately exemplify why or how specific teratogens that induce oxidative stress could cause a specific malformation. While untimely cell death during differentiation can have serious repercussions on the developing embryo, generalized cellular oxidation and subsequent apoptosis do not sufficiently describe specificity of malformations seen with most teratogens. To qualify as a plausible teratogenic mechanism, oxidative stress must be a controlled, specific event that alters cell function and/ or signal transduction pathways that would in turn cause specific dysmorphogenesis.

During particular periods in development, the embryo is more or less susceptible to oxidative stress. In early development, one-cell embryo relies on the Krebs cycle, whereas the blastocyst relies on glycolysis and anaerobic pathways as does the embryo during early organogenesis. Once the circulatory system is established, there is a higher reliance on oxidative and aerobic metabolism and more ROS are produced by mitochondria. Conversely, more antioxidants are available at this period to counteract and detoxify these reactive oxygen specieses (Hansen, 2006). Over the course of development, the delicate balance between oxidants and antioxidants can be disrupted by exogenous agents that simulate ROS production leading to oxidative stress.

Thalidomide is associated with multiple birth defects, including phocomelia (Lecutier, 1962; Taussig, 1962). The most sensitive organ to thalidomide toxicity is the limb. Although the mechanism of teratogenesis and determinants of risk remain unclear, related teratogenic xenobiotics are bioactivated by embryonic prostaglandin H synthase (PHS) producing reactive oxygen species, which cause oxidative damage to DNA and other cellular macromolecules. Similarly, thalidomide is bioactivated by horseradish peroxidase, and oxidizes DNA and glutathione, indicating free radical-mediated oxidative stress. Furthermore, thalidomide teratogenicity is reduced by the PHS inhibitor acetylsalicylic acid, indicating PHS-catalyzed bioactivation. This appears to be regulated through redox shift resulting from depletion of GSH and increased GSSG in the nucleus, and this may imply

inflammatory cytokines. Therefore, aberrations in any one of these factors, along with the temporal and spatial regulation of blood flow in the intervillous space has the potential to result in altered gene expression and trophoblast phenotype leading to fail of implantation.

Basic principles of teratogenesis state that a teratogen must cause a specific malformation through a specific mechanism during a period in which the conceptus is susceptible to said mechanism (Karnofsky, 1965). Different mechanisms are responsible for malformations that

A mechanism that has not been well described in teratology is oxidant induced or redox misregulation of developmental signals en route to dysmorphogenesis. The paucity of teratogenic study of redox misregulation is partially due to oxidative stress (Sies, 1985). Oxidizing and reducing equivalent imbalance in turn, leads to macromolecule damage, namely protein modification, lipid peroxidation, and DNA oxidation, and can lead to cell death. Unspecific oxidation of cellular components does not apply to basic principles of teratology or adequately explain the manifestation of teratogenic effects. If oxidative stress is the random, unspecific oxidation of cellular molecules, it does not adequately exemplify why or how specific teratogens that induce oxidative stress could cause a specific malformation. While untimely cell death during differentiation can have serious repercussions on the developing embryo, generalized cellular oxidation and subsequent apoptosis do not sufficiently describe specificity of malformations seen with most teratogens. To qualify as a plausible teratogenic mechanism, oxidative stress must be a controlled, specific event that alters cell function and/ or signal transduction pathways that

During particular periods in development, the embryo is more or less susceptible to oxidative stress. In early development, one-cell embryo relies on the Krebs cycle, whereas the blastocyst relies on glycolysis and anaerobic pathways as does the embryo during early organogenesis. Once the circulatory system is established, there is a higher reliance on oxidative and aerobic metabolism and more ROS are produced by mitochondria. Conversely, more antioxidants are available at this period to counteract and detoxify these reactive oxygen specieses (Hansen, 2006). Over the course of development, the delicate balance between oxidants and antioxidants can be disrupted by exogenous agents that

Thalidomide is associated with multiple birth defects, including phocomelia (Lecutier, 1962; Taussig, 1962). The most sensitive organ to thalidomide toxicity is the limb. Although the mechanism of teratogenesis and determinants of risk remain unclear, related teratogenic xenobiotics are bioactivated by embryonic prostaglandin H synthase (PHS) producing reactive oxygen species, which cause oxidative damage to DNA and other cellular macromolecules. Similarly, thalidomide is bioactivated by horseradish peroxidase, and oxidizes DNA and glutathione, indicating free radical-mediated oxidative stress. Furthermore, thalidomide teratogenicity is reduced by the PHS inhibitor acetylsalicylic acid, indicating PHS-catalyzed bioactivation. This appears to be regulated through redox shift resulting from depletion of GSH and increased GSSG in the nucleus, and this may imply

**3. The role of oxidative stress in embryo and fetal malformation** 

are in agreement with these basic scientific principles.

would in turn cause specific dysmorphogenesis.

simulate ROS production leading to oxidative stress.

that various transcription factors are affected by thalidomide through redox regulation (Hansen et al., 2001, 2002).

Exposure to the anticonvulsant valproic acid during the first trimester of pregnancy is associated with an increased risk of congenital malformations including heart defects, craniofacial abnormalities, skeletal and limb defects, and most frequently, neural tube defects (NTDs). The mechanisms by which valproic acid induces teratogenic effects are not fully understood, although previous studies support a role for oxidative stress. Valproic acid can alter cell signaling through gene expression changes mediated through histone deacetylase inhibition (Phiel et al., 2001), and is a direct inhibitor of class I and II histone deacetylases. Several laboratories have shown that embryonic histone acetylation levels are increased following exposure to valproic acid (Menegola et al., 2005; Tung and Winn, 2010). Furthermore, studies have supported a role for histone deacetylase inhibition as a mechanism of teratogenesis as analogs of valproic acid that lack histone deacetylase inhibitory activity are less teratogenic (Gurvich et al., 2005). Gene microarray studies have also demonstrated that valproic acid targets genes regulated by histone deacetylase, including *Mt1* and *Mt2*, both of which are ROS-sensitive (Jergil et al., 2009). In addition, histone deacetylase inhibitors have also been shown to increase ROS production and induce apoptosis in several cancer cell lines (Carew et al., 2008). Therefore, alterations in gene expression and/or increases in ROS formation mediated by histone deacetylase inhibition during development may induce teratogenesis.

The widely used anticonvulsant, phenytoin, can double the incidence of structural and functional birth defects when used in pregnancy (Kaneko et al., 1991). It can induce vascular disruption, which leads to hypoxia and hypoperfusion (Danielsson et al.,1995). In addition, phenytoin results in oxidative DNA damage and dysmorphogenesis, which can be eliminated by antioxidants (Winn and Wells, 1995). Phenytoin also selectively increased NF-kB activity in targeted tissues. Blocking of these signaling events with p65 antisense oligonucleotides eliminated the associated embryopathies (Kennedy et al., 2004). Further evidence that oxidative stress is important in phenytoin mediated toxicity is exemplified by the fact that treatment with polyethylene-modified superoxide dismutase enhances embryo toxicity whereas antioxidant levels were modulated with phenytoin (Winn and Wells, 1999).

Chronic ethanol consumption can lead to the generation of ROS and, as a consequence, teratogenicity. Prolonged ethanol exposure lead to increased production of lipid peroxides and decreased expression of antioxidant enzymes. Ascorbic acid can prevent against ethanol toxicity through inhibition of ROS formation and NF-kB activation (Peng et al.,2005). Zebrafish embryos exposed to ethanol with lipoic acid and/or only partially attenuated ethanol embryo toxicity, suggesting that other mechanisms are also involved (Reimers et al., 2006).

#### **4. Oxidative stress in pathological pregnancies**

#### **4.1 Preeclampsia**

Preeclampsia is a complex multisystem disorder that occurs during the pregnancy. The disease affects 5-8% of all pregnancies and is one of the leading couses of maternal and fetal

The Role of Oxidative Stress in Female Reproduction and Pregnancy 321

 **Endothelial dysfunction**

**PREECLAMPSIA**

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important enzyme that generates superoxide anion radical localized in the placenta syncytial microvillous membrane (Matsubara & Sato, 2001; Raijmakers et al., 2004). NADPH oxidase may play a role in placental lipid peroxidation by generating increased amounts of the superoxide anion radical. Poor antioxidant reserves can also tilt the balance in favor of prooxidation. Lipid peroxidation results in formation of primary lipid peroxidation products such as lipid hydroperoxides and secondary products such as malondialdehyde (MDA) and lipid peroxides. Lipid hydroperoxides are formed and bind to lipoproteins. They are then carried to distant sites where the hydroperoxides can cause ongoing lipid peroxidation and result in systemic oxidative stress. Increased placental production of lipid peroxides and thromboxane was demonstrated from both the trophoblast and the villous core components

Studies of women undergoing cesarean section showed significantly higher contents of lipid hydroperoxides, phospholipids, cholesterol and free 8-iso-prostaglandin F2α (8-iso-PGF2α), but not the total (free plus esterified) 8-iso-PGF2α in decidual tissues from women with preeclampsia as compared with tissues from normal pregnancies (Staff et al., 1999).

**Hypertension Proteinuria Edema**

Fig. 2. Supposed oxidative stress pathway in preeclampsia

of placentas in patients with preeclampsia (Walsh & Wang, 1995).

**ANTIOXIDANTS Activity of leukocytes Proinflammatory cytokines**

**Platelet activation**

**OXIDANTS Superoxid Dysmutase Peroxynitrine**

morbidity and mortality. It is characterized with hypertension and proteinuria. The systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥ 90 mmHg and the proteinuria at least 300 mg in 24 h urine collection. Women with mild proteinuria generally have no symptoms. However, women with severe preeclampsia (blood presure ≥160/110 mmHg, proteinuria >2-5g/24h) may have simptoms such as renal insufficiency, liver disease, haematological and neurological disturbances. Preeclampsia is charactized by vasospasm, reduced placental perfusion and abnormal placentation. The main couse of fetal compromise is the disturbance in uteroplacental perfusion. The only cure is the delivery of the baby. With antihypertensive treatment may prolong the pregnancy, increasing the chances of the baby to survive. If the blood presure cannot be controlled, or the laboratory parameters entry in a critical value the baby must be delivered. Preeclampsia has been proposed as a two-stage disorder. In first stage the placenta produces cytotoxic factors, in the second stage the maternal response to the placental factors occurs. There are several theories regarding to the main couse of disorder: abnormal placentation, immunological background, abnormal inflamatory response, etc. Assuming preeclampsia literature we can conclude that all the theories are part of disorders etiology.

#### **4.1.1 Oxidative agents**

Some reserches suggest that the placental oxidative stress may be involved in the ethiopathogenesis of preeclampisa. As there was mentioned above, the oxidatives stress is described as an imbalance in the production of reactive oxygen specieses and the ability of atioxidant defense to scavenge them. Pregnancy is a state of oxidative stress arrising from the increased metabolic activity in the placenta and reduced scavenging power of antioxidants (Wisdom et al. 1991). During the gestation the oxygenation of the uteroplacental unit is changing. The placenta and fetus exist in a hypoxic enviromet during early pregnancy as the uterine oxygen tension is extremly low till 8-10. weeks of gestation (pO2<20 mmHg, 5%O2), prior to estabilishment of the blood flow into intervillous space. The onset of blood flow is processig from the periphery to the center of placental disc, with villous regression in the placental periphery envolving into the chorion leave (Jauniaux et al., 2003). The developing chorioallantoic villous trees are exposed to a marked increase of pO2 in a range of 40-80 mm Hg (Sjostedt et al., 1960; Shaaps et al., 2005; Rooth et al., 1961). This reoxygenation of the uteroplacental unit results an oxidative burst, controlled by antioxidant mechanisms.

Proposed effect of oxidative stress on placental fatty acid metabolism.

As a consequence of abnormal trophoblast invasion, and maternofetal barrier preeclampsia is characterized by induced oxidative stress and decreased antioxidants (Patil et al., 2009). In preeclamptic women, maternal circulating levels, placental tissue levels and production rate of lipid peroxides are increased and several antioxidants are markedly decreased (Serdar et al., 2003; Orhan et al., 2003). Normal pregnancy is associated with physiological hyperlipidemia (Belo et al., 2004). Physiological alterations are manifested by increased levels of triglycerides and cholesterol in pregnancy, which decreases rapidly after delivery. Preeclampsia is characterized by further elevation of serum triglycerides and serum free fatty acids (Hubel et al., 1996). (Figure 2.) Increased lipid peroxidation has been reported in preeclampsia, IUGR (Liu et al., 2005; Gupta et al., 2004, Bretelle et al., 2004).

morbidity and mortality. It is characterized with hypertension and proteinuria. The systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥ 90 mmHg and the proteinuria at least 300 mg in 24 h urine collection. Women with mild proteinuria generally have no symptoms. However, women with severe preeclampsia (blood presure ≥160/110 mmHg, proteinuria >2-5g/24h) may have simptoms such as renal insufficiency, liver disease, haematological and neurological disturbances. Preeclampsia is charactized by vasospasm, reduced placental perfusion and abnormal placentation. The main couse of fetal compromise is the disturbance in uteroplacental perfusion. The only cure is the delivery of the baby. With antihypertensive treatment may prolong the pregnancy, increasing the chances of the baby to survive. If the blood presure cannot be controlled, or the laboratory parameters entry in a critical value the baby must be delivered. Preeclampsia has been proposed as a two-stage disorder. In first stage the placenta produces cytotoxic factors, in the second stage the maternal response to the placental factors occurs. There are several theories regarding to the main couse of disorder: abnormal placentation, immunological background, abnormal inflamatory response, etc. Assuming preeclampsia literature we can

Some reserches suggest that the placental oxidative stress may be involved in the ethiopathogenesis of preeclampisa. As there was mentioned above, the oxidatives stress is described as an imbalance in the production of reactive oxygen specieses and the ability of atioxidant defense to scavenge them. Pregnancy is a state of oxidative stress arrising from the increased metabolic activity in the placenta and reduced scavenging power of antioxidants (Wisdom et al. 1991). During the gestation the oxygenation of the uteroplacental unit is changing. The placenta and fetus exist in a hypoxic enviromet during early pregnancy as the uterine oxygen tension is extremly low till 8-10. weeks of gestation (pO2<20 mmHg, 5%O2), prior to estabilishment of the blood flow into intervillous space. The onset of blood flow is processig from the periphery to the center of placental disc, with villous regression in the placental periphery envolving into the chorion leave (Jauniaux et al., 2003). The developing chorioallantoic villous trees are exposed to a marked increase of pO2 in a range of 40-80 mm Hg (Sjostedt et al., 1960; Shaaps et al., 2005; Rooth et al., 1961). This reoxygenation of the uteroplacental unit results an oxidative burst, controlled by

As a consequence of abnormal trophoblast invasion, and maternofetal barrier preeclampsia is characterized by induced oxidative stress and decreased antioxidants (Patil et al., 2009). In preeclamptic women, maternal circulating levels, placental tissue levels and production rate of lipid peroxides are increased and several antioxidants are markedly decreased (Serdar et al., 2003; Orhan et al., 2003). Normal pregnancy is associated with physiological hyperlipidemia (Belo et al., 2004). Physiological alterations are manifested by increased levels of triglycerides and cholesterol in pregnancy, which decreases rapidly after delivery. Preeclampsia is characterized by further elevation of serum triglycerides and serum free fatty acids (Hubel et al., 1996). (Figure 2.) Increased lipid peroxidation has been reported in

conclude that all the theories are part of disorders etiology.

Proposed effect of oxidative stress on placental fatty acid metabolism.

preeclampsia, IUGR (Liu et al., 2005; Gupta et al., 2004, Bretelle et al., 2004).

**4.1.1 Oxidative agents** 

antioxidant mechanisms.

Fig. 2. Supposed oxidative stress pathway in preeclampsia

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important enzyme that generates superoxide anion radical localized in the placenta syncytial microvillous membrane (Matsubara & Sato, 2001; Raijmakers et al., 2004). NADPH oxidase may play a role in placental lipid peroxidation by generating increased amounts of the superoxide anion radical. Poor antioxidant reserves can also tilt the balance in favor of prooxidation. Lipid peroxidation results in formation of primary lipid peroxidation products such as lipid hydroperoxides and secondary products such as malondialdehyde (MDA) and lipid peroxides. Lipid hydroperoxides are formed and bind to lipoproteins. They are then carried to distant sites where the hydroperoxides can cause ongoing lipid peroxidation and result in systemic oxidative stress. Increased placental production of lipid peroxides and thromboxane was demonstrated from both the trophoblast and the villous core components of placentas in patients with preeclampsia (Walsh & Wang, 1995).

Studies of women undergoing cesarean section showed significantly higher contents of lipid hydroperoxides, phospholipids, cholesterol and free 8-iso-prostaglandin F2α (8-iso-PGF2α), but not the total (free plus esterified) 8-iso-PGF2α in decidual tissues from women with preeclampsia as compared with tissues from normal pregnancies (Staff et al., 1999).

The Role of Oxidative Stress in Female Reproduction and Pregnancy 323

preeclampsia (Kharb et al., 2000). In patients with preeclampsia, antioxidants scavenge the increased free radicals, resulting in lowered antioxidant levels. Water-soluble antioxidants may function as a first line of antioxidants to scavenge excess of reactive oxygen species in plasma, whereas lipid soluble antioxidants such as tocopherol and carotene scavenge

The activities of placental superoxide dismutase and glucose-6-phosphate dehydrogenase are decreased in preeclampsia compared to normal pregnancy (Poranen et al., 1996). Moreover, the activity and mRNA expression of Cu,Zn-SOD, glutathione peroxidase, and tissue levels of vitamin E are significantly lower in placental tissues from preeclampsia than from normal pregnancy (Wnag & Walsh, 1996). Glutathione and its related enzymes are antioxidants that help detoxify the increased generation of free radicals. Significantly reduced whole blood glutathione levels have been reported in women with preeclampsia

The other main caracteristics of preeclampsia is the exacerbated inflamatory state (Bretelle et al., 2004; Holthe et al., 2005; Redman et al., 1999). Activated leukocytes, both monocytes and granulocytes, generate excess reactive oxygen specieses resulting in oxidative stress (Holthe et al., 2004). Compared with normotensive pregnant women, women with preeclampsia have higher levels of calprotectin, a protein involved in various physiological inflammatory processes, which is indicative of leukocyte activation (Holthe et al., 2005). The expression of surface adhesion molecules on cord blood neutrophils was significantly higher in infants born to women with preeclampsia than in infants born to the control subjects. Increased TNF secretion by leukocytes was detected in blood from patients with preeclampsia, providing further evidence of leukocyte activation (Beckman et al., 2004). TNF-α can activate the endothelial cells and upregulate the gene expression of numerous molecules such as platelet-derived growth factor, cell adhesion molecules, endothelin-1 and PAI-1. These molecules have been reported to have detrimental effects on the vasculature and also characterize preeclamptic pregnancy (Hajjar et al., 1987; van Hinsbrgh et al., 1988). Furthermore, chronic infusion of TNF-α into rats during late pregnancy results in a significant increase in renal vascular resistance and arterial pressure (Alexander et al., 2002;

Endothelial dysfunction is also one of the main pathogenic features of preeclampsia. The markers of endothelial dysfunction such as tissue plasminogen activator, von Willebrand factor, sE-selectin, and fibronectin are elevated in patients with preeclampsia (Aydin et al., 2004; Stubbs et al., 1984; Halligan et al., 1994). Although the exact mechanisms of vascular endothelial damage in preeclampsia are unclear, increased lipid peroxidation may lead to endothelial cell dysfunction (Davidge et al., 1996). Tumor necrosis factor (TNF), tissue factor (TF) of placental origin, endothelial nitric oxide synthase (NOS), and excessive activity of the enzyme polymerase may contribute to endothelial dysfunction. Compared with normotensive pregnant women, women with preeclampsia have reduced expression of constitutive nitrite oxidative stress -mRNA, and this lead to reduced production of NO.

reactive oxygen species affecting the membrane lipids (Mikhail et al., 1994).

and HELPP syndrome (Madazil et al., 2002; Knapen et al., 1998).

**4.1.3 Leukocyte activation** 

Giardina et al., 2002).

**4.1.4 Endothelial cell dysfunction** 

Moreover, tissue levels of free and total 8-iso-PGF2α are significantly higher in preeclamptic placenta than in normal placenta (Walsh et al., 2000). Isoprostanes like 8-iso-PGF2α are produced specifically by free radical-catalyzed peroxidation of arachidonic acid (Morrow et al., 1990). Free 8-iso-PGF2α has activities of relevance to preeclampsia, being a potent vasoconstrictor in kidney (Morrow et al., 1990) and placenta (Kwek et al., 1990), platelet activator (Minzu et al., 2002), and inducer of the release of endothelin from endothelial cells (Yura et al. 1999).

An increase in diastolic pressure correlates significantly with an increase in lipid peroxide levels, indicating that the severity of hypertension is correlated with the extent of lipid peroxidation (Aydin et al., 2004; Jain & Wise, 1995; Gupta et al., 2006). Women with preeclampsia have significantly higher mean plasma levels of malonaldehyde and significantly lower superoxide dismutase levels compared with normotensive pregnant women, (Aydin et al., 2004). The decrease in nitric oxide (NO) and superoxide dismutase (SOD) levels followed by a concomitant increase in levels of malonaledehyde, fibronectin, endothelin-1 (ET-1), and soluble-E selectin (sE-selectin) correlate with an increase in diastolic blood pressure (Aydin et al., 2004). In further studies, malonaldehyde levels in maternal erythrocytes were significantly elevated in women with developed preeclampsia. The risk of developing preeclampsia was 24-fold higher when malonaldehyde levels were above the cutoff value of 36 nmol/g (Basbug et al., 2003).

The cord plasma malonaldehyde and vitamin E levels were higher in patients with eclampsia than in patients with preeclampsia and in normotensive pregnant patients (Bowen et al. 2001).

Hyperhomocystinemia and altered eicosanoid synthesis has also been implicated in the pathophysiology of preeclampsia. Eicosanoids have vasoactive properties and enhance lipid peroxidation and decrease prostacyclin synthesis. The generation of the eicosanoid, 15 hydroxyeicosateranoic acid by the placenta was higher in women with preeclampsia than in normotensive control subjects. In preeclampsia, there is increased synthesis of thromboxane and reduced synthesis of prostacyclin. Lipid peroxides may also stimulate the cyclooxygenase enzyme to produce more thromboxane, resulting in a hypercoagulable state (Walsh, 2004).

#### **4.1.2 Antioxidant agents**

Antioxidants can be enzymatic or nonenzymatic. The enzymatic antioxidants are superoxide dismutase, thioredoxin, thioredoxin reductase, and glutathione peroxidase. The nonenzymatic antioxidants can be lipid-soluble such as vitamin E or water-soluble such as vitamin C. Serum levels of vitamin E and beta carotene (Serdar et al., 2003; Akyol et al., 2000), serum coenzyme Q10 and tocopherol levels (Palan et al., 2004), ascorbic acid were significantly reduced in pregnancies complicated by mild or severe preeclampsia, and the total antioxidant capacity was significantly reduced in pregnant women with mild and severe preeclampsia (Sagol et al., 1999). The balance between lipid peroxides and antioxidant vitamin E is tipped in favor of lipid peroxides in patients with mild and severe preeclampsia. A two-fold increase in the ratio between lipid peroxidation and antioxidant capacity was reported in the antepartum period in women with preeclampsia (Davidge et al., 1992). Significantly lower levels of vitamin C, E, and total thiol were seen in women with preeclampsia (Kharb et al., 2000). In patients with preeclampsia, antioxidants scavenge the increased free radicals, resulting in lowered antioxidant levels. Water-soluble antioxidants may function as a first line of antioxidants to scavenge excess of reactive oxygen species in plasma, whereas lipid soluble antioxidants such as tocopherol and carotene scavenge reactive oxygen species affecting the membrane lipids (Mikhail et al., 1994).

The activities of placental superoxide dismutase and glucose-6-phosphate dehydrogenase are decreased in preeclampsia compared to normal pregnancy (Poranen et al., 1996). Moreover, the activity and mRNA expression of Cu,Zn-SOD, glutathione peroxidase, and tissue levels of vitamin E are significantly lower in placental tissues from preeclampsia than from normal pregnancy (Wnag & Walsh, 1996). Glutathione and its related enzymes are antioxidants that help detoxify the increased generation of free radicals. Significantly reduced whole blood glutathione levels have been reported in women with preeclampsia and HELPP syndrome (Madazil et al., 2002; Knapen et al., 1998).

#### **4.1.3 Leukocyte activation**

322 Oxidative Stress and Diseases

Moreover, tissue levels of free and total 8-iso-PGF2α are significantly higher in preeclamptic placenta than in normal placenta (Walsh et al., 2000). Isoprostanes like 8-iso-PGF2α are produced specifically by free radical-catalyzed peroxidation of arachidonic acid (Morrow et al., 1990). Free 8-iso-PGF2α has activities of relevance to preeclampsia, being a potent vasoconstrictor in kidney (Morrow et al., 1990) and placenta (Kwek et al., 1990), platelet activator (Minzu et al., 2002), and inducer of the release of endothelin from endothelial cells

An increase in diastolic pressure correlates significantly with an increase in lipid peroxide levels, indicating that the severity of hypertension is correlated with the extent of lipid peroxidation (Aydin et al., 2004; Jain & Wise, 1995; Gupta et al., 2006). Women with preeclampsia have significantly higher mean plasma levels of malonaldehyde and significantly lower superoxide dismutase levels compared with normotensive pregnant women, (Aydin et al., 2004). The decrease in nitric oxide (NO) and superoxide dismutase (SOD) levels followed by a concomitant increase in levels of malonaledehyde, fibronectin, endothelin-1 (ET-1), and soluble-E selectin (sE-selectin) correlate with an increase in diastolic blood pressure (Aydin et al., 2004). In further studies, malonaldehyde levels in maternal erythrocytes were significantly elevated in women with developed preeclampsia. The risk of developing preeclampsia was 24-fold higher when malonaldehyde levels were

The cord plasma malonaldehyde and vitamin E levels were higher in patients with eclampsia than in patients with preeclampsia and in normotensive pregnant patients

Hyperhomocystinemia and altered eicosanoid synthesis has also been implicated in the pathophysiology of preeclampsia. Eicosanoids have vasoactive properties and enhance lipid peroxidation and decrease prostacyclin synthesis. The generation of the eicosanoid, 15 hydroxyeicosateranoic acid by the placenta was higher in women with preeclampsia than in normotensive control subjects. In preeclampsia, there is increased synthesis of thromboxane and reduced synthesis of prostacyclin. Lipid peroxides may also stimulate the cyclooxygenase enzyme to produce more thromboxane, resulting in a hypercoagulable state

Antioxidants can be enzymatic or nonenzymatic. The enzymatic antioxidants are superoxide dismutase, thioredoxin, thioredoxin reductase, and glutathione peroxidase. The nonenzymatic antioxidants can be lipid-soluble such as vitamin E or water-soluble such as vitamin C. Serum levels of vitamin E and beta carotene (Serdar et al., 2003; Akyol et al., 2000), serum coenzyme Q10 and tocopherol levels (Palan et al., 2004), ascorbic acid were significantly reduced in pregnancies complicated by mild or severe preeclampsia, and the total antioxidant capacity was significantly reduced in pregnant women with mild and severe preeclampsia (Sagol et al., 1999). The balance between lipid peroxides and antioxidant vitamin E is tipped in favor of lipid peroxides in patients with mild and severe preeclampsia. A two-fold increase in the ratio between lipid peroxidation and antioxidant capacity was reported in the antepartum period in women with preeclampsia (Davidge et al., 1992). Significantly lower levels of vitamin C, E, and total thiol were seen in women with

above the cutoff value of 36 nmol/g (Basbug et al., 2003).

(Yura et al. 1999).

(Bowen et al. 2001).

(Walsh, 2004).

**4.1.2 Antioxidant agents** 

The other main caracteristics of preeclampsia is the exacerbated inflamatory state (Bretelle et al., 2004; Holthe et al., 2005; Redman et al., 1999). Activated leukocytes, both monocytes and granulocytes, generate excess reactive oxygen specieses resulting in oxidative stress (Holthe et al., 2004). Compared with normotensive pregnant women, women with preeclampsia have higher levels of calprotectin, a protein involved in various physiological inflammatory processes, which is indicative of leukocyte activation (Holthe et al., 2005). The expression of surface adhesion molecules on cord blood neutrophils was significantly higher in infants born to women with preeclampsia than in infants born to the control subjects. Increased TNF secretion by leukocytes was detected in blood from patients with preeclampsia, providing further evidence of leukocyte activation (Beckman et al., 2004). TNF-α can activate the endothelial cells and upregulate the gene expression of numerous molecules such as platelet-derived growth factor, cell adhesion molecules, endothelin-1 and PAI-1. These molecules have been reported to have detrimental effects on the vasculature and also characterize preeclamptic pregnancy (Hajjar et al., 1987; van Hinsbrgh et al., 1988). Furthermore, chronic infusion of TNF-α into rats during late pregnancy results in a significant increase in renal vascular resistance and arterial pressure (Alexander et al., 2002; Giardina et al., 2002).

#### **4.1.4 Endothelial cell dysfunction**

Endothelial dysfunction is also one of the main pathogenic features of preeclampsia. The markers of endothelial dysfunction such as tissue plasminogen activator, von Willebrand factor, sE-selectin, and fibronectin are elevated in patients with preeclampsia (Aydin et al., 2004; Stubbs et al., 1984; Halligan et al., 1994). Although the exact mechanisms of vascular endothelial damage in preeclampsia are unclear, increased lipid peroxidation may lead to endothelial cell dysfunction (Davidge et al., 1996). Tumor necrosis factor (TNF), tissue factor (TF) of placental origin, endothelial nitric oxide synthase (NOS), and excessive activity of the enzyme polymerase may contribute to endothelial dysfunction. Compared with normotensive pregnant women, women with preeclampsia have reduced expression of constitutive nitrite oxidative stress -mRNA, and this lead to reduced production of NO.

The Role of Oxidative Stress in Female Reproduction and Pregnancy 325

Fig. 3. Mechanisms by which hyperglycemia induces cellular dysfunction and damage.

carbohydtrates and the free amino groups on proteins, lipids, and nucleic acids. Elevated concentration of glucose leads to enhanced formation glycolytic products which together with the tricarboxylic acid (TCA) cycle intermediates provide glycation of intracellular proteins. The interaction of aldehyde groups of glucose with free amino groups on proteins generates a Schiff's base. Extracellular AGE can bind to the AGE receptor (RAGE), a multiligand member of the immunoglobulin superfamily. Engagement of RAGE by AGE results in activation of intracellular signaling molecules resulting in oxidative stress and inflammation. Since oxidative stress induction and inflammation are closely associated with gestational diabetes (Coughlan et al., 2001; Lappas et al., 2004), it is plausible that the AGE-RAGE system could play a role in the pathogenesis of this metabolic disease. During pregnancy, the AGE-RAGE axis may be involved in oxidative and inflammatory responses. Specifically, AGE-BSA stimulated the release of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α and prostaglandins PGE2 and PGF2α. NF-κB and MAPK activate expression of several pro-inflammatory genes, including pro-inflammatory cytokines, the adhesion molecules vascular cell adhesion molecule (VCAM)-1, and intercellular cell adhesion molecule (ICAM)-1, and RAGE causing cellular inflammation. This is consistent with gestational diabetes being closely associated with low-grade inflammation (Kirwan et al., 2002) and atherosclerosis (Anastasiou et al., 1998; Hannemann et al., 2002). Additionally, the activation of cytokines by AGE in human placenta may also be involved in insulin resistance associated with gestational diabetes (Colomiere et al., 2009).

Hyperglycemia also activats hexosamine biosynthetic pathway (Rajapakse et al., 2009). This pathway of glucose metabolism uses fructose-6-phosphate derived from glycolysis to metabolize glucosamine-6-phosphate by glucosamine-6-phosphate amidotransferase.

NOS inhibition consequence is the increased endothelial permeability and an abnormal response of the endothelial cells to the stress (Wang et al., 2004). In preeclampsia greater nitrotyrosine immunostaining were found in placental villous vascular endothelium and its surrounding smooth muscle cells, and also in villous stromal cells compared to normal pregnant controls (Myatt et al., 2006). Moreover particularly intense immunoreactivity of nitrotyrosine was measured within the invasive cytotrophoblasts in placental biopsies and vascular endothelium in the floating villi obtained from women with preeclampsia (Many et al., 2000).

#### **4.1.5 Vascular developement**

Aberrant placental vasculature development and abnormal placental blood flow are characterized by increased impedance in Doppler velocimetry (Farag et al., 2004). These abnormalities significantly correlated with expression of tissue factor in the placenta of women with severe preeclampsia (Di Paolo et al., 2003). The expression of tissue factor was found to be markedly increased in the endothelial cells within the basal deciduus. Doppler impedance modifications were significantly correlated to the endothelial cell activation. Tumor necrosis factor, a circulating cytokine, has also been implicated as causing endothelial dysfunction in preeclampsia (Hung et al., 2004). Significantly higher tissue levels of tumor necrosis factor were demonstrated in the placenta from women with preeclampsia (Wang et al., 1996). Higher levels of tumor necrosis factor lead to increased generation of Eselectin, a marker of endothelial activation of umbilical endothelial cells.

#### **4.2 Oxidative stress in gestational diabetes (GDM)**

Gestational diabetes is defined as a carbohydrate intolerance of variable severity, which begins, or is identified during the pregnancy (Lopez et al. 2011). The prevalence of gestational diabetes mellitus is around 5% of all pregnancies (Ben Haroush et al., 2004). The presence of this disease increases the risk of macrosomia, perinatal morbido-mortality (Ostlund et al., 2003) and subsequent developement of type 2 diabetes mellitus. The pathophysiology of gestational diabetes remain unclear. Pregnant women with gestational diabetes have a reduction in insulin sensitivity (Catalano et al., 1999), hypergicemia, and hyperlipidemia. Oxidative stress implication in developement of the disease is a result of imbalance between the increase in the formation of reactive oxidative substances (Brownlee, 2001; Maddux et al., 2001) and the insufficience of antioxidative defence mechanisms (Chen et al., 2003).

#### **4.2.1 Induction of oxidative stress in gestational diaetes pathways**

Hyperglycemia induces oxidative stress and cell and tissue damage through several metabolic mechanisms.

These include the formation of advanced glycation endproducts (AGE), activation of protein kinase C (PKC), the hexosamine pathway, and increased reactive oxygen specieses production in the mitochondria. An important source of free radicals in diabetes is the interaction of glucose with proteins. Maillard reaction that form by nonenzymatic glycation through covalent attachment of highly reactive aldehyde or ketone groups of reducing

NOS inhibition consequence is the increased endothelial permeability and an abnormal response of the endothelial cells to the stress (Wang et al., 2004). In preeclampsia greater nitrotyrosine immunostaining were found in placental villous vascular endothelium and its surrounding smooth muscle cells, and also in villous stromal cells compared to normal pregnant controls (Myatt et al., 2006). Moreover particularly intense immunoreactivity of nitrotyrosine was measured within the invasive cytotrophoblasts in placental biopsies and vascular endothelium in the floating villi obtained from women with preeclampsia (Many et

Aberrant placental vasculature development and abnormal placental blood flow are characterized by increased impedance in Doppler velocimetry (Farag et al., 2004). These abnormalities significantly correlated with expression of tissue factor in the placenta of women with severe preeclampsia (Di Paolo et al., 2003). The expression of tissue factor was found to be markedly increased in the endothelial cells within the basal deciduus. Doppler impedance modifications were significantly correlated to the endothelial cell activation. Tumor necrosis factor, a circulating cytokine, has also been implicated as causing endothelial dysfunction in preeclampsia (Hung et al., 2004). Significantly higher tissue levels of tumor necrosis factor were demonstrated in the placenta from women with preeclampsia (Wang et al., 1996). Higher levels of tumor necrosis factor lead to increased generation of E-

Gestational diabetes is defined as a carbohydrate intolerance of variable severity, which begins, or is identified during the pregnancy (Lopez et al. 2011). The prevalence of gestational diabetes mellitus is around 5% of all pregnancies (Ben Haroush et al., 2004). The presence of this disease increases the risk of macrosomia, perinatal morbido-mortality (Ostlund et al., 2003) and subsequent developement of type 2 diabetes mellitus. The pathophysiology of gestational diabetes remain unclear. Pregnant women with gestational diabetes have a reduction in insulin sensitivity (Catalano et al., 1999), hypergicemia, and hyperlipidemia. Oxidative stress implication in developement of the disease is a result of imbalance between the increase in the formation of reactive oxidative substances (Brownlee, 2001; Maddux et al., 2001) and the insufficience of antioxidative defence mechanisms (Chen

Hyperglycemia induces oxidative stress and cell and tissue damage through several

These include the formation of advanced glycation endproducts (AGE), activation of protein kinase C (PKC), the hexosamine pathway, and increased reactive oxygen specieses production in the mitochondria. An important source of free radicals in diabetes is the interaction of glucose with proteins. Maillard reaction that form by nonenzymatic glycation through covalent attachment of highly reactive aldehyde or ketone groups of reducing

selectin, a marker of endothelial activation of umbilical endothelial cells.

**4.2.1 Induction of oxidative stress in gestational diaetes pathways** 

**4.2 Oxidative stress in gestational diabetes (GDM)** 

al., 2000).

et al., 2003).

metabolic mechanisms.

**4.1.5 Vascular developement** 

Fig. 3. Mechanisms by which hyperglycemia induces cellular dysfunction and damage.

carbohydtrates and the free amino groups on proteins, lipids, and nucleic acids. Elevated concentration of glucose leads to enhanced formation glycolytic products which together with the tricarboxylic acid (TCA) cycle intermediates provide glycation of intracellular proteins. The interaction of aldehyde groups of glucose with free amino groups on proteins generates a Schiff's base. Extracellular AGE can bind to the AGE receptor (RAGE), a multiligand member of the immunoglobulin superfamily. Engagement of RAGE by AGE results in activation of intracellular signaling molecules resulting in oxidative stress and inflammation. Since oxidative stress induction and inflammation are closely associated with gestational diabetes (Coughlan et al., 2001; Lappas et al., 2004), it is plausible that the AGE-RAGE system could play a role in the pathogenesis of this metabolic disease. During pregnancy, the AGE-RAGE axis may be involved in oxidative and inflammatory responses. Specifically, AGE-BSA stimulated the release of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α and prostaglandins PGE2 and PGF2α. NF-κB and MAPK activate expression of several pro-inflammatory genes, including pro-inflammatory cytokines, the adhesion molecules vascular cell adhesion molecule (VCAM)-1, and intercellular cell adhesion molecule (ICAM)-1, and RAGE causing cellular inflammation. This is consistent with gestational diabetes being closely associated with low-grade inflammation (Kirwan et al., 2002) and atherosclerosis (Anastasiou et al., 1998; Hannemann et al., 2002). Additionally, the activation of cytokines by AGE in human placenta may also be involved in insulin resistance associated with gestational diabetes (Colomiere et al., 2009).

Hyperglycemia also activats hexosamine biosynthetic pathway (Rajapakse et al., 2009). This pathway of glucose metabolism uses fructose-6-phosphate derived from glycolysis to metabolize glucosamine-6-phosphate by glucosamine-6-phosphate amidotransferase.

The Role of Oxidative Stress in Female Reproduction and Pregnancy 327

increases in NO production in different tissues. Reduction in NO-induced stress is related to diabetes-induced endothelial dysfunction, NOS activation can also be induced by diabetes. NO induces the expression of antioxidant enzymes Mn- and Cu,Zn-SODs, and heme oxygenase-1 and increases intracellular glutathione concentration (Moellering et al., 1999). Although NO stimulates O2−-induced lipoperoxidation in membranes, it can also mediate protective reactions to inhibit O2− and ONOO− induced lipoperoxidation (Rubbo et al., 1994). NO production has been found increased in the placenta, placental veins and arteries, and in umbilical vein endothelial cells from estational diabetes mellitus patients (Figueroa et al., 2000; vonMadach et al., 2003). NOS expression is also altered, as NOS has been found overexpressed in the placenta and eNoxidative stress increased in umbilical vein endothelial cells from estational diabetes mellitus patients (SanMartin et al., 2006). In estational diabetes mellitus, increases in reactive oxygen specieses and NO production, evident in the placenta and umbilical vessels, lead to peroxynitrite formation. In platelets from estational diabetes mellitus patients, elevated NOS activity and peroxynitrite production have been reported, possibly associated with platelet dysfunction and membrane damage due to increased lipid peroxidation (Mazzanti et al. , 2004). Strong protein nitration is found in term placentas from diabetic rats. Collectively, these data provide evidence of reactive nitrogen species (RNS)-induced damage in estational diabetes mellitus in the placenta and the vasculature of the mother, the placenta, and the umbilical cord, produced as a resulting consequence of

**4.2.4 Role of oxidative stress in gestational diabetes-induced teratogenesis** 

Diabetes in pregnancy is associated with suboptimal decidualization (Garris et al., 1988). NO plays a key role in decidualization and embryo implantation (Norwitz et al., 2001). It increases vascular permeability, vasodilation, and blood flow in the uterus, and is a component of the decidual cell reaction (Valdes et al., 2009). Diabetes during pregnancy is associated with embryonic dysmorphogenesis. Due to its capacity to regulate cell survival, apoptosis, differentiation, oxidative and nitrosative stresses play a significant role in embryo organogenesis. Low and high levels of NO can lead to embryonic maldevelopment, possibly due to an improper regulation of apoptotic events. During embryo and fetal development, NO has been found to be relevant in regulating differentiation of lung branching morphogenesis, cephalic morphogenesis, heart development, and nephrogenesis (Bloch et

Transcription factors, as paired box (PAX)-3 and peroxisome proliferator-activated receptor (PPAR) δ, has been found to be involved in the induction of both neural tube and heart malformations, the most common malformations in gestational diabetes pregnants (Higa et al., 2007; Loeken et al., 2006). Different antioxidants such as α-tocopherol and gluthatione ethyl ester increase expression of *PAX-3* and prevent apoptosis and the induction of hyperglycemia-induced neural tube and heart defects (Chang et al., 2003; Morgan et al. 2008). The higher 8-isoprostane levels observed in the offspring of diabetic animals (Wentzel & Erikkson, 2002; Wentzel et al., 1999) have its own teratogenic potency. Diabetic embryopathy is also associated with inhibition of GAPDH activity resulting from an excess of reactive oxygen speciesesin the embryo (Wentzel et al., 2002). Oxidative glucose metabolism is low and about 80% of the glucose used by the placenta. The effect of oxidative stress on placental glucose metabolism is not known. However, in nongestational tissues,

exacerbated NO and ROS production.

al., 1999; Tain et al., 2010).

Glucosamine-6-phosphate is a competitive inhibitor of glucose-6-phosphate dehydrogenase (G6PDH). Glucosamine-6 phosphate produced in the hexosamine pathway, leads to decreased NAPDH concentrations, diminished cellular GSH levels, and elevated oxidative stress. The activity of G6PDH also rapidly increases in response to intracellular reactive oxygen specieses production (Jian et al., 2003).

NADPH oxidase is a membrane enzyme complex accounting for ROS generation by electron transport chain and the enzyme is especially important in redox signaling. Under diabetic conditions it can be stimulated by AGE, insulin, and angiotensin II. Hypoxia possibly induces all these stimuli, which can activate NADPH oxidase. Once activated in response to high glucose levels, NADPH oxidase catalyzes the transfer of electrons from NADPH to molecular oxygen to produce O2,- convetred to H2O2. High glucose levels lead to generation of reactive oxygen specieses by stimulation of NADPH oxidase (Gao et al., 2009; Gupte et al., 2010).

Under physiological conditions reactive oxygen specieses are eliminated by cellular defense mechanisms, including diverse enzymes and vitamins. Imbalance of reactive oxygen specieses production and antioxidant systems of a cell can lead to an upregulation of expression of antioxidant enzyme encoding genes. Hyperglycemia causes excessive reactive oxygen specieses formation, thus activating the Nrf2/ARE pathway (Xue et al., 2008). NADPH oxidase was shown to be higher expressed and activated in endothelial cells of pregnant women with GDM (Sankaralingam et al., 2009). Protein kinase C (PKC) promotes the activation of mitochondrial NADPH oxidase, thereby leading to oxidative stress events. Once stimulated, NADPH oxidase reduces glutathione levels and impairs the cellular antioxidant defense systems (King et al., 2004).

#### **4.2.2 Oxidant species**

Maternal MDA levels in serum and plasma are increased in gestational diabete mellitus women compared to normal glucose tolerant (NGT) pregnant women (Chaudhari et al., 2003; Surapanieni et al., 2008). Higher levels of lipid peroxidation are evident in patients with poor glycemic control. Proteins undergo oxidative damage, they become increasingly susceptible to proteolytic degradation. Erythrocytes contain proteolytic enzymes that can degrade oxidatively damaged proteins such as hemoglobin, thus preventing the accumulation of nonfunctional proteins and protein fragments. GDM is associated with higher levels of maternal erythrocyte proteolytic activity than NGT controls (Kamath et al., 1998).

#### **4.2.3 Antioxidants**

The level of total superoxide dismutase in placental tissues of gestational diabetes mellitus (both diet- and insulin-controlled) patients is lower (Kinalski et al., 2001) or did not significantly change (Biri et al., 2006; Lappas et al., 2010). Relative ratio of Cu,Zn-SOD to 8 isoprostane or protein carbonyl was lower in gestational diabetes mellitus placentas, suggesting that the increase in superoxide deismutase is not sufficient to compensate for the developed oxidative stress (Coughlan et al., 2004).

Oxidative stress plays a significant role in both NO overproduction and loss of NO bioavailability (Gloire et al., 2006; Xia et al., 2007). Oxidative stress leads to NOS-dependent

Glucosamine-6-phosphate is a competitive inhibitor of glucose-6-phosphate dehydrogenase (G6PDH). Glucosamine-6 phosphate produced in the hexosamine pathway, leads to decreased NAPDH concentrations, diminished cellular GSH levels, and elevated oxidative stress. The activity of G6PDH also rapidly increases in response to intracellular reactive

NADPH oxidase is a membrane enzyme complex accounting for ROS generation by electron transport chain and the enzyme is especially important in redox signaling. Under diabetic conditions it can be stimulated by AGE, insulin, and angiotensin II. Hypoxia possibly induces all these stimuli, which can activate NADPH oxidase. Once activated in response to high glucose levels, NADPH oxidase catalyzes the transfer of electrons from NADPH to molecular oxygen to produce O2,- convetred to H2O2. High glucose levels lead to generation of reactive oxygen specieses by stimulation of NADPH oxidase (Gao et al., 2009; Gupte et

Under physiological conditions reactive oxygen specieses are eliminated by cellular defense mechanisms, including diverse enzymes and vitamins. Imbalance of reactive oxygen specieses production and antioxidant systems of a cell can lead to an upregulation of expression of antioxidant enzyme encoding genes. Hyperglycemia causes excessive reactive oxygen specieses formation, thus activating the Nrf2/ARE pathway (Xue et al., 2008). NADPH oxidase was shown to be higher expressed and activated in endothelial cells of pregnant women with GDM (Sankaralingam et al., 2009). Protein kinase C (PKC) promotes the activation of mitochondrial NADPH oxidase, thereby leading to oxidative stress events. Once stimulated, NADPH oxidase reduces glutathione levels and impairs the cellular

Maternal MDA levels in serum and plasma are increased in gestational diabete mellitus women compared to normal glucose tolerant (NGT) pregnant women (Chaudhari et al., 2003; Surapanieni et al., 2008). Higher levels of lipid peroxidation are evident in patients with poor glycemic control. Proteins undergo oxidative damage, they become increasingly susceptible to proteolytic degradation. Erythrocytes contain proteolytic enzymes that can degrade oxidatively damaged proteins such as hemoglobin, thus preventing the accumulation of nonfunctional proteins and protein fragments. GDM is associated with higher levels of

The level of total superoxide dismutase in placental tissues of gestational diabetes mellitus (both diet- and insulin-controlled) patients is lower (Kinalski et al., 2001) or did not significantly change (Biri et al., 2006; Lappas et al., 2010). Relative ratio of Cu,Zn-SOD to 8 isoprostane or protein carbonyl was lower in gestational diabetes mellitus placentas, suggesting that the increase in superoxide deismutase is not sufficient to compensate for the

Oxidative stress plays a significant role in both NO overproduction and loss of NO bioavailability (Gloire et al., 2006; Xia et al., 2007). Oxidative stress leads to NOS-dependent

maternal erythrocyte proteolytic activity than NGT controls (Kamath et al., 1998).

oxygen specieses production (Jian et al., 2003).

antioxidant defense systems (King et al., 2004).

developed oxidative stress (Coughlan et al., 2004).

**4.2.2 Oxidant species** 

**4.2.3 Antioxidants** 

al., 2010).

increases in NO production in different tissues. Reduction in NO-induced stress is related to diabetes-induced endothelial dysfunction, NOS activation can also be induced by diabetes. NO induces the expression of antioxidant enzymes Mn- and Cu,Zn-SODs, and heme oxygenase-1 and increases intracellular glutathione concentration (Moellering et al., 1999). Although NO stimulates O2 <sup>−</sup>-induced lipoperoxidation in membranes, it can also mediate protective reactions to inhibit O2− and ONOO− induced lipoperoxidation (Rubbo et al., 1994). NO production has been found increased in the placenta, placental veins and arteries, and in umbilical vein endothelial cells from estational diabetes mellitus patients (Figueroa et al., 2000; vonMadach et al., 2003). NOS expression is also altered, as NOS has been found overexpressed in the placenta and eNoxidative stress increased in umbilical vein endothelial cells from estational diabetes mellitus patients (SanMartin et al., 2006). In estational diabetes mellitus, increases in reactive oxygen specieses and NO production, evident in the placenta and umbilical vessels, lead to peroxynitrite formation. In platelets from estational diabetes mellitus patients, elevated NOS activity and peroxynitrite production have been reported, possibly associated with platelet dysfunction and membrane damage due to increased lipid peroxidation (Mazzanti et al. , 2004). Strong protein nitration is found in term placentas from diabetic rats. Collectively, these data provide evidence of reactive nitrogen species (RNS)-induced damage in estational diabetes mellitus in the placenta and the vasculature of the mother, the placenta, and the umbilical cord, produced as a resulting consequence of exacerbated NO and ROS production.

#### **4.2.4 Role of oxidative stress in gestational diabetes-induced teratogenesis**

Diabetes in pregnancy is associated with suboptimal decidualization (Garris et al., 1988). NO plays a key role in decidualization and embryo implantation (Norwitz et al., 2001). It increases vascular permeability, vasodilation, and blood flow in the uterus, and is a component of the decidual cell reaction (Valdes et al., 2009). Diabetes during pregnancy is associated with embryonic dysmorphogenesis. Due to its capacity to regulate cell survival, apoptosis, differentiation, oxidative and nitrosative stresses play a significant role in embryo organogenesis. Low and high levels of NO can lead to embryonic maldevelopment, possibly due to an improper regulation of apoptotic events. During embryo and fetal development, NO has been found to be relevant in regulating differentiation of lung branching morphogenesis, cephalic morphogenesis, heart development, and nephrogenesis (Bloch et al., 1999; Tain et al., 2010).

Transcription factors, as paired box (PAX)-3 and peroxisome proliferator-activated receptor (PPAR) δ, has been found to be involved in the induction of both neural tube and heart malformations, the most common malformations in gestational diabetes pregnants (Higa et al., 2007; Loeken et al., 2006). Different antioxidants such as α-tocopherol and gluthatione ethyl ester increase expression of *PAX-3* and prevent apoptosis and the induction of hyperglycemia-induced neural tube and heart defects (Chang et al., 2003; Morgan et al. 2008). The higher 8-isoprostane levels observed in the offspring of diabetic animals (Wentzel & Erikkson, 2002; Wentzel et al., 1999) have its own teratogenic potency. Diabetic embryopathy is also associated with inhibition of GAPDH activity resulting from an excess of reactive oxygen speciesesin the embryo (Wentzel et al., 2002). Oxidative glucose metabolism is low and about 80% of the glucose used by the placenta. The effect of oxidative stress on placental glucose metabolism is not known. However, in nongestational tissues,

The Role of Oxidative Stress in Female Reproduction and Pregnancy 329

Clinical and research centers are investigating the usefulness of antioxidant supplementation and their role in prevention of pathological pregnancies. Antioxidant supplementation, for example vitamin C and vitamin E, has been shown to have beneficial effects in preventing luteal phase deficiency and resultant increased pregnancy rate (Hemi et al., 2003; Crha et al., 2003). Meta-analysis investigating the intervention of vitamin-C supplementation in pregnancy was inconclusive (Rumbold et al, 2005). Another metaanalysis of women taking any of the vitamin supplements started prior to 20 weeks' gestation revealed no reduction in total fetal losses, or in early and late miscarriage, having used the fixed-effects model. Improved pregnancy rates were also reported with combination therapy with the antioxidants pentoxifylline and vitamin-E supplementation for 6 months in patients with thin endometria who were undergoing in vitro fertilization with oocyte donation (Ledee-Bataille et al., 2002). Supplementation with vitamin E has also been reported to prevent the deleterious effects of ethanol toxicity on cerebral development in the animal model (Peng et al., 2005). There are essential differences among the population groups and the dosage and duration of supplementation for prevention of preeclampsia. Although many advances are being made in the field of antioxidants therapy, the data are still debatable and need further controlled evaluations in larger populations (Ashok et al.,

The establishment of pregnancy requires a harmonic hormonal, ovarial and fallopian tube function, a receptive uterus able to respond to a variety of biochemical and molecular signals produced by the developing conceptus, as well as specific interactions between the uterine endometrium and the extra-embryonic membranes. Therefore, the fetal, placental development and function are prerequisites for an adequate supply of nutrients and oxygen to the fetus and successful establishment of pregnancy. Oxidative stress is a complex system, affecting in a complex way the female fertility, and pregnancy outcome. The inbalance of the oxidative agents and antioxidants has been proposed as the causative agents of female sterility, recurrent pregnancy loss and several pregnancy-related disorders, most notably preeclampsia, intra-uterine growth restriction (IUGR) and

Preeclampsia is characterized by increased oxidative stress due to the imbalance between lipid peroxidation and antioxidant defense mechanisms, leading to endothelial dysfunction and free radical mediated cell injury. Other maternal factors including activated neutrophils and imbalance between anticoagulants and procoagulants aggravate the oxidative stress and endothelial dysfunction this plays crucial role in developement of the disease. There is no doubt that both hypoxia and hypoxia-reperfusion lead to reactive oxygen species production, but both may also arise from the same underlying problem of impaired conversion of the spiral arteries. The effects of the reduced trophoblast invasion associated with complicated pregnancies can easily be superimposed on this basic model. Reduced invasion will leave the spiral arteries vasoreactive, and thus more likely to undergo spontaneous transient vasoconstriction. They will be more responsive to endogenous and

**5. Role of antioxidant supplementation in pregnancy** 

2006).

**6. Conclusions** 

gestational diabetes.

there is certainly evidence demonstrating oxidative stress regulates GLUT-1 and/or GLUT-3 dependent glucose uptake and transport. On the other hand estational diabetes mellitus placenta is less sensitive to oxidative stress due to the heightened level of antioxidants. Under normal conditions, physiological levels of reactive oxygen speciesespromote and stimulate adequate insulin signaling. The insulin signaling pathway leads to low levels of reactive oxygen species production itself and ROS act as second messengers of which disposal impairs insulin signaling. Insulin-induced reactive oxygen specieses production is accounted for by activation of the NADPH oxidase NOX4 through PI3K. The reactive oxygen speciesespathway subsequently activates kinases or induces gene expression by redox-sensitive transcription (Omroy, 2007).

#### **4.3 Intrauterine Growth Restriction (IUGR)**

Fetal growth depends on the interactions of genetic and epigenetic determinants functioning against an environment of maternal, fetal, and placental influences (Gardosi *et al.,* 1992). Intrauterine growth restriction (IUGR) manifests as a variable syndrome of suboptimal growth and body disproportions rather than a well-defined etiologic entity. Causes for IUGR are diverse and include aneuploidies, non-aneuploid syndromes, infections, metabolic factors and placental disorders. IUGR places the fetus and neonate at risk of death or disability in the perinatal period (Baschat *et al.* 2000; Bernstein *et al.* 2000) and predisposes the child to a lifelong increased risk for hypertension, cardiovascular disorders and renal disease, among others (Murphy *et al.*, 2006). A common definition is an estimated fetal weight less than the 10th percent for gestational age. Diminished fetal arterial and venous Doppler flows in key vascular beds predict worsening fetal acid base status (Rizzo et al., 2001; Baschat *et al.* 2004) and such findings frequently lead to delivery of a markedly premature baby to avoid *in utero* demise.

A diverse number of stimuli and mediators contribute to the observed injury to the chorioallantoic villi but oxidative stress is high on the list as an injurious agent (Hung *et al.*, 2002). The production of reactive oxygen species during oxidative stress is linked with tissue injury in many diseases (Ryter *et al.,* 2007). Paper shows that the placentas of pregnancies with IUGR exhibit overt signs of oxidative stress, with reduced protein translation and particular reductions in key signalling proteins pathways (Yung *et al.,* 2008). Moreover, the syncytiotrophoblast shows signs of endoplasmatic reticulum (ER) stress by activating the unfolded protein response, which leads to an ER signal for enhanced apoptosis. The identified dysregulation of protein translation, signalling pathways and trophoblast turnover in placentas of pregnancies with IUGR (Burton *et al.*, 2009). Hypoxia, ischaemia/reperfusion, or both may contribute to placental injury through mechanisms other than reactive oxygen species generation, as variable blood flow to organs also activates the complement cascade (Levy *et al.,* 2000; Hung *et al.*, 2002; Heazell *et al,.* 2008). Activation of the complement cascade injure the feto-placental unit (Girardi *et al,.* 2003).

Clarifying the role of complement activation in pregnancies complicated by IUGR, and in placental dysfunction generally, may lead to new approaches to treatment for IUGR, as therapeutic options to modulate complement receptors and complement activity are on the horizon.

#### **5. Role of antioxidant supplementation in pregnancy**

Clinical and research centers are investigating the usefulness of antioxidant supplementation and their role in prevention of pathological pregnancies. Antioxidant supplementation, for example vitamin C and vitamin E, has been shown to have beneficial effects in preventing luteal phase deficiency and resultant increased pregnancy rate (Hemi et al., 2003; Crha et al., 2003). Meta-analysis investigating the intervention of vitamin-C supplementation in pregnancy was inconclusive (Rumbold et al, 2005). Another metaanalysis of women taking any of the vitamin supplements started prior to 20 weeks' gestation revealed no reduction in total fetal losses, or in early and late miscarriage, having used the fixed-effects model. Improved pregnancy rates were also reported with combination therapy with the antioxidants pentoxifylline and vitamin-E supplementation for 6 months in patients with thin endometria who were undergoing in vitro fertilization with oocyte donation (Ledee-Bataille et al., 2002). Supplementation with vitamin E has also been reported to prevent the deleterious effects of ethanol toxicity on cerebral development in the animal model (Peng et al., 2005). There are essential differences among the population groups and the dosage and duration of supplementation for prevention of preeclampsia. Although many advances are being made in the field of antioxidants therapy, the data are still debatable and need further controlled evaluations in larger populations (Ashok et al., 2006).

#### **6. Conclusions**

328 Oxidative Stress and Diseases

there is certainly evidence demonstrating oxidative stress regulates GLUT-1 and/or GLUT-3 dependent glucose uptake and transport. On the other hand estational diabetes mellitus placenta is less sensitive to oxidative stress due to the heightened level of antioxidants. Under normal conditions, physiological levels of reactive oxygen speciesespromote and stimulate adequate insulin signaling. The insulin signaling pathway leads to low levels of reactive oxygen species production itself and ROS act as second messengers of which disposal impairs insulin signaling. Insulin-induced reactive oxygen specieses production is accounted for by activation of the NADPH oxidase NOX4 through PI3K. The reactive oxygen speciesespathway subsequently activates kinases or induces gene expression by

Fetal growth depends on the interactions of genetic and epigenetic determinants functioning against an environment of maternal, fetal, and placental influences (Gardosi *et al.,* 1992). Intrauterine growth restriction (IUGR) manifests as a variable syndrome of suboptimal growth and body disproportions rather than a well-defined etiologic entity. Causes for IUGR are diverse and include aneuploidies, non-aneuploid syndromes, infections, metabolic factors and placental disorders. IUGR places the fetus and neonate at risk of death or disability in the perinatal period (Baschat *et al.* 2000; Bernstein *et al.* 2000) and predisposes the child to a lifelong increased risk for hypertension, cardiovascular disorders and renal disease, among others (Murphy *et al.*, 2006). A common definition is an estimated fetal weight less than the 10th percent for gestational age. Diminished fetal arterial and venous Doppler flows in key vascular beds predict worsening fetal acid base status (Rizzo et al., 2001; Baschat *et al.* 2004) and such findings frequently lead to delivery of a markedly

A diverse number of stimuli and mediators contribute to the observed injury to the chorioallantoic villi but oxidative stress is high on the list as an injurious agent (Hung *et al.*, 2002). The production of reactive oxygen species during oxidative stress is linked with tissue injury in many diseases (Ryter *et al.,* 2007). Paper shows that the placentas of pregnancies with IUGR exhibit overt signs of oxidative stress, with reduced protein translation and particular reductions in key signalling proteins pathways (Yung *et al.,* 2008). Moreover, the syncytiotrophoblast shows signs of endoplasmatic reticulum (ER) stress by activating the unfolded protein response, which leads to an ER signal for enhanced apoptosis. The identified dysregulation of protein translation, signalling pathways and trophoblast turnover in placentas of pregnancies with IUGR (Burton *et al.*, 2009). Hypoxia, ischaemia/reperfusion, or both may contribute to placental injury through mechanisms other than reactive oxygen species generation, as variable blood flow to organs also activates the complement cascade (Levy *et al.,* 2000; Hung *et al.*, 2002; Heazell *et al,.* 2008). Activation of the complement cascade injure the feto-placental unit (Girardi *et al,.* 2003).

Clarifying the role of complement activation in pregnancies complicated by IUGR, and in placental dysfunction generally, may lead to new approaches to treatment for IUGR, as therapeutic options to modulate complement receptors and complement activity are on the

redox-sensitive transcription (Omroy, 2007).

**4.3 Intrauterine Growth Restriction (IUGR)** 

premature baby to avoid *in utero* demise.

horizon.

The establishment of pregnancy requires a harmonic hormonal, ovarial and fallopian tube function, a receptive uterus able to respond to a variety of biochemical and molecular signals produced by the developing conceptus, as well as specific interactions between the uterine endometrium and the extra-embryonic membranes. Therefore, the fetal, placental development and function are prerequisites for an adequate supply of nutrients and oxygen to the fetus and successful establishment of pregnancy. Oxidative stress is a complex system, affecting in a complex way the female fertility, and pregnancy outcome. The inbalance of the oxidative agents and antioxidants has been proposed as the causative agents of female sterility, recurrent pregnancy loss and several pregnancy-related disorders, most notably preeclampsia, intra-uterine growth restriction (IUGR) and gestational diabetes.

Preeclampsia is characterized by increased oxidative stress due to the imbalance between lipid peroxidation and antioxidant defense mechanisms, leading to endothelial dysfunction and free radical mediated cell injury. Other maternal factors including activated neutrophils and imbalance between anticoagulants and procoagulants aggravate the oxidative stress and endothelial dysfunction this plays crucial role in developement of the disease. There is no doubt that both hypoxia and hypoxia-reperfusion lead to reactive oxygen species production, but both may also arise from the same underlying problem of impaired conversion of the spiral arteries. The effects of the reduced trophoblast invasion associated with complicated pregnancies can easily be superimposed on this basic model. Reduced invasion will leave the spiral arteries vasoreactive, and thus more likely to undergo spontaneous transient vasoconstriction. They will be more responsive to endogenous and

The Role of Oxidative Stress in Female Reproduction and Pregnancy 331

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1–32.

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Association of valproate-induced teratogenesis with histone deacetylase inhibition

exogenous vasoactive stimuli. Partial obliteration of their lumens by atherotic changes will also impair flow. Excessive production of inflammatory cytokines, deportation of apoptotic microvillous placental fragments, activation of maternal leukocytes and platelets, or depletion of NO production may then cause or contribute to the maternal endothelial response. The degree of the oxidative stress will likely reflect the extent of the maternal vascular pathology.

There are a number of pathways that may contribute to oxidative stress observed in the gestational diabetes mellitus placenta. In the placenta, reactive oxygen species and reactive nitrite spesies are an important source of growth and signaling factors, and are susceptible to ROS-mediated apoptosis. The placenta is endowed with many antioxidants, some of which are increased in gestational diabetes mellitus. However, there is much data to indicate that maternal diabetes during pregnancy may induce oxidative stress in the newborn that may entail biochemical disturbances of the fetus (Hung et al., 2006). Given that The placenta provides the interface of the maternal and fetal circulations, it may play a crucial role in protecting the fetus from adverse effects of the maternal diabetic milieu (Lappas et al., 2011).

The investigation of oxidtive stress is inevitabile for better understanding of aerobe organism function. Evaluation of environmental factors effect on oxidative stress molecular pathways can sereve possible solutions for female reproductive malfunctions. The are several fertility and pregnancy related disease, as unexplained infertility, preeclampsia, HELLP syndrome where the reactive oxidative species and antioxidant mechanism play key role in pathogenesis of the disease. The antioxidant supplementation, avoidance of different enviromental factors, as polluted comestibles may lead to decrease of infertility rate and incidence of pregnancy related disorders.

#### **7. References**


exogenous vasoactive stimuli. Partial obliteration of their lumens by atherotic changes will also impair flow. Excessive production of inflammatory cytokines, deportation of apoptotic microvillous placental fragments, activation of maternal leukocytes and platelets, or depletion of NO production may then cause or contribute to the maternal endothelial response. The degree of the oxidative stress will likely reflect the extent of the maternal

There are a number of pathways that may contribute to oxidative stress observed in the gestational diabetes mellitus placenta. In the placenta, reactive oxygen species and reactive nitrite spesies are an important source of growth and signaling factors, and are susceptible to ROS-mediated apoptosis. The placenta is endowed with many antioxidants, some of which are increased in gestational diabetes mellitus. However, there is much data to indicate that maternal diabetes during pregnancy may induce oxidative stress in the newborn that may entail biochemical disturbances of the fetus (Hung et al., 2006). Given that The placenta provides the interface of the maternal and fetal circulations, it may play a crucial role in protecting the fetus from adverse effects of the maternal diabetic milieu

The investigation of oxidtive stress is inevitabile for better understanding of aerobe organism function. Evaluation of environmental factors effect on oxidative stress molecular pathways can sereve possible solutions for female reproductive malfunctions. The are several fertility and pregnancy related disease, as unexplained infertility, preeclampsia, HELLP syndrome where the reactive oxidative species and antioxidant mechanism play key role in pathogenesis of the disease. The antioxidant supplementation, avoidance of different enviromental factors, as polluted comestibles may lead to decrease of infertility rate and

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**15** 

 *Serbia* 

Zorica Jovanović

**Effects of Oxidative Stress** 

**on the Electrophysiological** 

*Department of Pathological Physiology, Faculty of Medicine, Kragujevac,* 

**Function of Neuronal Membranes** 

Numerous experimental and clinical observations suggest that reactive oxygen species (ROS) play a significant role in several pathological conditions of the central nervous system where they directly injure tissue and where their formation may also be a consequence of tissue injury. Reactive oxygen metabolites are particularly active in the brain and neuronal tissue, and they are involved in numerous cellular functions, including cell death and survival. In comparison with other organs of the body, the brain may, for a number of biochemical, physiological and anatomical reasons, be especially vulnerable to oxidative stress and ROS mediated injury. A high metabolic rate (the brain consumes approximately 20% of total-body oxygen) and an abundant supply of transition metals, make the brain an ideal target for free radical attack (Facchinetti et al., 1998; Gutowicz, 2011). In addition, the brain has high susceptibility to oxidative stress due to high polyunsaturated fatty acid content and relatively lower regenerative capacity in comparison with other tissues. On the other hand, the brain is poor in catalytic activity and has moderate amounts of glutathion peroxidase and superoxyde dismutase. Of all the cell types in the body, neuronal cells may be among the most vulnerable to oxidative stress. These cells are continuously exposed to ROS. Accumulating evidence demonstrating that the defense of nerve cells against ROSmediated oxidative damage is essential for maintaining functionality of nerve cells. Because hydrogen peroxide (H2O2) is the peroxide generated in the highest quantity in the brain, the defense against the oxidative stress appears to be particularly important. When production exceeds antioxidant protection, oxidative stress leads to molecular damage. An important component of the cellular detoxification of ROS is the antioxidant glutathione (GSH) (Dringen & Gutterer, 2002; Dringen & Hirrlinger, 2003). Because neurons have limited antioxidant capacity, they rely heavily on their metabolic coupling with astrocytes to combat oxidative stress. Evidence is growing that glutathione plays an important role in the detoxification of H2O2 and organic hydroperoxides in the brain and that glutathione is the main antioxidant molecule in neurons (Aoyama et al., 2008; Haskew-Layton et al., 2010; Limon-Pacheco & Gonsebat, 2010). Ongoing studies have shown that neuron-glial compartmentalization of antioxidants is critical for neuronal signaling by H2O2, as well as neuronal protection. The neurons are more vulnerable to oxidative stress than astrocytes,

**1. Introduction** 

endothelial cells induced by hyperglycemia linked to vascular disease. *Diabetes* 57, 2809–2817.

Zimmerman EF, Potturi RB, Resnick E, Fisher JE. (1994). Role of oxygen free radicals in cocaine-induced vascular disruption in mice. *Teratology*, 49, 192–201.

### **Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes**

Zorica Jovanović *Department of Pathological Physiology, Faculty of Medicine, Kragujevac, Serbia* 

#### **1. Introduction**

336 Oxidative Stress and Diseases

Zimmerman EF, Potturi RB, Resnick E, Fisher JE. (1994). Role of oxygen free radicals in cocaine-induced vascular disruption in mice. *Teratology*, 49, 192–201.

2809–2817.

endothelial cells induced by hyperglycemia linked to vascular disease. *Diabetes* 57,

Numerous experimental and clinical observations suggest that reactive oxygen species (ROS) play a significant role in several pathological conditions of the central nervous system where they directly injure tissue and where their formation may also be a consequence of tissue injury. Reactive oxygen metabolites are particularly active in the brain and neuronal tissue, and they are involved in numerous cellular functions, including cell death and survival. In comparison with other organs of the body, the brain may, for a number of biochemical, physiological and anatomical reasons, be especially vulnerable to oxidative stress and ROS mediated injury. A high metabolic rate (the brain consumes approximately 20% of total-body oxygen) and an abundant supply of transition metals, make the brain an ideal target for free radical attack (Facchinetti et al., 1998; Gutowicz, 2011). In addition, the brain has high susceptibility to oxidative stress due to high polyunsaturated fatty acid content and relatively lower regenerative capacity in comparison with other tissues. On the other hand, the brain is poor in catalytic activity and has moderate amounts of glutathion peroxidase and superoxyde dismutase. Of all the cell types in the body, neuronal cells may be among the most vulnerable to oxidative stress. These cells are continuously exposed to ROS. Accumulating evidence demonstrating that the defense of nerve cells against ROSmediated oxidative damage is essential for maintaining functionality of nerve cells. Because hydrogen peroxide (H2O2) is the peroxide generated in the highest quantity in the brain, the defense against the oxidative stress appears to be particularly important. When production exceeds antioxidant protection, oxidative stress leads to molecular damage. An important component of the cellular detoxification of ROS is the antioxidant glutathione (GSH) (Dringen & Gutterer, 2002; Dringen & Hirrlinger, 2003). Because neurons have limited antioxidant capacity, they rely heavily on their metabolic coupling with astrocytes to combat oxidative stress. Evidence is growing that glutathione plays an important role in the detoxification of H2O2 and organic hydroperoxides in the brain and that glutathione is the main antioxidant molecule in neurons (Aoyama et al., 2008; Haskew-Layton et al., 2010; Limon-Pacheco & Gonsebat, 2010). Ongoing studies have shown that neuron-glial compartmentalization of antioxidants is critical for neuronal signaling by H2O2, as well as neuronal protection. The neurons are more vulnerable to oxidative stress than astrocytes,

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 339

regulatory proteins or indirectly via peroxidation of membrane lipids. The nature and sequence of events that lead to the disruptions of these ion transport pathways are not fully understood. Early studies revealed that the effects of ROS on membrane properties could be deduced from electrophysiological parameters of the membrane. These include changes in membrane potential and current, ionic gradients, action potential duration and amplitude, spontaneous activity and excitability (Tarr et al., 1995; Tarr & Valenzeno, 1989; Beleslin et al., 1998). Oxygen-derived free radicals are thought to induce alterations in nervous electrical activity, however, the underlying membrane ionic currents affected by ROS and the mechanisms by which ROS induce their effects on ion channels in the nerve cells are not well defined. Considering neuronal function, ROS can attack ion channels and transporters directly, or indirectly by causing lipid peroxidation (Kourie, 1998; Carmeliet, 1999) and affecting associated signaling molecules (Hool, 2006). The mechanism of initiation of ROS peroxidation is not understood completely. The hydroxyl radical (HO•), a highly reactive oxidant, has been proposed as the initiating species. The ability of the HO• to initiate lipid peroxidation has been questioned by some investigators. In addition to initiating lipid peroxidation, the HO• has been implicated in direct cellular damage. Peroxidation of membrane phospholipids has been demonstrated to affect various transmembrane processes, such as receptor activation and formation of intracellular second messengers and Ca2+ homeostasis. Ca2+ ions also play a central role in the control of neuronal excitability. ROS oxidatively modify numerous membrane-bound proteins including ion channels. ROS can also react with proteins directly and in this case seem to have a prevalence for SH groups or disulfide bridges on the ion transport proteins (Van der Vliet & Bast, 1992). Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms (Su et al., 2007). The mechanism of ROS-induced modifications in ion transport pathways involves the inhibition of membrane-bound regulatory enzymes and modification

Studies have demonstrated that oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. In the CNS, cells respond to oxidative stress by initiating endogenous protective cascades often regulated at the transcriptional level. The transcription factor, nuclear factor erythroid 2 related factor 2 (Nrf2) plays an integral role in astrocyte-mediated protection of neurons from oxidative stress. Previous studies have reported that MAPKs may play a role in the induction and regulation of the Nrf2 system in the brain (Clark & Simon, 2009). When cells are exposed to oxidative stress, the Nrf2 binds to the antioxidant responsive element (ARE). The Nrf2–ARE pathway elicits transcriptional activation of antioxidant genes and detoxifying genes that protect cells and organisms from oxidative stress. Activation of this pathway protects cells from oxidative stress-induced cell death (Hur and Gray, 2011). The NRF2/KEAP1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox state (Stepkowski & Kruszewski, 2011). The Nrf2-mediated GSH biosynthesis and release from astrocytes protects neurons from oxidative stress (Shih et al., 2003). Increased levels of GSH may be a major component of the protection observed by Nrf2 activation. In the CNS, Nrf2 plays an integral role in astrocytemediated protection of neurons from oxidative stress. Neuronal viability is enhanced significantly by an increased supply of GSH precursors from Nrf2-overexpressing glia. Thus Nrf2-dependent enhancement of glial GSH release appears to be necessary and sufficient for

of the oxidative phosphorylation and ATP levels.

due to an insufficient detoxification of ROS via their glutathione system (Dringen et al., 1999; Martin & Teismann, 2009). But, the concentration of glutathione is in relatively lesser quantities in the brain in comparison to the other organs of the body (Skaper et al., 1999). In contrast to other ROS, H2O2 is neither a free radical nor an ion, which limits its reactivity (Cohen, 1994). However, in the presence of transition metals such as iron or copper, H2O2 can give rise to the indiscriminately reactive and toxic hydroxyl radical (HO•) by Fenton chemistry. H2O2 is able to diffuse across biological membranes, and therefore can diffuse freely from a site of generation (Bienert et al., 2007; Makino et al., 2004) so that it is well-suited as a diffusible messenger. Increasing evidence indicates that H2O2 is a particularly intriguing candidate as an intracellular and intercellular signaling molecule because it is neutral and membrane-permeable (Nistico et al., 2008; Forman et al., *2010)*. Recent research into mechanisms of ROS-induced modifications in ion transport pathways involves: oxidation of sulfhydryl (SH) groups on the ion transport proteins, lipid peroxidation, and alterations of calcium (Ca2+) homeostasis, a major second messenger system (Kourie, 1998). Increases in Ca2+ initiate inappropriate activation of several enzyme systems e.g., nitric oxide synthase and phospholipase A2. Overactivation of these enzymes results in the breakdown of proteins and phospholipids and initiates several cascades that damage cells (Lee et al., 1999). It has been described that elevation in cytoplasmic Ca2+ levels activates the mitogen-activated protein kinase (MAPK) cascade (Liu & Templeton, 2008; Son et al., 2011) and the phosphatidylinositol 3′-kinase (PI3K)-Akt pathway (Cheng et al., 2003). ROS produce cell damage through multiple mechanisms, including excitotoxicity, metabolic dysfunction and disturbance of intracellular homeostasis of Ca2+ (Halliwell & Gutteridge, 1984; Del Maestro et al., 1980; Bracci, 1992). Activation of glutamate ionotropic receptors promptly triggers membrane depolarization and Ca2+ influx, resulting in the activation of several different protein kinases (Ca2+-calmodulindependent kinase, protein kinase C and MAPK) and transcription factors, such as cyclic AMP response element binding protein (CREB). Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of apurinic endonuclease 1 (APE1) (Yang et al., 2011).

Studies have demonstrated that ROS can induce or mediate the activation of the MAPK pathways (McCubrey et al., 2006). The mechanisms by which ROS can activate MAPK pathways are unclear. Because ROS can alter protein structure and function by modifying critical amino acid residues of proteins (Thannickal & Fanburg, 2000), the oxidative modification of signaling proteins by ROS may be one of the plausible mechanisms for the activation of MAPK pathways. However, the precise molecular target(s) of ROS is unknown. The prevention of oxidative stress by antioxidants blocks MAPK activation after cell stimulation with cellular stimuli indicating the involvement of ROS in activation of MAPK pathways. The recent observations provide a strong argument for activation of MAPK pathways by direct exposure of cells to exogenous H2O2 (Ruffels et al., 2004; Son et al., 2011).

The cell membrane would seem of special interest because of its large surface area and because of the susceptibility of membrane unsaturated fatty acids and proteins containing oxidizable amino acids (such as cysteine and methionine) to oxidant attack. Oxidative stress affects cellular membrane lipids and proteins. Cell membranes are either a source of neurotoxic lipid oxidation products or the target of pathogenic processes that cause permeability changes or ion channel formation (Axelsen, 2011). Reactive oxygen metabolites modify ion transport mechanisms either directly via ion transport pathway proteins and

due to an insufficient detoxification of ROS via their glutathione system (Dringen et al., 1999; Martin & Teismann, 2009). But, the concentration of glutathione is in relatively lesser quantities in the brain in comparison to the other organs of the body (Skaper et al., 1999). In contrast to other ROS, H2O2 is neither a free radical nor an ion, which limits its reactivity (Cohen, 1994). However, in the presence of transition metals such as iron or copper, H2O2 can give rise to the indiscriminately reactive and toxic hydroxyl radical (HO•) by Fenton chemistry. H2O2 is able to diffuse across biological membranes, and therefore can diffuse freely from a site of generation (Bienert et al., 2007; Makino et al., 2004) so that it is well-suited as a diffusible messenger. Increasing evidence indicates that H2O2 is a particularly intriguing candidate as an intracellular and intercellular signaling molecule because it is neutral and membrane-permeable (Nistico et al., 2008; Forman et al., *2010)*. Recent research into mechanisms of ROS-induced modifications in ion transport pathways involves: oxidation of sulfhydryl (SH) groups on the ion transport proteins, lipid peroxidation, and alterations of calcium (Ca2+) homeostasis, a major second messenger system (Kourie, 1998). Increases in Ca2+ initiate inappropriate activation of several enzyme systems e.g., nitric oxide synthase and phospholipase A2. Overactivation of these enzymes results in the breakdown of proteins and phospholipids and initiates several cascades that damage cells (Lee et al., 1999). It has been described that elevation in cytoplasmic Ca2+ levels activates the mitogen-activated protein kinase (MAPK) cascade (Liu & Templeton, 2008; Son et al., 2011) and the phosphatidylinositol 3′-kinase (PI3K)-Akt pathway (Cheng et al., 2003). ROS produce cell damage through multiple mechanisms, including excitotoxicity, metabolic dysfunction and disturbance of intracellular homeostasis of Ca2+ (Halliwell & Gutteridge, 1984; Del Maestro et al., 1980; Bracci, 1992). Activation of glutamate ionotropic receptors promptly triggers membrane depolarization and Ca2+ influx, resulting in the activation of several different protein kinases (Ca2+-calmodulindependent kinase, protein kinase C and MAPK) and transcription factors, such as cyclic AMP response element binding protein (CREB). Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of apurinic

Studies have demonstrated that ROS can induce or mediate the activation of the MAPK pathways (McCubrey et al., 2006). The mechanisms by which ROS can activate MAPK pathways are unclear. Because ROS can alter protein structure and function by modifying critical amino acid residues of proteins (Thannickal & Fanburg, 2000), the oxidative modification of signaling proteins by ROS may be one of the plausible mechanisms for the activation of MAPK pathways. However, the precise molecular target(s) of ROS is unknown. The prevention of oxidative stress by antioxidants blocks MAPK activation after cell stimulation with cellular stimuli indicating the involvement of ROS in activation of MAPK pathways. The recent observations provide a strong argument for activation of MAPK pathways by direct exposure of cells to exogenous H2O2 (Ruffels et al., 2004; Son et al., 2011). The cell membrane would seem of special interest because of its large surface area and because of the susceptibility of membrane unsaturated fatty acids and proteins containing oxidizable amino acids (such as cysteine and methionine) to oxidant attack. Oxidative stress affects cellular membrane lipids and proteins. Cell membranes are either a source of neurotoxic lipid oxidation products or the target of pathogenic processes that cause permeability changes or ion channel formation (Axelsen, 2011). Reactive oxygen metabolites modify ion transport mechanisms either directly via ion transport pathway proteins and

endonuclease 1 (APE1) (Yang et al., 2011).

regulatory proteins or indirectly via peroxidation of membrane lipids. The nature and sequence of events that lead to the disruptions of these ion transport pathways are not fully understood. Early studies revealed that the effects of ROS on membrane properties could be deduced from electrophysiological parameters of the membrane. These include changes in membrane potential and current, ionic gradients, action potential duration and amplitude, spontaneous activity and excitability (Tarr et al., 1995; Tarr & Valenzeno, 1989; Beleslin et al., 1998). Oxygen-derived free radicals are thought to induce alterations in nervous electrical activity, however, the underlying membrane ionic currents affected by ROS and the mechanisms by which ROS induce their effects on ion channels in the nerve cells are not well defined. Considering neuronal function, ROS can attack ion channels and transporters directly, or indirectly by causing lipid peroxidation (Kourie, 1998; Carmeliet, 1999) and affecting associated signaling molecules (Hool, 2006). The mechanism of initiation of ROS peroxidation is not understood completely. The hydroxyl radical (HO•), a highly reactive oxidant, has been proposed as the initiating species. The ability of the HO• to initiate lipid peroxidation has been questioned by some investigators. In addition to initiating lipid peroxidation, the HO• has been implicated in direct cellular damage. Peroxidation of membrane phospholipids has been demonstrated to affect various transmembrane processes, such as receptor activation and formation of intracellular second messengers and Ca2+ homeostasis. Ca2+ ions also play a central role in the control of neuronal excitability. ROS oxidatively modify numerous membrane-bound proteins including ion channels. ROS can also react with proteins directly and in this case seem to have a prevalence for SH groups or disulfide bridges on the ion transport proteins (Van der Vliet & Bast, 1992). Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms (Su et al., 2007). The mechanism of ROS-induced modifications in ion transport pathways involves the inhibition of membrane-bound regulatory enzymes and modification of the oxidative phosphorylation and ATP levels.

Studies have demonstrated that oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. In the CNS, cells respond to oxidative stress by initiating endogenous protective cascades often regulated at the transcriptional level. The transcription factor, nuclear factor erythroid 2 related factor 2 (Nrf2) plays an integral role in astrocyte-mediated protection of neurons from oxidative stress. Previous studies have reported that MAPKs may play a role in the induction and regulation of the Nrf2 system in the brain (Clark & Simon, 2009). When cells are exposed to oxidative stress, the Nrf2 binds to the antioxidant responsive element (ARE). The Nrf2–ARE pathway elicits transcriptional activation of antioxidant genes and detoxifying genes that protect cells and organisms from oxidative stress. Activation of this pathway protects cells from oxidative stress-induced cell death (Hur and Gray, 2011). The NRF2/KEAP1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox state (Stepkowski & Kruszewski, 2011). The Nrf2-mediated GSH biosynthesis and release from astrocytes protects neurons from oxidative stress (Shih et al., 2003). Increased levels of GSH may be a major component of the protection observed by Nrf2 activation. In the CNS, Nrf2 plays an integral role in astrocytemediated protection of neurons from oxidative stress. Neuronal viability is enhanced significantly by an increased supply of GSH precursors from Nrf2-overexpressing glia. Thus Nrf2-dependent enhancement of glial GSH release appears to be necessary and sufficient for

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 341

and penetration of the cells was performed in the cage under a stereomicroscope. Retzius neurons were identified based on the position within the ganglion, the size and the bioelectrical properties of the cells. The Retzius cells, are the largest neurons (40-60 µm in diameter) situated on the ventral side of the ganglia. It is well known that the resting potential of Retzius nerve cells of medical and horse leeches are lower than in other neurons (Hagiwara & Morita, 1962; Beleslin, 1985). Theoretically, this can be due to the low resting potassium permeability or the high membrane permeability to sodium. The Retzius cell is spontaneously active and responds to depolarization with an increased firing rate proportional to the depolarization (Lent, 1977). In leech Retzius nerve cells three classes of K+ channels (fast, slow calcium-activated and late voltage-regulated) have been identified (Beleslin et al., 1982; Beleslin, 1985). To change the solution, the chamber was flushed continuously with a volume of fluid at least five times that of the chamber volume. The perfusion rate was kept low so that implanted microelectrodes remained inside the cells during the perfusion. The bath volume was 2 ml and the solution changes were completed

In this study, we investigated the time-dependent changes in action potential configuration and changes in steady-state membrane currents in leech Retzius nerve cells. The spontaneous spike activity was recorded with a conventional 3 M KCl microelectrode. Membrane voltage and current were recorded using voltage-clamp techniques. This was shown in voltage-clamped neurons by long depolarizing steps (to 500 ms) from the holding potential which was more negative than –40 mV in a sodium free leech Ringer, in order to induce fast and slow K+ outward currents. The recording electrodes were prepared from 1.5 mm borosilicate capillaries (Clark Electromedical Instruments, UK) and filled with a 3 M KCl-containing solution. The pipette resistance ranged from 5 to 10 MΩ (when filled with 3M KCl solution). Microelectrodes with a tip potential less than 5 mV in an artificial solution, were selected for use. Usually the microelectrode was connected through a Ringer bridge and Ag-AgCl electrode via a negative capacitance high input resistant amplifier Bioelectric Instrument DS2C to a computer. Command pulses were derived from a Tektronix 161 pulse generator. The signals were digitized by the use of an analog-todigital converter (Digidata 1200; Axon Instruments) and saved in a computer for off-line

Pharmacological agents were prepared and dissolved immediately before application in the physiological salt solution at the concentrations stated. H2O2, cumene hydroperoxide (CHP) and reduced glutathione (GSH), were added to the leech or Tris-Ringer. All drugs were administered sufficient to reach a steady-state response (up to 30 min). The bath volume was 2 ml and the solution changes were completed within 30 sec. The ganglia were bathed in a leech Ringer containing (mM): 115 NaCl, 4 KCl, 2 CaCl2, 1.2 Na2HPO4, 0.3 NaH2PO4 (pH 7.2). In the Na+-free Ringer, 115 mM NaCl was completely replaced with an equal amount of Tris (hydroxy-methyl)aminomethane-Cl (Tris Ringer) and Na2HPO4 and NaH2PO4 were

within 30 sec.

analysis.

omitted.

**2.2 Solutions** 

**2.1 Electrical methods** 

neuronal protection. The observations of Correa et al. (2011) suggest that activated microglia can stimulate or reduce the GSH-related anti-oxidant defense in cultured astrocytes. Recently, Zou et al. (2011) reported that overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells.

Ion channels and transporters are susceptible to oxidative stress. For example, voltagedependent Na+, K+ and Ca2+ channels, Ca2+-activated K+ channels, and KATP channels have all been identified as targets for ROS (Hool, 2006). Several previous studies indicate that H2O2 alters energy metabolism, ATP- sensitive K+currents, L-type Ca2+currents (Goldhaber & Liu, 1994; Racay et al., 1997), as well as delayed rectifier K+ currents (Goldhaber et al., 1989). However in literature the data concerning the effect of ROS on potassium current are controversal. For example, Cerbai et al. (1991) and Ward & Giles (1997) did not observe any effect, in contrast to Tarr & Valenzeno (1989) who obtained a decrease in the outward, delayed rectifier potassium current. The results of Hasan et al. (2007) suggest that oxidative stress, which inhibits the delayed-rectifier current, can alter neural activity. Ward and Giles (1997) studied the effects of H2O2 on action potentials and underlying ionic currents in isolated rat ventricular myocytes. They showed that H2O2 caused no significant changes in either the Ca2+-independent transient outward K+ current (Ito) or the inwardly rectifying K+ current (IK1). The most prominent effect of H2O2 on the ionic currents which underlie the action potential is a slowing of inactivation of the INa. Potassium channels constitute a highly diverse class of ion channel and thus participate in multiple modulatory functions. Although altered potassium dynamics play a major role in this type of neuronal activity (Dudek et al., 1998) the role of K+ channels is still incompletely understood. The voltageactivated K+ channels are responsible for the establishment of the resting membrane potential, repolarization during action potentials and regulation of action potential frequency (Vacher et al., 2008). Three principal K+ currents were identified in LRNC (Stewart et al., 1989). These differed in their time courses of activation and inactivation and in their responses to Ca2+ channel blockers. K+ currents of the A-type (IA) with rapid activation and inactivation kinetics, were not affected by Ca2+ channel blockers. The A-type K+ currents were a minor component of the outward current in LRNC. A Ca2+ activated K+ current (ICa), that inactivated more slowly and was reduced by Ca2+ channel blockers, constituted the major outward current in LRNC. The third K+ current resembled the delayed rectifier currents (IK1 and IK2) of squid axons, activated and inactivated slowly. Modifications of K+ channel activity by ROS in the brain would lead to drastic changes in the electrical excitability of neuronal cells and could easily explain a tendency to brain hyperexcitability, or even neuronal death.

#### **2. Materials and methods**

All experiments were carried out at room temperature (22-25 C) on the Retzius nerve cells of isolated abdominal segmental ganglia of the ventral nerve cord of the horse leech *Haemopis saguisuga.* The dissection procedure, the recording method and point voltage clamp technique were employed as described previously (Beleslin et al., 1988). Dissected segments of 4 ganglia were immediately transferred to a 2.5 ml plastic chamber with a leech Ringer and fixed by means of fine steel clips. The plastic chamber was then placed in a grounded Faraday's cage mounted on a fixed table in a manner that prevents vibrations. Identification and penetration of the cells was performed in the cage under a stereomicroscope. Retzius neurons were identified based on the position within the ganglion, the size and the bioelectrical properties of the cells. The Retzius cells, are the largest neurons (40-60 µm in diameter) situated on the ventral side of the ganglia. It is well known that the resting potential of Retzius nerve cells of medical and horse leeches are lower than in other neurons (Hagiwara & Morita, 1962; Beleslin, 1985). Theoretically, this can be due to the low resting potassium permeability or the high membrane permeability to sodium. The Retzius cell is spontaneously active and responds to depolarization with an increased firing rate proportional to the depolarization (Lent, 1977). In leech Retzius nerve cells three classes of K+ channels (fast, slow calcium-activated and late voltage-regulated) have been identified (Beleslin et al., 1982; Beleslin, 1985). To change the solution, the chamber was flushed continuously with a volume of fluid at least five times that of the chamber volume. The perfusion rate was kept low so that implanted microelectrodes remained inside the cells during the perfusion. The bath volume was 2 ml and the solution changes were completed within 30 sec.

#### **2.1 Electrical methods**

340 Oxidative Stress and Diseases

neuronal protection. The observations of Correa et al. (2011) suggest that activated microglia can stimulate or reduce the GSH-related anti-oxidant defense in cultured astrocytes. Recently, Zou et al. (2011) reported that overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss and apoptosis

Ion channels and transporters are susceptible to oxidative stress. For example, voltagedependent Na+, K+ and Ca2+ channels, Ca2+-activated K+ channels, and KATP channels have all been identified as targets for ROS (Hool, 2006). Several previous studies indicate that H2O2 alters energy metabolism, ATP- sensitive K+currents, L-type Ca2+currents (Goldhaber & Liu, 1994; Racay et al., 1997), as well as delayed rectifier K+ currents (Goldhaber et al., 1989). However in literature the data concerning the effect of ROS on potassium current are controversal. For example, Cerbai et al. (1991) and Ward & Giles (1997) did not observe any effect, in contrast to Tarr & Valenzeno (1989) who obtained a decrease in the outward, delayed rectifier potassium current. The results of Hasan et al. (2007) suggest that oxidative stress, which inhibits the delayed-rectifier current, can alter neural activity. Ward and Giles (1997) studied the effects of H2O2 on action potentials and underlying ionic currents in isolated rat ventricular myocytes. They showed that H2O2 caused no significant changes in either the Ca2+-independent transient outward K+ current (Ito) or the inwardly rectifying K+ current (IK1). The most prominent effect of H2O2 on the ionic currents which underlie the action potential is a slowing of inactivation of the INa. Potassium channels constitute a highly diverse class of ion channel and thus participate in multiple modulatory functions. Although altered potassium dynamics play a major role in this type of neuronal activity (Dudek et al., 1998) the role of K+ channels is still incompletely understood. The voltageactivated K+ channels are responsible for the establishment of the resting membrane potential, repolarization during action potentials and regulation of action potential frequency (Vacher et al., 2008). Three principal K+ currents were identified in LRNC (Stewart et al., 1989). These differed in their time courses of activation and inactivation and in their responses to Ca2+ channel blockers. K+ currents of the A-type (IA) with rapid activation and inactivation kinetics, were not affected by Ca2+ channel blockers. The A-type K+ currents were a minor component of the outward current in LRNC. A Ca2+ activated K+ current (ICa), that inactivated more slowly and was reduced by Ca2+ channel blockers, constituted the major outward current in LRNC. The third K+ current resembled the delayed rectifier currents (IK1 and IK2) of squid axons, activated and inactivated slowly. Modifications of K+ channel activity by ROS in the brain would lead to drastic changes in the electrical excitability of neuronal cells and could easily explain a tendency to brain hyperexcitability,

in cultured human retinal pigment epithelial cells.

or even neuronal death.

**2. Materials and methods** 

All experiments were carried out at room temperature (22-25

isolated abdominal segmental ganglia of the ventral nerve cord of the horse leech *Haemopis saguisuga.* The dissection procedure, the recording method and point voltage clamp technique were employed as described previously (Beleslin et al., 1988). Dissected segments of 4 ganglia were immediately transferred to a 2.5 ml plastic chamber with a leech Ringer and fixed by means of fine steel clips. The plastic chamber was then placed in a grounded Faraday's cage mounted on a fixed table in a manner that prevents vibrations. Identification

C) on the Retzius nerve cells of

In this study, we investigated the time-dependent changes in action potential configuration and changes in steady-state membrane currents in leech Retzius nerve cells. The spontaneous spike activity was recorded with a conventional 3 M KCl microelectrode. Membrane voltage and current were recorded using voltage-clamp techniques. This was shown in voltage-clamped neurons by long depolarizing steps (to 500 ms) from the holding potential which was more negative than –40 mV in a sodium free leech Ringer, in order to induce fast and slow K+ outward currents. The recording electrodes were prepared from 1.5 mm borosilicate capillaries (Clark Electromedical Instruments, UK) and filled with a 3 M KCl-containing solution. The pipette resistance ranged from 5 to 10 MΩ (when filled with 3M KCl solution). Microelectrodes with a tip potential less than 5 mV in an artificial solution, were selected for use. Usually the microelectrode was connected through a Ringer bridge and Ag-AgCl electrode via a negative capacitance high input resistant amplifier Bioelectric Instrument DS2C to a computer. Command pulses were derived from a Tektronix 161 pulse generator. The signals were digitized by the use of an analog-todigital converter (Digidata 1200; Axon Instruments) and saved in a computer for off-line analysis.

#### **2.2 Solutions**

Pharmacological agents were prepared and dissolved immediately before application in the physiological salt solution at the concentrations stated. H2O2, cumene hydroperoxide (CHP) and reduced glutathione (GSH), were added to the leech or Tris-Ringer. All drugs were administered sufficient to reach a steady-state response (up to 30 min). The bath volume was 2 ml and the solution changes were completed within 30 sec. The ganglia were bathed in a leech Ringer containing (mM): 115 NaCl, 4 KCl, 2 CaCl2, 1.2 Na2HPO4, 0.3 NaH2PO4 (pH 7.2). In the Na+-free Ringer, 115 mM NaCl was completely replaced with an equal amount of Tris (hydroxy-methyl)aminomethane-Cl (Tris Ringer) and Na2HPO4 and NaH2PO4 were omitted.

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 343

Table 1 summarizes the values of the APD in a leech Ringer and after adding CHP (0.25, 1 and 1.5 mM). Table 1 shows that CHP caused a concentration-dependent increase in APD.

10.450.98 12.331.74 13.502.13 13.501.39 14.40.80

9.660.52 13.174.02 25.172.91 31.055.96 35.3313.60

9.662.18 16.093.15 41.648.27 68.7213.4 127.8015.95

Table 1. Values of the APD (in ms) of LRNC before, 5, 15, 20 and 30 minutes after the adding of CHP (0.25, 1 and 1.5 mM) and during the recovery. Data are expressed as mean ± SEM;

**3.2 Effects of hydrogen peroxide on the spontaneous spike potential of leech Retzius** 

Previous investigations have shown that H2O2 is involved in cascades of pathological events affecting neural cells. The background of this study were the findings that 1 mM CHP treatment caused an extreme change in the duration of the action potential and suppression of Ca2+ activated outward K+ currents of LRNC. The aim of the present experiments was to examine the effect of the higher concentrations of H2O2 on LRNC. H2O2 in concentrations up to 10 mM in the reaction mixture had no effect on spontaneous spike potential. Extracellular application of H2O2 (1, 5 and 10 mM) did not significantly change (P > 0.05) the duration of the action potential of the LRNC. H2O2 is ineffective in generating either cardiac-like action

**3.3 Effects of glutathione on the cumene hydroperoxide-induced change of the** 

The background of this study were the findings that the hydroxyl radical scavenger, mannitol (5 mM) significantly reduced neurotoxic effect of CHP on the spontaneous spike

Taking in to consideration that it has been proved that CHP affects the lasting action potentials of Retzius nerve cells, the possibility of recovering the changes by the effect of antioxidant, GSH was also examined. Firstly, Retzius cells were exposed to the effect of CHP (1 mM) then GSH was added in a concentration of 0.2 mM to the Leech Ringer solution. The application of GSH, a free radical scavenger, to a bathing solution reverses the CHP effects. The GSH, largely inhibited the effects of CHP on the APD. In the presence of the GSH the APD has been extended by 9.22±1.14 ms (in controlled conditions, before the application of

Ringer 5 min 15 min 20 min 30 min Recovery

20 min

26.508.38

23.434.61

P>0.05 15.661.05

P≤0.01

P≤0.01

Leech

0.25 mM CHP n= 6

1 mM CHP n= 6

1.5 mM CHP n= 11

n=number of cells.

**nerve cells** 

 *repetitive firing* 

potential or early after depolarization in LRNC.

electrogenesis of LRNC (Jovanović, 2010).

**spontaneous spike potential of leech Retzius nerve cells** 

Solutions containing H2O2 were prepared freshly before their use from 30% H2O2 solution (Zorka Pharma, Sabac) and added to the Ringer solution (or Tris-Ringer) at final concentrations of 1, 5 and 10 mM. CHP and GSH were added to the normal or Tris-Ringer solution. The CHP was obtained from Sigma (St. Louis, U.S.A.), dissolved in 0.01% dimethyl sulfoxide (Sigma, St. Louis, U.S.A.) and added to the Ringer solution (or Tris-Ringer) in a concentrations of 0.25, 1 and 1.5 mM. A GSH (Sigma, St. Louis, U.S.A.) was added to the Ringer solution to produce a final concentration of 0.2 mM.

#### **2.3 Data analysis**

Data are expressed as mean ± SEM. Comparisons between the mean values were made with a Student's t-analysis. P values <0.05 were considered significant.

#### **3. Results**

#### **3.1 Effects of cumene hydroperoxide on the spontaneous spike potential of leech Retzius nerve cells**

In our work we used CHP to stimulate lipid peroxidation as the mechanism of free radicalinduced cell membrane damage. We investigated the time-dependent changes in action potential configuration and changes in steady-state membrane currents in LRNC. Superfusion of leech abdominal ganglia with CHP (0.25, 1 and 1.5 mM) caused an extreme change to the shape and action potential duration (APD) in LRNC. Exposure of LRNC to CHP prolonged the duration of the action potentials of the LRNC in a concentrationdependent manner. Figure 1 illustrates the representative record obtained 15, 20, 25 and 30 min after the exposure of an isolated ganglia to 1 mM of CHP. A cardiac-like action potential with a rapid depolarization, followed by a sustained depolarization or plateau and fast repolarization was recorded. During the 20 min of exposure with leech Ringer containing 1 mM CHP, early after depolarization was recorded. Higher concentration of CHP led to appearance of repetitive firing only a few minutes after application of CHP, which was followed by loss of excitability of leech Retzius nerve cells.

Fig. 1*.* Early after depolarization and repetitive firing recorded in LRNC after exposure of isolated ganglion to 1 mM CHP

Solutions containing H2O2 were prepared freshly before their use from 30% H2O2 solution (Zorka Pharma, Sabac) and added to the Ringer solution (or Tris-Ringer) at final concentrations of 1, 5 and 10 mM. CHP and GSH were added to the normal or Tris-Ringer solution. The CHP was obtained from Sigma (St. Louis, U.S.A.), dissolved in 0.01% dimethyl sulfoxide (Sigma, St. Louis, U.S.A.) and added to the Ringer solution (or Tris-Ringer) in a concentrations of 0.25, 1 and 1.5 mM. A GSH (Sigma, St. Louis, U.S.A.) was

Data are expressed as mean ± SEM. Comparisons between the mean values were made with

**3.1 Effects of cumene hydroperoxide on the spontaneous spike potential of leech** 

In our work we used CHP to stimulate lipid peroxidation as the mechanism of free radicalinduced cell membrane damage. We investigated the time-dependent changes in action potential configuration and changes in steady-state membrane currents in LRNC. Superfusion of leech abdominal ganglia with CHP (0.25, 1 and 1.5 mM) caused an extreme change to the shape and action potential duration (APD) in LRNC. Exposure of LRNC to CHP prolonged the duration of the action potentials of the LRNC in a concentrationdependent manner. Figure 1 illustrates the representative record obtained 15, 20, 25 and 30 min after the exposure of an isolated ganglia to 1 mM of CHP. A cardiac-like action potential with a rapid depolarization, followed by a sustained depolarization or plateau and fast repolarization was recorded. During the 20 min of exposure with leech Ringer containing 1 mM CHP, early after depolarization was recorded. Higher concentration of CHP led to appearance of repetitive firing only a few minutes after application of CHP, which was

20 mV

20 mV

10 ms

10 ms

20 mV

10 ms

20 min

added to the Ringer solution to produce a final concentration of 0.2 mM.

a Student's t-analysis. P values <0.05 were considered significant.

followed by loss of excitability of leech Retzius nerve cells.

Leech Ringer 5 min 15 min 20 min

25 min 30 min Recovery

Fig. 1*.* Early after depolarization and repetitive firing recorded in LRNC after exposure of

**2.3 Data analysis** 

**Retzius nerve cells** 

isolated ganglion to 1 mM CHP

**3. Results** 


Table 1 summarizes the values of the APD in a leech Ringer and after adding CHP (0.25, 1 and 1.5 mM). Table 1 shows that CHP caused a concentration-dependent increase in APD.

Table 1. Values of the APD (in ms) of LRNC before, 5, 15, 20 and 30 minutes after the adding of CHP (0.25, 1 and 1.5 mM) and during the recovery. Data are expressed as mean ± SEM; n=number of cells.  *repetitive firing* 

#### **3.2 Effects of hydrogen peroxide on the spontaneous spike potential of leech Retzius nerve cells**

Previous investigations have shown that H2O2 is involved in cascades of pathological events affecting neural cells. The background of this study were the findings that 1 mM CHP treatment caused an extreme change in the duration of the action potential and suppression of Ca2+ activated outward K+ currents of LRNC. The aim of the present experiments was to examine the effect of the higher concentrations of H2O2 on LRNC. H2O2 in concentrations up to 10 mM in the reaction mixture had no effect on spontaneous spike potential. Extracellular application of H2O2 (1, 5 and 10 mM) did not significantly change (P > 0.05) the duration of the action potential of the LRNC. H2O2 is ineffective in generating either cardiac-like action potential or early after depolarization in LRNC.

#### **3.3 Effects of glutathione on the cumene hydroperoxide-induced change of the spontaneous spike potential of leech Retzius nerve cells**

The background of this study were the findings that the hydroxyl radical scavenger, mannitol (5 mM) significantly reduced neurotoxic effect of CHP on the spontaneous spike electrogenesis of LRNC (Jovanović, 2010).

Taking in to consideration that it has been proved that CHP affects the lasting action potentials of Retzius nerve cells, the possibility of recovering the changes by the effect of antioxidant, GSH was also examined. Firstly, Retzius cells were exposed to the effect of CHP (1 mM) then GSH was added in a concentration of 0.2 mM to the Leech Ringer solution. The application of GSH, a free radical scavenger, to a bathing solution reverses the CHP effects. The GSH, largely inhibited the effects of CHP on the APD. In the presence of the GSH the APD has been extended by 9.22±1.14 ms (in controlled conditions, before the application of

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 345

Fig. 2. Patterns of voltage clamp current record obtained from Retzius nerve cell in Tris-Ringer, after adding 1 mM CHP and again in an Na-free fluid (recovery) during

outward current recovered by approximately 30% and 40% within 15 min.



Figure 3 shows the current-voltage relationship, separately, for the peak and established a steady level of depolarizing K+ outward current. At the test potential of +4 mV the fast and late steady part of the K+ outward current dropped from 60 to 36 nA (40%) and from 33 to 23 nA (31%). These results demonstrate the marked electrophysiological effects of CHP in leech Retzius nerve cells. Upon washout of the CHP, the fast and slow steady part of the K+




10

30

50

70

I (nA)


10

30

50

70

I (nA)

TRIS Ringer 1mM CHP

displacement of holding potential from -56 mV to +4 mV.

TRIS Ringer

CHP (1 mM)




+4 mV

Recovery TRIS Ringer

> \_\_\_ \_\_\_\_ 100 ms

50 nA

Em (mV)

Em (mV)

CHP and GSH) to 12.45±1.56 ms (30 min after adding CHP and GSH to the Leech Ringer solution), which did not have any significant statistical result (Table 2). In the presence of GSH repetitive firings has not been registered in any examined cells.


Table 2*.* Effects of GSH on CHP-induced prolongation of the APD in LRNC.

The APD of LRNC before and after the adding of CHP (1 mM) and GSH (0.2 mM). Data are expressed as mean ± SEM; n=number of cells

The application of 0.2 mM GSH solution significantly decreased the bursting frequency, duration and amplitude of depolarization plateaus, and the number of spikes per plateau. These observations point out the significance of GSH in the protection of SH groups of membrane proteins as well as lipids in oxidative stress caused by CHP.

#### **3.4 Modulation of Ca2+ activated K+ current in leech Retzius nerve cells by cumene hydroperoxide**

The elongation of action potentials by CHP suggested that CHP decreased the magnitude of ion currents needed for the repolarization phase of action potentials. The action potentials of **leech** Retzius nerve cells elongated after the exposure to 1mM of CHP, suggested that CHP modified the outward K+ currents that form action potentials together with the Na+ current. In order to explore the ionic mechanism by which CHP prolongs spike potential, we examined its effects on membrane K+currents. Several studies have reported the possibility that ROS alter ionic channel function. The K+ channels, key regulators of neuronal excitability, are targets of ROS. It is well known that outward K+ current are essential for maintaining normal APD. The K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-clamped Retzius neurones. Modifications of voltage-sensitive K+ channel activity by ROS would lead to changes in APD and the electrical excitability of neuronal cells. To test this hypothesis, the effect of CHP on Ca2+ activated K+ current was studied.

This was shown in voltage-clamped neurons by long depolarizing steps (to 500 ms) from the holding potential which was more negative than –40 mV in the sodium free leech Ringer in order to induce fast and slow K+ outward currents. Figure 2 illustrates the typical outward currents elicited in a CHP responsive neuron, depolarized in steps from a holding potential of -56 mV to +4 mV. Both components of the delayed outward K+ current, Ikr and Iks were inhibited by external CHP.

Application of CHP (in a concentration of 1 mM) caused suppression of fast and slow Ca2+ activated outward K+ currents. The fast and slow steady part of the K+ outward current was reduced by 40% and 31%, respectively. Figure 2 shows K+ current amplitudes measured before and during exposure to CHP.

CHP and GSH) to 12.45±1.56 ms (30 min after adding CHP and GSH to the Leech Ringer solution), which did not have any significant statistical result (Table 2). In the presence of

The APD of LRNC before and after the adding of CHP (1 mM) and GSH (0.2 mM). Data are

The application of 0.2 mM GSH solution significantly decreased the bursting frequency, duration and amplitude of depolarization plateaus, and the number of spikes per plateau. These observations point out the significance of GSH in the protection of SH groups of

**3.4 Modulation of Ca2+ activated K+ current in leech Retzius nerve cells by cumene** 

The elongation of action potentials by CHP suggested that CHP decreased the magnitude of ion currents needed for the repolarization phase of action potentials. The action potentials of **leech** Retzius nerve cells elongated after the exposure to 1mM of CHP, suggested that CHP modified the outward K+ currents that form action potentials together with the Na+ current. In order to explore the ionic mechanism by which CHP prolongs spike potential, we examined its effects on membrane K+currents. Several studies have reported the possibility that ROS alter ionic channel function. The K+ channels, key regulators of neuronal excitability, are targets of ROS. It is well known that outward K+ current are essential for maintaining normal APD. The K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-clamped Retzius neurones. Modifications of voltage-sensitive K+ channel activity by ROS would lead to changes in APD and the electrical excitability of neuronal cells. To test this hypothesis, the

This was shown in voltage-clamped neurons by long depolarizing steps (to 500 ms) from the holding potential which was more negative than –40 mV in the sodium free leech Ringer in order to induce fast and slow K+ outward currents. Figure 2 illustrates the typical outward currents elicited in a CHP responsive neuron, depolarized in steps from a holding potential of -56 mV to +4 mV. Both components of the delayed outward K+ current, Ikr and Iks were

Application of CHP (in a concentration of 1 mM) caused suppression of fast and slow Ca2+ activated outward K+ currents. The fast and slow steady part of the K+ outward current was reduced by 40% and 31%, respectively. Figure 2 shows K+ current amplitudes measured

Ringer 5 min 15 min 20 min 30 min Recovery

20 min

P>0.05 10.341.21

12.451.56

GSH repetitive firings has not been registered in any examined cells.

n=10 9.221.14 8.872.34 9.671.44 10.762.32

membrane proteins as well as lipids in oxidative stress caused by CHP.

Table 2*.* Effects of GSH on CHP-induced prolongation of the APD in LRNC.

Leech

expressed as mean ± SEM; n=number of cells

effect of CHP on Ca2+ activated K+ current was studied.

CHP+GSH

**hydroperoxide** 

inhibited by external CHP.

before and during exposure to CHP.

Fig. 2. Patterns of voltage clamp current record obtained from Retzius nerve cell in Tris-Ringer, after adding 1 mM CHP and again in an Na-free fluid (recovery) during displacement of holding potential from -56 mV to +4 mV.

Figure 3 shows the current-voltage relationship, separately, for the peak and established a steady level of depolarizing K+ outward current. At the test potential of +4 mV the fast and late steady part of the K+ outward current dropped from 60 to 36 nA (40%) and from 33 to 23 nA (31%). These results demonstrate the marked electrophysiological effects of CHP in leech Retzius nerve cells. Upon washout of the CHP, the fast and slow steady part of the K+ outward current recovered by approximately 30% and 40% within 15 min.

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 347



Fig. 4. Current-voltage relationship for the same cell measured at the peak of the K+ outward current (open circles) and at the end of stimulation (solid circles) in Tris-Ringer, 25 min after

Ikr- rapid Ca2+ activated K+ current; Iks- slow Ca2+ activated K+ current; Ileak - passive leak

**3.6 Effects of glutathione on cumene hydroperoxide-induced suppression of the Ca2+** 

Reduced glutathione applied in a concentration of 0.2 mM partially blocked the effect of CHP on Ca2+ activated outward K+ currents. Figure 5 illustrates the effect of GSH on Ca2+ activated K+ currents. The application of the GSH reduced fast and slow K+ outward currents in the leech Ringer. At the test potential of -17 mV from the holding potential of -57 mV, the fast and late steady part of the K+ outward current dropped by 21% and 12%,


10

30

50

70

I (nA)




10

Em (mV)

Em (mV)

Ikr Iks Ileak

30

50

70

I (nA)

H2O2 (5 mM)

Recovery (TRIS Ringer)

adding 5 mM H2O2 and during the recovery.

**activated K+ current of leech Retzius nerve cells** 

current

respectively.

Fig. 3. The current-voltage relationship for the same cell, measured at the peak of the K+ outward current (open circles) and at the end of stimulation (solid circles) in the Tris-Ringer, 25 min after adding 1 mM CHP and during the recovery.

#### **3.5 Effects of hydrogen peroxide on the Ca2+ activated K+ current of leech Retzius nerve cells**

Modification of on membrane potassium currents by H2O2, a membrane-permeable form of ROS, in LRNC was examined using the voltage clamp technique. Using a two-electrode voltage clamp, we examined the H2O2 effect on the K+ outward current. In contrast to the effect of CHP, application of the H2O2 failed to inhibit fast and slow outward K+ currents in leech Ringer. At the test potential of -14 mV from holding potential of -77 mV, the fast and late steady part of K+ outward current dropped by 7.48% and 6.07%, respectively. In the current-voltage relationship (Fig. 4) there were no significant changes on the early or late part of the K+ outward current in the presence of H2O2. Voltage clamp experiments using double microelectrode methods revealed that H2O2 reduced a fast and slow K+ outward current by 7.48% and 6.07% respectively at the test potential of -14 mV, after 25 min.

Ikr- rapid Ca2+ activated K+ current; Iks- slow Ca2+ activated K+ current; Ileak - passive leak current



Fig. 3. The current-voltage relationship for the same cell, measured at the peak of the K+ outward current (open circles) and at the end of stimulation (solid circles) in the Tris-Ringer,

**3.5 Effects of hydrogen peroxide on the Ca2+ activated K+ current of leech Retzius** 

current by 7.48% and 6.07% respectively at the test potential of -14 mV, after 25 min.


Ikr- rapid Ca2+ activated K+ current; Iks- slow Ca2+ activated K+ current; Ileak - passive leak



10

30

50

70

I (nA)

Modification of on membrane potassium currents by H2O2, a membrane-permeable form of ROS, in LRNC was examined using the voltage clamp technique. Using a two-electrode voltage clamp, we examined the H2O2 effect on the K+ outward current. In contrast to the effect of CHP, application of the H2O2 failed to inhibit fast and slow outward K+ currents in leech Ringer. At the test potential of -14 mV from holding potential of -77 mV, the fast and late steady part of K+ outward current dropped by 7.48% and 6.07%, respectively. In the current-voltage relationship (Fig. 4) there were no significant changes on the early or late part of the K+ outward current in the presence of H2O2. Voltage clamp experiments using double microelectrode methods revealed that H2O2 reduced a fast and slow K+ outward

I (nA)

Em (mV)

Em (mV)

Ikr Iks Ileak

Recovery (TRIS Ringer)

25 min after adding 1 mM CHP and during the recovery.

**nerve cells** 

current

TRIS Ringer

Fig. 4. Current-voltage relationship for the same cell measured at the peak of the K+ outward current (open circles) and at the end of stimulation (solid circles) in Tris-Ringer, 25 min after adding 5 mM H2O2 and during the recovery.

Ikr- rapid Ca2+ activated K+ current; Iks- slow Ca2+ activated K+ current; Ileak - passive leak current

#### **3.6 Effects of glutathione on cumene hydroperoxide-induced suppression of the Ca2+ activated K+ current of leech Retzius nerve cells**

Reduced glutathione applied in a concentration of 0.2 mM partially blocked the effect of CHP on Ca2+ activated outward K+ currents. Figure 5 illustrates the effect of GSH on Ca2+ activated K+ currents. The application of the GSH reduced fast and slow K+ outward currents in the leech Ringer. At the test potential of -17 mV from the holding potential of -57 mV, the fast and late steady part of the K+ outward current dropped by 21% and 12%, respectively.

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 349


Fig. 6. Current-voltage relationship for the same cell measured at the peak of the potassium outward current (open circles) and at the end of stimulation (solid circles) in the Tris-Ringer,

Ikr- rapid Ca2+ activated K+ current; Iks- slow Ca2+ activated K+ current; Ileak - passive leak

The interest in H2O2 as a biologically active oxygen-derived intermediate is evident, because it is associated to a series of alterations and effects in different types of cells. The present data show that H2O2 did not significantly change, within 30 min, the shape of the amplitude of spontaneous spike potentials of LRNC. In the voltage clamp experiments, H2O2 was ineffective in the supression of fast and slow Ca2+ activated K+ currents. The background of this study were the findings that a 1 mM H2O2 treatment with or without FeCl2 did not significantly change the resting membrane potential of LRNC (Jovanović & Beleslin, 1996;

The present results suggest that leech ganglia have an efficient system against oxidative stress. There are several explanations why leech Retzius nerve cells should be resistant to H2O2. The simplest could be that leech Retzius nerve cells have a low concentration of polyunsaturated fatty acids, which are very sensitive to radical injury. This possibility is unlikely since neuronal membrane are rich in lipids (Whittemore et al., 1995; Wilson, 1997). Another explanation could be that they have an efficient scavenging enzyme system which reacts rapidly with H2O2. Peroxidation of lipids that inactivates membrane-associated enzymatic protein, increases membrane permeability. However, since we have insignificant changes in action potential with H2O2, it is reasonable to suppose that lipid membrane peroxidation did not occur. On the other hand, since changes in the action potential were not significant, it could further be expected that H2O2 was decomposed by a number of enzymatic and nonenzymatic antioxidant systems. A possible explanation for weak responses of LRNC to H2O2 is that the extracellular H2O2 application, results in an intracellular concentration some 7–10-fold below that of extracellular (Stone, 2004). It is well known that concentration as well as time of exposure plays an important role in the



25 min after adding 1 mM CHP and 0.2 mM GSH, and during the recovery.

Jovanović & Beleslin, 1997; Jovanović & Beleslin, 2004).

10

Em (mV)

Ikr Iks Ileak

30

50

70

I (nA)

Recovery (TRIS Ringer)

current

**4. Discussion** 

Fig. 5. Patterns of the voltage clamp current record obtained from Retzius nerve cell in the Tris-Ringer, after adding 1 mM CHP and 0.2 mM GSH, and in Na-free fluid (recovery) during displacement of the holding potential from -57 mV to -17 mV.

In the corresponding current-voltage relationship (Fig. 6) there were no significant changes on the early or late part of the K+ outward current in the presence of 1 mM CHP and 0.2 mM GSH. At the test potential of -17 mV the fast and late steady part of the K+ outward current dropped from 65 to 51 nA (21%) and from 46 to 38 nA (12%).

Fig. 6. Current-voltage relationship for the same cell measured at the peak of the potassium outward current (open circles) and at the end of stimulation (solid circles) in the Tris-Ringer, 25 min after adding 1 mM CHP and 0.2 mM GSH, and during the recovery.

Ikr- rapid Ca2+ activated K+ current; Iks- slow Ca2+ activated K+ current; Ileak - passive leak current

#### **4. Discussion**

348 Oxidative Stress and Diseases

TRIS Ringer CHP + GSH Recovery

Fig. 5. Patterns of the voltage clamp current record obtained from Retzius nerve cell in the Tris-Ringer, after adding 1 mM CHP and 0.2 mM GSH, and in Na-free fluid (recovery)

In the corresponding current-voltage relationship (Fig. 6) there were no significant changes on the early or late part of the K+ outward current in the presence of 1 mM CHP and 0.2 mM GSH. At the test potential of -17 mV the fast and late steady part of the K+ outward current





10

30

50

70

I (nA)

I (nA)

during displacement of the holding potential from -57 mV to -17 mV.

dropped from 65 to 51 nA (21%) and from 46 to 38 nA (12%).

CHP (1m M) + GSH (0.2 mM)

TRIS Ringer




\_\_\_\_\_ \_\_\_

50 nA

TRIS Ringer

Em (mV)

Em (mV)

100 ms

The interest in H2O2 as a biologically active oxygen-derived intermediate is evident, because it is associated to a series of alterations and effects in different types of cells. The present data show that H2O2 did not significantly change, within 30 min, the shape of the amplitude of spontaneous spike potentials of LRNC. In the voltage clamp experiments, H2O2 was ineffective in the supression of fast and slow Ca2+ activated K+ currents. The background of this study were the findings that a 1 mM H2O2 treatment with or without FeCl2 did not significantly change the resting membrane potential of LRNC (Jovanović & Beleslin, 1996; Jovanović & Beleslin, 1997; Jovanović & Beleslin, 2004).

The present results suggest that leech ganglia have an efficient system against oxidative stress. There are several explanations why leech Retzius nerve cells should be resistant to H2O2. The simplest could be that leech Retzius nerve cells have a low concentration of polyunsaturated fatty acids, which are very sensitive to radical injury. This possibility is unlikely since neuronal membrane are rich in lipids (Whittemore et al., 1995; Wilson, 1997). Another explanation could be that they have an efficient scavenging enzyme system which reacts rapidly with H2O2. Peroxidation of lipids that inactivates membrane-associated enzymatic protein, increases membrane permeability. However, since we have insignificant changes in action potential with H2O2, it is reasonable to suppose that lipid membrane peroxidation did not occur. On the other hand, since changes in the action potential were not significant, it could further be expected that H2O2 was decomposed by a number of enzymatic and nonenzymatic antioxidant systems. A possible explanation for weak responses of LRNC to H2O2 is that the extracellular H2O2 application, results in an intracellular concentration some 7–10-fold below that of extracellular (Stone, 2004). It is well known that concentration as well as time of exposure plays an important role in the

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 351

al., 2010; Whyte et al., 2009). Nakaya et al. (1992 ) examined the mechanism of membrane depolarization induced by CHP in guinea-pig papillary muscles, using ion-selective microelectrode and patch clamp techniques. They demonstrated that the depolarization of the resting membrane is, at least in part, due to the inhibition of inward rectifier K+ channel activity, and may play an important role in the genesis of reperfusion-induced arrhythmias. There are conflicting descriptions of the current changes induced by ROS, and an incomplete understanding of which is responsible for the observed changes in action potential duration. For example, the inward rectifying K+ current has been reported to be either unaffected (Cerbai et al., 1991) or decreased (Tarr & Valenzeno, 1991). The electrophysiological effects of ROS generally consist of a reduction in action potential amplitude and an increase in action potential duration followed by a reduction (Tarr and Valenzeno, 1989; Barrington, 1994; Satoh and Matsui, 1997), although either only a reduction (Goldhaber et al., 1989; Hayashi et al., 1989; Coetzee et al., 1990) or only an increase in action potential duration (Barrington, 1994) have also been reported. Nakaya et al. (1991) reported that ROS-induced shortening of the action potential duration of guinea-pig isolated ventricular myocytes. The underlying mechanisms of the action potential shortening are undoubtedly complex, and changes in membrane currents other than the outward current through the ATP-sensitive K+ channels may also contribute to the action potential shortening. Matsuura and Shattock (1991) demonstrated that oxidant stress induces a decrease in the resting potassium conductance and an increase in Ca2+ activated membrane conductance. Both factors may underlie the depolarization of resting membrane potential, prolongation of the action potential and automaticity. Tokube et al. (1996) reported biphasic changes in the action potential duration, with initial lengthening of the action potential and subsequent shortening. In voltage-clamp experiments, ROS suppressed the L-type calcium current, the delayed rectifier K+ current and the inward rectifier K+ current. A recent paper reported that relatively low concentrations of CHP (100 μM) led to a significant decrease in

the cellular content of ATP and reduced glutathione (Vimard et al., 2010).

K+ channels are a family of ion channels that govern the intrinsic electrical properties of neurons in the brain (Lujan, 2010). K+ currents control action potential duration, Ca2+ dependent synaptic plasticity, the release of neurotransmitters and epileptiform burst activity. Enhanced excitability of K+ channels (via downmodulation, or changes in biophysical properties such as inactivation kinetics and voltage dependence), could all result in enhanced Ca2+ responses to firing activity (Pongs, 1999). Ca2+ activated K+ channels are a large family of K+ channels that are found throughout the central nervous system and in many other cell types, but its *in vivo* physiological functions have not been fully defined. Ca2+ dependent K+ channels are activated by both depolarization and increases in intracellular free [Ca2+]. Ca2+ dependent K+ currents contribute to the repolarization of neurons to resting membrane potential (Hille, 2001). Thus, Ca2+ dependent K+ channels determine the shape of the action potential and help in regulating cell excitability (Goodman, 2008). Outward currents play a principal but not a sole role in repolarization in many types of excitable cells. The excitability of neurons is governed by the input they receive from their neighbours and the intrinsic excitability of the neuron. Electrophysiological studies have revealed that the voltage gated ion channels are important in determining the intrinsic excitability of neurons in the CNS. The voltage gated ion channels are critical in producing hyperexcitability such as that associated with seizure discharges (Errington, 2005) and causing epilepsy in humans (Du et al., 2005; Ez-Sampedro

response generated by ROS. The range of [H2O2] used by different authors varies from 0.1 to 50 mM (Kourie, 1998).

When cells are exposed to external H2O2, the intracellular consumption of H2O2 catalyzed by anti-oxidants and enzymes is able to generate a gradient of H2O2 across membranes, which makes the intracellular H2O2 concentration lower than the extracellular one. Previous studies have reported that H2O2 did not affect channel activity when added to the extracellular side (Soto, 2002). In particular, oxidation of free SH groups of cysteines, present in a greater proportion at the intracellular side, could explain the observed difference. These results provide evidence for an intracellular site(s) of H2O2 action. It has been recently demonstrated that H2O2 activates TRPC6 channels via modification of thiol groups of intracellular proteins (Graham et al., 2010). H2O2 is a weaker oxidizing agent than other ROS. H2O2 is not by itself reactive enough to oxidize organic molecules in an aqueous environment. Nevertheless, the biological importance of H2O2 stems from its participation in the production of more reactive chemical species such as HO• and its role as a source of free radicals has been emphasized rather than its chemical reactivity. However, because of its extremely short half-life, HO• is effective only near the locus of its production.

The results reported in this paper show that an alkyl-hydroperoxide, CHP is a more efficient oxidant than H2O2. In contrast to H2O2, CHP, induced dose and time dependent membrane depolarization with a marked prolongation of spontaneous repetitive activity. These actions appear to underlie the prolongation of the action potential, and contribute to repetitive firing. Several mechanisms have been proposed for the plateau of the prolonged action potential, such as sustained inward Na+ current, block of Ca2+ activated K+ channels, modification of Ca2+ channels or its transformation in Na+ channels. Our findings support the second proposal. A possible explanation is that CHP form free radicals that are more stable than the HO•. H2O2 and organic hydroperoxides, generate distinct ROS. HO• generated by one-electron reduction of the H2O2, damages adjacent molecules at diffusion controlled rates. By contrast, the organic hydroperoxide triggers the generation of the free radical intermediates peroxyl and alkoxyl radicals, which can cross cellular membranes and evoke the production of the HO• (Hwang et al., 2009). It was well known that HO• generated from H2O2 could cause peroxidation of lipids that inactivates membraneassociated protein, increasing membrane permeability. This metabolic and physico-chemical alteration of a cell membrane would produce intracellular Ca2+ overload. CHP is more hydrophobic than H2O2. The most important finding of the present study is that CHP modulates Ca2+ activated K+ channels in leech Retzius nerve cells. In the voltage clamp experiments, fast and slow Ca2+ activated outward K+ currents were suppressed with CHP. The present results support the view that CHP stimulates lipid peroxidation, as the mechanism of ROS-induced cell membrane injury.

Although several previous investigations have described electrophysiological effects of H2O2 and CHP, the literature describing these effects is sometimes contradictory. For example, Cerbai et al. (1991) and Ward and Giles (1997) did not observe any effect, in contrast to Tarr and Valenzeno (1989) who obtained a decrease in the rectifying current. Vega-Saenz de Miera and Rudy (1992) reported that H2O2 inhibited three cloned voltagegated K+ channels expressed in Xenopus oocytes. A recent paper reported that ROS donors (H2O2 and t-BHP) reduced the voltage operated Ca2+ current but increased the amplitude of the delayed rectifier K+ current in adult rat intracardiac ganglion neurons (Dyavanapalli et

response generated by ROS. The range of [H2O2] used by different authors varies from 0.1 to

When cells are exposed to external H2O2, the intracellular consumption of H2O2 catalyzed by anti-oxidants and enzymes is able to generate a gradient of H2O2 across membranes, which makes the intracellular H2O2 concentration lower than the extracellular one. Previous studies have reported that H2O2 did not affect channel activity when added to the extracellular side (Soto, 2002). In particular, oxidation of free SH groups of cysteines, present in a greater proportion at the intracellular side, could explain the observed difference. These results provide evidence for an intracellular site(s) of H2O2 action. It has been recently demonstrated that H2O2 activates TRPC6 channels via modification of thiol groups of intracellular proteins (Graham et al., 2010). H2O2 is a weaker oxidizing agent than other ROS. H2O2 is not by itself reactive enough to oxidize organic molecules in an aqueous environment. Nevertheless, the biological importance of H2O2 stems from its participation in the production of more reactive chemical species such as HO• and its role as a source of free radicals has been emphasized rather than its chemical reactivity. However, because of its

extremely short half-life, HO• is effective only near the locus of its production.

mechanism of ROS-induced cell membrane injury.

The results reported in this paper show that an alkyl-hydroperoxide, CHP is a more efficient oxidant than H2O2. In contrast to H2O2, CHP, induced dose and time dependent membrane depolarization with a marked prolongation of spontaneous repetitive activity. These actions appear to underlie the prolongation of the action potential, and contribute to repetitive firing. Several mechanisms have been proposed for the plateau of the prolonged action potential, such as sustained inward Na+ current, block of Ca2+ activated K+ channels, modification of Ca2+ channels or its transformation in Na+ channels. Our findings support the second proposal. A possible explanation is that CHP form free radicals that are more stable than the HO•. H2O2 and organic hydroperoxides, generate distinct ROS. HO• generated by one-electron reduction of the H2O2, damages adjacent molecules at diffusion controlled rates. By contrast, the organic hydroperoxide triggers the generation of the free radical intermediates peroxyl and alkoxyl radicals, which can cross cellular membranes and evoke the production of the HO• (Hwang et al., 2009). It was well known that HO• generated from H2O2 could cause peroxidation of lipids that inactivates membraneassociated protein, increasing membrane permeability. This metabolic and physico-chemical alteration of a cell membrane would produce intracellular Ca2+ overload. CHP is more hydrophobic than H2O2. The most important finding of the present study is that CHP modulates Ca2+ activated K+ channels in leech Retzius nerve cells. In the voltage clamp experiments, fast and slow Ca2+ activated outward K+ currents were suppressed with CHP. The present results support the view that CHP stimulates lipid peroxidation, as the

Although several previous investigations have described electrophysiological effects of H2O2 and CHP, the literature describing these effects is sometimes contradictory. For example, Cerbai et al. (1991) and Ward and Giles (1997) did not observe any effect, in contrast to Tarr and Valenzeno (1989) who obtained a decrease in the rectifying current. Vega-Saenz de Miera and Rudy (1992) reported that H2O2 inhibited three cloned voltagegated K+ channels expressed in Xenopus oocytes. A recent paper reported that ROS donors (H2O2 and t-BHP) reduced the voltage operated Ca2+ current but increased the amplitude of the delayed rectifier K+ current in adult rat intracardiac ganglion neurons (Dyavanapalli et

50 mM (Kourie, 1998).

al., 2010; Whyte et al., 2009). Nakaya et al. (1992 ) examined the mechanism of membrane depolarization induced by CHP in guinea-pig papillary muscles, using ion-selective microelectrode and patch clamp techniques. They demonstrated that the depolarization of the resting membrane is, at least in part, due to the inhibition of inward rectifier K+ channel activity, and may play an important role in the genesis of reperfusion-induced arrhythmias.

There are conflicting descriptions of the current changes induced by ROS, and an incomplete understanding of which is responsible for the observed changes in action potential duration. For example, the inward rectifying K+ current has been reported to be either unaffected (Cerbai et al., 1991) or decreased (Tarr & Valenzeno, 1991). The electrophysiological effects of ROS generally consist of a reduction in action potential amplitude and an increase in action potential duration followed by a reduction (Tarr and Valenzeno, 1989; Barrington, 1994; Satoh and Matsui, 1997), although either only a reduction (Goldhaber et al., 1989; Hayashi et al., 1989; Coetzee et al., 1990) or only an increase in action potential duration (Barrington, 1994) have also been reported. Nakaya et al. (1991) reported that ROS-induced shortening of the action potential duration of guinea-pig isolated ventricular myocytes. The underlying mechanisms of the action potential shortening are undoubtedly complex, and changes in membrane currents other than the outward current through the ATP-sensitive K+ channels may also contribute to the action potential shortening. Matsuura and Shattock (1991) demonstrated that oxidant stress induces a decrease in the resting potassium conductance and an increase in Ca2+ activated membrane conductance. Both factors may underlie the depolarization of resting membrane potential, prolongation of the action potential and automaticity. Tokube et al. (1996) reported biphasic changes in the action potential duration, with initial lengthening of the action potential and subsequent shortening. In voltage-clamp experiments, ROS suppressed the L-type calcium current, the delayed rectifier K+ current and the inward rectifier K+ current. A recent paper reported that relatively low concentrations of CHP (100 μM) led to a significant decrease in the cellular content of ATP and reduced glutathione (Vimard et al., 2010).

K+ channels are a family of ion channels that govern the intrinsic electrical properties of neurons in the brain (Lujan, 2010). K+ currents control action potential duration, Ca2+ dependent synaptic plasticity, the release of neurotransmitters and epileptiform burst activity. Enhanced excitability of K+ channels (via downmodulation, or changes in biophysical properties such as inactivation kinetics and voltage dependence), could all result in enhanced Ca2+ responses to firing activity (Pongs, 1999). Ca2+ activated K+ channels are a large family of K+ channels that are found throughout the central nervous system and in many other cell types, but its *in vivo* physiological functions have not been fully defined. Ca2+ dependent K+ channels are activated by both depolarization and increases in intracellular free [Ca2+]. Ca2+ dependent K+ currents contribute to the repolarization of neurons to resting membrane potential (Hille, 2001). Thus, Ca2+ dependent K+ channels determine the shape of the action potential and help in regulating cell excitability (Goodman, 2008). Outward currents play a principal but not a sole role in repolarization in many types of excitable cells. The excitability of neurons is governed by the input they receive from their neighbours and the intrinsic excitability of the neuron. Electrophysiological studies have revealed that the voltage gated ion channels are important in determining the intrinsic excitability of neurons in the CNS. The voltage gated ion channels are critical in producing hyperexcitability such as that associated with seizure discharges (Errington, 2005) and causing epilepsy in humans (Du et al., 2005; Ez-Sampedro

Effects of Oxidative Stress on the Electrophysiological Function of Neuronal Membranes 353

Electrophysiological analyses showed that oxidative modification of K+ channels might represent a fundamental pathogenic mechanism in the mammalian brain during normal aging, as well as in neurodegenerative diseases such as Alzheimer's and Parkinson's. Therefore, it is probable that the action of ROS on K+ channels might play a role in changes in electrical identity of neurons produced by ischemia and of course in neuronal death.

Considering that K+ channels and ROS are universal players in the biological game, we put forward the hypothesis that the oxidation of voltage-gated K+ channels may represent a general pathogenic mechanism in biological organisms. Obviously, more work is needed to establish the possible involvement of ion channels and of their modulation by ROS as important mechanisms in several pathological conditions in the brain. In addition, such knowledge may help to explain pathophysiological alterations in neurological diseases and to develop new strategies for therapeutic intervention that aim at preventing oxidative

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et al., 2006; Jorge et al., 2011). Ca2+ activated K+ channels are essential for the production of bursting activity in mammalian cortical neurons (Jin et al., 2000) and they can also influence rhythmic firing patterns and bursting output (Gu et al., 2007). Voltage gated K+ currents play crucial roles in modifying neuronal cellular and network excitability and activity (Muller & Connor, 1991).

Results of our study demonstrate that SH reducing agent, GSH, incompletely inhibited the effect of CHP on calcium-activated potassium channels in LRNC. The SH groups are known to be important for the function of many membrane transport systems. These include also various potassium channels (Lee et al., 1994; Han et al., 1996). Redox modification of cysteine SH groups may also be an important mechanism of controlling ion channel function. There are several explanations for the incomplete recovery of calcium-activated potassium channels. The simplest could be that CHP treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Cysteine and methionine residues are particularly sensitive to oxidation by almost all forms of ROS. In addition, the localization of the critical SH groups (responsible for the inhibitory action of the oxidative agents), could explain the observed partly protective effects of glutathione against cumene hydroperoxide-induced toxicity. The part of changes in channel properties depend on cysteine residues located on the cytoplasmic domains of the calcium-activated potassium channels in LRNC. The activity of potassium channels is dependent on the redox status of one or more SH groups on the channel protein, or an associated regulatory protein. Of course, it is possible that the oxidant agent affects other components associated to the membrane or to the channel (the target of the oxidizing agent could be a β-subunit or some membrane-bound enzyme able to promote channel phosphorylation).

#### **5. Conclusions and implications**

The present data show that the CHP is a more efficient neurotoxin and oxidant than H2O2, as well as that the suppression of Ca2+ activated outward K+ currents is responsible for the prolongation of spike potential in leech Retzius nerve cells. Here we discuss the implications that free radicals can have a significant role in the appearance of spontaneous repetitive activity in Retzius nerve cells by interrupting the process of repolarization.

What is the pathophysiological relevance of a block of voltage-gated K+ channels? In the past few years it has become more appreciated that a block of voltage-gated K+ channels by ROS contributes to increased neuronal excitability and repetitive firing. These data indicate that a block of voltage gated K+ channels contributes to an increase in neuronal excitability such as that associated with seizure discharges and causing epilepsy in humans. The interaction of ROS with K+ channels may cause modifications of membrane currents and potentials thereby leading to neuronal dysfunction. The suppression of Ca2+ dependent K+ currents proposed in this paper might have a broader significance, pertaining not only to leeches, but mammalian neurons as well. Leech ganglia are good models for studying the cellular basis for epileptiform activity. The largest neurons in the leech nervous system are Retzius cells which exhibit stable resting membrane potential and which are non-bursting neurons with a low spontaneous firing rate. An understanding of ion mechanisms epilepsy will provide insight into the molecular events of epileptogenesis, improve molecular diagnostic utility, and identify novel therapeutic targets for improved treatment of human epilepsy.

Electrophysiological analyses showed that oxidative modification of K+ channels might represent a fundamental pathogenic mechanism in the mammalian brain during normal aging, as well as in neurodegenerative diseases such as Alzheimer's and Parkinson's. Therefore, it is probable that the action of ROS on K+ channels might play a role in changes in electrical identity of neurons produced by ischemia and of course in neuronal death.

Considering that K+ channels and ROS are universal players in the biological game, we put forward the hypothesis that the oxidation of voltage-gated K+ channels may represent a general pathogenic mechanism in biological organisms. Obviously, more work is needed to establish the possible involvement of ion channels and of their modulation by ROS as important mechanisms in several pathological conditions in the brain. In addition, such knowledge may help to explain pathophysiological alterations in neurological diseases and to develop new strategies for therapeutic intervention that aim at preventing oxidative stress in the brain.

#### **6. References**

352 Oxidative Stress and Diseases

et al., 2006; Jorge et al., 2011). Ca2+ activated K+ channels are essential for the production of bursting activity in mammalian cortical neurons (Jin et al., 2000) and they can also influence rhythmic firing patterns and bursting output (Gu et al., 2007). Voltage gated K+ currents play crucial roles in modifying neuronal cellular and network excitability and activity

Results of our study demonstrate that SH reducing agent, GSH, incompletely inhibited the effect of CHP on calcium-activated potassium channels in LRNC. The SH groups are known to be important for the function of many membrane transport systems. These include also various potassium channels (Lee et al., 1994; Han et al., 1996). Redox modification of cysteine SH groups may also be an important mechanism of controlling ion channel function. There are several explanations for the incomplete recovery of calcium-activated potassium channels. The simplest could be that CHP treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Cysteine and methionine residues are particularly sensitive to oxidation by almost all forms of ROS. In addition, the localization of the critical SH groups (responsible for the inhibitory action of the oxidative agents), could explain the observed partly protective effects of glutathione against cumene hydroperoxide-induced toxicity. The part of changes in channel properties depend on cysteine residues located on the cytoplasmic domains of the calcium-activated potassium channels in LRNC. The activity of potassium channels is dependent on the redox status of one or more SH groups on the channel protein, or an associated regulatory protein. Of course, it is possible that the oxidant agent affects other components associated to the membrane or to the channel (the target of the oxidizing agent could be a β-subunit or some

The present data show that the CHP is a more efficient neurotoxin and oxidant than H2O2, as well as that the suppression of Ca2+ activated outward K+ currents is responsible for the prolongation of spike potential in leech Retzius nerve cells. Here we discuss the implications that free radicals can have a significant role in the appearance of spontaneous repetitive

What is the pathophysiological relevance of a block of voltage-gated K+ channels? In the past few years it has become more appreciated that a block of voltage-gated K+ channels by ROS contributes to increased neuronal excitability and repetitive firing. These data indicate that a block of voltage gated K+ channels contributes to an increase in neuronal excitability such as that associated with seizure discharges and causing epilepsy in humans. The interaction of ROS with K+ channels may cause modifications of membrane currents and potentials thereby leading to neuronal dysfunction. The suppression of Ca2+ dependent K+ currents proposed in this paper might have a broader significance, pertaining not only to leeches, but mammalian neurons as well. Leech ganglia are good models for studying the cellular basis for epileptiform activity. The largest neurons in the leech nervous system are Retzius cells which exhibit stable resting membrane potential and which are non-bursting neurons with a low spontaneous firing rate. An understanding of ion mechanisms epilepsy will provide insight into the molecular events of epileptogenesis, improve molecular diagnostic utility, and identify novel therapeutic targets for improved treatment of human

membrane-bound enzyme able to promote channel phosphorylation).

activity in Retzius nerve cells by interrupting the process of repolarization.

**5. Conclusions and implications** 

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mesangial cells by Cd2+: involvement of p38 kinase and CaMK-II. *Journal of Cellular* 

H2O2 elimination by mammalian cells including H2O2 permeation through cytoplasmic and peroxisomal membranes: comparison with experimental data.

currents in isilated ventricular myocytes. *American Journal of Physiology,* Vol. 261,

activation of the MAP kinase signaling pathways. *Antioxidants and Redox Signaling,* 

blockers on the action potential shortening in hypoxic and ischaemic myocardium.


**16** 

**Circulating Advanced Oxidation Protein** 

**Pro-Inflammatory Cytokines in Patients** 

Eugenia Murawska-Cialowicz2 and Monika Pazgan-Simon3 *1Department of Pharmaceutical Biochemistry, Wroclaw Medical University,* 

*3Clinic of Infectious Diseases, Liver Diseases and Acquired Immune Deficiency,* 

*2Department of Physiology and Biochemistry, University of Physical Education, Wroclaw,* 

Patients with chronic liver disease are characterized by hepatic inflammation and the destruction of hepatocytes. Viral antigen-specific cytotoxic T lymphocytes, polyclonal cytokines, immune modulators, and oxidized biomolecules have been shown to induce damage and destruction of hepatocytes in these patients (Tsutsui *et al*., 2003). The contribution of oxidative stress *per se* to the chronic inflammatory state has been suggested, and consistent evidence has been afforded that both monocyte/macrophage activation and a defect in antioxidant systems occur early in the course of chronic liver failure and gradually increase with its progression to end-stage liver disease (Kirkham, 2007; Videla, 2009). Oxidative stress lead to formation of glycoxidation products, including advanced glycation endproducts (AGEs - among them Nε- (carboxymethyl)lysine (CML) is best known), and advanced oxidation protein products (AOPPs). AOPPs can be formed *in vitro* by exposure of serum albumin to hypochlorous acid. *In vivo*, plasma AOPPs are mainly carried by albumin and their concentrations are closely correlated with the levels of dityrosine. Within the heterogeneous group of AGEs, Nε-(carboxymethyl)lysine has been identified as a major AGEs *in vivo* (Reddy *et al*., 1995). Plasma concentrations of AGEs (closely correlating with AOPPs levels) increase with progression of chronic diseases (Witko-Sarsat *et al.*, 1996; 1998), therefore CML has been considered as liver disease-related biomarker for oxidative stress (Sebeková *et al*., 2002;

The receptor for advanced glycation endproducts (RAGE) is a signal transduction receptor that binds both AGEs and AOPPs. RAGE is expressed by various cell types, including

**1. Introduction** 

Yagmur *et al*., 2006).

**with Liver Cirrhosis: Correlations** 

**with Clinical Parameters** 

Jolanta Zuwala-Jagiello1,

*Wroclaw Medical University,* 

*Poland* 

**Products, Nε-(Carboxymethyl) Lysine and** 


### **Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters**

Jolanta Zuwala-Jagiello1,

Eugenia Murawska-Cialowicz2 and Monika Pazgan-Simon3 *1Department of Pharmaceutical Biochemistry, Wroclaw Medical University, 2Department of Physiology and Biochemistry, University of Physical Education, Wroclaw, 3Clinic of Infectious Diseases, Liver Diseases and Acquired Immune Deficiency, Wroclaw Medical University, Poland* 

#### **1. Introduction**

358 Oxidative Stress and Diseases

Takeuchi, K. & Yoshii, K. (2008). Superoxide modifies AMPA receptors and voltage-gated K+ channels of mouse hippocampal neurons. *Brain Research*, Vol.21, No.1236, pp. 49-56 Tarr, M. & Valenzeno, D.P. (1989). Modification of cardiac action potential by

Tarr, M. & Valenzeno, D.P. (1991). Modification of cardiac ionic currents by photosensiter-

Tarr, M.; Arriaga, E. & Valenzeno, D. (1995). Progression ofcardiac potassium current

Thannickal, V.J. & Fanburg, B.L. (2000). Reactive oxygen species in cell signaling. *American* 

Tokube, K.; Kiyosue, T. & Arita, M. (1996). Openings of cardiac KATP channel by oxygen free

Vacher, H.; Mohapatra, D.P. & Trimmer, J.S. (2008). Localization and targeting of voltage-

Van der Vliet, A. & Bast, A. (1992). Effect of oxidative stress on receptors and signal transmission. *Chemico-Biological Interactions,* Vol.85, No.2-3, pp. 95-116 Vega-Saenz de Miera, E. & Rudy, B. (1992). Modulation of K+ channels by hydrogen

Vimard, F.; Saucet, M.; Nicole, O.; Feuilloley, M. & Duval, D. (2010). Toxicity induced by

Ward, C.A. & Giles, W.R. (1997). Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes. *Journal of Physiology,* Vol.500, No.3, pp. 631-642 Wilson, J.X. (1997). Antioxidant defense of the brain: a role for astrocytes. *Canadian Journal of* 

Whittemore, E.R.; Loo, D.T.; Watt, J.A. & Cotman, C.W. (1995). A detailed analysis of

Whyte, K.A.; Hogg, R.C.; Dyavanapalli, J.; Harper, A.A. & Adams, D.J. (2009). Reactive

Yang, J.L.; Sykora, P.; Wilson, D.M.; Mattson, M.P. & Bohr, V.A. (2011). The excitatory

Zou, X.; Feng, Z.; Li, Y.; Wang, Y.; Wertz, K.; Weber, P.; Fu, Y. & Liu, J. (2011). Stimulation of

*Mechanisms of Ageing and Development*, Vol.132, No.8-9, pp. 405-411

pathways. *Journal of Nutritional Biochemistry,* [Epub ahead of print]

*Biochemical and Molecular Toxicology,* Vol.25, No.4, pp.205-215

*Physiology and Pharmacology,* Vol.7, No.10-11, pp. 1149-1163

*Cardiology,* Vol.21, pp. 539–543

*Cardiology,* Vol.27, pp. 1099–1109

Vol.271, No.2, pp. H478-489

Vol.88, No.4, pp. 1407-1447

Vol.67, No.4, pp. 921-993

*Autonomic Neuroscience,* Vol.150, pp. 45–52

639-649

L1005-1028

1681-1687

photosensitizer-generated reactive oxygen. *Journal of Molecular and Cellular* 

generated reactive oxygen. *Journal of Molecular and Cellular Cardiology,* Vol.23, pp.

modification after brief exposure to reactive oxygen. *Journal of Molecular and Cellular* 

*Journal of Physiology: Lung Cellular and Molecular Physiology,* Vol*.*279, No.6, pp.

radicals produced by xanthine oxidase reaction. *American Journal of Physiology,* 

dependent ion channels in mammalian central neurons. Physiological Reviews,

peroxide. *Biochemical and Biophysical Research Communications,* Vol.186, No.3, pp.

cumene hydroperoxide in PC12 cells: Protective role of thiol donors. *Journal of* 

hydrogen peroxide-induced cell death in primary neuronal culture. *Neuroscience,*

oxygen species modulate neuronal excitability in rat intrinsic cardiac ganglia.

neurotransmitter glutamate stimulates DNA repair to increase neuronal resiliency.

GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: activation of Nrf2 and JNK-p62/SQSTM1 Patients with chronic liver disease are characterized by hepatic inflammation and the destruction of hepatocytes. Viral antigen-specific cytotoxic T lymphocytes, polyclonal cytokines, immune modulators, and oxidized biomolecules have been shown to induce damage and destruction of hepatocytes in these patients (Tsutsui *et al*., 2003). The contribution of oxidative stress *per se* to the chronic inflammatory state has been suggested, and consistent evidence has been afforded that both monocyte/macrophage activation and a defect in antioxidant systems occur early in the course of chronic liver failure and gradually increase with its progression to end-stage liver disease (Kirkham, 2007; Videla, 2009). Oxidative stress lead to formation of glycoxidation products, including advanced glycation endproducts (AGEs - among them Nε- (carboxymethyl)lysine (CML) is best known), and advanced oxidation protein products (AOPPs). AOPPs can be formed *in vitro* by exposure of serum albumin to hypochlorous acid. *In vivo*, plasma AOPPs are mainly carried by albumin and their concentrations are closely correlated with the levels of dityrosine. Within the heterogeneous group of AGEs, Nε-(carboxymethyl)lysine has been identified as a major AGEs *in vivo* (Reddy *et al*., 1995). Plasma concentrations of AGEs (closely correlating with AOPPs levels) increase with progression of chronic diseases (Witko-Sarsat *et al.*, 1996; 1998), therefore CML has been considered as liver disease-related biomarker for oxidative stress (Sebeková *et al*., 2002; Yagmur *et al*., 2006).

The receptor for advanced glycation endproducts (RAGE) is a signal transduction receptor that binds both AGEs and AOPPs. RAGE is expressed by various cell types, including

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

Healthy controls

(19–56)

45 (36–57)

(20-28)

(23-30)

(0.6-0.9)

(25.3-27.8)

(50-130)

0.8 (0.7-1.0)

140 (138-141)

international ratio; MELD, model of end-stage liver disease.

Age (years) 55

ALT (U/L) 24

AST (U/L) 27

Bilirubin (mg/dL) 0.7

γGT (U/L) 26

AP (U/L) 90

Serum creatinine

Serum sodium (mEq/L)

(mg/dL)

Etiology (n) Virus hepatitis Alcohol Biliary

Albumin (g/L)

biochemical characteristics of the study group are reported in detail in Table 1.

(*n*) 40 129 41 88

Male:Female ratio 23:17 68:61 15:26 53:35

66 (18–74)

62 54 13

34 (16–45)

40 (16-79)

70 (19-150)

0.98 (1.0-3.6)

70 (41-106)

125 (100-163)

1.4 (0.7-2.4)

137 (129-142)

Table 1. Clinical and biochemical characteristics of the study subjects. Statistical significance: \**P* < 0.05; \*\* *P* < 0.01 vs. healthy controls. AST, aspartate aminotransferase; ALT, alanine aminotransferase; AP, alkaline phosphatase; γGT, γ-glutamyltransferase; INR, normalised

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 361

Physiology and Biochemistry, University of Physical Education in Wroclaw. Clinical and

Non-cirrhotic patients

56 (18–69)

18 21 2

37\* (29–49)

28 (24-33)

41 (19-64)

0.92 (0.90-0.95)

48 (41-56)

105 (100-147)

0.96 (0.9-1.2)

136 (129-138) Cirrhotic patients

55 (21-74)

44 33 11

30\* (16-45)

47\*\* (16-79)

79\*\* (19-150)

1.6\* (1.0-3.6)

92\*\* (78-106)

152\*\* (141-163)

1.39\* (0.7-2.4)

130\*\* (129-142)

All patients

monocytes/macrophages, endothelial cells, smooth muscle cells and renal cells (Miyata *et al*., 1994). Advanced glycation endproducts have been found to act as pro-inflammatory factors (Sparvero *et al*., 2009). Nevertheless, AOPPs are believed to be more closely related to inflammation (Alderman *et al*., 2002; Baskol *et al*., 2006; Fialova *et al*., 2006; Witko-Sarsat *et al*., 2003; Yazici *et al*., 2004) than AGEs, whose receptor for advanced glycation endproducts participates in AOPPs-mediated signal transduction (Kalousová *et al*., 2003; 2005). These interactions enhance reactive oxygen species formation, with activation of nuclear factor NFκB and release of pro-inflammatory cytokines (Bierhaus *et al.*, 2006; Hyogo & Yamagishi, 2008; Saito & Ishii, 2004). Moreover, the monocyte/macrophage RAGE can be up-regulated by tumor necrosis factor-α (TNF-α) (Miyata *et al.*, 1994). Peripheral blood monocytes showed activity and elevated expression of TNF-α which correlated with liver disease severity (Hanck *et al.*, 2000). The concentrations of advanced oxidation protein products are high in liver cirrhosis of various etiologies (Zuwala-Jagiello *et al*., 2009) and can reflect hemodynamic alterations in the liver (Guo *et al.*, 2008). This is accompanied by the activation of monocytes and increased expression of TNF-α (Giron-González *et al.*, 2004). High serum levels of TNF-α and interleukin-6 (IL-6) have been found in cirrhotic patients with ascites in the absence of demonstrable infection (Tilg *et al.*, 1992; Zeni *et al.*, 1993).

The accumulation of AGEs has been linked to vascular lesions in diabetes, chronic renal insufficiency, and atherosclerosis. Activation of NF-κB, mediated by RAGE, promotes expression of the cytokines, as well as pro-inflammatory adhesion molecules (Basta *et al*., 2002; Bierhaus *et al.,* 1998; Esposito *et al*., 1989), what may enhance interaction of cirrhotic vasculature with circulating monocytes (Cybulsky *et al*., 1991; Li *et al.,* 1993). Recently, it has also been shown that AOPPs activates vascular endothelial cells *via* RAGE-mediated signals (Guo *et al.*, 2008).

Endothelial activation plays an active role in the modifications of circulatory status of cirrhotic patients (Genesca *et al.*, 1999). The circulatory changes are more evident in advanced stages of liver cirrhosis, such as those represented by the presence of ascites or hepatorenal syndrome (Porcel *et al.*, 2002). We have demonstrated that elevated levels of AOPPs modified–albumin (AOPPs-albumin) are related to the severity of liver cirrhosis (Zuwala-Jagiello *et al.*, 2009; 2011). The role of AOPPs-albumin in liver cirrhosis and portal hypertension has not yet been studied. The effects of pro-inflammatory cytokines on the vessels and on liver function would influence the liver cirrhosis, with higher plasma levels of AOPPs indicating a poor prognosis. In the present study, plasma levels of AOPPsalbumin, as well as of Nε (carboxymethyl)lysine modified-albumin (CML-albumin) and proinflammatory cytokines, such as TNF-α and IL-6, have been analyzed in cirrhotic patients and were found to be correlated with clinical parameters of liver dysfunction.

#### **2. Patients and methods**

#### **2.1 Patients**

This study was performed on 129 patients with chronic liver disease admitted to the Clinic of Infectious Diseases, Liver Diseases and Acquired Immune Deficiency for evaluation. The experimental group consisted of 68 men and 61 women with age of 18–74 years (median age was 66). The control group contained 40 healthy subjects (23 men and 17 women) with age of 19–56 (median age was 55). Blood samples were collected in the Department of

monocytes/macrophages, endothelial cells, smooth muscle cells and renal cells (Miyata *et al*., 1994). Advanced glycation endproducts have been found to act as pro-inflammatory factors (Sparvero *et al*., 2009). Nevertheless, AOPPs are believed to be more closely related to inflammation (Alderman *et al*., 2002; Baskol *et al*., 2006; Fialova *et al*., 2006; Witko-Sarsat *et al*., 2003; Yazici *et al*., 2004) than AGEs, whose receptor for advanced glycation endproducts participates in AOPPs-mediated signal transduction (Kalousová *et al*., 2003; 2005). These interactions enhance reactive oxygen species formation, with activation of nuclear factor NFκB and release of pro-inflammatory cytokines (Bierhaus *et al.*, 2006; Hyogo & Yamagishi, 2008; Saito & Ishii, 2004). Moreover, the monocyte/macrophage RAGE can be up-regulated by tumor necrosis factor-α (TNF-α) (Miyata *et al.*, 1994). Peripheral blood monocytes showed activity and elevated expression of TNF-α which correlated with liver disease severity (Hanck *et al.*, 2000). The concentrations of advanced oxidation protein products are high in liver cirrhosis of various etiologies (Zuwala-Jagiello *et al*., 2009) and can reflect hemodynamic alterations in the liver (Guo *et al.*, 2008). This is accompanied by the activation of monocytes and increased expression of TNF-α (Giron-González *et al.*, 2004). High serum levels of TNF-α and interleukin-6 (IL-6) have been found in cirrhotic patients with ascites in

The accumulation of AGEs has been linked to vascular lesions in diabetes, chronic renal insufficiency, and atherosclerosis. Activation of NF-κB, mediated by RAGE, promotes expression of the cytokines, as well as pro-inflammatory adhesion molecules (Basta *et al*., 2002; Bierhaus *et al.,* 1998; Esposito *et al*., 1989), what may enhance interaction of cirrhotic vasculature with circulating monocytes (Cybulsky *et al*., 1991; Li *et al.,* 1993). Recently, it has also been shown that AOPPs activates vascular endothelial cells *via* RAGE-mediated signals

Endothelial activation plays an active role in the modifications of circulatory status of cirrhotic patients (Genesca *et al.*, 1999). The circulatory changes are more evident in advanced stages of liver cirrhosis, such as those represented by the presence of ascites or hepatorenal syndrome (Porcel *et al.*, 2002). We have demonstrated that elevated levels of AOPPs modified–albumin (AOPPs-albumin) are related to the severity of liver cirrhosis (Zuwala-Jagiello *et al.*, 2009; 2011). The role of AOPPs-albumin in liver cirrhosis and portal hypertension has not yet been studied. The effects of pro-inflammatory cytokines on the vessels and on liver function would influence the liver cirrhosis, with higher plasma levels of AOPPs indicating a poor prognosis. In the present study, plasma levels of AOPPsalbumin, as well as of Nε (carboxymethyl)lysine modified-albumin (CML-albumin) and proinflammatory cytokines, such as TNF-α and IL-6, have been analyzed in cirrhotic patients

This study was performed on 129 patients with chronic liver disease admitted to the Clinic of Infectious Diseases, Liver Diseases and Acquired Immune Deficiency for evaluation. The experimental group consisted of 68 men and 61 women with age of 18–74 years (median age was 66). The control group contained 40 healthy subjects (23 men and 17 women) with age of 19–56 (median age was 55). Blood samples were collected in the Department of

and were found to be correlated with clinical parameters of liver dysfunction.

the absence of demonstrable infection (Tilg *et al.*, 1992; Zeni *et al.*, 1993).

(Guo *et al.*, 2008).

**2. Patients and methods** 

**2.1 Patients** 

Physiology and Biochemistry, University of Physical Education in Wroclaw. Clinical and biochemical characteristics of the study group are reported in detail in Table 1.


Table 1. Clinical and biochemical characteristics of the study subjects. Statistical significance: \**P* < 0.05; \*\* *P* < 0.01 vs. healthy controls. AST, aspartate aminotransferase; ALT, alanine aminotransferase; AP, alkaline phosphatase; γGT, γ-glutamyltransferase; INR, normalised international ratio; MELD, model of end-stage liver disease.

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

**2.3 Determination of circulating Nε-(carboxymethyl) lysine** 

micrograms of CML per gram of albumin (μg/g).

**2.4 Laboratory determinations** 

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 363

absorbance at 340 nm was linear within the range of 0 to 100 μmol/L. The ratio of AOPPs concentration to albumin level (AOPPs-albumin) was expressed in micromoles of AOPPs per gram of albumin (μmol/g). The ratio of AOPPs to albumin content allows the evaluation of whether the proportion of oxidatively modified albumin is altered. Coefficient of variation (CV) served as an indicator of precision. Intra-day and inter-day CV values were <10%.

Plasma Nε-(carboxymethyl)lysine (CML) levels were determined using a specific competitive ELISA kit [CircuLex CML/Nε-(carboxymethyl)lysine ELISA Kit (CycLex Co., Ltd, Nagano, Japan)]. Measurements were performed in duplicate and the results were averaged. The ratio of CML concentration to albumin level (CML-albumin) was expressed in

Biochemical parameters were measured using routine laboratory methods. Serum highsensitivity C-reactive protein (hs-CRP) level was determined with a high-sensitivity nephelometric method using the Beckman Image Immunochemistry system (Beckman Instruments, Fullerton, CA), which has a minimum level of detection of 0.2 mg/L. Serum levels of TNF-α and IL-6 were assayed with enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. The minimum levels of detection were 1.6 pg/mL and < 0.70 pg/mL for TNF-α and IL-6, respectively. The intra- and interassay coefficients of variation for measurements of CRP, IL-6, and TNF-α were 2.7%, 4.3%, and 5.0%, respectively, and 3.0%, 5.5, and 6.9%, respectively.

Aldosterone (Aldoctk-2-P2714; Sorin Biomedica Diagnostics, Barcelona, Spain. Normal values, 35-150 pg/mL) and plasma renin activity (Clinical Assays, Baxter, Cambridge, Mass., USA. Normal values, 0.4-2.3 ng mL-1 h-1) were measured by specific radioimmunoassays. Antidiuretic hormone was also tested by a commercial radioimmunoassay (Buhlman

The plasma antioxidant capacity was measured using a commercially available total antioxidant status TAS kit (Randox Laboratories, Crumlin, UK). The TAS assay is based on the inhibition by antioxidants of the absorbance of the radical cation of 2,2'-azinobis-(3 ethylbenzothiazoline-6-sulfonate) (ABTS) formed by the interaction of ABTS with ferrylmyoglobin radical species. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the plasma, preventing the color change and thereby providing an effective measure of the total antioxidant capacity of the plasma. The assay has excellent

The model for end-stage liver disease (MELD) score was calculated from the following

Laboratories, Basel, Switzerland. Normal values, less than 1 pg/mL).

precision values, lower than 3%, and the results are expressed as mmol/L.

**2.5 Measurement of the total antioxidant status of plasma** 

**2.6 Model for End-stage Liver Disease (MELD) score** 

equation:

The diagnosis of liver cirrhosis was based on clinical, laboratory and ultrasonographic findings or histological criteria. Alcohol-related liver cirrhosis was diagnosed in 33 of patients, primary biliary cirrhosis in 11, cirrhosis caused by hepatitis C virus in 30, whereas cirrhosis caused by hepatitis B virus in 14 patients. The Child-Pugh score was used to assess the severity of liver disease. Three biochemical variables [serum albumin, bilirubin, and prothrombin time (international normalized ratio, INR)] in addition to the two clinical characteristics (presence or absence of ascites and clinical signs of encephalopathy) were determined. Patients were scored as follows: 5–6 as class (group) A, 7–9 as class (group) B and 10–15 as class (group) C. The patients with cirrhosis were divided into compensated (Child-Pugh class A) and decompensated (Child-Pugh classes B and C) groups. At the time of the study no Child-Pugh A patients showed clinical features of decompensated liver cirrhosis (ascites or hepatic encephalopathy). At enrollment, esophageal varices were detected by endoscopy in 88% of patients, ascites and hepatic encephalopathy grade were present by physical examination in 53 (60%) and 23 (26%) patients, respectively.

Exclusion criteria were concurrent use of antioxidant drugs; co-existing diseases like diabetes mellitus, chronic kidney disease, cardiovascular disease, hepatocellular carcinoma; gastrointestinal bleeding, bacterial infection, and blood transfusion within previous two weeks.

Patients had not been receiving diuretic, antibiotic, vasoactive drug (nitrates, β-blockers), and lactulose or lactitiol therapy during the eight days before inclusion in the study. After 2 h of bed rest, blood pressure was determined with an automatic digital sphygmomanometer and blood samples were collected in ice-cooled, ethylenediaminetetraacetic acid (EDTA) containing tubes for the determination of plasma renin activity, antidiuretic hormone, and plasma AOPPs or Nε-(carboxymethyl)lysine, in tubes with no additive for routine biochemical study and aldosterone and cytokine concentrations. All samples were separated immediately by centrifugation at 4°C and stored at -80°C until further analysis.

The consent of the Bioethics Committee of the Wroclaw Medical University was obtained and all patients were informed about the character of analyses made. Studies were conducted in compliance with the ethical standards formulated in the Helsinki Declaration of 1975 (revised in 1983).

#### **2.2 Determination of circulating AOPPs**

*In vivo* plasma levels of AOPPs closely correlate with levels of dityrosine, a hallmark of oxidized proteins and with pentosidine, a marker of protein glycation closely related to oxidative stress. A new chromogen is found which caused increased absorbance at 340 nm and its spectrophotometric determination is proposed as a novel index of oxidative stress measuring the level of AOPPs (Witko-Sarsat *et al*., 1996). Two- hundred microliters of plasma diluted 1:5 in 20 mM phosphate buffer pH 7.4 containing 0.9% sodium chloride (PBS), or chloramine-T standard solutions (0 to 100 μmol/L), were placed in each well of a 96-well microtiter plate (Becton Dickinson Labware, Lincoln Park, NJ, USA), followed by 20 μL of 10% acetic acid. Ten microliters of 1.16 M potassium iodide (Sigma-Aldrich Co. LLC, Canada) were then added, followed by 20 μL of 10% acetic acid. The absorbance of the reaction mixture was immediately read at 340 nm in a microplate reader against a blank containing 200 μL of PBS, 10 μL of KI and 20 μL of 10% acetic acid. The chloramine-T absorbance at 340 nm was linear within the range of 0 to 100 μmol/L. The ratio of AOPPs concentration to albumin level (AOPPs-albumin) was expressed in micromoles of AOPPs per gram of albumin (μmol/g). The ratio of AOPPs to albumin content allows the evaluation of whether the proportion of oxidatively modified albumin is altered. Coefficient of variation (CV) served as an indicator of precision. Intra-day and inter-day CV values were <10%.

#### **2.3 Determination of circulating Nε-(carboxymethyl) lysine**

Plasma Nε-(carboxymethyl)lysine (CML) levels were determined using a specific competitive ELISA kit [CircuLex CML/Nε-(carboxymethyl)lysine ELISA Kit (CycLex Co., Ltd, Nagano, Japan)]. Measurements were performed in duplicate and the results were averaged. The ratio of CML concentration to albumin level (CML-albumin) was expressed in micrograms of CML per gram of albumin (μg/g).

#### **2.4 Laboratory determinations**

362 Oxidative Stress and Diseases

The diagnosis of liver cirrhosis was based on clinical, laboratory and ultrasonographic findings or histological criteria. Alcohol-related liver cirrhosis was diagnosed in 33 of patients, primary biliary cirrhosis in 11, cirrhosis caused by hepatitis C virus in 30, whereas cirrhosis caused by hepatitis B virus in 14 patients. The Child-Pugh score was used to assess the severity of liver disease. Three biochemical variables [serum albumin, bilirubin, and prothrombin time (international normalized ratio, INR)] in addition to the two clinical characteristics (presence or absence of ascites and clinical signs of encephalopathy) were determined. Patients were scored as follows: 5–6 as class (group) A, 7–9 as class (group) B and 10–15 as class (group) C. The patients with cirrhosis were divided into compensated (Child-Pugh class A) and decompensated (Child-Pugh classes B and C) groups. At the time of the study no Child-Pugh A patients showed clinical features of decompensated liver cirrhosis (ascites or hepatic encephalopathy). At enrollment, esophageal varices were detected by endoscopy in 88% of patients, ascites and hepatic encephalopathy grade were

present by physical examination in 53 (60%) and 23 (26%) patients, respectively.

immediately by centrifugation at 4°C and stored at -80°C until further analysis.

weeks.

of 1975 (revised in 1983).

**2.2 Determination of circulating AOPPs** 

Exclusion criteria were concurrent use of antioxidant drugs; co-existing diseases like diabetes mellitus, chronic kidney disease, cardiovascular disease, hepatocellular carcinoma; gastrointestinal bleeding, bacterial infection, and blood transfusion within previous two

Patients had not been receiving diuretic, antibiotic, vasoactive drug (nitrates, β-blockers), and lactulose or lactitiol therapy during the eight days before inclusion in the study. After 2 h of bed rest, blood pressure was determined with an automatic digital sphygmomanometer and blood samples were collected in ice-cooled, ethylenediaminetetraacetic acid (EDTA) containing tubes for the determination of plasma renin activity, antidiuretic hormone, and plasma AOPPs or Nε-(carboxymethyl)lysine, in tubes with no additive for routine biochemical study and aldosterone and cytokine concentrations. All samples were separated

The consent of the Bioethics Committee of the Wroclaw Medical University was obtained and all patients were informed about the character of analyses made. Studies were conducted in compliance with the ethical standards formulated in the Helsinki Declaration

*In vivo* plasma levels of AOPPs closely correlate with levels of dityrosine, a hallmark of oxidized proteins and with pentosidine, a marker of protein glycation closely related to oxidative stress. A new chromogen is found which caused increased absorbance at 340 nm and its spectrophotometric determination is proposed as a novel index of oxidative stress measuring the level of AOPPs (Witko-Sarsat *et al*., 1996). Two- hundred microliters of plasma diluted 1:5 in 20 mM phosphate buffer pH 7.4 containing 0.9% sodium chloride (PBS), or chloramine-T standard solutions (0 to 100 μmol/L), were placed in each well of a 96-well microtiter plate (Becton Dickinson Labware, Lincoln Park, NJ, USA), followed by 20 μL of 10% acetic acid. Ten microliters of 1.16 M potassium iodide (Sigma-Aldrich Co. LLC, Canada) were then added, followed by 20 μL of 10% acetic acid. The absorbance of the reaction mixture was immediately read at 340 nm in a microplate reader against a blank containing 200 μL of PBS, 10 μL of KI and 20 μL of 10% acetic acid. The chloramine-T Biochemical parameters were measured using routine laboratory methods. Serum highsensitivity C-reactive protein (hs-CRP) level was determined with a high-sensitivity nephelometric method using the Beckman Image Immunochemistry system (Beckman Instruments, Fullerton, CA), which has a minimum level of detection of 0.2 mg/L. Serum levels of TNF-α and IL-6 were assayed with enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. The minimum levels of detection were 1.6 pg/mL and < 0.70 pg/mL for TNF-α and IL-6, respectively. The intra- and interassay coefficients of variation for measurements of CRP, IL-6, and TNF-α were 2.7%, 4.3%, and 5.0%, respectively, and 3.0%, 5.5, and 6.9%, respectively.

Aldosterone (Aldoctk-2-P2714; Sorin Biomedica Diagnostics, Barcelona, Spain. Normal values, 35-150 pg/mL) and plasma renin activity (Clinical Assays, Baxter, Cambridge, Mass., USA. Normal values, 0.4-2.3 ng mL-1 h-1) were measured by specific radioimmunoassays. Antidiuretic hormone was also tested by a commercial radioimmunoassay (Buhlman Laboratories, Basel, Switzerland. Normal values, less than 1 pg/mL).

#### **2.5 Measurement of the total antioxidant status of plasma**

The plasma antioxidant capacity was measured using a commercially available total antioxidant status TAS kit (Randox Laboratories, Crumlin, UK). The TAS assay is based on the inhibition by antioxidants of the absorbance of the radical cation of 2,2'-azinobis-(3 ethylbenzothiazoline-6-sulfonate) (ABTS) formed by the interaction of ABTS with ferrylmyoglobin radical species. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the plasma, preventing the color change and thereby providing an effective measure of the total antioxidant capacity of the plasma. The assay has excellent precision values, lower than 3%, and the results are expressed as mmol/L.

#### **2.6 Model for End-stage Liver Disease (MELD) score**

The model for end-stage liver disease (MELD) score was calculated from the following equation:

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 365

Fig. 1. **(A)** AOPPs-albumin serum concentrations in 129 patients with chronic liver disease, according to Child's stage of cirrhosis, and in an control group of 40 healthy blood donors. *P*  values are given in the table. Comparisons between subgroups are illustrated with box plot graphics, where the dotted line indicates the median per group, the box represents 50% of the values, and horizontal lines show minimum and maximum values of the calculated nonoutlier values; asterisks and open circles indicate outlier values. **(B)** AOPPs-albumin serum concentrations in patients with cirrhosis are correlated with the MELD (model of end-stage liver disease) score (*r* = 0.43, *P* < 0.01, Spearman rank correlation test). **(C)** CML-albumin serum concentrations increase with the stage of liver cirrhosis in patients with chronic liver

disease. *P* values are given in the table.

9.57 × loge (creatinine mg/dL) + 3.78 × loge (total bilirubin mg/dL) + 11.2 × loge (international normalized ratio-INR) + 6.43 (constant for liver disease etiology).

The maximal creatinine concentration considered in the MELD score is 4.0 mg/dL (Huo *et al.,* 2006).

#### **2.7 Statistical analysis**

Results are expressed as median (25th percentile–75th percentile). Frequency data were compared using the χ2 test or the Fischer's exact test when necessary. Because many of the variables analyzed did not have a normal distribution as determined by the Kolmogorov-Smirnov test, nonparametric tests were used for comparison of data. The Mann- Whitney U test and the Kruskal-Wallis test were used to analyze differences among two or more groups, respectively. Multivariate analysis by conditional logistical regression with a forward stepwise method was performed to find independent variables associated with the presence of ascites and low mean arterial pressure. Regression analysis to determine significant correlations among different parameters was performed using the Spearman correlation coefficient. Statistical significance was established at *P* < 0.05

#### **3. Results**

#### **3.1 AOPPs-albumin plasma concentrations in patients with chronic liver disease and healthy controls**

We analyzed 129 patients (68 males/61 females, median age 66 years, range 18–74 years) with chronic liver disease. The distribution of the stages of liver cirrhosis as defined according to the Child–Pugh score, and measurements of AOPPs-albumin and CMLalbumin plasma concentrations are presented in Fig.1. The concentration of AOPPs-albumin

9.57 × loge (creatinine mg/dL) + 3.78 × loge (total bilirubin mg/dL) + 11.2 × loge

The maximal creatinine concentration considered in the MELD score is 4.0 mg/dL (Huo *et* 

Results are expressed as median (25th percentile–75th percentile). Frequency data were compared using the χ2 test or the Fischer's exact test when necessary. Because many of the variables analyzed did not have a normal distribution as determined by the Kolmogorov-Smirnov test, nonparametric tests were used for comparison of data. The Mann- Whitney U test and the Kruskal-Wallis test were used to analyze differences among two or more groups, respectively. Multivariate analysis by conditional logistical regression with a forward stepwise method was performed to find independent variables associated with the presence of ascites and low mean arterial pressure. Regression analysis to determine significant correlations among different parameters was performed using the Spearman

**3.1 AOPPs-albumin plasma concentrations in patients with chronic liver disease and** 

We analyzed 129 patients (68 males/61 females, median age 66 years, range 18–74 years) with chronic liver disease. The distribution of the stages of liver cirrhosis as defined according to the Child–Pugh score, and measurements of AOPPs-albumin and CMLalbumin plasma concentrations are presented in Fig.1. The concentration of AOPPs-albumin

(international normalized ratio-INR) + 6.43 (constant for liver disease etiology).

correlation coefficient. Statistical significance was established at *P* < 0.05

*al.,* 2006).

**3. Results** 

**healthy controls** 

**2.7 Statistical analysis** 

Fig. 1. **(A)** AOPPs-albumin serum concentrations in 129 patients with chronic liver disease, according to Child's stage of cirrhosis, and in an control group of 40 healthy blood donors. *P*  values are given in the table. Comparisons between subgroups are illustrated with box plot graphics, where the dotted line indicates the median per group, the box represents 50% of the values, and horizontal lines show minimum and maximum values of the calculated nonoutlier values; asterisks and open circles indicate outlier values. **(B)** AOPPs-albumin serum concentrations in patients with cirrhosis are correlated with the MELD (model of end-stage liver disease) score (*r* = 0.43, *P* < 0.01, Spearman rank correlation test). **(C)** CML-albumin serum concentrations increase with the stage of liver cirrhosis in patients with chronic liver disease. *P* values are given in the table.

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

Non-cirrhotic patients

56 (18–69)

40 (29–49)

0.92 (0.90-0.95)

2.1\* (0.9-3.0)

14.1 (9.8-20.1)

0.29 (0.21-0.33)

45.2 (31.9-58.6)

1.1 (0.9-1.3)


(*n*) 40 41 34 34 20

Healthy controls

(19–56)

45 (36–57)

0.7 (0.6-0.9)

1.7 (0.8-2.7)

10.7 (9.0–13.5)

0.35 (0.25-0.39)

54.3 (38.3-70.3)

1.3 (1.1-1.5)

INR 0.8-1.1 - 0.9

Age (years) 55

Albumin (g/L)

Bilirubin (mg/dL)

AOPPs-albumin (μmol/g)

CML-albumin (μg/g)

Uric acid (mmol/L)

Vitamin C (µmol/L)

MELD score 6-8

disease score.

TAS (mmol/L)

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 367

The MELD scores were determined in the 88 patients with liver cirrhosis (Table 3). These were higher in the Child-Pugh C cirrhotic patients than in the Child-Pugh A cirrhotic patients (p<0.01). Significant correlations between AOPPs levels and MELD scores (r = 0.43, *P* < 0.01; Fig. 1B) were observed among the cirrhotic patients belonging to all three Child-Pugh classes.

> Patients of class A

51 (21–74)

34\* (28–45)

1.01 (1.02-1.03)

2.8\* (1.3-4.4)

12.6\* (8.3-18.2)

0.31 (0.26-0.34)

47.1 (28.3-64.0)

0.9 (0.6-1.0)

7.8 (5.2-10.3)

Table 3. Plasma concentrations of AOPPs-albumin, CML-albumin and antioxidant parameters in patients with chronic liver disease without cirrhosis and in patients with cirrhosis. Significance between groups: \**P* < 0.05; \*\* *P* < 0.01 vs. healthy controls; + *P* < 0.05; ++*P* < 0.01 vs. patients of class A. AOPPs, advanced oxidation protein products; CML, Nε- (carboxymethyl)lysine; TAS, total antioxidant status; MELD, model for end-stage liver

(0.8-1.09)

Patients of class B

58 (24–71)

30\* (20–40)

1.56\* (1.0-2.0)

3.2 (1.9-4.5)

15.8\*\* (11.2–25.5)

0.22 (0.16-0.24)

33.6 (20.2-45.7)

0.63 (0.5-0.76)

1.2 (1.1-1.3)

15.9\* (8.7-23.1) Patients of class C

56 (29–69)

25\* (16–32)

2.15\* (1.1-3.6)

4.1\*\*+ (2.3–5.2)

18.3\*\* (9.7–23.8)

0.18\* (0.19-0.31)

36.9 (29.2-54.2)

0.53\*\*+ (0.5-0.8)

2.3 (1.6-2.9)

24.1\*+ <sup>+</sup> (14.4-28.4)

in healthy subjects was 1.7 μmol/g (range 0.8-2.7 μmol/g, P < 0.05). In patients with chronic liver disease, AOPPs-albumin plasma concentrations were 1.3-fold higher. In healthy controls, the plasma AOPPs or CML were similar to those in control groups in other studies (Sebeková *et al*., 2002; Kalousová *et al.,* 2003).

#### **3.2 AOPPs-albumin and liver cirrhosis**

AOPPs-albumin plasma concentration was significantly higher in patients with liver cirrhosis (n = 88, median 2.4 μmol/g, range 1.3-5.6 μmol/g) compared to patients with chronic liver disease without cirrhosis (n = 41, median 2.1 μmol/g, range 0.9-3.0 μmol/g) (*P* < 0.05, Fig.1A). Patients with Child-Pugh class C exhibited significantly higher plasma concentrations of AOPPs-albumin than patients with Child-Pugh class A and controls (*P* < 0.05, *P* < 0.01, respectively) (Fig.1A). There was no significant difference in AOPPs concentrations between control subjects and Child-Pugh B cirrhotic patients.

Differences in plasma AOPPs-albumin or CML-albumin were not significant in patients with liver cirrhosis of various etiologies (Table 2). Only in the group with primary biliary cirrhosis AOPPs-albumin were decreased (n = 11, median 1.3 μmol/g, range 0.80-2.2 μmol/g), though it should be consider with caution since small number of subjects included in this group.


Biliary etiology shows lower AOPPs-albumin levels compared with other etiologies of liver disease. Significance levels between groups: \*P < 0.05 vs. healthy controls.

AOPPs, advanced oxidation protein products; CML, Nε-(carboxymethyl)lysine; TAS, total antioxidant status.

Table 2. Plasma AOPPs-albumin, CML-albumin and TAS in liver cirrhosis patients of various etiologies.

in healthy subjects was 1.7 μmol/g (range 0.8-2.7 μmol/g, P < 0.05). In patients with chronic liver disease, AOPPs-albumin plasma concentrations were 1.3-fold higher. In healthy controls, the plasma AOPPs or CML were similar to those in control groups in other studies

AOPPs-albumin plasma concentration was significantly higher in patients with liver cirrhosis (n = 88, median 2.4 μmol/g, range 1.3-5.6 μmol/g) compared to patients with chronic liver disease without cirrhosis (n = 41, median 2.1 μmol/g, range 0.9-3.0 μmol/g) (*P* < 0.05, Fig.1A). Patients with Child-Pugh class C exhibited significantly higher plasma concentrations of AOPPs-albumin than patients with Child-Pugh class A and controls (*P* < 0.05, *P* < 0.01, respectively) (Fig.1A). There was no significant difference in AOPPs

Differences in plasma AOPPs-albumin or CML-albumin were not significant in patients with liver cirrhosis of various etiologies (Table 2). Only in the group with primary biliary cirrhosis AOPPs-albumin were decreased (n = 11, median 1.3 μmol/g, range 0.80-2.2 μmol/g), though it should be consider with caution since small number of subjects included

> CML-albumin (μg/g)

10.7 (9.0–13.5)

13.3\* (11.6-18.1)

16.3\* (13.3-25.5)

11.8 (8.3-12.5) TAS (mmol/L)

1.31 (1.12-1.5)

0.65\* (0.48-0.75)

0.71\* (0.60-0.73)

0.98 (0.76-0.83)

concentrations between control subjects and Child-Pugh B cirrhotic patients.

AOPPs-albumin (μmol/g)

1.7 (0.80-2.7)

3.09\* (1.5-5.2)

2.9\* (1.6-4.3)

1.3 (0.80-2.2)

Significance levels between groups: \*P < 0.05 vs. healthy controls.

Biliary etiology shows lower AOPPs-albumin levels compared with other etiologies of liver disease.

Table 2. Plasma AOPPs-albumin, CML-albumin and TAS in liver cirrhosis patients of

AOPPs, advanced oxidation protein products; CML, Nε-(carboxymethyl)lysine; TAS, total antioxidant

(Sebeková *et al*., 2002; Kalousová *et al.,* 2003).

**3.2 AOPPs-albumin and liver cirrhosis** 

in this group.

Healthy controls

Viral hepatitis-related

Alcohol-related cirrhosis

(n=40)

cirrhosis (n=44)

(n=33)

status.

Primary biliary cirrhosis (n=11)

various etiologies.

The MELD scores were determined in the 88 patients with liver cirrhosis (Table 3). These were higher in the Child-Pugh C cirrhotic patients than in the Child-Pugh A cirrhotic patients (p<0.01). Significant correlations between AOPPs levels and MELD scores (r = 0.43, *P* < 0.01; Fig. 1B) were observed among the cirrhotic patients belonging to all three Child-Pugh classes.


Table 3. Plasma concentrations of AOPPs-albumin, CML-albumin and antioxidant parameters in patients with chronic liver disease without cirrhosis and in patients with cirrhosis. Significance between groups: \**P* < 0.05; \*\* *P* < 0.01 vs. healthy controls; + *P* < 0.05; ++*P* < 0.01 vs. patients of class A. AOPPs, advanced oxidation protein products; CML, Nε- (carboxymethyl)lysine; TAS, total antioxidant status; MELD, model for end-stage liver disease score.

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

AOPPs-albumin (μmol/g)

Albumin (g/L) r = -0.38, *P* < 0.05 r = - 0.26, *P* = 0.07

ALT (U/L) r = 0.10, *P* = 0.54 r = -0.14, *P* = 0.31

AST (U/L) r = -0.20, *P* = 0.11 r = -0.15, *P* = 0.26

Bilirubin (mg/dL) r = 0.23, *P* = 0.07 r = 0.24, *P* = 0.06

MELD score r = 0.43, *P* < 0.01 r = 0.23, *P* = 0.07

aspartate aminotransferase; MELD, model for end-stage liver disease score.

Overall

(n=88)

2.4\* (1.3-5.6)

15.7\* (8.3-25.5)

31\*\* (16-35.4)

41.5\* (37.6-64.0)

13.3\*\* (6.4-39.9)

5.3\*\* (4.9-11.0)

Healthy controls (n=40)

1.7 (0.8-2.7)

10.7 (9.0–13.5)

45 (36–57)

32.9 (31.0-35.2)

(5.4-6.8)

1.5 (0.63-2.0)

Table 4. Correlations between plasma AOPPs-albumin and CML-albumin and selected biochemical indices of liver function and injury. ALT, alanine aminotransferase; AST,

r = -0.25, *P* < 0.05 r = - 0.43, *P* < 0.001

Patients of class A (n=34)

2.8\* (1.3-4.4)

12.6\* (8.3-18.2)

34\* (28–45)

36.9\* (37.7-45.6)

8.8\* (6.4-34.6)

4.8\* (5.5-7.0) Patients of class B (n=34)

3.2 (1.9-4.5)

15.8\*\* (11.2–25.5)

30\* (20–40)

42.0\* (37.6-47.2)

12.3\* (6.8-33.9)

5.2\*\* (4.9-7.7) Patients of class C (n=20)

4.1\*+ (2.3–5.2)

18.3\*\* (9.7–23.8)

25\* (16–32)

51.7\*+ (48.7-58.3)

18.9\* (9.0-39.9)

6.3\*+ (6.8-10.1)

Prothrombin ratio

AOPPs-albumin (μmol/g)

CML-albumin (μg/g)

IL-6 (pg/mL) 5.9

Albumin (g/L)

TNF-α (pg/mL)

hsCRP (mg/L)

(%)

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 369

CML-albumin (μg/g)

#### **3.3 CML-albumin plasma concentrations in patients with CLD and healthy controls**

In patients with chronic liver disease, CML-albumin had a median value of 14.1 μg/g (range 9.8-20.1 μg/g). Plasma CML-albumin concentrations were higher in Child-Pugh A to C cirrhotic patients (n = 88, median 15.7 μg/g, range 8.3-25.5 μg/g) than in patients without cirrhosis, but this difference was not statistically significant (Fig. 1C). The levels of plasma CML-albumin in all liver cirrhotic patients were higher than those of the controls and this difference was statistically significant (Fig. 1C). Plasma CML-albumin in patients with Child-Pugh class C cirrhosis was only slightly elevated compared with those in Child-Pugh class A cirrhosis (*P* = 0.17) (Fig. 1C). There was no statistically significant correlation between CML-albumin levels and the Child-Pugh score in cirrhotic patients.

#### **3.4 Antioxidant parameters and liver cirrhosis**

As it is seen in Table 3, while all individual parameters of the antioxidant status tend to decrease, only the decrease of uric acid was statistically significant. There was a markedly decreased total antioxidant status (TAS) in patients with Child-Pugh class C cirrhosis compared to those with Child-Pugh class A cirrhosis or controls (*P* < 0.05, *P* < 0.01, respectively). Although differences between cirrhosis and chronic liver disease (n = 41, median 1.1, mmol/L, range 0.9-1.3 mmol/L) were not statistically significant, weak but significant correlation was observed between TAS and plasma AOPPs-albumin (r =-0.31, *P* < 0.05). We failed to find, however, any relation between circulating CML-albumin levels and TAS (r = -0.22, *P* = 0.059).

#### **3.5 Markers of oxidative stress and hepatic function**

The serum albumin concentration was determined in all patients (n = 129, median 34 g/L, range 16–45 g/L) and healthy control subjects (n = 40, median 45 g/L, range 36–57 g/L). Level of albumin, the main substrate in both AOPPs and CML formation (Kalousová *et al.*; 2005), was significantly depleted both in chronic liver disease (n = 41, median 37 g/L, range 29–49 g/L) and in cirrhosis (median 30 g/L, range 16-45 g/L) (Table 1). Plasma CML-albumin and foremost AOPPs-albumin showed significant associations with biochemical indices of liver function (albumin, prothrombin time, bilirubin concentration) but not with markers of liver injury – aminotransferases (Table 4). As expected, in patients with cirrhosis, AOPPs-albumin weakly but significantly correlated with the serum albumin (r = -0.38, *P* < 0.05).

#### **3.6 AOPPs-albumin, CML-albumin and chronic inflammatory state in cirrhotic patients**

We assessed the levels of several inflammatory markers and their association with the levels of AOPPs-albumin and CML-albumin. Serum high-sensitivity C-reactive protein (hs-CRP) levels and white blood cells (WBC) counts were significantly elevated in cirrhotic patients (Table 5). Serum TNF-α levels were higher in the Child-Pugh class C cirrhosis patients than in the Child-Pugh class A cirrhosis patients (*P* < 0.05) (Table 5). Moreover, TNF-α concentrations were weakly but significantly correlated with Child-Pugh score in cirrhotic group (r = 0.31, *P* < 0.05). The levels of serum IL-6 in cirrhotic patients were higher than those of the control group and this difference was statistically significant (*P* < 0.05) (Table 5). The levels of serum IL-6 in patients with Child-Pugh class C cirrhosis were higher than those in Child-Pugh class A cirrhosis, but this difference was not statistically significant.

As it is seen in Table 3, while all individual parameters of the antioxidant status tend to decrease, only the decrease of uric acid was statistically significant. There was a markedly decreased total antioxidant status (TAS) in patients with Child-Pugh class C cirrhosis compared to those with Child-Pugh class A cirrhosis or controls (*P* < 0.05, *P* < 0.01, respectively). Although differences between cirrhosis and chronic liver disease (n = 41, median 1.1, mmol/L, range 0.9-1.3 mmol/L) were not statistically significant, weak but significant correlation was observed between TAS and plasma AOPPs-albumin (r =-0.31, *P* < 0.05). We failed to find, however, any relation between circulating CML-albumin levels and

The serum albumin concentration was determined in all patients (n = 129, median 34 g/L, range 16–45 g/L) and healthy control subjects (n = 40, median 45 g/L, range 36–57 g/L). Level of albumin, the main substrate in both AOPPs and CML formation (Kalousová *et al.*; 2005), was significantly depleted both in chronic liver disease (n = 41, median 37 g/L, range 29–49 g/L) and in cirrhosis (median 30 g/L, range 16-45 g/L) (Table 1). Plasma CML-albumin and foremost AOPPs-albumin showed significant associations with biochemical indices of liver function (albumin, prothrombin time, bilirubin concentration) but not with markers of liver injury – aminotransferases (Table 4). As expected, in patients with cirrhosis, AOPPs-albumin

**3.6 AOPPs-albumin, CML-albumin and chronic inflammatory state in cirrhotic patients**  We assessed the levels of several inflammatory markers and their association with the levels of AOPPs-albumin and CML-albumin. Serum high-sensitivity C-reactive protein (hs-CRP) levels and white blood cells (WBC) counts were significantly elevated in cirrhotic patients (Table 5). Serum TNF-α levels were higher in the Child-Pugh class C cirrhosis patients than in the Child-Pugh class A cirrhosis patients (*P* < 0.05) (Table 5). Moreover, TNF-α concentrations were weakly but significantly correlated with Child-Pugh score in cirrhotic group (r = 0.31, *P* < 0.05). The levels of serum IL-6 in cirrhotic patients were higher than those of the control group and this difference was statistically significant (*P* < 0.05) (Table 5). The levels of serum IL-6 in patients with Child-Pugh class C cirrhosis were higher than those in Child-Pugh class A cirrhosis, but this difference was not statistically significant.

weakly but significantly correlated with the serum albumin (r = -0.38, *P* < 0.05).

**3.3 CML-albumin plasma concentrations in patients with CLD and healthy controls**  In patients with chronic liver disease, CML-albumin had a median value of 14.1 μg/g (range 9.8-20.1 μg/g). Plasma CML-albumin concentrations were higher in Child-Pugh A to C cirrhotic patients (n = 88, median 15.7 μg/g, range 8.3-25.5 μg/g) than in patients without cirrhosis, but this difference was not statistically significant (Fig. 1C). The levels of plasma CML-albumin in all liver cirrhotic patients were higher than those of the controls and this difference was statistically significant (Fig. 1C). Plasma CML-albumin in patients with Child-Pugh class C cirrhosis was only slightly elevated compared with those in Child-Pugh class A cirrhosis (*P* = 0.17) (Fig. 1C). There was no statistically significant correlation

between CML-albumin levels and the Child-Pugh score in cirrhotic patients.

**3.4 Antioxidant parameters and liver cirrhosis** 

**3.5 Markers of oxidative stress and hepatic function** 

TAS (r = -0.22, *P* = 0.059).


Table 4. Correlations between plasma AOPPs-albumin and CML-albumin and selected biochemical indices of liver function and injury. ALT, alanine aminotransferase; AST, aspartate aminotransferase; MELD, model for end-stage liver disease score.


Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 371

Fig. 2. Receiver operating characteristic (ROC) curve and optimal cut-off levels of advanced oxidation protein products (AOPPs), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) for distinguishing cirrhotic patients from healthy controls; AUC, area under the curve.

AOPPs-albumin, TNF-a, and IL-6, compared with patients without ascites. Cirrhotic patients who had ascites showed higher AOPPs-albumin levels (n = 53, median 3.6 μmol/g, range 1.5-5.3 μmol/g) than patients without ascites (n = 35, median 2.2 μmol/g, range 1.0- 3.4 μmol/g) (*P* < 0.05, Table 6). AOPPs-albumin levels were not different between cirrhotic patients without ascites and controls, while patients with ascites had higher AOPPs-albumin levels than controls (median 1.7 μmol/g, range 0.8-2.7 μmol/g) (*P* < 0.01). High IL-6 levels

To differentiate cirrhotic patients with a more intense hemodynamic alteration (vasodilatation), we divided patients according to those with low mean arterial pressure (MAP ≤83 mm Hg) and high mean arterial pressure (MAP >83 mm Hg) (Table 7). Only IL-6 levels were significantly higher in patients with more severe vasodilatation; this association

Plasma levels of AOPPs-albumin very weakly but significantly correlated with MAP (r = - 0.25, *P* < 0.01; Fig.3). Furthermore, IL-6 levels had a significant correlation with several

showed an independent association with the presence of ascites.

was independent of other factors.


Table 5. Plasma concentrations of AOPPs-albumin, CML-albumin and inflammatory markers in healthy controls and in patients with liver cirrhosis. Significance levels between groups: \**P* < 0.05; \*\* *P* < 0.01 vs. healthy controls; + *P* < 0.05, ++*P* < 0.01 vs. patients of class A. AOPPs, advanced oxidation protein products; CML, Nε-(carboxymethyl)lysine; TNF, tumor necrosis factor; IL, interleukin; CRP, C-reactive protein; WBC, white blood cells; MELD, model for end-stage liver disease score.

The association study revealed only a tendency toward an extremely weak but significant correlation between AOPPs-albumin and WBC in all cirrhotic patients (r = 0.23, *P* < 0.05). In turn, a weak but significant correlation between AOPPs-albumin levels and hs-CRP was observed among the cirrhotic patients belonging to all three Child-Pugh classes (r = 0.33, *P* < 0.05). There was a significant correlation between the IL-6 and the AOPPs-albumin level (r = 0.42, *P* < 0.05) and MELD score (r = 0.38, *P* < 0.05) in cirrhotic patients. As it was expected, a significant correlation between AOPPs-albumin levels and TNF-α (r = 0.48, *P* < 0.05) was observed in Child-Pugh class A cirrhosis patients. In the multivariate analysis the relationship between plasma AOPPs-albumin, TNF-α and Child-Pugh score was independent of age, sex and liver cirrhosis etiology (data not shown).

There was no statistically significant correlation between CML-albumin level and hs-CRP or cytokines levels in all liver cirrhotic patients (data not shown).

The ROC curve analyses are shown in Fig. 2 (sensitivity versus 1-specificity). The cut-off values of plasma AOPPs-albumin, TNF-α and IL-6 to separate cirrhotic patients from healthy controls were 3.71 μmol/g, 37.2 pg/mL, and 8.95 pg/mL, respectively.

#### **3.7 Hemodynamic characteristics of the patients with liver cirrhosis**

Hemodynamic characteristics of patients are shown in Tables 1 and 6. Cirrhotic patients had significantly lower values of mean arterial pressure (MAP) when compared with controls (Table 6). Parameters related to hemodynamic disturbances such as decreased mean arterial pressure and increased plasma renin activity and aldosterone levels deteriorated with increasing of Child-Pugh score. However, similar values of antidiuretic hormone were detected in all patients grouped according to the Child-Pugh classification.

Among clinical parameters of liver dysfunction ascites revealed significant association with plasma AOPPs-albumin, TNF-α and IL-6 (Table 6). By contrast, patients presented similar AOPPs-albumin levels when classified according to the presence or absence of encephalopathy grade I (data not shown). Ascitic patients had a more intense alteration of hemodynamic parameters (plasma renin activity, aldosterone), along with higher levels of

Patients of class A (n=34)

4.7 (1.1-8.6)

7.8 (5.2-10.3) Patients of class B (n=34)

5.2\* (3.0-7.8)

15.9\* (8.7-23.1) Patients of class C (n=20)

5.4\*\*+ (2.7-8.2)

24.1\*+ <sup>+</sup> (14.4-28.4)

Overall

(n=88)

(5.2-28.4)

Table 5. Plasma concentrations of AOPPs-albumin, CML-albumin and inflammatory markers in healthy controls and in patients with liver cirrhosis. Significance levels between groups: \**P* < 0.05; \*\* *P* < 0.01 vs. healthy controls; + *P* < 0.05, ++*P* < 0.01 vs. patients of class A. AOPPs, advanced oxidation protein products; CML, Nε-(carboxymethyl)lysine; TNF, tumor necrosis factor; IL, interleukin; CRP, C-reactive protein; WBC, white blood cells; MELD,

The association study revealed only a tendency toward an extremely weak but significant correlation between AOPPs-albumin and WBC in all cirrhotic patients (r = 0.23, *P* < 0.05). In turn, a weak but significant correlation between AOPPs-albumin levels and hs-CRP was observed among the cirrhotic patients belonging to all three Child-Pugh classes (r = 0.33, *P* < 0.05). There was a significant correlation between the IL-6 and the AOPPs-albumin level (r = 0.42, *P* < 0.05) and MELD score (r = 0.38, *P* < 0.05) in cirrhotic patients. As it was expected, a significant correlation between AOPPs-albumin levels and TNF-α (r = 0.48, *P* < 0.05) was observed in Child-Pugh class A cirrhosis patients. In the multivariate analysis the relationship between plasma AOPPs-albumin, TNF-α and Child-Pugh score was

There was no statistically significant correlation between CML-albumin level and hs-CRP or

The ROC curve analyses are shown in Fig. 2 (sensitivity versus 1-specificity). The cut-off values of plasma AOPPs-albumin, TNF-α and IL-6 to separate cirrhotic patients from

Hemodynamic characteristics of patients are shown in Tables 1 and 6. Cirrhotic patients had significantly lower values of mean arterial pressure (MAP) when compared with controls (Table 6). Parameters related to hemodynamic disturbances such as decreased mean arterial pressure and increased plasma renin activity and aldosterone levels deteriorated with increasing of Child-Pugh score. However, similar values of antidiuretic hormone were

Among clinical parameters of liver dysfunction ascites revealed significant association with plasma AOPPs-albumin, TNF-α and IL-6 (Table 6). By contrast, patients presented similar AOPPs-albumin levels when classified according to the presence or absence of encephalopathy grade I (data not shown). Ascitic patients had a more intense alteration of hemodynamic parameters (plasma renin activity, aldosterone), along with higher levels of

healthy controls were 3.71 μmol/g, 37.2 pg/mL, and 8.95 pg/mL, respectively.

**3.7 Hemodynamic characteristics of the patients with liver cirrhosis** 

detected in all patients grouped according to the Child-Pugh classification.

5.0 (1.1-8.6)

independent of age, sex and liver cirrhosis etiology (data not shown).

cytokines levels in all liver cirrhotic patients (data not shown).

Healthy controls (n=40)

(4.2-5.0)

MELD score 6-8 13.5\*

model for end-stage liver disease score.

WBC (x109/L) 4.0


Fig. 2. Receiver operating characteristic (ROC) curve and optimal cut-off levels of advanced oxidation protein products (AOPPs), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) for distinguishing cirrhotic patients from healthy controls; AUC, area under the curve.

AOPPs-albumin, TNF-a, and IL-6, compared with patients without ascites. Cirrhotic patients who had ascites showed higher AOPPs-albumin levels (n = 53, median 3.6 μmol/g, range 1.5-5.3 μmol/g) than patients without ascites (n = 35, median 2.2 μmol/g, range 1.0- 3.4 μmol/g) (*P* < 0.05, Table 6). AOPPs-albumin levels were not different between cirrhotic patients without ascites and controls, while patients with ascites had higher AOPPs-albumin levels than controls (median 1.7 μmol/g, range 0.8-2.7 μmol/g) (*P* < 0.01). High IL-6 levels showed an independent association with the presence of ascites.

To differentiate cirrhotic patients with a more intense hemodynamic alteration (vasodilatation), we divided patients according to those with low mean arterial pressure (MAP ≤83 mm Hg) and high mean arterial pressure (MAP >83 mm Hg) (Table 7). Only IL-6 levels were significantly higher in patients with more severe vasodilatation; this association was independent of other factors.

Plasma levels of AOPPs-albumin very weakly but significantly correlated with MAP (r = - 0.25, *P* < 0.01; Fig.3). Furthermore, IL-6 levels had a significant correlation with several

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

(n=51)

1.7 (0.46-5.5)

25.6 (10.6-40.5)

4.7 (3.7-6.7)

3.3 (1.4-4.8)

11.0+ (9.5-12.8)

47.9 (43.4-58.0)

groups: +*P* < 0.01 by multivariate analysis.

Table 7. Comparison between cirrhotic patients classified according to the finding of low (≤ 83 mm Hg) and high (>83 mm Hg) mean arterial pressure (MAP). Significance between

Fig. 3. AOPPs-albumin concentrations are very weakly but significantly correlated with the mean arterial pressure (MAP) in patients with chronic liver disease (r = -0.25, *P* < 0.01).

Plasma renin activity

Antidiuretic hormone

AOPPs-albumin (μmol/g)

(ng mL-1 h-1)

Aldosterone (ng/dL)

(pg/mL)

IL-6 (pg/mL)

TNF-α (pg/mL)

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 373

Mean arterial pressure (MAP) > 83 mm Hg

(n=37)

1.4 (0.37-4.4)

13.7 (5.7-21.6)

4.2 (3.3-5.9)

3.01 (1.2-4.5)

6.3 (5.4-7.0)

49.8 (45.1-60.3)

Mean arterial pressure (MAP) ≤ 83 mm Hg


parameters, including plasma renin activity (r = 0.39, *P* < 0.05) and MAP (r = -0.38, *P* < 0.01), in addition to albumin (r = - 0.51, *P* < 0.001). Neither TNF-α levels nor the CML-albumin levels correlated significantly with hemodynamic parameters.

Significance between groups: \**P* < 0.05; \*\**P* < 0.001 vs. patients of class A. +*P* < 0.05; ++*P* < 0.01, +++*P* < 0.001 vs. liver cirrhosis without ascites. AOPPs, advanced oxidation protein products; TNF, tumor necrosis factor; IL, interleukin.

Table 6. Blood pressure, plasma renin activity and plasma concentrations of aldosterone, antidiuretic hormone and AOPPs-albumin according to the Child–Pugh class or presence of ascites.

parameters, including plasma renin activity (r = 0.39, *P* < 0.05) and MAP (r = -0.38, *P* < 0.01), in addition to albumin (r = - 0.51, *P* < 0.001). Neither TNF-α levels nor the CML-albumin

> Patients of class B

Patients of class C Cirrhosis without ascites (n=35)

88 (85-93)

0.48 (0.13-1.6)

13.2 (5.5-20.9)

4.6 (3.0-6.8)

2.2 (1.0-3.4)

37.5 (35.4-46.5)

6.4 (5.6-7.3) Cirrhosis with ascites n=53)

77+ (73-89)

1.9++ (0.51-6.2)

33.0+++ (13.7- 52.2)

4.5 (3.6-6.4)

3.6+ (1.5-5.3)

57.5+ (52.1-69.7)

10.8++ (8.5-12.4)

(n=20)

76\* (73-81)

6.0\*\* 1.2-7.6)

30.0\* (21.0-71.0)

4.8 (3.8-6.5)

4.1\* (2.3–5.2)

51.7\* (48.7-58.3)

18.9 (9.0-39.9

(n=34)

83 (77-91)

1.9\* (0.95-4.8)

20.6 (14.0-/51.0)

4.5 (3.7-6.2)

3.2 (1.9-4.5)

42.0 (37.6-47.2)

12.3 (6.8-33.9)

Significance between groups: \**P* < 0.05; \*\**P* < 0.001 vs. patients of class A. +*P* < 0.05; ++*P* < 0.01, +++*P* < 0.001 vs. liver cirrhosis without ascites. AOPPs, advanced oxidation protein products; TNF, tumor

Table 6. Blood pressure, plasma renin activity and plasma concentrations of aldosterone, antidiuretic hormone and AOPPs-albumin according to the Child–Pugh class or presence of

levels correlated significantly with hemodynamic parameters.

Patients of class A

(n=34)

89 (85-93)

0.37 (0.1-1.2)

12.0 (5.0-19.0)

3.8 (2.5-5.7)

2.8 (1.3-4.4)

36.9 (37.7-45.6)

8.8 (6.4-34.6)

Overall

(n=88)

83 (76-93)

0.6 (0.1-7.6)

15.0 (5.0-71.0)

4.9 (2.5-6.5)

2.4 (1.3-5.2)

41.5 (37.6-64.0)

13.3 (6.4-39.9)

necrosis factor; IL, interleukin.

Mean arterial pressure-MAP (mmHg)

Plasma renin activity (ng mL-1 h-1)

Aldosterone (ng/dL) 20.6 (14.0-51.0)

Antidiuretic hormone (pg/mL)

AOPPsalbumin (μmol/g)

TNF-α (pg/mL)

IL-6 (pg/mL)

ascites.


Table 7. Comparison between cirrhotic patients classified according to the finding of low (≤ 83 mm Hg) and high (>83 mm Hg) mean arterial pressure (MAP). Significance between groups: +*P* < 0.01 by multivariate analysis.

Fig. 3. AOPPs-albumin concentrations are very weakly but significantly correlated with the mean arterial pressure (MAP) in patients with chronic liver disease (r = -0.25, *P* < 0.01).

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

cirrhosis of various etiologies (Zuwala-Jagiello *et al.,* 2011).

and hemodynamic changes in cirrhotic patients.

The adverse effects of oxidative stress on the progression of cirrhosis may be categorized into effects on protein modifications and inflammatory response. Figure 4 presents a summary of the effects of AOPPs or AGEs and oxidative stress on markers of inflammation

Very recently, advanced glycation endproducts have been found to act as pro-inflammatory factors (Sparvero *et al*., 2009). Nevertheless, AOPPs are believed to be more closely related to inflammation (Fialova *et al*., 2006) than AGEs, whose RAGE participates in AOPPs-mediated signal transduction (Kalousová *et al*., 2005; 2006). These interactions enhance reactive oxygen species formation, with activation of nuclear factor NF-κB and release of proinflammatory cytokines (Bierhaus *et al.*, 2006; Hyogo & Yamagishi, 2008; Saito & Ishii, 2004) (Fig. 4). Moreover, the macrophage RAGE can be up-regulated by TNF-α (Miyata *et al.*,

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 375

increasingly being used as markers instead of lipid peroxidation products in demonstrating oxidative stress (Dalle-Donne *et al.*, 2003). AOPPs measurements reflect the reactive species generation and the degree of protein oxidation (Witko-Sarsat *et al.*, 1996). It was reported that AOPPs generated by different oxidation patterns lead to the production of either hydrogen peroxide or nitric oxide (Servettaz *et al.,* 2007). Nitric oxide can interact with superoxide anion-radical forming reactive nitrogen species such as peroxynitrite. These reactive nitrogen species secondarily promote important reactions such as nitrosation, oxidation or nitration, leading to impaired cellular functions and enhanced inflammatory reactions (Friedman, 2008; Iwakiri & Groszmann, 2007). AOPPs are referred to as markers of oxidative stress as well as markers of neutrophil activation in chronic disease (Witko-Sarsat *et al.,* 2003). It has thus been shown that chlorinated oxidants of neutrophil origin may lead to oxidative stress, notably protein oxidation. Once formed, such AOPPs foci create a nidus for the amplification of oxidative stress. In addition to increased formation, decreased removal/detoxification of AOPPs may contribute to the stress. There is increasing evidence that the liver plays important roles in the elimination of AOPPs (Iwao *et al*., 2006). In patients with chronic liver diseases, constriction of the sinusoidal blood stream leads to the development of portal hypertension with portocaval shunts (Svistounov & Smedsrød, 2004). The hindrance of substance exchange between hepatocytes and the sinusoidal blood stream could increase plasma level of AOPPs in these patients. Therefore, the liver, especially in cirrhotic patients, cannot prevent the accumulation of AOPPs effectively. Finally, our findings extended the results of Oettl *et al*. (2008) which suggested that albumin is oxidatively modified in patients with advanced liver disease depending on its severity. The present finding that AOPPs-albumin accumulation coexists with decreased TAS, while the plasma concentration of CML-modified albumin remains stable, supports the contention that AOPPs-albumin is more accurate marker of oxidative stress than glycoxidation products in cirrhotic patients. An increase in reactive species formation, manifested by increased hepatic and plasma levels of AOPPs (Gorka *et al*., 2008; Sebeková *et al*., 2002; Yagmur *et al*., 2006; Zuwala−Jagiello *et al*., 2006;) and as well as decreased antioxidant levels (Jain *et al*., 2002; Zuwala−Jagiello *et al*., 2009) have been reported in patients with liver cirrhosis. Finally, our previous study found that the patients with cirrhosis were exposed to oxidative stress and the level of AOPPs was significantly related to the severity of liver

#### **4. Discussion**

Cirrhosis is characterized by inflammation of the liver, often caused by a rise in oxygenderived free radicals within the liver. Under normal circumstances, the liver maintains a supply of internal antioxidants to neutralize the reactive species generated in response to viral infection and during metabolism of various endo- and exogenous compounds processed in the liver. However, when the liver antioxidants are low, or when the liver is undergone to continued oxidative insults (e.g., long-lasting alcohol abuse or infection with different hepatitis viruses), the damage from reactive species (Halliwell, 2007) may increase, resulting in inflammation and the formation of scar tissue (fibrosis) (Valko *et al*., 2007). The progressive decrease of antioxidant reserves, the dysfunction of liver microcirculation through nitric oxide-mediated pathways, may determine the shift to liver cirrhosis. Advanced glycation and oxidation endproducts (AGEs and AOPPs, respectively) cause oxidative stress and trigger cytokine driven inflammatory reactions *in vitro*. (reviewed in Yan *et al.,* 2010). The net effects on markers of inflammation and hemodynamic changes in cirrhotic patients are unknown.

Advanced glycation endproducts are formed from the reaction of glucose and other reducing sugars with amino acid groups of proteins. This interaction generates a labile Schiff base followed by rearrangement to more stable Amadori-products, and subsequently these early glycation products may undergo further chemical rearrangements resulting in various irreversibly formed AGEs (Baynes & Thorpe, 1999). Three different mechanisms have been proposed by which AGEs lead to cirrhosis complications: 1) the binding of AGEs to the receptors for advanced glycation endproducts on different cell types including monocytes/macrophages, T lymphocytes, endothelial cells, smooth muscle cells, and activation of cell signaling pathways with subsequent modulation of gene expression, 2) intracellular AGEs formation leading to impaired cell function, and 3) the accumulation of AGEs in the extracellular matrix. CML-albumin levels (as prototype of the AGEs) were higher in cirrhosis groups than in the controls. In agreement with two previous reports (Zuwala-Jagiello *et al*., 2009; 2011) these results indicate that reactive oxygen species are overproduced in patients with liver cirrhosis. CML may lead to progression of cirrhosis by interaction with receptors that induce production of reactive species followed by a release of inflammatory cytokines in different cell types (Bierhaus *et al*., 1998; Raj *et al*., 2000) such as IL-6, ultimately leading to the production of CRP by the liver. A correlation to high sensitive C-reactive protein (hs-CRP) could not be demonstrated, and the suggestion that high levels of CML may activate an inflammatory response was not demonstrated by serum markers (TNF-α and IL-6). It is likely that the blood load of CML resembles only a small fraction of body's AGEs content and that the serum levels reflect particular changes in the body's AGEs pool. Thus, circulating CML-albumin may not be an adequate parameter for demonstrating effects on inflammatory response in cirrhotic patients. Most likely the focus should be on intracellular AGEs (Thornalley *et al*., 2003).

A role of oxidative stress in the pathogenesis of chronic liver disease has been proposed by several authors (Bandara *et al.,* 2005; Nagata *et al.,* 2007; Nakhjavani *et al.,* 2011; Serejo *et al.,* 2003; Zuwala-Jagiello *et al.,* 2007). Studies have shown increased plasma levels of markers of lipid peroxidation and reduced plasma antioxidant content. Protein oxidation products are

Cirrhosis is characterized by inflammation of the liver, often caused by a rise in oxygenderived free radicals within the liver. Under normal circumstances, the liver maintains a supply of internal antioxidants to neutralize the reactive species generated in response to viral infection and during metabolism of various endo- and exogenous compounds processed in the liver. However, when the liver antioxidants are low, or when the liver is undergone to continued oxidative insults (e.g., long-lasting alcohol abuse or infection with different hepatitis viruses), the damage from reactive species (Halliwell, 2007) may increase, resulting in inflammation and the formation of scar tissue (fibrosis) (Valko *et al*., 2007). The progressive decrease of antioxidant reserves, the dysfunction of liver microcirculation through nitric oxide-mediated pathways, may determine the shift to liver cirrhosis. Advanced glycation and oxidation endproducts (AGEs and AOPPs, respectively) cause oxidative stress and trigger cytokine driven inflammatory reactions *in vitro*. (reviewed in Yan *et al.,* 2010). The net effects on markers of inflammation and hemodynamic changes in

Advanced glycation endproducts are formed from the reaction of glucose and other reducing sugars with amino acid groups of proteins. This interaction generates a labile Schiff base followed by rearrangement to more stable Amadori-products, and subsequently these early glycation products may undergo further chemical rearrangements resulting in various irreversibly formed AGEs (Baynes & Thorpe, 1999). Three different mechanisms have been proposed by which AGEs lead to cirrhosis complications: 1) the binding of AGEs to the receptors for advanced glycation endproducts on different cell types including monocytes/macrophages, T lymphocytes, endothelial cells, smooth muscle cells, and activation of cell signaling pathways with subsequent modulation of gene expression, 2) intracellular AGEs formation leading to impaired cell function, and 3) the accumulation of AGEs in the extracellular matrix. CML-albumin levels (as prototype of the AGEs) were higher in cirrhosis groups than in the controls. In agreement with two previous reports (Zuwala-Jagiello *et al*., 2009; 2011) these results indicate that reactive oxygen species are overproduced in patients with liver cirrhosis. CML may lead to progression of cirrhosis by interaction with receptors that induce production of reactive species followed by a release of inflammatory cytokines in different cell types (Bierhaus *et al*., 1998; Raj *et al*., 2000) such as IL-6, ultimately leading to the production of CRP by the liver. A correlation to high sensitive C-reactive protein (hs-CRP) could not be demonstrated, and the suggestion that high levels of CML may activate an inflammatory response was not demonstrated by serum markers (TNF-α and IL-6). It is likely that the blood load of CML resembles only a small fraction of body's AGEs content and that the serum levels reflect particular changes in the body's AGEs pool. Thus, circulating CML-albumin may not be an adequate parameter for demonstrating effects on inflammatory response in cirrhotic patients. Most likely the focus should be on

A role of oxidative stress in the pathogenesis of chronic liver disease has been proposed by several authors (Bandara *et al.,* 2005; Nagata *et al.,* 2007; Nakhjavani *et al.,* 2011; Serejo *et al.,* 2003; Zuwala-Jagiello *et al.,* 2007). Studies have shown increased plasma levels of markers of lipid peroxidation and reduced plasma antioxidant content. Protein oxidation products are

**4. Discussion** 

cirrhotic patients are unknown.

intracellular AGEs (Thornalley *et al*., 2003).

increasingly being used as markers instead of lipid peroxidation products in demonstrating oxidative stress (Dalle-Donne *et al.*, 2003). AOPPs measurements reflect the reactive species generation and the degree of protein oxidation (Witko-Sarsat *et al.*, 1996). It was reported that AOPPs generated by different oxidation patterns lead to the production of either hydrogen peroxide or nitric oxide (Servettaz *et al.,* 2007). Nitric oxide can interact with superoxide anion-radical forming reactive nitrogen species such as peroxynitrite. These reactive nitrogen species secondarily promote important reactions such as nitrosation, oxidation or nitration, leading to impaired cellular functions and enhanced inflammatory reactions (Friedman, 2008; Iwakiri & Groszmann, 2007). AOPPs are referred to as markers of oxidative stress as well as markers of neutrophil activation in chronic disease (Witko-Sarsat *et al.,* 2003). It has thus been shown that chlorinated oxidants of neutrophil origin may lead to oxidative stress, notably protein oxidation. Once formed, such AOPPs foci create a nidus for the amplification of oxidative stress. In addition to increased formation, decreased removal/detoxification of AOPPs may contribute to the stress. There is increasing evidence that the liver plays important roles in the elimination of AOPPs (Iwao *et al*., 2006). In patients with chronic liver diseases, constriction of the sinusoidal blood stream leads to the development of portal hypertension with portocaval shunts (Svistounov & Smedsrød, 2004). The hindrance of substance exchange between hepatocytes and the sinusoidal blood stream could increase plasma level of AOPPs in these patients. Therefore, the liver, especially in cirrhotic patients, cannot prevent the accumulation of AOPPs effectively. Finally, our findings extended the results of Oettl *et al*. (2008) which suggested that albumin is oxidatively modified in patients with advanced liver disease depending on its severity. The present finding that AOPPs-albumin accumulation coexists with decreased TAS, while the plasma concentration of CML-modified albumin remains stable, supports the contention that AOPPs-albumin is more accurate marker of oxidative stress than glycoxidation products in cirrhotic patients. An increase in reactive species formation, manifested by increased hepatic and plasma levels of AOPPs (Gorka *et al*., 2008; Sebeková *et al*., 2002; Yagmur *et al*., 2006; Zuwala−Jagiello *et al*., 2006;) and as well as decreased antioxidant levels (Jain *et al*., 2002; Zuwala−Jagiello *et al*., 2009) have been reported in patients with liver cirrhosis. Finally, our previous study found that the patients with cirrhosis were exposed to oxidative stress and the level of AOPPs was significantly related to the severity of liver cirrhosis of various etiologies (Zuwala-Jagiello *et al.,* 2011).

The adverse effects of oxidative stress on the progression of cirrhosis may be categorized into effects on protein modifications and inflammatory response. Figure 4 presents a summary of the effects of AOPPs or AGEs and oxidative stress on markers of inflammation and hemodynamic changes in cirrhotic patients.

Very recently, advanced glycation endproducts have been found to act as pro-inflammatory factors (Sparvero *et al*., 2009). Nevertheless, AOPPs are believed to be more closely related to inflammation (Fialova *et al*., 2006) than AGEs, whose RAGE participates in AOPPs-mediated signal transduction (Kalousová *et al*., 2005; 2006). These interactions enhance reactive oxygen species formation, with activation of nuclear factor NF-κB and release of proinflammatory cytokines (Bierhaus *et al.*, 2006; Hyogo & Yamagishi, 2008; Saito & Ishii, 2004) (Fig. 4). Moreover, the macrophage RAGE can be up-regulated by TNF-α (Miyata *et al.*,

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 377

Fig. 4. Summary of the effects of AOPPs or AGEs and oxidative stress on markers of inflammation and hemodynamic changes in cirrhotic patients. AOPPs or AGEs bind with RAGE on the surface of endothelial cells lining blood vessels. AOPPS, AGEs ligands of RAGE sustain stimulation of RAGE. One consequence of RAGE signaling is the activation of NADPH oxidase and production of reactive oxygen species. Once formed, reactive oxygen species activate key transcription factor such as NF-κB, which results in the transcriptional activation of genes relevant for inflammation. Consequences include increased migration and activation of RAGE-expressing macrophages. This results in release of the proinflammatory cytokines. In this inflammatory environment, *via* interaction with RAGE on the surface of macrophages, AOPPs or AGEs magnify activation of NF-κB and other factors, thereby amplifying cellular stress and hepatic damage. In the aggregate, these processes may contribute to propagation of inflammation and vascular perturbation in liver cirrhosis. AGEs, advanced glycation endproducts; AOPPs, advanced oxidation protein products; CRP,

C-reactive protein; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MAP,

TNF-α, tumour necrosis factor α; VCAM-1, vascular cell adhesion molecule.

mitogen activated protein; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NF-κB, nuclear factor κB; RAGE, receptor for AGEs; ROS, reactive oxygen species;

1994). This is accompanied by the activation of macrophages and increased expression of TNF-α (Giron-González *et al.*, 2004). TNF-α production is also stimulated by macrophage sensing of intestinal microflora pathogen associated molecular patterns by toll-like receptors (Riordan *et al.*, 2003). It could therefore be consistent with observed increase levels of both AOPPs-albumin and TNF-α at an early stage of liver cirrhosis*.*

The hyperdynamic circulatory state associated with liver cirrhosis is characterized by vasodilatation and increased cardiac output; the arterial hypotension and relative hypovolemia caused by vasodilatation activate a number of vasoactive and neurohumoral systems (Wiest & Groszmann, 2002). TNF-α induces an endothelial activation, which can be detected by increased synthesis of nitric oxide (Spitzer, 1994). Endogenous nitric oxide, a powerful endothelium-derived vasodilator, has been implicated in hemodynamic changes present in cirrhotic patients (Garc'a-Tsao *et al*., 1998). Treatment with both specific anti-TNF polyclonal antibodies and thalidomide, an inhibitor of TNF-α production, significantly prevent the development of the hyperdynamic circulation and reduces portal pressure (Gatta *et al*., 2008). TNF-α levels in our patients were clearly different from control group and were much higher in ascitic patients. However, no differences existed between patients with high and low mean arterial pressure, and no significant correlations with hemodynamic values were found. These data suggest that, although TNF-α might be one of the inducers of nitric oxide generation in cirrhotic patients, other factors acting through different pathways probably exist.

AOPPs derived from *in vivo* sources stimulated endothelial cell generation of reactive oxygen species, in particular superoxide anion (Guo *et al.,* 2008), at least in part through NADPH-oxidase (Wautier *et al.,* 2001). However, the exact mechanisms and sources by which reactive oxygen species are generated in the vasculature are not yet known in detail. It has been observed in several experimental animal models, that the endothelium is one of the major sources for the generation of reactive oxygen species. In parallel with the vascular dysfunction the formation of superoxide anions became augmented, and removal of endothelium completely abolished the production of reactive oxygen species. (reviewed in Wright *et al*., 2006). In another report, Rhee *et al.* (2003) demonstrated that growth factorinduced H2O2 production (e.g. PDGF, EGF) requires the activation of phosphoinositide 3 kinase. The essential role of phosphoinositide 3-kinase is likely to provide phosphatidylinositol (3, 4, 5)-trisphosphate that recruits and activates a guanine nucleotide exchange factor of Rac, which is required for the activation of NADPH-oxidase. Thus, the generation of reactive oxygen species is largely dependent on the activation of NADPHoxidase that is present in endothelial cell. AOPPs stimulated endothelial cell activation of the following signaling mediators: NADPH oxidase and NF-κB, the factors linked to increased expression of pro-inflammatory adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) (Kim *et al.,* 2001; Yan *et al*., 2010) (Fig. 4). Endothelial activation plays an active role in the modifications of circulatory status of cirrhotic patients (Grangé & Amiot, 2004). Elevated levels of AOPPsalbumin were detected in the early stages of liver dysfunction: plasma concentrations were increased in patients in Child Pugh class A, with higher values found in those in class B or C. Plasma concentrations of AOPPs-albumin were very weakly correlated with hemodynamic alterations (mean arterial pressure).

#### Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 377

376 Oxidative Stress and Diseases

1994). This is accompanied by the activation of macrophages and increased expression of TNF-α (Giron-González *et al.*, 2004). TNF-α production is also stimulated by macrophage sensing of intestinal microflora pathogen associated molecular patterns by toll-like receptors (Riordan *et al.*, 2003). It could therefore be consistent with observed increase levels of both

The hyperdynamic circulatory state associated with liver cirrhosis is characterized by vasodilatation and increased cardiac output; the arterial hypotension and relative hypovolemia caused by vasodilatation activate a number of vasoactive and neurohumoral systems (Wiest & Groszmann, 2002). TNF-α induces an endothelial activation, which can be detected by increased synthesis of nitric oxide (Spitzer, 1994). Endogenous nitric oxide, a powerful endothelium-derived vasodilator, has been implicated in hemodynamic changes present in cirrhotic patients (Garc'a-Tsao *et al*., 1998). Treatment with both specific anti-TNF polyclonal antibodies and thalidomide, an inhibitor of TNF-α production, significantly prevent the development of the hyperdynamic circulation and reduces portal pressure (Gatta *et al*., 2008). TNF-α levels in our patients were clearly different from control group and were much higher in ascitic patients. However, no differences existed between patients with high and low mean arterial pressure, and no significant correlations with hemodynamic values were found. These data suggest that, although TNF-α might be one of the inducers of nitric oxide generation in cirrhotic patients, other factors acting through

AOPPs derived from *in vivo* sources stimulated endothelial cell generation of reactive oxygen species, in particular superoxide anion (Guo *et al.,* 2008), at least in part through NADPH-oxidase (Wautier *et al.,* 2001). However, the exact mechanisms and sources by which reactive oxygen species are generated in the vasculature are not yet known in detail. It has been observed in several experimental animal models, that the endothelium is one of the major sources for the generation of reactive oxygen species. In parallel with the vascular dysfunction the formation of superoxide anions became augmented, and removal of endothelium completely abolished the production of reactive oxygen species. (reviewed in Wright *et al*., 2006). In another report, Rhee *et al.* (2003) demonstrated that growth factorinduced H2O2 production (e.g. PDGF, EGF) requires the activation of phosphoinositide 3 kinase. The essential role of phosphoinositide 3-kinase is likely to provide phosphatidylinositol (3, 4, 5)-trisphosphate that recruits and activates a guanine nucleotide exchange factor of Rac, which is required for the activation of NADPH-oxidase. Thus, the generation of reactive oxygen species is largely dependent on the activation of NADPHoxidase that is present in endothelial cell. AOPPs stimulated endothelial cell activation of the following signaling mediators: NADPH oxidase and NF-κB, the factors linked to increased expression of pro-inflammatory adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) (Kim *et al.,* 2001; Yan *et al*., 2010) (Fig. 4). Endothelial activation plays an active role in the modifications of circulatory status of cirrhotic patients (Grangé & Amiot, 2004). Elevated levels of AOPPsalbumin were detected in the early stages of liver dysfunction: plasma concentrations were increased in patients in Child Pugh class A, with higher values found in those in class B or C. Plasma concentrations of AOPPs-albumin were very weakly correlated with

AOPPs-albumin and TNF-α at an early stage of liver cirrhosis*.*

different pathways probably exist.

hemodynamic alterations (mean arterial pressure).

Fig. 4. Summary of the effects of AOPPs or AGEs and oxidative stress on markers of inflammation and hemodynamic changes in cirrhotic patients. AOPPs or AGEs bind with RAGE on the surface of endothelial cells lining blood vessels. AOPPS, AGEs ligands of RAGE sustain stimulation of RAGE. One consequence of RAGE signaling is the activation of NADPH oxidase and production of reactive oxygen species. Once formed, reactive oxygen species activate key transcription factor such as NF-κB, which results in the transcriptional activation of genes relevant for inflammation. Consequences include increased migration and activation of RAGE-expressing macrophages. This results in release of the proinflammatory cytokines. In this inflammatory environment, *via* interaction with RAGE on the surface of macrophages, AOPPs or AGEs magnify activation of NF-κB and other factors, thereby amplifying cellular stress and hepatic damage. In the aggregate, these processes may contribute to propagation of inflammation and vascular perturbation in liver cirrhosis. AGEs, advanced glycation endproducts; AOPPs, advanced oxidation protein products; CRP, C-reactive protein; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MAP, mitogen activated protein; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NF-κB, nuclear factor κB; RAGE, receptor for AGEs; ROS, reactive oxygen species; TNF-α, tumour necrosis factor α; VCAM-1, vascular cell adhesion molecule.

Circulating Advanced Oxidation Protein Products, Nε-(Carboxymethyl) Lysine and

present in cirrhotic patients (Fig. 4).

AOPPs stimulation (Li et al., 2007).

**5. Conclusion** 

Pro-Inflammatory Cytokines in Patients with Liver Cirrhosis: Correlations with Clinical Parameters 379

demonstrated fair associations with other markers of oxidative stress, such as lipid peroxidation products and F2-isoprostanes. Furthermore, results from a recent study demonstrate that glycosylated and oxidized proteins indirectly up-regulate CRP expression in hepatocytes by stimulating monocytes to produce IL-6 (Li *et al.*, 2007). It seem, based on our study, even though there was functional loss of hepatocytes in patients with hepatic cirrhosis, the serum CRP level was still maintained in high level and dependent of AOPPsalbumin level with its significant correlation. The remaining viable hepatocytes may still contribute to this result. IL-6 is the main stimulant for hepatic production of CRP but also has other important roles leading to increased endothelial cell adhesiveness by upregulating ICAM-1, and VCAM-1 and releasing inflammatory mediators, including IL-6 itself (Szmitko *et al.*, 2003). Finally, the significant correlation between the levels of AOPPs and IL-6 supports the existence of a link between AOPPs and hemodynamic changes

Portal hypertension is characterized by intrahepatic vascular resistance causing an increase of portal vein pressure, and leads to the development of ascite (Møller *et al.*, 2008). IL-6 levels in our cirrhotic patients were different from those in controls, increased with the severity of liver disease, were independently associated to the presence of ascites. IL-6 increased in serum of patients with ascites compared to compensated (Child-Pugh class A) patients without ascites, and similar results were obtained in a study where serum IL-6 was also analyzed (Zhang *et al.*, 2002). In a simplistic view, portal hypertension leads to the formation of portosystemic collateral veins in liver cirrhosis and the resulting shunting (Cichoz-Lach *et al.*, 2008) also contributes to impaired hepatic uptake of IL-6. It has been convincingly shown that hepatic uptake of IL-6 is significantly impaired in patients with liver cirrhosis, and this may at least in part explain elevated serum levels in these patients (Soresi *et al.*, 2006). In our study, IL-6 levels increase significantly in association with the severity of liver cirrhosis according to the MELD score. Moreover, IL-6 levels in all cirrhotic patients were independently associated to the presence of low mean arterial pressure, and showed significant correlations with parameters related to hemodynamic abnormalities. The mechanism by which IL-6 could cause vasodilatation is unknown. However, the effect appears to be independent of nitric oxide, possibly due to an important role of prostacyclin synthesis (Dagher & Moore, 2001). It is then possible that IL-6 would produces vasodilatation by inducing prostacyclin synthesis; the effect of IL-6 would be potentiated by

Further, investigators have tried to find more noninvasive biomarkers for cirrhotic patients for years (Schuppan & Afdhal, 2008). We observed good abilities of plasma AOPPs-albumin, TNF-α and IL-6 levels to distinguish cirrhotic patients from healthy controls, with good sensitivities and specificities by ROC analysis. Additionally, these parameters also were found to be elevated in concordance with the severity of cirrhosis. Thereby, it is possible that plasma levels of AOPPs-albumin, TNF-α and IL-6 levels could be evaluated as candidate

In conclusion, there are differences between advanced oxidation protein products modified– albumin (AOPPs-albumin), which act as a pure oxidative stress marker, and Nε (carboxymethyl)lysine modified-albumin (CML-albumin; as prototype of the advanced

biomarkers for initial and long-term assessment of liver cirrhosis.

These results suggested that this association of AOPPs with hemodynamic disturbances is dependent of the severity of cirrhosis. Additionally, AOPPs levels could be elevated as a result of insufficient renal elimination. However, the precise mechanism by which AOPPs is cleared from plasma is currently unknown. In addition, we found no correlation between AOPPs-albumin and serum creatinine levels. In any case, the values of AOPPs-albumin in patients with a low Child-Pugh score and absence of ascites suggests that AOPPs might have a role in the late stages of cirrhosis by aggravating the already initiated vasodilatation. Indeed, the presence of ascites, one of the major complications of cirrhosis and closely related to the hemodynamic disturbances of cirrhotic patients, was found in patients with higher levels of AOPPs. Finally, the extremely weak correlation between AOPPs-albumin levels and mean arterial pressure may suggest an indirect contribution of AOPPs to arterial vasodilatation through other mediators.

Structure and function of albumin are impaired in advanced liver disease by different mechanisms: plasma levels are decreased due to reduced synthesis and albumin is oxidatively modified. In this context, AOPPs-albumin may shows altered binding capacities for several substances. Decreased bilirubin binding was reported for *in vitro* oxidized albumin (Oettl & Stauber, 2007). As bilirubin is preferentially bound by the fully reduced form of albumin, impaired binding of bilirubin and other ligands (e.g. nitric oxide) is likely to occur in liver cirrhosis. Theoretically, increased circulating AOPPs-albumin may indirectly lead to elevation of nitric oxide which can, in turn, contribute to oxidative stress in cells (La Villa & Gentilini, 2008). Finally, nitric oxide plays a major key role in the development of hyperdynamic circulation and portal hypertension in cirrhosis (Iwakiri & Groszmann, 2007). Infusion of albumin (Garcovich *et al*., 2009) as well as albumin dialysis has been shown to improve the circulatory dysfunction as evidenced by an increase in mean blood pressure and systemic vascular resistance (Mitzner *et al*., 2001). This improvement in systemic hemodynamics might be due to a reduction in vasodilation following removal of nitric oxide which results in deactivation of the neurohormonal systems and a decrease in plasma levels of renin, aldosterone, norepinephrine and vasopressin (Chen *et al*., 2009). However, other findings of the present study imply that AOPPs are not only the factors responsible for the hemodynamic changes observed in cirrhotic patients. The AOPPsalbumin levels did not correlate significantly with the parameters that accompany important hemodynamic alterations in cirrhotic patients, such as plasma renin activity and aldosterone. Finally, when cirrhotic patients in our study were divided according to high and low mean arterial pressure, we found similar AOPPs-albumin levels in both groups.

Portal hypertension and cirrhosis can increase gut permeability to endotoxin and impair reticuloendothelial function of the liver that may result in increased serum endotoxin concentrations (Cariello *et al*., 2010). This may be a stimulus for the production of proinflammatory cytokines, resulting in the increased production of acute phase proteins such as C-reactive protein. In turn, CRP is capable of stimulating IL-6 and TNF-α production by monocytes (Ballou & Lozanski, 1992) and reactive oxygen species formation (Wang *et al*., 2003). Advanced oxidation protein products, as pro-inflammatory factors, accumulated in cirrhotic patients (Zuwala−Jagiello *et al*., 2006, 2009, 2011) and played an important role in the occurrence and progression of complications such as dysfunction of endothelial cells (Witko-Sarsat *et al*., 1998). AOPPs correlate well with certain cytokines (Kalousová *et al*., 2005) as well as with some markers of inflammation, including fibrinogen, orosomucoid. Even if the correlation between AOPPs-albumin and hs-CRP were poor, other studies demonstrated fair associations with other markers of oxidative stress, such as lipid peroxidation products and F2-isoprostanes. Furthermore, results from a recent study demonstrate that glycosylated and oxidized proteins indirectly up-regulate CRP expression in hepatocytes by stimulating monocytes to produce IL-6 (Li *et al.*, 2007). It seem, based on our study, even though there was functional loss of hepatocytes in patients with hepatic cirrhosis, the serum CRP level was still maintained in high level and dependent of AOPPsalbumin level with its significant correlation. The remaining viable hepatocytes may still contribute to this result. IL-6 is the main stimulant for hepatic production of CRP but also has other important roles leading to increased endothelial cell adhesiveness by upregulating ICAM-1, and VCAM-1 and releasing inflammatory mediators, including IL-6 itself (Szmitko *et al.*, 2003). Finally, the significant correlation between the levels of AOPPs and IL-6 supports the existence of a link between AOPPs and hemodynamic changes present in cirrhotic patients (Fig. 4).

Portal hypertension is characterized by intrahepatic vascular resistance causing an increase of portal vein pressure, and leads to the development of ascite (Møller *et al.*, 2008). IL-6 levels in our cirrhotic patients were different from those in controls, increased with the severity of liver disease, were independently associated to the presence of ascites. IL-6 increased in serum of patients with ascites compared to compensated (Child-Pugh class A) patients without ascites, and similar results were obtained in a study where serum IL-6 was also analyzed (Zhang *et al.*, 2002). In a simplistic view, portal hypertension leads to the formation of portosystemic collateral veins in liver cirrhosis and the resulting shunting (Cichoz-Lach *et al.*, 2008) also contributes to impaired hepatic uptake of IL-6. It has been convincingly shown that hepatic uptake of IL-6 is significantly impaired in patients with liver cirrhosis, and this may at least in part explain elevated serum levels in these patients (Soresi *et al.*, 2006). In our study, IL-6 levels increase significantly in association with the severity of liver cirrhosis according to the MELD score. Moreover, IL-6 levels in all cirrhotic patients were independently associated to the presence of low mean arterial pressure, and showed significant correlations with parameters related to hemodynamic abnormalities. The mechanism by which IL-6 could cause vasodilatation is unknown. However, the effect appears to be independent of nitric oxide, possibly due to an important role of prostacyclin synthesis (Dagher & Moore, 2001). It is then possible that IL-6 would produces vasodilatation by inducing prostacyclin synthesis; the effect of IL-6 would be potentiated by AOPPs stimulation (Li et al., 2007).

Further, investigators have tried to find more noninvasive biomarkers for cirrhotic patients for years (Schuppan & Afdhal, 2008). We observed good abilities of plasma AOPPs-albumin, TNF-α and IL-6 levels to distinguish cirrhotic patients from healthy controls, with good sensitivities and specificities by ROC analysis. Additionally, these parameters also were found to be elevated in concordance with the severity of cirrhosis. Thereby, it is possible that plasma levels of AOPPs-albumin, TNF-α and IL-6 levels could be evaluated as candidate biomarkers for initial and long-term assessment of liver cirrhosis.

#### **5. Conclusion**

378 Oxidative Stress and Diseases

These results suggested that this association of AOPPs with hemodynamic disturbances is dependent of the severity of cirrhosis. Additionally, AOPPs levels could be elevated as a result of insufficient renal elimination. However, the precise mechanism by which AOPPs is cleared from plasma is currently unknown. In addition, we found no correlation between AOPPs-albumin and serum creatinine levels. In any case, the values of AOPPs-albumin in patients with a low Child-Pugh score and absence of ascites suggests that AOPPs might have a role in the late stages of cirrhosis by aggravating the already initiated vasodilatation. Indeed, the presence of ascites, one of the major complications of cirrhosis and closely related to the hemodynamic disturbances of cirrhotic patients, was found in patients with higher levels of AOPPs. Finally, the extremely weak correlation between AOPPs-albumin levels and mean arterial pressure may suggest an indirect contribution of AOPPs to arterial

Structure and function of albumin are impaired in advanced liver disease by different mechanisms: plasma levels are decreased due to reduced synthesis and albumin is oxidatively modified. In this context, AOPPs-albumin may shows altered binding capacities for several substances. Decreased bilirubin binding was reported for *in vitro* oxidized albumin (Oettl & Stauber, 2007). As bilirubin is preferentially bound by the fully reduced form of albumin, impaired binding of bilirubin and other ligands (e.g. nitric oxide) is likely to occur in liver cirrhosis. Theoretically, increased circulating AOPPs-albumin may indirectly lead to elevation of nitric oxide which can, in turn, contribute to oxidative stress in cells (La Villa & Gentilini, 2008). Finally, nitric oxide plays a major key role in the development of hyperdynamic circulation and portal hypertension in cirrhosis (Iwakiri & Groszmann, 2007). Infusion of albumin (Garcovich *et al*., 2009) as well as albumin dialysis has been shown to improve the circulatory dysfunction as evidenced by an increase in mean blood pressure and systemic vascular resistance (Mitzner *et al*., 2001). This improvement in systemic hemodynamics might be due to a reduction in vasodilation following removal of nitric oxide which results in deactivation of the neurohormonal systems and a decrease in plasma levels of renin, aldosterone, norepinephrine and vasopressin (Chen *et al*., 2009). However, other findings of the present study imply that AOPPs are not only the factors responsible for the hemodynamic changes observed in cirrhotic patients. The AOPPsalbumin levels did not correlate significantly with the parameters that accompany important hemodynamic alterations in cirrhotic patients, such as plasma renin activity and aldosterone. Finally, when cirrhotic patients in our study were divided according to high and low mean arterial pressure, we found similar AOPPs-albumin levels in both groups.

Portal hypertension and cirrhosis can increase gut permeability to endotoxin and impair reticuloendothelial function of the liver that may result in increased serum endotoxin concentrations (Cariello *et al*., 2010). This may be a stimulus for the production of proinflammatory cytokines, resulting in the increased production of acute phase proteins such as C-reactive protein. In turn, CRP is capable of stimulating IL-6 and TNF-α production by monocytes (Ballou & Lozanski, 1992) and reactive oxygen species formation (Wang *et al*., 2003). Advanced oxidation protein products, as pro-inflammatory factors, accumulated in cirrhotic patients (Zuwala−Jagiello *et al*., 2006, 2009, 2011) and played an important role in the occurrence and progression of complications such as dysfunction of endothelial cells (Witko-Sarsat *et al*., 1998). AOPPs correlate well with certain cytokines (Kalousová *et al*., 2005) as well as with some markers of inflammation, including fibrinogen, orosomucoid. Even if the correlation between AOPPs-albumin and hs-CRP were poor, other studies

vasodilatation through other mediators.

In conclusion, there are differences between advanced oxidation protein products modified– albumin (AOPPs-albumin), which act as a pure oxidative stress marker, and Nε (carboxymethyl)lysine modified-albumin (CML-albumin; as prototype of the advanced

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**17** 

**Oxidative Stress in Parkinson's Disease;** 

Anwar Norazit1,2, George Mellick1 and Adrian C. B. Meedeniya1,3\*

Reactive oxygen species (ROS) are byproducts generated primarily from the breakdown of oxygen during aerobic metabolism. ROS can be found as free radicals containing highly reactive unpaired electrons (super oxide, nitric oxide, hydroxyl radical) or as other molecules (hydrogen peroxide, peroxynitrate). Under normal physiological conditions, ROS are neutralized by antioxidants. However, an increased production of ROS, namely, oxidative stress, can occur under pathophysiological conditions. Oxidative stress can be defined as an imbalance between oxidants and antioxidants where the oxidants are

The brain is highly susceptible to oxidative stress due to its high metabolic rate and limited regeneration capability (Andersen 2004). Oxidative stress has been implicated in a variety of neurodegenerative disease, including Parkinson's disease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis. However, presence of oxidative stress as a cause or

This review focuses on the pathogenesis of PD in relation to oxidative stress and the current animal models used to mimic the pathophysiology of human PD. We will also compare the animal and human data for PD like neurodegeneration with cell models of PD. This will also include a review on current experimentation and antioxidant therapies for counteracting

PD was first described in the early 19th century in the monologue "An essay on the shaking palsy" (Parkinson 2002). The three cardinal symptoms of PD are bradykinesia, rigidity, and

favoured, potentially leading to cellular damage (Sies 1985, Sies 1986, Sies 1991).

consequence of neurodegeneration, remains to be determined.

**1. Introduction** 

oxidative stress.

 \*

**2. Parkinson's disease** 

Corresponding Author

*3Griffith Health Institute, Griffith University, Gold Coast, Queensland,* 

**Parallels Between Current Animal Models, Human Studies and Cells** 

*1Eskitis Institute for Cell and Molecular Therapies,* 

*2Department of Molecular Medicine, Faculty of Medicine,* 

*Griffith University, Nathan, Queensland* 

*University of Kuala Lumpur,* 

*1,3Australia 2Malaysia* 

cirrhotic patients with spontaneous bacterial peritonitis. *Clinical Infectious Diseases*, Vol.17, No.2, (August 1993), pp. 218-223, ISSN 1058-4838


### **Oxidative Stress in Parkinson's Disease; Parallels Between Current Animal Models, Human Studies and Cells**

Anwar Norazit1,2, George Mellick1 and Adrian C. B. Meedeniya1,3\* *1Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland 2Department of Molecular Medicine, Faculty of Medicine, University of Kuala Lumpur, 3Griffith Health Institute, Griffith University, Gold Coast, Queensland, 1,3Australia 2Malaysia* 

#### **1. Introduction**

386 Oxidative Stress and Diseases

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protein products and inflammatory markers in liver cirrhosis, a comparison between alcohol-related and HCV-related cirrhosis. *Acta Biochimica Polonica*, Vol.58,

> Reactive oxygen species (ROS) are byproducts generated primarily from the breakdown of oxygen during aerobic metabolism. ROS can be found as free radicals containing highly reactive unpaired electrons (super oxide, nitric oxide, hydroxyl radical) or as other molecules (hydrogen peroxide, peroxynitrate). Under normal physiological conditions, ROS are neutralized by antioxidants. However, an increased production of ROS, namely, oxidative stress, can occur under pathophysiological conditions. Oxidative stress can be defined as an imbalance between oxidants and antioxidants where the oxidants are favoured, potentially leading to cellular damage (Sies 1985, Sies 1986, Sies 1991).

> The brain is highly susceptible to oxidative stress due to its high metabolic rate and limited regeneration capability (Andersen 2004). Oxidative stress has been implicated in a variety of neurodegenerative disease, including Parkinson's disease (PD), Alzheimer's disease, and amyotrophic lateral sclerosis. However, presence of oxidative stress as a cause or consequence of neurodegeneration, remains to be determined.

> This review focuses on the pathogenesis of PD in relation to oxidative stress and the current animal models used to mimic the pathophysiology of human PD. We will also compare the animal and human data for PD like neurodegeneration with cell models of PD. This will also include a review on current experimentation and antioxidant therapies for counteracting oxidative stress.

#### **2. Parkinson's disease**

PD was first described in the early 19th century in the monologue "An essay on the shaking palsy" (Parkinson 2002). The three cardinal symptoms of PD are bradykinesia, rigidity, and

<sup>\*</sup> Corresponding Author

Oxidative Stress in Parkinson's Disease;

dopaminergic structures (Levy 2007).

**2.2.2 Environmental toxins** 

**2.2.3 Genetic determinants** 

oxidative stress.

damage.

Parallels Between Current Animal Models, Human Studies, and Cells

superposition of a topographic gradient of neuronal loss in brainstem and basal forebrain structures related to the disease process and an age-related temporal gradient. Clinical progression of PD is determined by advancing age and not by disease duration; and a biological interaction is involved in the effects of the disease process and aging on non

Pesticide exposure is implicated as an environmental risk factor for PD (Herishanu et al. 2001, Le Couteur et al. 1999, Tanner 1989, Ho, Woo and Lee 1989). The susceptibility of humans to these pesticides has been reported to be linked to genetic factors (Menegon et al. 1998, Drozdzik et al. 2003). Many of these pesticides have a major site of action along the mitochondrial electron transport chain (Degli Esposti 1998), which results in increasing

The role of genetic factors in PD has been the subject of intense scrutiny. The first clue of familial PD was provided in 1907 when Gowers reported approximately 15% of his PD patients reported an affected family member (Gowers 1902) Since then many genes have been identified as causing familial PD (Schapira 2008). The products associated with the gene mutations are either mitochondrial proteins or are associated with mitochondria. Namely, proteins that interface with the pathways of oxidative stress and free radical

**Gene Locus Inheritance Gene product PARK1/4** 4q21 Autosomal dominant α-synuclein **PARK2** 6q25 Autosomal recessive Parkin **PARK3** 2p13 Autosomal dominant -

**UCH-L1** 4p15 Autosomal dominant Ubiquitin thiolesterase **PINK1** 1p35 Autosomal recessive PTEN-induced putative kinase 1

**LRRK2** 12p Autosomal dominant Leucine-rich repeat kinase 2 **ATP13A2** 1p36 Autosomal recessive ATPase type 13A2

The Braak hypothesis suggests that the initial event in sporadic PD may be an infectious assault on susceptible neuronal types in the olfactory or enteric nervous system (Braak et al. 2003a, Hawkes, Del Tredici and Braak 2007, Braak et al. 2003b). The Lewy neuritis and Lewy bodies' progress rostrally in stages into the lower brainstem region (medulla oblongata and pontine tegmentum; stages 1 and 2), followed by the midbrain (substantia nigra; stage 3) and the basal prosencephalon and mesocortex (stage 4), and eventually the neocortex (stage 5 and 6) (Braak et al. 2003a, Braak et al. 2003b). Stages 1 to 3 have been characterised as the

**PARK7** 1p36 Autosomal recessive DJ-1

**PARK10** 1p32 Autosomal recessive - **PARK11** 2q36-q37 - -

Table 1. Genetic causes of PD (modified from Schapira, 2008)

**2.2.4 Sporadic Parkinson's disease** 

389

postural instability (Gash et al. 1996). The clinical diagnosis of PD is usually based on the United Kingdom Parkinson's Disease Society Brain Research Centre criteria (Gibb and Lees 1988), with the accuracy of this diagnosis being high (90%) (Hughes, Daniel and Lees 2001). During the first 10 years of the disease, patients usually exhibit slowness of movement, mild gait hypokinesia, resting tremor, micrographic handwriting and reduced speech volume (Morris 2000). During the latter stages of the disease, festination, dyskinesia, akinesia, marked hypokinesia, postural instability and falls are usually more pronounced (Morris 2000). The symptoms of this condition only show after 80% of the striatal innervations and 60% of the substantia nigra par compacta dopaminergic neurons are lost, suggestive of a pathological process initiated at the synaptic end of the nigral neurons, with neuronal death as a result from a 'dying back' process (Dauer and Przedborski 2003).

#### **2.1 Parkinson's disease pathology**

PD can be defined as a progressive neurodegenerative disorder characterised histopathologically by the degeneration of the dopaminergic nigrostriatal pathway (Watts et al. 1997). Post-mortem analyses of the substantia nigra in PD patients have shown effects of oxidative stress with a decrease in glutathione (GSH) levels, increased levels of iron, neuromelanin associated redox-active iron, lipid peroxidation, protein oxidation and DNA damage (Jenner and Olanow 1998, Faucheux et al. 2003, Dexter et al. 1989). These changes may directly induce nigral cell degeneration via oxidative stress or render neurons susceptible to the actions of toxins.

The neurotrophic factor milieu of the brain is affected in PD patients. A decrease in brain derived neurotrophic factor (BDNF) in the post-mortem brains of clinically and pathologically diagnosed PD patients, compared to normal controls (Mogi et al. 1999, Parain et al. 1999). A decrease in BDNF mRNA expression in the substantia nigra of PD patients has also been demonstrated (Howells et al. 2000). A decrease in glial cell derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) also occurs in the brains of PD patients (Chauhan, Siegel and Lee 2001). The resultant inability to up-regulate neurotrophic factors in response to injury or stress may compromised defence mechanisms of the brain, thus contributing to cell degeneration (Olanow and Tatton 1999).

#### **2.2 Pathogenic role of host/exogenous factors**

There is increasing evidence of host and /or exogenous factors playing a role in the pathogenesis of PD. Many of these factors negatively impact mitochondrial function.

#### **2.2.1 Age**

The incidence of PD is related to increasing age (de Rijk et al. 1997, Mayeux et al. 1992, Van Den Eeden et al. 2003). With age, high levels of mitochondrial DNA deletion (Bender et al. 2006); increase in α-synuclein (Chu and Kordower 2007); decrease in dopamine transporter mRNA (Bannon et al. 1992); decrease in neurotrophic factor gene expression (Lee, Weindruch and Prolla 2000); reduced response to growth factors (Smith 1996); decrease in brain peroxidase and catalase activity (Ambani, Van Woert and Murphy 1975); and a decrease in dopamine binding sites (Severson et al. 1982) are apparent in the neuronal population of the substantia nigra. The relationship between PD and aging includes a superposition of a topographic gradient of neuronal loss in brainstem and basal forebrain structures related to the disease process and an age-related temporal gradient. Clinical progression of PD is determined by advancing age and not by disease duration; and a biological interaction is involved in the effects of the disease process and aging on non dopaminergic structures (Levy 2007).

#### **2.2.2 Environmental toxins**

388 Oxidative Stress and Diseases

postural instability (Gash et al. 1996). The clinical diagnosis of PD is usually based on the United Kingdom Parkinson's Disease Society Brain Research Centre criteria (Gibb and Lees 1988), with the accuracy of this diagnosis being high (90%) (Hughes, Daniel and Lees 2001). During the first 10 years of the disease, patients usually exhibit slowness of movement, mild gait hypokinesia, resting tremor, micrographic handwriting and reduced speech volume (Morris 2000). During the latter stages of the disease, festination, dyskinesia, akinesia, marked hypokinesia, postural instability and falls are usually more pronounced (Morris 2000). The symptoms of this condition only show after 80% of the striatal innervations and 60% of the substantia nigra par compacta dopaminergic neurons are lost, suggestive of a pathological process initiated at the synaptic end of the nigral neurons, with neuronal death

PD can be defined as a progressive neurodegenerative disorder characterised histopathologically by the degeneration of the dopaminergic nigrostriatal pathway (Watts et al. 1997). Post-mortem analyses of the substantia nigra in PD patients have shown effects of oxidative stress with a decrease in glutathione (GSH) levels, increased levels of iron, neuromelanin associated redox-active iron, lipid peroxidation, protein oxidation and DNA damage (Jenner and Olanow 1998, Faucheux et al. 2003, Dexter et al. 1989). These changes may directly induce nigral cell degeneration via oxidative stress or render neurons

The neurotrophic factor milieu of the brain is affected in PD patients. A decrease in brain derived neurotrophic factor (BDNF) in the post-mortem brains of clinically and pathologically diagnosed PD patients, compared to normal controls (Mogi et al. 1999, Parain et al. 1999). A decrease in BDNF mRNA expression in the substantia nigra of PD patients has also been demonstrated (Howells et al. 2000). A decrease in glial cell derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) also occurs in the brains of PD patients (Chauhan, Siegel and Lee 2001). The resultant inability to up-regulate neurotrophic factors in response to injury or stress may compromised defence mechanisms

There is increasing evidence of host and /or exogenous factors playing a role in the

The incidence of PD is related to increasing age (de Rijk et al. 1997, Mayeux et al. 1992, Van Den Eeden et al. 2003). With age, high levels of mitochondrial DNA deletion (Bender et al. 2006); increase in α-synuclein (Chu and Kordower 2007); decrease in dopamine transporter mRNA (Bannon et al. 1992); decrease in neurotrophic factor gene expression (Lee, Weindruch and Prolla 2000); reduced response to growth factors (Smith 1996); decrease in brain peroxidase and catalase activity (Ambani, Van Woert and Murphy 1975); and a decrease in dopamine binding sites (Severson et al. 1982) are apparent in the neuronal population of the substantia nigra. The relationship between PD and aging includes a

pathogenesis of PD. Many of these factors negatively impact mitochondrial function.

of the brain, thus contributing to cell degeneration (Olanow and Tatton 1999).

as a result from a 'dying back' process (Dauer and Przedborski 2003).

**2.1 Parkinson's disease pathology** 

susceptible to the actions of toxins.

**2.2.1 Age** 

**2.2 Pathogenic role of host/exogenous factors** 

Pesticide exposure is implicated as an environmental risk factor for PD (Herishanu et al. 2001, Le Couteur et al. 1999, Tanner 1989, Ho, Woo and Lee 1989). The susceptibility of humans to these pesticides has been reported to be linked to genetic factors (Menegon et al. 1998, Drozdzik et al. 2003). Many of these pesticides have a major site of action along the mitochondrial electron transport chain (Degli Esposti 1998), which results in increasing oxidative stress.

#### **2.2.3 Genetic determinants**

The role of genetic factors in PD has been the subject of intense scrutiny. The first clue of familial PD was provided in 1907 when Gowers reported approximately 15% of his PD patients reported an affected family member (Gowers 1902) Since then many genes have been identified as causing familial PD (Schapira 2008). The products associated with the gene mutations are either mitochondrial proteins or are associated with mitochondria. Namely, proteins that interface with the pathways of oxidative stress and free radical damage.


Table 1. Genetic causes of PD (modified from Schapira, 2008)

#### **2.2.4 Sporadic Parkinson's disease**

The Braak hypothesis suggests that the initial event in sporadic PD may be an infectious assault on susceptible neuronal types in the olfactory or enteric nervous system (Braak et al. 2003a, Hawkes, Del Tredici and Braak 2007, Braak et al. 2003b). The Lewy neuritis and Lewy bodies' progress rostrally in stages into the lower brainstem region (medulla oblongata and pontine tegmentum; stages 1 and 2), followed by the midbrain (substantia nigra; stage 3) and the basal prosencephalon and mesocortex (stage 4), and eventually the neocortex (stage 5 and 6) (Braak et al. 2003a, Braak et al. 2003b). Stages 1 to 3 have been characterised as the

Oxidative Stress in Parkinson's Disease;

**3.1.3 Paraquat and Maneb** 

to environmental factors.

**3.1.4 Rotenone** 

model of PD.

Parallels Between Current Animal Models, Human Studies, and Cells

intracellular calcium homeostasis (Sedelis, Schwarting and Huston 2001). When MPTP is delivered systemically, MPTP produces bilateral lesions of the dopamine neurons (Sedelis et al. 2001). Following systemic administration and collateral damage to the ventral tegmental area, an external source of dopamine is needed to stimulate adequate food and water uptake (Petzinger and Langston 1998). Bilateral lesions also have a high morbidity and mortality rate. The other difficulty with using MPTP lesioned animal models is that MPTP has varying effects on different animal models and strains due to differences in visceral functions (Betarbet et al. 2002). These drawbacks in mice are being looked at with the behavioural phenotyping of a MPTP mouse animal model for PD (Sedelis et al. 2001).

1,1-dimethyl-4,4-bipyridinium, better known as paraquat is a herbicide that has a similar structure to MPP+, making it a putative risk factor for PD (Dawson and Dawson 2002). Paraquat when delivered systemically can pass the blood brain barrier (Brooks et al. 1999). Paraquat has a high affinity to the nigrostriatal dopaminergic system (Thiruchelvam et al. 2000) and exposure in mice can cause up-regulation and aggregation of α-synuclein, a pathological sign of PD in humans (Manning-Bog et al. 2002). Due to its structural similarity to MPP+, paraquat's mechanism of action is believed to involve oxidative stress and its toxic effect via the mitochondria (Betarbet et al. 2002). A link between paraquat and other types of herbicide/pesticides with a increased incidence of PD has been demonstrated (Liou et al. 1997). The effects of paraquat on the dopaminergic system can be increased when mixed with Maneb (manganese ethylenebisdithiocarbamate) (Thiruchelvam et al. 2000). Maneb is a fungicide that has been implicated in an increased incidence of PD in humans (Ferraz et al. 1988, Meco et al. 1994). This animal model is of use when examining PD like syndromes due

Rotenone is a naturally occurring root extract of *Lonchocarpus utilis* and *Lonchocarpus urucu* used as an insecticide as well as a piscicide (Caboni et al. 2004). Rotenone is a high-affinity inhibitor of complex 1 of the mitochondrial electron transport chain (Sherer et al. 2003b). Rotenone cytotoxicity is not dependent on the dopamine transporter (Hirata et al. 2008). Complex 1 inhibition by rotenone can cause the production of ROS that causes oxidative

When neurons are exposed to rotenone in cell culture, they produce ROS and superoxides, with dopaminergic neurons showing higher susceptibility to rotenone compared to other neurons (Radad, Rausch and Gille 2006, Ahmadi et al. 2003, Moon et al. 2005). Over time, the increase in ROS and superoxides produced by rotenone exposure leads to cell death (Ahmadi et al. 2003, Moon et al. 2005). Thus rotenone is a relevant toxin for developing a rat

In general, rotenone blocks the electron transfer between the Complex I-associated ironsulfur clusters and ubiquinone binding site (Grivennikova et al. 1997) (Figure 1). Specifically, rotenone acts as a semiquinone antagonist and displaces the ubisemiquinone intermediate at the ubiquinone binding site (Degli Esposti 1998). By inhibiting the ubiquinone binding site, rotenone alters the state of complex I, leading to higher superoxide

damage in dopaminergic neurons (Testa, Sherer and Greenamyre 2005).

391

pre-symptomatic phase while stage 4 to 6 has been characterised as the symptomatic phase. The sequential ascending topography reported by Braak (2003b) has been reported to be only partially in line with the latest imaging of PD (Brooks 2010). It may instead reflect the more primitive regions of the nervous system (and perhaps the more active) showing a greater susceptibility.

#### **3. Parkinson's disease animal models**

There are both toxin and genetic animal models popularly used to represent PD. Both of these models increase ROS production directly or indirectly to induce the degeneration of the nigrostriatal pathway in laboratory animals.

#### **3.1 Neurotoxins**

There are four main toxin induced models popularly used to produce PD like symptoms in rodents. The neurotoxins used are 6-OHDA, MPTP, paraquat in combination with Maneb, and rotenone.

#### **3.1.1 6-Hydroxydopamine (6-OHDA)**

The neurotoxin 6-OHDA destroys catecholaminergic neurons by the combined effect of reactive oxygen species (ROS) and quinines (Cohen and Heikkila 1984). To specifically induce PD in an animal model, 6-OHDA is injected stereotaxically into the substantia nigra, the medial forebrain bundle, or the striatum (Javoy et al. 1976). 6-OHDA is delivered directly into the brain by stereotaxic means as 6-OHDA does not cross the blood-brain barrier, thus ruling out systemic injections (Bove et al. 2005). The administration of 6-OHDA into the substantia nigra or the medial forebrain bundle mediates its uptake anterogradely, while administration into the striatum causes the uptake of the chemical retrogradely. 6- OHDA is transported into dopaminergic neurons via their high-affinity catecholaminergic uptake system (Zigmond, Hastings and Abercrombie 1992). 6-OHDA is usually administered unilaterally to produce a unilateral lesion, allowing the unlesioned side to act as an internal control and to minimise morbidity and mortality (Betarbet, Sherer and Greenamyre 2002). The 6-OHDA lesioned rat has been extensively characterised behaviourally and pathologically, making it one of the models of choice when investigating PD (Schwarting and Huston 1996). However, the effects of 6-OHDA are acute and do not show the same cellular pathology (Lewy bodies) as seen in PD (Dawson and Dawson 2002).

#### **3.1.2 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine (MPTP)**

MPTP was accidentally produced during the illegal production of 1-methyl-4-phenyl-4 propionoxypiperidine (MPPP), a synthetic opioid drug, causing heroin addicts to display Parkinson-like symptoms (Langston 1996). MPTP readily crosses the blood-brain-barrier and is converted by the enzyme monoamine oxidase B (MAO-B) to 1-methyl-4-phenyl-2, 3 dihydropyridium (MPDP+) that then deprotonates to generate the corresponding pyridium species, MPP+ (Smeyne and Jackson-Lewis 2005). MPP+ has a high affinity to the dopamine transporter, thus it is highly selective to dopaminergic neurons (Javitch et al. 1985). Its selective uptake leads to severe damage to the nigrostriatal dopaminergic system, acting as a neurotoxin that inhibits mitochondrial complex I, producing oxidative stress and disturbing intracellular calcium homeostasis (Sedelis, Schwarting and Huston 2001). When MPTP is delivered systemically, MPTP produces bilateral lesions of the dopamine neurons (Sedelis et al. 2001). Following systemic administration and collateral damage to the ventral tegmental area, an external source of dopamine is needed to stimulate adequate food and water uptake (Petzinger and Langston 1998). Bilateral lesions also have a high morbidity and mortality rate. The other difficulty with using MPTP lesioned animal models is that MPTP has varying effects on different animal models and strains due to differences in visceral functions (Betarbet et al. 2002). These drawbacks in mice are being looked at with the behavioural phenotyping of a MPTP mouse animal model for PD (Sedelis et al. 2001).

#### **3.1.3 Paraquat and Maneb**

390 Oxidative Stress and Diseases

pre-symptomatic phase while stage 4 to 6 has been characterised as the symptomatic phase. The sequential ascending topography reported by Braak (2003b) has been reported to be only partially in line with the latest imaging of PD (Brooks 2010). It may instead reflect the more primitive regions of the nervous system (and perhaps the more active) showing a

There are both toxin and genetic animal models popularly used to represent PD. Both of these models increase ROS production directly or indirectly to induce the degeneration of

There are four main toxin induced models popularly used to produce PD like symptoms in rodents. The neurotoxins used are 6-OHDA, MPTP, paraquat in combination with Maneb,

The neurotoxin 6-OHDA destroys catecholaminergic neurons by the combined effect of reactive oxygen species (ROS) and quinines (Cohen and Heikkila 1984). To specifically induce PD in an animal model, 6-OHDA is injected stereotaxically into the substantia nigra, the medial forebrain bundle, or the striatum (Javoy et al. 1976). 6-OHDA is delivered directly into the brain by stereotaxic means as 6-OHDA does not cross the blood-brain barrier, thus ruling out systemic injections (Bove et al. 2005). The administration of 6-OHDA into the substantia nigra or the medial forebrain bundle mediates its uptake anterogradely, while administration into the striatum causes the uptake of the chemical retrogradely. 6- OHDA is transported into dopaminergic neurons via their high-affinity catecholaminergic uptake system (Zigmond, Hastings and Abercrombie 1992). 6-OHDA is usually administered unilaterally to produce a unilateral lesion, allowing the unlesioned side to act as an internal control and to minimise morbidity and mortality (Betarbet, Sherer and Greenamyre 2002). The 6-OHDA lesioned rat has been extensively characterised behaviourally and pathologically, making it one of the models of choice when investigating PD (Schwarting and Huston 1996). However, the effects of 6-OHDA are acute and do not show the same cellular pathology (Lewy bodies) as seen in PD (Dawson and Dawson 2002).

MPTP was accidentally produced during the illegal production of 1-methyl-4-phenyl-4 propionoxypiperidine (MPPP), a synthetic opioid drug, causing heroin addicts to display Parkinson-like symptoms (Langston 1996). MPTP readily crosses the blood-brain-barrier and is converted by the enzyme monoamine oxidase B (MAO-B) to 1-methyl-4-phenyl-2, 3 dihydropyridium (MPDP+) that then deprotonates to generate the corresponding pyridium species, MPP+ (Smeyne and Jackson-Lewis 2005). MPP+ has a high affinity to the dopamine transporter, thus it is highly selective to dopaminergic neurons (Javitch et al. 1985). Its selective uptake leads to severe damage to the nigrostriatal dopaminergic system, acting as a neurotoxin that inhibits mitochondrial complex I, producing oxidative stress and disturbing

greater susceptibility.

**3.1 Neurotoxins** 

and rotenone.

**3. Parkinson's disease animal models** 

the nigrostriatal pathway in laboratory animals.

**3.1.2 1-Methyl-4-Phenyl-1,2,5,6-Tetrahydropyridine (MPTP)** 

**3.1.1 6-Hydroxydopamine (6-OHDA)** 

1,1-dimethyl-4,4-bipyridinium, better known as paraquat is a herbicide that has a similar structure to MPP+, making it a putative risk factor for PD (Dawson and Dawson 2002). Paraquat when delivered systemically can pass the blood brain barrier (Brooks et al. 1999). Paraquat has a high affinity to the nigrostriatal dopaminergic system (Thiruchelvam et al. 2000) and exposure in mice can cause up-regulation and aggregation of α-synuclein, a pathological sign of PD in humans (Manning-Bog et al. 2002). Due to its structural similarity to MPP+, paraquat's mechanism of action is believed to involve oxidative stress and its toxic effect via the mitochondria (Betarbet et al. 2002). A link between paraquat and other types of herbicide/pesticides with a increased incidence of PD has been demonstrated (Liou et al. 1997). The effects of paraquat on the dopaminergic system can be increased when mixed with Maneb (manganese ethylenebisdithiocarbamate) (Thiruchelvam et al. 2000). Maneb is a fungicide that has been implicated in an increased incidence of PD in humans (Ferraz et al. 1988, Meco et al. 1994). This animal model is of use when examining PD like syndromes due to environmental factors.

#### **3.1.4 Rotenone**

Rotenone is a naturally occurring root extract of *Lonchocarpus utilis* and *Lonchocarpus urucu* used as an insecticide as well as a piscicide (Caboni et al. 2004). Rotenone is a high-affinity inhibitor of complex 1 of the mitochondrial electron transport chain (Sherer et al. 2003b). Rotenone cytotoxicity is not dependent on the dopamine transporter (Hirata et al. 2008). Complex 1 inhibition by rotenone can cause the production of ROS that causes oxidative damage in dopaminergic neurons (Testa, Sherer and Greenamyre 2005).

When neurons are exposed to rotenone in cell culture, they produce ROS and superoxides, with dopaminergic neurons showing higher susceptibility to rotenone compared to other neurons (Radad, Rausch and Gille 2006, Ahmadi et al. 2003, Moon et al. 2005). Over time, the increase in ROS and superoxides produced by rotenone exposure leads to cell death (Ahmadi et al. 2003, Moon et al. 2005). Thus rotenone is a relevant toxin for developing a rat model of PD.

In general, rotenone blocks the electron transfer between the Complex I-associated ironsulfur clusters and ubiquinone binding site (Grivennikova et al. 1997) (Figure 1). Specifically, rotenone acts as a semiquinone antagonist and displaces the ubisemiquinone intermediate at the ubiquinone binding site (Degli Esposti 1998). By inhibiting the ubiquinone binding site, rotenone alters the state of complex I, leading to higher superoxide

Oxidative Stress in Parkinson's Disease;

disease (Qin et al. 1998, Panet et al. 2001).

Parallels Between Current Animal Models, Human Studies, and Cells

production at the same site (Lambert and Brand 2004). The inhibition of Complex I may overwhelm the mitochondria antioxidant system that consist of superoxide dismutase production with manganese (SOD) at the active site and GSH (Fridovich 1995, Betarbet et al. 2002), increasing the ROS concentration in the mitochondrial matrix. The increase of ROS activates the apoptotic intrinsic pathway, which increases the permeability of the outer membrane via the opening of transition pores (Turrens 2003). This allows cytochrome c to move from the intermembrane space into the cells cytoplasm, allowing it to bind with Apaf-1 (apoptopic protease activating factor) (Zou et al. 1997). In the presence of ATP or dATP, the Apaf-1 – cytochrome c complex changes its configuration, exposing the Apaf-1's CARD (caspase recruitment domain) to allow for the recruitment of procaspase-9 (Li et al. 1997). This interaction changes procaspase-9 into caspase-9 that in turn cleaves procaspase-3 into caspase-3. Cells contain an inhibitor of apoptosis (IAP) to prevent accidental caspase-9 activation. XIAP binds to the activated N terminus of caspase-9, making it inactive (Shiozaki et al. 2003). This process can be reversed by the mitochondrial proteins Diablo/Smac and HtrA2/Omi that are released during apoptosis (Vaux and Silke 2003), leading to caspase-3 activation. Caspase-3 cleaves the 45 kDa subunit of a two unit protein in two places producing a DNA Fragmentation Factor (DFF) (Liu et al. 1997). The increase in oxidative stress can also activate nuclear factor kappa β (NF-κβ) (Panet et al. 2001), which has been implicated in the beginning of a pro-apoptopic gene expression programme which may play a role in neurodegenerative

Rotenone has the ability to activate microglia *in vivo* (Sherer et al. 2003a). This activation causes the release of tumour necrosis factor α (TNF- α), interleukin 1β (IL-1β), and interferon γ (IFN- γ). These cytokines can bind to their respective receptors and activate transduction pathways, leading to NF-κβ activation and ultimately, apoptosis (Gao, Liu and Hong 2003). It was reported that NF-κβ is present before caspase-3 (Wang et al. 2002), which suggests that superoxides and cytokines produced by the activation of microglia by rotenone play a role in the activation of NF-κβ before complex I inhibition initiates the activation of caspase-3 apoptosis. However, excess ROS from the cytoplasm due to the Complex I inhibition could result in ROS crossing over into the cytoplasm via voltagedependent anion channels (Han et al. 2003), which could also help activate NF-κβ. Excess ROS in the cytoplasm could also be complemented by the superoxides produced by the activated microglia via NADPH oxidase (Gao et al. 2002). The superoxides produced by NADPH oxidase can also interact with nitric oxide (NO) to produce peroxinitrate (ONOO-), a potent oxidant (Gao et al. 2003) implicated with being cytotoxic and able to damage the neuron's cell membrane (Hirsch 2000, Imao et al. 1998). NO is produced via the inducible nitric oxide synthase that is activated by CD23 which in turn is induced by the presence of

Initial studies of the effect of rotenone used systemically in the rat showed a decrease in brain dopamine levels, decreases in tyrosine hydroxylase immunoreactivity in the substantia nigra, and motor deficits similar to those seen in PD (Betarbet et al. 2000, Sherer et al. 2003c, Alam and Schmidt 2002). Rotenone treatment also leads to intracytoplasmic inclusions within dopaminergic neurons thereby mimicking some aspects of PD histopathology (Betarbet et al. 2002). Unfortunately, systemic delivery of rotenone results in high mortality due to the systemic toxicity resulting in liver failure and inconsistent lesions of the substantia nigra (Dawson and Dawson 2002). These problems were alleviated with intra-peritoneal

TNF- α and IFN- γ (Munoz-Fernandez and Fresno 1998, Hirsch 2000).

393

Fig. 1. Mechanism of action of rotenone

Fig. 1. Mechanism of action of rotenone

production at the same site (Lambert and Brand 2004). The inhibition of Complex I may overwhelm the mitochondria antioxidant system that consist of superoxide dismutase production with manganese (SOD) at the active site and GSH (Fridovich 1995, Betarbet et al. 2002), increasing the ROS concentration in the mitochondrial matrix. The increase of ROS activates the apoptotic intrinsic pathway, which increases the permeability of the outer membrane via the opening of transition pores (Turrens 2003). This allows cytochrome c to move from the intermembrane space into the cells cytoplasm, allowing it to bind with Apaf-1 (apoptopic protease activating factor) (Zou et al. 1997). In the presence of ATP or dATP, the Apaf-1 – cytochrome c complex changes its configuration, exposing the Apaf-1's CARD (caspase recruitment domain) to allow for the recruitment of procaspase-9 (Li et al. 1997). This interaction changes procaspase-9 into caspase-9 that in turn cleaves procaspase-3 into caspase-3. Cells contain an inhibitor of apoptosis (IAP) to prevent accidental caspase-9 activation. XIAP binds to the activated N terminus of caspase-9, making it inactive (Shiozaki et al. 2003). This process can be reversed by the mitochondrial proteins Diablo/Smac and HtrA2/Omi that are released during apoptosis (Vaux and Silke 2003), leading to caspase-3 activation. Caspase-3 cleaves the 45 kDa subunit of a two unit protein in two places producing a DNA Fragmentation Factor (DFF) (Liu et al. 1997). The increase in oxidative stress can also activate nuclear factor kappa β (NF-κβ) (Panet et al. 2001), which has been implicated in the beginning of a pro-apoptopic gene expression programme which may play a role in neurodegenerative disease (Qin et al. 1998, Panet et al. 2001).

Rotenone has the ability to activate microglia *in vivo* (Sherer et al. 2003a). This activation causes the release of tumour necrosis factor α (TNF- α), interleukin 1β (IL-1β), and interferon γ (IFN- γ). These cytokines can bind to their respective receptors and activate transduction pathways, leading to NF-κβ activation and ultimately, apoptosis (Gao, Liu and Hong 2003). It was reported that NF-κβ is present before caspase-3 (Wang et al. 2002), which suggests that superoxides and cytokines produced by the activation of microglia by rotenone play a role in the activation of NF-κβ before complex I inhibition initiates the activation of caspase-3 apoptosis. However, excess ROS from the cytoplasm due to the Complex I inhibition could result in ROS crossing over into the cytoplasm via voltagedependent anion channels (Han et al. 2003), which could also help activate NF-κβ. Excess ROS in the cytoplasm could also be complemented by the superoxides produced by the activated microglia via NADPH oxidase (Gao et al. 2002). The superoxides produced by NADPH oxidase can also interact with nitric oxide (NO) to produce peroxinitrate (ONOO-), a potent oxidant (Gao et al. 2003) implicated with being cytotoxic and able to damage the neuron's cell membrane (Hirsch 2000, Imao et al. 1998). NO is produced via the inducible nitric oxide synthase that is activated by CD23 which in turn is induced by the presence of TNF- α and IFN- γ (Munoz-Fernandez and Fresno 1998, Hirsch 2000).

Initial studies of the effect of rotenone used systemically in the rat showed a decrease in brain dopamine levels, decreases in tyrosine hydroxylase immunoreactivity in the substantia nigra, and motor deficits similar to those seen in PD (Betarbet et al. 2000, Sherer et al. 2003c, Alam and Schmidt 2002). Rotenone treatment also leads to intracytoplasmic inclusions within dopaminergic neurons thereby mimicking some aspects of PD histopathology (Betarbet et al. 2002). Unfortunately, systemic delivery of rotenone results in high mortality due to the systemic toxicity resulting in liver failure and inconsistent lesions of the substantia nigra (Dawson and Dawson 2002). These problems were alleviated with intra-peritoneal

Oxidative Stress in Parkinson's Disease;

factors (unpublished data).

DAPI (blue) (scale bar=10 μm)

**3.2 Cellular based models** 

Parallels Between Current Animal Models, Human Studies, and Cells

medial forebrain bundle (Hashimoto et al. 2002, Quilty et al. 2006). This model has now been successfully used to test the neuroprotective properties of chronic exposure to neurotrophic

Fig. 2. Dopamine cell pathophysiology of the substantia nigra, induced by 0.5-μg rotenone delivered focally into the medial forebrain bundle and harvested after 14 days. a) The unilateral lesion is manifest as a subtle yet significant reduction in the number of dopaminergic neurons and dendritic arbour within the substantia nigra (asterisk) and a reduction in the dopaminergic innervation of the striatum (data not shown). The

nigrostriatal circuitry contralateral to the lesion is uneffected by the rotenone treatment. b) Ras-GAP SH3 domain binding protein (G3BP) immunoreactivity (red, filled arrowhead) was expressed in tyrosine hydroxylase immunoreactive (green, filled arrowhead) dopaminergic neurons located in the rotenone exposed substantia nigra. c) Superoxide dismutase (SOD2) immunoreactivity (red, filled arrowhead) was also expressed in the rotenone exposed tyrosine hydroxylase immunoreactive (green, filled arrowhead)

dopaminergic neurons. Nuclei for all sections are counterstained with the nuclear marker

Complete human disease phenotype is rarely observed in animal models introduced with human gene mutations. Thus, relevant human tissue is required to study the disease process

395

administration of rotenone in a specialised vehicle of a medium-chain triglyceride, although this model developed a debilitating behavioural phenotype in a relatively short time and is not appropriate as a slow onset chronic model (Cannon et al. 2009).

The side effects of peripheral rotenone treatment can be reduced or avoided by the infusion of a lower dose directly into the striatum, medial forebrain bundle or substantia nigra (Ravenstijn et al. 2008, Xiong et al. 2009). Bilateral infusion of rotenone into the medial forebrain bundle causes reductions in striatal dopamine and disruptions in motor behaviours (Alam, Mayerhofer and Schmidt 2004). However, bilateral infusions lead to weight loss and require specialised diets to maintain the animals (Betarbet et al. 2000, Sherer et al. 2003c), possibly due to bilateral lesioning of the ventral tegmental area. These problems are ameliorated by unilateral lesions which reduce dopamine signalling, dopamine level, DOPAC (3,4-dihydroxyphenylacetic acid) level and dopaminergic innervation, while increasing oxidative stress (hydroxyl radicals, GSH level, superoxide dismutase levels), as well as up-regulating α-synuclein expression in the ipsilateral substantia nigra (Sindhu et al. 2006, Antkiewicz-Michaluk et al. 2004, Saravanan, Sindhu and Mohanakumar 2005, Sindhu, Saravanan and Mohanakumar 2005, Ravenstijn et al. 2008, Xiong et al. 2009). Unilateral rotenone lesioned animals have shown differences in several behavioural indices, including rotarod and amphetamine or apomorphine-induced rotation, demonstrating the unilateral functional motor deficits associated with substantia nigra dopamine neuron loss (Sindhu et al. 2006, Sindhu et al. 2005, Ravenstijn et al. 2008, Xiong et al. 2009). Behavioural indices are also influenced by the lesion of the ventral tegmental area, as can occur with larger doses of rotenone in the medial forebrain bundle (Sindhu et al. 2005, Xiong et al. 2009, Thomas et al. 1994).

Our laboratory has recently reported that a low dose of rotenone injected into the medial forebrain pathway in adult rats caused progressive loss of dopaminergic neurons with the remaining neurons displaying pathophysiological hallmark of human PD (Norazit et al. 2010)(Figure 2). Unlike the complete lesion of dopaminergic neurons induced by focal 6- OHDA injection, rotenone injection into the medial forebrain bundle induced the upregulation of markers of oxidative stress and markers of cell stress in dopaminergic neurons (Sindhu et al. 2005, Norazit et al. 2010). 0.5 μg of rotenone caused negligible necrosis, inflammation and a diffused glial response. A progressive loss of dopaminergic neurons in the substantia nigra and loss of striatal innervation was shown. The low dose of rotenone mediated dopaminergic cell death by oxidative stress as previously demonstrated (Rodrigues, Gomide and Chadi 2004). An increase in astrocytes and microglia occurs in human PD, where they have been ascribed both a neuroprotective and deleterious role (Imamura et al. 2003, Ishida et al. 2006, Vila et al. 2001). The study presented direct support for the hypothesis that rotenone induces a chronic state of oxidative stress in dopaminergic neurons. Rotenone exposure increased SOD2 immunoreactivity within surviving tyrosine hydroxylase positive neurons. The toxin leads to high super-oxide production, activating the apoptotic pathway as shown in the study with increased caspase-3 immunoreactivity (Esposti 1998, Lambert and Brand 2004, Turrens 2003). Under oxidative stress, cells generate G3BP positive cytoplasmic stress granules (Cande et al. 2004, Kedersha and Anderson 2002). This proposal was further validated by the presence of α-synuclein in tyrosine hydroxylase positive neurons 60 days following rotenone exposure (Norazit et al. 2010). α-synuclein is up-regulated in neurons subject to chronic oxidative stress, and plays a neuroprotective role and is expressed sporadically in the substantia nigra 28 days after rotenone infusion into the

administration of rotenone in a specialised vehicle of a medium-chain triglyceride, although this model developed a debilitating behavioural phenotype in a relatively short time and is not

The side effects of peripheral rotenone treatment can be reduced or avoided by the infusion of a lower dose directly into the striatum, medial forebrain bundle or substantia nigra (Ravenstijn et al. 2008, Xiong et al. 2009). Bilateral infusion of rotenone into the medial forebrain bundle causes reductions in striatal dopamine and disruptions in motor behaviours (Alam, Mayerhofer and Schmidt 2004). However, bilateral infusions lead to weight loss and require specialised diets to maintain the animals (Betarbet et al. 2000, Sherer et al. 2003c), possibly due to bilateral lesioning of the ventral tegmental area. These problems are ameliorated by unilateral lesions which reduce dopamine signalling, dopamine level, DOPAC (3,4-dihydroxyphenylacetic acid) level and dopaminergic innervation, while increasing oxidative stress (hydroxyl radicals, GSH level, superoxide dismutase levels), as well as up-regulating α-synuclein expression in the ipsilateral substantia nigra (Sindhu et al. 2006, Antkiewicz-Michaluk et al. 2004, Saravanan, Sindhu and Mohanakumar 2005, Sindhu, Saravanan and Mohanakumar 2005, Ravenstijn et al. 2008, Xiong et al. 2009). Unilateral rotenone lesioned animals have shown differences in several behavioural indices, including rotarod and amphetamine or apomorphine-induced rotation, demonstrating the unilateral functional motor deficits associated with substantia nigra dopamine neuron loss (Sindhu et al. 2006, Sindhu et al. 2005, Ravenstijn et al. 2008, Xiong et al. 2009). Behavioural indices are also influenced by the lesion of the ventral tegmental area, as can occur with larger doses of rotenone in the medial forebrain bundle (Sindhu et al.

Our laboratory has recently reported that a low dose of rotenone injected into the medial forebrain pathway in adult rats caused progressive loss of dopaminergic neurons with the remaining neurons displaying pathophysiological hallmark of human PD (Norazit et al. 2010)(Figure 2). Unlike the complete lesion of dopaminergic neurons induced by focal 6- OHDA injection, rotenone injection into the medial forebrain bundle induced the upregulation of markers of oxidative stress and markers of cell stress in dopaminergic neurons (Sindhu et al. 2005, Norazit et al. 2010). 0.5 μg of rotenone caused negligible necrosis, inflammation and a diffused glial response. A progressive loss of dopaminergic neurons in the substantia nigra and loss of striatal innervation was shown. The low dose of rotenone mediated dopaminergic cell death by oxidative stress as previously demonstrated (Rodrigues, Gomide and Chadi 2004). An increase in astrocytes and microglia occurs in human PD, where they have been ascribed both a neuroprotective and deleterious role (Imamura et al. 2003, Ishida et al. 2006, Vila et al. 2001). The study presented direct support for the hypothesis that rotenone induces a chronic state of oxidative stress in dopaminergic neurons. Rotenone exposure increased SOD2 immunoreactivity within surviving tyrosine hydroxylase positive neurons. The toxin leads to high super-oxide production, activating the apoptotic pathway as shown in the study with increased caspase-3 immunoreactivity (Esposti 1998, Lambert and Brand 2004, Turrens 2003). Under oxidative stress, cells generate G3BP positive cytoplasmic stress granules (Cande et al. 2004, Kedersha and Anderson 2002). This proposal was further validated by the presence of α-synuclein in tyrosine hydroxylase positive neurons 60 days following rotenone exposure (Norazit et al. 2010). α-synuclein is up-regulated in neurons subject to chronic oxidative stress, and plays a neuroprotective role and is expressed sporadically in the substantia nigra 28 days after rotenone infusion into the

appropriate as a slow onset chronic model (Cannon et al. 2009).

2005, Xiong et al. 2009, Thomas et al. 1994).

medial forebrain bundle (Hashimoto et al. 2002, Quilty et al. 2006). This model has now been successfully used to test the neuroprotective properties of chronic exposure to neurotrophic factors (unpublished data).

Fig. 2. Dopamine cell pathophysiology of the substantia nigra, induced by 0.5-μg rotenone delivered focally into the medial forebrain bundle and harvested after 14 days. a) The unilateral lesion is manifest as a subtle yet significant reduction in the number of dopaminergic neurons and dendritic arbour within the substantia nigra (asterisk) and a reduction in the dopaminergic innervation of the striatum (data not shown). The nigrostriatal circuitry contralateral to the lesion is uneffected by the rotenone treatment. b) Ras-GAP SH3 domain binding protein (G3BP) immunoreactivity (red, filled arrowhead) was expressed in tyrosine hydroxylase immunoreactive (green, filled arrowhead) dopaminergic neurons located in the rotenone exposed substantia nigra. c) Superoxide dismutase (SOD2) immunoreactivity (red, filled arrowhead) was also expressed in the rotenone exposed tyrosine hydroxylase immunoreactive (green, filled arrowhead) dopaminergic neurons. Nuclei for all sections are counterstained with the nuclear marker DAPI (blue) (scale bar=10 μm)

#### **3.2 Cellular based models**

Complete human disease phenotype is rarely observed in animal models introduced with human gene mutations. Thus, relevant human tissue is required to study the disease process

Oxidative Stress in Parkinson's Disease;

Whitworth et al. 2005).

**3.3.3 α -Synuclein** 

**3.3.2 PINK1** 

Parallels Between Current Animal Models, Human Studies, and Cells

knockout drosophila animal model (Clark et al. 2006, Park et al. 2006).

(Casarejos et al. 2006). In some of the mouse knockout models, subtle abnormalities of the nigrostriatal pathway or the locus coeruleus noradrenergic system have been observed (Goldberg et al. 2003, von Coelln et al. 2006). Conversely, over-expression of mutant parkin produces a progressive loss of dopaminergic neurons in the nigrostriatal pathway in both mice and drosophila (Lu et al. 2009, Sang et al. 2007, Wang et al. 2007). This suggests that some parkin mutants may act in a dominant negative fashion. The parkin animal model exhibits several movement indices in both drosophila and mice (Greene et al. 2003,

Similar to parkin knockouts, PINK1 knockouts also have mild mitochondrial defects (Gautier, Kitada and Shen 2008, Palacino et al. 2004).The PINK1 product is transcribed in the nucleus, translated in the cytoplasm, and imported intact into the mitochondria, with subsequent processing and intra mitochondrial sorting (Schapira 2008). The lack of PINK1 in transgenic mice causes enlargement of mitochondria as well as a decrease in mitochondrial numbers in dopaminergic neurons of the nigrostriatal pathway (Gautier et al. 2008, Gispert et al. 2009, Kitada et al. 2007). Although these animals do not exhibit any changes in the nigrostriatal pathway, a deficit in dopamine neurotransmission has been observed (Kitada et al. 2007). Changes in several behavioral indices have also been shown in the PINK1

It is interesting to point out that while both the knockdown of parkin and PINK1 have been linked to mitochondrial dysfunction, the expression of parkin ameliorates PINK1-related abnormalities but not vice versa (Clark et al. 2006, Park et al. 2006). This suggests that parkin and PINK1 are part of a common pathway with PINK1 functioning upstream from parkin.

α -synuclein is a protein aggregate that is the main part of Lewy bodies in human PD. The function of α-synuclein is as yet unclear, however, there appears to be a reciprocal relationship between this protein and oxidative stress (Henchcliffe and Beal 2008). αsynuclein is up-regulated in neurons subject to chronic oxidative stress and expressed sporadically in the substantia nigra (Hashimoto et al. 2002, Quilty et al. 2006, Norazit et al. 2010). The association between the presence of α-synuclein and PD has led to the development of a variety of animal models (Table 1). The over-expression of α-synuclein increased the loss in dopaminergic neurons in both drosophila and C. elegans models. (Feany and Bender 2000, Kuwahara et al. 2006, Lakso et al. 2003). However, only the dopaminergic loss in the drosophila model is progressive. A loss of dopaminergic neurons with the over-expression of α-synuclein has been demonstrated in mice; however the phenotypic outcome depends on the promoters used to drive transgene expression (Chesselet 2008). Transgenic mice presented with several functional abnormalities in the nigrostriatal system, some of which are dopamine responsive (Chesselet 2008). However, the loss of dopaminergic neurons is not progressive. α-synuclein toxicity is induced though mitochondrial dysfunction, proteasomal and lysosomal impairments, and disruption of ER-Golgi trafficking (Cooper et al. 2006, Cuervo et al. 2004, Martin et al. 2006, Tanaka et al. 2001). The link between mitochondrial dysfunction and α-synuclein aggregation suggests a

397

and potential therapies for neurodegenerative disorders such as PD. Pathological human samples are often confounded by difficult-to-control artefacts resulting from the disease process itself (Sutherland et al. 2009), biased sampling, and the necessity to process tissue in a timely manner following death (Atz et al. 2007, Marcotte, Srivastava and Quirion 2003, Preece and Cairns 2003). Together with rare foetal derived tissue, patient derived olfactory stem cells and induced pluripotent stem cells are currently used as models for PD.

#### **3.2.1 Patient derived olfactory stem cells**

Procuring relevant neural cells from patients with central nervous system disorders is difficult. Therefore, we have developed olfactory stem cells as a model for PD. Neural stem cells from adult human olfactory mucosa may be harvested and expanded to enrich for the stem cells, which are then frozen, banked, thawed, and regrown in quantity for gene and protein expression analyses and functional investigations. Assays on olfactory stem cell function have shown a reduction in gluthathione while pathway analysis has demonstrated significantly dysregulated pathways associated with mitochondrial function and oxidative stress (Matigian et al. 2010). The ease of patient derived olfactory stem cells propagation and banking allows them to be used for extended genomic, proteomic, and functional studies, including drug and biomarker discovery.

#### **3.2.2 Patient derived induced pluripotent stem cells**

Induced pluripotent stem (iPS) cells are pluripotent cells derived from differentiated cells, for example by introducing key transcription factor genes, as demonstrated in adult mouse fibroblasts (Takahashi and Yamanaka 2006). iPS cells have successfully been used to generate neurons from patients with sporadic PD (Soldner et al. 2009). Notably, despite these iPS cells being derived from LRRK2 mutation carriers, no phenotypic differences between sporadic PD iPS and control iPS cells were demonstrated (Nguyen et al. 2011). Patient-derived iPS cells have the potential to be used to identify changes in neural cell biology associated with the identified mutations.

#### **3.3 Genetic animal models**

Transgenic models have been developed as genetic factors linked to PD have been identified. Several autosomal dominant and recessive genes linked to mitochondrial dysfunction have been identified in humans as reviewed by Schapira (Schapira 2008). This created an opportunity for transgenic animals to mimic familial forms of PD (Fleming, Fernagut and Chesselet 2005).

#### **3.3.1 PARK2 (PARKIN)**

PARK 2 (Parkin) is transcribed in the mitochondria (Schapira 2008). The function of parkin remains to be elucidated; however the direct association between parkin and the mitochondria is of interest and warrants further study. In the knockout transgenic mouse model, animals had a decrease in mitochondrial respiratory chain function in the striatum and reductions in specific respiratory chain and antioxidant proteins (Palacino et al. 2004). Midbrain neuronal cultures obtained from PARK2 knockout mice had an increased sensitivity to rotenone, suggesting an effect on the mitochondrial respiratory chain (Casarejos et al. 2006). In some of the mouse knockout models, subtle abnormalities of the nigrostriatal pathway or the locus coeruleus noradrenergic system have been observed (Goldberg et al. 2003, von Coelln et al. 2006). Conversely, over-expression of mutant parkin produces a progressive loss of dopaminergic neurons in the nigrostriatal pathway in both mice and drosophila (Lu et al. 2009, Sang et al. 2007, Wang et al. 2007). This suggests that some parkin mutants may act in a dominant negative fashion. The parkin animal model exhibits several movement indices in both drosophila and mice (Greene et al. 2003, Whitworth et al. 2005).

#### **3.3.2 PINK1**

396 Oxidative Stress and Diseases

and potential therapies for neurodegenerative disorders such as PD. Pathological human samples are often confounded by difficult-to-control artefacts resulting from the disease process itself (Sutherland et al. 2009), biased sampling, and the necessity to process tissue in a timely manner following death (Atz et al. 2007, Marcotte, Srivastava and Quirion 2003, Preece and Cairns 2003). Together with rare foetal derived tissue, patient derived olfactory

Procuring relevant neural cells from patients with central nervous system disorders is difficult. Therefore, we have developed olfactory stem cells as a model for PD. Neural stem cells from adult human olfactory mucosa may be harvested and expanded to enrich for the stem cells, which are then frozen, banked, thawed, and regrown in quantity for gene and protein expression analyses and functional investigations. Assays on olfactory stem cell function have shown a reduction in gluthathione while pathway analysis has demonstrated significantly dysregulated pathways associated with mitochondrial function and oxidative stress (Matigian et al. 2010). The ease of patient derived olfactory stem cells propagation and banking allows them to be used for extended genomic, proteomic, and functional studies,

Induced pluripotent stem (iPS) cells are pluripotent cells derived from differentiated cells, for example by introducing key transcription factor genes, as demonstrated in adult mouse fibroblasts (Takahashi and Yamanaka 2006). iPS cells have successfully been used to generate neurons from patients with sporadic PD (Soldner et al. 2009). Notably, despite these iPS cells being derived from LRRK2 mutation carriers, no phenotypic differences between sporadic PD iPS and control iPS cells were demonstrated (Nguyen et al. 2011). Patient-derived iPS cells have the potential to be used to identify changes in neural cell

Transgenic models have been developed as genetic factors linked to PD have been identified. Several autosomal dominant and recessive genes linked to mitochondrial dysfunction have been identified in humans as reviewed by Schapira (Schapira 2008). This created an opportunity for transgenic animals to mimic familial forms of PD (Fleming,

PARK 2 (Parkin) is transcribed in the mitochondria (Schapira 2008). The function of parkin remains to be elucidated; however the direct association between parkin and the mitochondria is of interest and warrants further study. In the knockout transgenic mouse model, animals had a decrease in mitochondrial respiratory chain function in the striatum and reductions in specific respiratory chain and antioxidant proteins (Palacino et al. 2004). Midbrain neuronal cultures obtained from PARK2 knockout mice had an increased sensitivity to rotenone, suggesting an effect on the mitochondrial respiratory chain

stem cells and induced pluripotent stem cells are currently used as models for PD.

**3.2.1 Patient derived olfactory stem cells** 

including drug and biomarker discovery.

**3.2.2 Patient derived induced pluripotent stem cells** 

biology associated with the identified mutations.

**3.3 Genetic animal models** 

Fernagut and Chesselet 2005).

**3.3.1 PARK2 (PARKIN)** 

Similar to parkin knockouts, PINK1 knockouts also have mild mitochondrial defects (Gautier, Kitada and Shen 2008, Palacino et al. 2004).The PINK1 product is transcribed in the nucleus, translated in the cytoplasm, and imported intact into the mitochondria, with subsequent processing and intra mitochondrial sorting (Schapira 2008). The lack of PINK1 in transgenic mice causes enlargement of mitochondria as well as a decrease in mitochondrial numbers in dopaminergic neurons of the nigrostriatal pathway (Gautier et al. 2008, Gispert et al. 2009, Kitada et al. 2007). Although these animals do not exhibit any changes in the nigrostriatal pathway, a deficit in dopamine neurotransmission has been observed (Kitada et al. 2007). Changes in several behavioral indices have also been shown in the PINK1 knockout drosophila animal model (Clark et al. 2006, Park et al. 2006).

It is interesting to point out that while both the knockdown of parkin and PINK1 have been linked to mitochondrial dysfunction, the expression of parkin ameliorates PINK1-related abnormalities but not vice versa (Clark et al. 2006, Park et al. 2006). This suggests that parkin and PINK1 are part of a common pathway with PINK1 functioning upstream from parkin.

#### **3.3.3 α -Synuclein**

α -synuclein is a protein aggregate that is the main part of Lewy bodies in human PD. The function of α-synuclein is as yet unclear, however, there appears to be a reciprocal relationship between this protein and oxidative stress (Henchcliffe and Beal 2008). αsynuclein is up-regulated in neurons subject to chronic oxidative stress and expressed sporadically in the substantia nigra (Hashimoto et al. 2002, Quilty et al. 2006, Norazit et al. 2010). The association between the presence of α-synuclein and PD has led to the development of a variety of animal models (Table 1). The over-expression of α-synuclein increased the loss in dopaminergic neurons in both drosophila and C. elegans models. (Feany and Bender 2000, Kuwahara et al. 2006, Lakso et al. 2003). However, only the dopaminergic loss in the drosophila model is progressive. A loss of dopaminergic neurons with the over-expression of α-synuclein has been demonstrated in mice; however the phenotypic outcome depends on the promoters used to drive transgene expression (Chesselet 2008). Transgenic mice presented with several functional abnormalities in the nigrostriatal system, some of which are dopamine responsive (Chesselet 2008). However, the loss of dopaminergic neurons is not progressive. α-synuclein toxicity is induced though mitochondrial dysfunction, proteasomal and lysosomal impairments, and disruption of ER-Golgi trafficking (Cooper et al. 2006, Cuervo et al. 2004, Martin et al. 2006, Tanaka et al. 2001). The link between mitochondrial dysfunction and α-synuclein aggregation suggests a

Oxidative Stress in Parkinson's Disease;

**4.1.2 Desferrioxamine** 

**4.2 Plant polyphenols** 

Jankovic and Le 2003).

**4.3 Antioxidant therapy** 

**4.4 Coenzyme Q10 (CoQ10)** 

treatment.

suggested to ameliorate these effects.

Parallels Between Current Animal Models, Human Studies, and Cells

Previously, desferrioxamine was used to treat iron overload disease (Mandel et al. 2007). The use of Desferrioxamine has been reported to be neuroprotective *in vivo* and *in vitro* (Ben-Shachar et al. 1991, Jiang et al. 2006, Sangchot et al. 2002, Youdim, Stephenson and Ben Shachar 2004). Unlike hydroxyquinolines, desferrioxamine does not cross the blood brain

Polyphenols have been reported to have antioxidant properties, thus making them a candidate for antioxidant therapies (Malesev and Kuntic 2007). Green tea, cranberry, traditional chinese tea, and tumeric are sources of a variety of polyphenols (Reto et al. 2007, Ramassamy 2006, Perez, Wei and Guo 2009, Tan, Meng and Hostettmann 2000). Neuroprotective and neurorescue properties of plant polyphenols have been demonstrated *in vivo* and *in vitro* (Mandel, Maor and Youdim 2004, Mercer et al. 2005, Chen et al. 2006, Zbarsky et al. 2005, Mandel et al. 2006). Polyphenols exert their antioxidant effect via scavenging of free radicals and inhibition of the Fenton reaction (Perez et al. 2009, Pan,

The effects of oxidative stress are demonstrated in PD patients who have decreased GSH levels, increased levels of iron, neuromelanin associated redox-active iron, lipid peroxidation, protein oxidation and DNA damage in the substantia nigra (Jenner and Olanow 1998, Faucheux et al. 2003, Dexter et al. 1989). Antioxidant therapy has been

CoQ10, also known as ubiquinone is a cofactor that accepts electrons from Complex I and II of the electron transport chain in the mitochiondria (Beyer 1992, Dallner and Sindelar 2000). CoQ10 mediates its antioxidant effect via its interaction with *α*-tocopherol (Beyer 1992, Noack, Kube and Augustin 1994), inhibiting the activation of mitochondrial permeability, and acting independently of its free radical scavenging activity. Thus it blocks apoptosis (Papucci *et al.*, 2003), acting as a co-factor of mitochondrial uncoupling proteins which reduces mitochondrial-free radical generation (Echtay, Winkler and Klingenberg 2000, Echtay et al. 2002). Neuroprotection has been associated with the ability of CoQ10 to induce mitochondrial uncoupling in the substantia nigra of primates, after MPTP toxicity (Horvath et al. 2003). The use of CoQ10 as an antioxidant has been translated into phase II clinical trials (Shults et al. 2002) which showed a slowing of disease progression following 16 months of

**4.5 Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP)** 

Deprenyl and tocopherol antioxidative therapy have been clinically trialed to explore its potential as therapy for PD (Shoulson et al. 2002). Deprenyl treatment delayed the initiation of levodopa therapy. Continued deprenyl treated subjects exhibited slower motor decline

barrier, thus removing the option of oral administration (Aouad et al. 2002).

399

feed-forward loop that has the potential to initiate the progressive loss of dopaminergic neurons in the nigrostriatal system due to oxidative stress.

#### **3.3.4 LRRK2**

The LRRK2 protein functions as a serine-thereonine kinase, a known affector of mitochondrial function. A small percentage (10%) of LRRK2 is located in the outer mitochondrial membrane (West et al. 2005). Although the precise function of mitochondriallocated LRRK2 is not known, it has been suggested that it interacts with parkin. The current transgenic LRRK2 animal models are not robust enough to be used as a PD animal model due to the lack of loss of dopaminergic neurons of the nigrostriatal pathway (Li et al. 2010, Li et al. 2009, Lin et al. 2009, Wang et al. 2008, Tong et al. 2009). However, these animals do show several abnormalities in DA neurotransmission or in dopamine responsive behavior. It has been suggested that the mouse LRRK2 transgenic models do not exhibit more substantial pathology as LRRK2 mutations in humans are only partially penetrant, with further genetic and/or environmental insult required to induce the degeneration of dopaminergic neurons (Dawson, Ko and Dawson 2010).

#### **4. Experimental therapies**

#### **4.1 Metal chelation therapy**

Increased iron levels have been detected in the midbrain of PD patients suggesting that the increased levels of iron may be part of the disease pathology (Andersen 2004). Notably, iron participates as a catalyst to produce ROS.

#### **4.1.1 8-Hydroxyquinolines**

Previously the hydroxyquinoline clioquinol had been examined as a metal chelator in a clinical trial for Alzeimer's disease (Ritchie et al. 2003). The use of a variety of hydroxyquinolines to chelate iron into a form that does not catalyze ROS has shown promise in both *in vivo* and *in vitro* models (Table 2).


Table 2. Hydroxyquinolines shown to chelate iron in both *in vivo* and *in vitro* models

Hydroxyquinolines are able to cross the blood brain barrier, allowing for oral administration. Hydroxyquinolines have also shown inhibitory effect on the activity of enzyme MAO-B (Yassin et al. 2000, Youdim, Fridkin and Zheng 2005).

#### **4.1.2 Desferrioxamine**

398 Oxidative Stress and Diseases

feed-forward loop that has the potential to initiate the progressive loss of dopaminergic

The LRRK2 protein functions as a serine-thereonine kinase, a known affector of mitochondrial function. A small percentage (10%) of LRRK2 is located in the outer mitochondrial membrane (West et al. 2005). Although the precise function of mitochondriallocated LRRK2 is not known, it has been suggested that it interacts with parkin. The current transgenic LRRK2 animal models are not robust enough to be used as a PD animal model due to the lack of loss of dopaminergic neurons of the nigrostriatal pathway (Li et al. 2010, Li et al. 2009, Lin et al. 2009, Wang et al. 2008, Tong et al. 2009). However, these animals do show several abnormalities in DA neurotransmission or in dopamine responsive behavior. It has been suggested that the mouse LRRK2 transgenic models do not exhibit more substantial pathology as LRRK2 mutations in humans are only partially penetrant, with further genetic and/or environmental insult required to induce the degeneration of

Increased iron levels have been detected in the midbrain of PD patients suggesting that the increased levels of iron may be part of the disease pathology (Andersen 2004). Notably, iron

Previously the hydroxyquinoline clioquinol had been examined as a metal chelator in a clinical trial for Alzeimer's disease (Ritchie et al. 2003). The use of a variety of hydroxyquinolines to chelate iron into a form that does not catalyze ROS has shown

**Hydroxyquinoline Model Reference** 

OHDA

enzyme MAO-B (Yassin et al. 2000, Youdim, Fridkin and Zheng 2005).

Table 2. Hydroxyquinolines shown to chelate iron in both *in vivo* and *in vitro* models

*Clioquinol* MPTP mouse model (Kaur et al. 2003)

exposuded to 6-OHDA (Zheng et al. 2005)

In vitro (Gal et al. 2005, Gal et al. 2006)

(Gal et al. 2006, Zheng, Blat and Fridkin 2006)

**VK-28** 6-OHDA rat model (Tsubaki, Honma and Hoshi 1971)

**M10** PC12 cells exposed to 6-OHDA (Ritchie et al. 2003)

Hydroxyquinolines are able to cross the blood brain barrier, allowing for oral administration. Hydroxyquinolines have also shown inhibitory effect on the activity of

neurons in the nigrostriatal system due to oxidative stress.

dopaminergic neurons (Dawson, Ko and Dawson 2010).

**4. Experimental therapies 4.1 Metal chelation therapy** 

**4.1.1 8-Hydroxyquinolines** 

participates as a catalyst to produce ROS.

promise in both *in vivo* and *in vitro* models (Table 2).

**HLA20** P19 cells

**M30** MPTP mouse model

**M98 and M99** SH-SY5Y and PC12 exposed to 6-

**3.3.4 LRRK2** 

Previously, desferrioxamine was used to treat iron overload disease (Mandel et al. 2007). The use of Desferrioxamine has been reported to be neuroprotective *in vivo* and *in vitro* (Ben-Shachar et al. 1991, Jiang et al. 2006, Sangchot et al. 2002, Youdim, Stephenson and Ben Shachar 2004). Unlike hydroxyquinolines, desferrioxamine does not cross the blood brain barrier, thus removing the option of oral administration (Aouad et al. 2002).

#### **4.2 Plant polyphenols**

Polyphenols have been reported to have antioxidant properties, thus making them a candidate for antioxidant therapies (Malesev and Kuntic 2007). Green tea, cranberry, traditional chinese tea, and tumeric are sources of a variety of polyphenols (Reto et al. 2007, Ramassamy 2006, Perez, Wei and Guo 2009, Tan, Meng and Hostettmann 2000). Neuroprotective and neurorescue properties of plant polyphenols have been demonstrated *in vivo* and *in vitro* (Mandel, Maor and Youdim 2004, Mercer et al. 2005, Chen et al. 2006, Zbarsky et al. 2005, Mandel et al. 2006). Polyphenols exert their antioxidant effect via scavenging of free radicals and inhibition of the Fenton reaction (Perez et al. 2009, Pan, Jankovic and Le 2003).

#### **4.3 Antioxidant therapy**

The effects of oxidative stress are demonstrated in PD patients who have decreased GSH levels, increased levels of iron, neuromelanin associated redox-active iron, lipid peroxidation, protein oxidation and DNA damage in the substantia nigra (Jenner and Olanow 1998, Faucheux et al. 2003, Dexter et al. 1989). Antioxidant therapy has been suggested to ameliorate these effects.

#### **4.4 Coenzyme Q10 (CoQ10)**

CoQ10, also known as ubiquinone is a cofactor that accepts electrons from Complex I and II of the electron transport chain in the mitochiondria (Beyer 1992, Dallner and Sindelar 2000). CoQ10 mediates its antioxidant effect via its interaction with *α*-tocopherol (Beyer 1992, Noack, Kube and Augustin 1994), inhibiting the activation of mitochondrial permeability, and acting independently of its free radical scavenging activity. Thus it blocks apoptosis (Papucci *et al.*, 2003), acting as a co-factor of mitochondrial uncoupling proteins which reduces mitochondrial-free radical generation (Echtay, Winkler and Klingenberg 2000, Echtay et al. 2002). Neuroprotection has been associated with the ability of CoQ10 to induce mitochondrial uncoupling in the substantia nigra of primates, after MPTP toxicity (Horvath et al. 2003). The use of CoQ10 as an antioxidant has been translated into phase II clinical trials (Shults et al. 2002) which showed a slowing of disease progression following 16 months of treatment.

#### **4.5 Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP)**

Deprenyl and tocopherol antioxidative therapy have been clinically trialed to explore its potential as therapy for PD (Shoulson et al. 2002). Deprenyl treatment delayed the initiation of levodopa therapy. Continued deprenyl treated subjects exhibited slower motor decline

Oxidative Stress in Parkinson's Disease;

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and lower likelihood of developing freezing of gait. However, this treatment increased the likelihood of developing dyskinesia.

#### **5. Conclusion**

There is increasing evidence that many factors including age, environmental toxins, genetic determinants, and lifestyle factors influence the risk for PD. Many of these impact on oxidative stress related pathways. Animal and cellular models have been developed to mimic the disease pathology in humans. Currently, antioxidant therapies are being investigated in both animals and human clinical trials, with promising outcomes. Notably, antioxidant therapies appear to delay the onset of disease and disease progression, although they do not prevent or reverse disease progression.

#### **6. Acknowledgment**

The authors would like to thank Suzita Mohd Noor, Maria Nguyen, Charlotte Dickson, and Brenton Cavanagh for their technical and editing support.

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**18** 

 *México*

**The Relationship Between Thyroid States,** 

The thyroid hormones play an important role in many physiological processes, such as differentiation, growth, development, and the physiology of all cells. One of the most studied effects of the thyroid hormone is the control of the basal metabolic rate. Modifications in its levels can produce several alterations including modifications in the ROS steady-state and the REDOX environment in the cells. There is much evidence that show both hyperthyroidism and hypothyroidism are related to oxidative stress and cellular damage. For hypothyroidism, there are other findings that point to its protective effects. In this chapter we show both findings and propose that hypothyroidism is a protective state

Thyroid hormones (THs) T4 (thyroxine or 3´,5´,3,5-L-tetra-iodothyronine) and T3 (3´,3,5 triiodothyronine) are synthesized in the thyroid gland located in the anterior part of the trachea, just below the larynx. It consists of two lobes joined in the middle by a narrow portion of the gland. The major thyroid-secretor cells, known as follicular cells, are arranged into hollow spheres, each of which it forms a functional unit called a follicle. On a microscopic section, rings of follicular cells enclosing an inner lumen filled with colloid form

The principal constituent of the colloid is a large protein molecule, thyroglobulin, where thyroid hormones are incorporated in their various stages of synthesis. The follicular cells produce the two iodine-containing hormones derived from the amino acid tyrosine; T4 and T3, the thyroid hormones. The mechanism involved in thyroid hormone syntheses and their release from thyrolobulin are shown in figure 2. Iodine, an essential element of the thyroid

at the basolateral membrane of the thyrocyte and it diffuses by an exchanger, known as pendrin (PDS, encoded by the *SLC26A4* gene) to the lumen at the apical membrane. At the extracellular apical membrane, thyroperoxidase (TPO, EC 1.11.1.8) with hydrogen peroxide (H2O2), generated by dual oxidase 2 (DUOX2, EC 1.6.3.1), oxidizes and binds iodine

**1. Introduction** 

against toxic agents.

**2. Thyroid hormones** 

the follicles (figure 1).

molecule, is actively transported by the Na+-**I-**

**Oxidative Stress and Cellular Damage**

Cano-Europa, Blas-Valdivia Vanessa,

 *Biológicas del Instituto Politécnico Nacional,* 

symporter (NIS, encoded by the *SLC5A5* gene)

*Escuela Nacional de Ciencias* 

Franco-Colin Margarita and Ortiz-Butron Rocio


### **The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage**

Cano-Europa, Blas-Valdivia Vanessa, Franco-Colin Margarita and Ortiz-Butron Rocio *Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional, México*

#### **1. Introduction**

412 Oxidative Stress and Diseases

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homologous to C. elegans CED-4, participates in cytochrome c-dependent

The thyroid hormones play an important role in many physiological processes, such as differentiation, growth, development, and the physiology of all cells. One of the most studied effects of the thyroid hormone is the control of the basal metabolic rate. Modifications in its levels can produce several alterations including modifications in the ROS steady-state and the REDOX environment in the cells. There is much evidence that show both hyperthyroidism and hypothyroidism are related to oxidative stress and cellular damage. For hypothyroidism, there are other findings that point to its protective effects. In this chapter we show both findings and propose that hypothyroidism is a protective state against toxic agents.

#### **2. Thyroid hormones**

Thyroid hormones (THs) T4 (thyroxine or 3´,5´,3,5-L-tetra-iodothyronine) and T3 (3´,3,5 triiodothyronine) are synthesized in the thyroid gland located in the anterior part of the trachea, just below the larynx. It consists of two lobes joined in the middle by a narrow portion of the gland. The major thyroid-secretor cells, known as follicular cells, are arranged into hollow spheres, each of which it forms a functional unit called a follicle. On a microscopic section, rings of follicular cells enclosing an inner lumen filled with colloid form the follicles (figure 1).

The principal constituent of the colloid is a large protein molecule, thyroglobulin, where thyroid hormones are incorporated in their various stages of synthesis. The follicular cells produce the two iodine-containing hormones derived from the amino acid tyrosine; T4 and T3, the thyroid hormones. The mechanism involved in thyroid hormone syntheses and their release from thyrolobulin are shown in figure 2. Iodine, an essential element of the thyroid molecule, is actively transported by the Na+-**I** symporter (NIS, encoded by the *SLC5A5* gene) at the basolateral membrane of the thyrocyte and it diffuses by an exchanger, known as pendrin (PDS, encoded by the *SLC26A4* gene) to the lumen at the apical membrane. At the extracellular apical membrane, thyroperoxidase (TPO, EC 1.11.1.8) with hydrogen peroxide (H2O2), generated by dual oxidase 2 (DUOX2, EC 1.6.3.1), oxidizes and binds iodine

The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 415

The role played by the deiodinases is physiologically relevant. They have a role in various aspects of mammalian physiology, such as the maintenance of plasma T3 concentration (Bianco et al., 2002), TSH and TRH feedback regulation (Christoffolete et al., 2006; Larsen,

D2 Brain, pituitary gland, BAT, thyroid, muscle D3 Developing tissues and placenta, adult skin, brain

Because the thyroid hormones have a crucial role in the function of every tissue in the body, their levels must be maintained relatively constant around an optimum level. This homeostatic-control mechanism primarily operates on the principle of negative feedback (figure 3). In this homeostatic-control mechanism, the hypothalamic thyrotropin-releasing hormone (TRH), the thyroid-stimulating hormone (TSH), and thyroid hormone all together form the hypothalamus-pituitary-thyroid axis. Thus, TRH in trophic fashion turns on the TSH secretion by the anterior pituitary, whereas thyroid hormone, in negative feedback fashion, turns off the TSH secretion. In the hypothalamus-pituitary-thyroid axis, inhibition is exerted primarily at the level of the anterior pituitary. As with other negative feedback loops, the one between thyroid hormone and TSH tends to maintain a stable thyroid

Fig. 3. Hypothalamus-pituitary-thyroid axes. In this image is shown the negative feedback

1982), and the clearance of sulfated iodothyronines (Schneider et al., 2006).

Table 1. Tissue distribution of deiodinases in humans (St.Germain et al., 2009).

D1 Liver, thyroid, kidney

Deiodinase Tissue

**2.1 Thyroid hormone regulation levels** 

hormone output (Hulbert, 2000).

exerted by a high T3 concentration.

Fig. 1. Histological thyroid and the thyroid hormone structure. There are thyroid follicles with different activity. A: Thyroid follicle resting. B: Follicle with high activity.

covalently to tyrosyl residues, producing monoiodotyrosine (MIT) and diiodotyrosine (DIT) within the thyrogloblin macromolecule. The enzyme thyroperoxidase catalyzes the coupling of two iodotyrosine residues to produce the prohormone T4 and smaller amounts of the active hormone T3. After endocytosis, iodinated thyroglobulin is hydrolyzed in the lysosomes by cathepsins and the thyroid hormone is released from the thyroglobulin backbone. The released MIT and DIT are deiodinated by a specific iodotyrosine deiodinase (IYD, or DEHAL1, EC 1.22.1.1), and the released iodine is recycled within the cell. The mechanism involved in the last step in the process, the thyroid hormone secretion, remains unknown (Di Cosmo et al., 2010). About 90% of the secretory product released from the thyroid gland is in the form of T4 though T3 is about four times more potent in its biologic activity. Most of the secreted T4 is converted into T3 by a group of enzymes known as iodothyronine deiodinases (D1 and D2, EC 1.97.1.10), which also include an inactivating deiodinase, the type 3 deiodinase (D3), that inactivates both T4 and T3 (Bianco et al., 2002)(table 1).

Fig. 2. Thyroid hormone syntheses in a follicular thyroid cell. Based on the model proposed by Di Cosmo (Di Cosmo et al., 2010)

The role played by the deiodinases is physiologically relevant. They have a role in various aspects of mammalian physiology, such as the maintenance of plasma T3 concentration (Bianco et al., 2002), TSH and TRH feedback regulation (Christoffolete et al., 2006; Larsen, 1982), and the clearance of sulfated iodothyronines (Schneider et al., 2006).


Table 1. Tissue distribution of deiodinases in humans (St.Germain et al., 2009).

#### **2.1 Thyroid hormone regulation levels**

414 Oxidative Stress and Diseases

Fig. 1. Histological thyroid and the thyroid hormone structure. There are thyroid follicles

covalently to tyrosyl residues, producing monoiodotyrosine (MIT) and diiodotyrosine (DIT) within the thyrogloblin macromolecule. The enzyme thyroperoxidase catalyzes the coupling of two iodotyrosine residues to produce the prohormone T4 and smaller amounts of the active hormone T3. After endocytosis, iodinated thyroglobulin is hydrolyzed in the lysosomes by cathepsins and the thyroid hormone is released from the thyroglobulin backbone. The released MIT and DIT are deiodinated by a specific iodotyrosine deiodinase (IYD, or DEHAL1, EC 1.22.1.1), and the released iodine is recycled within the cell. The mechanism involved in the last step in the process, the thyroid hormone secretion, remains unknown (Di Cosmo et al., 2010). About 90% of the secretory product released from the thyroid gland is in the form of T4 though T3 is about four times more potent in its biologic activity. Most of the secreted T4 is converted into T3 by a group of enzymes known as iodothyronine deiodinases (D1 and D2, EC 1.97.1.10), which also include an inactivating deiodinase, the type 3 deiodinase (D3), that

Fig. 2. Thyroid hormone syntheses in a follicular thyroid cell. Based on the model proposed

with different activity. A: Thyroid follicle resting. B: Follicle with high activity.

inactivates both T4 and T3 (Bianco et al., 2002)(table 1).

by Di Cosmo (Di Cosmo et al., 2010)

Because the thyroid hormones have a crucial role in the function of every tissue in the body, their levels must be maintained relatively constant around an optimum level. This homeostatic-control mechanism primarily operates on the principle of negative feedback (figure 3). In this homeostatic-control mechanism, the hypothalamic thyrotropin-releasing hormone (TRH), the thyroid-stimulating hormone (TSH), and thyroid hormone all together form the hypothalamus-pituitary-thyroid axis. Thus, TRH in trophic fashion turns on the TSH secretion by the anterior pituitary, whereas thyroid hormone, in negative feedback fashion, turns off the TSH secretion. In the hypothalamus-pituitary-thyroid axis, inhibition is exerted primarily at the level of the anterior pituitary. As with other negative feedback loops, the one between thyroid hormone and TSH tends to maintain a stable thyroid hormone output (Hulbert, 2000).

Fig. 3. Hypothalamus-pituitary-thyroid axes. In this image is shown the negative feedback exerted by a high T3 concentration.

The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 417

Fig. 4. Nuclear gene expressions by T3 to the thyroid hormone receptors TR and TR. Heterodimers must bind to specific thyroid response elements (TREs) in the promoters of

The existence of cell-surface receptors for thyroid hormones has been acknowledged. The presence of binding sites for thyroid hormones on the cell surface has been known for many years in the red blood-cell membrane (Davis et al., 1983) and in the synaptosome (Giguere et al., 1996; Giguere et al., 1992). The identity of the proteins involved in membrane binding of hormones was not established in these studies and there has been a reluctance to believe that integrin av3, containing a binding site for the thyroid hormones, is an initiation site for complex hormone-directed cellular events, such as cell division and angiogenesis (Davis et

Integrins are ubiquitous heterodimeric structural proteins of the cell membrane that convey signals from the cell interior to the extracellular matrix (ECM) (inside-out) and from the ECM to the cell (outside-in). The integrin purified from the plasma membrane bound radiolabeled thyroid hormones and with high affinity. This integrin contains a binding site for thyroid hormones caused by the functional consequences of the binding activation of MAPK (figure 5). The receptor has been located at the Arg-Gly-Asp (RGD) recognition site on the integrin that is important to the binding of a number of extracellular-matrix proteins and growth factors. From this site, the thyroid hormone signals are transduced by MAPK (ERK1-2) in angiogenesis in endothelial cells and the cell proliferation of tumor cell lines. The T4 in concentrations that are physiological (10-10 M free T4) and T3 in supraphysiological concentrations cause ERK-dependent cell proliferation. It is now clear that the hormone receptor domain on the integrin is more complex than initially thought. There is a T3-specific

T3-target genes and activate or repress transcription in response to hormone.

al., 2005).

#### **2.2 Mechanism of action of the thyroid hormones**

Because thyroid hormones have been considered as lipophilic in their intracellular action, passive hormone diffusion through the lipid bilayer has been accepted. Because of the existence of nuclear receptors for thyroid hormones, it has long been believed that the THs caused effects only via genomic effects, however since the late 1980s it has been proposed that the THs may cause effects independently of genetic mechanisms, affecting membrane lipid composition or activation of enzymes (Lazar, 1993). Receptors for thyroid hormones (TRs) are proteins that act as transcription factors that belong to the superfamily of nuclear receptors, which includes steroids, vitamin D, retinoic acid, fatty acids, prostaglandins, and orphan receptors (Zhang & Lazar, 2000). The TRs have regions; the DNA binding domain (DBD), ligand binding domain (LBD), hinge region (HR), and amino terminal domain (A-B) (Sap et al., 1986). The THs cross the lipid membrane because of their hydrophobic nature. In the cytoplasm they can bind to newly synthesized TRs, however most THs bind to nuclear receptors. The TRs bound to the THs may regulate the transcription process by modifying the structure of chromatin, allowing other factors to exert their action on elements of the TH responses (figure 4). In addition, the THs interact, directly or indirectly, through bridge or coactivator molecules, with the transcriptional machinery of the process. A critical aspect in regulating the transcription process by the TRs is the conformational change that T3 exerts on the receiver itself. The T3 decreases the ability of the hydrophobic TRs and can modify the way in which the TRs, either as dimer or heterodimer, bind to DNA. The TRs bind to regulate transcription as a monomer, homodimer, heterodimer, or heteromultimer. The thyroid hormones binding to the TRs cause changes in the structure of these complexes thus modulating the interaction with other elements of the transcriptional apparatus to determine the type of response, enhancing or inhibiting (Cheng et al., 2010; Yen, 2001).

Because thyroid hormones have been considered as lipophilic in their intracellular action, passive hormone diffusion through the lipid bilayer has been accepted. Because of the existence of nuclear receptors for thyroid hormones, it has long been believed that the THs caused effects only via genomic effects, however since the late 1980s it has been proposed that the THs may cause effects independently of genetic mechanisms, affecting membrane lipid composition or activation of enzymes (Lazar, 1993). Receptors for thyroid hormones (TRs) are proteins that act as transcription factors that belong to the superfamily of nuclear receptors, which includes steroids, vitamin D, retinoic acid, fatty acids, prostaglandins, and orphan receptors (Zhang & Lazar, 2000). The TRs have regions; the DNA binding domain (DBD), ligand binding domain (LBD), hinge region (HR), and amino terminal domain (A-B) (Sap et al., 1986). The THs cross the lipid membrane because of their hydrophobic nature. In the cytoplasm they can bind to newly synthesized TRs, however most THs bind to nuclear receptors. The TRs bound to the THs may regulate the transcription process by modifying the structure of chromatin, allowing other factors to exert their action on elements of the TH responses (figure 4). In addition, the THs interact, directly or indirectly, through bridge or coactivator molecules, with the transcriptional machinery of the process. A critical aspect in regulating the transcription process by the TRs is the conformational change that T3 exerts on the receiver itself. The T3 decreases the ability of the hydrophobic TRs and can modify the way in which the TRs, either as dimer or heterodimer, bind to DNA. The TRs bind to regulate transcription as a monomer, homodimer, heterodimer, or heteromultimer. The thyroid hormones binding to the TRs cause changes in the structure of these complexes thus modulating the interaction with other elements of the transcriptional apparatus to determine the type of response, enhancing or inhibiting (Cheng et al., 2010; Yen, 2001).

**2.2 Mechanism of action of the thyroid hormones** 

Fig. 4. Nuclear gene expressions by T3 to the thyroid hormone receptors TR and TR. Heterodimers must bind to specific thyroid response elements (TREs) in the promoters of T3-target genes and activate or repress transcription in response to hormone.

The existence of cell-surface receptors for thyroid hormones has been acknowledged. The presence of binding sites for thyroid hormones on the cell surface has been known for many years in the red blood-cell membrane (Davis et al., 1983) and in the synaptosome (Giguere et al., 1996; Giguere et al., 1992). The identity of the proteins involved in membrane binding of hormones was not established in these studies and there has been a reluctance to believe that integrin av3, containing a binding site for the thyroid hormones, is an initiation site for complex hormone-directed cellular events, such as cell division and angiogenesis (Davis et al., 2005).

Integrins are ubiquitous heterodimeric structural proteins of the cell membrane that convey signals from the cell interior to the extracellular matrix (ECM) (inside-out) and from the ECM to the cell (outside-in). The integrin purified from the plasma membrane bound radiolabeled thyroid hormones and with high affinity. This integrin contains a binding site for thyroid hormones caused by the functional consequences of the binding activation of MAPK (figure 5). The receptor has been located at the Arg-Gly-Asp (RGD) recognition site on the integrin that is important to the binding of a number of extracellular-matrix proteins and growth factors. From this site, the thyroid hormone signals are transduced by MAPK (ERK1-2) in angiogenesis in endothelial cells and the cell proliferation of tumor cell lines. The T4 in concentrations that are physiological (10-10 M free T4) and T3 in supraphysiological concentrations cause ERK-dependent cell proliferation. It is now clear that the hormone receptor domain on the integrin is more complex than initially thought. There is a T3-specific

The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 419

It has been proposed that the thyroid hormones influence the ROS steady-state and REDOX environment in the cell. The most common idea is that hyperthyroidism enhances the ROS production that perturbs the ROS steady-state and changes the REDOX environment to facilitate cell damage. A hypometabolic state caused by hypothyroidism could be a

Because thyroid hormones modulate many functions, if thyroid hormone levels change, many cellular processes could be altered, including modifications in the REDOX environment. Related to this issue we can ask what happens in a hyperthyroid condition?. Because one of the most studied effects of the thyroid hormone is the control of the basal metabolic rate, a hypermetabolic state produces a modification of the REDOX environment (Venditti & Di Meo, 2006). It is well-known that a higher T3 level, a hypermetabolic state, causes calorigenesis in two ways. The first is a short-term signaling mechanism with the allosteric activation of cytochrome-C oxidase and the second is a long-term pathway producing nuclear and mitochondrial gene transcription through T3 signaling, thus stimulating basal thermogenesis (Oppenheimer et al., 1994). This last mechanism causes the synthesis of the enzymes involved in energy metabolism and the components of the respiratory-chain apparatus, leading to a higher capacity of oxidative phosphorylation (Videla, 2000; Soboll, 1993). These short- and long-term pathways are mainly responsible for the increased cellular respiration caused by the hyperthyroid state. Other processes may also play a role, namely 1) energy expenditure caused by a higher active cation transport, 2) loss of energy from futile cycles caused by increases in catabolic and anabolic pathways of intermediary metabolism, 3) higher activity of membrane-bound enzymes associated with electron transfer and metabolite carriers caused by changes in the lipid composition of mitochondrial membranes (Soboll, 1993), and 4) O2 equivalents related to oxidative stress (Videla, 2000), a REDOX imbalance that leads to various pathological events in several organs as the liver (Jaeschke et al., 2002). In these pathologies, the cellular damage occurs when the balance between oxidant and antioxidants is disturbed and the antioxidant system does not balance the oxidants, thus altering the ROS steady-state level (Lushchak, 2011). An enhanced ROS causes lipid peroxidation, enhancement of reactive oxygen species, nitration,

**3. Relationship between alterations of the thyroid hormones and the ROS-**

protective state. In the next part of this chapter we show the evidence for this.

carbonylation, or glutathionylation of proteins, and fragmentation of DNA.

Fernandez et al. found that thyroid calorigenesis is a hormonal stimulus for the REDOX activation of NFB, a response that is triggered in Kupffer cells having higher respiratoryburst activities (Fernandez et al., 2006). These findings are in agreement with studies showing that the NFB activation can be achieved by physiological levels of the ROS, which are produced during the respiratory burst after stimulation of isolated or cultured macrophages (Kaul & Forman, 1996) and in carbon-stimulated Kupffer cells in the isolated, perfused rat liver (Romanque et al., 2003). This may be caused by the damage produced by the oxidative stress generated by an excess of thyroid hormones. There are data indicating that excess thyroid hormones act at multiple levels to cause apoptosis, because this higher level enhances the expression of several death receptors and their ligands, such as TNF-,

**3.1 Hyperthyroidism and the ROS-steady state** 

**steady state** 

Fig. 5. Nongenomic mechanisms of action of thyroid hormones mediated by integrine-type membranal receptors.

site in the domain and a site at which both T4 and T3 may act. The T3-specific site activates PI3K and is linked not to cell proliferation, but to trafficking of certain intracellular proteins such as shuttling of TRs from the cytoplasm to nucleus and to the transcription of specific genes, such as hypoxia-inducible factor-1 (HIF-1). T4 is unable to activate PI3K (Cheng et al., 2010).

#### **2.3 Thyroid hormone actions**

The T3 is recognized as a key metabolic hormone of the body. It has many physiological actions and it modulates all metabolic pathways through alterations in oxygen consumption and changes in protein, lipid, carbohydrate, and vitamin metabolism. Through its direct manipulation of protein expression associated with such pathways, T3 affects the synthesis and degradation of many other hormones and growth factors and indirectly influences additional endocrine signaling. To see more details of the thyroid hormone action, check the review of Hulbert (Hulbert, 2000).

#### **2.4 Abnormalities of the thyroid function**

Abnormalities of the thyroid function are among the most common of all endocrine disorders. They fall into two categories, hypothyroidism and hyperthyroidism, reflecting deficient and excess thyroid hormone secretion. There are many causes that generate these conditions. Whatever the cause, the consequences of too little or too much thyroid hormone secretion are largely predictable, given the knowledge of the functions of the thyroid hormones.

#### **3. Relationship between alterations of the thyroid hormones and the ROSsteady state**

It has been proposed that the thyroid hormones influence the ROS steady-state and REDOX environment in the cell. The most common idea is that hyperthyroidism enhances the ROS production that perturbs the ROS steady-state and changes the REDOX environment to facilitate cell damage. A hypometabolic state caused by hypothyroidism could be a protective state. In the next part of this chapter we show the evidence for this.

#### **3.1 Hyperthyroidism and the ROS-steady state**

418 Oxidative Stress and Diseases

Fig. 5. Nongenomic mechanisms of action of thyroid hormones mediated by integrine-type

site in the domain and a site at which both T4 and T3 may act. The T3-specific site activates PI3K and is linked not to cell proliferation, but to trafficking of certain intracellular proteins such as shuttling of TRs from the cytoplasm to nucleus and to the transcription of specific genes, such as hypoxia-inducible factor-1 (HIF-1). T4 is unable to activate PI3K (Cheng et al.,

The T3 is recognized as a key metabolic hormone of the body. It has many physiological actions and it modulates all metabolic pathways through alterations in oxygen consumption and changes in protein, lipid, carbohydrate, and vitamin metabolism. Through its direct manipulation of protein expression associated with such pathways, T3 affects the synthesis and degradation of many other hormones and growth factors and indirectly influences additional endocrine signaling. To see more details of the thyroid hormone action, check the

Abnormalities of the thyroid function are among the most common of all endocrine disorders. They fall into two categories, hypothyroidism and hyperthyroidism, reflecting deficient and excess thyroid hormone secretion. There are many causes that generate these conditions. Whatever the cause, the consequences of too little or too much thyroid hormone secretion are largely predictable, given the knowledge of the functions of the thyroid

membranal receptors.

**2.3 Thyroid hormone actions** 

review of Hulbert (Hulbert, 2000).

**2.4 Abnormalities of the thyroid function** 

2010).

hormones.

Because thyroid hormones modulate many functions, if thyroid hormone levels change, many cellular processes could be altered, including modifications in the REDOX environment. Related to this issue we can ask what happens in a hyperthyroid condition?. Because one of the most studied effects of the thyroid hormone is the control of the basal metabolic rate, a hypermetabolic state produces a modification of the REDOX environment (Venditti & Di Meo, 2006). It is well-known that a higher T3 level, a hypermetabolic state, causes calorigenesis in two ways. The first is a short-term signaling mechanism with the allosteric activation of cytochrome-C oxidase and the second is a long-term pathway producing nuclear and mitochondrial gene transcription through T3 signaling, thus stimulating basal thermogenesis (Oppenheimer et al., 1994). This last mechanism causes the synthesis of the enzymes involved in energy metabolism and the components of the respiratory-chain apparatus, leading to a higher capacity of oxidative phosphorylation (Videla, 2000; Soboll, 1993). These short- and long-term pathways are mainly responsible for the increased cellular respiration caused by the hyperthyroid state. Other processes may also play a role, namely 1) energy expenditure caused by a higher active cation transport, 2) loss of energy from futile cycles caused by increases in catabolic and anabolic pathways of intermediary metabolism, 3) higher activity of membrane-bound enzymes associated with electron transfer and metabolite carriers caused by changes in the lipid composition of mitochondrial membranes (Soboll, 1993), and 4) O2 equivalents related to oxidative stress (Videla, 2000), a REDOX imbalance that leads to various pathological events in several organs as the liver (Jaeschke et al., 2002). In these pathologies, the cellular damage occurs when the balance between oxidant and antioxidants is disturbed and the antioxidant system does not balance the oxidants, thus altering the ROS steady-state level (Lushchak, 2011). An enhanced ROS causes lipid peroxidation, enhancement of reactive oxygen species, nitration, carbonylation, or glutathionylation of proteins, and fragmentation of DNA.

Fernandez et al. found that thyroid calorigenesis is a hormonal stimulus for the REDOX activation of NFB, a response that is triggered in Kupffer cells having higher respiratoryburst activities (Fernandez et al., 2006). These findings are in agreement with studies showing that the NFB activation can be achieved by physiological levels of the ROS, which are produced during the respiratory burst after stimulation of isolated or cultured macrophages (Kaul & Forman, 1996) and in carbon-stimulated Kupffer cells in the isolated, perfused rat liver (Romanque et al., 2003). This may be caused by the damage produced by the oxidative stress generated by an excess of thyroid hormones. There are data indicating that excess thyroid hormones act at multiple levels to cause apoptosis, because this higher level enhances the expression of several death receptors and their ligands, such as TNF-,

The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 421

elevated G6PD activity indicates that severe hyperthyroidism may compromise the cellular ability to maintain the redox state. Lombardi et al. have demonstrated that injection of T3 into hypothyroid rats caused an increase in both enzyme activity and mRNA expression of G6PD in the liver. Nevertheless, the reduced activities of GPx, GR, and GST in the hyperthyroid liver prevent optimum GSH use and recycling. Accumulation of GSSG can lead to protein modifications because of interactions with –SH groups (Reed, 1990). Though the T3 exerted a positive stimulatory effect on the NADPH supply, it was not sufficient to compensate for the massive GSH depletion and this probably explains the negative regulatory impact of T3 on activities of GSH-dependent enzymes such as GPx and GR in the rat liver. Under such conditions, the cellular redox-status is disturbed, as reflected in the

In brief, thyroid calorigenesis resulting from acceleration of energy metabolism and secondary electron-transfer processes lead to a higher generation of ROS in the target tissue. This prooxidant condition enhances the oxidative-stress status of the organs when the

 a) substantial oxidative deterioration of biomolecules, with loss of their functions that may compromise cell viability, b) activities of GPx, GR, GST, catalase, and superoxide dismutase are considerably impaired, c) total GSH equivalents and GSH and GSSG pools were increasingly depleted, d) a higher susceptibility of the liver to toxic stimuli that exacerbate liver injury, e) upregulation of gene expression, f) apoptosis, g) shorter lifespan, and h)

Hypothyroidism has been related to some diseases because it causes a hypometabolic state. This condition can be beneficial. Why can we make this assertion? There are many findings to support this suggestion. It is well-known that a deficiency of the thyroid hormones results in decreased metabolism and lowering of the basal metabolic rate (BMR). There is evidence that supports the lower cell stress in the hypothyroidism condition. Tenorio-Velázquez et al. have demonstrated that hypothyroidism attenuates oxidative stress and renal injury caused by ischemia-reperfusion, produced by an increase in the ROS and reactive nitrogen species (Tenorio-Velásquez et al., 2005). Most research has been done in the kidney and liver models of ischemia (Swaroop & Ramasarma, 1985; Paller, 1986). The postulated mechanism in such organs has been either a decrease in the general metabolic rate or a reduced free radical scavenging response after ischemia. The lipid peroxidation in hypothyroid animals with renal ischemia was decreased (Paller, 1986). The content of malondialdehyde, which is an indirect measure of the generation of oxygen free radicals, was decreased and the cortical content of glutathione, a free radical scavenger, was increased in the hypothyroid, ischemic animals. Similarly, in the liver model of hypothyroid-ischemic injury, lipid peroxidation and free-radical generation were decreased in the hypothyroid animals (Swaroop & Ramasarma, 1985). These investigators have shown a significant decrease in hydrogen peroxide, a measure of the oxygen free-radical status, in the liver mitochondrion in the hypothyroid animals. Hypothyroidism attenuates not only renal but also cardiac damage caused by ischemia and reperfusion. Bobadilla et al. have shown that hypothyroidism conferred protection against reperfusion arrhythmias and the cardiac release of creatine kinase and

decrease in the antioxidant potential is not adequately compensated for, leading to

high oxidative-stress index of hyperthyroid rats.

**3.2 Hypothyrodism and the ROS-steady state** 

myelin deficit.

FasL, proNGF, and proBDNF, resulting in activation of apical caspase-8, which is further amplified through the activation of the p75NRT-mediated pathways (Kumar et al., 2007). Hyperthyroid animals appear to have a shorter lifespan and, at an advanced age, have a myelin deficit (Carageorgiou et al., 2005). It is known that hyperthyroidism increases hepatic protein oxidation, as evidenced by a significant 88% increase in the content of protein hydrazone derivatives 3 days after a T3 treatment. This effect may be caused by the increased generation of ROS generated by T3 (Fernández et al., 1985; Fernandez & Videla, 1993) leading to the formation of carbonyl derivatives mainly occurring at the arginyl, prolyl, lysyl, and histidyl residues in proteins (Stadtman, 1990; Reznick & Packer, 1994). The T3-caused ROS formation can cause the conversion of cysteinyl residues to protein-protein disulfide conjugates or to mixed-disulfide derivatives (Stadtman, 1990), whereas T3-caused NO generation (Fernandez et al., 1997) may lead to protein oxidation or nitration through peroxynitrite formation (Alvarez & Radi, 2001). The biological significance of the oxidative modification of proteins in T3-caused liver oxidative stress can be visualized on two levels; 1) loss of protein function and 2) increased protein degradation. Under high rates of ROS and RNS input, the oxidative modification of enzymes can occur with the consequent reduction in enzyme activity (Stadtman, 1990; Lissi et al., 1991). The inactivation of hepatic antioxidant enzymes has been described in several conditions in vivo involving oxidative stress in the tissue, including hyperthyroidism (Lissi et al., 1991), which determines a decrease in the activity of superoxide dismutase and catalase (Fernandez et al., 1988) and in the content of cytochrome P450 (Fernández et al., 1985). In agreement with this contention, inactivation of superoxide dismutase by H2O2 (Bray et al., 1974) and of catalase by O2**.**- (Kono & Fridovich, 1982) has been reported in conditions in vitro. In addition to enzyme inactivation, thyrotoxicosis in mammals results in the stimulation of both synthesis and degradation of protein, with a predominance of degradation, as shown by the increase in protein catabolism, negative nitrogen balance, and the loss of protein from muscle and other body stores (Loeb, 1996). These findings are in accordance with those of Tapia et al. who found a higher oxidation of the liver protein and an increase in lipid peroxidation levels in hyperthyroid rats (Tapia et al., 2010).

Interestingly, the activities of both GPx-1 and GR are decreased in hyperthyroid rats. It is remarkable that both enzymatic activities are strongly GSH-dependent. The GPx-1 catalyzes the reduction of H2O2 and lipid hydroperoxides coupled with oxidation of GSH into GSSG whereas the GR replenishes the GSH pool with the help of NADPH principally provided by the pentose-phosphate pathway. The intracellular GSH status appears to be a sensitive indicator of the cellular ability to resist ROS. Furthermore, it has been found that total GSH equivalents and the GSH and GSSG pools were increasingly depleted by T3 over time (Chattopadhyay et al., 2007). The liver is especially rich in GST that metabolizes xenobiotics by conjugating with GSH. In fact, the GST-catalyzed conjugation of GSH with exogenous compounds and endogenous metabolites such as 4-hydroxynonenal is regarded as a major cellular-defense mechanism against toxicity (Cheng et al., 2001). The activities of GST were considerably impaired with the progression of the T3 treatment (Chattopadhyay et al., 2007). Because recycling of oxidized glutathione consumes NADPH, the cellular levels of NADPH and its synthesis represent the rate limiting factors of H2O2 consumption by catalasedeficient tissues (Ho et al., 2004). Moreover, prolonged hyperthyroidism diminishes GR but

FasL, proNGF, and proBDNF, resulting in activation of apical caspase-8, which is further amplified through the activation of the p75NRT-mediated pathways (Kumar et al., 2007). Hyperthyroid animals appear to have a shorter lifespan and, at an advanced age, have a myelin deficit (Carageorgiou et al., 2005). It is known that hyperthyroidism increases hepatic protein oxidation, as evidenced by a significant 88% increase in the content of protein hydrazone derivatives 3 days after a T3 treatment. This effect may be caused by the increased generation of ROS generated by T3 (Fernández et al., 1985; Fernandez & Videla, 1993) leading to the formation of carbonyl derivatives mainly occurring at the arginyl, prolyl, lysyl, and histidyl residues in proteins (Stadtman, 1990; Reznick & Packer, 1994). The T3-caused ROS formation can cause the conversion of cysteinyl residues to protein-protein disulfide conjugates or to mixed-disulfide derivatives (Stadtman, 1990), whereas T3-caused NO generation (Fernandez et al., 1997) may lead to protein oxidation or nitration through peroxynitrite formation (Alvarez & Radi, 2001). The biological significance of the oxidative modification of proteins in T3-caused liver oxidative stress can be visualized on two levels; 1) loss of protein function and 2) increased protein degradation. Under high rates of ROS and RNS input, the oxidative modification of enzymes can occur with the consequent reduction in enzyme activity (Stadtman, 1990; Lissi et al., 1991). The inactivation of hepatic antioxidant enzymes has been described in several conditions in vivo involving oxidative stress in the tissue, including hyperthyroidism (Lissi et al., 1991), which determines a decrease in the activity of superoxide dismutase and catalase (Fernandez et al., 1988) and in the content of cytochrome P450 (Fernández et al., 1985). In agreement with this contention,

inactivation of superoxide dismutase by H2O2 (Bray et al., 1974) and of catalase by O2

hyperthyroid rats (Tapia et al., 2010).

& Fridovich, 1982) has been reported in conditions in vitro. In addition to enzyme inactivation, thyrotoxicosis in mammals results in the stimulation of both synthesis and degradation of protein, with a predominance of degradation, as shown by the increase in protein catabolism, negative nitrogen balance, and the loss of protein from muscle and other body stores (Loeb, 1996). These findings are in accordance with those of Tapia et al. who found a higher oxidation of the liver protein and an increase in lipid peroxidation levels in

Interestingly, the activities of both GPx-1 and GR are decreased in hyperthyroid rats. It is remarkable that both enzymatic activities are strongly GSH-dependent. The GPx-1 catalyzes the reduction of H2O2 and lipid hydroperoxides coupled with oxidation of GSH into GSSG whereas the GR replenishes the GSH pool with the help of NADPH principally provided by the pentose-phosphate pathway. The intracellular GSH status appears to be a sensitive indicator of the cellular ability to resist ROS. Furthermore, it has been found that total GSH equivalents and the GSH and GSSG pools were increasingly depleted by T3 over time (Chattopadhyay et al., 2007). The liver is especially rich in GST that metabolizes xenobiotics by conjugating with GSH. In fact, the GST-catalyzed conjugation of GSH with exogenous compounds and endogenous metabolites such as 4-hydroxynonenal is regarded as a major cellular-defense mechanism against toxicity (Cheng et al., 2001). The activities of GST were considerably impaired with the progression of the T3 treatment (Chattopadhyay et al., 2007). Because recycling of oxidized glutathione consumes NADPH, the cellular levels of NADPH and its synthesis represent the rate limiting factors of H2O2 consumption by catalasedeficient tissues (Ho et al., 2004). Moreover, prolonged hyperthyroidism diminishes GR but

**.**- (Kono

elevated G6PD activity indicates that severe hyperthyroidism may compromise the cellular ability to maintain the redox state. Lombardi et al. have demonstrated that injection of T3 into hypothyroid rats caused an increase in both enzyme activity and mRNA expression of G6PD in the liver. Nevertheless, the reduced activities of GPx, GR, and GST in the hyperthyroid liver prevent optimum GSH use and recycling. Accumulation of GSSG can lead to protein modifications because of interactions with –SH groups (Reed, 1990). Though the T3 exerted a positive stimulatory effect on the NADPH supply, it was not sufficient to compensate for the massive GSH depletion and this probably explains the negative regulatory impact of T3 on activities of GSH-dependent enzymes such as GPx and GR in the rat liver. Under such conditions, the cellular redox-status is disturbed, as reflected in the high oxidative-stress index of hyperthyroid rats.

In brief, thyroid calorigenesis resulting from acceleration of energy metabolism and secondary electron-transfer processes lead to a higher generation of ROS in the target tissue. This prooxidant condition enhances the oxidative-stress status of the organs when the decrease in the antioxidant potential is not adequately compensated for, leading to

 a) substantial oxidative deterioration of biomolecules, with loss of their functions that may compromise cell viability, b) activities of GPx, GR, GST, catalase, and superoxide dismutase are considerably impaired, c) total GSH equivalents and GSH and GSSG pools were increasingly depleted, d) a higher susceptibility of the liver to toxic stimuli that exacerbate liver injury, e) upregulation of gene expression, f) apoptosis, g) shorter lifespan, and h) myelin deficit.

#### **3.2 Hypothyrodism and the ROS-steady state**

Hypothyroidism has been related to some diseases because it causes a hypometabolic state. This condition can be beneficial. Why can we make this assertion? There are many findings to support this suggestion. It is well-known that a deficiency of the thyroid hormones results in decreased metabolism and lowering of the basal metabolic rate (BMR). There is evidence that supports the lower cell stress in the hypothyroidism condition. Tenorio-Velázquez et al. have demonstrated that hypothyroidism attenuates oxidative stress and renal injury caused by ischemia-reperfusion, produced by an increase in the ROS and reactive nitrogen species (Tenorio-Velásquez et al., 2005). Most research has been done in the kidney and liver models of ischemia (Swaroop & Ramasarma, 1985; Paller, 1986). The postulated mechanism in such organs has been either a decrease in the general metabolic rate or a reduced free radical scavenging response after ischemia. The lipid peroxidation in hypothyroid animals with renal ischemia was decreased (Paller, 1986). The content of malondialdehyde, which is an indirect measure of the generation of oxygen free radicals, was decreased and the cortical content of glutathione, a free radical scavenger, was increased in the hypothyroid, ischemic animals. Similarly, in the liver model of hypothyroid-ischemic injury, lipid peroxidation and free-radical generation were decreased in the hypothyroid animals (Swaroop & Ramasarma, 1985). These investigators have shown a significant decrease in hydrogen peroxide, a measure of the oxygen free-radical status, in the liver mitochondrion in the hypothyroid animals. Hypothyroidism attenuates not only renal but also cardiac damage caused by ischemia and reperfusion. Bobadilla et al. have shown that hypothyroidism conferred protection against reperfusion arrhythmias and the cardiac release of creatine kinase and

The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 423

stress markers (ROS and lipid peroxidation) that were not compensated for by the antioxidant system. The catalase activity is reduced in hepatic tissue and this allows H2O2 caused hepatic damage (Cano-Europa et al., 2010). The increase of the glutathione-cycle enzymes was insufficient to prevent oxidative-stress markers (Ortiz-Butron et al., 2011). All these findings together pointed out that methimazole and not the hypothyroidism is responsible for the cell damage. The tissues evaluated, especially the kidney and liver, have a high metabolic activity that generates ROS. Under physiological conditions the presence of antioxidant enzymes, in particular peroxidases and dismutases, prevent oxidative stress and tissue damage (Halliwell & Gutteridge, 2007; Angermuller et al., 2009). Some drugs, such as methimazole, disturb the physiological steady state. Methimazole alters the intracellular REDOX environment and causes cellular damage because of oxidant generation and ROS, and consequently the lipid peroxidation is not completely neutralized by the antioxidant system. We suggest that the central mechanism of the methimazole-caused cell damage is based on the reduction of catalase activity caused by a methimazole-inactivated catalytic

Other investigators, like Bergman and Brittebo, have demonstrated this anthytiroid-caused damage in other models, i.e. an olfactory mucosa model. They found that this drug covalently binds to the tissue, and pretreatment with the cytochrome-P450 inhibitor metyrapone prevented both the covalent binding and the toxicity of methimazole in this tissue. They suggest a cytochrome P450-dependent metabolic activation of methimazole to a reactive and toxic intermediate at this site (Bergman & Brittebo, 1999). The pretreatment with thyroxin did not protect against the methimazole-caused necrosis, suggesting that this lesion is not related to a transient decrease in thyroid hormone levels. The covalent binding shown by methimazole in this tissue has been found in other tissues, such as the bronchial epithelium and the centrilobular parts of the liver. It is possible that methimazole suffers activation at these sites. Further, this drug is metabolized stepwise to the corresponding sulfenic and sulfinic acids with a concurrent formation of reactive intermediates (Poulsen et al., 1974). It is known that methimazole produces a decrease of P450 at the hepatic level (Decker & Doerge, 1992). In rodents given the methimazole analogs 1-methy-imidazole, 4-methylimidazol, or methyl pyrrole, which are devoid of a thiol group, no morphological changes were observed in the olfactory mucosa (Brittebo, 1995). The thiol group in methimazole seems to be important for the methimazole-caused toxicity, suggesting that enzyme-catalyzed changes of the thiol

Other methimazole-caused damage mechanisms are associated with its chemical structure and its biotransformation. Some investigators suggest that this drug binds covalently to the hepatocytes, mainly those next to the hepatic triad (Decker & Doerge, 1992; Lee & Neal, 1978). For biotransformation, methimazole may be oxidized by the P450 enzymes to form the 4,5-epoxide. The enzymatic or nonenzymatic hydrolysis of the epoxide formed would produce an unstable hemiketal-like intermediate, which it is expected to undergo spontaneous ring cleavage to form glyoxal and N-methylthiourea. The metabolism of Nmethylthiourea is complex, but it is believed that sulfur oxidation, mediated mainly by flavin-monooxigenase (FMO, EC.EC 1.14.13.8), proceeds primarly to the sulfenic acids and then possibly to the sulfinic acids. It is known that this step is necessary in the bioactivation of thioureas resulting in protein binding, enzyme inactivation, and organ toxicity (Mizutani

center (Bandyopadhyay et al., 1995; Bandyopadhyay et al., 2002).

group will give rise to an intermediate toxin in the tissue.

et al., 1994; Neal & Halpert, 1982).

aspartate amino transferase and preserved the normal structure of the myocardial tissue (Bobadilla et al., 2002). It has been proposed that hypothyroidism protects against pore opening and heart reperfusion (Chávez et al., 1998). This may be relevant to the protective effect of hypothyroidism in ischemia and reperfusion because it has been recognized that the mitochondria play a key role in cell-death pathways by activating the mitochondrialpermeability transition pore and causing the release of cytochrome C, proapoptotic factors, and the Ca2+ overload that causes a nonselective permeability of the inner membrane. The prolonged opening of the membrane-permeability transition pore during the first few minutes of reperfusion is a critical determinant of cell death, and pharmacological inhibition of the pore at the time of reperfusion protects the cell (Halestrap et al., 2004). It has been found that there is a decreased glutamate release during hypothyroidism and this is correlated to a protection in cerebral ischemia (Shuaib et al., 1994). The reason why hypothyroidism results in a decreased release of glutamate is as yet unknown. It is possible that the hypothyroidism affects the release mechanisms in the presynaptic receptors. It is also possible that the hypothyroid state results in an increase in the reuptake mechanism for glutamate.

We have noted the protector effect of hypothyroidism, but many investigators use methimazole to cause it. There are some indications that antithyroid-caused hypothyroidism can produce cellular damage. Although, some results indicate that this drug causes cellular protection because of its chemical structure (Bruck et al., 2007; Tutuncu et al., 2007). In addition, there is evidence of extrathyroidal effects of antithyroid drugs, such as thionamides, in humans and animals (Bandyopadhyay et al., 2002). One of the effects of thionamides is the contribution to oxidative stress and cellular damage. These effects can produce an increase of oxidant species that causes lipid peroxidation, nitration, carbonylation, or glutathionylation of proteins, and fragmentation of DNA (Halliwell & Gutteridge, 2007; Valko et al., 2007). Because of this, we determined if methimazole or hypothyroidism causes cellular damage in several organs. After producing a hypothyroid animal caused by thyroidectomy or methimazole administration, the spleen, heart, liver, lung, and kidney were obtained. A portion of these tissues was processed for histological study and another portion was used for the biochemical assay for determining oxidative stress. Histologically, we demonstrated that only methimazole-caused hypothyroidism causes cellular damage in the kidney, lung, liver, heart, and spleen. Animals with methimazole and with T4 supplementation showed cellular damage in the lung, spleen, and renal medulla with lesser damage in the liver, renal cortex, and heart. Hypothyroidism did not produce cellular damage in any organs except the lung. The thyroidectomy group showed no other tissue alterations (Cano-Europa et al., 2011). These results are in accordance with what others have observed in animals and humans. Five percent of patients with hyperthyroidism treated with antithyroid drugs, including methimazole, are reported to have liver (Casallo Blanco et al., 2007; Woeber, 2002), lung (Tsai et al., 2001) and kidney damage (Calañas-Continente et al., 2005). The methimazole-caused hypothyroidism in animals has tumorigenic effects (Jemec, 1977) and modifies the pulmonary function (Liu & Ng, 1991). No tissue damage was seen in a model of hypothyroidism caused by a thyroidectomy (Tenorio-Velásquez et al., 2005). We also compared, over a time-course, markers of oxidative stress, the REDOX environment, and the antioxidant enzymatic system in the liver and the spleen of rats with methimazole- or thyroidectomy-caused hypothyroidism. We found that the cell damage was related with an increase of oxidative

aspartate amino transferase and preserved the normal structure of the myocardial tissue (Bobadilla et al., 2002). It has been proposed that hypothyroidism protects against pore opening and heart reperfusion (Chávez et al., 1998). This may be relevant to the protective effect of hypothyroidism in ischemia and reperfusion because it has been recognized that the mitochondria play a key role in cell-death pathways by activating the mitochondrialpermeability transition pore and causing the release of cytochrome C, proapoptotic factors, and the Ca2+ overload that causes a nonselective permeability of the inner membrane. The prolonged opening of the membrane-permeability transition pore during the first few minutes of reperfusion is a critical determinant of cell death, and pharmacological inhibition of the pore at the time of reperfusion protects the cell (Halestrap et al., 2004). It has been found that there is a decreased glutamate release during hypothyroidism and this is correlated to a protection in cerebral ischemia (Shuaib et al., 1994). The reason why hypothyroidism results in a decreased release of glutamate is as yet unknown. It is possible that the hypothyroidism affects the release mechanisms in the presynaptic receptors. It is also possible that the hypothyroid state results in an increase in the reuptake mechanism for

We have noted the protector effect of hypothyroidism, but many investigators use methimazole to cause it. There are some indications that antithyroid-caused hypothyroidism can produce cellular damage. Although, some results indicate that this drug causes cellular protection because of its chemical structure (Bruck et al., 2007; Tutuncu et al., 2007). In addition, there is evidence of extrathyroidal effects of antithyroid drugs, such as thionamides, in humans and animals (Bandyopadhyay et al., 2002). One of the effects of thionamides is the contribution to oxidative stress and cellular damage. These effects can produce an increase of oxidant species that causes lipid peroxidation, nitration, carbonylation, or glutathionylation of proteins, and fragmentation of DNA (Halliwell & Gutteridge, 2007; Valko et al., 2007). Because of this, we determined if methimazole or hypothyroidism causes cellular damage in several organs. After producing a hypothyroid animal caused by thyroidectomy or methimazole administration, the spleen, heart, liver, lung, and kidney were obtained. A portion of these tissues was processed for histological study and another portion was used for the biochemical assay for determining oxidative stress. Histologically, we demonstrated that only methimazole-caused hypothyroidism causes cellular damage in the kidney, lung, liver, heart, and spleen. Animals with methimazole and with T4 supplementation showed cellular damage in the lung, spleen, and renal medulla with lesser damage in the liver, renal cortex, and heart. Hypothyroidism did not produce cellular damage in any organs except the lung. The thyroidectomy group showed no other tissue alterations (Cano-Europa et al., 2011). These results are in accordance with what others have observed in animals and humans. Five percent of patients with hyperthyroidism treated with antithyroid drugs, including methimazole, are reported to have liver (Casallo Blanco et al., 2007; Woeber, 2002), lung (Tsai et al., 2001) and kidney damage (Calañas-Continente et al., 2005). The methimazole-caused hypothyroidism in animals has tumorigenic effects (Jemec, 1977) and modifies the pulmonary function (Liu & Ng, 1991). No tissue damage was seen in a model of hypothyroidism caused by a thyroidectomy (Tenorio-Velásquez et al., 2005). We also compared, over a time-course, markers of oxidative stress, the REDOX environment, and the antioxidant enzymatic system in the liver and the spleen of rats with methimazole- or thyroidectomy-caused hypothyroidism. We found that the cell damage was related with an increase of oxidative

glutamate.

stress markers (ROS and lipid peroxidation) that were not compensated for by the antioxidant system. The catalase activity is reduced in hepatic tissue and this allows H2O2 caused hepatic damage (Cano-Europa et al., 2010). The increase of the glutathione-cycle enzymes was insufficient to prevent oxidative-stress markers (Ortiz-Butron et al., 2011). All these findings together pointed out that methimazole and not the hypothyroidism is responsible for the cell damage. The tissues evaluated, especially the kidney and liver, have a high metabolic activity that generates ROS. Under physiological conditions the presence of antioxidant enzymes, in particular peroxidases and dismutases, prevent oxidative stress and tissue damage (Halliwell & Gutteridge, 2007; Angermuller et al., 2009). Some drugs, such as methimazole, disturb the physiological steady state. Methimazole alters the intracellular REDOX environment and causes cellular damage because of oxidant generation and ROS, and consequently the lipid peroxidation is not completely neutralized by the antioxidant system. We suggest that the central mechanism of the methimazole-caused cell damage is based on the reduction of catalase activity caused by a methimazole-inactivated catalytic center (Bandyopadhyay et al., 1995; Bandyopadhyay et al., 2002).

Other investigators, like Bergman and Brittebo, have demonstrated this anthytiroid-caused damage in other models, i.e. an olfactory mucosa model. They found that this drug covalently binds to the tissue, and pretreatment with the cytochrome-P450 inhibitor metyrapone prevented both the covalent binding and the toxicity of methimazole in this tissue. They suggest a cytochrome P450-dependent metabolic activation of methimazole to a reactive and toxic intermediate at this site (Bergman & Brittebo, 1999). The pretreatment with thyroxin did not protect against the methimazole-caused necrosis, suggesting that this lesion is not related to a transient decrease in thyroid hormone levels. The covalent binding shown by methimazole in this tissue has been found in other tissues, such as the bronchial epithelium and the centrilobular parts of the liver. It is possible that methimazole suffers activation at these sites. Further, this drug is metabolized stepwise to the corresponding sulfenic and sulfinic acids with a concurrent formation of reactive intermediates (Poulsen et al., 1974). It is known that methimazole produces a decrease of P450 at the hepatic level (Decker & Doerge, 1992). In rodents given the methimazole analogs 1-methy-imidazole, 4-methylimidazol, or methyl pyrrole, which are devoid of a thiol group, no morphological changes were observed in the olfactory mucosa (Brittebo, 1995). The thiol group in methimazole seems to be important for the methimazole-caused toxicity, suggesting that enzyme-catalyzed changes of the thiol group will give rise to an intermediate toxin in the tissue.

Other methimazole-caused damage mechanisms are associated with its chemical structure and its biotransformation. Some investigators suggest that this drug binds covalently to the hepatocytes, mainly those next to the hepatic triad (Decker & Doerge, 1992; Lee & Neal, 1978). For biotransformation, methimazole may be oxidized by the P450 enzymes to form the 4,5-epoxide. The enzymatic or nonenzymatic hydrolysis of the epoxide formed would produce an unstable hemiketal-like intermediate, which it is expected to undergo spontaneous ring cleavage to form glyoxal and N-methylthiourea. The metabolism of Nmethylthiourea is complex, but it is believed that sulfur oxidation, mediated mainly by flavin-monooxigenase (FMO, EC.EC 1.14.13.8), proceeds primarly to the sulfenic acids and then possibly to the sulfinic acids. It is known that this step is necessary in the bioactivation of thioureas resulting in protein binding, enzyme inactivation, and organ toxicity (Mizutani et al., 1994; Neal & Halpert, 1982).

The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 425

because it occurs in lower concentrations in plasma and has a lower Km (Aoyama et al., 2008). Once the amino acids have entered the cell, the g-glutamylcysteine synthetase (-GCS, EC 6.3.2.2) forms the g-glutamylcysteine. The formation of the product involves two steps. The first is the interaction between glutamate and ATP in the presence of Mg+2 to form g glutamylphosphate and the second involves the interaction of this intermediate with the cysteine and with ADP release (Griffith & Mulcahy, 1999; Griffith, 1999). This first step is the most important in the formation of GSH because -GCS is the limiting enzyme in the synthesis of GSH. The -GCS is an heterodimeric enzyme composed of a catalytic subunit known as the heavy subunit (-GCSH Mr, ≈ 73 kDa) and a regulatory or light subunit (- GCSL Mr, ≈ 31 kDa). The -GCS activity depends primarily on the substrates and is inhibited by GSH. The -GCSL activity is under the control of kinases such as protein kinase

Two processes can occur thermodynamically once -glutamyl-cysteinyl is formed. The compound may be used by the GSH synthetase (GS, EC 6.3.2.3) to form GSH when conjugated with glycine or it may interact with g -glutamyl cyclotransferase to form 5-oxo-L-proline and L-cysteine. The pathway that prevails depends of the Km of each enzyme. Under physiological conditions the Km of GS is 12 times greater than the -glutamyl cyclotransferase so it favors the formation of GSH in more than 95% (Weber, 1999). Once

1. In the hydrolysis of plasma GSH to synthesize GSH de novo for another cell. For example, if a hepatocyte secretes GSH, another cell can hydrolyze such a compound into its precursors (cysteinylglycine and glutamate) by the g-glutamyl transpeptidase (-GT, EC 2.3.2.2) expressed on the outside of the plasmatic membrane. The cysteinylglycine compounds or their S-conjugates can be hydrolyzed by dipeptidases to yield free amino acids that can be introduced into the cell and start the formation of

2. In the detoxification of electrophiles by conjugating these with a-carbonyls and by bunsaturation by glutathion-S transferase (GST, EC. 2.5.1.18). This reaction results in the elimination of the electrophile by the consequent metabolism of the glutathione Sconjugate by the -GT enzymes and the cysteinylglycine dypeptidase. This process is not always in the favor for the cell, because it can sometimes create more toxic species

3. In the detoxification of hydrogen peroxide by the action of the glutathione peroxidase

5. In intracellular communication processes as a modulator of diverse signaling pathways

6. In the modulation of membrane receptors as for NMDA receptors in the central nervous

The mitochondrial concentration of GSH is approximately 11-15 mM. The entry of GSH into the mitochondria depends on the electroneutral transporters, such as the tricarboxylic or dicarboxylic acids (Lash, 2006). In general, the ratio GSH/GSSG is greater than 10 for the cells and organelles, such as mitochondria and nucleus, whereas the endoplasmic reticulum

7. In the transport of metals such as Cu+2, Hg+2, Pb+2, and Zn+2 (Filomeni et al., 2002).

enzymes (GPX, EC 1.11.1.19) (Beckett & Arthur, 2005).

4. In maintaining ascorbic acid and vitamin E (Van Acker et al., 1993).

GSH has been synthesized there are different processes in which it participates;

A (PKA) and PKC (Griffith, 1999)

GSH (Weber, 1999).

(Weber, 1999).

(Cruz et al., 2003).

system (Oja et al., 2000).

The thyroidectomy group examinee showed no other tissue alterations, except for the lung. There is some evidence that demonstrates molecular mechanisms by which hypothyroidism itself may produce a protected state of the tissues, such as reducing the enzyme activity associated with the mitochondrial-respiratory chain (Paradies et al., 1994), the decrease in adenine nucleotide translocase (Schonfeld et al., 1997), reduced activity of cytochrome-C oxidase (Paradies et al., 1997), and the resistance to forming the permeability transition-pore formation of the inner mitochondrial membrane (Chávez et al., 1998).

With all this evidence it is important to develop other therapies or antithyroid drugs with fewer side effects. We suggest that hypothyroidism is a protective state against toxic agents and it is related to an increase of reduced glutathione or –L-glutamyl- cysteinyl-glycine (GSH) synthesis and a mild immunosuppression.

#### **3.3 Enhanced GSH synthesis in the hypothyroid state, a mechanism of cell protection**

Before we show evidence of the relationship between hypothyroidism and high –Lglutamyl- cysteinyl-glycine (GSH) concentration, we need to know more about GSH. The synthesis of reduced glutathione or GSH involves two ATP-dependent enzymatic steps made in the cell cytoplasm. Figure 6 shows the cycle of GSH.

Fig. 6. GSH cycle. GR is glutathione reductase; GST glutathione S-transferase, and GSSG oxidized glutathione.

The synthesis of GSH starts with the entry into the cells of its amino acid precursors: glutamate, cysteine, and glycine. Glutamate and glycine can enter the cell by secondary active transport. Some of the glutamate cotransport carriers transfer cysteine. For the cysteine, entry into the cell may also be caused by the transporters of the neutral amino acid system. It is believed that cysteine is the limiting amino acid for the synthesis of GSH

The thyroidectomy group examinee showed no other tissue alterations, except for the lung. There is some evidence that demonstrates molecular mechanisms by which hypothyroidism itself may produce a protected state of the tissues, such as reducing the enzyme activity associated with the mitochondrial-respiratory chain (Paradies et al., 1994), the decrease in adenine nucleotide translocase (Schonfeld et al., 1997), reduced activity of cytochrome-C oxidase (Paradies et al., 1997), and the resistance to forming the permeability transition-pore

With all this evidence it is important to develop other therapies or antithyroid drugs with fewer side effects. We suggest that hypothyroidism is a protective state against toxic agents and it is related to an increase of reduced glutathione or –L-glutamyl- cysteinyl-glycine

**3.3 Enhanced GSH synthesis in the hypothyroid state, a mechanism of cell protection**  Before we show evidence of the relationship between hypothyroidism and high –Lglutamyl- cysteinyl-glycine (GSH) concentration, we need to know more about GSH. The synthesis of reduced glutathione or GSH involves two ATP-dependent enzymatic steps

Fig. 6. GSH cycle. GR is glutathione reductase; GST glutathione S-transferase, and GSSG

The synthesis of GSH starts with the entry into the cells of its amino acid precursors: glutamate, cysteine, and glycine. Glutamate and glycine can enter the cell by secondary active transport. Some of the glutamate cotransport carriers transfer cysteine. For the cysteine, entry into the cell may also be caused by the transporters of the neutral amino acid system. It is believed that cysteine is the limiting amino acid for the synthesis of GSH

formation of the inner mitochondrial membrane (Chávez et al., 1998).

(GSH) synthesis and a mild immunosuppression.

oxidized glutathione.

made in the cell cytoplasm. Figure 6 shows the cycle of GSH.

because it occurs in lower concentrations in plasma and has a lower Km (Aoyama et al., 2008). Once the amino acids have entered the cell, the g-glutamylcysteine synthetase (-GCS, EC 6.3.2.2) forms the g-glutamylcysteine. The formation of the product involves two steps. The first is the interaction between glutamate and ATP in the presence of Mg+2 to form g glutamylphosphate and the second involves the interaction of this intermediate with the cysteine and with ADP release (Griffith & Mulcahy, 1999; Griffith, 1999). This first step is the most important in the formation of GSH because -GCS is the limiting enzyme in the synthesis of GSH. The -GCS is an heterodimeric enzyme composed of a catalytic subunit known as the heavy subunit (-GCSH Mr, ≈ 73 kDa) and a regulatory or light subunit (- GCSL Mr, ≈ 31 kDa). The -GCS activity depends primarily on the substrates and is inhibited by GSH. The -GCSL activity is under the control of kinases such as protein kinase A (PKA) and PKC (Griffith, 1999)

Two processes can occur thermodynamically once -glutamyl-cysteinyl is formed. The compound may be used by the GSH synthetase (GS, EC 6.3.2.3) to form GSH when conjugated with glycine or it may interact with g -glutamyl cyclotransferase to form 5-oxo-L-proline and L-cysteine. The pathway that prevails depends of the Km of each enzyme. Under physiological conditions the Km of GS is 12 times greater than the -glutamyl cyclotransferase so it favors the formation of GSH in more than 95% (Weber, 1999). Once GSH has been synthesized there are different processes in which it participates;


The mitochondrial concentration of GSH is approximately 11-15 mM. The entry of GSH into the mitochondria depends on the electroneutral transporters, such as the tricarboxylic or dicarboxylic acids (Lash, 2006). In general, the ratio GSH/GSSG is greater than 10 for the cells and organelles, such as mitochondria and nucleus, whereas the endoplasmic reticulum

The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 427

catalytic and modulator subunit of -GCS genes have been seen. However, we need to do further experiments to demonstrate this. If this does occur then hypothyroidism could be a protective state against chemical- or physical-caused oxidative stress and cell damage. At present, we are evaluated if the hypothyroid state protects against ethylene glycol-caused oxidative stress and renal damage. At this respect, the results are in accordance with the

There is evidence of the thyroid hormones and immune systems and their development and function from amphibious animals to mammals (Rollins-Smith & Blair, 1990; Lam et al.,

In zebra fish the thyroid state participates in thymus development and lymphopoiesis (Lam et al., 2005). In humans and other mammals, clinical hyperthyroidism increased the size and cellularity of the thymus, particularly a larger number of thymus nurse cells, Thy1+ thymocytes, and the CD4-CD8- and CD44-positive cells (Villa-Verde et al., 1993; Scheiff et al., 1977). Hyperthyroidism increases T cells in the spleen and thymus with high levels of NK cells only in the spleen (Watanabe et al., 1995). Hypothyroidism reduces the cellularity in the spleen and thymus (Bendyug et al., 2003). In neonatal hypothyroidism, it has been observed that the NK cells and regulatory T cells (CD4+CD25+) are enhanced in thymus, spleen, and peripheral blood. The dendritic cells integrate signals from several pathways and receptors, including those arising from engagement of uptake and pattern recognition receptors, proinflammatory and antiinflammatory cytokines, chemokines, and hormones like THs. The T3 promotes the dendritic-cell maturation and Th1-type cytokine secretion (Mascanfroni et al., 2008). The dendritic cells are modulated by the THs because theT3-TR1 causes Akt signaling-pathway activation and NFB-dependence, but a PI3K-independent

There are reports that hypothyroidism decreases immune system activity and increases

All this evidence suggests the proposal that the hypothyroid condition decreases the immune response. This could be protective in the case of toxicant-caused oxidative stress and cell damage caused by the immune system activation by a substance like aniline. Aniline is a toxic, aromatic amine and it is an extensively used industrial chemical. Exposure to aniline is known to cause toxicity to the hematopoietic system. Aniline toxicity is generally characterized by methemoglobinemia, hemolysis, and hemolytic anemia and by the development of splenic hyperplasia, fibrosis, and a variety of primary sarcomas after chronic exposure in rats. The immunological system participates actively in aniline-caused oxidative stress and spleen damage (Wang et al., 2011; Wang et al., 2010; Wang et al., 2008). In our laboratory, we evaluated the participation of hypothyroidism and aniline-caused oxidative stress and spleen damage. We used male Wistar rats weighing 240 to 260 g divided into four groups; 1) euthyroid, 2) euthyroid + aniline, 3) hypothyroid, and 4) hypothyroid + aniline. The hypothyroidism was produced by thyroidectomy with implantation of the parathyroid gland. Two weeks after surgery, the animals were treated with 1 mmol/kg/d ig aniline for five days. On the fifth day, the animals were killed, the

infection in humans (Schoenfeld et al., 1995; Amadi et al., 2008).

idea that the hypothyroidism-enhanced REDOX environment (unpublished data).

**3.4 Thyroid hormone alteration levels and the immune response** 

2005; Watanabe et al., 1995; Nakamura et al., 2007).

pathway (Mascanfroni et al., 2010).

has the lowest GSH/GSSG ratio of 1 to 3. The best indicator of the REDOX environment is the GSH2/GSSG ratio because the REDOX environment involves the transfer of electrons, for which the theoretical model of Schafer and Buettner uses the Nernst equation (Schafer & Buettner, 2001). These authors proposed that other REDOX couples can participate in the REDOX environment maintaining the ratios of NADPH/NADP+, reduced thioredoxin/oxidized thioredoxin (TrxSH2/TrxSS), and GSH2/GSSG. These REDOX couples could participate in the maintaining of the REDOX environment because their pKas are above the physiological pH and the ratio of the reduced pair to its oxidized counterpart is 1:100, 1:1000, or greater. The GSH2/GSSG ratio is the most important couple in the REDOX environment because their chemical structures are not susceptible to any peptidase and their use is in the cell, particularly for cell antioxidant protection, and not in essential biosynthetic pathways. Also, the GSH2/GSSG ratio has the highest concentration of the three REDOX ratios mentioned, and this one best buffers the REDOX potential changes between -300 and - 100 mV, despite varying the concentration of the GSH. The change in the half-cell reduction potential of this REDOX couple is related to the processes such as cell proliferation, differentiation, apoptosis, and necrosis in biological experiments (Cai & Jones, 1998; Cai et al., 2000; Hwang et al., 1992; Jones et al., 1995; Kirlin et al., 1999).

In our group we are studying the effect of the hypothyroid state and the GSH synthesis in various organs, with special interest in the liver and kidney. For that we used thyroidectomyzed rats with a parathyroid gland reimplant (only to affect thyroid hormone system). Two weeks postsurgery we determined the GSH content by a fluorometric method and the -GCS by a spectophotometric method as described (Cano-Europa et al., 2010; Ortiz-Butron et al., 2011). Figure 7 shows that hypothyroid animals have a higher GSH content than euthyroid animals because they have an enhanced -GCS activity.

Fig. 7. Effect of hypothyroidism on GSH content (A) and -GCS activity in liver and kidney. Values are the mean ± SE. (\*) *P* < 0.05 vs. euthyroid (*n* = 5 for each group).

It is possible that some intracellular signals modify the -GCS activity. It is also probable that the thyroid hormone-receptor complex acts as a negative regulator for -GCS because the putative thyroid-hormone response-element sequences in the promoter regions of both the

has the lowest GSH/GSSG ratio of 1 to 3. The best indicator of the REDOX environment is the GSH2/GSSG ratio because the REDOX environment involves the transfer of electrons, for which the theoretical model of Schafer and Buettner uses the Nernst equation (Schafer & Buettner, 2001). These authors proposed that other REDOX couples can participate in the REDOX environment maintaining the ratios of NADPH/NADP+, reduced thioredoxin/oxidized thioredoxin (TrxSH2/TrxSS), and GSH2/GSSG. These REDOX couples could participate in the maintaining of the REDOX environment because their pKas are above the physiological pH and the ratio of the reduced pair to its oxidized counterpart is 1:100, 1:1000, or greater. The GSH2/GSSG ratio is the most important couple in the REDOX environment because their chemical structures are not susceptible to any peptidase and their use is in the cell, particularly for cell antioxidant protection, and not in essential biosynthetic pathways. Also, the GSH2/GSSG ratio has the highest concentration of the three REDOX ratios mentioned, and this one best buffers the REDOX potential changes between -300 and - 100 mV, despite varying the concentration of the GSH. The change in the half-cell reduction potential of this REDOX couple is related to the processes such as cell proliferation, differentiation, apoptosis, and necrosis in biological experiments (Cai & Jones, 1998; Cai et

In our group we are studying the effect of the hypothyroid state and the GSH synthesis in various organs, with special interest in the liver and kidney. For that we used thyroidectomyzed rats with a parathyroid gland reimplant (only to affect thyroid hormone system). Two weeks postsurgery we determined the GSH content by a fluorometric method and the -GCS by a spectophotometric method as described (Cano-Europa et al., 2010; Ortiz-Butron et al., 2011). Figure 7 shows that hypothyroid animals have a higher GSH content

**Kidney**

\*

**0**

**5**

**10**

**-glutamylcysteine**

Fig. 7. Effect of hypothyroidism on GSH content (A) and -GCS activity in liver and kidney.

It is possible that some intracellular signals modify the -GCS activity. It is also probable that the thyroid hormone-receptor complex acts as a negative regulator for -GCS because the putative thyroid-hormone response-element sequences in the promoter regions of both the

**synthetase activity**

**(P5+ formed/mg**

**proteins/h)**

**15** B

**Liver**

\*

al., 2000; Hwang et al., 1992; Jones et al., 1995; Kirlin et al., 1999).

than euthyroid animals because they have an enhanced -GCS activity.

\*

**Liver**

Values are the mean ± SE. (\*) *P* < 0.05 vs. euthyroid (*n* = 5 for each group).

**Kidney**

Euthyroid Hypothyroid

\*

**0**

**50**

**100**

**GSH concentration**

**(g GSH/mg proteins)**

**150**

A

catalytic and modulator subunit of -GCS genes have been seen. However, we need to do further experiments to demonstrate this. If this does occur then hypothyroidism could be a protective state against chemical- or physical-caused oxidative stress and cell damage. At present, we are evaluated if the hypothyroid state protects against ethylene glycol-caused oxidative stress and renal damage. At this respect, the results are in accordance with the idea that the hypothyroidism-enhanced REDOX environment (unpublished data).

#### **3.4 Thyroid hormone alteration levels and the immune response**

There is evidence of the thyroid hormones and immune systems and their development and function from amphibious animals to mammals (Rollins-Smith & Blair, 1990; Lam et al., 2005; Watanabe et al., 1995; Nakamura et al., 2007).

In zebra fish the thyroid state participates in thymus development and lymphopoiesis (Lam et al., 2005). In humans and other mammals, clinical hyperthyroidism increased the size and cellularity of the thymus, particularly a larger number of thymus nurse cells, Thy1+ thymocytes, and the CD4-CD8- and CD44-positive cells (Villa-Verde et al., 1993; Scheiff et al., 1977). Hyperthyroidism increases T cells in the spleen and thymus with high levels of NK cells only in the spleen (Watanabe et al., 1995). Hypothyroidism reduces the cellularity in the spleen and thymus (Bendyug et al., 2003). In neonatal hypothyroidism, it has been observed that the NK cells and regulatory T cells (CD4+CD25+) are enhanced in thymus, spleen, and peripheral blood. The dendritic cells integrate signals from several pathways and receptors, including those arising from engagement of uptake and pattern recognition receptors, proinflammatory and antiinflammatory cytokines, chemokines, and hormones like THs. The T3 promotes the dendritic-cell maturation and Th1-type cytokine secretion (Mascanfroni et al., 2008). The dendritic cells are modulated by the THs because theT3-TR1 causes Akt signaling-pathway activation and NFB-dependence, but a PI3K-independent pathway (Mascanfroni et al., 2010).

There are reports that hypothyroidism decreases immune system activity and increases infection in humans (Schoenfeld et al., 1995; Amadi et al., 2008).

All this evidence suggests the proposal that the hypothyroid condition decreases the immune response. This could be protective in the case of toxicant-caused oxidative stress and cell damage caused by the immune system activation by a substance like aniline. Aniline is a toxic, aromatic amine and it is an extensively used industrial chemical. Exposure to aniline is known to cause toxicity to the hematopoietic system. Aniline toxicity is generally characterized by methemoglobinemia, hemolysis, and hemolytic anemia and by the development of splenic hyperplasia, fibrosis, and a variety of primary sarcomas after chronic exposure in rats. The immunological system participates actively in aniline-caused oxidative stress and spleen damage (Wang et al., 2011; Wang et al., 2010; Wang et al., 2008).

In our laboratory, we evaluated the participation of hypothyroidism and aniline-caused oxidative stress and spleen damage. We used male Wistar rats weighing 240 to 260 g divided into four groups; 1) euthyroid, 2) euthyroid + aniline, 3) hypothyroid, and 4) hypothyroid + aniline. The hypothyroidism was produced by thyroidectomy with implantation of the parathyroid gland. Two weeks after surgery, the animals were treated with 1 mmol/kg/d ig aniline for five days. On the fifth day, the animals were killed, the

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Amadi,K., Sabo,A.M., Ogunkeye,O.O. & Oluwole,F.S. (2008). Thyroid hormone: a "prime

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blood obtained to determine the lymphocyte count and the spleen was dissected to assess lipid peroxidation and the quantification of reactive oxygen species as preliminary results. In figure 8 are the results. It was shown that hypothyroid rats had decreased ROS concentration, lipid peroxidation, and the lymphocyte counts in aniline-treated rats compared with euthyroid rats.

Fig. 8. Effect of hypothyroidism on ROS concentration (A), lipid peroxidation (B), and lymphocyte count (C) in the spleen of rats treated with aniline. Values are the mean ± SE. (\*) *P* < 0.05 vs. euthyroid (*n* = 5 for each group).

The hypothyroid state can be protective in this situation because it decreased the ROS production, increased the GSH, and decreased the lymphocyte count. Although we need to do more experiments to demonstrate the idea of a mild immunosuppression participating in cell protection, these preliminary results suggest it.

#### **4. Final remarks**

In earlier years it was believed that the hypo- and hyperthyroid conditions modifying the ROS steady state caused cell damage. Presently, there is much evidence that only hyperthyroidism does this. Hypothyroidism is believed to be a protective state because scientists did not believe the drug-caused hypothyroidism modified the ROS steady state. On this point, by studying hypothyroidism in animal models that modify only TH concentrations, such as thyroidectomy, we can observe cell protection against chemical- and physical-caused oxidative stress. Right now, we can describe two different pathways by which the hypothyroid state can protect: GSH synthesis and mild immunosuppression. This is now an open field in which to study these possibilities.

#### **5. Acknowledgement**

This study was partially supported by SIP-IPN 20110283 y 20110336. Thanks to Dr. Ellis Glazier for editing this English-language text.

#### **6. References**

Alvarez,B. & Radi,R. (2001). Peroxynitrite decay in the presence of hydrogen peroxide, mannitol and ethanol: a reappraisal. *Free Radical Research* Vol. 34, No. 5, pp. 467-475 (2001) ISSN 1071-5762

blood obtained to determine the lymphocyte count and the spleen was dissected to assess lipid peroxidation and the quantification of reactive oxygen species as preliminary results. In figure 8 are the results. It was shown that hypothyroid rats had decreased ROS concentration, lipid peroxidation, and the lymphocyte counts in aniline-treated rats

\*

**Euthyroid Hypothyroid**

**0 1.0104 2.0104 3.0104 4.0104 5.0104**

**Lynfocytes (mm3**

**)**

Vehicle Aniline <sup>C</sup>

\*

**Euthyroid Hypothyroid**

\*

compared with euthyroid rats.

\*

**Euthyroid Hypothyroid**

*P* < 0.05 vs. euthyroid (*n* = 5 for each group).

cell protection, these preliminary results suggest it.

is now an open field in which to study these possibilities.

Glazier for editing this English-language text.

(2001) ISSN 1071-5762

**0.00 0.05 0.10 0.15 0.20 0.5 1.0 1.5 2.0** B

Fig. 8. Effect of hypothyroidism on ROS concentration (A), lipid peroxidation (B), and lymphocyte count (C) in the spleen of rats treated with aniline. Values are the mean ± SE. (\*)

The hypothyroid state can be protective in this situation because it decreased the ROS production, increased the GSH, and decreased the lymphocyte count. Although we need to do more experiments to demonstrate the idea of a mild immunosuppression participating in

In earlier years it was believed that the hypo- and hyperthyroid conditions modifying the ROS steady state caused cell damage. Presently, there is much evidence that only hyperthyroidism does this. Hypothyroidism is believed to be a protective state because scientists did not believe the drug-caused hypothyroidism modified the ROS steady state. On this point, by studying hypothyroidism in animal models that modify only TH concentrations, such as thyroidectomy, we can observe cell protection against chemical- and physical-caused oxidative stress. Right now, we can describe two different pathways by which the hypothyroid state can protect: GSH synthesis and mild immunosuppression. This

This study was partially supported by SIP-IPN 20110283 y 20110336. Thanks to Dr. Ellis

Alvarez,B. & Radi,R. (2001). Peroxynitrite decay in the presence of hydrogen peroxide,

mannitol and ethanol: a reappraisal. *Free Radical Research* Vol. 34, No. 5, pp. 467-475

**Lipid peroxidation**

**URF/mg proteins**

**4. Final remarks** 

**5. Acknowledgement** 

**6. References** 

**Quatification of ROS**

**(ng of DCF formed/**

**mg proteins/h)**


The Relationship Between Thyroid States, Oxidative Stress and Cellular Damage 431

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**19** 

Martina Škurlová

*Czech Republic* 

**Oxidative Stress in Human** 

**Autoimmune Joint Diseases** 

*Department of Normal, Pathological, and Clinical Physiology, Third Faculty of Medicine, Charles University in Prague,* 

Living with oxygen is basically unsafe, but vital. During evolution, oxygen originally a waste product of the metabolism in primitive unicellular organisms became normal product of the metabolism in higher animal species involving humans. Even when oxidative reactions are toxic, and destructive, they are tolerated by all organisms to some extent. The fact has opened the discussion about efficiency of antioxidant mechanisms. The classical enzyme antioxidant defence alone does not explain high tolerance of the organism for oxygen. Moreover, enzyme antioxidant mechanisms are not hundred percent effective in

The pathogenesis of autoimmune joint inflammatory diseases is related to activation of native immune system. At site of inflammation, activated neutrophils and macrophages consume large amounts of oxygen, whose corollary is the increase of reactive oxygen species (ROS) production. There are several mechanisms how oxidative stress is involved into the pathogenesis of autoimmune joint inflammatory diseases. Excess production of ROS in the joint area encourages process of re-oxygenation, which then promotes joint inflammation. ROS further inhibit connective tissue cell proliferation, in some cases ROS have been shown

Cellular responses to oxidative stress depend on the cellular redox status. When the oxidants' level does not exceed the redox capacities in a cell, oxidants are beneficial to the cell controlling cellular functions such as signal transduction. In contrast, when the cellular antioxidant capacity is insufficient, the production of oxidants exceeds the capacity to neutralize them (Hitchon & El- Gabalawy, 2004). Insufficient oxidative defence mechanisms shift the balance between oxidants and antioxidants in the direction of oxidants leading to oxidative stress. Insufficient oxidative defence mechanisms result from depletion of enzymatic (e.g., superoxide dismuthase, catalase, glutathione peroxidase), and nonenzymatic (e.g., glutathione, vitamins A, C, and E, and selenium) antioxidants (Hovatta et al., 2010). The pro-oxidant conditions of the ´*internal milieu*´, due to low redox status of the

preventing oxidation what allows oxidative damage to continue.

to induce cell death to these cells inducing apoptosis.

**1. Introduction** 

**2. Oxidative stress** 

**2.1 Biology of oxidative stress** 


### **Oxidative Stress in Human Autoimmune Joint Diseases**

Martina Škurlová

*Department of Normal, Pathological, and Clinical Physiology, Third Faculty of Medicine, Charles University in Prague, Czech Republic* 

#### **1. Introduction**

436 Oxidative Stress and Diseases

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thyroid hormone on lymphocyte subsets in spleens and thymuses of mice.

of the glutathione cycle. *Neuroscience and Biobehavioral Rev*iews 23, No.8 (December

Living with oxygen is basically unsafe, but vital. During evolution, oxygen originally a waste product of the metabolism in primitive unicellular organisms became normal product of the metabolism in higher animal species involving humans. Even when oxidative reactions are toxic, and destructive, they are tolerated by all organisms to some extent. The fact has opened the discussion about efficiency of antioxidant mechanisms. The classical enzyme antioxidant defence alone does not explain high tolerance of the organism for oxygen. Moreover, enzyme antioxidant mechanisms are not hundred percent effective in preventing oxidation what allows oxidative damage to continue.

The pathogenesis of autoimmune joint inflammatory diseases is related to activation of native immune system. At site of inflammation, activated neutrophils and macrophages consume large amounts of oxygen, whose corollary is the increase of reactive oxygen species (ROS) production. There are several mechanisms how oxidative stress is involved into the pathogenesis of autoimmune joint inflammatory diseases. Excess production of ROS in the joint area encourages process of re-oxygenation, which then promotes joint inflammation. ROS further inhibit connective tissue cell proliferation, in some cases ROS have been shown to induce cell death to these cells inducing apoptosis.

#### **2. Oxidative stress**

#### **2.1 Biology of oxidative stress**

Cellular responses to oxidative stress depend on the cellular redox status. When the oxidants' level does not exceed the redox capacities in a cell, oxidants are beneficial to the cell controlling cellular functions such as signal transduction. In contrast, when the cellular antioxidant capacity is insufficient, the production of oxidants exceeds the capacity to neutralize them (Hitchon & El- Gabalawy, 2004). Insufficient oxidative defence mechanisms shift the balance between oxidants and antioxidants in the direction of oxidants leading to oxidative stress. Insufficient oxidative defence mechanisms result from depletion of enzymatic (e.g., superoxide dismuthase, catalase, glutathione peroxidase), and nonenzymatic (e.g., glutathione, vitamins A, C, and E, and selenium) antioxidants (Hovatta et al., 2010). The pro-oxidant conditions of the ´*internal milieu*´, due to low redox status of the

Oxidative Stress in Human Autoimmune Joint Diseases 439

Reactive nitrogen species are produced by phagocytes during the reaction of NO with O2•\_,

reaction between ON• and O2•\_ , both of which are free radicals. Peroxynitrite then undergoes a secondary reaction to produce an agent that is able to nitrate tyrosine. The precise composition of the agent is not known, but several candidates have been proposed. Among them are various derivatives of ONOO-. Major cellular sites of ROS generation include the mitochondria, and non- mitochondrial membrane bound enzyme systems

Articular cartilage is a unique tissue for its constituent cells, the chondrocytes, which maintain the cartilage matrix through their continual synthesis and degradation. The environment is avascular, hyperosmotic and acidic. Cartilage cells are synoviocytes, and chondrocytes. Chondrocytes display a metabolism adapted to anaerobic conditions. Synoviocytes supply the avascular cartilage tissue with nutrients via synovial fluid. Because cartilage is an avascular environment, the oxygen tension in the area is usually low. In pathological conditions, like inflammation, oxygen tension is subject of fluctuations. These variations of oxygen tension force chondrocytes to produce reactive species. The main reactive species produced by chondrocytes are O2•\_ radical, and NO that generate other derivative radicals, including ONOO- and H2O2 (Hiran et al., 1997; Stefanovic- Racic et al., 1997). The effects of free radicals on articular cartilage are dual. "Chondrocyte- derived" free radical levels are important for the maintenance of ion homeostasis. Natrium hydrogen (Na+ - H+) - exchanger (NHE- activity) and free radical levels exhibit a significant positive correlation. How exactly highly reactive species alter ion transport is not known, although

Except beneficial effect free radicals may have on articular cartilage when exceed in a cell, free radicals damage both chondrocytes, and extracellular matrix (ECM) components of articular cartilage. Free radicals shift the redox balance in articular cartilage in direction of oxidants. Hypochlorous acid, singlet oxygen, and peroxynitrite radicals balance the

ROS and RNS damage articular cartilage directly or indirectly by up- regulating the mediators of the ECM degradation. Reactive oxygen/ nitrogen species, e.g. ONOO-

have been shown to degrade aggrecan, a major component of ECM, and this degradation is one of the initial events in the process of cartilage destruction (Billinghurst *et al.,* 1997). The incidence of sulfated glycosaminoglycans (GAGs) reflects the ratio of aggrecan degradation

In seeking for a role of oxidative radicals in cartilage metabolism, it has been noted that endogenously generated NO suppresses the biosynthesis of aggrecan, a major macromolecular component of the cartilaginous matrix (Cao et al., 1997). Furthermore, it was discovered that oxygen radicals fragment hyaluronan and chondroitin sulphate

Collagen, which provides tensile strength and forms a network that resists the swelling pressure of aggrecan- hyaluronate aggregates, can be altered directly by oxygen radicals. Free radicals prime collagen to proteolytic enzymes. Incubation of cartilage slices with

, formed in a

, also

and other oxidizing species. The first characterized of these species is ONOO-

interference with protein phosphorylation is possible (Gibson et al., 2008).

ascorbate, an antioxidant vitamin, from cartilage (Hajdigogos et al., 2003).

**3.2 Reactive oxidant species in the joint tissue** 

(Babior, 2000).

in the cartilage.

(Kennett & Davies, 2009).

cell led to a new definition of oxidative stress. Oxidative stress is defined as ˮ disruption between ROS production and elimination leading to their enhanced steady- state in the body ˮ (Lushchak, 2011).

Reactive oxygen species destroy not only intracellular components, but also cell membranes, and extracellular components. ROS modify proteins by oxidation, nitrosylation, nitration or chlorination of specific amino acids, leading to their impaired biological activity, changes in protein structure and accumulation of damaged proteins in the tissue. During lipid peroxidation, which is a marker of oxidative stress, polyunsaturated fatty lipids are oxidized and produce lipid peroxyl radicals that in turn up-regulate oxidation, and cell membrane damage (Hitchon & El- Gabalawy, 2004). Genotoxic effects of oxidative stress involve direct breakage of DNA and DNA repair mechanisms. Oxidative stress may also cause cell death. Cellular content containing oxidized molecules when released into the extracellular environment may contribute to the exacerbation of synovial inflammation as newly formed ROS and degradation products form a vicious inflammatory circle (Henrontin et al., 2003).

#### **3. Reactive oxidant species**

Free radicals are very reactive chemical species that have unpaired valence shell electrons in their outer orbitals (Afonso et al., 2007). Highly reactive and partly reduced oxygen metabolites are a by-product of oxidative phosphorylation process, which takes part in mitochondria. These metabolites called ROS include oxygen radicals [superoxide (O2•\_), hydroxyl (HO•), peroxyl (O2R•), and alkoxyl (OR•)] and certain non radicals that are either oxidizing agents or are easily converted into radicals, such as hypochlorous acid (HOCl), ozone (O3), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Other oxidants, generated by interactions with these molecules, include reactive nitrogen species (RNS), as an example nitric oxide (NO), peroxynitrite (ONOO- ).

#### **3.1 Generation of reactive oxidant species**

Generation of ROS is generally cascade of reactions that starts with the production of O2•\_. Superoxide rapidly dismutates to H2O2 either spontaneously, particularly at low pH, or catalyzed by superoxide dismutase (SOD) enzyme. Other steps in the cascade of ROS generation include the reaction of O2•\_ with ON• to form ONOO -, the peroxidase-catalyzed formation of HOCl from H2O2, and the iron-catalyzed Fenton reaction leading to the generation of HO•. Hydroxyl radical is one of the most reactive oxygen radicals. In biologic systems, the HO• is formed by the reaction between H2O2 and iron or copper in a low valence state. The oxidized halogens are almost as diverse group of reactive oxidants as are the free radicals. They consist of HOCl and the vast number of chloramines. Chloramines can be produced from the reaction of HOCl with the many amines that are found in biological systems. Like HOCl, the chloramines are oxidizing species. Chloramine (NH2Cl) is formed by the reaction of HOCl with ammonia (NH3). The reaction of HOCl with amino acids leads through chloramines to aldehydes. Altogether, the oxidized halogens represent probably the most important microbicidal oxidants produced by neutrophils. Oxygen (O2) alone is a diradical with two unpaired electrons. There is also a much more reactive form of oxygen known as 1O2, in which those two electrons are paired. The 1O2 is produced by neutrophils, which manufacture it by the reaction between H2O2 and an oxidized halogen.

cell led to a new definition of oxidative stress. Oxidative stress is defined as ˮ disruption between ROS production and elimination leading to their enhanced steady- state in the

Reactive oxygen species destroy not only intracellular components, but also cell membranes, and extracellular components. ROS modify proteins by oxidation, nitrosylation, nitration or chlorination of specific amino acids, leading to their impaired biological activity, changes in protein structure and accumulation of damaged proteins in the tissue. During lipid peroxidation, which is a marker of oxidative stress, polyunsaturated fatty lipids are oxidized and produce lipid peroxyl radicals that in turn up-regulate oxidation, and cell membrane damage (Hitchon & El- Gabalawy, 2004). Genotoxic effects of oxidative stress involve direct breakage of DNA and DNA repair mechanisms. Oxidative stress may also cause cell death. Cellular content containing oxidized molecules when released into the extracellular environment may contribute to the exacerbation of synovial inflammation as newly formed ROS and degradation products form a vicious inflammatory circle (Henrontin et al., 2003).

Free radicals are very reactive chemical species that have unpaired valence shell electrons in their outer orbitals (Afonso et al., 2007). Highly reactive and partly reduced oxygen metabolites are a by-product of oxidative phosphorylation process, which takes part in mitochondria. These metabolites called ROS include oxygen radicals [superoxide (O2•\_), hydroxyl (HO•), peroxyl (O2R•), and alkoxyl (OR•)] and certain non radicals that are either oxidizing agents or are easily converted into radicals, such as hypochlorous acid (HOCl), ozone (O3), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Other oxidants, generated by interactions with these molecules, include reactive nitrogen species (RNS), as an example

Generation of ROS is generally cascade of reactions that starts with the production of O2•\_. Superoxide rapidly dismutates to H2O2 either spontaneously, particularly at low pH, or catalyzed by superoxide dismutase (SOD) enzyme. Other steps in the cascade of ROS generation include the reaction of O2•\_ with ON• to form ONOO -, the peroxidase-catalyzed formation of HOCl from H2O2, and the iron-catalyzed Fenton reaction leading to the generation of HO•. Hydroxyl radical is one of the most reactive oxygen radicals. In biologic systems, the HO• is formed by the reaction between H2O2 and iron or copper in a low valence state. The oxidized halogens are almost as diverse group of reactive oxidants as are the free radicals. They consist of HOCl and the vast number of chloramines. Chloramines can be produced from the reaction of HOCl with the many amines that are found in biological systems. Like HOCl, the chloramines are oxidizing species. Chloramine (NH2Cl) is formed by the reaction of HOCl with ammonia (NH3). The reaction of HOCl with amino acids leads through chloramines to aldehydes. Altogether, the oxidized halogens represent probably the most important microbicidal oxidants produced by neutrophils. Oxygen (O2) alone is a diradical with two unpaired electrons. There is also a much more reactive form of oxygen known as 1O2, in which those two electrons are paired. The 1O2 is produced by neutrophils, which manufacture it by the reaction between H2O2 and an oxidized halogen.

body ˮ (Lushchak, 2011).

**3. Reactive oxidant species** 

nitric oxide (NO), peroxynitrite (ONOO-).

**3.1 Generation of reactive oxidant species** 

Reactive nitrogen species are produced by phagocytes during the reaction of NO with O2•\_, and other oxidizing species. The first characterized of these species is ONOO- , formed in a reaction between ON• and O2•\_ , both of which are free radicals. Peroxynitrite then undergoes a secondary reaction to produce an agent that is able to nitrate tyrosine. The precise composition of the agent is not known, but several candidates have been proposed. Among them are various derivatives of ONOO-. Major cellular sites of ROS generation include the mitochondria, and non- mitochondrial membrane bound enzyme systems (Babior, 2000).

#### **3.2 Reactive oxidant species in the joint tissue**

Articular cartilage is a unique tissue for its constituent cells, the chondrocytes, which maintain the cartilage matrix through their continual synthesis and degradation. The environment is avascular, hyperosmotic and acidic. Cartilage cells are synoviocytes, and chondrocytes. Chondrocytes display a metabolism adapted to anaerobic conditions. Synoviocytes supply the avascular cartilage tissue with nutrients via synovial fluid. Because cartilage is an avascular environment, the oxygen tension in the area is usually low. In pathological conditions, like inflammation, oxygen tension is subject of fluctuations. These variations of oxygen tension force chondrocytes to produce reactive species. The main reactive species produced by chondrocytes are O2•\_ radical, and NO that generate other derivative radicals, including ONOO and H2O2 (Hiran et al., 1997; Stefanovic- Racic et al., 1997). The effects of free radicals on articular cartilage are dual. "Chondrocyte- derived" free radical levels are important for the maintenance of ion homeostasis. Natrium hydrogen (Na+ - H+) - exchanger (NHE- activity) and free radical levels exhibit a significant positive correlation. How exactly highly reactive species alter ion transport is not known, although interference with protein phosphorylation is possible (Gibson et al., 2008).

Except beneficial effect free radicals may have on articular cartilage when exceed in a cell, free radicals damage both chondrocytes, and extracellular matrix (ECM) components of articular cartilage. Free radicals shift the redox balance in articular cartilage in direction of oxidants. Hypochlorous acid, singlet oxygen, and peroxynitrite radicals balance the ascorbate, an antioxidant vitamin, from cartilage (Hajdigogos et al., 2003).

ROS and RNS damage articular cartilage directly or indirectly by up- regulating the mediators of the ECM degradation. Reactive oxygen/ nitrogen species, e.g. ONOO- , also have been shown to degrade aggrecan, a major component of ECM, and this degradation is one of the initial events in the process of cartilage destruction (Billinghurst *et al.,* 1997). The incidence of sulfated glycosaminoglycans (GAGs) reflects the ratio of aggrecan degradation in the cartilage.

In seeking for a role of oxidative radicals in cartilage metabolism, it has been noted that endogenously generated NO suppresses the biosynthesis of aggrecan, a major macromolecular component of the cartilaginous matrix (Cao et al., 1997). Furthermore, it was discovered that oxygen radicals fragment hyaluronan and chondroitin sulphate (Kennett & Davies, 2009).

Collagen, which provides tensile strength and forms a network that resists the swelling pressure of aggrecan- hyaluronate aggregates, can be altered directly by oxygen radicals. Free radicals prime collagen to proteolytic enzymes. Incubation of cartilage slices with

Oxidative Stress in Human Autoimmune Joint Diseases 441

sulfate are extensively depolymerized by hydroxyl and carbonyl radicals, which may be formed from ONOO-. Polymer fragmentation is shown to be dependent on the radical flux (Kennett & Davies, 2009). NO, and O2.- inhibit type II collagen and proteoglycans synthesis

In autoimmune joint diseases systemic inflammation exists long before it exerts local effects on synovial membrane. At one point, systemic inflammation is translocated into synovium where it initiates the inflammatory response often leading to oxidative burst. Oxidative burst in rheumatoid joints is a result of the activation of innate immune system cells. Activated phagocytic cells such as neutrophils, and macrophages both produce free radicals in the joint area. Activated phagocytes produce reactive oxidants by enzymes: the NADPH- oxidase, and the nitric oxide synthase (NOS). Mechanism of free radicals production differs between these cell groups. While macrophages are stimulated by the ˮNADPH- oxidaseˮ system to produce free radicals, the presence of NOS accompanied by the NADPH- oxidase is necessary for neutrophils to secrete free radicals. RA neutrophils also generate enhanced amount of ONOOby NOS (El Benna et al., 2002). Chemiluminescence assays demonstrated significant activation of the neutrophil myeloperoxidase H2O2 system in synovial fluids from patients with RA further suggesting that oxidative stress may contribute to the cyclic, self- perpetuating nature of rheumatoid inflammation. ROS, produced by activated phagocytes alters the antigenic behaviour of immunoglobulin G (IgG). Radical-exposed IgG is able to bind rheumatoid factor and results in the generation of C3alpha complement component. This reaction may be selfperpetuating within the rheumatoid joint, suggesting that free radicals play a role in the

Pro-inflammatory cytokines´ presence and activity undoubtedly subjects to rheumatoid synovitis governing a variety of pathological processes including cell activation, cell proliferation, tissue resorption and chemotaxis (Schett et al., 2000). Experimental evidence confirms cytokine- induced oxidative stress in rheumatoid synovium. Thioredoxin, a cellular catalyst induced by oxidative stress, is found in high amounts in RA synovial cells and tissue. Thioredoxin acts as a co- factor for tumor necrosis factor- alpha (TNF- induced synthesis of interleukins (IL-6 and IL-8) in synovial fibroblastlike cells (Yoshida et al., 1999). Edaravone, which is a clinically available antioxidant, suppresses IL-1β- induced synovial cells proliferation and migration under *in vitro* conditions (Arii et al., 2006). Nacetylcytein, a known thiol antioxidant, abrogated L-6 - induced proliferation of RA

ROS are documented as mediators of synovial inflammation. Excessive production of ROS at the site of inflammation contributes to the inflammatory process in general, by induction of the expression of adhesion molecules, pro-inflammatory cytokines, and chemoattractants. Furthermore, ROS can directly increase tissue destruction through inactivation of the major inhibitor of degrading enzymes, 1-antiproteinase, which subsequently leads to the activation of extracellular matrix-degrading metalloproteinases (MMPs) (Maurice et al., 1997). 3-nitrotyrosine (3- NT) has been identified as a stable end product and marker of

and the sulfation of newly synthesized GaGs (Hickery & Bayliss, 1998).

**4. Inflammatory synovitis: May oxidative stress be a cause?** 

chronicity of rheumatoid inflammation (Newkirk et al., 2002).

patients synovial fibroblasts (Ji-Yeon et al., 2000).

**4.1 ROS in inflammatory synovium** 

xanthine-oxidase- generated O2•\_ anion degrades type I collagen and fibril formation by this collagen. Hydroxyl radical in the presence of oxygen fragments collagen into small peptides. The cleavage seems to be specific to proline or 4- hydroxyproline residues (Monboisse & Borel, 1992). Interestingly, free radicals may destruct collagen synthesis indirectly. NO inhibits collagen synthesis via interleukin-1 (IL-1) (Cao et al., 1997). H2O2 inhibits cartilage proteoglycan synthesis interfering with adenosine triphosphate (ATP) synthesis, in part by inhibiting the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase in chondrocytes. ONOO and HOCl may facilitate cartilage damage by inactivating tissue inhibitor of metalloproteinases (TIMPs). TIMP-1 inhibits stromelysins, collagenases and gelatinases (Henrontin et al., 2003). Moreover it was observed, that treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, up- regulates the collagenase mRNA levels, which is one of MMPs (Lo et al., 1998).

Exposure of the chondrocytes to H2O2 inhibits proteoglycan synthesis (Henrontin et al., 2003). Chondrocytes in arthritic cartilage respond poorly to insulin- like growth factor 1 (IGF-1) what may lead to abrogation of cartilage repair. In this context, ROS may participate in reducing the capacity of chondrogenic precursor cells to migrate and proliferate within joint area. Nitric oxide radical was also demonstrated to inhibit chondrocyte migration and attachment to fibronectin via modification of the actin cytoskeleton. Chondrocytes produce high levels of NO, which is a mediator of anti- proliferative effects of IL-1 in these articular cells (Blanco Lotz, 1994). In addition, it was discovered that IL-1 induces apoptosis to chondrocytes via NO. Combination of IL-1 with the radical scavengers like N-acetyl cysteine, dimethyl sulfoxide, or 5, 5'-dimetylpyrroline 1-oxide induced apoptosis, which was inhibited in a dose dependent manner by the NO synthase inhibitor N-monomethyl Larginine (Blanco et al., 1995). Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, was completely blocked in p47phox-/- mice confirming the "Nicotinamide adenine dinucleotide phosphate" (NADPH)- oxidaseˮ driven oxygen radical production in mediating these effects (Lem van Lent et al., 2005).

Synoviocytes are the second kind of articular cells. These cells consume larger amount of oxygen when compared to chondrocytes (Schneider et al., 2005). An indirect evidence of oxidative stress in synoviocytes is the incidence of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase in these cells (Mattey et al., 1993). Oxidative stress makes synoviocytes to undergo a cell death of an apoptotic nature (Galleron et al., 1999). The ROS scavenger system of synoviocytes protects chondrocytes from toxic effects of free radicals. In a co-culture of these cells synoviocytes reduced toxic effects of H2O2 on chondrocyte cell damage (Kurz et al., 1999). NO is the primary inducer of apoptosis in human articular chondrocytes (Blanco et al., 1995). NO- mediated chondrocyte cell death requires generation of additional reactive species like ONOO- and O2 .- (Del Carlo Jr. & Loeser, 2002).

Joint fluid is produced as a transudate of plasma from synovial cells and provides nutrition to the articular cartilage by diffusion of oxygen and other molecules. The primary catalytic antioxidant of the joint fluid is the extracellular SOD (Regan et al., 2008). SOD type III accounts for 80% of the enzyme's activity in the joint fluid (Afonso et al., 2007). Glycosaminoglycans (long-chain polysaccharides) are major components of the extracellular matrix, glycocalyx, and synovial fluid. Modifications to these materials are linked to multiple human pathologies including autoimmune diseases. Hyaluronan and chondroitin sulfate are extensively depolymerized by hydroxyl and carbonyl radicals, which may be formed from ONOO-. Polymer fragmentation is shown to be dependent on the radical flux (Kennett & Davies, 2009). NO, and O2.- inhibit type II collagen and proteoglycans synthesis and the sulfation of newly synthesized GaGs (Hickery & Bayliss, 1998).

#### **4. Inflammatory synovitis: May oxidative stress be a cause?**

#### **4.1 ROS in inflammatory synovium**

440 Oxidative Stress and Diseases

collagen. Hydroxyl radical in the presence of oxygen fragments collagen into small peptides. The cleavage seems to be specific to proline or 4- hydroxyproline residues (Monboisse & Borel, 1992). Interestingly, free radicals may destruct collagen synthesis indirectly. NO inhibits collagen synthesis via interleukin-1 (IL-1) (Cao et al., 1997). H2O2 inhibits cartilage proteoglycan synthesis interfering with adenosine triphosphate (ATP) synthesis, in part by inhibiting the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase in

inhibitor of metalloproteinases (TIMPs). TIMP-1 inhibits stromelysins, collagenases and gelatinases (Henrontin et al., 2003). Moreover it was observed, that treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, up- regulates

Exposure of the chondrocytes to H2O2 inhibits proteoglycan synthesis (Henrontin et al., 2003). Chondrocytes in arthritic cartilage respond poorly to insulin- like growth factor 1 (IGF-1) what may lead to abrogation of cartilage repair. In this context, ROS may participate in reducing the capacity of chondrogenic precursor cells to migrate and proliferate within joint area. Nitric oxide radical was also demonstrated to inhibit chondrocyte migration and attachment to fibronectin via modification of the actin cytoskeleton. Chondrocytes produce high levels of NO, which is a mediator of anti- proliferative effects of IL-1 in these articular cells (Blanco Lotz, 1994). In addition, it was discovered that IL-1 induces apoptosis to chondrocytes via NO. Combination of IL-1 with the radical scavengers like N-acetyl cysteine, dimethyl sulfoxide, or 5, 5'-dimetylpyrroline 1-oxide induced apoptosis, which was inhibited in a dose dependent manner by the NO synthase inhibitor N-monomethyl Larginine (Blanco et al., 1995). Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, was completely blocked in p47phox-/- mice confirming the "Nicotinamide adenine dinucleotide phosphate" (NADPH)- oxidaseˮ driven oxygen radical

Synoviocytes are the second kind of articular cells. These cells consume larger amount of oxygen when compared to chondrocytes (Schneider et al., 2005). An indirect evidence of oxidative stress in synoviocytes is the incidence of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase in these cells (Mattey et al., 1993). Oxidative stress makes synoviocytes to undergo a cell death of an apoptotic nature (Galleron et al., 1999). The ROS scavenger system of synoviocytes protects chondrocytes from toxic effects of free radicals. In a co-culture of these cells synoviocytes reduced toxic effects of H2O2 on chondrocyte cell damage (Kurz et al., 1999). NO is the primary inducer of apoptosis in human articular chondrocytes (Blanco et al., 1995). NO- mediated chondrocyte cell death requires generation of additional reactive species like ONOO- and O2.- (Del Carlo Jr. &

Joint fluid is produced as a transudate of plasma from synovial cells and provides nutrition to the articular cartilage by diffusion of oxygen and other molecules. The primary catalytic antioxidant of the joint fluid is the extracellular SOD (Regan et al., 2008). SOD type III accounts for 80% of the enzyme's activity in the joint fluid (Afonso et al., 2007). Glycosaminoglycans (long-chain polysaccharides) are major components of the extracellular matrix, glycocalyx, and synovial fluid. Modifications to these materials are linked to multiple human pathologies including autoimmune diseases. Hyaluronan and chondroitin

the collagenase mRNA levels, which is one of MMPs (Lo et al., 1998).

production in mediating these effects (Lem van Lent et al., 2005).

•\_ anion degrades type I collagen and fibril formation by this

and HOCl may facilitate cartilage damage by inactivating tissue

xanthine-oxidase- generated O2

chondrocytes. ONOO-

Loeser, 2002).

In autoimmune joint diseases systemic inflammation exists long before it exerts local effects on synovial membrane. At one point, systemic inflammation is translocated into synovium where it initiates the inflammatory response often leading to oxidative burst. Oxidative burst in rheumatoid joints is a result of the activation of innate immune system cells. Activated phagocytic cells such as neutrophils, and macrophages both produce free radicals in the joint area. Activated phagocytes produce reactive oxidants by enzymes: the NADPH- oxidase, and the nitric oxide synthase (NOS). Mechanism of free radicals production differs between these cell groups. While macrophages are stimulated by the ˮNADPH- oxidaseˮ system to produce free radicals, the presence of NOS accompanied by the NADPH- oxidase is necessary for neutrophils to secrete free radicals. RA neutrophils also generate enhanced amount of ONOOby NOS (El Benna et al., 2002). Chemiluminescence assays demonstrated significant activation of the neutrophil myeloperoxidase H2O2 system in synovial fluids from patients with RA further suggesting that oxidative stress may contribute to the cyclic, self- perpetuating nature of rheumatoid inflammation. ROS, produced by activated phagocytes alters the antigenic behaviour of immunoglobulin G (IgG). Radical-exposed IgG is able to bind rheumatoid factor and results in the generation of C3alpha complement component. This reaction may be selfperpetuating within the rheumatoid joint, suggesting that free radicals play a role in the chronicity of rheumatoid inflammation (Newkirk et al., 2002).

Pro-inflammatory cytokines´ presence and activity undoubtedly subjects to rheumatoid synovitis governing a variety of pathological processes including cell activation, cell proliferation, tissue resorption and chemotaxis (Schett et al., 2000). Experimental evidence confirms cytokine- induced oxidative stress in rheumatoid synovium. Thioredoxin, a cellular catalyst induced by oxidative stress, is found in high amounts in RA synovial cells and tissue. Thioredoxin acts as a co- factor for tumor necrosis factor- alpha (TNF- induced synthesis of interleukins (IL-6 and IL-8) in synovial fibroblastlike cells (Yoshida et al., 1999). Edaravone, which is a clinically available antioxidant, suppresses IL-1β- induced synovial cells proliferation and migration under *in vitro* conditions (Arii et al., 2006). Nacetylcytein, a known thiol antioxidant, abrogated L-6 - induced proliferation of RA patients synovial fibroblasts (Ji-Yeon et al., 2000).

ROS are documented as mediators of synovial inflammation. Excessive production of ROS at the site of inflammation contributes to the inflammatory process in general, by induction of the expression of adhesion molecules, pro-inflammatory cytokines, and chemoattractants. Furthermore, ROS can directly increase tissue destruction through inactivation of the major inhibitor of degrading enzymes, 1-antiproteinase, which subsequently leads to the activation of extracellular matrix-degrading metalloproteinases (MMPs) (Maurice et al., 1997). 3-nitrotyrosine (3- NT) has been identified as a stable end product and marker of

Oxidative Stress in Human Autoimmune Joint Diseases 443

partner HIF-1β. In rheumatic patients, joint movement in a ratio of normally functional joint increases the pO2 leading to re-oxygenation. In RA cycling transient episodes of hypoxia/reoxygenation increase levels of ROS. Increased mitochondrial ROS levels stabilize the transcription factor HIF- 1 (Chandel et al., 2000). Of particular relevance to RA was the marked attenuation of synovitis and articular damage in an adjuvant arthritis model when HIF- 1 was absent. In RA synovitis, HIF-1a protein accumulates and translocates to the nucleus and directly activates transcription of pro-angiogenic factor like VEGF. VEGF is highly inducible by hypoxia which occurs in the inflamed joints of RA. The HIF-1 translocates and binds to core DNA motif in the hypoxia responsive elements (HRE) which are associated with target genes such as VEGF and induces its gene expression and thereby angiogenesis. HIF-1 is expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone (Hollander et al., 2001). The synovial expression of HIF-1 also showed a mixed nuclear and cytoplasmic pattern mostly seen in lining cells, stromal cells, mononuclear cells, and blood vessels (Giatromanolaki et al., 2002). The number of HIF-1 -positive cells correlated strongly with the number of blood vessels in RA synovial tissue and with inflammatory endothelial cell infiltration (blood vessels), cell proliferation (Ki67) and the synovitis score (Brouwer et al., 2009). Reduction in the degradation rate of HIFs (HIF-1, and HIF-2) as occurs under hypoxic stress throughout arthritis, results in increased steady- state level of HIFs proteins and up-regulation of the neo- angiogenic process. Neo-angiogenic VEGF/ KDR pathway was shown persistently increased in RA, as indeed was microvessel density and the expression of PD- ECGF, irrespective of the extent of HIF expression (Giatromanolaki et al., 2002). The direct link exists between accumulation of HIFs and overexpression of VEGF in RA. Neo-angiogenesis contributes to pathogenesis of RA encouraging synovitis, pannus formation and articular cartilage destruction. Concluding, the HIF seems to be a promising

factor that targets both synovitis and angiogenesis in RA.

compared to healthy controls. Production rates of O2

**5.1 Rheumatoid arthritis** 

**5. Oxidative stress in human autoimmune joint diseases** 

**5.1.1 Hematopoietic cells and tissue as oxidative stress targets in RA** 

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease primarily affecting synovial membranes of joints. Pathogenesis of RA is a multistep process where cellular and humoral interactions mediated by lymphocytes (T and B cells) and non hematopoietic cells like fibroblasts, connective tissue cells, and bone cells play a role. At site of inflammation, activation of T cells and macrophages leads to a large increase in oxygen consumption, whose corollary is increased release of ROS (Afonso et al., 2007). Both, hematopoietic, and connective tissue cells are subject of oxidative stress process in arthritis.

Activity of NADPH- oxidase is enhanced in circulating neutrophils and monocytes of RA patients. These phagocytic cells synthesize two- to eight- fold higher amounts of O2•\_ when

rheumatic patients positively correlate with the plasma levels of TNF- (Miesel et al., 1996). Other parameters of oxidative stress like decrease in neutrophil SOD activity and an increase in the levels of "loose" iron in the plasmalemma of RA neutrophils and monocytes

•\_ in neutrophils and monocytes from

inflammation and RNS production. Nitrated proteins are generated in inflamed tissues by inflammatory cells producing ONOO- , a naturally occurring nitrating agent. A study by Khan & Siddiqui (2006) further investigated the binding characteristics of naturally occurring antibodies to 3- NT present in synovial fluid. Antibodies to 3- NT were found higher in the synovial fluid of RA patients.

*Ex-vivo* cultured rheumatoid synovium produces a significant amount of nitrite, and the addition of N- methylarginine (L-NMMA) significantly inhibits NO production. *In vitro* study revealed that NO production from freshly isolated synovial cells was up- regulated by stimulation with a combination of IL-1, TNF- and lipopolysaccharide. Inducible NOS expression was induced when human chondrocytes were stimulated with IL-1, TNF- or endotoxin in a dose- and time-dependent manner. Inflammatory reaction in the synovium of RA patients could be augmented by the autocrine or other cytokine-induced production of IL-6 with subsequent generation of ROS in the synoviocytes. Fibroblast- like synoviocytes proliferation by IL-6 was inhibited by N- acetylcysteine. Oxidative stress of RA synovial tissue can cause DNA damage and suppress the DNA mismatch repair (MMR) system in cultured synoviocytes. DNA MMR enzyme expression is greatest in the synovial intimal lining layer, where maximal oxidative stress in RA occurs (Šimelyte et al., 2004).

#### **4.2 Cycles of hypoxia/ reoxygenation in inflammatory synovium**

Angiogenesis in the synovial membrane is an important early step in pathogenesis of RA, in the perpetuation of the disease and may precede other pathological features. Elevated levels of pro-angiogenic factor, the vascular endothelial growth factor VEGF, are expressed in synovium, synovial fluids, and serum of RA patients. An increased VEGF level in RA is responsible for subsequent joint destruction. The synovium of RA is hypoxic as a result of synovial tissue proliferation outpacing in angiogenesis. Hypoxia is a potent inducer of cytokines, matrix degrading enzymes, angiogenic factors that play a central role in the inflammatory response. Hypoxia- inducible factor- 1 (HIF-1) is a key transcription factor which is highly inducible by hypoxia and expressed predominantly in synovium of RA. Expression of HIF-1 is critical for joint inflammation. RA synovial fluids are hypoxic, acidic with low glucose and elevated lactate concentrations. This biochemical profile is indicative of a chronically hypoxic microenvironment that compensates by anaerobic metabolism. The oxygen partial pressure (pO2) is extremely low in RA synovial fluids, and correlates with elevated plasma levels of lactate. Inflamed synovitis is a hallmark of RA which is hypoxic in nature (Shankar et al., 2009). The biological basis of this process relates to hypoxia- inducible factor (HIF). The response of mammalian cells to hypoxia is mediated partly through stabilization of certain transcription factors including HIF-1 and HIF-2. These oxygen sensitive transcription factors are multifunctional. Firstly, they program the cells to anaerobic metabolism, secondly, they enhance cell survival by inhibiting apoptosis, and thirdly they improve the supply of oxygen by promoting angiogenesis and increase oxygencarrying capacity. In view of the crucial role of HIF-1 in cellular adaptation to hypoxia, its regulation needs to be rapidly responsive to changes in the cellular oxygen supply. Inhibition of degradation is the primary mechanism by which hypoxia directly regulates HIF- 1. In the absence of oxygen critical step of degradation process of HIF-1, the hydroxylation of proline and asparagine residues, becomes rate limiting, thus preventing HIF-1 from being degraded and leaving it free to bind to its constitutively expressed

inflammation and RNS production. Nitrated proteins are generated in inflamed tissues by

Khan & Siddiqui (2006) further investigated the binding characteristics of naturally occurring antibodies to 3- NT present in synovial fluid. Antibodies to 3- NT were found

*Ex-vivo* cultured rheumatoid synovium produces a significant amount of nitrite, and the addition of N- methylarginine (L-NMMA) significantly inhibits NO production. *In vitro* study revealed that NO production from freshly isolated synovial cells was up- regulated by stimulation with a combination of IL-1, TNF- and lipopolysaccharide. Inducible NOS expression was induced when human chondrocytes were stimulated with IL-1, TNF- or endotoxin in a dose- and time-dependent manner. Inflammatory reaction in the synovium of RA patients could be augmented by the autocrine or other cytokine-induced production of IL-6 with subsequent generation of ROS in the synoviocytes. Fibroblast- like synoviocytes proliferation by IL-6 was inhibited by N- acetylcysteine. Oxidative stress of RA synovial tissue can cause DNA damage and suppress the DNA mismatch repair (MMR) system in cultured synoviocytes. DNA MMR enzyme expression is greatest in the synovial intimal

lining layer, where maximal oxidative stress in RA occurs (Šimelyte et al., 2004).

Angiogenesis in the synovial membrane is an important early step in pathogenesis of RA, in the perpetuation of the disease and may precede other pathological features. Elevated levels of pro-angiogenic factor, the vascular endothelial growth factor VEGF, are expressed in synovium, synovial fluids, and serum of RA patients. An increased VEGF level in RA is responsible for subsequent joint destruction. The synovium of RA is hypoxic as a result of synovial tissue proliferation outpacing in angiogenesis. Hypoxia is a potent inducer of cytokines, matrix degrading enzymes, angiogenic factors that play a central role in the inflammatory response. Hypoxia- inducible factor- 1 (HIF-1) is a key transcription factor which is highly inducible by hypoxia and expressed predominantly in synovium of RA. Expression of HIF-1 is critical for joint inflammation. RA synovial fluids are hypoxic, acidic with low glucose and elevated lactate concentrations. This biochemical profile is indicative of a chronically hypoxic microenvironment that compensates by anaerobic metabolism. The oxygen partial pressure (pO2) is extremely low in RA synovial fluids, and correlates with elevated plasma levels of lactate. Inflamed synovitis is a hallmark of RA which is hypoxic in nature (Shankar et al., 2009). The biological basis of this process relates to hypoxia- inducible factor (HIF). The response of mammalian cells to hypoxia is mediated partly through stabilization of certain transcription factors including HIF-1 and HIF-2. These oxygen sensitive transcription factors are multifunctional. Firstly, they program the cells to anaerobic metabolism, secondly, they enhance cell survival by inhibiting apoptosis, and thirdly they improve the supply of oxygen by promoting angiogenesis and increase oxygencarrying capacity. In view of the crucial role of HIF-1 in cellular adaptation to hypoxia, its regulation needs to be rapidly responsive to changes in the cellular oxygen supply. Inhibition of degradation is the primary mechanism by which hypoxia directly regulates HIF- 1. In the absence of oxygen critical step of degradation process of HIF-1, the hydroxylation of proline and asparagine residues, becomes rate limiting, thus preventing HIF-1 from being degraded and leaving it free to bind to its constitutively expressed

**4.2 Cycles of hypoxia/ reoxygenation in inflammatory synovium** 

, a naturally occurring nitrating agent. A study by

inflammatory cells producing ONOO-

higher in the synovial fluid of RA patients.

partner HIF-1β. In rheumatic patients, joint movement in a ratio of normally functional joint increases the pO2 leading to re-oxygenation. In RA cycling transient episodes of hypoxia/reoxygenation increase levels of ROS. Increased mitochondrial ROS levels stabilize the transcription factor HIF- 1 (Chandel et al., 2000). Of particular relevance to RA was the marked attenuation of synovitis and articular damage in an adjuvant arthritis model when HIF- 1 was absent. In RA synovitis, HIF-1a protein accumulates and translocates to the nucleus and directly activates transcription of pro-angiogenic factor like VEGF. VEGF is highly inducible by hypoxia which occurs in the inflamed joints of RA. The HIF-1 translocates and binds to core DNA motif in the hypoxia responsive elements (HRE) which are associated with target genes such as VEGF and induces its gene expression and thereby angiogenesis. HIF-1 is expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone (Hollander et al., 2001). The synovial expression of HIF-1 also showed a mixed nuclear and cytoplasmic pattern mostly seen in lining cells, stromal cells, mononuclear cells, and blood vessels (Giatromanolaki et al., 2002). The number of HIF-1 -positive cells correlated strongly with the number of blood vessels in RA synovial tissue and with inflammatory endothelial cell infiltration (blood vessels), cell proliferation (Ki67) and the synovitis score (Brouwer et al., 2009). Reduction in the degradation rate of HIFs (HIF-1, and HIF-2) as occurs under hypoxic stress throughout arthritis, results in increased steady- state level of HIFs proteins and up-regulation of the neo- angiogenic process. Neo-angiogenic VEGF/ KDR pathway was shown persistently increased in RA, as indeed was microvessel density and the expression of PD- ECGF, irrespective of the extent of HIF expression (Giatromanolaki et al., 2002). The direct link exists between accumulation of HIFs and overexpression of VEGF in RA. Neo-angiogenesis contributes to pathogenesis of RA encouraging synovitis, pannus formation and articular cartilage destruction. Concluding, the HIF seems to be a promising factor that targets both synovitis and angiogenesis in RA.

#### **5. Oxidative stress in human autoimmune joint diseases**

#### **5.1 Rheumatoid arthritis**

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease primarily affecting synovial membranes of joints. Pathogenesis of RA is a multistep process where cellular and humoral interactions mediated by lymphocytes (T and B cells) and non hematopoietic cells like fibroblasts, connective tissue cells, and bone cells play a role. At site of inflammation, activation of T cells and macrophages leads to a large increase in oxygen consumption, whose corollary is increased release of ROS (Afonso et al., 2007). Both, hematopoietic, and connective tissue cells are subject of oxidative stress process in arthritis.

#### **5.1.1 Hematopoietic cells and tissue as oxidative stress targets in RA**

Activity of NADPH- oxidase is enhanced in circulating neutrophils and monocytes of RA patients. These phagocytic cells synthesize two- to eight- fold higher amounts of O2•\_ when compared to healthy controls. Production rates of O2 •\_ in neutrophils and monocytes from rheumatic patients positively correlate with the plasma levels of TNF- (Miesel et al., 1996). Other parameters of oxidative stress like decrease in neutrophil SOD activity and an increase in the levels of "loose" iron in the plasmalemma of RA neutrophils and monocytes

Oxidative Stress in Human Autoimmune Joint Diseases 445

In RA oxidative stress also features by oxidation of low- density lipoproteins (LDL). Oxidized LDLs promote inflammatory changes including local up- regulation of adhesion

RA tissue has evidence of microsatellite instability reflecting ongoing mutagenesis. Such mutagenesis is normally corrected by DNA repair systems including the mismatch repair

> activated phagocytic cells in connective tissue

Fig. 1. Connective tissue and cells as targets of oxidative stress in rheumatoid arthritis.

Systemic lupus erythematosus (SLE) is a prototype autoimmune, multisystem and multifactorial disease characterized by the presence of auto-antibodies to a variety of nuclear antigens such as DNA and histones, as well as protein antigens and protein–nucleic acid complexes. The initial immunizing antigen(s) that drive the development of SLE are unknown, but characteristics of the immune response in SLE suggest that it is an antigendriven condition. Multiorgan inflammatory lesions also involve the joints. Immune complex deposits in the synovium are associated with mild inflammation and cartilage destruction is seldom severe. The arthritis of SLE is described as non-destructive and non-deforming. Immune complex deposits in the synovium trigger inflammatory reaction, which led continuously to cartilage destruction (Khan Siddiqui, 2006). Free radicals synthesize autoantigens, which contribute to disease development. Oxidative stress and inflammation

INFLAMMATORY SYNOVIUM •DNA oxidative damage •Lipid peroxidation • Synovial phagocytes secrete large amounts of superoxide •hyporesponsive T cells • increased VEGF level

> CARTILAGE DAMAGE •Lipid peroxidation • Nitration of proteins sehyporesponsive T cells • increased VEGF level

hypoxia

BONE DAMAGE • activated osteoclasts secrete superoxide

O2 - O2 - O2 -

system, which is defective in RA, probably due to oxidative stress.

O2 -

O2 secretion of superoxide

O2 -

molecules and chemokines.

activation

OXIDATIVE STRESS IN RHEUMATOID ARTHRITIS

**6. Systemic lupus erythematosus** 

were also observed (Ostrakhovitch Afanas´ev, 2001). A characteristic feature of RA ROSproducing neutrophils is their functional state. The production of O2 •\_ by blood neutrophils from RA patients in response to N-formyl-methionylleucyl-phenylalanine, a stimulatory agent, was greater in arthritic than control blood neutrophils (Eggleton et al., 1995). The study suggested that stimulated sub-population of neutrophils are source of ROS in arthritis.

#### **5.1.2 Connective cells and tissue as oxidative stress targets in RA**

Studies of RA synovial fluid (SF) and tissue have demonstrated oxidative damage to the tissue. Signs of oxidative stress such as DNA oxidative damage, and lipid peroxidation are present in the inflammatory synovium of RA patients. Immunohistochemical analysis revealed increased staining of 8- oxo- 7,8- dihydro- 2´- deoxyguanine, a marker of DNA oxidative damage, and increased staining of 4-hydroxy-2-nonenal in the lining and sublining layers of the RA inflammatory synovium (Šimelyte et al., 2004). Furthermore, it was shown that increased lipid peroxidation damage in RA inflammatory synovium is proportional to the levels of hypoxia in the joint, disease activity and angiogenic marker expression (Biniecka et al., 2009). Synovial fluids macrophages produce increased amounts of O2•\_. Also neutrophils from synovial fluids of rheumatic patients generate increased amounts of O2 •\_ possibly because of their exposure to cytokines present in synovial fluids (Hitchon El-Gabalawy, 2004). Chronic oxidative stress contributes to functional hyporesponsiveness of synovial T lymphocytes. The impaired mitogenic responses of SF T lymphocytes correlated with a significant decrease in the levels of the intracellular redoxregulating agent glutathione (GSH) (Maurice et al., 1997).

Indirect evidence for ROS implication in cartilage degradation comes from the presence of lipid peroxidation products, nitrite, nitrotyrosine, a nitrated type II collagen peptide, modified low-density lipoprotein (LDL) and oxidized IgG in the biological fluids of patients with arthritis. Furthermore, nitrotyrosine, nitrated proteins and oxidized LDL (ox-LDL) have been found to be accumulated in cartilage of arthritic patients demonstrating the direct implication of ROS in some joint diseases. Rheumatoid arthritis is characterized by irreversible damage to the cartilage matrix caused by enzymatic degradation of the proteins, e.g., collagen type II (CII), and proteoglycans of cartilage (e.g., aggrecan) (Billinghurst et al., 1997). As a result of the breakdown of the proteins and proteoglycans, CII degradation products and sulfated glycosaminoglycans (GAGs) appear in SFs of the affected joints. The level of GAGs in SF indicates the extent of proteoglycan degradation (Lark et al., 1997). Hydrogene peroxide, and singlet oxygen accelerate bone resorption by osteoclasts. Osteoclasts generate ROS through ˮNADPH- oxidase dependent mechanismsˮ. Studies involving assays of nitrotyrosine residues in synovial tissues from patients with RA or exposure of chondrocytes to synthetic peroxynitrite *in vitro* have established that combination of the O2 .- anion to nitric oxide (NO) causes cartilage damage (Abramson et al., 2001).

The cytokine network is involved in the pathogenesis of RA. IL-1 is a key mediator of bone resorption and cartilage destruction in arthritis. The cytokine activates bone resorption through its effects on osteoclast differentiation and activation. IL-1 destructs cartilage by stimulating release of MMPs from fibroblasts and chondrocytes. Neutralizing ROS activity significantly attenuated IL-1- induced collagenase gene expression in bovine chondrocytes (Lo et al., 1998).

were also observed (Ostrakhovitch Afanas´ev, 2001). A characteristic feature of RA ROS-

from RA patients in response to N-formyl-methionylleucyl-phenylalanine, a stimulatory agent, was greater in arthritic than control blood neutrophils (Eggleton et al., 1995). The study suggested that stimulated sub-population of neutrophils are source of ROS in

Studies of RA synovial fluid (SF) and tissue have demonstrated oxidative damage to the tissue. Signs of oxidative stress such as DNA oxidative damage, and lipid peroxidation are present in the inflammatory synovium of RA patients. Immunohistochemical analysis revealed increased staining of 8- oxo- 7,8- dihydro- 2´- deoxyguanine, a marker of DNA oxidative damage, and increased staining of 4-hydroxy-2-nonenal in the lining and sublining layers of the RA inflammatory synovium (Šimelyte et al., 2004). Furthermore, it was shown that increased lipid peroxidation damage in RA inflammatory synovium is proportional to the levels of hypoxia in the joint, disease activity and angiogenic marker expression (Biniecka et al., 2009). Synovial fluids macrophages produce increased amounts of O2•\_. Also neutrophils from synovial fluids of rheumatic patients generate increased amounts of O2•\_ possibly because of their exposure to cytokines present in synovial fluids (Hitchon El-Gabalawy, 2004). Chronic oxidative stress contributes to functional hyporesponsiveness of synovial T lymphocytes. The impaired mitogenic responses of SF T lymphocytes correlated with a significant decrease in the levels of the intracellular redox-

Indirect evidence for ROS implication in cartilage degradation comes from the presence of lipid peroxidation products, nitrite, nitrotyrosine, a nitrated type II collagen peptide, modified low-density lipoprotein (LDL) and oxidized IgG in the biological fluids of patients with arthritis. Furthermore, nitrotyrosine, nitrated proteins and oxidized LDL (ox-LDL) have been found to be accumulated in cartilage of arthritic patients demonstrating the direct implication of ROS in some joint diseases. Rheumatoid arthritis is characterized by irreversible damage to the cartilage matrix caused by enzymatic degradation of the proteins, e.g., collagen type II (CII), and proteoglycans of cartilage (e.g., aggrecan) (Billinghurst et al., 1997). As a result of the breakdown of the proteins and proteoglycans, CII degradation products and sulfated glycosaminoglycans (GAGs) appear in SFs of the affected joints. The level of GAGs in SF indicates the extent of proteoglycan degradation (Lark et al., 1997). Hydrogene peroxide, and singlet oxygen accelerate bone resorption by osteoclasts. Osteoclasts generate ROS through ˮNADPH- oxidase dependent mechanismsˮ. Studies involving assays of nitrotyrosine residues in synovial tissues from patients with RA or exposure of chondrocytes to synthetic

The cytokine network is involved in the pathogenesis of RA. IL-1 is a key mediator of bone resorption and cartilage destruction in arthritis. The cytokine activates bone resorption through its effects on osteoclast differentiation and activation. IL-1 destructs cartilage by stimulating release of MMPs from fibroblasts and chondrocytes. Neutralizing ROS activity significantly attenuated IL-1- induced collagenase gene expression in bovine chondrocytes

•\_ by blood neutrophils

.- anion to nitric oxide (NO)

producing neutrophils is their functional state. The production of O2

**5.1.2 Connective cells and tissue as oxidative stress targets in RA** 

regulating agent glutathione (GSH) (Maurice et al., 1997).

peroxynitrite *in vitro* have established that combination of the O2

causes cartilage damage (Abramson et al., 2001).

(Lo et al., 1998).

arthritis.

In RA oxidative stress also features by oxidation of low- density lipoproteins (LDL). Oxidized LDLs promote inflammatory changes including local up- regulation of adhesion molecules and chemokines.

RA tissue has evidence of microsatellite instability reflecting ongoing mutagenesis. Such mutagenesis is normally corrected by DNA repair systems including the mismatch repair system, which is defective in RA, probably due to oxidative stress.

Fig. 1. Connective tissue and cells as targets of oxidative stress in rheumatoid arthritis.

#### **6. Systemic lupus erythematosus**

Systemic lupus erythematosus (SLE) is a prototype autoimmune, multisystem and multifactorial disease characterized by the presence of auto-antibodies to a variety of nuclear antigens such as DNA and histones, as well as protein antigens and protein–nucleic acid complexes. The initial immunizing antigen(s) that drive the development of SLE are unknown, but characteristics of the immune response in SLE suggest that it is an antigendriven condition. Multiorgan inflammatory lesions also involve the joints. Immune complex deposits in the synovium are associated with mild inflammation and cartilage destruction is seldom severe. The arthritis of SLE is described as non-destructive and non-deforming. Immune complex deposits in the synovium trigger inflammatory reaction, which led continuously to cartilage destruction (Khan Siddiqui, 2006). Free radicals synthesize autoantigens, which contribute to disease development. Oxidative stress and inflammation

Oxidative Stress in Human Autoimmune Joint Diseases 447

products (AOPP), which form by the reaction between chlorinated oxidants (HOCl/OCl\_) and proteins. AOPP are defined as dityrosine-containing cross-linked proteins. MDA is one of the end products of lipid peroxidation induced by ROS and marker of oxidative stress in lipids. 8-Hydroxy-2´-deoxyguanosine (8-OHdG) has been recognized as a biomarker of oxidative DNA damage by endogenously generated oxygen radicals. In addition, oxidative stress may be estimated by levels of enzymes like thioredoxin, a protein with reduction / oxidation active disulfide / dithiol groups in its active site. Signs of oxidative stress are apparent on cells and tissue affected by arthritis. Advanced glycation end products (AGEs), which are formed during the Maillard reaction by non-enzymatic glycation, and oxidation of proteins were detected in the synovial lining, sublining, and endothelium in RA patiens. CML showed positive immunostaining in some RA macrophages (CD68+) and T cells (CD45RO+) (Drinda et al., 2002). High levels of protein carbonyl (PCO), and AGE products were found in serum of collagen – induced arthritis mice (Choi, 2007). Plasma MDA concentrations are significantly higher in RA patients (Sarban et al., 2005). An excessive degree of oxidative stress in RA patients confirm decline of protein thiol levels and lower activity of antioxidant enzymes like glutathione (GSH), GPx, and CuZn SOD in blood of these patients (Seven et al., 2008). The study by Jikimoto (Jikimoto et al., 2001) discovered elevated levels of TRX in plasma, and synovial fluid of RA patients. Plasma TRX correlated with urinary excretion of 8-hydroxy-29-deoxyguanosine (8-OHdG). Serum MDA levels were increased in SLE patients, while serum antioxidant levels were decreased in these

patients what confirms oxidative stress in the pathology of SLE (Taysi et al., 2002 a).

**8. Oxidative stress in autoimmune joint diseases: Beneficial or harmful?** 

The recent research describes dual role of ROS in autoimmune- joint diseases. The ROS production by the activated NOX- family (NADPH) of oxidases has a principal function throughout the priming phase of the development of autoimmune joint diseases. The NADPH oxidase- deficient knockout mice developed a serious inflammatory arthritis with extensive bone erosions and a massive osteolysis (van de Loo et al., 2003). ROS might reduce arthritis development influencing circulating inflammatory cells before they reach the joint. ROS limit T cell responses to self- antigens inducing their apoptosis, what inhibits disease development (Olofsson et al., 2003). Arthritis development correlates with functionality of NADPH- oxidase complex, which is determined by the neutrophil cytosolic factor 1 (Ncf 1), a phagocytic oxidase. The naturally occurring polymorphism of Ncf1 allele limiting the NADPH functionality promotes activation of arthritogenic CD4+ cells. An expansion of arthritogenic T cells owing to the lower activity of the NADPH- oxidase complex was observed in the DA Ncf 1 allele (Olofsson et al., 2003). An increased severity of arthritis was observed in animals with either loss of function mutations or deletions in components of the phagocyte NADPH- oxidase like the p47phox (van de Loo et al., 2003). Furthermore, the effect of NOX-derived ROS might depend on the arthritis model: in IFNgamma (INF- ) enhanced imine complex arthritis, the p47phox-deficient animals showed a less severe joint destruction and decreased chondrocyte death (Lem van Lent et al., 2005).

The NADPH- oxidase- derived superoxide is not exclusive effector molecule in arthritis. Superoxide dismuthase (SOD) catalyses the dismutation of superoxide anion to oxygen and hydrogen peroxide. Three SOD enzyme isoforms have been characterized in humans. SOD I, and SOD III are copper, zinc, enzyme isoforms. SOD II is a manganese enzyme isoform.

are interrelated in SLE. Malondialdehyd (MDA) levels positively correlated with levels of interferon- (INF-), and interleukin- 12 (IL- 12) in lupus disease (Shah et al., 2010).

#### **6.1 Hematopoietic cells and tissue as oxidative stress targets in SLE**

Oxidative damage to red blood cells and leukocytes are hallmarks of oxidative stress in SLE. Lipid peroxidation, in SLE erythrocytes, and generation of O2 .- and H2O2 in leukocytes were increased in SLE patients when compared to healthy controls (Tewthanom et al., 2008). Destroying effects of free radicals in lupus disease are apparent on the reactions with serum proteins. Oxidation makes the proteins more fragile. Modified proteins become autoantigens, and more, their presence enhances oxidative stress in serum. 4-hydroxy-2 nonenal (4- HNE), which is a marker of lipid peroxidation in SLE, after binding protein forms dangerous protein adducts. The target of 4- HNE modification reported was catalase, a membrane protein of red blood cells (D' Souza et al., 2008). Hydroxyl radical binds human serum albumin. The increase in total serum protein carbonyl levels in the SLE patients was largely due to an increase in oxidized albumin (Shjekh et al., 2007).

In lupus disease, oxidative stress presents by lipid peroxidation mostly. Moreover it was discovered that lipid peroxidation influences pathogenesis of the disease. The disease activity score (SLEDAI) positively correlates with serum levels of MDA. T-lymphocyte apoptosis and MDA were positively associated with disease activity (Shah et al., 2011). MDA- modified proteins as the catalase, and SOD are targets of IgG circulated autoantibodies promoting disease development. On the other hand, SLEDAI score correlates negatively with serum antioxidant enzymes as SOD, and glutathione peroxidase (GPx) (Taysi et al., 2002 b). Auto- antibodies against plasma lipoproteins have been reported in SLE patients (Batuca et al., 2007). The study further documented incidence of antibodies toward high density lipoproteins (HDL) in patients with SLE, and identified Apo A-I as a target of oxidative damage. The incidence of auto-antibodies against lipid particles correlated with reduced activity of paraoxonase (PON), which is the most active antioxidant enzyme in lipids, and also prevents lipid peroxidation of low density lipoproteins (LDL).

#### **6.2 Connective cells and tissue as oxidative stress targets in SLE**

The arthritis of SLE is described as non- destructive and non- deforming. Immune complex deposits in the synovium are associated with mild inflammation and cartilage destruction is seldom severe. In SLE patients, NO and its intermediates may be mediators of inflammatory arthritis. Antibodies against 3- nitrotyrosine (3- NT) were found elevated in sera, and synovial fluid of SLE patients. Interestingly, the sera levels were much higher than in synovial fluid (Khan & Siddiqui, 2006).

#### **7. Markers of oxidative stress in human autoimmune joint diseases**

A short lifetime of free radicals in body fluids restricts their direct estimation instead effects of oxygen on lipids, proteins, and nucleic acids molecules are used. There are many chemical modifiers of protein, and lipid structures, whose activity is accelerated by oxidative stress. *N*-carboxymethyllysine (CML) represents a chemically modified amino acid and originates *in vivo* from carbohydrate as well as from lipid derived precursors. Oxidative damage to proteins is also reflected by increased levels of advanced oxidation protein

are interrelated in SLE. Malondialdehyd (MDA) levels positively correlated with levels of

Oxidative damage to red blood cells and leukocytes are hallmarks of oxidative stress in SLE. Lipid peroxidation, in SLE erythrocytes, and generation of O2.- and H2O2 in leukocytes were increased in SLE patients when compared to healthy controls (Tewthanom et al., 2008). Destroying effects of free radicals in lupus disease are apparent on the reactions with serum proteins. Oxidation makes the proteins more fragile. Modified proteins become autoantigens, and more, their presence enhances oxidative stress in serum. 4-hydroxy-2 nonenal (4- HNE), which is a marker of lipid peroxidation in SLE, after binding protein forms dangerous protein adducts. The target of 4- HNE modification reported was catalase,

serum albumin. The increase in total serum protein carbonyl levels in the SLE patients was

In lupus disease, oxidative stress presents by lipid peroxidation mostly. Moreover it was discovered that lipid peroxidation influences pathogenesis of the disease. The disease activity score (SLEDAI) positively correlates with serum levels of MDA. T-lymphocyte apoptosis and MDA were positively associated with disease activity (Shah et al., 2011). MDA- modified proteins as the catalase, and SOD are targets of IgG circulated autoantibodies promoting disease development. On the other hand, SLEDAI score correlates negatively with serum antioxidant enzymes as SOD, and glutathione peroxidase (GPx) (Taysi et al., 2002 b). Auto- antibodies against plasma lipoproteins have been reported in SLE patients (Batuca et al., 2007). The study further documented incidence of antibodies toward high density lipoproteins (HDL) in patients with SLE, and identified Apo A-I as a target of oxidative damage. The incidence of auto-antibodies against lipid particles correlated with reduced activity of paraoxonase (PON), which is the most active antioxidant enzyme in lipids, and also prevents lipid peroxidation of low density lipoproteins (LDL).

The arthritis of SLE is described as non- destructive and non- deforming. Immune complex deposits in the synovium are associated with mild inflammation and cartilage destruction is seldom severe. In SLE patients, NO and its intermediates may be mediators of inflammatory arthritis. Antibodies against 3- nitrotyrosine (3- NT) were found elevated in sera, and synovial fluid of SLE patients. Interestingly, the sera levels were much higher than in

A short lifetime of free radicals in body fluids restricts their direct estimation instead effects of oxygen on lipids, proteins, and nucleic acids molecules are used. There are many chemical modifiers of protein, and lipid structures, whose activity is accelerated by oxidative stress. *N*-carboxymethyllysine (CML) represents a chemically modified amino acid and originates *in vivo* from carbohydrate as well as from lipid derived precursors. Oxidative damage to proteins is also reflected by increased levels of advanced oxidation protein

Souza et al., 2008). Hydroxyl radical binds human

interferon- (INF-), and interleukin- 12 (IL- 12) in lupus disease (Shah et al., 2010).

**6.1 Hematopoietic cells and tissue as oxidative stress targets in SLE** 

largely due to an increase in oxidized albumin (Shjekh et al., 2007).

**6.2 Connective cells and tissue as oxidative stress targets in SLE** 

**7. Markers of oxidative stress in human autoimmune joint diseases** 

a membrane protein of red blood cells (D'

synovial fluid (Khan & Siddiqui, 2006).

products (AOPP), which form by the reaction between chlorinated oxidants (HOCl/OCl\_) and proteins. AOPP are defined as dityrosine-containing cross-linked proteins. MDA is one of the end products of lipid peroxidation induced by ROS and marker of oxidative stress in lipids. 8-Hydroxy-2´-deoxyguanosine (8-OHdG) has been recognized as a biomarker of oxidative DNA damage by endogenously generated oxygen radicals. In addition, oxidative stress may be estimated by levels of enzymes like thioredoxin, a protein with reduction / oxidation active disulfide / dithiol groups in its active site. Signs of oxidative stress are apparent on cells and tissue affected by arthritis. Advanced glycation end products (AGEs), which are formed during the Maillard reaction by non-enzymatic glycation, and oxidation of proteins were detected in the synovial lining, sublining, and endothelium in RA patiens. CML showed positive immunostaining in some RA macrophages (CD68+) and T cells (CD45RO+) (Drinda et al., 2002). High levels of protein carbonyl (PCO), and AGE products were found in serum of collagen – induced arthritis mice (Choi, 2007). Plasma MDA concentrations are significantly higher in RA patients (Sarban et al., 2005). An excessive degree of oxidative stress in RA patients confirm decline of protein thiol levels and lower activity of antioxidant enzymes like glutathione (GSH), GPx, and CuZn SOD in blood of these patients (Seven et al., 2008). The study by Jikimoto (Jikimoto et al., 2001) discovered elevated levels of TRX in plasma, and synovial fluid of RA patients. Plasma TRX correlated with urinary excretion of 8-hydroxy-29-deoxyguanosine (8-OHdG). Serum MDA levels were increased in SLE patients, while serum antioxidant levels were decreased in these patients what confirms oxidative stress in the pathology of SLE (Taysi et al., 2002 a).

#### **8. Oxidative stress in autoimmune joint diseases: Beneficial or harmful?**

The recent research describes dual role of ROS in autoimmune- joint diseases. The ROS production by the activated NOX- family (NADPH) of oxidases has a principal function throughout the priming phase of the development of autoimmune joint diseases. The NADPH oxidase- deficient knockout mice developed a serious inflammatory arthritis with extensive bone erosions and a massive osteolysis (van de Loo et al., 2003). ROS might reduce arthritis development influencing circulating inflammatory cells before they reach the joint. ROS limit T cell responses to self- antigens inducing their apoptosis, what inhibits disease development (Olofsson et al., 2003). Arthritis development correlates with functionality of NADPH- oxidase complex, which is determined by the neutrophil cytosolic factor 1 (Ncf 1), a phagocytic oxidase. The naturally occurring polymorphism of Ncf1 allele limiting the NADPH functionality promotes activation of arthritogenic CD4+ cells. An expansion of arthritogenic T cells owing to the lower activity of the NADPH- oxidase complex was observed in the DA Ncf 1 allele (Olofsson et al., 2003). An increased severity of arthritis was observed in animals with either loss of function mutations or deletions in components of the phagocyte NADPH- oxidase like the p47phox (van de Loo et al., 2003). Furthermore, the effect of NOX-derived ROS might depend on the arthritis model: in IFNgamma (INF- ) enhanced imine complex arthritis, the p47phox-deficient animals showed a less severe joint destruction and decreased chondrocyte death (Lem van Lent et al., 2005).

The NADPH- oxidase- derived superoxide is not exclusive effector molecule in arthritis. Superoxide dismuthase (SOD) catalyses the dismutation of superoxide anion to oxygen and hydrogen peroxide. Three SOD enzyme isoforms have been characterized in humans. SOD I, and SOD III are copper, zinc, enzyme isoforms. SOD II is a manganese enzyme isoform.

Oxidative Stress in Human Autoimmune Joint Diseases 449

2g/kg body weight) applied to arthritic Lewis rats, delayed incidence of paw oedema in these rats. Histological studies revealed a decreased inflammatory cells infiltration of superficial layer of synovium, and decreased synovial SOD activity in these rats (Sakai et al., 1999). In addition, administration of rutin (vitamin P) strongly inhibited spontaneous ROS production in RA neutrophils (Ostrakhovitch Afanas´ev, 2001). Vitamin E exerts analgetic effects. The results from a double blind clinical trials suggested that a dose of 400–1200 mg of -tocopherol daily was effective with respect to various pain parameters such as pain on pressure, pain at rest, and pain on movement. The postulated analgesic properties of vitamin E seem to be correlated to vitamin E plasma concentrations. Patients with active RA supplemented with a combination of conjugated fatty acids and vitamin E improved their disease activity score. The diet in these patients relieved from night and activity pain, and morning stiffness (Aryaeian et al., 2009). The analgesic effect of vitamin E is independent of a peripheral anti-inflammatory action, thereby suggesting a central rather than peripheral action. Dietary antioxidant micronutrients act as scavengers of reactive oxygen radicals and may protect against free radical mediated tissue damage in an inflamed joint. Vitamin E supplemented in soyabean oil reduced anti- double- stranded DNA IgG antibodies (Chia-

Eicosapentaenoic acid and docosahexaenoic acid (EPA and DHA respectively) exert potent antioxidant properties. Supplementation with these acids to lupus patients restored levels of antioxidant enzymes as the GPx, and SOD to baseline levels (Mohan, Das, 1997). Lupus autoimmune female mice fed with fish oil exhibited significantly higher liver of catalase, SOD, and GPx. The diet was shown to be beneficial against oxidative damage of hepatic tissue (Bhattacharya et al., 2003). Sera of lupus mice treated with conjugated linoleic acid (CLA) contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, gammaGCL, glutathione Stransferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. The activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in

In all cell types, oxygen metabolism can lead to the production of reactive oxygen / nitrogen species. Reactive species are known for their dual role as both beneficial and harmful. Deleterious effects of oxidative stress on cell components (proteins, lipids, DNA) include changes in their structure, and function. On the other hand, beneficial effects of ROS/RNS involve physiological cellular responses to noxious agents, e.g., in defense against infectious agents, in a number of cellular signaling pathways and in the induction of a mitogenic response. Oxidants even protect cells against oxidative stress and re-establish or maintain

Oxygen metabolism has an important role in the pathogenesis of autoimmune joint diseases. Reactive oxygen/nitrogen species are documented as mediators of synovial inflammation. Their excessive production at the site of inflammation contributes to inflammatory process

Chien Bi- Fong, 2005).

lupus mice (Bergamo et al., 2007).

redox homeostasis (Ortona et al., 2008).

**10. Conclusions and future perspectives** 

**9.3 Fatty acids** 

Whereas type I SOD is found mostly in the cytoplasm, nucleus, and intermembrane space of mitochondria, type III of SOD is extracellular. Manganese is cofactor for SOD type II, which is a mitochondrial enzyme. The enzyme is involved in the pathogenesis of inflammatory joint disease. In adjuvant model of arthritis the enzyme suppressed swelling, and retarded bone destruction (Shingu et al., 1994). An intra-articular injection of SOD significantly reduced synovitis in streptococcal wall cell (SWC)- model of arthritis. Intra- aricular injection of native SOD (bovine orgotein) produced greater clinical improvements than did intra- articular aspirin in patients with RA involving the knee (Afonso et al., 2007). Available data suggest a protective role of SOD in inflammatory joint disease. In mice that are genetically deficient in SOD III, both the severity of collagen-induced arthritis and the production of pro-inflammatory cytokines are increased. SODIII gene transferred subcutaneously or intra- articularly decreased the severity of experimental arthritis in rodents.

#### **9. Antioxidants as therapeutic possibilities in human autoimmune joint diseases**

#### **9.1 Antioxidant enzymes**

SODs exert protective effects in animal models of inflammation. In mice, genetically deficient in SOD III, both the severity of collagen- induced arthritis and the production of pro- inflammatory cytokines are increased (Ross et al., 2004). SOD III gene transfer into the knee decreased the severity of experimental arthritis in rodents. In humans, serum SOD III levels correlated negatively with disease activity. Despite conflicting results of native SOD (orgotein) in RA patients, SOD mimetics have shown beneficial effects. Until now, the most promising are nitroxide (tempol) and Mn (II) pentaazamacrocyclic ligand (M40403). *In vitro*, tempol diminishes hydroxyl radical production, and decreases the cytotoxic effects of hydrogen peroxide and peroxynitrite. Tempol decreased collagen- induced arthritis in rats (Cuzzocrea et al., 2001). The anti-inflammatory effects of M40403 ligand are related to superoxide elimination, and restraint of nitration of tyrosine residues in proteins. M40403 ligand diminishes the expression of adhesion molecules such as integrins or selectins decreasing influx of neutrophils to inflammatory site. M40403 ligand inhibits TNF- via blocking the nuclear factor kappa B (NF- B). Its beneficial effects have been reported in rats with collagen- induced arthritis (Cuzzocrea et al., 2005).

#### **9.2 Vitamins**

Micronutrient supplementation has been recommended to arthritic patients based on findings that the antioxidant status is very low in these patients. It has been documented that the mean synovial fluid concentrations of α-tocopherol are significantly decreased in arthritic patients compared to healthy controls. Also, levels of vitamin E in peripheral blood cell were significantly decreased in patients with active arthritis than in healthy subjects (Vasanthi et al., 2009). Concentrations of other antioxidant vitamins such as A, C are of lower order in RA and SLE patients than in normal subjects too. Based on animal studies, there is evidence that antioxidant vitamins may prevent arthritis, and increase antioxidant status. Daily oral administration of vitamin E to male arthritic rats restored levels of thiol (- SH) groups to pre- arthritic levels (Kheir- Eldin et al., 1992). High doses of vitamin C (12g/kg body weight) applied to arthritic Lewis rats, delayed incidence of paw oedema in these rats. Histological studies revealed a decreased inflammatory cells infiltration of superficial layer of synovium, and decreased synovial SOD activity in these rats (Sakai et al., 1999). In addition, administration of rutin (vitamin P) strongly inhibited spontaneous ROS production in RA neutrophils (Ostrakhovitch Afanas´ev, 2001). Vitamin E exerts analgetic effects. The results from a double blind clinical trials suggested that a dose of 400–1200 mg of -tocopherol daily was effective with respect to various pain parameters such as pain on pressure, pain at rest, and pain on movement. The postulated analgesic properties of vitamin E seem to be correlated to vitamin E plasma concentrations. Patients with active RA supplemented with a combination of conjugated fatty acids and vitamin E improved their disease activity score. The diet in these patients relieved from night and activity pain, and morning stiffness (Aryaeian et al., 2009). The analgesic effect of vitamin E is independent of a peripheral anti-inflammatory action, thereby suggesting a central rather than peripheral action. Dietary antioxidant micronutrients act as scavengers of reactive oxygen radicals and may protect against free radical mediated tissue damage in an inflamed joint. Vitamin E supplemented in soyabean oil reduced anti- double- stranded DNA IgG antibodies (Chia-Chien Bi- Fong, 2005).

#### **9.3 Fatty acids**

448 Oxidative Stress and Diseases

Whereas type I SOD is found mostly in the cytoplasm, nucleus, and intermembrane space of mitochondria, type III of SOD is extracellular. Manganese is cofactor for SOD type II, which is a mitochondrial enzyme. The enzyme is involved in the pathogenesis of inflammatory joint disease. In adjuvant model of arthritis the enzyme suppressed swelling, and retarded bone destruction (Shingu et al., 1994). An intra-articular injection of SOD significantly reduced synovitis in streptococcal wall cell (SWC)- model of arthritis. Intra- aricular injection of native SOD (bovine orgotein) produced greater clinical improvements than did intra- articular aspirin in patients with RA involving the knee (Afonso et al., 2007). Available data suggest a protective role of SOD in inflammatory joint disease. In mice that are genetically deficient in SOD III, both the severity of collagen-induced arthritis and the production of pro-inflammatory cytokines are increased. SODIII gene transferred subcutaneously or intra- articularly decreased the severity of experimental arthritis in

**9. Antioxidants as therapeutic possibilities in human autoimmune joint** 

SODs exert protective effects in animal models of inflammation. In mice, genetically deficient in SOD III, both the severity of collagen- induced arthritis and the production of pro- inflammatory cytokines are increased (Ross et al., 2004). SOD III gene transfer into the knee decreased the severity of experimental arthritis in rodents. In humans, serum SOD III levels correlated negatively with disease activity. Despite conflicting results of native SOD (orgotein) in RA patients, SOD mimetics have shown beneficial effects. Until now, the most promising are nitroxide (tempol) and Mn (II) pentaazamacrocyclic ligand (M40403). *In vitro*, tempol diminishes hydroxyl radical production, and decreases the cytotoxic effects of hydrogen peroxide and peroxynitrite. Tempol decreased collagen- induced arthritis in rats (Cuzzocrea et al., 2001). The anti-inflammatory effects of M40403 ligand are related to superoxide elimination, and restraint of nitration of tyrosine residues in proteins. M40403 ligand diminishes the expression of adhesion molecules such as integrins or selectins decreasing influx of neutrophils to inflammatory site. M40403 ligand inhibits TNF- via blocking the nuclear factor kappa B (NF- B). Its beneficial effects have been reported in rats

Micronutrient supplementation has been recommended to arthritic patients based on findings that the antioxidant status is very low in these patients. It has been documented that the mean synovial fluid concentrations of α-tocopherol are significantly decreased in arthritic patients compared to healthy controls. Also, levels of vitamin E in peripheral blood cell were significantly decreased in patients with active arthritis than in healthy subjects (Vasanthi et al., 2009). Concentrations of other antioxidant vitamins such as A, C are of lower order in RA and SLE patients than in normal subjects too. Based on animal studies, there is evidence that antioxidant vitamins may prevent arthritis, and increase antioxidant status. Daily oral administration of vitamin E to male arthritic rats restored levels of thiol (- SH) groups to pre- arthritic levels (Kheir- Eldin et al., 1992). High doses of vitamin C (1-

rodents.

**diseases** 

**9.2 Vitamins**

**9.1 Antioxidant enzymes** 

with collagen- induced arthritis (Cuzzocrea et al., 2005).

Eicosapentaenoic acid and docosahexaenoic acid (EPA and DHA respectively) exert potent antioxidant properties. Supplementation with these acids to lupus patients restored levels of antioxidant enzymes as the GPx, and SOD to baseline levels (Mohan, Das, 1997). Lupus autoimmune female mice fed with fish oil exhibited significantly higher liver of catalase, SOD, and GPx. The diet was shown to be beneficial against oxidative damage of hepatic tissue (Bhattacharya et al., 2003). Sera of lupus mice treated with conjugated linoleic acid (CLA) contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, gammaGCL, glutathione Stransferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. The activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in lupus mice (Bergamo et al., 2007).

#### **10. Conclusions and future perspectives**

In all cell types, oxygen metabolism can lead to the production of reactive oxygen / nitrogen species. Reactive species are known for their dual role as both beneficial and harmful. Deleterious effects of oxidative stress on cell components (proteins, lipids, DNA) include changes in their structure, and function. On the other hand, beneficial effects of ROS/RNS involve physiological cellular responses to noxious agents, e.g., in defense against infectious agents, in a number of cellular signaling pathways and in the induction of a mitogenic response. Oxidants even protect cells against oxidative stress and re-establish or maintain redox homeostasis (Ortona et al., 2008).

Oxygen metabolism has an important role in the pathogenesis of autoimmune joint diseases. Reactive oxygen/nitrogen species are documented as mediators of synovial inflammation. Their excessive production at the site of inflammation contributes to inflammatory process

Oxidative Stress in Human Autoimmune Joint Diseases 451

The work was supported by VZ 0021620816 and by 262708/SVV/2011 and by CN LC 554.

Abramson, S.B.; Amin, A.R.; Clancy, R.M. & Attur, M. (2001). The role of nitric oxide in

Arii, K.; Kumon, Y.; Ikeda, Y; Suehiro, T. &Hashimoto, K. (2006). Edaravone inhibits

Aryaeian, N.; Shahram, F.; Djalali, M.; Eshragian, M.R.; Djazayeri, A.; Sarrafnejad, A.;

Babior, B.M. (2000). Phagocytes and oxidative stress. *Am J Med,* Vol. 109, No. 1, pp. 33-44. Batuca, J.R.; Ames, P.R.J.; Isenberg, D.A. & Delgado Aves, J. (2007). Antibodies toward

tissue destruction. *Best Practice & Research Clinical Rheumatology,* Vol. 15, No.5, pp.

rheumatoid synovial cell proliferation and migration. *Free Radic Res,* Vol. 40, No. 2,

Salimzadeh, A.; Naderi, N.; Maryam, Ch. (2009). Effect of conjugated linoleic acids, vitamin E and thein combination on the clinical outcome of Iranian adults with active rheumatoid arthritis. *International Journal of Rheumatic Diseases,* Vol. 12, pp. 20–28. Afonso, V.; Champy, R.; Mitrovic, D.; Collin, P. & Lomri, A. (2007). Reactive oxygen species

and superoxide dismutases: Role in joint diseases. *Joint Bone Spine,* Vol. 74, No. 4 ,

high/densitz lipoprotein components inhibit paraoxonase activity in patients with systemic lupus erythematosus. *Ann. N.Y.Acad. Sci.* Vol. 1108, No. 1, pp. 137- 146. Bergamo, P.; Maurano, F. & Rossi, M. (2007). Phase 2 enzyme induction by conjugated

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Oxidative damage in synovial tissue is associated with in vivo hypoxic status in the

J. ; Bos, R. ; Limburg, P.C. ; Kallenberg, C.G. & Westra J. (2009). Hypoxia inducible factor- 1- alpha (HIF- 1alpha) is related to both angiogenesis and inflammation in

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Biniecka, M. ; Kennedy, A. ; Fearon, U. ; Ng, Ch. T.. ; Veale, D.J. & O' Sullivan. (2009).

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**11. Acknowledgements** 

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**12. References** 

in general, by induction of local production of chemoattractants, adhesion molecules, and pro- inflammatory cytokines. In addition, oxidative stress may contribute to the cyclic, selfperpetuating nature of autoimmune inflammation. Immune cells when "affected" by oxidants became auto- antigens intensifying auto- immune responses. Persisting autoinflammation, and oxidative stress in joint area leads to damages of connective tissue and the ECM directly or indirectly. *In vitro,* lipid peroxidation, ONOO- formation is associated with decreased production of type II collagen and aggrecan, and with diminished chondrocyte responses to growth factors. Moreover, ONOO- interferes with metabolism of matrix enhancing the expression of matrix degrading enzymes inhibiting the production and activity of tissue inhibiting enzymes. Hydrogen peroxide and singlet oxygen accelerate bone resorption. Level of oxidative stress in rheumatoid synovium is proportional to the levels of hypoxia and angiogenesis in the joint area.

Oxidative stress generated within an inflammatory joint can produce autoimmune phenomena and connective tissue destruction in rheumatoid synovitis (Vasanthi et al., 2009). Swelling stiffness pain in rest immobility are common symptoms of autoimmune joint diseases. Rationale of antioxidant therapy should relieve from objective complications. Polyunsaturated fatty acids (PUFA) can modulate oxidative stress and may have a role as regulators in the synthesis of antioxidant enzymes (Mohan Das, 1997). Arthritic patients taking these acids showed biochemical and chemical improvement. Dietary supplementation significantly decreased joint pain index. In combination with other dietary modifications modest improvement in morning stiffness and in the number of painful joints were reported. Clinical improvements were connected with anti- inflammatory effects as a decreased synthesis of leukotriens by neutrophils and lower synthesis of IL-1 by macrophages. Significant clinical benefit has been claimed in SLE patients given a low- fat diet with PUFA (reviewed in Darlington Stone, 2001). The clinical work with diets containing PUFA has clearly demonstrated their anti-inflammatory effects, but it was also shown that these effects were attributed to omega- 3 (-3) rather than -6 PUFA. As there is no doubt about dietary fatty acids do decrease the generation of inflammatory agents, opposite results have been obtained on free radical formation. PUFA are especially potent at increasing levels of oxidative stress. On the other hand, EPA increases mitochondrial Mn-SOD mitochondrial activity.

The hypothesis about oxidative stress promotes arthritic process was challenged when oxidants were shown to decrease disease severity in mouse and rat arthritis models. Certain oils with an alkane structure such as phytol besides its oxidative effects protect against arthritis development. Its subcutaneous administration prevented development of pristaneinduced arthritis (Hultqvist et al., 2006). Rats treated with phytol in acute phases of pristane arthritis showed no signs of inflammation. A decrease in COMP, a measurement of ongoing cartilage destruction, was prevented during chronic phases of the disease. The efficiency of phytol in preventing arthritis was compared to methotrexate and/or etanecerpt. Etanecerpt, TNF- blocker, was highly effective in reducing collagen- induced arthritis. In pristaneinduced arthritis, the preventive effects of phytol was more pronounced than that of etanecerpt. Also, in comparison to MTX, phytol was valid as a potential therapeutic agent. Concluded, ROS-promoting substances such as phytol represent a promising class of therapeutics for treatment of autoimmune joint inflammatory diseases what needs further research.

#### **11. Acknowledgements**

The work was supported by VZ 0021620816 and by 262708/SVV/2011 and by CN LC 554.

#### **12. References**

450 Oxidative Stress and Diseases

in general, by induction of local production of chemoattractants, adhesion molecules, and pro- inflammatory cytokines. In addition, oxidative stress may contribute to the cyclic, selfperpetuating nature of autoimmune inflammation. Immune cells when "affected" by oxidants became auto- antigens intensifying auto- immune responses. Persisting autoinflammation, and oxidative stress in joint area leads to damages of connective tissue and the ECM directly or indirectly. *In vitro,* lipid peroxidation, ONOO- formation is associated with decreased production of type II collagen and aggrecan, and with diminished chondrocyte responses to growth factors. Moreover, ONOO- interferes with metabolism of matrix enhancing the expression of matrix degrading enzymes inhibiting the production and activity of tissue inhibiting enzymes. Hydrogen peroxide and singlet oxygen accelerate bone resorption. Level of oxidative stress in rheumatoid synovium is proportional to the

Oxidative stress generated within an inflammatory joint can produce autoimmune phenomena and connective tissue destruction in rheumatoid synovitis (Vasanthi et al., 2009). Swelling stiffness pain in rest immobility are common symptoms of autoimmune joint diseases. Rationale of antioxidant therapy should relieve from objective complications. Polyunsaturated fatty acids (PUFA) can modulate oxidative stress and may have a role as regulators in the synthesis of antioxidant enzymes (Mohan Das, 1997). Arthritic patients taking these acids showed biochemical and chemical improvement. Dietary supplementation significantly decreased joint pain index. In combination with other dietary modifications modest improvement in morning stiffness and in the number of painful joints were reported. Clinical improvements were connected with anti- inflammatory effects as a decreased synthesis of leukotriens by neutrophils and lower synthesis of IL-1 by macrophages. Significant clinical benefit has been claimed in SLE patients given a low- fat diet with PUFA (reviewed in Darlington Stone, 2001). The clinical work with diets containing PUFA has clearly demonstrated their anti-inflammatory effects, but it was also shown that these effects were attributed to omega- 3 (-3) rather than -6 PUFA. As there is no doubt about dietary fatty acids do decrease the generation of inflammatory agents, opposite results have been obtained on free radical formation. PUFA are especially potent at increasing levels of oxidative stress. On the other hand, EPA increases mitochondrial Mn-

The hypothesis about oxidative stress promotes arthritic process was challenged when oxidants were shown to decrease disease severity in mouse and rat arthritis models. Certain oils with an alkane structure such as phytol besides its oxidative effects protect against arthritis development. Its subcutaneous administration prevented development of pristaneinduced arthritis (Hultqvist et al., 2006). Rats treated with phytol in acute phases of pristane arthritis showed no signs of inflammation. A decrease in COMP, a measurement of ongoing cartilage destruction, was prevented during chronic phases of the disease. The efficiency of phytol in preventing arthritis was compared to methotrexate and/or etanecerpt. Etanecerpt, TNF- blocker, was highly effective in reducing collagen- induced arthritis. In pristaneinduced arthritis, the preventive effects of phytol was more pronounced than that of etanecerpt. Also, in comparison to MTX, phytol was valid as a potential therapeutic agent. Concluded, ROS-promoting substances such as phytol represent a promising class of therapeutics for treatment of autoimmune joint inflammatory diseases what needs further

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**20** 

 *Japan* 

**Oxidative Stress in Multiple Organ** 

**Damage in Hypertension, Diabetes** 

*Department of Clinical Laboratory, Faculty of Medicine, University of Tokyo* 

Hypertension, diabetes, hypercholesterolemia and chronic kidney disease (CKD) lead to cardiovascular (CV) events and cardiovascular death consists of main cause in mortality of those diseases. Understanding of pathophysiology that links them and CV events has been vigorously studied and several factors are believed to play roles such as NO, reninangiotensin system, and oxidative stress. It has been shown that those factors affect endothelial function and consequently organ circulation as well as function and viability of cells and organs. Despite overwhelming evidences in the consequences of experimental models of ROS-induced organ damage, large-scale clinical trials of former antioxidant therapies, such as vitamin C, vitamin E or -carotene, could not demonstrate satisfactory benefit to patients and they seemed to be harmful in some cases (Hennekens *et al.*, 1996; Omenn *et al.*, 1996; Virtamo *et al.*, 1998; Hercberg *et al.*, 1999; Lee *et al.*, 1999; Yusuf *et al.*, 2000; de Gaetano, 2001; Heart, 2002; Vivekananthan *et al.*, 2003; Hercberg *et al.*, 2004; Kris-Etherton *et al.*, 2004; Lonn *et al.*, 2005). Several studies concluded that β-carotene supplementation increased the relative risk of death in patients with some types of cancer and had no benefit on patients with cardiovascular disease. Another study said vitamin E increased hemorrhagic stroke. Even antioxidant cocktails increased in all-cause motality (Omenn *et al.*, 1996; Rosen *et al.*, 2001a). So far, supplementation with vitamins C and E, either alone or in combination with each other or with other antioxidant vitamins, does not appear to be efficacious for the treatment of cardiovascular disease (Lonn *et al.*, 2005). We investigated role of oxidative stress in consequences of multiple organ damages in mouse

\* Tomoyo Kaneko2, Xu Qingyou1, Yusei Miyamoto3, Mu Shengyu2, Hong Wang2, Sayoko Ogura2, Rika Jimbo2, Bohumil Majtan2, Yuzaburo Uetake2, Daigoro Hirohama2, Fumiko Kawakami-Mori2,

*2 Department of Endocrinology and Nephrology, Faculty of Medicinem, Japan 3 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Japan*

<sup>1</sup>*Department of Clinical Laboratory, Faculty of Medicine, University of Tokyo, Japan* 

**1. Introduction** 

and possible new therapeutic agent.

Toshiro Fujita2 and Yutaka Yatomi1

**and CKD, Mechanisms and New** 

**Therapeutic Possibilities** 

Tatsuo Shimosawa\* et al.


### **Oxidative Stress in Multiple Organ Damage in Hypertension, Diabetes and CKD, Mechanisms and New Therapeutic Possibilities**

Tatsuo Shimosawa\* et al. *Department of Clinical Laboratory, Faculty of Medicine, University of Tokyo Japan* 

#### **1. Introduction**

456 Oxidative Stress and Diseases

Taysi, T.; Gul, M.; Saris, R.A.; Akcay, F. & Bakan, N. (2002 a). Serum oxidant/ antioxidant

Taysi, T.; Polat, F.; Gul, M.; Saris, R.A. Bakan, N. (2002 b). Lipid peroxidation, some

Tewthanom, K.; Janwityanuchit, S.; Totemchockchyakarn, K. & Panomvana, D. (2008).

Van de Loo, F.A.J.; Bennink, M.B.; Arntz, O.J.; Smeets, R.L.; Lubberts, E.; Joosten, L.A.B.; van

experimental arthritis model. *Am J Pathol,* Vol. 163, No. 4, pp. 1525- 1536. Vasanthi, P.; Nalini, G. & Rajasekhar, G. (2009). Status of oxidative stress in rheumatoid

Yoshida, S.; Katoh, T.; Tetsuka, T.; Uno, K.; Matsui, N. & Okamoto, T. (1999). Involvement of

arthritis. *Int J Rheum Dis,* Vol. 12, No. 1 , pp. 29- 33.

rheumatoid arthritis. *Rheumatol Int,* Vol. 21, No. 5, pp. 200- 204.

No. 7, pp. 684- 688.

11, No. 3, pp. 30-34.

No. 1, pp. 351-358.

status of patients with systemic lupus erythematosus. *Clin Chem Lab Med,* Vol. 40,

extracellular antioxidants, and antioxidant enzymes in serum of patients with

Correlation of lipid peroxidation and glutatione levels with severity of systemic lupus erythematosus: A pilot study from single center. *J Pharm Pharmaceut Sci,* Vol.

Lent, P.L.E.M.; Coenen- de Roo, Ch. J.J.; Cuzzocrea, S,; Segal, B.H.; Holland, S.M. & van den Berg, W. B. (2003). Deficiency of NADPH oxidase components p47phox and gp91phox caused granulomatosus synovitis and increased tissue destruction in

thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF- - induced production of IL-6 and IL-8 from cultured synovial fibroblasts. *Immunol,* Vol. 163, Hypertension, diabetes, hypercholesterolemia and chronic kidney disease (CKD) lead to cardiovascular (CV) events and cardiovascular death consists of main cause in mortality of those diseases. Understanding of pathophysiology that links them and CV events has been vigorously studied and several factors are believed to play roles such as NO, reninangiotensin system, and oxidative stress. It has been shown that those factors affect endothelial function and consequently organ circulation as well as function and viability of cells and organs. Despite overwhelming evidences in the consequences of experimental models of ROS-induced organ damage, large-scale clinical trials of former antioxidant therapies, such as vitamin C, vitamin E or -carotene, could not demonstrate satisfactory benefit to patients and they seemed to be harmful in some cases (Hennekens *et al.*, 1996; Omenn *et al.*, 1996; Virtamo *et al.*, 1998; Hercberg *et al.*, 1999; Lee *et al.*, 1999; Yusuf *et al.*, 2000; de Gaetano, 2001; Heart, 2002; Vivekananthan *et al.*, 2003; Hercberg *et al.*, 2004; Kris-Etherton *et al.*, 2004; Lonn *et al.*, 2005). Several studies concluded that β-carotene supplementation increased the relative risk of death in patients with some types of cancer and had no benefit on patients with cardiovascular disease. Another study said vitamin E increased hemorrhagic stroke. Even antioxidant cocktails increased in all-cause motality (Omenn *et al.*, 1996; Rosen *et al.*, 2001a). So far, supplementation with vitamins C and E, either alone or in combination with each other or with other antioxidant vitamins, does not appear to be efficacious for the treatment of cardiovascular disease (Lonn *et al.*, 2005). We investigated role of oxidative stress in consequences of multiple organ damages in mouse and possible new therapeutic agent.

<sup>\*</sup> Tomoyo Kaneko2, Xu Qingyou1, Yusei Miyamoto3, Mu Shengyu2, Hong Wang2, Sayoko Ogura2, Rika Jimbo2, Bohumil Majtan2, Yuzaburo Uetake2, Daigoro Hirohama2, Fumiko Kawakami-Mori2,

Toshiro Fujita2 and Yutaka Yatomi1

<sup>1</sup>*Department of Clinical Laboratory, Faculty of Medicine, University of Tokyo, Japan* 

*<sup>2</sup> Department of Endocrinology and Nephrology, Faculty of Medicinem, Japan 3 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Japan*

Oxidative Stress in Multiple Organ Damage in Hypertension,

Modified from Shimosawa *et al*. 2002.

Diabetes and CKD, Mechanisms and New Therapeutic Possibilities 459

We measured urinary excretion of 8-OHdG (a) and isoprostane (b) as footsteps of oxidative stress in whole body. As for confirming local oxidative stress, we immunostained coronary artery with 3-nitrotyrosine (c) that reflects NO-related oxidative stress and is a good marker for oxidative stress at the endothelial layer (arrow). For evaluation of real time production of oxidative stress, we measured ESR signal in the heart (d). On the left of panel d, time dependent of decay of ESR signal were plotted and on the right panel, the decay ratio of ESR signal that reflects production of oxidative stress was calculated. All these data suggest angiotensin II and salt-loaded AM+/- mice has higher oxidative stress than wild type mice.

Hypoxia is known to cause pulmonary hypertension and pulmonary vascular remodeling. With this model, evidence is accumulating that ROS are the upstream signals of chronic hypoxia-induced pulmonary vasoconstriction and the development of vascular remodeling. It has been reported that ROS generated by hypoxia can induce calcium release from sarcoplasmic reticulum stores, which is followed by pulmonary vasoconstriction (Waypa *et al.*, 2002). Moreover, ROS activated several growth factors such as vascular endothelial growth factor and platelet-activating factor (PAF) (Hartung *et al.*, 1983; Ono *et al.*, 1992; Chandel *et al.*, 1998), which induce vascular remodeling. In the early phase of hypoxic conditions, ROS may act as a trigger of this signaling cascade in the process of vascular remodeling. In our experiments, 10% O2 conditions caused higher ROS in AM-knockout mice compared with wild-type mice. Concomitant with increased oxidative stress in the pulmonary artery, AM-knockout mice showed marked vascular remodeling and higher mortality (Matsui *et al.*, 2004), which were reversed by AM supplementation or by treatment with 4-Hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxyl-TEMPO), a mimetic of superoxide dismutase. Based on this basic research, translational research studies have been conducted in patients with pulmonary hypertension, and potent therapeutic effects of inhaling AM have been reported (Nagaya *et al.*, 2004). As the mortality among patients with pulmonary hypertension is high even treatment with nitric oxide and prostaglandins, AM, or other agents may be novel and promising agents for the treatment of these patients.

**2.2 Renal effects and possible role in CKD and diabetic kidney disease** 

Renal dysfunction or chronic kidney disease (CKD) draws high interests among nephrologists as well as cardiologists and other specialities. There are several factors implicated to connect renal dysfunction and cardiovascular events, endothelial dysfunction, renin-angiotensin-aldosterone axis, oxidative stress, and unknown toxic agents. In order to investigate if renin-angiotensin axis and oxidative stress can be therapeutical target in CKD, we used AM knockout mice. We found increased oxidative stress and local reninangiotensin system in the ureter-obstructed kidney of AM knockout mice. Concordance with the increase of oxidative stress and renin-angiotensin system, severe interstitial fibrosis, and cell proliferation were prominent in AM-knockout mice. The interstitial fibrosis can be partly reversed by hydroxyl-TEMPO, angiotensin receptor blockers, or the systemic replacement of AM (unpublished observation). When dual treatment with angiotensin receptor blocker and hydroxyl-TEMPO were given, pathological changes are more effectively reversed compared with that by each agent alone. Angiotensin II is known to induce oxidative stress via activating NADPH oxidase and AM can block this pathway via

#### **2. Adrenomedullin**

#### **2.1 Cardiovascular effects**

Adrenomedullin is a 52-amino-acid peptide and was originally isolated from pheochromocytoma cell but is also produced and secreted in endothelial cells and potent hypotensive peptide (Shimosawa & Fujita, 2005). We generated its deficient mice and proved that it is an endogenous antioxidants.

We examined the antioxidant properties of AM in both angiotensin II-salt (Shimosawa *et al.*, 2002) and pulmonary hypertension models (Matsui *et al.*, 2004). We showed that AMknockout mice had higher oxidative stress through 3 ways: the urinary excretion of oxidative stress markers, such as 8-hydroxydeoxyguanosine (Fig. 1a) and isoprostane (Fig. 1b) by ELISA method, the immunostaining of 3-nitrotyrosine to localize oxidative stress (Fig. 1c), and real-time oxidant production measurement by the electron spin resonance (ESR) method (Fig. 1d). Pathological changes in the heart, such as periarterial fibrosis and coronary artery occlusion, were prominent in the knockout mice, and further investigation revealed that high oxidative stress caused coronary artery damage in this model.

Fig. 1. Evaluation of oxidative stress in AM+/- mice after angiotensin II and salt loading.

Adrenomedullin is a 52-amino-acid peptide and was originally isolated from pheochromocytoma cell but is also produced and secreted in endothelial cells and potent hypotensive peptide (Shimosawa & Fujita, 2005). We generated its deficient mice and

We examined the antioxidant properties of AM in both angiotensin II-salt (Shimosawa *et al.*, 2002) and pulmonary hypertension models (Matsui *et al.*, 2004). We showed that AMknockout mice had higher oxidative stress through 3 ways: the urinary excretion of oxidative stress markers, such as 8-hydroxydeoxyguanosine (Fig. 1a) and isoprostane (Fig. 1b) by ELISA method, the immunostaining of 3-nitrotyrosine to localize oxidative stress (Fig. 1c), and real-time oxidant production measurement by the electron spin resonance (ESR) method (Fig. 1d). Pathological changes in the heart, such as periarterial fibrosis and coronary artery occlusion, were prominent in the knockout mice, and further investigation

revealed that high oxidative stress caused coronary artery damage in this model.

Fig. 1. Evaluation of oxidative stress in AM+/- mice after angiotensin II and salt loading.

**2. Adrenomedullin** 

**2.1 Cardiovascular effects** 

proved that it is an endogenous antioxidants.

We measured urinary excretion of 8-OHdG (a) and isoprostane (b) as footsteps of oxidative stress in whole body. As for confirming local oxidative stress, we immunostained coronary artery with 3-nitrotyrosine (c) that reflects NO-related oxidative stress and is a good marker for oxidative stress at the endothelial layer (arrow). For evaluation of real time production of oxidative stress, we measured ESR signal in the heart (d). On the left of panel d, time dependent of decay of ESR signal were plotted and on the right panel, the decay ratio of ESR signal that reflects production of oxidative stress was calculated. All these data suggest angiotensin II and salt-loaded AM+/- mice has higher oxidative stress than wild type mice. Modified from Shimosawa *et al*. 2002.

Hypoxia is known to cause pulmonary hypertension and pulmonary vascular remodeling. With this model, evidence is accumulating that ROS are the upstream signals of chronic hypoxia-induced pulmonary vasoconstriction and the development of vascular remodeling. It has been reported that ROS generated by hypoxia can induce calcium release from sarcoplasmic reticulum stores, which is followed by pulmonary vasoconstriction (Waypa *et al.*, 2002). Moreover, ROS activated several growth factors such as vascular endothelial growth factor and platelet-activating factor (PAF) (Hartung *et al.*, 1983; Ono *et al.*, 1992; Chandel *et al.*, 1998), which induce vascular remodeling. In the early phase of hypoxic conditions, ROS may act as a trigger of this signaling cascade in the process of vascular remodeling. In our experiments, 10% O2 conditions caused higher ROS in AM-knockout mice compared with wild-type mice. Concomitant with increased oxidative stress in the pulmonary artery, AM-knockout mice showed marked vascular remodeling and higher mortality (Matsui *et al.*, 2004), which were reversed by AM supplementation or by treatment with 4-Hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxyl-TEMPO), a mimetic of superoxide dismutase. Based on this basic research, translational research studies have been conducted in patients with pulmonary hypertension, and potent therapeutic effects of inhaling AM have been reported (Nagaya *et al.*, 2004). As the mortality among patients with pulmonary hypertension is high even treatment with nitric oxide and prostaglandins, AM, or other agents may be novel and promising agents for the treatment of these patients.

#### **2.2 Renal effects and possible role in CKD and diabetic kidney disease**

Renal dysfunction or chronic kidney disease (CKD) draws high interests among nephrologists as well as cardiologists and other specialities. There are several factors implicated to connect renal dysfunction and cardiovascular events, endothelial dysfunction, renin-angiotensin-aldosterone axis, oxidative stress, and unknown toxic agents. In order to investigate if renin-angiotensin axis and oxidative stress can be therapeutical target in CKD, we used AM knockout mice. We found increased oxidative stress and local reninangiotensin system in the ureter-obstructed kidney of AM knockout mice. Concordance with the increase of oxidative stress and renin-angiotensin system, severe interstitial fibrosis, and cell proliferation were prominent in AM-knockout mice. The interstitial fibrosis can be partly reversed by hydroxyl-TEMPO, angiotensin receptor blockers, or the systemic replacement of AM (unpublished observation). When dual treatment with angiotensin receptor blocker and hydroxyl-TEMPO were given, pathological changes are more effectively reversed compared with that by each agent alone. Angiotensin II is known to induce oxidative stress via activating NADPH oxidase and AM can block this pathway via

Oxidative Stress in Multiple Organ Damage in Hypertension,

cardiovascular events or mortality in CKD patients.

investigated the possible therapeutic effect of platinum-nano-particle.

damage in metabolic syndrome model by eliminating ROS.

metabolic biomarkers of mice.

**3. Platinum nanoparticle** 

Diabetes and CKD, Mechanisms and New Therapeutic Possibilities 461

Recent clinical trial in diabetic CKD patients revealed bardoxolone methyl effectively improved in the estimated GFR independent from blood pressure which is the most important risk in CKD (Pergola *et al.*, 2011). Also, it improved insulin resistance by increasing glucose uptake in skeletal muscle (Saha *et al.*), which is compatible with our findings in AM knockout mice as described above. Bardoxolone methyl is an oral antioxidant inflammation modulator and activates the Keap1–Nrf2 pathway. Keap1-Nrf2 pathway regulates inflammation and oxidative stress (Dinkova-Kostova *et al.*, 2005) via increased expression of heme oxygenase 1 (Wu *et al.*, 2011). This compound is so far the only clinically available antioxidants that show organ protective effect, however, it requires further assessment if bardoxolone methyl can prevent hard end-point such as

In diabetic-CKD, possible clinical usefulness of bardoxolone methyl was shown as mentioned above, so far few antioxidant agents are proved to be effective in cardiovascular protection. Although in pulmonary hypertension AM showed its clinical usefulness by inhaling, AM is a peptide and its clinical use is limited due to its short half life. We next

In recent years, a few reports about new drug delivery system using nanotechnology have been released (Muro *et al.*, 2006; Kajita *et al.*, 2007; Shimizu et al., 2010). Among them platinum nanoparticles as both superoxide dismutase (SOD) mimetic and catalase mimetic (Kajita *et al.*, 2007; Watanabe *et al.*, 2009). In vivo experiments shows its effectiveness in smoking-induced lung damage (Onizawa *et al.*, 2009) or stroke model mice (Takamiya et al.*et al.*, 2011). Platinum nanoparticles (PAA-Pt) was administered intranasally, which were then exposed to cigarette smoking for 3 days. Cigarette smoking induced NFkappaB activation, and neutrophilic inflammation in the lungs of mice, and intranasal administration of PAA-Pt inhibited these changes. Moreover in in vitro experiments, treatment of alveolar-type-II-like A549 cells with PAA-Pt inhibited cell death after exposure to a cigarette smoke extract. Transient middle cerebral artery occlusion (tMCAO) and reperfusion model was used in this study. PAA-Pt was administered intravenously. PAA-Pt dramatically reduced oxidative stress in the brain and significantly improved the motor function and greatly reduced the infarct volume, especially in the cerebral cortex with preserved collagen IV and a remarkable suppression of MMP-9. By these acute models the antioxidant effect and concomitant organ protection by PAA-Pt were established. We investigated the chronic effect of PAA-Pt and studied if PAA-Pt reverse cardiovascular

Metabolic syndrome model was established by angiotensin II and high salt diet on diabetic 8-week-old male db/db mice. 10 M platinum nanoparticles were given orally. The oxidative stress was measured in two ways. First, the urinary excretion of isoprostane reflects the state of systemic oxidative stress. Ang II and salt loading increased isoprostane excretion in db/db mice. PAA-Pt reduced isoprostane excretion, suggesting that PAA-Pt would be an effective antioxidant (Fig 3). Blood pressure was elevated by angiotensin II and high salt diet and PAA-Pt treatment did not have any effects on blood pressure and other

c-Src (Liu *et al.*, 2007). In the kidney, oxidative stress is generated not only by NADPH oxidase but from mitochondria and xanthine oxidase and thus AM or angiotensin II blocker alone therapy is not sufficient and together with hydroxyl-TEMPO that can totally block oxidative stress is effective.

Multiple lines of study have shown that diabetic patients have increased oxidative stress and the resultant organ damage (Rosen *et al.*, 2001b). In turn, it is hypothesized that oxidative stress can induce diabetes by series of studies; oxidative stress impairs insulin internalization (Bertelsen *et al.*, 2001), blocks insulin receptor substrate phosphorylation, impairs phosphoinositide-3 kinase activity (Najib & Sanchez-Margalet, 2001), induces protein glycation and as a consequence of advanced glycation end-products-receptor binding that leads to cytotoxicity in pancreatic beta cells and reduces the translocation of glucose transporter type-4 (Rudich *et al.*, 1997; Rudich *et al.*, 1998). An in vivo study also showed that the administration of oxidative stress aggravated diabetes in diabetes-prone obese-Zucker rats (Laight *et al.*, 2000). Antioxidan supplementation studies have shown conflicting resuts; some reported beneficial effects in endothelial function, retinal blood flow and renal function outcomes (Blum *et al.*; Ziegler *et al.*, 2004; Lopes de Jesus *et al.*, 2008), in contrast, the recent metaanalysis of antioxidants, vitamin C and E, supplementation trials revealed ineffectiveness in glycemic control (Akbar *et al.*, 2011). On the other hand, in clinical settings, diabetics have higher oxidative stress and AM levels (Hayashi *et al.*, 1997), which led us to assume that AM is upregulated in order to antagonize oxidative stress. In fact, aged or angiotensin II-treated AM-knockout mice showed insulin resistance, and this was reversed by AM supplementation (Shimosawa *et al.*, 2003; Xing *et al.*, 2004) (Fig. 2). In this experiment, we showed that oxidative stress directly impaired insulin signaling by interfering with insulin receptor substrate 1 and 2 phosphorylations. By in vitro experiments, we found that oxidative stress not only impairs insulin signaling, but also reduces glucose transporter 4 transcription (unpublished observation).

Fig. 2. Insulin resistance in skeletal muscle of aged AM+/- mice which was recovered by AM supplement.

Insulin resistance was measured by ex vivo experiments. Isolated soleus muscle was incubated with insulin and 12C-deoxyglucose. Deoxyglucose uptake was evaluated as insulin sensitivity. Aged male AM+/- mice (55 weeks old) showed insulin resistance and AM supplementation for a month successfully reversed insulin resistant state. Modified from Shimosawa *et al*. 2003.

Recent clinical trial in diabetic CKD patients revealed bardoxolone methyl effectively improved in the estimated GFR independent from blood pressure which is the most important risk in CKD (Pergola *et al.*, 2011). Also, it improved insulin resistance by increasing glucose uptake in skeletal muscle (Saha *et al.*), which is compatible with our findings in AM knockout mice as described above. Bardoxolone methyl is an oral antioxidant inflammation modulator and activates the Keap1–Nrf2 pathway. Keap1-Nrf2 pathway regulates inflammation and oxidative stress (Dinkova-Kostova *et al.*, 2005) via increased expression of heme oxygenase 1 (Wu *et al.*, 2011). This compound is so far the only clinically available antioxidants that show organ protective effect, however, it requires further assessment if bardoxolone methyl can prevent hard end-point such as cardiovascular events or mortality in CKD patients.

#### **3. Platinum nanoparticle**

460 Oxidative Stress and Diseases

c-Src (Liu *et al.*, 2007). In the kidney, oxidative stress is generated not only by NADPH oxidase but from mitochondria and xanthine oxidase and thus AM or angiotensin II blocker alone therapy is not sufficient and together with hydroxyl-TEMPO that can totally block

Multiple lines of study have shown that diabetic patients have increased oxidative stress and the resultant organ damage (Rosen *et al.*, 2001b). In turn, it is hypothesized that oxidative stress can induce diabetes by series of studies; oxidative stress impairs insulin internalization (Bertelsen *et al.*, 2001), blocks insulin receptor substrate phosphorylation, impairs phosphoinositide-3 kinase activity (Najib & Sanchez-Margalet, 2001), induces protein glycation and as a consequence of advanced glycation end-products-receptor binding that leads to cytotoxicity in pancreatic beta cells and reduces the translocation of glucose transporter type-4 (Rudich *et al.*, 1997; Rudich *et al.*, 1998). An in vivo study also showed that the administration of oxidative stress aggravated diabetes in diabetes-prone obese-Zucker rats (Laight *et al.*, 2000). Antioxidan supplementation studies have shown conflicting resuts; some reported beneficial effects in endothelial function, retinal blood flow and renal function outcomes (Blum *et al.*; Ziegler *et al.*, 2004; Lopes de Jesus *et al.*, 2008), in contrast, the recent metaanalysis of antioxidants, vitamin C and E, supplementation trials revealed ineffectiveness in glycemic control (Akbar *et al.*, 2011). On the other hand, in clinical settings, diabetics have higher oxidative stress and AM levels (Hayashi *et al.*, 1997), which led us to assume that AM is upregulated in order to antagonize oxidative stress. In fact, aged or angiotensin II-treated AM-knockout mice showed insulin resistance, and this was reversed by AM supplementation (Shimosawa *et al.*, 2003; Xing *et al.*, 2004) (Fig. 2). In this experiment, we showed that oxidative stress directly impaired insulin signaling by interfering with insulin receptor substrate 1 and 2 phosphorylations. By in vitro experiments, we found that oxidative stress not only impairs insulin signaling, but also

reduces glucose transporter 4 transcription (unpublished observation).

Fig. 2. Insulin resistance in skeletal muscle of aged AM+/- mice which was recovered by AM

Insulin resistance was measured by ex vivo experiments. Isolated soleus muscle was incubated with insulin and 12C-deoxyglucose. Deoxyglucose uptake was evaluated as insulin sensitivity. Aged male AM+/- mice (55 weeks old) showed insulin resistance and AM supplementation for a month successfully reversed insulin resistant state. Modified from Shimosawa *et al*. 2003.

oxidative stress is effective.

supplement.

In diabetic-CKD, possible clinical usefulness of bardoxolone methyl was shown as mentioned above, so far few antioxidant agents are proved to be effective in cardiovascular protection. Although in pulmonary hypertension AM showed its clinical usefulness by inhaling, AM is a peptide and its clinical use is limited due to its short half life. We next investigated the possible therapeutic effect of platinum-nano-particle.

In recent years, a few reports about new drug delivery system using nanotechnology have been released (Muro *et al.*, 2006; Kajita *et al.*, 2007; Shimizu et al., 2010). Among them platinum nanoparticles as both superoxide dismutase (SOD) mimetic and catalase mimetic (Kajita *et al.*, 2007; Watanabe *et al.*, 2009). In vivo experiments shows its effectiveness in smoking-induced lung damage (Onizawa *et al.*, 2009) or stroke model mice (Takamiya et al.*et al.*, 2011). Platinum nanoparticles (PAA-Pt) was administered intranasally, which were then exposed to cigarette smoking for 3 days. Cigarette smoking induced NFkappaB activation, and neutrophilic inflammation in the lungs of mice, and intranasal administration of PAA-Pt inhibited these changes. Moreover in in vitro experiments, treatment of alveolar-type-II-like A549 cells with PAA-Pt inhibited cell death after exposure to a cigarette smoke extract. Transient middle cerebral artery occlusion (tMCAO) and reperfusion model was used in this study. PAA-Pt was administered intravenously. PAA-Pt dramatically reduced oxidative stress in the brain and significantly improved the motor function and greatly reduced the infarct volume, especially in the cerebral cortex with preserved collagen IV and a remarkable suppression of MMP-9. By these acute models the antioxidant effect and concomitant organ protection by PAA-Pt were established. We investigated the chronic effect of PAA-Pt and studied if PAA-Pt reverse cardiovascular damage in metabolic syndrome model by eliminating ROS.

Metabolic syndrome model was established by angiotensin II and high salt diet on diabetic 8-week-old male db/db mice. 10 M platinum nanoparticles were given orally. The oxidative stress was measured in two ways. First, the urinary excretion of isoprostane reflects the state of systemic oxidative stress. Ang II and salt loading increased isoprostane excretion in db/db mice. PAA-Pt reduced isoprostane excretion, suggesting that PAA-Pt would be an effective antioxidant (Fig 3). Blood pressure was elevated by angiotensin II and high salt diet and PAA-Pt treatment did not have any effects on blood pressure and other metabolic biomarkers of mice.

Oxidative Stress in Multiple Organ Damage in Hypertension,

and high salt diet and was reversed by PAA-Pt.

Fig. 5. Molecular markers for cardiac damage and fibrosis.

preventing organ damage in metabolic syndrome.

Pt.

Diabetes and CKD, Mechanisms and New Therapeutic Possibilities 463

histopathological findings. We examined mRNA expression of ANP and procollagen type I in the heart as molecular markers of cardiac damage and we found that those molecular markers were apparently up-regulated by angiotensin II and salt loading (Fig 5). PAA-Pt reduced these markers expressions to the almost same extent with the control db/db mice

Pericoronary fibrosis were evaluated by Azan staining. Fibrosis was robust by angiotensin II

mRNA expression of ANP and procollagen type I were evaluated. Concomitant with figure 4, angiotensin II and high salt diet aggravated cardiac damage and it was reversed by PAA-

PAA-Pt behaves as an SOD mimetic and possesses a catalase activity, then its efficacy can last longer than vitamin C (Kajita *et al.*, 2007). It is mainly because the catalytic property of PAA-Pt which is distinguished from vitamin C that is quickly consumed. Therefore we examined the effect of PAA-Pt in metabolic syndrome model mice to reduce oxidative stress and protect the cardiovascular system. Concordance with in vitro study, PAA-Pt effectually

We found the extension of cardiovascular damages was much severer and the level of systemic oxidative stress, which was evaluated by urinary excretion of isoprostane, was much higher in db/db mice. Then, it is indicated that the 'metabolic-syndrome-status' is equal to 'high-oxidative-stress-status' and this status seems to be the trigger of the vicious cycle of organ damage. Thus, the scavenging ROS would be a promising approach to

PAA-Pt also has the possibility to solve the problems of former antioxidants as vitamin C, E or -carotene. It is said the one of the reasons why those former antioxidants would have failed to be effective against organ damage that they are short-acting and could not eliminate intracellular ROS, including mitochondrial ROS. PAA-Pt possesses long-lasting activity (means catalytic effect) and dual properties as SOD and catalase mimetics. The

reduced ROS and prevented cardiovascular damage in hypertensive db/db mice.

and these results support our findings in the histopathological examinations.

#### Fig. 3. PAA-Pt effect on oxidative stress

Db/db mice were treated with angiotensin II and high salt diet and urinary isoprostane was measured as a marker of oxidative stress. PAA-Pt effectively reduced oxidative stress.

Consistent with AM knockout mice, angiotensin II and high salt diet induced pericoronary fibrosis in db/db mice (Fig. 4a). The degree of deterioration of vascular damage was much severer in the db/db mice than wild type mice. PAA-Pt therapy reversed the vascular damage in angiotensin II/salt loaded db/db mice. The area of fibrous changes was calculated for each group (Fig 4b). The results of RT-PCR supported the aforesaid

Fig. 4. Histopathological changes of db/db mice by angiotensin II/high salt diet and effect of PAA-Pt.

Db/db mice were treated with angiotensin II and high salt diet and urinary isoprostane was measured as a marker of oxidative stress. PAA-Pt effectively reduced oxidative stress.

Consistent with AM knockout mice, angiotensin II and high salt diet induced pericoronary fibrosis in db/db mice (Fig. 4a). The degree of deterioration of vascular damage was much severer in the db/db mice than wild type mice. PAA-Pt therapy reversed the vascular damage in angiotensin II/salt loaded db/db mice. The area of fibrous changes was calculated for each group (Fig 4b). The results of RT-PCR supported the aforesaid

Fig. 4. Histopathological changes of db/db mice by angiotensin II/high salt diet and effect

Fig. 3. PAA-Pt effect on oxidative stress

of PAA-Pt.

histopathological findings. We examined mRNA expression of ANP and procollagen type I in the heart as molecular markers of cardiac damage and we found that those molecular markers were apparently up-regulated by angiotensin II and salt loading (Fig 5). PAA-Pt reduced these markers expressions to the almost same extent with the control db/db mice and these results support our findings in the histopathological examinations.

Pericoronary fibrosis were evaluated by Azan staining. Fibrosis was robust by angiotensin II and high salt diet and was reversed by PAA-Pt.

Fig. 5. Molecular markers for cardiac damage and fibrosis.

mRNA expression of ANP and procollagen type I were evaluated. Concomitant with figure 4, angiotensin II and high salt diet aggravated cardiac damage and it was reversed by PAA-Pt.

PAA-Pt behaves as an SOD mimetic and possesses a catalase activity, then its efficacy can last longer than vitamin C (Kajita *et al.*, 2007). It is mainly because the catalytic property of PAA-Pt which is distinguished from vitamin C that is quickly consumed. Therefore we examined the effect of PAA-Pt in metabolic syndrome model mice to reduce oxidative stress and protect the cardiovascular system. Concordance with in vitro study, PAA-Pt effectually reduced ROS and prevented cardiovascular damage in hypertensive db/db mice.

We found the extension of cardiovascular damages was much severer and the level of systemic oxidative stress, which was evaluated by urinary excretion of isoprostane, was much higher in db/db mice. Then, it is indicated that the 'metabolic-syndrome-status' is equal to 'high-oxidative-stress-status' and this status seems to be the trigger of the vicious cycle of organ damage. Thus, the scavenging ROS would be a promising approach to preventing organ damage in metabolic syndrome.

PAA-Pt also has the possibility to solve the problems of former antioxidants as vitamin C, E or -carotene. It is said the one of the reasons why those former antioxidants would have failed to be effective against organ damage that they are short-acting and could not eliminate intracellular ROS, including mitochondrial ROS. PAA-Pt possesses long-lasting activity (means catalytic effect) and dual properties as SOD and catalase mimetics. The

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pharmacokinetics of PAA-Pt in detail have not been revealed yet that PAA-Pt should enter to systemic circulation from digestive apparatus, and be excreted from urine. Also we suggestively expect the novel antioxidant PAA-Pt enables to eliminate intracellular ROS. Further studies in pharmacokinetics and safety of PAA-Pt in chronic use are required. Specially, PAA-Pt contains platinum, which is sometimes toxic to the kidney, we must be careful about its nephrotoxicity before clinical applications. In site of some assignments such as its safety or full internal dynamism must be proven before clinical use, this novel agent will be certain way to open the door to interdisciplinary treatments between nanotechnology and medicine.

#### **4. Perspectives**

Piles of evidence accumulated that oxidative stress is a new therapeutic targets in preventing cardiovascular events and its risks. Among risks, hypertension, glucose metabolism and lipid accumulation in the vasculature are closely related with oxidative stress, however, some but not all antioxidant lower blood pressure or prevent atherosclerosis. Blood pressure is regulated by both circulating blood volume and vascular resistance and most of the data suggest that oxidative stress may affect vascular resistance but not blood volume, which can explain antioxidant therapy is not consistent in lowering blood pressure. For lipid disorders and consecutive atherosclerosis, oxidized LDL as well as its receptor is important. In mice LDL level are almost negligible however, oxidized LDL receptor such as LOX-1 overexpression leads to vascular damage (Inoue *et al.*, 2005). It suggests that ligand regulation by targeting oxidative stress is not sufficient but we should consider receptor regulation as well. On the other hand, insulin resistance or glucose metabolism can be a good target of antioxidant therapy. Insulin signaling is impaired by oxidative stress and it is reversed by antioxidants. Our and others' basic trials as well as recent data by bardoxolone methyl are promising, although the optimal glucose regulation level to prevent cardiovascular complications in diabetes are still under debate. The 'lower the better' of blood glucose is controversial because of hypoglycemia induces poor prognosis in cardiovascular event, however, targeting oxidative stress recovers impairedinsulin resistance and does not stimulate insulin release, therefore, it would be safer than insulin replacement or insulin secretion stimulating agents such as sulphonylurea. Further clinical trials are required.

#### **5. Conclusion**

AM, PAA-PT or bardoxolone are possibly promising antioxidants in treating or reducing risks for chronic metabolic disease and organ damages. Other possible agents are also under investigations and novel technology will lead us to invent antioxidants with more efficacy and less side effect. Oxidative stress is indispensable for host defence and appropriate reduction of oxidative stress is required when considering antioxidants in clinical use.

#### **6. References**

Akbar S, Bellary S & Griffiths HR. (2011). Dietary Antioxidant Interventions in Type 2 Diabetes Patients A Meta-analysis. *Br J Diabetes Vasc Dis* 11, 62-68.

pharmacokinetics of PAA-Pt in detail have not been revealed yet that PAA-Pt should enter to systemic circulation from digestive apparatus, and be excreted from urine. Also we suggestively expect the novel antioxidant PAA-Pt enables to eliminate intracellular ROS. Further studies in pharmacokinetics and safety of PAA-Pt in chronic use are required. Specially, PAA-Pt contains platinum, which is sometimes toxic to the kidney, we must be careful about its nephrotoxicity before clinical applications. In site of some assignments such as its safety or full internal dynamism must be proven before clinical use, this novel agent will be certain way to open the door to interdisciplinary treatments between

Piles of evidence accumulated that oxidative stress is a new therapeutic targets in preventing cardiovascular events and its risks. Among risks, hypertension, glucose metabolism and lipid accumulation in the vasculature are closely related with oxidative stress, however, some but not all antioxidant lower blood pressure or prevent atherosclerosis. Blood pressure is regulated by both circulating blood volume and vascular resistance and most of the data suggest that oxidative stress may affect vascular resistance but not blood volume, which can explain antioxidant therapy is not consistent in lowering blood pressure. For lipid disorders and consecutive atherosclerosis, oxidized LDL as well as its receptor is important. In mice LDL level are almost negligible however, oxidized LDL receptor such as LOX-1 overexpression leads to vascular damage (Inoue *et al.*, 2005). It suggests that ligand regulation by targeting oxidative stress is not sufficient but we should consider receptor regulation as well. On the other hand, insulin resistance or glucose metabolism can be a good target of antioxidant therapy. Insulin signaling is impaired by oxidative stress and it is reversed by antioxidants. Our and others' basic trials as well as recent data by bardoxolone methyl are promising, although the optimal glucose regulation level to prevent cardiovascular complications in diabetes are still under debate. The 'lower the better' of blood glucose is controversial because of hypoglycemia induces poor prognosis in cardiovascular event, however, targeting oxidative stress recovers impairedinsulin resistance and does not stimulate insulin release, therefore, it would be safer than insulin replacement or insulin secretion stimulating agents such as sulphonylurea. Further

AM, PAA-PT or bardoxolone are possibly promising antioxidants in treating or reducing risks for chronic metabolic disease and organ damages. Other possible agents are also under investigations and novel technology will lead us to invent antioxidants with more efficacy and less side effect. Oxidative stress is indispensable for host defence and appropriate reduction of oxidative stress is required when considering antioxidants in clinical use.

Akbar S, Bellary S & Griffiths HR. (2011). Dietary Antioxidant Interventions in Type 2

Diabetes Patients A Meta-analysis. *Br J Diabetes Vasc Dis* 11, 62-68.

nanotechnology and medicine.

clinical trials are required.

**5. Conclusion** 

**6. References** 

**4. Perspectives** 


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**21** 

*USA* 

**Retinal Vein Occlusion Induced by a MEK** 

The retina is a highly specialized sensory organ that transduces light energy into neural signal. It also has high energy requirement and an extensive vascular network. Reactive oxygen species (ROS) generated via light exposure, normal energy production, phagocytosis of spent photoreceptor membranes by retinal pigment epithelium (RPE) cells, and circulating toxins render retina at an increased risk for oxidative stress (Hardy et al., 2005; Siu et al., 2008). To cope with the high oxidant load, the retina is equipped with various antioxidant defense mechanisms, such as the expression of glutathione peroxidase and superoxide dismutase, the production of glutathione by Müller cells, high levels of vitamins C and E, and the presence of free radical scavenger melanin in RPE cells (Siu et al., 2008). However, when the redox balance is disrupted, retinal pathologies could result, and one of the consequences is impairment of the blood retinal barrier (BRB). Indeed, several retinal diseases have been shown or postulated to be linked to a state of oxidative stress and

Previously, we investigated the molecular mechanisms towards the development of retinal vein occlusion (RVO) in cancer patients treated with a mitogen-activated protein kinase kinase (MEK) inhibitor, PD0325901 (LoRusso et al., 2010). Through gene expression profiling analysis, we identified several mechanisms relevant to the development of RVO, including oxidative stress response, acute phase and inflammatory response, blood-retinal barrier

This chapter aims to provide an overview of BRB structures and functions, the role of oxidative stress in BRB disruption and development of retinal pathologies, a detailed overview of RVO, and finally, a description of proposed mechanisms of PD0325901-induced RVO, highlighting several important cellular and molecular processes relevant to this pathology.

The BRB has an endothelial and an epithelial component, namely the tight junctions between the endothelial cells of the inner retinal vessels, and those between cells of the RPE;

(BRB) breakdown, leukostasis, and coagulation cascade (Huang et al., 2009).

**1. Introduction** 

resulting BRB dysfunction.

**2. Blood-retinal barrier** 

**Inhibitor – Impact of Oxidative Stress** 

**on the Blood-Retinal Barrier** 

Amy H. Yang and Wenhu Huang *Drug Safety Research & Development, Pfizer Inc., La Jolla Laboratories,* 


### **Retinal Vein Occlusion Induced by a MEK Inhibitor – Impact of Oxidative Stress on the Blood-Retinal Barrier**

Amy H. Yang and Wenhu Huang *Drug Safety Research & Development, Pfizer Inc., La Jolla Laboratories, USA* 

#### **1. Introduction**

468 Oxidative Stress and Diseases

Yusuf S, Dagenais G, Pogue J, Bosch J & Sleight P. (2000). Vitamin E supplementation and

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cardiovascular events in high-risk patients. The Heart Outcomes Prevention

diabetic polyneuropathy with the antioxidant alpha-lipoic acid: a meta-analysis.

The retina is a highly specialized sensory organ that transduces light energy into neural signal. It also has high energy requirement and an extensive vascular network. Reactive oxygen species (ROS) generated via light exposure, normal energy production, phagocytosis of spent photoreceptor membranes by retinal pigment epithelium (RPE) cells, and circulating toxins render retina at an increased risk for oxidative stress (Hardy et al., 2005; Siu et al., 2008). To cope with the high oxidant load, the retina is equipped with various antioxidant defense mechanisms, such as the expression of glutathione peroxidase and superoxide dismutase, the production of glutathione by Müller cells, high levels of vitamins C and E, and the presence of free radical scavenger melanin in RPE cells (Siu et al., 2008). However, when the redox balance is disrupted, retinal pathologies could result, and one of the consequences is impairment of the blood retinal barrier (BRB). Indeed, several retinal diseases have been shown or postulated to be linked to a state of oxidative stress and resulting BRB dysfunction.

Previously, we investigated the molecular mechanisms towards the development of retinal vein occlusion (RVO) in cancer patients treated with a mitogen-activated protein kinase kinase (MEK) inhibitor, PD0325901 (LoRusso et al., 2010). Through gene expression profiling analysis, we identified several mechanisms relevant to the development of RVO, including oxidative stress response, acute phase and inflammatory response, blood-retinal barrier (BRB) breakdown, leukostasis, and coagulation cascade (Huang et al., 2009).

This chapter aims to provide an overview of BRB structures and functions, the role of oxidative stress in BRB disruption and development of retinal pathologies, a detailed overview of RVO, and finally, a description of proposed mechanisms of PD0325901-induced RVO, highlighting several important cellular and molecular processes relevant to this pathology.

#### **2. Blood-retinal barrier**

The BRB has an endothelial and an epithelial component, namely the tight junctions between the endothelial cells of the inner retinal vessels, and those between cells of the RPE;

Retinal Vein Occlusion Induced by a MEK Inhibitor –

Impact of Oxidative Stress on the Blood-Retinal Barrier 471

Fig. 1. The blood-retinal barrier (A) The retina is a multi-layered tissue in the posterior segment of the eye, and is shown by the H&E stained micrograph on the left. The cell types comprising the inner BRB (endothelial cells, pericytes, astrocytes, and Müller cells) and outer BRB (retinal pigment epithelial cells) are overlaid on the retinal micrograph, and are magnified on the right. Tight junctions between retinal capillary endothelial cells and retinal

pigment epithelial cells form the basis of the inner and outer BRB, respectively. The endothelial cells of inner retinal capillaries are not fenestrated, whereas those of the

the intercellular junctions. Sources: Kaur et al., 2008; Niessen, 2007; http://www.landesbioscience.com/curie/images/chapters/Hosoya1.jpg

choroidal capillaries are (depicted below the retinal micrograph). (B) Protein components of

these cell types comprise integral components of the inner and outer BRB, respectively (Siu et al., 2008; Fig. 1A). The BRB regulates the transport of fluid and molecules between the retinal tissue and vasculature, hence playing an important role in maintaining the homeostatsis of the retinal microenvironment (Kaur et al., 2008; Siu et al., 2008).

#### **2.1 Inner blood-retinal barrier**

The inner BRB is composed of endothelial cells, astrocytes, Müller cells, and pericytes (Fig. 1A). Tight junctions between capillary endothelial cells form the basis of the inner BRB (Fig. 1B). Astrocytes, Müller cells and pericytes, all closely associated with the endothelial cells of the inner BRB, contribute to proper BRB functions.

The endothelial cells of inner retinal capillaries are not fenestrated, contributing to their low permeability. Solutes traverse the retinal endothelium via both the transcellular and paracellular pathways: the former involves vesicle-mediated transport of macromolecules, and the latter, passage through minute intercellular space safeguarded by junctional proteins (Vandenbroucke et al., 2008; Fig. 1B). Tight junctions consist of occludins, claudins, and junctional adhesion molecules (JAMs), all of which form complexes between adjacent endothelial cells. Zonula occludens (ZO) proteins link occludins and claudins to the endothelial actin cytoskeleton via cingulin. In addition to tight junctions, adherens junctions (AJ) also contribute to the endothelial barrier, where vascular endothelial (VE) cadherins on adjacent endothelial cells form a homophilic complex (Garrido-Urbani et al., 2008; Vandenbroucke et al., 2008). The C-terminal domain of VE-cadherin binds -catenins and catenins, linking the AJs to the actin cytoskeleton. Several lines of evidence show that the regulation of actin cytoskeletal dynamics is central to the proper functioning of the endothelial barrier (Houle and Huot, 2006; Houle et al., 2003; Huot et al., 1998; Lum and Roebuck, 2001). It has also been reported that retinal endothelial cells are more susceptible to oxidative damage, leading to increased permeability, than endothelial cells at other sites. Indeed, ROS are also known to induce the expression of vascular endothelial growth factor (VEGF), a well-known endothelial mitogen and permeability factor, which contributes to the breakdown of BRB in experimental diabetes models (Chua et al., 1998; El-Remessy et al., 2003).

Pericytes line the outer surface of endothelial cells (Fig. 1A) and are contractile in nature, expressing actin, myosin, and tropomyosin (Kaur et al., 2008). They contract in response to signals such as hypoxia, endothelin-1, and angiontensin II, and relax on exposure to carbon dioxide, nitric oxide and adenosine. Therefore, pericytes regulate the vascular tone and blood flow. Under normoxia, they maintain the integrity of the inner BRB by inducing mRNA and protein expression of occludin and ZO-1, and by partially reversing the occludin decrease under hypoxia. Loss of pericytes and disruption of inner BRB are early events in diabetes.

Müller cells are the principal glial cells of the retina, and a functional link between neurons and vessels (Reichenbach et al., 2007). They span the inner and outer limiting membranes of the retina, with their foot processes in close contact with the retinal endothelial cells (Fig. 1A). Under physiological conditions, Müller cells contribute to the integrity of the BRB; however, when exposed to cellular stress they impair the barrier function. Under normoxia, Müller cells secrete pigment epithelium-derived factor (PEDF),

these cell types comprise integral components of the inner and outer BRB, respectively (Siu et al., 2008; Fig. 1A). The BRB regulates the transport of fluid and molecules between the retinal tissue and vasculature, hence playing an important role in maintaining the

The inner BRB is composed of endothelial cells, astrocytes, Müller cells, and pericytes (Fig. 1A). Tight junctions between capillary endothelial cells form the basis of the inner BRB (Fig. 1B). Astrocytes, Müller cells and pericytes, all closely associated with the endothelial cells of

The endothelial cells of inner retinal capillaries are not fenestrated, contributing to their low permeability. Solutes traverse the retinal endothelium via both the transcellular and paracellular pathways: the former involves vesicle-mediated transport of macromolecules, and the latter, passage through minute intercellular space safeguarded by junctional proteins (Vandenbroucke et al., 2008; Fig. 1B). Tight junctions consist of occludins, claudins, and junctional adhesion molecules (JAMs), all of which form complexes between adjacent endothelial cells. Zonula occludens (ZO) proteins link occludins and claudins to the endothelial actin cytoskeleton via cingulin. In addition to tight junctions, adherens junctions (AJ) also contribute to the endothelial barrier, where vascular endothelial (VE) cadherins on adjacent endothelial cells form a homophilic complex (Garrido-Urbani et al., 2008; Vandenbroucke et al., 2008). The C-terminal domain of VE-cadherin binds -catenins and catenins, linking the AJs to the actin cytoskeleton. Several lines of evidence show that the regulation of actin cytoskeletal dynamics is central to the proper functioning of the endothelial barrier (Houle and Huot, 2006; Houle et al., 2003; Huot et al., 1998; Lum and Roebuck, 2001). It has also been reported that retinal endothelial cells are more susceptible to oxidative damage, leading to increased permeability, than endothelial cells at other sites. Indeed, ROS are also known to induce the expression of vascular endothelial growth factor (VEGF), a well-known endothelial mitogen and permeability factor, which contributes to the breakdown of BRB in experimental diabetes models (Chua et al., 1998; El-Remessy et al.,

Pericytes line the outer surface of endothelial cells (Fig. 1A) and are contractile in nature, expressing actin, myosin, and tropomyosin (Kaur et al., 2008). They contract in response to signals such as hypoxia, endothelin-1, and angiontensin II, and relax on exposure to carbon dioxide, nitric oxide and adenosine. Therefore, pericytes regulate the vascular tone and blood flow. Under normoxia, they maintain the integrity of the inner BRB by inducing mRNA and protein expression of occludin and ZO-1, and by partially reversing the occludin decrease under hypoxia. Loss of pericytes and disruption of inner BRB are early events in

Müller cells are the principal glial cells of the retina, and a functional link between neurons and vessels (Reichenbach et al., 2007). They span the inner and outer limiting membranes of the retina, with their foot processes in close contact with the retinal endothelial cells (Fig. 1A). Under physiological conditions, Müller cells contribute to the integrity of the BRB; however, when exposed to cellular stress they impair the barrier function. Under normoxia, Müller cells secrete pigment epithelium-derived factor (PEDF),

homeostatsis of the retinal microenvironment (Kaur et al., 2008; Siu et al., 2008).

**2.1 Inner blood-retinal barrier** 

2003).

diabetes.

the inner BRB, contribute to proper BRB functions.

Fig. 1. The blood-retinal barrier (A) The retina is a multi-layered tissue in the posterior segment of the eye, and is shown by the H&E stained micrograph on the left. The cell types comprising the inner BRB (endothelial cells, pericytes, astrocytes, and Müller cells) and outer BRB (retinal pigment epithelial cells) are overlaid on the retinal micrograph, and are magnified on the right. Tight junctions between retinal capillary endothelial cells and retinal pigment epithelial cells form the basis of the inner and outer BRB, respectively. The endothelial cells of inner retinal capillaries are not fenestrated, whereas those of the choroidal capillaries are (depicted below the retinal micrograph). (B) Protein components of the intercellular junctions. Sources: Kaur et al., 2008; Niessen, 2007; http://www.landesbioscience.com/curie/images/chapters/Hosoya1.jpg

Retinal Vein Occlusion Induced by a MEK Inhibitor –

signaling transduction pathways.

**3.2.2 Retinopathy of prematurity** 

**3.2.3 Age-related macular degeneration** 

**3.2.1 Diabetic retinopathy** 

Impact of Oxidative Stress on the Blood-Retinal Barrier 473

and lipoxygenase pathways (Frey and Antonetti, 2011; Hardy et al., 2005; Kunsch and Medford, 1999). ROS can also act as intracellular second messengers and activate various

Diabetic retinopathy is a significant cause of blindness. Tissue hypoxia and hyperglycemia are generally regarded as contributors to diabetic retinopathy, but how these lead to the disease state is unclear. Current hypotheses, which are not mutually exclusive, for pathogenic mechanisms leading to diabetic retinopathy include oxidative stress, hemodynamic changes, inflammation and the activation of microglia, and increased leukocyte adhesion to the endothelial cells and entrapment (leukostasis) (Chibber et al., 2007). Many of these processes in fact have an association with excessive production of ROS (Yang et al., 2010b). For instance, growing evidence supports an important role for leukostasis in the development of diabetic retinopathy, with downstream consequences including capillary occlusion and localized production of ROS, resulting in endothelial cell damage, BRB breakdown, and increased vascular permeability (Chibber et al., 2007). Inflammatory cytokines interact with cell surface receptors in various cell types to activate signaling pathways that mediate responses of cell adhesion, permeability and apoptosis; they also increase the production of ROS by mitochondria (Busik et al., 2008; Sprague and Khalil, 2009). There are several models that recapitulate various aspects of diabetic retinopathy, including streptozotocin-induced diabetes and ischemia-reperfusion injury.

Retinopathy of prematurity (ROP) is a vasoproliferative disease that often develops when premature infants are given supplemental oxygen, and is a leading cause of blindness in children (Hardy et al., 2005; Uno et al., 2010). The developing eye is at an increased risk for oxidative injury from hyperoxia, as the retinal vasculature in premature infants lacks fullydeveloped mechanisms to auto-regulate oxygen tension (Hardy et al., 2005). ROP develops in two phases (Hardy et al., 2005). Hyperoxia in the retina leads to cessation of vascular development, resulting in endothelial cell death, vaso-obliteration and consequently, ischemia. To re-establish retinal perfusion, the retina mounts an exaggerated intravitreal preretinal neovascularization, which may ultimately result in retinal detachment and vision loss. Many features of ROP are recapitulated in oxygen induced retinopathy (OIR) (Brafman et al., 2004; Gu et al., 2002; Uno et al., 2010), in which neonatal animals are exposed to hyperoxia, leading to the generation of ROS, which have been postulated to be causal for vaso-obliteration, death of endothelial cells, and consequently, impairment of the BRB.

One of the major causes of blindness in the elderly population, age-related macular degeneration (AMD) is characterized by regional degeneration of photoreceptors and the RPE, lipofuscin accumulation in RPE cells, chronic inflammation, and drusen formation. Chronic oxidative stress has also been suggested to be an important factor to the

**3.2 Ocular pathologies associated with oxidative stress and BRB dysfunction** 

which downregulates VEGF expression and decreases vascular permeability (Yafai et al., 2007). Under hypoxia (Kaur et al., 2008) and oxidative stress (Yoshida et al., 2009), PEDF expression is decreased in Müller cells, thus favoring the secretion of VEGF and breakdown of the inner BRB. In addition, Müller cells are a source of matrix metalloproteinases, which proteolytically degrade the tight junction protein occludin, impairing the barrier function of retinal endothelial cells under cellular stress conditions. Müller cells also play a vital role in maintaining the retinal fluid balance (Reichenbach et al., 2007). Under physiological conditions, Müller cells carry out transcellular water transport from the retinal interstitial space into the blood, thus preventing excess fluid buildup within the retina. The transcellular water transport is osmotically coupled to the transport of potassium ions. When exposed to oxidative stress and inflammation, Müller cells have been shown to contribute to retinal edema through a disturbed intracellular fluid transport. Finally, Müller cells also respond to oxidative stress by increasing their production of the antioxidant glutathione (Siu et al., 2008).

Similar to pericytes and Müller cells, astrocytes are closely associated with the retinal vessels (Fig. 1A). They help maintain the BRB integrity by increasing the expression of the tight junction protein ZO-1 and modifying endothelial morphology (Kaur et al., 2008). Dysfunction of astrocytes has been linked to inner BRB breakdown and vasogenic edema.

#### **2.2 Outer blood-retinal barrier**

The outer BRB consists of tight junctions between RPE cells (Fig. 1A). The RPE is a monolayer of cells between the neuroretina and the choroid, and regulates access of blood components to the retina. Similar to the endothelium, movement across RPE is both transcellular and paracellular. The RPE cells exhibit a polarized morphology, with apical microvilli in contact with photoreceptor outer segments, and basal infoldings adjacent to the Bruch's membrane that separates the retina from choroidal capillaries called choriocapillaris (Kaur et al., 2008). Unlike the capillaries of the inner retina, the choroidal capillaries are fenestrated (Fig. 1A) and therefore do not contribute to the outer BRB (Kaur et al., 2008; Siu et al., 2008). Na+,K+-ATPase and aquaporin 1 (AQP1) expressed on the apical surface regulate movement of sodium, potassium, and water molecules across the RPE. Tight junctions, located at the apical side of the lateral membrane of the RPE cells, restrict paracellular movement between neighboring RPE cells. In addition to its function to regulate molecular transport integral to the outer BRB, RPE is responsible for phagocytizing photoreceptor outer segment membranes, which are digested by an extensive lysosome system, whose waste products are removed by the adjacent choriocapillaris (Burke, 2008; Siu et al., 2008). In pigmented animals, RPE cells also express melanin, a free radical scavenger that is also capable of absorbing stray light, and is thought to contribute to the retinal antioxidant mechanisms (Siu et al., 2008).

#### **3. Oxidative stress, BRB dysfunction and ocular diseases**

#### **3.1 Sources of ROS in the Retina**

In addition to the mitochondria, cellular sources of ROS in retina include endothelial cell xanthine oxidase, NAD(P)H oxidase, cyclooxygenase (COX), nitric oxide synthase (NOS), and lipoxygenase pathways (Frey and Antonetti, 2011; Hardy et al., 2005; Kunsch and Medford, 1999). ROS can also act as intracellular second messengers and activate various signaling transduction pathways.

#### **3.2 Ocular pathologies associated with oxidative stress and BRB dysfunction**

#### **3.2.1 Diabetic retinopathy**

472 Oxidative Stress and Diseases

which downregulates VEGF expression and decreases vascular permeability (Yafai et al., 2007). Under hypoxia (Kaur et al., 2008) and oxidative stress (Yoshida et al., 2009), PEDF expression is decreased in Müller cells, thus favoring the secretion of VEGF and breakdown of the inner BRB. In addition, Müller cells are a source of matrix metalloproteinases, which proteolytically degrade the tight junction protein occludin, impairing the barrier function of retinal endothelial cells under cellular stress conditions. Müller cells also play a vital role in maintaining the retinal fluid balance (Reichenbach et al., 2007). Under physiological conditions, Müller cells carry out transcellular water transport from the retinal interstitial space into the blood, thus preventing excess fluid buildup within the retina. The transcellular water transport is osmotically coupled to the transport of potassium ions. When exposed to oxidative stress and inflammation, Müller cells have been shown to contribute to retinal edema through a disturbed intracellular fluid transport. Finally, Müller cells also respond to oxidative stress by increasing their

Similar to pericytes and Müller cells, astrocytes are closely associated with the retinal vessels (Fig. 1A). They help maintain the BRB integrity by increasing the expression of the tight junction protein ZO-1 and modifying endothelial morphology (Kaur et al., 2008). Dysfunction of astrocytes has been linked to inner BRB breakdown and vasogenic edema.

The outer BRB consists of tight junctions between RPE cells (Fig. 1A). The RPE is a monolayer of cells between the neuroretina and the choroid, and regulates access of blood components to the retina. Similar to the endothelium, movement across RPE is both transcellular and paracellular. The RPE cells exhibit a polarized morphology, with apical microvilli in contact with photoreceptor outer segments, and basal infoldings adjacent to the Bruch's membrane that separates the retina from choroidal capillaries called choriocapillaris (Kaur et al., 2008). Unlike the capillaries of the inner retina, the choroidal capillaries are fenestrated (Fig. 1A) and therefore do not contribute to the outer BRB (Kaur et al., 2008; Siu et al., 2008). Na+,K+-ATPase and aquaporin 1 (AQP1) expressed on the apical surface regulate movement of sodium, potassium, and water molecules across the RPE. Tight junctions, located at the apical side of the lateral membrane of the RPE cells, restrict paracellular movement between neighboring RPE cells. In addition to its function to regulate molecular transport integral to the outer BRB, RPE is responsible for phagocytizing photoreceptor outer segment membranes, which are digested by an extensive lysosome system, whose waste products are removed by the adjacent choriocapillaris (Burke, 2008; Siu et al., 2008). In pigmented animals, RPE cells also express melanin, a free radical scavenger that is also capable of absorbing stray light, and is thought to contribute to the

In addition to the mitochondria, cellular sources of ROS in retina include endothelial cell xanthine oxidase, NAD(P)H oxidase, cyclooxygenase (COX), nitric oxide synthase (NOS),

production of the antioxidant glutathione (Siu et al., 2008).

**2.2 Outer blood-retinal barrier** 

retinal antioxidant mechanisms (Siu et al., 2008).

**3.1 Sources of ROS in the Retina** 

**3. Oxidative stress, BRB dysfunction and ocular diseases** 

Diabetic retinopathy is a significant cause of blindness. Tissue hypoxia and hyperglycemia are generally regarded as contributors to diabetic retinopathy, but how these lead to the disease state is unclear. Current hypotheses, which are not mutually exclusive, for pathogenic mechanisms leading to diabetic retinopathy include oxidative stress, hemodynamic changes, inflammation and the activation of microglia, and increased leukocyte adhesion to the endothelial cells and entrapment (leukostasis) (Chibber et al., 2007). Many of these processes in fact have an association with excessive production of ROS (Yang et al., 2010b). For instance, growing evidence supports an important role for leukostasis in the development of diabetic retinopathy, with downstream consequences including capillary occlusion and localized production of ROS, resulting in endothelial cell damage, BRB breakdown, and increased vascular permeability (Chibber et al., 2007). Inflammatory cytokines interact with cell surface receptors in various cell types to activate signaling pathways that mediate responses of cell adhesion, permeability and apoptosis; they also increase the production of ROS by mitochondria (Busik et al., 2008; Sprague and Khalil, 2009). There are several models that recapitulate various aspects of diabetic retinopathy, including streptozotocin-induced diabetes and ischemia-reperfusion injury.

#### **3.2.2 Retinopathy of prematurity**

Retinopathy of prematurity (ROP) is a vasoproliferative disease that often develops when premature infants are given supplemental oxygen, and is a leading cause of blindness in children (Hardy et al., 2005; Uno et al., 2010). The developing eye is at an increased risk for oxidative injury from hyperoxia, as the retinal vasculature in premature infants lacks fullydeveloped mechanisms to auto-regulate oxygen tension (Hardy et al., 2005). ROP develops in two phases (Hardy et al., 2005). Hyperoxia in the retina leads to cessation of vascular development, resulting in endothelial cell death, vaso-obliteration and consequently, ischemia. To re-establish retinal perfusion, the retina mounts an exaggerated intravitreal preretinal neovascularization, which may ultimately result in retinal detachment and vision loss. Many features of ROP are recapitulated in oxygen induced retinopathy (OIR) (Brafman et al., 2004; Gu et al., 2002; Uno et al., 2010), in which neonatal animals are exposed to hyperoxia, leading to the generation of ROS, which have been postulated to be causal for vaso-obliteration, death of endothelial cells, and consequently, impairment of the BRB.

#### **3.2.3 Age-related macular degeneration**

One of the major causes of blindness in the elderly population, age-related macular degeneration (AMD) is characterized by regional degeneration of photoreceptors and the RPE, lipofuscin accumulation in RPE cells, chronic inflammation, and drusen formation. Chronic oxidative stress has also been suggested to be an important factor to the

Retinal Vein Occlusion Induced by a MEK Inhibitor –

causing complications.

**4.2 Risk factors of RVO** 

which may induce RVO.

Impact of Oxidative Stress on the Blood-Retinal Barrier 475

therefore most of the retina is affected. In BRVO, when macular venules are occluded, a vision decrease can occur depending on the amount of ischemia and edema. When one of the vein's two trunks is blocked and half of the retina is affected, it is called hemi-central retinal vein occlusion (HRVO). According to several RVO epidemiology studies, the prevalence of both CRVO and BRVO increases significantly with age, more in middle-aged and elderly populations and uncommon in young adults under the age of 40. Most patients with CRVO are male and over 65 years of age, but there seems to be no gender difference for BRVO. Most CRVO cases are unilateral and painless and only 6-14% of cases are found to be bilateral (Cheung et al., 2008; Klein et al., 2008; Lim et al., 2008; Marcucci et al., 2011; Xu et al., 2007). A recent combined world-wide data pool, containing 68,751 individuals with ages ranging from 30 to 101 years, suggested that approximately 16 million people are affected by RVO with 5.2 per 1000 for any RVO, 4.42 per 1000 for BRVO and 0.8 per 1000 for CRVO. The incidence of CRVO was lower than that of BRVO in all ethnic populations (Rogers et al., 2010). However, CRVO is the most clinically relevant RVO as it is associated with severe vision loss, especially for the ischemic (non-perfused or hemorrhagic retinopathy) RVO. Among the complications of RVO, the devastating neovascular glaucoma resulting from anterior segment neovascularization is seen only in ischemic CRVO. Fortunately, most cases (81%) (Hayreh et al., 1994) are of the non-ischemic type that rarely develops blindness-

CRVO and BRVO have different symptoms, risk factors, pathogenesis, and therefore treatment. The pathogenesis for RVO is multifactorial and still under investigation. Anatomical positions of retinal veins play an important role in the pathogenesis of RVO (Fraenkl et al., 2010). The central retinal artery and vein share a common adventitial sheath in the optic nerve head. In CRVO, the tract of central retinal vein passing through the narrowing lamina cribrosa is the most frequent site of occlusion. In BRVO, vein occlusions occur at the junction of retinal vein and artery crossings in the retina. The mechanical compression of the veins at the narrowing passage or arteriovenous crossings predispose retinal veins to thrombus formation by various factors, including slowed or disturbed blood flow, endothelial damage in the vessel wall, changes in the blood viscosity, perivascular changes such as in lamina cribrosa (Albon et al., 1995), and sclerotic changes in the retinal arteries. Ocular risk factors associated with RVO include glaucoma or ocular hypertension. In glaucoma, increased intraocular pressure causes mechanical compression of retinal veins,

RVO has often been associated with a variety of systemic vascular disorders including arterial hypertension, arteriosclerosis, diabetes mellitus, dyslipidemia, and systemic vasculitis (The Eye Disease Case-Control Study Group, 1993, 1996; Koizumi et al., 2007; Mitchell et al., 1996; Sperduto et al., 1998). The increased rigidity of arterial wall affiliated

Abnormal blood viscosity, platelets, and coagulation have also been suggested to be involved in RVO pathogenesis (Trope et al., 1983). Hematological dysfunction, such as increased plasma fibrinogen and disruption of the thrombosis-fibrinolysis balance, have been implicated in the development of RVO (Rehak and Rehak, 2008). Increased fibrinogen

with these diseases may result in compression of retinal veins.

pathogenesis of AMD. As alluded to earlier, RPE may be at a high risk for oxidative stress due to its location and function. The RPE is in an oxygen-rich environment adjacent to the choriocapillaris, is continuously exposed to light, sometimes at phototoxic wavelengths, and is responsible for the renewal of photoreceptor outer segments via phagocytosis (Burke, 2008). The high content of polyunsaturated fatty acids of these membrane segments make them susceptible to lipid peroxidation and subsequent free radical formation. Experimental evidence supporting a role of oxidative stress in AMD showed that supplementation of antioxidants in AMD patients has a protective effect, and that cigarette smoking, known to be a source of exogenous free radicals, is a risk factor for AMD (Burke, 2008). Given the central role RPE plays in AMD pathogenesis, a commonly used experimental model to study the link between oxidative stress and AMD involves the use of cultured human RPE (ARPE-19) cells. Common endpoints include cell survival, morphology, activation of signaling cascades, and cytokine production (Chan et al., 2008; Dong et al., 2011; Glotin et al., 2006; Jiang et al., 2009; Klettner and Roider, 2009; Qin et al., 2006; Tsao et al., 2006; Wang et al., 1998; Wu et al., 2010).

#### **4. Retinal vein occlusion**

Retinal vein occlusion is a vascular disorder of the retina that occurs when one or more of the retinal veins are blocked and the circulation of retinal blood becomes obstructed. This ocular pathology can be a primary lesion or secondary to other retinal diseases. With the blockage, poor venous drainage and increased retinal capillary pressure and permeability ultimately lead to retinal ischemia and edema. Retinal ischemia could lead to the generation of ROS, impacting the integrity of the BRB. Diabetic retinopathy and RVO are the two most common causes of inner BRB breakdown. Among complications found in the clinical examination are hemorrhages, edema, ischemia, neovascularization of the retina as well as increased intraocular pressure. Depending on the location and severity, loss of visual acuity can range from very mild to severe. While some patients with RVO may not have any symptoms, some patients may complain of blurred vision or visual field defects. In severe cases, RVO can lead to vision loss in the affected eye. The most common cause of decreased vision is macular degeneration secondary to RVO, which occurs when leakage within macula leads to macular edema and ischemia. Neovascularization and neovascular glaucoma are the other vision-threatening complications that are devastating for patients with RVO and that should be promptly diagnosed and treated. Vein occlusion is commonly diagnosed by examining the fundus with ophthalmoscope for characteristic morphological changes such as venous tortuosity, cotton-wool spots, dot and flame hemorrhage, and edematous optic nerves, and by fluorescein angiography for vasculature blood flow obstruction, leakage in the retina, retinal ischemia, aneurysm, neovascularization, and macular edema. Sometimes optical coherence tomography (OCT) is used to measure retinal thickness for the determination of the presence of macular edema. Central and peripheral visual disturbance should be evaluated by functional tests in the physical examination.

#### **4.1 Classification of RVO**

Retinal vein occlusion is primarily classified into central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO) based on the location of obstruction. In CRVO, the occlusion of the central retinal vein can slow or stop blood from leaving the retina and

pathogenesis of AMD. As alluded to earlier, RPE may be at a high risk for oxidative stress due to its location and function. The RPE is in an oxygen-rich environment adjacent to the choriocapillaris, is continuously exposed to light, sometimes at phototoxic wavelengths, and is responsible for the renewal of photoreceptor outer segments via phagocytosis (Burke, 2008). The high content of polyunsaturated fatty acids of these membrane segments make them susceptible to lipid peroxidation and subsequent free radical formation. Experimental evidence supporting a role of oxidative stress in AMD showed that supplementation of antioxidants in AMD patients has a protective effect, and that cigarette smoking, known to be a source of exogenous free radicals, is a risk factor for AMD (Burke, 2008). Given the central role RPE plays in AMD pathogenesis, a commonly used experimental model to study the link between oxidative stress and AMD involves the use of cultured human RPE (ARPE-19) cells. Common endpoints include cell survival, morphology, activation of signaling cascades, and cytokine production (Chan et al., 2008; Dong et al., 2011; Glotin et al., 2006; Jiang et al., 2009; Klettner and Roider, 2009; Qin et al., 2006; Tsao et al., 2006; Wang

Retinal vein occlusion is a vascular disorder of the retina that occurs when one or more of the retinal veins are blocked and the circulation of retinal blood becomes obstructed. This ocular pathology can be a primary lesion or secondary to other retinal diseases. With the blockage, poor venous drainage and increased retinal capillary pressure and permeability ultimately lead to retinal ischemia and edema. Retinal ischemia could lead to the generation of ROS, impacting the integrity of the BRB. Diabetic retinopathy and RVO are the two most common causes of inner BRB breakdown. Among complications found in the clinical examination are hemorrhages, edema, ischemia, neovascularization of the retina as well as increased intraocular pressure. Depending on the location and severity, loss of visual acuity can range from very mild to severe. While some patients with RVO may not have any symptoms, some patients may complain of blurred vision or visual field defects. In severe cases, RVO can lead to vision loss in the affected eye. The most common cause of decreased vision is macular degeneration secondary to RVO, which occurs when leakage within macula leads to macular edema and ischemia. Neovascularization and neovascular glaucoma are the other vision-threatening complications that are devastating for patients with RVO and that should be promptly diagnosed and treated. Vein occlusion is commonly diagnosed by examining the fundus with ophthalmoscope for characteristic morphological changes such as venous tortuosity, cotton-wool spots, dot and flame hemorrhage, and edematous optic nerves, and by fluorescein angiography for vasculature blood flow obstruction, leakage in the retina, retinal ischemia, aneurysm, neovascularization, and macular edema. Sometimes optical coherence tomography (OCT) is used to measure retinal thickness for the determination of the presence of macular edema. Central and peripheral visual disturbance should be evaluated by functional tests in the physical examination.

Retinal vein occlusion is primarily classified into central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO) based on the location of obstruction. In CRVO, the occlusion of the central retinal vein can slow or stop blood from leaving the retina and

et al., 1998; Wu et al., 2010).

**4. Retinal vein occlusion** 

**4.1 Classification of RVO** 

therefore most of the retina is affected. In BRVO, when macular venules are occluded, a vision decrease can occur depending on the amount of ischemia and edema. When one of the vein's two trunks is blocked and half of the retina is affected, it is called hemi-central retinal vein occlusion (HRVO). According to several RVO epidemiology studies, the prevalence of both CRVO and BRVO increases significantly with age, more in middle-aged and elderly populations and uncommon in young adults under the age of 40. Most patients with CRVO are male and over 65 years of age, but there seems to be no gender difference for BRVO. Most CRVO cases are unilateral and painless and only 6-14% of cases are found to be bilateral (Cheung et al., 2008; Klein et al., 2008; Lim et al., 2008; Marcucci et al., 2011; Xu et al., 2007). A recent combined world-wide data pool, containing 68,751 individuals with ages ranging from 30 to 101 years, suggested that approximately 16 million people are affected by RVO with 5.2 per 1000 for any RVO, 4.42 per 1000 for BRVO and 0.8 per 1000 for CRVO. The incidence of CRVO was lower than that of BRVO in all ethnic populations (Rogers et al., 2010). However, CRVO is the most clinically relevant RVO as it is associated with severe vision loss, especially for the ischemic (non-perfused or hemorrhagic retinopathy) RVO. Among the complications of RVO, the devastating neovascular glaucoma resulting from anterior segment neovascularization is seen only in ischemic CRVO. Fortunately, most cases (81%) (Hayreh et al., 1994) are of the non-ischemic type that rarely develops blindnesscausing complications.

#### **4.2 Risk factors of RVO**

CRVO and BRVO have different symptoms, risk factors, pathogenesis, and therefore treatment. The pathogenesis for RVO is multifactorial and still under investigation. Anatomical positions of retinal veins play an important role in the pathogenesis of RVO (Fraenkl et al., 2010). The central retinal artery and vein share a common adventitial sheath in the optic nerve head. In CRVO, the tract of central retinal vein passing through the narrowing lamina cribrosa is the most frequent site of occlusion. In BRVO, vein occlusions occur at the junction of retinal vein and artery crossings in the retina. The mechanical compression of the veins at the narrowing passage or arteriovenous crossings predispose retinal veins to thrombus formation by various factors, including slowed or disturbed blood flow, endothelial damage in the vessel wall, changes in the blood viscosity, perivascular changes such as in lamina cribrosa (Albon et al., 1995), and sclerotic changes in the retinal arteries. Ocular risk factors associated with RVO include glaucoma or ocular hypertension. In glaucoma, increased intraocular pressure causes mechanical compression of retinal veins, which may induce RVO.

RVO has often been associated with a variety of systemic vascular disorders including arterial hypertension, arteriosclerosis, diabetes mellitus, dyslipidemia, and systemic vasculitis (The Eye Disease Case-Control Study Group, 1993, 1996; Koizumi et al., 2007; Mitchell et al., 1996; Sperduto et al., 1998). The increased rigidity of arterial wall affiliated with these diseases may result in compression of retinal veins.

Abnormal blood viscosity, platelets, and coagulation have also been suggested to be involved in RVO pathogenesis (Trope et al., 1983). Hematological dysfunction, such as increased plasma fibrinogen and disruption of the thrombosis-fibrinolysis balance, have been implicated in the development of RVO (Rehak and Rehak, 2008). Increased fibrinogen

Retinal Vein Occlusion Induced by a MEK Inhibitor –

hemorrhages and cotton wool spots.

predispose patients to weakened BRB.

**4.4.3 MEK inhibitor PD0325901** 

**4.4.2 Tumor necrosis factor** 

**4.4.1 Interferon-α** 

Impact of Oxidative Stress on the Blood-Retinal Barrier 477

In addition to RVO that arises due to pathophysiological causes described above, this ocular disorder can also develop as an adverse event from treatment with certain therapeutics.

Interferon-α (IFN-α) is used for the treatment of many cancers and chronic hepatitis C. Interferon-associated retinopathy has been documented since the 1990s, most commonly characterized by hemorrhage and cotton-wool spots, and sometimes by macular edema, retinal vascular occlusion, and retinal ischemia. The RVO could involve either the vein or artery, or both, and in most cases is reversible. The exact pathophysiological mechanism of interferon-induced retinopathy is unknown, although there are similarities with early stages of diabetic retinopathy (Bajaire et al., 2011; Esmaeli et al., 2001). Several risk factors have been suggested, including hypertension, hyperlipidemia, a hypercoagulable state, and diabetes (Nadir et al., 2000). In addition, IFN-α is known to cause the development of autoantibodies in 10% of the patients receiving treatment, and to exacerbate certain systemic autoimmune diseases. It is speculated that IFN-α therapy might cause deposition of immune complexes in retinal vasculature, with sequelae of retinal ischemia,

Tumor necrosis factor (TNF) is a proinflammatory cytokine that has been implicated in various diseases, including autoimmune diseases, diabetes, and cancer. In a phase II trial of recombinant TNF in patients with advanced colon cancer, TNF was administered by i.v. infusions twice daily for 5 consecutive days every other week for 8 weeks (Kemeny et al., 1990). Two out of 16 patients developed retinal vein thrombosis several weeks following completion of therapy. This finding is consistent with the known role of TNF in vascular leakage and blood-retinal barrier breakdown in diabetic retinopathy (Frey and Antonetti, 2011). In support of this, a patient with macular edema secondary to BRVO saw an improvement in visual acuity and cessation of macular edema during treatment with infliximab, a TNF- antibody, administered for rheumatoid arthritis (Kachi et al., 2010). Paradoxically, infliximab therapy has also been linked in several case studies to the development of retinal vein thrombosis/occlusion in a patient being treated for ulcerative colitis (Veerappan et al., 2008), psoriasis (Vergou et al., 2010), or Crohn's disease (Puli and Benage, 2003). The temporal relationship between infliximab infusion and retinopathy suggested the two may be causally related. In two of the three cases, a medical history of myocardial infarction or hyperlipidemia was noted, both of which considered risk factors for RVO. Moreover, all three of these diseases are inflammatory in nature, and may

PD0325901 is a potent and selective MEK inhibitor, developed for the treatment of advanced cancer. MEK is a key molecule in the Ras-mitogen-activated protein kinase (MAPK) pathway, which has roles including cellular proliferation and survival, and its only known

**4.4 Therapeutics associated with the clinical presentation of RVO** 

has been associated with RVO in several clinical reports (Lip et al., 1998; Patrassi et al., 1987; Peduzzi et al., 1986). An increasing number of studies have sought to establish an association between RVO and thrombophilic abnormalities. Thrombophilic risk factors related to RVO include hyperhomocysteinemia, methylenetetrahydrofolate reductase (MTHFR) gene mutation, factor V Leiden mutation, protein C and S deficiency, antithrombin deficiency, prothrombin gene mutation, anticardiolipin antibodies and lupus anticoagulant. High levels of circulating homocysteine may damage the vascular endothelium by releasing free radicals, creating a hypercoagulable environment (Angayarkanni et al., 2008). It appears that there is an association between RVO and hyperhomocysteinaemia and anti-phospholipid antibodies. However, for the other thrombophilic risk factors, there is a lack of consistency among the studies and the association with RVO is inconclusive (Fegan, 2002; Janssen et al., 2005; Rehak and Rehak, 2008). More recently, elevated levels of soluble endothelial protein C receptor (sEPCR) emerged an important candidate risk factor especially for CRVO (Gumus et al., 2006).

Significantly increased concentrations of growth factors, cytokines, and chemokines such as VEGF, interleukin (IL)-6, IL-8, interferon-inducible 10-kDa protein (IP-10), monocytochemotactic protein-1 (MCP-1), and platelet-derived growth factor (PDGF)-AA were observed in vitreous or aqueous humor samples of patients with RVO (Funk et al., 2009; Noma et al., 2009; Yoshimura et al., 2009). Excessive production of VEGF and inflammatory cytokines can be induced by ischemic conditions. The levels of VEGF and inflammatory cytokines are correlated with severity of retinal ischemia and macular edema (Noma et al., 2006), as well as neovascularization. A close correlation between aqueous VEGF levels and iris neovascularization and vascular permeability in CRVO patients has been found (Boyd et al., 2002).

#### **4.3 Oxidative stress and RVO**

Retinal ischemia that occurs in some cases of RVO could lead to the generation of ROS, and compromise the integrity of the BRB. In fact, RVO is a common complication of diabetic retinopathy, in which hypoxia-ischemia is thought to play a role in its pathogenesis. Many of the risk factors for RVO described above, such as alterations in blood flow, systemic vascular disorders, hypercoagulability, and elevated levels of pro-inflammatory cytokines, may also be associated with a state of oxidative stress (Simoncini et al., 2005). Indeed, in a case-control prospective study in young adult CRVO patients, serum levels of paraoxonase-1 arylesterase (PON1-ARE) activity, reported to have antioxidant potential, were found to be negatively correlated with hyperhomocysteinemia and lipid peroxidation, an indicator of oxidative stress (Angayarkanni et al., 2008). Decreased levels of PON1-ARE activity as well as increased levels of the lipid peroxidation marker were shown to be risk factors for CRVO. In another case study, an individual with glucose-6-phosphate dehydrogenase(G6PD) deficiency was exposed to an oxidative stressor, and later developed CRVO (Kotwal et al., 2009). G6PD deficiency is known to increase erythrocyte vulnerability to oxidative stress, which may precipitate hemolysis, increased erythrocyte aggregation and erythrocyteendothelium interaction, leading to thrombosis (Kotwal et al., 2009). Anti-phospholipid antibodies have been associated with the development of RVO, and shown to induce oxidative stress in endothelial cells (Simoncini et al., 2005). Taken together, these lines of evidence suggest that a state of oxidative stress may predispose individuals to RVO.

#### **4.4 Therapeutics associated with the clinical presentation of RVO**

In addition to RVO that arises due to pathophysiological causes described above, this ocular disorder can also develop as an adverse event from treatment with certain therapeutics.

#### **4.4.1 Interferon-α**

476 Oxidative Stress and Diseases

has been associated with RVO in several clinical reports (Lip et al., 1998; Patrassi et al., 1987; Peduzzi et al., 1986). An increasing number of studies have sought to establish an association between RVO and thrombophilic abnormalities. Thrombophilic risk factors related to RVO include hyperhomocysteinemia, methylenetetrahydrofolate reductase (MTHFR) gene mutation, factor V Leiden mutation, protein C and S deficiency, antithrombin deficiency, prothrombin gene mutation, anticardiolipin antibodies and lupus anticoagulant. High levels of circulating homocysteine may damage the vascular endothelium by releasing free radicals, creating a hypercoagulable environment (Angayarkanni et al., 2008). It appears that there is an association between RVO and hyperhomocysteinaemia and anti-phospholipid antibodies. However, for the other thrombophilic risk factors, there is a lack of consistency among the studies and the association with RVO is inconclusive (Fegan, 2002; Janssen et al., 2005; Rehak and Rehak, 2008). More recently, elevated levels of soluble endothelial protein C receptor (sEPCR) emerged an important candidate risk factor especially for CRVO (Gumus et al., 2006).

Significantly increased concentrations of growth factors, cytokines, and chemokines such as VEGF, interleukin (IL)-6, IL-8, interferon-inducible 10-kDa protein (IP-10), monocytochemotactic protein-1 (MCP-1), and platelet-derived growth factor (PDGF)-AA were observed in vitreous or aqueous humor samples of patients with RVO (Funk et al., 2009; Noma et al., 2009; Yoshimura et al., 2009). Excessive production of VEGF and inflammatory cytokines can be induced by ischemic conditions. The levels of VEGF and inflammatory cytokines are correlated with severity of retinal ischemia and macular edema (Noma et al., 2006), as well as neovascularization. A close correlation between aqueous VEGF levels and iris neovascularization and vascular permeability in CRVO patients has

Retinal ischemia that occurs in some cases of RVO could lead to the generation of ROS, and compromise the integrity of the BRB. In fact, RVO is a common complication of diabetic retinopathy, in which hypoxia-ischemia is thought to play a role in its pathogenesis. Many of the risk factors for RVO described above, such as alterations in blood flow, systemic vascular disorders, hypercoagulability, and elevated levels of pro-inflammatory cytokines, may also be associated with a state of oxidative stress (Simoncini et al., 2005). Indeed, in a case-control prospective study in young adult CRVO patients, serum levels of paraoxonase-1 arylesterase (PON1-ARE) activity, reported to have antioxidant potential, were found to be negatively correlated with hyperhomocysteinemia and lipid peroxidation, an indicator of oxidative stress (Angayarkanni et al., 2008). Decreased levels of PON1-ARE activity as well as increased levels of the lipid peroxidation marker were shown to be risk factors for CRVO. In another case study, an individual with glucose-6-phosphate dehydrogenase(G6PD) deficiency was exposed to an oxidative stressor, and later developed CRVO (Kotwal et al., 2009). G6PD deficiency is known to increase erythrocyte vulnerability to oxidative stress, which may precipitate hemolysis, increased erythrocyte aggregation and erythrocyteendothelium interaction, leading to thrombosis (Kotwal et al., 2009). Anti-phospholipid antibodies have been associated with the development of RVO, and shown to induce oxidative stress in endothelial cells (Simoncini et al., 2005). Taken together, these lines of

evidence suggest that a state of oxidative stress may predispose individuals to RVO.

been found (Boyd et al., 2002).

**4.3 Oxidative stress and RVO** 

Interferon-α (IFN-α) is used for the treatment of many cancers and chronic hepatitis C. Interferon-associated retinopathy has been documented since the 1990s, most commonly characterized by hemorrhage and cotton-wool spots, and sometimes by macular edema, retinal vascular occlusion, and retinal ischemia. The RVO could involve either the vein or artery, or both, and in most cases is reversible. The exact pathophysiological mechanism of interferon-induced retinopathy is unknown, although there are similarities with early stages of diabetic retinopathy (Bajaire et al., 2011; Esmaeli et al., 2001). Several risk factors have been suggested, including hypertension, hyperlipidemia, a hypercoagulable state, and diabetes (Nadir et al., 2000). In addition, IFN-α is known to cause the development of autoantibodies in 10% of the patients receiving treatment, and to exacerbate certain systemic autoimmune diseases. It is speculated that IFN-α therapy might cause deposition of immune complexes in retinal vasculature, with sequelae of retinal ischemia, hemorrhages and cotton wool spots.

#### **4.4.2 Tumor necrosis factor**

Tumor necrosis factor (TNF) is a proinflammatory cytokine that has been implicated in various diseases, including autoimmune diseases, diabetes, and cancer. In a phase II trial of recombinant TNF in patients with advanced colon cancer, TNF was administered by i.v. infusions twice daily for 5 consecutive days every other week for 8 weeks (Kemeny et al., 1990). Two out of 16 patients developed retinal vein thrombosis several weeks following completion of therapy. This finding is consistent with the known role of TNF in vascular leakage and blood-retinal barrier breakdown in diabetic retinopathy (Frey and Antonetti, 2011). In support of this, a patient with macular edema secondary to BRVO saw an improvement in visual acuity and cessation of macular edema during treatment with infliximab, a TNF- antibody, administered for rheumatoid arthritis (Kachi et al., 2010). Paradoxically, infliximab therapy has also been linked in several case studies to the development of retinal vein thrombosis/occlusion in a patient being treated for ulcerative colitis (Veerappan et al., 2008), psoriasis (Vergou et al., 2010), or Crohn's disease (Puli and Benage, 2003). The temporal relationship between infliximab infusion and retinopathy suggested the two may be causally related. In two of the three cases, a medical history of myocardial infarction or hyperlipidemia was noted, both of which considered risk factors for RVO. Moreover, all three of these diseases are inflammatory in nature, and may predispose patients to weakened BRB.

#### **4.4.3 MEK inhibitor PD0325901**

PD0325901 is a potent and selective MEK inhibitor, developed for the treatment of advanced cancer. MEK is a key molecule in the Ras-mitogen-activated protein kinase (MAPK) pathway, which has roles including cellular proliferation and survival, and its only known

Retinal Vein Occlusion Induced by a MEK Inhibitor –

Impact of Oxidative Stress on the Blood-Retinal Barrier 479

Fig. 2. MAPK signaling pathway The MAPK cascade is a highly conserved module that is involved in various cellular functions, including cell proliferation, differentiation and migration. Mammals express at least four distinctly regulated groups of MAPKs, ERK1/2, JNK1/2/3, p38alpha/beta/gamma/delta and ERK5, that are activated by specific MAPKKs: MEK1/2 for ERK1/2, MKK3/6 for the p38, MKK4/7 (JNKK1/2) for the JNKs, and MEK5 for ERK5. Each MAPKK, however, can be activated by more than one MAPKKK, increasing the

complexity and diversity of MAPK signalling. Presumably each MAPKKK confers responsiveness to distinct stimuli. For example, activation of ERK1/2 by growth factors depends on the MAPKKK c-Raf, but other MAPKKKs may activate ERK1/2 in response to

pro-inflammatory stimuli. Source: KEGG (http://www.genome.jp/dbget-

bin/www\_bget?map04010) (Kanehisa, 2000, 2012)

substrate is the extracellular signal-regulated kinase (ERK), which in turn phosphorylates and activates downstream molecules in the pathway (Fig. 2). In the phase I dose escalation clinical trial of PD0325901, dose-limiting RVO was observed, characterized by the presence of cotton wool spots, hemorrhages, and vein occlusion. RVO developed in 2 patients after 3.5-4 months of 10 or 15 mg BID continuous treatment schedule, and in 1 patient after 9 months of 10 mg BID on a 5 days on/2 days off schedule, and was reversible upon treatment discontinuation (LoRusso et al., 2010). It was noted that at doses >= 4 mg BID, the systemic exposure of PD0325901 was equivalent to that in animal models that resulted in 90% phosphorylated ERK (pERK) suppression (LoRusso et al., 2010). Therefore, the ocular lesions could be related to the prolonged and/or significant levels of pERK suppression.

Two other MEK inhibitors, CI-1040 and selumetinib (AZD6244), also progressed to the clinic, but did not cause RVO. CI-1040 is a structural analogue of PD0325901. Insufficient clinical efficacy was reported due to poor bioavailability and metabolic instability (Rinehart et al., 2004). Selumetinib caused blurred vision in 12% of patients at >=100 mg BID in a Phase I trial (Adjei et al., 2008); this finding was not reported in subsequent Phase II trials at the 100 mg BID dose (Bekaii-Saab et al., 2011; Bodoky et al., 2011). Compared to PD0325901, selumetinib is approximately 10-fold less potent, and has a relatively poor bioavailability. Taken together, even though it is at present unclear whether PD0325901 caused RVO due to its deep inhibition of pERK, or to its chemotype, the above evidence suggests that the incidence of ocular lesions correlates with the efficacy of MEK inhibition.

#### **5. Molecular mechanisms of MEK inhibitor PD0325901-Induced RVO**

To develop an animal model of RVO to investigate mechanisms of toxicity, an in-life study was performed in rabbits, in which PD0325901 was administered by intravitreal injection at doses of 0.5 and 1 mg/eye, with an observation period of 2 weeks (Huang et al., 2009). The high dose was extrapolated to be a potentially toxic dose, while the low dose was chosen as a subtoxic dose, based on in vitro cytotoxicity data (Huang et al., 2009). As early as 1 day after treatment, the high dose produced hemorrhages and vascular leakage with branch occlusion. These lesions progressed to retinal detachment, edema, abnormal kinetic blood flow, and retinal vessel occlusion after 7 days. At the low dose, retinal vascular leakage was observed without vascular occlusion. Therefore, the rabbit model provided evidence that PD0325901 at sufficient ocular concentrations could lead to similar retinal lesions seen in the clinic.

The retinal vascular toxicity was not observed in preclinical safety studies where PD0325901 was administered orally in rats and dogs for up to 13 weeks (Huang et al., 2009). The difference in the level of ocular toxicity between rabbits and rats/dogs could be attributed to ocular drug concentration differences arising from local vs. systemic routes of administration. Since molecular events could precede overt signs of tissue injury, an investigative study was conducted in which rats were dosed orally for 3 or 5 days at 45 mg/kg/day, estimated to be at 70% maximal tolerated dose (Huang et al., 2009). No retinal toxicity was observed by ophthalmic examinations or fundus fluorescein angiography. Despite the absence of overt injury, global gene expression profiling on vehicle and PD0325901-treated retinas revealed several mechanisms relevant to the development of RVO, including oxidative stress response, acute phase and inflammatory response, BRB

substrate is the extracellular signal-regulated kinase (ERK), which in turn phosphorylates and activates downstream molecules in the pathway (Fig. 2). In the phase I dose escalation clinical trial of PD0325901, dose-limiting RVO was observed, characterized by the presence of cotton wool spots, hemorrhages, and vein occlusion. RVO developed in 2 patients after 3.5-4 months of 10 or 15 mg BID continuous treatment schedule, and in 1 patient after 9 months of 10 mg BID on a 5 days on/2 days off schedule, and was reversible upon treatment discontinuation (LoRusso et al., 2010). It was noted that at doses >= 4 mg BID, the systemic exposure of PD0325901 was equivalent to that in animal models that resulted in 90% phosphorylated ERK (pERK) suppression (LoRusso et al., 2010). Therefore, the ocular lesions could be related to the prolonged and/or significant levels of pERK suppression.

Two other MEK inhibitors, CI-1040 and selumetinib (AZD6244), also progressed to the clinic, but did not cause RVO. CI-1040 is a structural analogue of PD0325901. Insufficient clinical efficacy was reported due to poor bioavailability and metabolic instability (Rinehart et al., 2004). Selumetinib caused blurred vision in 12% of patients at >=100 mg BID in a Phase I trial (Adjei et al., 2008); this finding was not reported in subsequent Phase II trials at the 100 mg BID dose (Bekaii-Saab et al., 2011; Bodoky et al., 2011). Compared to PD0325901, selumetinib is approximately 10-fold less potent, and has a relatively poor bioavailability. Taken together, even though it is at present unclear whether PD0325901 caused RVO due to its deep inhibition of pERK, or to its chemotype, the above evidence suggests that the

incidence of ocular lesions correlates with the efficacy of MEK inhibition.

**5. Molecular mechanisms of MEK inhibitor PD0325901-Induced RVO** 

sufficient ocular concentrations could lead to similar retinal lesions seen in the clinic.

The retinal vascular toxicity was not observed in preclinical safety studies where PD0325901 was administered orally in rats and dogs for up to 13 weeks (Huang et al., 2009). The difference in the level of ocular toxicity between rabbits and rats/dogs could be attributed to ocular drug concentration differences arising from local vs. systemic routes of administration. Since molecular events could precede overt signs of tissue injury, an investigative study was conducted in which rats were dosed orally for 3 or 5 days at 45 mg/kg/day, estimated to be at 70% maximal tolerated dose (Huang et al., 2009). No retinal toxicity was observed by ophthalmic examinations or fundus fluorescein angiography. Despite the absence of overt injury, global gene expression profiling on vehicle and PD0325901-treated retinas revealed several mechanisms relevant to the development of RVO, including oxidative stress response, acute phase and inflammatory response, BRB

To develop an animal model of RVO to investigate mechanisms of toxicity, an in-life study was performed in rabbits, in which PD0325901 was administered by intravitreal injection at doses of 0.5 and 1 mg/eye, with an observation period of 2 weeks (Huang et al., 2009). The high dose was extrapolated to be a potentially toxic dose, while the low dose was chosen as a subtoxic dose, based on in vitro cytotoxicity data (Huang et al., 2009). As early as 1 day after treatment, the high dose produced hemorrhages and vascular leakage with branch occlusion. These lesions progressed to retinal detachment, edema, abnormal kinetic blood flow, and retinal vessel occlusion after 7 days. At the low dose, retinal vascular leakage was observed without vascular occlusion. Therefore, the rabbit model provided evidence that PD0325901 at

Fig. 2. MAPK signaling pathway The MAPK cascade is a highly conserved module that is involved in various cellular functions, including cell proliferation, differentiation and migration. Mammals express at least four distinctly regulated groups of MAPKs, ERK1/2, JNK1/2/3, p38alpha/beta/gamma/delta and ERK5, that are activated by specific MAPKKs: MEK1/2 for ERK1/2, MKK3/6 for the p38, MKK4/7 (JNKK1/2) for the JNKs, and MEK5 for ERK5. Each MAPKK, however, can be activated by more than one MAPKKK, increasing the complexity and diversity of MAPK signalling. Presumably each MAPKKK confers responsiveness to distinct stimuli. For example, activation of ERK1/2 by growth factors depends on the MAPKKK c-Raf, but other MAPKKKs may activate ERK1/2 in response to pro-inflammatory stimuli. Source: KEGG (http://www.genome.jp/dbgetbin/www\_bget?map04010) (Kanehisa, 2000, 2012)

Retinal Vein Occlusion Induced by a MEK Inhibitor –

to oxidative/ischemic stress (Wurm et al., 2011).

**5.1.1 MAPK activation and Inflammation in the retina** 

IL-6 and TNF- (Du et al., 2010; Larrayoz et al., 2010; Wang et al., 2010).

mg/kg/day.

Impact of Oxidative Stress on the Blood-Retinal Barrier 481

Ushio-Fukai and Alexander, 2004), and inhibition of the ERK1/2 has been shown to lead to endothelial apoptosis (Huot et al., 1998). In a porcine model of retinal ischemia-reperfusion, Müller cells exhibited increased levels of glial fibrillary acidic protein (GFAP) and phosphorylated ERK proteins, implicating ERK in the process of glial activation in response

The following sections highlight specific MAPK-mediated molecular and cellular events in response to oxidative stress that are important for the maintenance of BRB function. The published data at times reveal contradictory findings with regards to whether the MAPKs are protective or disruptive in modulating these cellular processes following oxidative stress, underscoring the complexicity of these signaling pathways. In each section, literature review is followed by a discussion of relevant gene expression profiling data from the rat investigative study in which PD0325901 was administered orally for 3 or 5 days at 45

Inflammation is a non-specific response to injury, and involves a plethora of cellular and molecular mediators. Chronic inflammatory processes are also an important source of ROS in the retina, and have been implicated in ocular diseases such as diabetic retinopathy and AMD. Both oxidative stress and inflammation negatively impact the integrity of the BRB. MAPKs mediate some of the downstream effects of proinflammatory cytokines such as IL-1,

In the 5-day rat investigative study involving PD0325901, hematology analysis revealed an increased number of phagocytes (neutrophils and monocytes) and increased plasma fibrinogen levels, indicating a mild inflammation in the compound treated animals. Consistently, the gene expression of many acute phase response proteins, such as lipocalin 2, fibronectin, fibrinogen, ferritin light chain, complement proteins, and coagulation factors, are significantly induced on day 5. In addition to being an acute phase response protein, fibrinogen is also a key player in the coagulation cascade; as alluded in Section 4.2, it has been associated with the development of RVO in several clinical reports. Notably, some studies show that fibrinogen may increase endothelial permeability and mediate vasoconstriction through activation of ERK1/2 (Sen et al., 2009; Tyagi et al., 2008). Though the expression levels of IL-1, IL-6, IL-8, and TNF- genes were not affected, induction of downstream genes within these signaling pathways, including TRAF6 (TNF receptor associated factor 6), TNF receptor, IκB kinase, signal transducer and activator of transcription (STAT) 3, c-Jun, collagen type I, intracellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM)-1, and cyclooxygenase (Cox)-2, suggests pathway activation downstream of these cytokines. TNF- has been shown to increase vascular permeability via modulation of tight junction proteins in diabetic retinopathy (Aveleira et al., 2010). IL-1 may have a role in mediating retinal capillary degradation in diabetic retinopathy (Frey and Antonetti, 2011). Intravitreal levels of IL-6 are correlated with macular edema in branch retinal vein occlusion (Noma et al., 2006). ICAM-1, VCAM-1 and Cox-2 are downstream effectors of NF-κB in the IL-8 signaling pathway. ICAM-1 and VCAM-1 are adhesions molecules expressed on vascular endothelial cells and their induction play a critical role in leukostasis and inflammation. Cox-2 mediates the production of

breakdown, leukostasis, and activation of coagulation cascade (Huang et al., 2009). Progressive induction of oxidative stress response genes was observed over time, suggesting the tissue was mounting a response against ongoing oxidative stress. The induced genes encoding for antioxidant proteins include heat shock protein 27 (HSP27), αβ-crystalline, and those involved in glutathione synthesis/metabolism (GCLM, GSS, GSTs), and adhesion molecules; the only repressed genes were glutaredoxin 2 and peroxiredoxins. Of the oxidative stress response genes induced in this study, HSP27 and αβ-crystalline show some of the greatest magnitude of induction. These are small heat shock proteins that have diverse cytoprotective functions, including modulation of ubiquitin-proteosome pathway, inhibition of apoptosis, and increased resistance to oxidative stress and inflammation (Arrigo et al., 2007). Notably, the ubiquitin-proteosome pathway was significantly perturbed on both day 3 and day 5, which could be a response to misfolded proteins arising from oxidative stress. Antigen presentation by retinal cells is also a cited response to oxidative stress (Tezel et al., 2007; Zhang et al., 2005), consistent with the induction of -2 microglobulin gene of the major histocompatibility complex (MHC) class I on both day 3 and day 5. MHC class I molecules are normally expressed in the vascular endothelium and RPE (Zhang et al., 1997).

Multiple lines of evidence demonstrate that ROS mediate activation of the MAPK signaling pathway, which in turn modulates inflammation, intercellular junction assembly, actin cytoskeleton reorganization, and water transport, all of which are of critical importance to the maintenance of the BRB integrity. Disruption of one of the key MAPK pathways by PD0325901 could contribute to impaired BRB integrity, ultimately leading to retinal edema and RVO.

#### **5.1 MAPK activation and oxidative stress response in the retina**

Three main MAPK groups have been identified – ERKs, p38 MAPKs, and c-Jun N-terminal kinases (JNK). Typically, activation of ERKs are associated with growth-related signals, whereas p38 MAPKs and JNKs become activated in response to stress stimuli, including inflammation and oxidative stress (Fig. 2). However, the exact responses of these kinases in different cell types and tissue microenvironment under various experimental stimuli have proven to be more dynamic and less dichotomous than characterized above. Some degree of cross-talk also exist between these pathways (Houle and Huot, 2006). In the retina, the dynamic balance and cross-talk of these MAPK signaling pathways in cell types comprising the BRB, chiefly RPE, endothelial, and Müller cells, has been shown in experimental systems to be critical for modulating the integrity of the BRB.

The role of the MAPK pathway has been extensively investigated in cultured RPE cells (ARPE-19) following experimentally induced oxidative stress. In response to oxidants such as hydrogen peroxide or tert-butyl hydroperoxide, MAPK (most notably ERKs and p38 MAPK) activation has been shown to either protect against or exacerbate oxidative injury, differentiated by the amount of RPE cell death (Chan et al., 2008; Dong et al., 2011; Glotin et al., 2006; Jiang et al., 2009; Klettner and Roider, 2009; Qin et al., 2006; Tsao et al., 2006; Wang et al., 1998; Wu et al., 2010). These findings also raise questions as to the impact of oxidative stress on the outer BRB when the MAPK pathway is modulated pharmacologically by a MEK inhibitor. In endothelial cells, ROS are known to modulate the expression of redoxsensitive signaling pathways, including the MAPK cascades (Kunsch and Medford, 1999;

breakdown, leukostasis, and activation of coagulation cascade (Huang et al., 2009). Progressive induction of oxidative stress response genes was observed over time, suggesting the tissue was mounting a response against ongoing oxidative stress. The induced genes encoding for antioxidant proteins include heat shock protein 27 (HSP27), αβ-crystalline, and those involved in glutathione synthesis/metabolism (GCLM, GSS, GSTs), and adhesion molecules; the only repressed genes were glutaredoxin 2 and peroxiredoxins. Of the oxidative stress response genes induced in this study, HSP27 and αβ-crystalline show some of the greatest magnitude of induction. These are small heat shock proteins that have diverse cytoprotective functions, including modulation of ubiquitin-proteosome pathway, inhibition of apoptosis, and increased resistance to oxidative stress and inflammation (Arrigo et al., 2007). Notably, the ubiquitin-proteosome pathway was significantly perturbed on both day 3 and day 5, which could be a response to misfolded proteins arising from oxidative stress. Antigen presentation by retinal cells is also a cited response to oxidative stress (Tezel et al., 2007; Zhang et al., 2005), consistent with the induction of -2 microglobulin gene of the major histocompatibility complex (MHC) class I on both day 3 and day 5. MHC class I molecules are normally expressed in the vascular endothelium and

Multiple lines of evidence demonstrate that ROS mediate activation of the MAPK signaling pathway, which in turn modulates inflammation, intercellular junction assembly, actin cytoskeleton reorganization, and water transport, all of which are of critical importance to the maintenance of the BRB integrity. Disruption of one of the key MAPK pathways by PD0325901 could contribute to impaired BRB integrity, ultimately leading to retinal edema

Three main MAPK groups have been identified – ERKs, p38 MAPKs, and c-Jun N-terminal kinases (JNK). Typically, activation of ERKs are associated with growth-related signals, whereas p38 MAPKs and JNKs become activated in response to stress stimuli, including inflammation and oxidative stress (Fig. 2). However, the exact responses of these kinases in different cell types and tissue microenvironment under various experimental stimuli have proven to be more dynamic and less dichotomous than characterized above. Some degree of cross-talk also exist between these pathways (Houle and Huot, 2006). In the retina, the dynamic balance and cross-talk of these MAPK signaling pathways in cell types comprising the BRB, chiefly RPE, endothelial, and Müller cells, has been shown in experimental systems

The role of the MAPK pathway has been extensively investigated in cultured RPE cells (ARPE-19) following experimentally induced oxidative stress. In response to oxidants such as hydrogen peroxide or tert-butyl hydroperoxide, MAPK (most notably ERKs and p38 MAPK) activation has been shown to either protect against or exacerbate oxidative injury, differentiated by the amount of RPE cell death (Chan et al., 2008; Dong et al., 2011; Glotin et al., 2006; Jiang et al., 2009; Klettner and Roider, 2009; Qin et al., 2006; Tsao et al., 2006; Wang et al., 1998; Wu et al., 2010). These findings also raise questions as to the impact of oxidative stress on the outer BRB when the MAPK pathway is modulated pharmacologically by a MEK inhibitor. In endothelial cells, ROS are known to modulate the expression of redoxsensitive signaling pathways, including the MAPK cascades (Kunsch and Medford, 1999;

**5.1 MAPK activation and oxidative stress response in the retina** 

to be critical for modulating the integrity of the BRB.

RPE (Zhang et al., 1997).

and RVO.

Ushio-Fukai and Alexander, 2004), and inhibition of the ERK1/2 has been shown to lead to endothelial apoptosis (Huot et al., 1998). In a porcine model of retinal ischemia-reperfusion, Müller cells exhibited increased levels of glial fibrillary acidic protein (GFAP) and phosphorylated ERK proteins, implicating ERK in the process of glial activation in response to oxidative/ischemic stress (Wurm et al., 2011).

The following sections highlight specific MAPK-mediated molecular and cellular events in response to oxidative stress that are important for the maintenance of BRB function. The published data at times reveal contradictory findings with regards to whether the MAPKs are protective or disruptive in modulating these cellular processes following oxidative stress, underscoring the complexicity of these signaling pathways. In each section, literature review is followed by a discussion of relevant gene expression profiling data from the rat investigative study in which PD0325901 was administered orally for 3 or 5 days at 45 mg/kg/day.

#### **5.1.1 MAPK activation and Inflammation in the retina**

Inflammation is a non-specific response to injury, and involves a plethora of cellular and molecular mediators. Chronic inflammatory processes are also an important source of ROS in the retina, and have been implicated in ocular diseases such as diabetic retinopathy and AMD. Both oxidative stress and inflammation negatively impact the integrity of the BRB. MAPKs mediate some of the downstream effects of proinflammatory cytokines such as IL-1, IL-6 and TNF- (Du et al., 2010; Larrayoz et al., 2010; Wang et al., 2010).

In the 5-day rat investigative study involving PD0325901, hematology analysis revealed an increased number of phagocytes (neutrophils and monocytes) and increased plasma fibrinogen levels, indicating a mild inflammation in the compound treated animals. Consistently, the gene expression of many acute phase response proteins, such as lipocalin 2, fibronectin, fibrinogen, ferritin light chain, complement proteins, and coagulation factors, are significantly induced on day 5. In addition to being an acute phase response protein, fibrinogen is also a key player in the coagulation cascade; as alluded in Section 4.2, it has been associated with the development of RVO in several clinical reports. Notably, some studies show that fibrinogen may increase endothelial permeability and mediate vasoconstriction through activation of ERK1/2 (Sen et al., 2009; Tyagi et al., 2008). Though the expression levels of IL-1, IL-6, IL-8, and TNF- genes were not affected, induction of downstream genes within these signaling pathways, including TRAF6 (TNF receptor associated factor 6), TNF receptor, IκB kinase, signal transducer and activator of transcription (STAT) 3, c-Jun, collagen type I, intracellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM)-1, and cyclooxygenase (Cox)-2, suggests pathway activation downstream of these cytokines. TNF- has been shown to increase vascular permeability via modulation of tight junction proteins in diabetic retinopathy (Aveleira et al., 2010). IL-1 may have a role in mediating retinal capillary degradation in diabetic retinopathy (Frey and Antonetti, 2011). Intravitreal levels of IL-6 are correlated with macular edema in branch retinal vein occlusion (Noma et al., 2006). ICAM-1, VCAM-1 and Cox-2 are downstream effectors of NF-κB in the IL-8 signaling pathway. ICAM-1 and VCAM-1 are adhesions molecules expressed on vascular endothelial cells and their induction play a critical role in leukostasis and inflammation. Cox-2 mediates the production of

Retinal Vein Occlusion Induced by a MEK Inhibitor –

permeability (Reichenbach et al., 2007).

disruption and impaired endothelial barrier integrity.

Impact of Oxidative Stress on the Blood-Retinal Barrier 483

In our 5-day rat investigative study, the expression of myosin light chain kinase (MLCK) was significantly increased as a result of NF-κB signaling. Phosphorylation of myosin light chain by MLCK leads to actin-mediated endothelial cell contraction and increases permeability of endothelial junctional barrier. Activation of phosphatidylinositol 3-kinase (PI3K), whose expression was induced on both day 3 and day 5, has also been shown to increase vascular permeability (Abid et al., 2004; Lee et al., 2006; Serban et al., 2008). In addition, induction of MMP14 was observed on day 5. Under inflammatory conditions, Müller cells are a source of matrix metalloproteinases which impair the barrier function of retinal endothelial cells by degradation of the tight junction protein occludin (Reichenbach et al., 2007). Taken together, these data suggest increased retinal endothelial cell permeability and impaired BRB function as a result of PD0325901 administration. On the other hand, our data set also uncovered induction of genes important for maintaining the vascular endothelial barrier function, presumably as feedback mechanism to counteract permeability increases, such as repression of RhoA and induction of Rac1 to facilitate reannealing of adherens junctions (Vandenbroucke et al., 2008); induction of tight junction components claudin 11 and JAM-2; and induction of PEDF, likely in Müller cells, which represses expression of VEGF and decreases vascular

**5.1.3 Regulation of actin dynamics by MAPK in response to oxidative stress** 

The remodeling of actin cytoskeleton is an important response in endothelial cells exposed to oxidative stress, and contributes to increased permeability of the endothelial barrier (Houle and Huot, 2006; Lum and Roebuck, 2001). Under physiological and pathological stress conditions, endothelial cells undergo cell shape change, intercellular gap formation, and remodeling of the actin cytoskeleton, characterized by stress fiber formation and reduced cortical actin band. The formation of stress fibers is dependent on actin polymerization, and increases the endothelial cells' capacity to resist stress. On the other hand, stress fibers also pull apart intercellular junctions, likely contributing to their

ROS-induced MAPK activation plays an important role in actin remodeling. ERKs, p38 and JNK have all been shown to regulate actin dynamics induced by oxidative stress in endothelial cells (El-Remessy et al., 2011; Houle and Huot, 2006; Houle et al., 2003; Huot et al., 1998; Schweitzer et al., 2011; Usatyuk and Natarajan, 2004). p38 MAPK activation leads to phosphorylation of HSP27, which promotes actin polymerization. ERK activation results in phosphorylation of tropomyosin-1, which contributes to focal adhesion assembly and stress fiber formation, and modulates cell contractility. Inhibition of ERK activity by the MEK inhibitor PD098059 led to misassembly of focal adhesions and membrane blebbing, ultimately resulting in apoptosis (Huot et al. 1998). Physio-pathological consequence of surface blebbing of endothelial cells includes narrowing of vascular lumen associated with increased vascular resistance. Bleb shedding may also contribute to obstruction of blood vessels. Consistent with this interplay of MAPKs and actin dynamics, treatment with PD0325901 in the 5-day rat investigative study led to induction of genes in actin cytoskeleton and focal adhesion signaling pathways on study day 5, supporting perturbation of actin dynamics, likely downstream of oxidative stress. These data also raise

proinflammatory prostaglandins. NF-κB, c-Jun and STAT3 are important regulators of many genes mediating mammalian inflammatory and immune responses. In addition, the expression of GFAP was induced on day 5, suggesting activation of Müller cells in response to retinal stress such as inflammation.

Conversely, glucocorticoids, which have anti-inflammatory properties, have been shown to have a positive impact on promoting barrier integrity. In a porcine model of RVO, triamcinolone treatment, a widely used glucocorticoid in ocular applications, reduced VEGF and increased tight junction occludin levels in the retina, suggesting increased BRB integrity (McAllister et al., 2009). A study conducted to examine the impact of Streptococcus suis infection on blood-CSF (cerebral spinal fluid) barrier showed that the glucocorticoid dexamethasone improved the barrier function by preventing tight junction protein reorganization and degradation, and attenuated ERK activation and matrix metalloproteinase 3 (MMP3) expression (Tenenbaum et al., 2008). Paradoxically, dexamethasone has also been shown to activate ERK and JNK, which in turn induce the expression of the anti-inflammatory mediator MAPK phosphatase 1 (MKP-1) in human umbilical vein endothelial cells (Furst et al., 2008). Activated MKP-1 is then able to terminate the activity of activated MAPKs in a negative feedback loop (Furst et al., 2008). These data further demonstrate the complex spatiotemporal signaling network in which the MAPKs participate.

In our gene expression analysis, the glucocorticoid receptor signaling pathway was one of the few pathways significantly perturbed on day 3 following PD0325901 administration at 45 mg/kg/day, and many genes within the pathway were induced. On day 5, many of the same pathway genes became repressed. This could indicate an adaptive response to ongoing inflammation in the retina. As alluded to earlier, HSP27 and -crystallin levels were also induced following PD0325901 administration. Their gene products have the ability to interfere with inflammatory signaling, such as attenuation of TNF-, NF-κB signaling pathways, and may represent another cellular response to inflammation.

#### **5.1.2 MAPK-mediated modulation of intercellular junctions in response to oxidative stress**

Intercellular junctions are of critical importance to the integrity of the BRB function (Fig. 1). Oxidative stress is known to disrupt the structure and function of tight junctions and adherens junctions through MAPK activation, in both endothelial cells (Niwa et al., 2001; Simoncini et al., 2005; Usatyuk and Natarajan, 2004; Usatyuk et al., 2006; Yuan, 2002) and epithelial cells (Basuroy et al., 2006; Gonzalez et al., 2009), and these adverse effects on the junctional complexes could be ameliorated with the application of specific MAPK inhibitors. The presence of ROS could also induce the expression of the vascular permeability factor VEGF in endothelial cells (Chua et al., 1998; El-Remessy et al., 2003), often associated with downstream MAPK activation (Yang et al., 2010a; Zheng et al., 2010). In Müller cells, oxidative stress leads to decreased PEDF expression (Yoshida et al., 2009), thus relieving its antagonistic effect on VEGF action and subsequent MAPK activation in endothelial cells (Yafai et al., 2007), contributing to increased vascular permeability and breakdown of the inner BRB.

proinflammatory prostaglandins. NF-κB, c-Jun and STAT3 are important regulators of many genes mediating mammalian inflammatory and immune responses. In addition, the expression of GFAP was induced on day 5, suggesting activation of Müller cells in response

Conversely, glucocorticoids, which have anti-inflammatory properties, have been shown to have a positive impact on promoting barrier integrity. In a porcine model of RVO, triamcinolone treatment, a widely used glucocorticoid in ocular applications, reduced VEGF and increased tight junction occludin levels in the retina, suggesting increased BRB integrity (McAllister et al., 2009). A study conducted to examine the impact of Streptococcus suis infection on blood-CSF (cerebral spinal fluid) barrier showed that the glucocorticoid dexamethasone improved the barrier function by preventing tight junction protein reorganization and degradation, and attenuated ERK activation and matrix metalloproteinase 3 (MMP3) expression (Tenenbaum et al., 2008). Paradoxically, dexamethasone has also been shown to activate ERK and JNK, which in turn induce the expression of the anti-inflammatory mediator MAPK phosphatase 1 (MKP-1) in human umbilical vein endothelial cells (Furst et al., 2008). Activated MKP-1 is then able to terminate the activity of activated MAPKs in a negative feedback loop (Furst et al., 2008). These data further demonstrate the complex spatiotemporal signaling network in which the MAPKs

In our gene expression analysis, the glucocorticoid receptor signaling pathway was one of the few pathways significantly perturbed on day 3 following PD0325901 administration at 45 mg/kg/day, and many genes within the pathway were induced. On day 5, many of the same pathway genes became repressed. This could indicate an adaptive response to ongoing inflammation in the retina. As alluded to earlier, HSP27 and -crystallin levels were also induced following PD0325901 administration. Their gene products have the ability to interfere with inflammatory signaling, such as attenuation of TNF-, NF-κB signaling

**5.1.2 MAPK-mediated modulation of intercellular junctions in response to oxidative** 

Intercellular junctions are of critical importance to the integrity of the BRB function (Fig. 1). Oxidative stress is known to disrupt the structure and function of tight junctions and adherens junctions through MAPK activation, in both endothelial cells (Niwa et al., 2001; Simoncini et al., 2005; Usatyuk and Natarajan, 2004; Usatyuk et al., 2006; Yuan, 2002) and epithelial cells (Basuroy et al., 2006; Gonzalez et al., 2009), and these adverse effects on the junctional complexes could be ameliorated with the application of specific MAPK inhibitors. The presence of ROS could also induce the expression of the vascular permeability factor VEGF in endothelial cells (Chua et al., 1998; El-Remessy et al., 2003), often associated with downstream MAPK activation (Yang et al., 2010a; Zheng et al., 2010). In Müller cells, oxidative stress leads to decreased PEDF expression (Yoshida et al., 2009), thus relieving its antagonistic effect on VEGF action and subsequent MAPK activation in endothelial cells (Yafai et al., 2007), contributing to increased vascular permeability and breakdown of the

pathways, and may represent another cellular response to inflammation.

to retinal stress such as inflammation.

participate.

**stress** 

inner BRB.

In our 5-day rat investigative study, the expression of myosin light chain kinase (MLCK) was significantly increased as a result of NF-κB signaling. Phosphorylation of myosin light chain by MLCK leads to actin-mediated endothelial cell contraction and increases permeability of endothelial junctional barrier. Activation of phosphatidylinositol 3-kinase (PI3K), whose expression was induced on both day 3 and day 5, has also been shown to increase vascular permeability (Abid et al., 2004; Lee et al., 2006; Serban et al., 2008). In addition, induction of MMP14 was observed on day 5. Under inflammatory conditions, Müller cells are a source of matrix metalloproteinases which impair the barrier function of retinal endothelial cells by degradation of the tight junction protein occludin (Reichenbach et al., 2007). Taken together, these data suggest increased retinal endothelial cell permeability and impaired BRB function as a result of PD0325901 administration. On the other hand, our data set also uncovered induction of genes important for maintaining the vascular endothelial barrier function, presumably as feedback mechanism to counteract permeability increases, such as repression of RhoA and induction of Rac1 to facilitate reannealing of adherens junctions (Vandenbroucke et al., 2008); induction of tight junction components claudin 11 and JAM-2; and induction of PEDF, likely in Müller cells, which represses expression of VEGF and decreases vascular permeability (Reichenbach et al., 2007).

#### **5.1.3 Regulation of actin dynamics by MAPK in response to oxidative stress**

The remodeling of actin cytoskeleton is an important response in endothelial cells exposed to oxidative stress, and contributes to increased permeability of the endothelial barrier (Houle and Huot, 2006; Lum and Roebuck, 2001). Under physiological and pathological stress conditions, endothelial cells undergo cell shape change, intercellular gap formation, and remodeling of the actin cytoskeleton, characterized by stress fiber formation and reduced cortical actin band. The formation of stress fibers is dependent on actin polymerization, and increases the endothelial cells' capacity to resist stress. On the other hand, stress fibers also pull apart intercellular junctions, likely contributing to their disruption and impaired endothelial barrier integrity.

ROS-induced MAPK activation plays an important role in actin remodeling. ERKs, p38 and JNK have all been shown to regulate actin dynamics induced by oxidative stress in endothelial cells (El-Remessy et al., 2011; Houle and Huot, 2006; Houle et al., 2003; Huot et al., 1998; Schweitzer et al., 2011; Usatyuk and Natarajan, 2004). p38 MAPK activation leads to phosphorylation of HSP27, which promotes actin polymerization. ERK activation results in phosphorylation of tropomyosin-1, which contributes to focal adhesion assembly and stress fiber formation, and modulates cell contractility. Inhibition of ERK activity by the MEK inhibitor PD098059 led to misassembly of focal adhesions and membrane blebbing, ultimately resulting in apoptosis (Huot et al. 1998). Physio-pathological consequence of surface blebbing of endothelial cells includes narrowing of vascular lumen associated with increased vascular resistance. Bleb shedding may also contribute to obstruction of blood vessels. Consistent with this interplay of MAPKs and actin dynamics, treatment with PD0325901 in the 5-day rat investigative study led to induction of genes in actin cytoskeleton and focal adhesion signaling pathways on study day 5, supporting perturbation of actin dynamics, likely downstream of oxidative stress. These data also raise

Retinal Vein Occlusion Induced by a MEK Inhibitor –

role in perturbing this equilibrium.

**5.2 MAPK, IFN- and RVO** 

**6. Conclusion** 

Impact of Oxidative Stress on the Blood-Retinal Barrier 485

fluid balance in the retina and the brain raises the possibility that MEK inhibition may play a

Given the complex signaling cascades and cross-talk between various MAPK pathways in multiple cell types comprising the BRB, it is conceivable that inhibition of ERK activation by a MEK inhibitor could lead to dysregulated BRB integrity and subsequent development of RVO. Intriguingly, IFN-α treatment, which is also associated with the development of RVO in the clinic (see Section 4.4.1), has been shown to inhibit the activation of ERK and the associated survival effects, and that MEK and ERK inhibitors enhance the anti-proliferative effect of IFNα in tumor cells or transformed epithelial cells (Battcock et al., 2006; Caraglia et al., 2005; Caraglia et al., 2003; Christian et al., 2009; Li et al., 2004; Romerio et al., 2000; Romerio and Zella, 2002). Cross-talk between the IFN-α and Ras-MAPK pathways converge on the STAT family of transcription factors. STAT proteins are involved in cytokine, hormone, and growth factor signal transduction, mediating biological processes as diverse as cell proliferation, differentiation, apoptosis, transformation, inflammation and immune response (Caraglia et al., 2005). Activated Ras/MEK has been shown to inhibit the antiviral response of IFN-α by reducing STAT2 levels (Christian et al., 2009). It is conceivable that clinical IFN-α usage may perturb the balance of MAPK signaling pathways in the retina, disrupt BRB function, and ultimately contribute to the development of RVO. That both IFN-α therapy and PD0325901 inhibit ERK activation and are linked to clinical development of RVO lends further support to the hypothesis that modulation of the Ras-MAPK pathway and subsequently BRB

permeability changes play a role in the pathogenesis of this ocular adverse event.

inhibitor therapy for signs of ocular adverse events.

Constance Benedict for preparation of Fig. 1.

**7. Acknowledgements** 

The BRB, consisting of an endothelial and an epithelial barrier, serves to regulate the bidirectional passage of macromolecules through the retina. Oxidative stress can negatively impact the equilibrium across the BRB, leading to cellular disruption and ocular disorders. MAPK pathways involving ERK, p38 and JNK play a central role in the oxidative stress response of the BRB, modulating inflammatory response, actin cytoskeletal dynamics, water transport, as well as inter-epithelial and inter-endothelial adhesion molecule expression and redistribution. Disruption of the ERK signaling pathway by the MEK inhibitor PD0325901 may disrupt the balance and cross-talk between interconnected signaling networks and produce unexpected cellular sequalae. PD0325901-induced RVO could arise as a consequence of disruption of these tightly regulated molecular processes vital for proper functioning of the BRB. The animal models employed in our study serves as an investigative or screening paradigm for pre-clinical compounds suspected of RVO-inducing potential. Finally, while a firm connection between MEK inhibition and the development of RVO has not been established, it would be prudent for clinicians to monitor patients on MEK

The authors would like to thank Patrick Lappin for critical reading of the manuscript, and

the possibility of membrane blebbing in retinal vasculature following PD0325901 treatment, contributing to the development of RVO.

#### **5.1.4 MAPK pathway and water permeability in response to oxidative stress**

Macular edema was observed in our animal model administered with PD0325901. Two factors contribute to the development of chronic edema in the retina: increased vascular permeability leading to excessive fluid buildup, and reduced fluid absorption from the retina back into the blood. Müller and RPE cells play an integral role in transcellular fluid equilibrium (Reichenbach et al., 2007). Aquaporin 4 (AQP4) expressed on Müller cells and AQP1 expressed on RPE cells facilitate bidirectional water movements to maintain the osmotic and hydrostatic equilibrium in the retina. The Müller cell-specific AQP4 is colocalized with the inwardly rectifying potassium channel Kir4.1. Together they mediate the co-transport of water and potassium ions from the retinal tissue into the blood under normal conditions. In various animal models of retinopathy, characterized by inflammatory or oxidative stress conditions, Kir4.1 channel becomes mislocalized, and its expression is decreased in some cases (Reichenbach et al., 2007). This may lead to an intracellular potassium overload, increased osmotic pressure, and consequently, Müller cell swelling. In a study employing a rat model of RVO, downregulation of AQP1, AQP4, and Kir4.1 were observed, in addition to an altered distribution of Kir4.1 protein. Consequently, Müller cells displayed a decrease in potassium currents and increased in size (Rehak et al., 2008).

Application triamcinolone, a glucocorticoid frequently used for diabetic macular edema due to its anti-inflammatory properties (Felinski and Antonetti, 2005), reduced Müller cell swelling in animal models of ischemia-reperfusion and diabetic retinopathy (Reichenbach et al., 2007). In a porcine model of RVO, triamcinolone treatment reduced the glial activation marker GFAP expression in Müller cells, and also increased BRB integrity, as evidenced by reduced VEGF and increased tight junction occludin levels, potentially contributing to the resolution of edema in the retina (McAllister et al., 2009).

MAPKs are known to play an important role in cellular osmotic stress regulation (Cowan and Storey, 2003; de Nadal et al., 2002). In RPE cells, ultraviolet radiation (UVB) and hydrogen peroxide treatment, both of which are oxidative stress inducers, resulted in AQP1 downregulation which was mediated by MEK/ERK activation (Jiang et al., 2009). In the brain, astrocyte swelling often accompanies vascular edema (Reichenbach et al., 2007). In astrocytes exposed to the oxidative stressor manganese or glial reactive injury, there was an altered expression of AQPs, mediated by the MEK/ERK and p38 MAPKs (McCoy and Sontheimer, 2010; Rao et al., 2010).

In our 5-day rat investigative study, the repression of the Müller cell-specific water channel AQP4 on both day 3 and day 5, coupled with the repression of the inwardly rectifying potassium channels (Kcnj5, Kcnj6), and sodium channels on day 5, signals impaired transcellular fluid transport. Given the evidence for inflammation, intercellular junction disruption and actin cytoskeleton changes in the retina following PD0325901 treatment, this fluid imbalance would contribute to the observed retinal edema in the study and further weaken BRB integrity. The documented involvement of MAPKs in regulating transcellular fluid balance in the retina and the brain raises the possibility that MEK inhibition may play a role in perturbing this equilibrium.

#### **5.2 MAPK, IFN- and RVO**

484 Oxidative Stress and Diseases

the possibility of membrane blebbing in retinal vasculature following PD0325901 treatment,

Macular edema was observed in our animal model administered with PD0325901. Two factors contribute to the development of chronic edema in the retina: increased vascular permeability leading to excessive fluid buildup, and reduced fluid absorption from the retina back into the blood. Müller and RPE cells play an integral role in transcellular fluid equilibrium (Reichenbach et al., 2007). Aquaporin 4 (AQP4) expressed on Müller cells and AQP1 expressed on RPE cells facilitate bidirectional water movements to maintain the osmotic and hydrostatic equilibrium in the retina. The Müller cell-specific AQP4 is colocalized with the inwardly rectifying potassium channel Kir4.1. Together they mediate the co-transport of water and potassium ions from the retinal tissue into the blood under normal conditions. In various animal models of retinopathy, characterized by inflammatory or oxidative stress conditions, Kir4.1 channel becomes mislocalized, and its expression is decreased in some cases (Reichenbach et al., 2007). This may lead to an intracellular potassium overload, increased osmotic pressure, and consequently, Müller cell swelling. In a study employing a rat model of RVO, downregulation of AQP1, AQP4, and Kir4.1 were observed, in addition to an altered distribution of Kir4.1 protein. Consequently, Müller cells displayed a decrease in potassium currents and increased in

Application triamcinolone, a glucocorticoid frequently used for diabetic macular edema due to its anti-inflammatory properties (Felinski and Antonetti, 2005), reduced Müller cell swelling in animal models of ischemia-reperfusion and diabetic retinopathy (Reichenbach et al., 2007). In a porcine model of RVO, triamcinolone treatment reduced the glial activation marker GFAP expression in Müller cells, and also increased BRB integrity, as evidenced by reduced VEGF and increased tight junction occludin levels, potentially contributing to the

MAPKs are known to play an important role in cellular osmotic stress regulation (Cowan and Storey, 2003; de Nadal et al., 2002). In RPE cells, ultraviolet radiation (UVB) and hydrogen peroxide treatment, both of which are oxidative stress inducers, resulted in AQP1 downregulation which was mediated by MEK/ERK activation (Jiang et al., 2009). In the brain, astrocyte swelling often accompanies vascular edema (Reichenbach et al., 2007). In astrocytes exposed to the oxidative stressor manganese or glial reactive injury, there was an altered expression of AQPs, mediated by the MEK/ERK and p38 MAPKs (McCoy and

In our 5-day rat investigative study, the repression of the Müller cell-specific water channel AQP4 on both day 3 and day 5, coupled with the repression of the inwardly rectifying potassium channels (Kcnj5, Kcnj6), and sodium channels on day 5, signals impaired transcellular fluid transport. Given the evidence for inflammation, intercellular junction disruption and actin cytoskeleton changes in the retina following PD0325901 treatment, this fluid imbalance would contribute to the observed retinal edema in the study and further weaken BRB integrity. The documented involvement of MAPKs in regulating transcellular

**5.1.4 MAPK pathway and water permeability in response to oxidative stress** 

contributing to the development of RVO.

size (Rehak et al., 2008).

Sontheimer, 2010; Rao et al., 2010).

resolution of edema in the retina (McAllister et al., 2009).

Given the complex signaling cascades and cross-talk between various MAPK pathways in multiple cell types comprising the BRB, it is conceivable that inhibition of ERK activation by a MEK inhibitor could lead to dysregulated BRB integrity and subsequent development of RVO. Intriguingly, IFN-α treatment, which is also associated with the development of RVO in the clinic (see Section 4.4.1), has been shown to inhibit the activation of ERK and the associated survival effects, and that MEK and ERK inhibitors enhance the anti-proliferative effect of IFNα in tumor cells or transformed epithelial cells (Battcock et al., 2006; Caraglia et al., 2005; Caraglia et al., 2003; Christian et al., 2009; Li et al., 2004; Romerio et al., 2000; Romerio and Zella, 2002). Cross-talk between the IFN-α and Ras-MAPK pathways converge on the STAT family of transcription factors. STAT proteins are involved in cytokine, hormone, and growth factor signal transduction, mediating biological processes as diverse as cell proliferation, differentiation, apoptosis, transformation, inflammation and immune response (Caraglia et al., 2005). Activated Ras/MEK has been shown to inhibit the antiviral response of IFN-α by reducing STAT2 levels (Christian et al., 2009). It is conceivable that clinical IFN-α usage may perturb the balance of MAPK signaling pathways in the retina, disrupt BRB function, and ultimately contribute to the development of RVO. That both IFN-α therapy and PD0325901 inhibit ERK activation and are linked to clinical development of RVO lends further support to the hypothesis that modulation of the Ras-MAPK pathway and subsequently BRB permeability changes play a role in the pathogenesis of this ocular adverse event.

#### **6. Conclusion**

The BRB, consisting of an endothelial and an epithelial barrier, serves to regulate the bidirectional passage of macromolecules through the retina. Oxidative stress can negatively impact the equilibrium across the BRB, leading to cellular disruption and ocular disorders. MAPK pathways involving ERK, p38 and JNK play a central role in the oxidative stress response of the BRB, modulating inflammatory response, actin cytoskeletal dynamics, water transport, as well as inter-epithelial and inter-endothelial adhesion molecule expression and redistribution. Disruption of the ERK signaling pathway by the MEK inhibitor PD0325901 may disrupt the balance and cross-talk between interconnected signaling networks and produce unexpected cellular sequalae. PD0325901-induced RVO could arise as a consequence of disruption of these tightly regulated molecular processes vital for proper functioning of the BRB. The animal models employed in our study serves as an investigative or screening paradigm for pre-clinical compounds suspected of RVO-inducing potential. Finally, while a firm connection between MEK inhibition and the development of RVO has not been established, it would be prudent for clinicians to monitor patients on MEK inhibitor therapy for signs of ocular adverse events.

#### **7. Acknowledgements**

The authors would like to thank Patrick Lappin for critical reading of the manuscript, and Constance Benedict for preparation of Fig. 1.

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**Section 6** 

**Cancer** 

Zheng, Z., Chen, H., Wang, H., Ke, B., Zheng, B., Li, Q., Li, P., Su, L., Gu, Q., and Xu, X. (2010). Improvement of retinal vascular injury in diabetic rats by statins is associated with the inhibition of mitochondrial reactive oxygen species pathway mediated by peroxisome proliferator-activated receptor gamma coactivator 1alpha. Diabetes *59*, 2315-2325.

## **Section 6**

494 Oxidative Stress and Diseases

Zheng, Z., Chen, H., Wang, H., Ke, B., Zheng, B., Li, Q., Li, P., Su, L., Gu, Q., and Xu, X.

Diabetes *59*, 2315-2325.

(2010). Improvement of retinal vascular injury in diabetic rats by statins is associated with the inhibition of mitochondrial reactive oxygen species pathway mediated by peroxisome proliferator-activated receptor gamma coactivator 1alpha.

**Cancer** 

**22** 

 *Spain* 

**Oxidative Therapy Against Cancer** 

*Departamento de Citología e Histología Normal y Patológica,* 

Although a moderate increase of reactive oxygen species (ROS) may induce cell proliferation, excessive amounts of ROS can cause oxidative damage to lipids, proteins, and DNA, provoking oncogenic transformation, increased metabolic activity, and mitochondrial dysfunction (Dreher and Junod, 1996; Behrend et al., 2003; Pelicano et al, 2003). Many reports suggest that cancer cells are under a continuous oxidative stress (Pervaiz and Clement, 2004; Schumacker et al., 2006; Kryston et al., 2011). Studies with human tumor cell lines clearly show that these cells produce ROS at a much higher rate than healthy cells (Oberley and Buettner, 1979; Lu et al., 2007). ROS have been established as important molecules involved in the multistage process of carcinogenesis (Klaunig and Kamendulis, 2004). Mitochondria are the major consumers of molecular oxygen in cells, representing an important source of ROS. It is well accepted that cancer cells present mitochondrial alterations which result in respiration injury. This mitochondrial dysfunction may induce a low coupling efficiency of the mitochondrial electron chain, increasing electron leakage and leading to enhanced ROS formation. The resulting oxidative stress may cause further damage to both mitochondrial DNA (mtDNA) and the respiratory chain, amplifying the

The higher oxidative stress observed in cancer cells can also result from a decrease or inactivation of antioxidants (Huang et al., 2003; Conklin, 2004). The majority of tumor cells usually present very few antioxidative enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, which are known to play a protective role against ROS in normal cells (Sato et al., 1992; Hasegawa et al., 2002; Pelicano et al., 2004). The lack of proper antioxidant defences makes tumor cells very vulnerable to oxidative stress. Nevertheless, some studies have revealed increased expression of antioxidants, probably as a consequence of selective pressure towards stress adaptation. The sources of ROS in cancer cells and the

consequences of oxidative stress in the carcinogenesis process are still under debate.

What seems accepted is that cancer cells have increased ROS steady state level and are likely to be more vulnerable to damage by further ROS insults induced by exogenous agents. Thus, manipulating ROS levels by redox modulation could be a way to selectively kill cancer cells without causing significant toxicity to normal cells. A promising anticancer strategy named "oxidation therapy" has been developed by inducing cytotoxic oxidative stress for cancer treatment. This could be achieved by two different methods: inducing the

**1. Introduction** 

ROS generation (Zorov et al., 2006).

Manuel de Miguel and Mario D. Cordero

 *Facultad de Medicina, Universidad de Sevilla, Sevilla* 

### **Oxidative Therapy Against Cancer**

Manuel de Miguel and Mario D. Cordero

*Departamento de Citología e Histología Normal y Patológica, Facultad de Medicina, Universidad de Sevilla, Sevilla Spain* 

#### **1. Introduction**

Although a moderate increase of reactive oxygen species (ROS) may induce cell proliferation, excessive amounts of ROS can cause oxidative damage to lipids, proteins, and DNA, provoking oncogenic transformation, increased metabolic activity, and mitochondrial dysfunction (Dreher and Junod, 1996; Behrend et al., 2003; Pelicano et al, 2003). Many reports suggest that cancer cells are under a continuous oxidative stress (Pervaiz and Clement, 2004; Schumacker et al., 2006; Kryston et al., 2011). Studies with human tumor cell lines clearly show that these cells produce ROS at a much higher rate than healthy cells (Oberley and Buettner, 1979; Lu et al., 2007). ROS have been established as important molecules involved in the multistage process of carcinogenesis (Klaunig and Kamendulis, 2004). Mitochondria are the major consumers of molecular oxygen in cells, representing an important source of ROS. It is well accepted that cancer cells present mitochondrial alterations which result in respiration injury. This mitochondrial dysfunction may induce a low coupling efficiency of the mitochondrial electron chain, increasing electron leakage and leading to enhanced ROS formation. The resulting oxidative stress may cause further damage to both mitochondrial DNA (mtDNA) and the respiratory chain, amplifying the ROS generation (Zorov et al., 2006).

The higher oxidative stress observed in cancer cells can also result from a decrease or inactivation of antioxidants (Huang et al., 2003; Conklin, 2004). The majority of tumor cells usually present very few antioxidative enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, which are known to play a protective role against ROS in normal cells (Sato et al., 1992; Hasegawa et al., 2002; Pelicano et al., 2004). The lack of proper antioxidant defences makes tumor cells very vulnerable to oxidative stress. Nevertheless, some studies have revealed increased expression of antioxidants, probably as a consequence of selective pressure towards stress adaptation. The sources of ROS in cancer cells and the consequences of oxidative stress in the carcinogenesis process are still under debate.

What seems accepted is that cancer cells have increased ROS steady state level and are likely to be more vulnerable to damage by further ROS insults induced by exogenous agents. Thus, manipulating ROS levels by redox modulation could be a way to selectively kill cancer cells without causing significant toxicity to normal cells. A promising anticancer strategy named "oxidation therapy" has been developed by inducing cytotoxic oxidative stress for cancer treatment. This could be achieved by two different methods: inducing the

Oxidative Therapy Against Cancer 499

marker of mitochondrial function (Haas et al., 2008). CoQ deficiency induces decreased mitochondrial respiratory enzymes activity, reduced expression of mitochondrial proteins involved in oxidative phosphorylation, decreased mitochondrial membrane potential, increased production of ROS, mitochondrial permeabilization, mitophagy of dysfunctional mitochondria, reduced growth rates and cell death (Rodriguez-Hernandez et al., 2009,

In addition to energy, mitochondrial oxidative phosphorylation also generates ROS. When the MRC becomes highly reduced, the excess electrons from complex I or complex III may increase substantially, passing directly to O2 to generate high amounts of superoxide anion

−). Superoxide is transformed to hydrogen peroxide (H2O2) by the detoxification enzymes manganese superoxide dismutase (MnSOD) or copper/zinc superoxide dismutase (Cu/Zn SOD), and then to water by catalase, glutathione peroxidase (GPx) or peroxiredoxin III (PRX III). However, when these enzymes cannot convert ROS such as the superoxide radical to water fast enough, oxidative damage occurs and accumulates in the mitochondria. If H2O2 encounters a reduced transition metal or is mixed with O2•−, the H2O2 can be further reduced to hydroxyl radical (HO•), the most potent oxidizing agent among ROS. Additionally, nitric oxide (•NO) is produced within the mitochondria by mitochondrial nitric oxide synthase (mtNOS) and also freely diffuses into the mitochondria from the

radicals as well as others can do great damage to mitochondria and other cellular

Under normal physiological conditions, ROS production is highly regulated. However, if the MRC is inhibited, or key mitochondrial components, such as CoQ, are deficient, then, electrons accumulate on the MRC carriers, greatly increasing the rate of a single electron

exceed the cellular antioxidant defense and the cumulative damage can ultimately destroy

Mitochondria play an important role in cell bioenergetics and life signaling. Mitochondria are necessary for cell survival but they can also trigger cell death, thus exerting decisive control over the biochemistry of several cascades that lead to cell death, specifically the intrinsic pathway of apoptosis. The particular biochemical properties of these organelles are closely related to their segmented structures, which provide an optimal environment for multiple pathways of biosynthesis and bioenergetics. Consequently, it is possible that these organelles are involved in the process of carcinogenesis through alterations in cell metabolism and cell death pathways (Pilkington et al., 2008). Cancer cells show alterations in mtDNA, in oxidative phosphorylation and in energetic metabolism, all triggered by a pro-oxidative change (Indo et al., 2007). The "respiratory damage" in cancer cells predicts low coupling efficiency in electron transfer at the inner membrane of mitochondria and,

2003). The superoxide anion will thus generate more free radicals. Generated ROS can be released into cytosol and trigger "ROS-induced ROS-release" (RIRR) in neighbouring mitochondria. This mitochondrion-to-mitochondrion ROS-signaling constitutes a positive feedback mechanism for enhanced ROS production potentially leading to significant mitochondrial injury (Zorov et al., 2006). Recent studies by a number of groups have demonstrated that ROS can directly modify signaling proteins through different

consequently, greater loss of electrons, leading to the formation of more O2

<sup>−</sup> to produce peroxynitrite (ONOO−). Together, these two

−. An excessive mitochondrial ROS production can

− (Pelicano et al.,

Cotan et al, 2011).

cytosol. •NO reacts with O2

components (Turrens, 2003).

being transferred to O2 to generate O2

the cell by necrosis or apoptosis.

(O2

generation of cytotoxic levels of ROS, and inhibiting the antioxidant system of tumor cells (Fang et al., 2007; Trachootham et al., 2009).

It is well known that ROS, such as hydrogen peroxide (H2O2) and superoxide anion, induce apoptosis to a wide range of tumor cells through the activation of the caspase cascade. It has been described that mitochondrial damage induced by the use of certain drugs provokes an increment of oxidative stress and cell death (Chandra et al., 2000; Conklin, 2004). Major ROS-modulating agents are based on the capacity to induce high ROS generation or to reduce the antioxidant defence machinery of cancer cells.

An interesting drug for oxidative cancer therapy is amitriptyline, a tricyclic antidepressant commonly prescribed for depression and therapeutic treatments of several neuropathic and inflammatory illnesses. Chlorimipramine, another tricyclic antidepressant, has been already proposed as a novel anticancer agent targeted to mitochondria as it induces caspase-3 dependent apoptosis (Daley et al., 2005). Several reports showed that the toxicity of this drug is due to an increase in oxidative stress by the generation of high amounts of ROS (Daley et al., 2005; Moreno-Fernández et al., 2008; Cordero et al., 2009). Therefore, amitriptyline has being proposed to be used for anticancer oxidant therapy against tumors that present significant oxidative stress and/or low antioxidant defences (Cordero et al., 2010).

#### **2. Mitochondria and Reactive Oxygen Species (ROS)**

Mitochondria are dynamic organelles that play a central role in many cellular functions including the generation of chemical energy (adenosine triphosphate, ATP), heat, and intracellular calcium homeostasis. They are also responsible for the formation of ROS and for triggering the programmed cell death or apoptosis (Turrens, 2003). The primary metabolic function of mitochondria is oxidative phosphorylation, an energy-generating process that couples oxidation of respiratory substrates to the synthesis of ATP (Pieczenik & Neustadt, 2007). The mitochondrial respiratory chain (MRC) is composed of five multisubunit enzyme complexes. Both the mtDNA and the nuclear DNA (nDNA) encode for polypeptide components of these complexes. Electron transport between MRC complexes I–IV is coupled to the extrusion of protons across the inner mitochondrial membrane by proton pump components of the respiratory chain. This movement of protons creates an electrochemical gradient (m) across the inner mitochondrial membrane. Protons return to the mitochondrial matrix by flowing through ATP synthase (complex V), which utilizes the energy thus produced to synthesize ATP from adenosine diphosphate (ADP) and inorganic phosphate. Both the mtDNA and the nDNA encode for polypeptide components of these complexes. As a consequence, mutations in either genome can cause MRC dysfunction that impairs transport of electrons and/or protons and decreases ATP synthesis. Primary or secondary genetic diseases affecting MRC or secondary mitochondrial dysfunctions usually affect brain and skeletal muscle because of their energy requirements. Besides MRC enzyme complexes, two electron carriers, coenzyme Q10 (CoQ) and cytochrome c, are essential for mitochondrial synthesis of ATP. CoQ transports electrons from complexes I and II to complex III and is essential for the stability of complex III. CoQ is a lipid-soluble component of virtually all cell membranes. CoQ also functions as an antioxidant that protects cells both by direct ROS scavenging and by regenerating other antioxidants such as vitamins C and E (Turunen et al., 2004). Given the critical role of CoQ in mitochondria function, it has been suggested that CoQ levels could be a useful biological

generation of cytotoxic levels of ROS, and inhibiting the antioxidant system of tumor cells

It is well known that ROS, such as hydrogen peroxide (H2O2) and superoxide anion, induce apoptosis to a wide range of tumor cells through the activation of the caspase cascade. It has been described that mitochondrial damage induced by the use of certain drugs provokes an increment of oxidative stress and cell death (Chandra et al., 2000; Conklin, 2004). Major ROS-modulating agents are based on the capacity to induce high ROS generation or to

An interesting drug for oxidative cancer therapy is amitriptyline, a tricyclic antidepressant commonly prescribed for depression and therapeutic treatments of several neuropathic and inflammatory illnesses. Chlorimipramine, another tricyclic antidepressant, has been already proposed as a novel anticancer agent targeted to mitochondria as it induces caspase-3 dependent apoptosis (Daley et al., 2005). Several reports showed that the toxicity of this drug is due to an increase in oxidative stress by the generation of high amounts of ROS (Daley et al., 2005; Moreno-Fernández et al., 2008; Cordero et al., 2009). Therefore, amitriptyline has being proposed to be used for anticancer oxidant therapy against tumors that present significant

Mitochondria are dynamic organelles that play a central role in many cellular functions including the generation of chemical energy (adenosine triphosphate, ATP), heat, and intracellular calcium homeostasis. They are also responsible for the formation of ROS and for triggering the programmed cell death or apoptosis (Turrens, 2003). The primary metabolic function of mitochondria is oxidative phosphorylation, an energy-generating process that couples oxidation of respiratory substrates to the synthesis of ATP (Pieczenik & Neustadt, 2007). The mitochondrial respiratory chain (MRC) is composed of five multisubunit enzyme complexes. Both the mtDNA and the nuclear DNA (nDNA) encode for polypeptide components of these complexes. Electron transport between MRC complexes I–IV is coupled to the extrusion of protons across the inner mitochondrial membrane by proton pump components of the respiratory chain. This movement of protons creates an electrochemical gradient (m) across the inner mitochondrial membrane. Protons return to the mitochondrial matrix by flowing through ATP synthase (complex V), which utilizes the energy thus produced to synthesize ATP from adenosine diphosphate (ADP) and inorganic phosphate. Both the mtDNA and the nDNA encode for polypeptide components of these complexes. As a consequence, mutations in either genome can cause MRC dysfunction that impairs transport of electrons and/or protons and decreases ATP synthesis. Primary or secondary genetic diseases affecting MRC or secondary mitochondrial dysfunctions usually affect brain and skeletal muscle because of their energy requirements. Besides MRC enzyme complexes, two electron carriers, coenzyme Q10 (CoQ) and cytochrome c, are essential for mitochondrial synthesis of ATP. CoQ transports electrons from complexes I and II to complex III and is essential for the stability of complex III. CoQ is a lipid-soluble component of virtually all cell membranes. CoQ also functions as an antioxidant that protects cells both by direct ROS scavenging and by regenerating other antioxidants such as vitamins C and E (Turunen et al., 2004). Given the critical role of CoQ in mitochondria function, it has been suggested that CoQ levels could be a useful biological

(Fang et al., 2007; Trachootham et al., 2009).

reduce the antioxidant defence machinery of cancer cells.

oxidative stress and/or low antioxidant defences (Cordero et al., 2010).

**2. Mitochondria and Reactive Oxygen Species (ROS)** 

marker of mitochondrial function (Haas et al., 2008). CoQ deficiency induces decreased mitochondrial respiratory enzymes activity, reduced expression of mitochondrial proteins involved in oxidative phosphorylation, decreased mitochondrial membrane potential, increased production of ROS, mitochondrial permeabilization, mitophagy of dysfunctional mitochondria, reduced growth rates and cell death (Rodriguez-Hernandez et al., 2009, Cotan et al, 2011).

In addition to energy, mitochondrial oxidative phosphorylation also generates ROS. When the MRC becomes highly reduced, the excess electrons from complex I or complex III may increase substantially, passing directly to O2 to generate high amounts of superoxide anion (O2 −). Superoxide is transformed to hydrogen peroxide (H2O2) by the detoxification enzymes manganese superoxide dismutase (MnSOD) or copper/zinc superoxide dismutase (Cu/Zn SOD), and then to water by catalase, glutathione peroxidase (GPx) or peroxiredoxin III (PRX III). However, when these enzymes cannot convert ROS such as the superoxide radical to water fast enough, oxidative damage occurs and accumulates in the mitochondria. If H2O2 encounters a reduced transition metal or is mixed with O2•−, the H2O2 can be further reduced to hydroxyl radical (HO•), the most potent oxidizing agent among ROS. Additionally, nitric oxide (•NO) is produced within the mitochondria by mitochondrial nitric oxide synthase (mtNOS) and also freely diffuses into the mitochondria from the cytosol. •NO reacts with O2 <sup>−</sup> to produce peroxynitrite (ONOO−). Together, these two radicals as well as others can do great damage to mitochondria and other cellular components (Turrens, 2003).

Under normal physiological conditions, ROS production is highly regulated. However, if the MRC is inhibited, or key mitochondrial components, such as CoQ, are deficient, then, electrons accumulate on the MRC carriers, greatly increasing the rate of a single electron being transferred to O2 to generate O2 −. An excessive mitochondrial ROS production can exceed the cellular antioxidant defense and the cumulative damage can ultimately destroy the cell by necrosis or apoptosis.

Mitochondria play an important role in cell bioenergetics and life signaling. Mitochondria are necessary for cell survival but they can also trigger cell death, thus exerting decisive control over the biochemistry of several cascades that lead to cell death, specifically the intrinsic pathway of apoptosis. The particular biochemical properties of these organelles are closely related to their segmented structures, which provide an optimal environment for multiple pathways of biosynthesis and bioenergetics. Consequently, it is possible that these organelles are involved in the process of carcinogenesis through alterations in cell metabolism and cell death pathways (Pilkington et al., 2008). Cancer cells show alterations in mtDNA, in oxidative phosphorylation and in energetic metabolism, all triggered by a pro-oxidative change (Indo et al., 2007). The "respiratory damage" in cancer cells predicts low coupling efficiency in electron transfer at the inner membrane of mitochondria and, consequently, greater loss of electrons, leading to the formation of more O2 − (Pelicano et al., 2003). The superoxide anion will thus generate more free radicals. Generated ROS can be released into cytosol and trigger "ROS-induced ROS-release" (RIRR) in neighbouring mitochondria. This mitochondrion-to-mitochondrion ROS-signaling constitutes a positive feedback mechanism for enhanced ROS production potentially leading to significant mitochondrial injury (Zorov et al., 2006). Recent studies by a number of groups have demonstrated that ROS can directly modify signaling proteins through different

Oxidative Therapy Against Cancer 501

whose role in the carcinogenic process is not related directly to DNA damage. These compounds modulate mechanisms of cell growth and death. Thus, the development process of a cancer would consist of the accumulation of multiple events, and the ROS could act at different levels and in all stages (Klauning and Kamendulis, 2004). Thereby, ROS can induce both genomic instability, caused by DNA damage, and alterations in cell signaling processes related to survival, proliferation, resistance to apoptosis, angiogenesis and metastasis, thus

Initiation involves a DNA mutation that is not lethal, but it produces a cell alteration followed by at least one round of DNA synthesis that allows fixing the damage done. At this point, the cell can stop its cycle temporally to undo DNA damage and then resume cell division. DNA damage may be done by ROS, like hydroxyl radicals formed by the Fenton´s reaction. Several studies have revealed an interesting correlation between tumor size and the amount of 8-OHdG (8-hydroxy-2'-desoxyguanosine; also known as 8-oxodeoxyguanosine, 8-oxo-dG), a nucleotide modified by the activity of free radicals (Kennedy et al., 1998; Yano et al., 2009). The promotion stage is characterized by the expansion of initiated cells, stimulating cell proliferation and/or apoptosis inhibition. As a result of this process, an identificable lesion is formed, thus requiring the constant presence of an agent that stimulates promotion. However, it is a reversible process. Many promoter agents have a strong inhibitory capacity against antioxidants like catalases, glutathione, SOD, etc. While a high level of oxidative stress is cytotoxic for cells and stops proliferation inducing apoptosis or even necrosis, moderate levels of oxidative stress may stimulate cell division and, therefore, stimulate tumor growth and promotion (Dreher and Junod, 1996). Progression is the third and last stage of the carcinogenic process. This stage involves cellular and molecular changes that occur from a pre-neoplastic state to a neoplastic state. This stage is irreversible and it is characterized by the accumulation of genetic damage that allows the

ROS are considered as carcinogenic potentials that facilitate cancer promotion and progression (Pelicano et al., 2004). The DNA molecule is one of the main targets of free radicals activity in the cell, and the modifications performed as a consequence of this activity are relevant for the loss of cell homeostasis. This loss may be extended in time due to the DNA functions of information reservoir. This is why the agents and mechanisms of damage by ROS are studied in depth, because its clarification would lead to elucidate the pathogeny of great morbidity and mortality diseases like cancer. There are different types of oxidative damage to DNA, like modifications and depurinations of DNA bases, DNA chain ruptures, and mutations, which tend to accumulate in an environment with high concentrations of ROS. It is known that DNA damage by ROS occurs spontaneously and there is a normal level of bases modified by ROS in cellular DNA (Okamoto, 2000). The most frequent modification of DNA linked to ROS is the formation of 8-OH-dG, resulting from attack of either a hydroxyl radical or singlet oxygen on deoxyguanosine. 8-OH-dG has a highly mutagenic potential, as 5-hydroxymethyl-2 deoxyuridine (Retel et al., 1993). With relative frequency, the cell will evade DNA damage using specific DNA polymerases and enter DNA replication creating mutations and chromosomal lesions. Alternatively, the presence of unrepaired DNA lesions can induce cell death through the apoptotic pathway. Chronic exposure to DNA lesions can lead to mutations and genomic instability (pre-cancerous state) and eventually to malignant transformations

contributing to cancer initiation, promotion and progression.

cell evolving from benign to malignant.

(cancerous state) (Kryston et al., 2011).

modifications, for example by nitrosylation, carbonylation, disulphide bond formation and glutathionylation. Moreover, redox modification of proteins permits further regulation of cell signaling pathways (England and Cotter, 2005).

#### **3. Oxidative stress and cancer**

#### **3.1 ROS and carcinogenesis**

In general, oxidative/nitrosative stress could be defined as an imbalance between the presence of high levels of ROS and reactive nitrogen species (RNS), and the antioxidative defense mechanisms. These toxic molecules are formed via oxidation-reduction reactions and are highly reactive since they have an odd number of electrons. ROS generated under physiological conditions are essential for life, as they are involved in bactericidal activity of phagocytes, and in signal transduction pathways, regulating cell growth and reduction– oxidation (redox) status (Davies, 1995). ROS includes free radicals, such as hydroxyl and superoxide radicals, and non-radicals, including hydrogen peroxide and singlet oxygen. Oxidative stress and generation of free radicals, as primary or secondary event, have been related in a great number of diseases, including cancer (Floyd, 1990).

At the beginning of the carcinogenic process, tumor cells accumulate mutations that allow them to proliferate in an uncontrolled way. Moreover, these alterations contribute to increase the susceptibility to accumulate additional genetic modifications, facilitating tumor progression and cancer development. Cancer could be defined as a cell-cycle desease, where its misregulation is considered an essential step (Sandhu et al., 2000). Carcinogenesis is a complex process of different sequencies that allow a cell to evolve from a healthy state into a pre-cancerous state and eventually reach a cancerous state. In this sense, there are several theories about the process of carcinogenesis. For instance, an increase of DNA synthesis and mitosis triggered by non-genotoxic agents could induce mutations in new cells. These mutations could spread through new cell divisions, evolving from an initial pre-neoplastic state into a neoplastic state. Another theory explains the existence of an imbalance between proliferation and cell death, where proliferation is favoured. If DNA damage is too high, there are important mechanisms, such as apoptosis, by which the altered cells are selectively eliminated. Protein p53 plays a fundamental role in this process, as it initiates mechanisms that eliminate, for example, those oxidized DNA bases that could cause mutations. If cell damage is too high, p53 initiates the mechanisms of apoptosis, although uncontrolled processes of apoptosis can be harmful for the organism, since healthy cells could also be eliminated. Therefore, there are systems for regulating apoptosis that consist of both proapoptotic and anti-apoptotic factors. Alterations that affect the function of gene p53 have been found in more than half of all types of cancer. This fact supports the idea that carcinogenesis would be caused by an imbalance between proliferation and cell death, in favour of proliferation.

Studies of epidemiology and animal experimentation have shown that carcinogenesis could occur in several stages characterized by different mechanisms. Thus, the model of carcinogenesis based on the hypothesis of three stages: initiation, promotion and progression, should be highlighted. Genotoxic agents are mainly chemical substances that damage DNA directly, inducing the generation of a mutation and/or a set of structural changes. On the other hand, there is a second category of non-genotoxic carcinogenic agents,

modifications, for example by nitrosylation, carbonylation, disulphide bond formation and glutathionylation. Moreover, redox modification of proteins permits further regulation of

In general, oxidative/nitrosative stress could be defined as an imbalance between the presence of high levels of ROS and reactive nitrogen species (RNS), and the antioxidative defense mechanisms. These toxic molecules are formed via oxidation-reduction reactions and are highly reactive since they have an odd number of electrons. ROS generated under physiological conditions are essential for life, as they are involved in bactericidal activity of phagocytes, and in signal transduction pathways, regulating cell growth and reduction– oxidation (redox) status (Davies, 1995). ROS includes free radicals, such as hydroxyl and superoxide radicals, and non-radicals, including hydrogen peroxide and singlet oxygen. Oxidative stress and generation of free radicals, as primary or secondary event, have been

At the beginning of the carcinogenic process, tumor cells accumulate mutations that allow them to proliferate in an uncontrolled way. Moreover, these alterations contribute to increase the susceptibility to accumulate additional genetic modifications, facilitating tumor progression and cancer development. Cancer could be defined as a cell-cycle desease, where its misregulation is considered an essential step (Sandhu et al., 2000). Carcinogenesis is a complex process of different sequencies that allow a cell to evolve from a healthy state into a pre-cancerous state and eventually reach a cancerous state. In this sense, there are several theories about the process of carcinogenesis. For instance, an increase of DNA synthesis and mitosis triggered by non-genotoxic agents could induce mutations in new cells. These mutations could spread through new cell divisions, evolving from an initial pre-neoplastic state into a neoplastic state. Another theory explains the existence of an imbalance between proliferation and cell death, where proliferation is favoured. If DNA damage is too high, there are important mechanisms, such as apoptosis, by which the altered cells are selectively eliminated. Protein p53 plays a fundamental role in this process, as it initiates mechanisms that eliminate, for example, those oxidized DNA bases that could cause mutations. If cell damage is too high, p53 initiates the mechanisms of apoptosis, although uncontrolled processes of apoptosis can be harmful for the organism, since healthy cells could also be eliminated. Therefore, there are systems for regulating apoptosis that consist of both proapoptotic and anti-apoptotic factors. Alterations that affect the function of gene p53 have been found in more than half of all types of cancer. This fact supports the idea that carcinogenesis would be caused by an imbalance between proliferation and cell death, in

Studies of epidemiology and animal experimentation have shown that carcinogenesis could occur in several stages characterized by different mechanisms. Thus, the model of carcinogenesis based on the hypothesis of three stages: initiation, promotion and progression, should be highlighted. Genotoxic agents are mainly chemical substances that damage DNA directly, inducing the generation of a mutation and/or a set of structural changes. On the other hand, there is a second category of non-genotoxic carcinogenic agents,

cell signaling pathways (England and Cotter, 2005).

related in a great number of diseases, including cancer (Floyd, 1990).

**3. Oxidative stress and cancer**

**3.1 ROS and carcinogenesis** 

favour of proliferation.

whose role in the carcinogenic process is not related directly to DNA damage. These compounds modulate mechanisms of cell growth and death. Thus, the development process of a cancer would consist of the accumulation of multiple events, and the ROS could act at different levels and in all stages (Klauning and Kamendulis, 2004). Thereby, ROS can induce both genomic instability, caused by DNA damage, and alterations in cell signaling processes related to survival, proliferation, resistance to apoptosis, angiogenesis and metastasis, thus contributing to cancer initiation, promotion and progression.

Initiation involves a DNA mutation that is not lethal, but it produces a cell alteration followed by at least one round of DNA synthesis that allows fixing the damage done. At this point, the cell can stop its cycle temporally to undo DNA damage and then resume cell division. DNA damage may be done by ROS, like hydroxyl radicals formed by the Fenton´s reaction. Several studies have revealed an interesting correlation between tumor size and the amount of 8-OHdG (8-hydroxy-2'-desoxyguanosine; also known as 8-oxodeoxyguanosine, 8-oxo-dG), a nucleotide modified by the activity of free radicals (Kennedy et al., 1998; Yano et al., 2009). The promotion stage is characterized by the expansion of initiated cells, stimulating cell proliferation and/or apoptosis inhibition. As a result of this process, an identificable lesion is formed, thus requiring the constant presence of an agent that stimulates promotion. However, it is a reversible process. Many promoter agents have a strong inhibitory capacity against antioxidants like catalases, glutathione, SOD, etc. While a high level of oxidative stress is cytotoxic for cells and stops proliferation inducing apoptosis or even necrosis, moderate levels of oxidative stress may stimulate cell division and, therefore, stimulate tumor growth and promotion (Dreher and Junod, 1996). Progression is the third and last stage of the carcinogenic process. This stage involves cellular and molecular changes that occur from a pre-neoplastic state to a neoplastic state. This stage is irreversible and it is characterized by the accumulation of genetic damage that allows the cell evolving from benign to malignant.

ROS are considered as carcinogenic potentials that facilitate cancer promotion and progression (Pelicano et al., 2004). The DNA molecule is one of the main targets of free radicals activity in the cell, and the modifications performed as a consequence of this activity are relevant for the loss of cell homeostasis. This loss may be extended in time due to the DNA functions of information reservoir. This is why the agents and mechanisms of damage by ROS are studied in depth, because its clarification would lead to elucidate the pathogeny of great morbidity and mortality diseases like cancer. There are different types of oxidative damage to DNA, like modifications and depurinations of DNA bases, DNA chain ruptures, and mutations, which tend to accumulate in an environment with high concentrations of ROS. It is known that DNA damage by ROS occurs spontaneously and there is a normal level of bases modified by ROS in cellular DNA (Okamoto, 2000). The most frequent modification of DNA linked to ROS is the formation of 8-OH-dG, resulting from attack of either a hydroxyl radical or singlet oxygen on deoxyguanosine. 8-OH-dG has a highly mutagenic potential, as 5-hydroxymethyl-2 deoxyuridine (Retel et al., 1993). With relative frequency, the cell will evade DNA damage using specific DNA polymerases and enter DNA replication creating mutations and chromosomal lesions. Alternatively, the presence of unrepaired DNA lesions can induce cell death through the apoptotic pathway. Chronic exposure to DNA lesions can lead to mutations and genomic instability (pre-cancerous state) and eventually to malignant transformations (cancerous state) (Kryston et al., 2011).

Oxidative Therapy Against Cancer 503

oxidative stress helps tumor to go on to malignancy. As an example, *in vitro* data suggest that in environments with certain levels of oxidative stress some cytokines, such as interleukin-2 (IL-2) or interferon α (IFNα), induce a decrease of T-lymphocytes or *natural killer* cells, which could suposse a tumor evasion to the immune response (Mantovani et al., 2003). Another source of free radical generation, usually underestimated but highlighted by Kryston et al. (2011), is the chronic exposure to viral infections; as in the case of hepatitis viruses, where there is a connection between chronic infection and induction of oxidative stress. There is a variety of viruses associated with increased ROS levels, DNA damage and mutagenic rate. The high intracellular oxidation status in viral infections consists of decreased antioxidant enzymes like catalase, glutathione peroxidase, glutathione reductase

Since some time already, an increase of ROS and an altered redox state has been observed in cancerous cells (Trachootham et al., 2009). It is known that ROS may serve as cellular messengers in the signal translation pathway and also an increase of ROS may trigger cell growth and proliferation, contributing to cancer development (Filomeno et al., 2005). Uncontrolled tumor cell proliferation requires the up-regulation of multiple intracellular signaling pathways, including cascades involved in survival, proliferation, and cell cycle progression. The most significant effects of oxidants on signaling pathways have been observed in the MAPK/AP-1 and NF-κB pathways (Muller et al., 2010). At the advanced stage of the disease, cancerous cells usually show genetic instability and a sharp increase of ROS, which induces to genetic mutations and failures in metabolic functioning, provoking a greater generation of ROS (Pelicano et al., 2004). Several mechanisms may lead to oxidative stress in cancer patients, such as altered energy metabolism, overproduction of cytokines, which in turn may increase ROS production, and the use of anti-neoplastic drugs. Altered energy metabolism in cancer may explain symptoms such as anorexia/cachexia, nausea and vomiting, which prevent normal nutrition. This deficit in the normal supply of nutrients like glucose, proteins, antioxidants and vitamins, leads eventually to the lack of antioxidant

Protein p53 is known as "the guardian of the genome" because it is essential for maintaining its integrity. This tumor suppressor protein has a fundamental role at detecting and eliminating oxidative damage in nuclear and mitochondrial DNA, at preventing mutations and at genetic instability. On the other hand, p53 acts also as a transcription factor that regulates the expression of many pro-oxidants and antioxidant genes. The functional loss of p53 is related to redox disequilibrium, increase of ROS, greater mutagenesis and tumor growth (Attardi and Donehower, 2005). This loss of p53 function is observed in many

There is evidence that the increase of ROS in tumor cells has a fundamental role in the acquisition of cancer characteristics (Hanahan and Weinberg, 2011): immortalization and transformation, cell proliferation and mitogenic signals, and cell survival and interruption of apoptotic death. Interestingly, in contrast to the effect of tumor promotion, recent studies suggest that the high ROS level has a role in the induction and maintenance of the senescence induced by tumor suppression, through the supported activation of the cell cycle inhibitor p16 (Ramsey and Sharpless, 2006; Takahashi et al., 2006). On the other hand, if the

as well as high level of hydroxyl radicals (Kryston et al., 2011).

**3.3 Influence of ROS in the cell cycle** 

defenses for controlling free radical production.

human cancers, especially in advanced stages (Bourdon, 2007).

#### **3.2 Sources of oxidative stress**

Cells from all organisms are exposed to several oxidizing and harmful agents. These attacks can be divided into two groups: exogenous and endogenous. Exogenous sources are related to environmental, medical, diagnostic ionizing and non-ionizing radiations (X- or γ-rays, αparticles from radon decay, UVA radiation) or chemical agents. Endogenous (intracelullar) sources of reactive species are primarily produced by O2 metabolism, immune responses and inflammation. These processes may result in the production of ROS and RNS that react with DNA and produce several lesions and indirect effects. Ionizing radiations can damage DNA also by direct energy deposition and ionizations (Kryston et al., 2011).

Oxygen metabolism is the major source of ROS in tumor tissues. ROS are continously formed in mitochondria as respiration byproducts. Cancerous cells are metabolically very active and require a great supply of ATP in order to maintain proliferation and cell growth under control. This high energy demand in the mitocondrial respiratory chain contributes to the generation of ROS. Usually, ATP is produced with high efficiency through oxidative phosphorylation in mitochondria. However, a malfunction of mitochondrial respiration is usually observed in neoplastic cells due to deletions/mutations in mtDNA, to the aberrant expression of some enzymes involved in energy metabolism and to hypoxia (Xu et al., 2005; Verrax et al., 2008). On the other hand, the increase of glycolysis in tumor cells has been shown to be related to the aggressiveness of the tumor (Cuezva et al., 2002). Acquiring a glycolytic phenotype represents a key element for cancer survival and progression, while glycolysis inhibition could be proposed as a goal in antitumor therapy (Pelicano et al., 2004). In this sense, glucose deprivation in laboratory models has been shown to be related to an increase of oxidative stress in tumor cells (Spitz et al., 2000). In cell lines of breast carcinoma, glucose deprivation leads to an intracellular increase of pro-oxidants, a decrease of free radical neutralization, and pyruvate depletion, which leads to an increase of oxidative stress (Spitz et al., 2000; Lee et al., 1999). As a compensation mechanism, glucose deprivation induces the expression of heme oxigenase-1 (HO-1), an enzyme that plays an important role in the antioxidant defense system of the organism and in Fe homeostasis. Chang and collegues (Chang et al., 2003) showed that the generation of ROS in mitochondria induces the overexpression of HO-1, which demonstrates that this is a common mechanism of regulation with the aim of protecting cells against oxidative damage.

As already mentioned, immune response and inflamation are other sources of ROS. It is known that oxidative stress activates inflammatory pathways leading to transformation of a normal cell to tumor cell, tumor cell survival, proliferation, insensitivity to anti-growth signaling, invasion, sustained angiogenesis, and stem cell survival (Reuter et al., 2010). Chronic inflammation is triggered by environmental (extrinsic) factors (eg, infection, tobacco, asbestos) and host mutations (intrinsic) factors (eg, Ras, Myc, p53). Activation of Ras, Myc, and p53 cause mitochondrial dysfunction, resulting in mitochondrial ROS production and downstream signaling (eg, NFkappaB, STAT3, etc.) that promote inflammation-associated cancer (Kamp at al., 2011).

On the other hand, during immune responses of many carcinogenetic processes, several immune-related cells play their roles, like intratumoral lymphocytes killing malignant cells and macrophages and neutrophils degrading cells by using oxidants and enzymes. One of these enzymes is NADPH oxidase, which activity produces ROS. The increased intensity of

Cells from all organisms are exposed to several oxidizing and harmful agents. These attacks can be divided into two groups: exogenous and endogenous. Exogenous sources are related to environmental, medical, diagnostic ionizing and non-ionizing radiations (X- or γ-rays, αparticles from radon decay, UVA radiation) or chemical agents. Endogenous (intracelullar) sources of reactive species are primarily produced by O2 metabolism, immune responses and inflammation. These processes may result in the production of ROS and RNS that react with DNA and produce several lesions and indirect effects. Ionizing radiations can damage

Oxygen metabolism is the major source of ROS in tumor tissues. ROS are continously formed in mitochondria as respiration byproducts. Cancerous cells are metabolically very active and require a great supply of ATP in order to maintain proliferation and cell growth under control. This high energy demand in the mitocondrial respiratory chain contributes to the generation of ROS. Usually, ATP is produced with high efficiency through oxidative phosphorylation in mitochondria. However, a malfunction of mitochondrial respiration is usually observed in neoplastic cells due to deletions/mutations in mtDNA, to the aberrant expression of some enzymes involved in energy metabolism and to hypoxia (Xu et al., 2005; Verrax et al., 2008). On the other hand, the increase of glycolysis in tumor cells has been shown to be related to the aggressiveness of the tumor (Cuezva et al., 2002). Acquiring a glycolytic phenotype represents a key element for cancer survival and progression, while glycolysis inhibition could be proposed as a goal in antitumor therapy (Pelicano et al., 2004). In this sense, glucose deprivation in laboratory models has been shown to be related to an increase of oxidative stress in tumor cells (Spitz et al., 2000). In cell lines of breast carcinoma, glucose deprivation leads to an intracellular increase of pro-oxidants, a decrease of free radical neutralization, and pyruvate depletion, which leads to an increase of oxidative stress (Spitz et al., 2000; Lee et al., 1999). As a compensation mechanism, glucose deprivation induces the expression of heme oxigenase-1 (HO-1), an enzyme that plays an important role in the antioxidant defense system of the organism and in Fe homeostasis. Chang and collegues (Chang et al., 2003) showed that the generation of ROS in mitochondria induces the overexpression of HO-1, which demonstrates that this is a common mechanism of

DNA also by direct energy deposition and ionizations (Kryston et al., 2011).

regulation with the aim of protecting cells against oxidative damage.

inflammation-associated cancer (Kamp at al., 2011).

As already mentioned, immune response and inflamation are other sources of ROS. It is known that oxidative stress activates inflammatory pathways leading to transformation of a normal cell to tumor cell, tumor cell survival, proliferation, insensitivity to anti-growth signaling, invasion, sustained angiogenesis, and stem cell survival (Reuter et al., 2010). Chronic inflammation is triggered by environmental (extrinsic) factors (eg, infection, tobacco, asbestos) and host mutations (intrinsic) factors (eg, Ras, Myc, p53). Activation of Ras, Myc, and p53 cause mitochondrial dysfunction, resulting in mitochondrial ROS production and downstream signaling (eg, NFkappaB, STAT3, etc.) that promote

On the other hand, during immune responses of many carcinogenetic processes, several immune-related cells play their roles, like intratumoral lymphocytes killing malignant cells and macrophages and neutrophils degrading cells by using oxidants and enzymes. One of these enzymes is NADPH oxidase, which activity produces ROS. The increased intensity of

**3.2 Sources of oxidative stress** 

oxidative stress helps tumor to go on to malignancy. As an example, *in vitro* data suggest that in environments with certain levels of oxidative stress some cytokines, such as interleukin-2 (IL-2) or interferon α (IFNα), induce a decrease of T-lymphocytes or *natural killer* cells, which could suposse a tumor evasion to the immune response (Mantovani et al., 2003). Another source of free radical generation, usually underestimated but highlighted by Kryston et al. (2011), is the chronic exposure to viral infections; as in the case of hepatitis viruses, where there is a connection between chronic infection and induction of oxidative stress. There is a variety of viruses associated with increased ROS levels, DNA damage and mutagenic rate. The high intracellular oxidation status in viral infections consists of decreased antioxidant enzymes like catalase, glutathione peroxidase, glutathione reductase as well as high level of hydroxyl radicals (Kryston et al., 2011).

#### **3.3 Influence of ROS in the cell cycle**

Since some time already, an increase of ROS and an altered redox state has been observed in cancerous cells (Trachootham et al., 2009). It is known that ROS may serve as cellular messengers in the signal translation pathway and also an increase of ROS may trigger cell growth and proliferation, contributing to cancer development (Filomeno et al., 2005). Uncontrolled tumor cell proliferation requires the up-regulation of multiple intracellular signaling pathways, including cascades involved in survival, proliferation, and cell cycle progression. The most significant effects of oxidants on signaling pathways have been observed in the MAPK/AP-1 and NF-κB pathways (Muller et al., 2010). At the advanced stage of the disease, cancerous cells usually show genetic instability and a sharp increase of ROS, which induces to genetic mutations and failures in metabolic functioning, provoking a greater generation of ROS (Pelicano et al., 2004). Several mechanisms may lead to oxidative stress in cancer patients, such as altered energy metabolism, overproduction of cytokines, which in turn may increase ROS production, and the use of anti-neoplastic drugs. Altered energy metabolism in cancer may explain symptoms such as anorexia/cachexia, nausea and vomiting, which prevent normal nutrition. This deficit in the normal supply of nutrients like glucose, proteins, antioxidants and vitamins, leads eventually to the lack of antioxidant defenses for controlling free radical production.

Protein p53 is known as "the guardian of the genome" because it is essential for maintaining its integrity. This tumor suppressor protein has a fundamental role at detecting and eliminating oxidative damage in nuclear and mitochondrial DNA, at preventing mutations and at genetic instability. On the other hand, p53 acts also as a transcription factor that regulates the expression of many pro-oxidants and antioxidant genes. The functional loss of p53 is related to redox disequilibrium, increase of ROS, greater mutagenesis and tumor growth (Attardi and Donehower, 2005). This loss of p53 function is observed in many human cancers, especially in advanced stages (Bourdon, 2007).

There is evidence that the increase of ROS in tumor cells has a fundamental role in the acquisition of cancer characteristics (Hanahan and Weinberg, 2011): immortalization and transformation, cell proliferation and mitogenic signals, and cell survival and interruption of apoptotic death. Interestingly, in contrast to the effect of tumor promotion, recent studies suggest that the high ROS level has a role in the induction and maintenance of the senescence induced by tumor suppression, through the supported activation of the cell cycle inhibitor p16 (Ramsey and Sharpless, 2006; Takahashi et al., 2006). On the other hand, if the

Oxidative Therapy Against Cancer 505

reaction of ROS with thiol groups in the protease catalytic domain, being also ROS involved

Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signaling (Diers et al., 2010). One such redox signaling molecule is the electrophilic cyclopentenone prostaglandin, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), which can affect redox signaling through the posttranslational modification of critical cysteine residues in proteins such as actin, vimentin, and tubulin. The fact that 15d-PGJ2 can alter the cytoskeleton coincides with decreased migration and increased focal-adhesion disassembly, which might have important implications in the inhibition of metastatic

There is sufficient evidence that cancer cells are under greater oxidative stress (Pervaiz and Clement, 2004; Kryston et al., 2011). High levels of oxidative stress have been found in cancer patients (McEligot et al., 2005; Lu et al., 2007), and it has been reported that different biomarkers of oxidative-stress-mediated events are elevated in cancer-prone tissues (Bartsch and Nair, 2000). *In vitro* studies clearly show that human tumor cell lines produce ROS at a much higher rate than non-transformed cells (Oberley and Buettner, 1979; Lu et al., 2007). In cancerous cells, a high level of oxidative stress is observed, which may result not only from the overproduction of ROS, but also from low levels or inactivation of antioxidants (Huang et al., 2003). Antioxidants are molecules capable of slowing down or even preventing the oxidation of other molecules and play an essential role at protecting cells against ROS aggression. Inhibition of these enzymes seriously endangers the capability of cells to face ROS activity. The first frontier of cellular defense against ROS damage consists of endogenous non-enzymatic radical scavengers like glutathione (GSH) and vitamins like C, E, antioxidant enzymes like SOD, catalase and GPx, as well as specific repair pathways

A decrease of mitochondrial activity and an overexpression of Mn-SOD with a greater

pancreas (Van-Driel et al., 1997; Cullen et al., 2003). The accumulation of anion O2

increase of ROS contributes to the growth and survival of many cancers.

stimulates cell growth by altering the redox states of transcription factors and regulators of the protein cell cycle. The increase of ROS in tumor cells may induce an increase of endogenous antioxidants in order to avoid intracellular lesions. On the contrary, a decrease of SOD activity was detected in blood cells of patients with cervical cancer. The decrease of SOD activity observed could be related to the generation of free radicals that cause direct damage to the enzyme by reticulation or mutation induction (Manoharan et al., 2004; Naidu et al., 2007). Besides, this decrease may be caused by an alteration of the oligoelements (essential trace elements) that act as cofactors of this enzyme, which may be considered as a risk factor of tumor growth or carcinogenesis (Naidu et al., 2007). On the other hand, it was observed that the increase in the intensity of oxidative stress in the blood of patients with ovarian cancer is accompained by a decrease of antioxidants like SOD, catalase, vitamin C and vitamin E (Senthil et al., 2004). A high percentage of tumors show low catalase activity, which means an advantageous adaptation for the tumor, which continues to benefit from the high levels of ROS. Although oxidant agents can be toxic to normal cells, the moderate

− have been detected in some colorectal carcinomas and tumor cells of the

−

processes such as invasion, intravasation, and extravasation (Diers et al., 2010).

in MMP gene expression (Rajagopalan et al., 1996).

**3.6 ROS adaptation by cancer cells** 

(Kryston et al., 2011).

production of O2

ROS level increases up to a specific threshold that is incompatible with cell survival, ROS can exert cytotoxic effects that lead to the death of malignant cells and, therefore, limit cancer progression. This double game of ROS effect on cancerous cells may be used for developing new antitumor drugs that provide an increase of lethal oxidative stress in tumor cells, which are more sensitive to this type of attack than normal cells.

#### **3.4 Angiogenesis induced by ROS**

In addition to alterations in the cell cycle, an important event in the growth of any tumor is the generation of a new blood supply system that feeds the malignant cells. Migration, proliferation and tubular formation by endothelial cells are essential events in the process of angiogenesis. It has been suggested that ROS play an important role in angiogenesis, although their molecular mechanism remains unknown (Ushio-Fukai, 2004). The vascular endothelial growth factor (VEGF) triggers angiogenesis by stimulating the proliferation of endothelial cells and their migration through the receptor 2 (Flk1/KDR) of VEGF, which has tyrosine kinase activity. ROS derived from NAD(P)H oxidase are important for in vitro VEGF signaling and for in vivo angiogenesis. Arbiser and collegues (Arbiser et al., 2002) showed that ROS increased the expression of the VEGF, triggering the promotion of vascularization mechanisms and the fast expansion of tumors. On the other hand, greater metastatic capacity has been associated with high ROS levels in most tumors (Ishikawa et al., 2008). So, exogenous administration of ROS could increase certain metastatic states, while a treatment with antioxidants could slow down the metastatic progression (Ferraro et al., 2006).

Hypoxia seems to be the most important mechanism for tumor progression through the activation of angiogenesis, which is essential for the tumor growth (Harris, 2002). The neoplastic cells respond to hypoxia with an increase of ROS level; this occurs in the first stages of tumor development as a consequence of the blood supply deficit (Denko et al., 2003). It was shown that despite hypoxia there was a greater production of ROS, a paradox, since the oxygen availability for the formation of O2 − would be limited. Perhaps the generation of ROS occurs later, throughout tumor development, at the first stages of angiogenesis. When the hypoxia of a tumor is followed by blood supply reperfusion, high levels of ROS are generated; something similar occurs in a myocardial infarction or a brain ischemia.

#### **3.5 ROS and tumor cell invasion**

Tumor invasion and metastasis are two important events in cancer in which oxidative stress has shown to have an important role. When mammalian carcinoma cells are treated with hydrogen peroxide before intravenous injection into mice, an increase in lung metastasis formation is observed (Kundu et al., 1995), probably due to a decreased attachment of tumor cells to the basal lamina or it could alternatively be due to the increased activity or expression of proteins that regulate cellular motility.

On the other hand, the matrix metalloproteinases (MMPs) have been involved in the invasion and metastasis of malignant tumors of various histogenetic origins, and are capable of cleaving most components of the basement membrane and extracellular matrix (Westermarck et al., 1999). The activation of MMPs, such as MMP-2, probably occurs by the

ROS level increases up to a specific threshold that is incompatible with cell survival, ROS can exert cytotoxic effects that lead to the death of malignant cells and, therefore, limit cancer progression. This double game of ROS effect on cancerous cells may be used for developing new antitumor drugs that provide an increase of lethal oxidative stress in tumor

In addition to alterations in the cell cycle, an important event in the growth of any tumor is the generation of a new blood supply system that feeds the malignant cells. Migration, proliferation and tubular formation by endothelial cells are essential events in the process of angiogenesis. It has been suggested that ROS play an important role in angiogenesis, although their molecular mechanism remains unknown (Ushio-Fukai, 2004). The vascular endothelial growth factor (VEGF) triggers angiogenesis by stimulating the proliferation of endothelial cells and their migration through the receptor 2 (Flk1/KDR) of VEGF, which has tyrosine kinase activity. ROS derived from NAD(P)H oxidase are important for in vitro VEGF signaling and for in vivo angiogenesis. Arbiser and collegues (Arbiser et al., 2002) showed that ROS increased the expression of the VEGF, triggering the promotion of vascularization mechanisms and the fast expansion of tumors. On the other hand, greater metastatic capacity has been associated with high ROS levels in most tumors (Ishikawa et al., 2008). So, exogenous administration of ROS could increase certain metastatic states, while a treatment with antioxidants could slow down the metastatic progression (Ferraro et

Hypoxia seems to be the most important mechanism for tumor progression through the activation of angiogenesis, which is essential for the tumor growth (Harris, 2002). The neoplastic cells respond to hypoxia with an increase of ROS level; this occurs in the first stages of tumor development as a consequence of the blood supply deficit (Denko et al., 2003). It was shown that despite hypoxia there was a greater production of ROS, a paradox,

generation of ROS occurs later, throughout tumor development, at the first stages of angiogenesis. When the hypoxia of a tumor is followed by blood supply reperfusion, high levels of ROS are generated; something similar occurs in a myocardial infarction or a brain

Tumor invasion and metastasis are two important events in cancer in which oxidative stress has shown to have an important role. When mammalian carcinoma cells are treated with hydrogen peroxide before intravenous injection into mice, an increase in lung metastasis formation is observed (Kundu et al., 1995), probably due to a decreased attachment of tumor cells to the basal lamina or it could alternatively be due to the increased activity or

On the other hand, the matrix metalloproteinases (MMPs) have been involved in the invasion and metastasis of malignant tumors of various histogenetic origins, and are capable of cleaving most components of the basement membrane and extracellular matrix (Westermarck et al., 1999). The activation of MMPs, such as MMP-2, probably occurs by the

− would be limited. Perhaps the

cells, which are more sensitive to this type of attack than normal cells.

since the oxygen availability for the formation of O2

expression of proteins that regulate cellular motility.

**3.4 Angiogenesis induced by ROS** 

al., 2006).

ischemia.

**3.5 ROS and tumor cell invasion** 

reaction of ROS with thiol groups in the protease catalytic domain, being also ROS involved in MMP gene expression (Rajagopalan et al., 1996).

Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signaling (Diers et al., 2010). One such redox signaling molecule is the electrophilic cyclopentenone prostaglandin, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), which can affect redox signaling through the posttranslational modification of critical cysteine residues in proteins such as actin, vimentin, and tubulin. The fact that 15d-PGJ2 can alter the cytoskeleton coincides with decreased migration and increased focal-adhesion disassembly, which might have important implications in the inhibition of metastatic processes such as invasion, intravasation, and extravasation (Diers et al., 2010).

#### **3.6 ROS adaptation by cancer cells**

There is sufficient evidence that cancer cells are under greater oxidative stress (Pervaiz and Clement, 2004; Kryston et al., 2011). High levels of oxidative stress have been found in cancer patients (McEligot et al., 2005; Lu et al., 2007), and it has been reported that different biomarkers of oxidative-stress-mediated events are elevated in cancer-prone tissues (Bartsch and Nair, 2000). *In vitro* studies clearly show that human tumor cell lines produce ROS at a much higher rate than non-transformed cells (Oberley and Buettner, 1979; Lu et al., 2007). In cancerous cells, a high level of oxidative stress is observed, which may result not only from the overproduction of ROS, but also from low levels or inactivation of antioxidants (Huang et al., 2003). Antioxidants are molecules capable of slowing down or even preventing the oxidation of other molecules and play an essential role at protecting cells against ROS aggression. Inhibition of these enzymes seriously endangers the capability of cells to face ROS activity. The first frontier of cellular defense against ROS damage consists of endogenous non-enzymatic radical scavengers like glutathione (GSH) and vitamins like C, E, antioxidant enzymes like SOD, catalase and GPx, as well as specific repair pathways (Kryston et al., 2011).

A decrease of mitochondrial activity and an overexpression of Mn-SOD with a greater production of O2 − have been detected in some colorectal carcinomas and tumor cells of the pancreas (Van-Driel et al., 1997; Cullen et al., 2003). The accumulation of anion O2 − stimulates cell growth by altering the redox states of transcription factors and regulators of the protein cell cycle. The increase of ROS in tumor cells may induce an increase of endogenous antioxidants in order to avoid intracellular lesions. On the contrary, a decrease of SOD activity was detected in blood cells of patients with cervical cancer. The decrease of SOD activity observed could be related to the generation of free radicals that cause direct damage to the enzyme by reticulation or mutation induction (Manoharan et al., 2004; Naidu et al., 2007). Besides, this decrease may be caused by an alteration of the oligoelements (essential trace elements) that act as cofactors of this enzyme, which may be considered as a risk factor of tumor growth or carcinogenesis (Naidu et al., 2007). On the other hand, it was observed that the increase in the intensity of oxidative stress in the blood of patients with ovarian cancer is accompained by a decrease of antioxidants like SOD, catalase, vitamin C and vitamin E (Senthil et al., 2004). A high percentage of tumors show low catalase activity, which means an advantageous adaptation for the tumor, which continues to benefit from the high levels of ROS. Although oxidant agents can be toxic to normal cells, the moderate increase of ROS contributes to the growth and survival of many cancers.

Oxidative Therapy Against Cancer 507

Fig. 1. Biological basis for therapeutic selectivity in oxidative therapy against cancer cells.

Anticancer therapies aimed at the mitochondria of tumor cells are being developed in present years, since these organelles are involved in cell death (Daley et al., 2005; Nguyen and Hussain, 2007; Ozben, 2007; Pilkington et al., 2008). Mitochondria are the greatest oxygen consumers in the cell and they are an important source of reactive oxygen mediators. A drug that damages these organelles would cause an increase in ROS production and cell death. Currently, there is an anticancer strategy at its peak known as "oxidative therapy", which consists of inducing high ROS steady state levels in tumor cells. This therapy may be carried out in two different ways: causing the generation of high levels of ROS in solid tumors and inhibiting the antioxidant system of tumor cells (Fang et al., 2007; Trachootham et al., 2009). It is well established that high levels of ROS, like H2O2 and

−, induce apoptosis in a wide variety of tumor cells activating the caspase cascade

Neoplastic cells are metabolically more active and require a high supply of ATP in order to keep cell growth and proliferation under control. This high energy demand in the MRC leads to the increase of ROS generation (Behrend et al., 2003). The excessive production of ROS may damage several cellular components like DNA, proteins, lipids and cell membranes. The oxidation of mitochondrial lipids and proteins causes the permeabilization of the mitochondrial membrane, which leads to an alteration of the coupling efficiency of the electron transport chain, resulting in the generation of more free radicals, and the release of cytochrome *c*, activating the process of programmed cell death (apoptosis) which depends on caspases (Conklin, 2004). There is evidence that the main mechanism by which oxidant agents may kill cells is the activation of apoptosis. In some cases, the high levels of ROS generated may inhibit apoptosis at a caspase level and divert the process toward

Adapted from Gupte et al., 2009 and Trachootham et al., 2009.

(Matsura et al., 1999; Yamakawa et al., 2000).

**4.2 Cell death by ROS increment** 

O2

The strategy adopted by each type of tumor to increase the intensity of oxidative stress may vary from an increase of ROS to a decrease of antioxidants, going through a combination of both. The concentration of ROS in the tumor cell must be compensated with that of antioxidants in order to obtain an increase of ROS steady state level without reaching lethal levels. However, alterations in the mechanisms of antioxidant production are considered as indicators of oxidative stress in several types of cancer (Schumacker, 2006). Cancerous cells may adapt to survive at certain levels of oxidative stress. For example, greater levels of O2 − and H2O2 were observed in cells transformed with H-ras, showing high levels of antioxidants like peroxiredoxin-3 and thioredoxin peroxidase, compared to normal cells (Young et al., 2004). These adaptive mechanisms keep ROS levels in a distribution space that allows cancerous cells to avoid serious oxidative damage and to survive ROS-mediated stress and mutations. Redox adaptation may be crucial not only for cancer development, but also for drug resistance (Pervaiz and Clement, 2004; Sullivan and Graham, 2008). Redox adaptation, through the increase of endogenous antioxidants and the activation of the cell survival pathway, may confer greater capacity for tolerating the action of exogenous stress, with capacity for increasing DNA repair and decreased apoptosis. Thus, although cancerous cells are under chronic oxidative stress, they possess remodeled antioxidant system which let them to avoid deleterious ROS effects.

#### **4. Antitumor oxidative therapy**

#### **4.1 The strategy**

The therapeutic selectivity and avoiding resistance to drugs are two important issues in the therapy against cancer. Strategies for improving therapeutic selectivity depend largely on understanding the biological difference between cancerous and normal cells. Tumor cells, compared to normal cells, are under greater oxidative stress related to the oncogenic transformation, to alterations in metabolic activity and to an increase of ROS generation (Toyokuni et al., 1995; Hileman et al., 2003; Behrend et al., 2003). The high concentration of ROS in cancerous cells may have beneficial consequencies, like the stimulation of cell proliferation, mutation multiplication, genetic instability and alterations in the sensitivity to agents against cancer. However, because ROS are chemically active and may inflict serious cell damage, the very fact that cancerous cells usually show greater intrinsic oxidative stress may itself provide a unique opportunity to kill tumor cells according to the vulnerability of ROS action.

In this sense, the high concentration of ROS could function as a double-edged sword. A moderate increase of ROS could trigger both proliferation and differentiation, as well as other tumor characteristics. However, when ROS levels increase up to the lethal threshold it may break through the antioxidant capacity of the cell and trigger its death, by apoptosis or necrosis, depending on the degree of oxidative damage. Under physiological conditions, normal cells maintain redox homeostasis with a low level of basal ROS by controlling the equilibrium between the generation of ROS (pro-oxidant) and their elimination (antioxidant capacity). Exogenous agents that increase the generation of ROS or decrease the antioxidant capacity of cancerous cells will move the redox equilibrium and induce a general increase of ROS levels, which will cause cell death when exceeding the tolerance threshold (Figure 1).

The strategy adopted by each type of tumor to increase the intensity of oxidative stress may vary from an increase of ROS to a decrease of antioxidants, going through a combination of both. The concentration of ROS in the tumor cell must be compensated with that of antioxidants in order to obtain an increase of ROS steady state level without reaching lethal levels. However, alterations in the mechanisms of antioxidant production are considered as indicators of oxidative stress in several types of cancer (Schumacker, 2006). Cancerous cells may adapt to survive at certain levels of oxidative stress. For example, greater levels of O2

and H2O2 were observed in cells transformed with H-ras, showing high levels of antioxidants like peroxiredoxin-3 and thioredoxin peroxidase, compared to normal cells (Young et al., 2004). These adaptive mechanisms keep ROS levels in a distribution space that allows cancerous cells to avoid serious oxidative damage and to survive ROS-mediated stress and mutations. Redox adaptation may be crucial not only for cancer development, but also for drug resistance (Pervaiz and Clement, 2004; Sullivan and Graham, 2008). Redox adaptation, through the increase of endogenous antioxidants and the activation of the cell survival pathway, may confer greater capacity for tolerating the action of exogenous stress, with capacity for increasing DNA repair and decreased apoptosis. Thus, although cancerous cells are under chronic oxidative stress, they possess remodeled antioxidant system which

The therapeutic selectivity and avoiding resistance to drugs are two important issues in the therapy against cancer. Strategies for improving therapeutic selectivity depend largely on understanding the biological difference between cancerous and normal cells. Tumor cells, compared to normal cells, are under greater oxidative stress related to the oncogenic transformation, to alterations in metabolic activity and to an increase of ROS generation (Toyokuni et al., 1995; Hileman et al., 2003; Behrend et al., 2003). The high concentration of ROS in cancerous cells may have beneficial consequencies, like the stimulation of cell proliferation, mutation multiplication, genetic instability and alterations in the sensitivity to agents against cancer. However, because ROS are chemically active and may inflict serious cell damage, the very fact that cancerous cells usually show greater intrinsic oxidative stress may itself provide a unique opportunity to kill tumor cells according to the vulnerability of

In this sense, the high concentration of ROS could function as a double-edged sword. A moderate increase of ROS could trigger both proliferation and differentiation, as well as other tumor characteristics. However, when ROS levels increase up to the lethal threshold it may break through the antioxidant capacity of the cell and trigger its death, by apoptosis or necrosis, depending on the degree of oxidative damage. Under physiological conditions, normal cells maintain redox homeostasis with a low level of basal ROS by controlling the equilibrium between the generation of ROS (pro-oxidant) and their elimination (antioxidant capacity). Exogenous agents that increase the generation of ROS or decrease the antioxidant capacity of cancerous cells will move the redox equilibrium and induce a general increase of ROS levels, which will cause cell death when exceeding the tolerance threshold (Figure 1).

let them to avoid deleterious ROS effects.

**4. Antitumor oxidative therapy** 

**4.1 The strategy** 

ROS action.

−

Fig. 1. Biological basis for therapeutic selectivity in oxidative therapy against cancer cells. Adapted from Gupte et al., 2009 and Trachootham et al., 2009.

Anticancer therapies aimed at the mitochondria of tumor cells are being developed in present years, since these organelles are involved in cell death (Daley et al., 2005; Nguyen and Hussain, 2007; Ozben, 2007; Pilkington et al., 2008). Mitochondria are the greatest oxygen consumers in the cell and they are an important source of reactive oxygen mediators. A drug that damages these organelles would cause an increase in ROS production and cell death. Currently, there is an anticancer strategy at its peak known as "oxidative therapy", which consists of inducing high ROS steady state levels in tumor cells. This therapy may be carried out in two different ways: causing the generation of high levels of ROS in solid tumors and inhibiting the antioxidant system of tumor cells (Fang et al., 2007; Trachootham et al., 2009). It is well established that high levels of ROS, like H2O2 and O2 −, induce apoptosis in a wide variety of tumor cells activating the caspase cascade (Matsura et al., 1999; Yamakawa et al., 2000).

#### **4.2 Cell death by ROS increment**

Neoplastic cells are metabolically more active and require a high supply of ATP in order to keep cell growth and proliferation under control. This high energy demand in the MRC leads to the increase of ROS generation (Behrend et al., 2003). The excessive production of ROS may damage several cellular components like DNA, proteins, lipids and cell membranes. The oxidation of mitochondrial lipids and proteins causes the permeabilization of the mitochondrial membrane, which leads to an alteration of the coupling efficiency of the electron transport chain, resulting in the generation of more free radicals, and the release of cytochrome *c*, activating the process of programmed cell death (apoptosis) which depends on caspases (Conklin, 2004). There is evidence that the main mechanism by which oxidant agents may kill cells is the activation of apoptosis. In some cases, the high levels of ROS generated may inhibit apoptosis at a caspase level and divert the process toward

Oxidative Therapy Against Cancer 509

2006). So, for instance, an increase in the expression of Mn-SOD has been observed in breast cancer and in blood samples from patients with different types of leukemia (Nishiura et al., 1992; Devi et al., 2000; Ray et al., 2000). This fact may reflect an adaptive mechanism by which cancerous cells respond to an increase of ROS levels produced in mitochondria. ROS can induce the over-regulation of Mn-SOD through the modulation of the redox states of the transcription factors (AP- 1, NF-kappaB). Due to its high expression in certain types of cancer, Mn-SOD has been considered as a tumor marker (Schadendorf et al., 1995). This expression of SOD protects tumor cells against a lethal increase of ROS levels. In fact, it has been demonstrated that the inhibition of SOD with 2-methoxyestradiol would induce apoptosis in leukemia cells through a mechanism mediated by free radicals, without

Antitumor therapies mediated by ROS show a promising therapeutic activity in clinical studies (Trachootham et al., 2009). However, some tumor cells, especially in advanced stages of the disease, have adapted to oxidative stress due to their antioxidant capacity. This redox adaptation does not only allow tumor cells surviving under high levels of ROS, it also provides an increase of survival molecules and a greater capacity for drug inactivation. Moreover, it has been suggested that resistance to the agents that induce intracellular ROS production, such as paclitaxel, doxorubicin or other drugs, is correlated to the increase of antioxidants (Glorieux et al., 2011). Thereby, the capability of certain drugs to inhibit or reduce the antioxidant machinery is very useful in oxidative therapy. These drugs could be used in combination with oxidant agents for greater efficiency in antitumor therapies.

Amitriptyline is a commonly prescribed tricyclic antidepressant drug that is well known to death investigators, forensic pathologists, and toxicologists. Amitriptyline has sedative effects and is frequently prescribed for patients experiencing symptoms of depression. Amitriptyline, have also been used for therapeutic treatment of neuropathic and inflammatory diseases such as fibromyalgia, chronic fatigue syndrome, migraine, irritable bowel syndrome, and atypical facial pain (Gruber et al., 1996). Besides its anxiolytic properties, amitriptyline has central anticholinergic effects. Amitriptyline inhibits serotonin and noradrenaline uptake in presynaptic nerve ending (Maubach et al., 1999). However, toxicity of amitriptyline has been observed during standard treatments, and frequently during suicidal or accidental overdosage. Tricyclic antidepressant overdosage has toxic effects over cardiovascular, autonomous nervous, and central nervous systems, and may result in cardiotoxicity, cardiac conduction delays, dysrhythmia, hypotension, altered

mental status, and seizures (Thanacoody and Thomas, 2005; Kiyan et al., 2006).

In vitro administration of amitriptyline to cell cultures induces several signs of toxicity. Amitriptyline treatment induces alteration of cellular permeability based on its detergent nature (Kitagawa et al., 2006). Furthermore, amitriptyline causes alterations in the glucidic metabolism of neurons resulting in a decrease of both uptake and transport of glucose (Mannerstrom and Tahti, 2004). Additionally, amitriptyline provokes an increase of intracellular lipid peroxidation in mouse 3T3 fibroblasts (Viola et al., 2000) and some mouse tissues (Bautista-Ferrufino et al., 2011), and many of these toxic effects are prevented by antioxidants (Slamon and Pentreath, 2000). Recently, our group has shown that

showing significant toxicity in normal lymphocytes (Zhou et al., 2003).

**5. Amitriptyline as an anti-cancer agent** 

necrosis (Chandra et al., 2000). The change from apoptosis to necrosis is critical in solid tumors and requires considerable amounts of ROS, a decrease of ATP and alterations in the mitochondrial electron-transport chain (Lee et al., 1999). The harmful consequences of this change lie in the inflammation caused by the rupture of necrotic cells and later release of enzymes that degrade the tissues. Thereby, death by apoptosis is preferred in antineoplastic therapies. Apoptotic cells cause the least damage to nearby tissues, since they do not release their content and are phagocytosed by macrophages.

Cancerous cells evolve with the mediation of endogenous and exogenous oxidative agents, depending on the cell type and the evolution state of the tumor. Tumors adapt to these conditions through the development of powerful antioxidant mechanisms and even by the use of endogenous ROS for proliferation. When ROS levels increase above the tolerance threshold, death of tumor cells is induced. Therefore, the fact that an excess of ROS causes cell damage and even death by apoptosis provides us with a strategy for eliminating cancerous cells, which are more sensitive to exogenous oxidative stress than normal cells, through the generation of free radicals, induced by oxidant pharmacological agents. In some cases, tumor cells attacked with antineoplastic therapies may gain resistance to oxidative stress, which is why combined therapies are promising, and are intended to converge toward improving the oxidative action above the critical threshold, or gathering different cytotoxic mechanisms.

Unfortunately, antitumor therapies may exert harmful effects on normal tissues, partially caused by ROS, which limits the application dose and its antitumor activity. Overcoming these secondary effects, without altering the therapy efficiency, is a priority and a challenge in biomedical research. In this sense, great importance is given to targeted therapies, using vehicles (liposomes, nanoparticles) that recognize specific molecules expressed in tumor cells.

#### **4.3 Antioxidants in oxidative therapy against cancer**

Antioxidants play an essential role in cell protection against ROS. The oxidation of antioxidant enzymes reduces the capacity of cells to eliminate free radicals. An important approach in the antitumor therapeutic strategies is to inhibit the antioxidant systems, like catalase, SOD and GPx, which are the main defense lines of the cell. The inhibitors of different antioxidant enzymes have been characterized by their capacity for eliminating neoplastic cells, alone or combined. It has also been described that many pharmacological agents may have more than one mechanism of action and affect multiple biological processes. There are agents that induce apoptosis which are oxidant and others that stimulate cell metabolism. On the other hand, there are apoptosis inhibitors that have antioxidant activities. In the absence of adequate antioxidant defenses, the damage from oxidative stress leads to the activation of the genes responsible for apoptosis.

There are conflicting data in the results obtained by different researchers regarding the levels of antioxidants in tumor tissue and in blood from cancer patients. In some cases, there are differences between different antioxidants in the same patient; some increase and others decrease (Ray et al., 2000). In breast cancer, for example, several studies describe an increase of lipid peroxidation and a decrease of antioxidants (Khanzode et al., 2004; Sener et al., 2007). However, other studies performed in neoplastic tissues have shown a greater presence of ROS and a high expression of antioxidants (Oltra et al., 2001; Gönenç et al.,

necrosis (Chandra et al., 2000). The change from apoptosis to necrosis is critical in solid tumors and requires considerable amounts of ROS, a decrease of ATP and alterations in the mitochondrial electron-transport chain (Lee et al., 1999). The harmful consequences of this change lie in the inflammation caused by the rupture of necrotic cells and later release of enzymes that degrade the tissues. Thereby, death by apoptosis is preferred in antineoplastic therapies. Apoptotic cells cause the least damage to nearby tissues, since they do not release

Cancerous cells evolve with the mediation of endogenous and exogenous oxidative agents, depending on the cell type and the evolution state of the tumor. Tumors adapt to these conditions through the development of powerful antioxidant mechanisms and even by the use of endogenous ROS for proliferation. When ROS levels increase above the tolerance threshold, death of tumor cells is induced. Therefore, the fact that an excess of ROS causes cell damage and even death by apoptosis provides us with a strategy for eliminating cancerous cells, which are more sensitive to exogenous oxidative stress than normal cells, through the generation of free radicals, induced by oxidant pharmacological agents. In some cases, tumor cells attacked with antineoplastic therapies may gain resistance to oxidative stress, which is why combined therapies are promising, and are intended to converge toward improving the oxidative action above the critical threshold, or gathering different

Unfortunately, antitumor therapies may exert harmful effects on normal tissues, partially caused by ROS, which limits the application dose and its antitumor activity. Overcoming these secondary effects, without altering the therapy efficiency, is a priority and a challenge in biomedical research. In this sense, great importance is given to targeted therapies, using vehicles (liposomes, nanoparticles) that recognize specific molecules expressed in tumor cells.

Antioxidants play an essential role in cell protection against ROS. The oxidation of antioxidant enzymes reduces the capacity of cells to eliminate free radicals. An important approach in the antitumor therapeutic strategies is to inhibit the antioxidant systems, like catalase, SOD and GPx, which are the main defense lines of the cell. The inhibitors of different antioxidant enzymes have been characterized by their capacity for eliminating neoplastic cells, alone or combined. It has also been described that many pharmacological agents may have more than one mechanism of action and affect multiple biological processes. There are agents that induce apoptosis which are oxidant and others that stimulate cell metabolism. On the other hand, there are apoptosis inhibitors that have antioxidant activities. In the absence of adequate antioxidant defenses, the damage from

There are conflicting data in the results obtained by different researchers regarding the levels of antioxidants in tumor tissue and in blood from cancer patients. In some cases, there are differences between different antioxidants in the same patient; some increase and others decrease (Ray et al., 2000). In breast cancer, for example, several studies describe an increase of lipid peroxidation and a decrease of antioxidants (Khanzode et al., 2004; Sener et al., 2007). However, other studies performed in neoplastic tissues have shown a greater presence of ROS and a high expression of antioxidants (Oltra et al., 2001; Gönenç et al.,

oxidative stress leads to the activation of the genes responsible for apoptosis.

their content and are phagocytosed by macrophages.

**4.3 Antioxidants in oxidative therapy against cancer** 

cytotoxic mechanisms.

2006). So, for instance, an increase in the expression of Mn-SOD has been observed in breast cancer and in blood samples from patients with different types of leukemia (Nishiura et al., 1992; Devi et al., 2000; Ray et al., 2000). This fact may reflect an adaptive mechanism by which cancerous cells respond to an increase of ROS levels produced in mitochondria. ROS can induce the over-regulation of Mn-SOD through the modulation of the redox states of the transcription factors (AP- 1, NF-kappaB). Due to its high expression in certain types of cancer, Mn-SOD has been considered as a tumor marker (Schadendorf et al., 1995). This expression of SOD protects tumor cells against a lethal increase of ROS levels. In fact, it has been demonstrated that the inhibition of SOD with 2-methoxyestradiol would induce apoptosis in leukemia cells through a mechanism mediated by free radicals, without showing significant toxicity in normal lymphocytes (Zhou et al., 2003).

Antitumor therapies mediated by ROS show a promising therapeutic activity in clinical studies (Trachootham et al., 2009). However, some tumor cells, especially in advanced stages of the disease, have adapted to oxidative stress due to their antioxidant capacity. This redox adaptation does not only allow tumor cells surviving under high levels of ROS, it also provides an increase of survival molecules and a greater capacity for drug inactivation. Moreover, it has been suggested that resistance to the agents that induce intracellular ROS production, such as paclitaxel, doxorubicin or other drugs, is correlated to the increase of antioxidants (Glorieux et al., 2011). Thereby, the capability of certain drugs to inhibit or reduce the antioxidant machinery is very useful in oxidative therapy. These drugs could be used in combination with oxidant agents for greater efficiency in antitumor therapies.

#### **5. Amitriptyline as an anti-cancer agent**

Amitriptyline is a commonly prescribed tricyclic antidepressant drug that is well known to death investigators, forensic pathologists, and toxicologists. Amitriptyline has sedative effects and is frequently prescribed for patients experiencing symptoms of depression. Amitriptyline, have also been used for therapeutic treatment of neuropathic and inflammatory diseases such as fibromyalgia, chronic fatigue syndrome, migraine, irritable bowel syndrome, and atypical facial pain (Gruber et al., 1996). Besides its anxiolytic properties, amitriptyline has central anticholinergic effects. Amitriptyline inhibits serotonin and noradrenaline uptake in presynaptic nerve ending (Maubach et al., 1999). However, toxicity of amitriptyline has been observed during standard treatments, and frequently during suicidal or accidental overdosage. Tricyclic antidepressant overdosage has toxic effects over cardiovascular, autonomous nervous, and central nervous systems, and may result in cardiotoxicity, cardiac conduction delays, dysrhythmia, hypotension, altered mental status, and seizures (Thanacoody and Thomas, 2005; Kiyan et al., 2006).

In vitro administration of amitriptyline to cell cultures induces several signs of toxicity. Amitriptyline treatment induces alteration of cellular permeability based on its detergent nature (Kitagawa et al., 2006). Furthermore, amitriptyline causes alterations in the glucidic metabolism of neurons resulting in a decrease of both uptake and transport of glucose (Mannerstrom and Tahti, 2004). Additionally, amitriptyline provokes an increase of intracellular lipid peroxidation in mouse 3T3 fibroblasts (Viola et al., 2000) and some mouse tissues (Bautista-Ferrufino et al., 2011), and many of these toxic effects are prevented by antioxidants (Slamon and Pentreath, 2000). Recently, our group has shown that

Oxidative Therapy Against Cancer 511

through a mechanism dependent on caspase-3 activation. Apoptosis percentage was significally higher in those cells treated with amitriptyline than in cells treated with CPT, Doxo or Metho (Figure 2A). Moreover, when the cell cycle of synchronized cultures was stopped at the G0/G1 phase by depriving cells from serum, the difference of apoptosis percentage among amitriptyline and the remaining drugs was significantly higher than in normal cultures (Figure 2B). These results suggest that the effect of amitriptyline does not depend on cell cycle stage, whereas CPT, Doxo, and Metho are more harmful in dividing cells, as most chemotherapeutic drugs. These data are of special interest for cancer treatment

Fig. 2. Comparative study of amitriptyline and different chemotherapeutic drugs for the evaluation of apoptosis. (A) Percentages of apoptotic cells in H460 cell cultures 24 h after

synchronized cultures stopped at the G0/G1 phase. Adapted from Cordero et al., 2010.

After treating cancer cells with amitriptyline, we have found increased ROS level and several signs of mitochondrial damage, as attenuated complex I+III activity, decreased protein levels of complex III, decreased membrane potential, and a significant reduction of the number of this organelle, shown by cytochrome c and citrate synthase determination, and electron microscopy (Figure 3). So, this tricyclic compound provokes oxidative stress in cancer cells, being mitochondria the target of its toxicity. None of the chemotherapeutic drugs tested seemed to damage mitochondria seriously. However, the chemotherapeutic drugs induced apoptosis and increased ROS production in tumor cells, although not with

According to our data, amitriptyline induces a mitochondrial damage characterized by a decrease of the expression levels of complexes I and III of the MRC as well as of cytochrome *c* and of CoQ levels, which suggests an alteration in the activity, organization and assembly of the mitochondrial complexes, being this reflected in a decrease of the electron flow as well as a decrease of the mitochondrial membrane potential and, therefore, an increase of intramitochondrial ROS prodution. The damage caused by the increase of mitochondrial ROS induces the opening of the mitochondrial permeability transition pore (MPT), thus increasing mitochondrial permeability with the consequent release of proapoptotic proteins to the cytosol such as cytochrome c, Smac/Diablo, etc., initiating the intrinsic pathway of

administration of drugs at different concentration. (B) Apoptosis assessment in

during the nongrowing phases of certain tumors.

the intensity of amitriptyline.

apoptosis dependent on caspase-3 (Figure 4).

amitriptyline induced toxicity is caused through a mitochondrial dysfunction, and increased ROS level (Moreno-Fernandez et al., 2008; Cordero et al., 2009). Amitriptyline reduced significantly the number of cultured cells; enhanced the production of stimulated lipid peroxidation, inverting the lipid reduced/oxidized ratio; decreased catalase protein levels, cytochrome *c*, ΔΨm, and citrate synthase activity; revealing mitochondrial damage. So, amitriptyline-induced toxicity is caused through mitochondrial dysfunction, and increased mitochondrial ROS production. Moreover, CoQ level was decreased by amitriptyline treatment and CoQ and alpha-tocopherol supplementation ameliorated amitriptylineinduced toxicity in both cultured human primary fibroblasts and zebrafish embryos (Cordero et al., 2009).

Other toxic effects attributed to amitriptyline lie in the alteration of neuron carbohydrate metabolism, which results in a decrease of glucose absorption and transport; causing a total loss of neuron viability in a cell line of neuroblastoma (Mannestrom et al., 2004).

Recent studies have shown that some antidepressants can kill cancerous cells. In fact, tricyclic antidepressants have shown to cause cell death in human normal lymphocytes (Karlson et al., 1998), Hodgkin´s lymphoma cells (Serafeim et al., 2003), neurons (Lirk et al., 2006), glioma cells (Xia et al., 1999; Daley et al., 2005; Levkovitz et al., 2005) and colorectal cancer cells (Arimochi y Morita, 2006). Chlorimipramine exerts its effect via the inhibition of complex III of the MRC (Daley et al., 2005). The same is valid for amitriptyline, as we have already reported (Cordero et al., 2009). We showed in fibroblasts treated with amitriptyline a decrease of expression level of proteins of complex I, complex III, cytochrome c, and reduced CoQ10 levels. Deficient mitochondrial protein expression levels and reduced levels of CoQ10 may impair normal mitochondrial electron flow and proton pumping, inducing a drop in respiratory complexes activity, and mitochondrial membrane potential. Our data showed that amitriptyline-treated fibroblasts have reduced NADH:cytochrome c reductase (complex I+III) activity, and lower mitochondrial membrane potential, which may contribute to impaired mitochondrial protein import and aggravate mitochondrial dysfunction, ROS production, and oxidative stress. It has been proposed that ROS damage can induce the mitochondrial permeability transition (MPT) by the opening of non-specific high conductance permeability transition (PT) pores in the mitochondrial inner membrane (England and Cotter, 2005). This, in turn, leads to a simultaneous collapse of mitochondrial membrane potential. The activation of MPTcauses mitochondria to become permeable to all solutes up to a molecular mass of about 1500 Da (Forte and Bernardi, 2005). After MPT, mitochondria undergo a dramatic swelling driven by colloid osmotic forces, which culminates in the rupture of the outer membrane and release of proapoptotic mitochondrial intermembrane proteins into the cytosol, such as cytochrome c, apoptosis inducing factor, Smac/Diablo, and others (Cordero et al.,2009).

We have also studied the effect of amitriptyline on tumor cell lines (Cordero et al., 2010). We observed that this drug induced important mitochondrial damage in tumor cell lines (H460: non-small cell lung cancer, HeLa: epithelial cervical cancer, and HepG2: hepatoma), generating high amounts of ROS and provoking apoptotic cell death. Moreover, amitriptyline effects have been compared with three antitumor drugs frequently used in cancer therapy: camptothecin (CPT), doxorubicin (Doxo), and methotrexate (Metho). Interestingly, amitriptyline induced significantly higher ROS generation in comparison with the other drugs, producing a dose-dependent increase of apoptosis in human cancer cells

amitriptyline induced toxicity is caused through a mitochondrial dysfunction, and increased ROS level (Moreno-Fernandez et al., 2008; Cordero et al., 2009). Amitriptyline reduced significantly the number of cultured cells; enhanced the production of stimulated lipid peroxidation, inverting the lipid reduced/oxidized ratio; decreased catalase protein levels, cytochrome *c*, ΔΨm, and citrate synthase activity; revealing mitochondrial damage. So, amitriptyline-induced toxicity is caused through mitochondrial dysfunction, and increased mitochondrial ROS production. Moreover, CoQ level was decreased by amitriptyline treatment and CoQ and alpha-tocopherol supplementation ameliorated amitriptylineinduced toxicity in both cultured human primary fibroblasts and zebrafish embryos

Other toxic effects attributed to amitriptyline lie in the alteration of neuron carbohydrate metabolism, which results in a decrease of glucose absorption and transport; causing a total

Recent studies have shown that some antidepressants can kill cancerous cells. In fact, tricyclic antidepressants have shown to cause cell death in human normal lymphocytes (Karlson et al., 1998), Hodgkin´s lymphoma cells (Serafeim et al., 2003), neurons (Lirk et al., 2006), glioma cells (Xia et al., 1999; Daley et al., 2005; Levkovitz et al., 2005) and colorectal cancer cells (Arimochi y Morita, 2006). Chlorimipramine exerts its effect via the inhibition of complex III of the MRC (Daley et al., 2005). The same is valid for amitriptyline, as we have already reported (Cordero et al., 2009). We showed in fibroblasts treated with amitriptyline a decrease of expression level of proteins of complex I, complex III, cytochrome c, and reduced CoQ10 levels. Deficient mitochondrial protein expression levels and reduced levels of CoQ10 may impair normal mitochondrial electron flow and proton pumping, inducing a drop in respiratory complexes activity, and mitochondrial membrane potential. Our data showed that amitriptyline-treated fibroblasts have reduced NADH:cytochrome c reductase (complex I+III) activity, and lower mitochondrial membrane potential, which may contribute to impaired mitochondrial protein import and aggravate mitochondrial dysfunction, ROS production, and oxidative stress. It has been proposed that ROS damage can induce the mitochondrial permeability transition (MPT) by the opening of non-specific high conductance permeability transition (PT) pores in the mitochondrial inner membrane (England and Cotter, 2005). This, in turn, leads to a simultaneous collapse of mitochondrial membrane potential. The activation of MPTcauses mitochondria to become permeable to all solutes up to a molecular mass of about 1500 Da (Forte and Bernardi, 2005). After MPT, mitochondria undergo a dramatic swelling driven by colloid osmotic forces, which culminates in the rupture of the outer membrane and release of proapoptotic mitochondrial intermembrane proteins into the cytosol, such as cytochrome c, apoptosis inducing factor,

We have also studied the effect of amitriptyline on tumor cell lines (Cordero et al., 2010). We observed that this drug induced important mitochondrial damage in tumor cell lines (H460: non-small cell lung cancer, HeLa: epithelial cervical cancer, and HepG2: hepatoma), generating high amounts of ROS and provoking apoptotic cell death. Moreover, amitriptyline effects have been compared with three antitumor drugs frequently used in cancer therapy: camptothecin (CPT), doxorubicin (Doxo), and methotrexate (Metho). Interestingly, amitriptyline induced significantly higher ROS generation in comparison with the other drugs, producing a dose-dependent increase of apoptosis in human cancer cells

loss of neuron viability in a cell line of neuroblastoma (Mannestrom et al., 2004).

(Cordero et al., 2009).

Smac/Diablo, and others (Cordero et al.,2009).

through a mechanism dependent on caspase-3 activation. Apoptosis percentage was significally higher in those cells treated with amitriptyline than in cells treated with CPT, Doxo or Metho (Figure 2A). Moreover, when the cell cycle of synchronized cultures was stopped at the G0/G1 phase by depriving cells from serum, the difference of apoptosis percentage among amitriptyline and the remaining drugs was significantly higher than in normal cultures (Figure 2B). These results suggest that the effect of amitriptyline does not depend on cell cycle stage, whereas CPT, Doxo, and Metho are more harmful in dividing cells, as most chemotherapeutic drugs. These data are of special interest for cancer treatment during the nongrowing phases of certain tumors.

Fig. 2. Comparative study of amitriptyline and different chemotherapeutic drugs for the evaluation of apoptosis. (A) Percentages of apoptotic cells in H460 cell cultures 24 h after administration of drugs at different concentration. (B) Apoptosis assessment in synchronized cultures stopped at the G0/G1 phase. Adapted from Cordero et al., 2010.

After treating cancer cells with amitriptyline, we have found increased ROS level and several signs of mitochondrial damage, as attenuated complex I+III activity, decreased protein levels of complex III, decreased membrane potential, and a significant reduction of the number of this organelle, shown by cytochrome c and citrate synthase determination, and electron microscopy (Figure 3). So, this tricyclic compound provokes oxidative stress in cancer cells, being mitochondria the target of its toxicity. None of the chemotherapeutic drugs tested seemed to damage mitochondria seriously. However, the chemotherapeutic drugs induced apoptosis and increased ROS production in tumor cells, although not with the intensity of amitriptyline.

According to our data, amitriptyline induces a mitochondrial damage characterized by a decrease of the expression levels of complexes I and III of the MRC as well as of cytochrome *c* and of CoQ levels, which suggests an alteration in the activity, organization and assembly of the mitochondrial complexes, being this reflected in a decrease of the electron flow as well as a decrease of the mitochondrial membrane potential and, therefore, an increase of intramitochondrial ROS prodution. The damage caused by the increase of mitochondrial ROS induces the opening of the mitochondrial permeability transition pore (MPT), thus increasing mitochondrial permeability with the consequent release of proapoptotic proteins to the cytosol such as cytochrome c, Smac/Diablo, etc., initiating the intrinsic pathway of apoptosis dependent on caspase-3 (Figure 4).

Oxidative Therapy Against Cancer 513

Evidence exists that cancer cells are under a continuous oxidative stress, facilitating ROS to act as carcinogen, as they were shown to be involved in mutagenesis, cancer promotion and progression. On the other hand, ROS may promote either cell proliferation or cell death, depending on the rate of ROS production and the activity of the antioxidant system. Accordingly, oxidative therapy against cancer arises as a ROS-generating strategy which

In our studies, the tricyclic antidepressant amitriptyline induced high ROS generation as a result of mitochondrial dysfunction, provoking a higher level of apoptosis of tumor cells than common chemotherapeutic drugs. Moreover, it inhibits important antioxidants of the cell-defense machinery, dramatically limiting tumor cell response to ROS production. Thus, amitriptyline, as well as other tricyclic compounds, is being assayed as an anti-cancer drug

Unfortunately, oxidative anti-cancer therapy may exert harmful effects on normal tissues, limiting the applicable dose of drugs and anti-tumor activity. So, differences in ROSinduced cell death and oxidative status between normal and neoplastic cells from different cancer types must be thoroughly investigated, taking into account the complexity of physiological and pathological pathways involved in redox balance. On the other hand, it is not strange that after ROS-generating therapies cancer cells achieve further resistance to oxidative stress. In consequence, combination therapies could be achieved in order to either

increase oxidative status above the critical threshold required for cell survival.

Fig. 4. Mechanism of apoptosis induction by amitriptyline.

**6. Conclusion** 

in oxidative therapy.

Fig. 3. Transmission electron microscopy showing damage and fewer mitochondria in H460 cells treated with 50 mmol/l of amitriptyline. Degenerating mitochondria (arrows) are observed in treated tumor cells. (A) Nontreated tumor cells. (B) Amitriptyline-treated tumor cells (Cordero et al., 2010).

In general, the increase of ROS production causes, as a response, an increase of the antioxidants activities. However, under the high input rate of ROS, enzyme inactivation prevails, which leads to the reduction of antioxidant enzymes activity and to the process of oxidative damage. Thus, tumor cells frequently possess very little antioxidative enzymes, such as catalase, SOD, and glutathione peroxidase, which are known to play a protective role against ROS in normal cells. We have observed in normal fibroblast treated with amitriptyline a decrease in protein expression of antioxidant enzymes (catalase and MnSOD) 16 h after the treatment, followed by restored levels after 24h, as a mechanism of antioxidant defense (Moreno-Fernández et al., 2008). Interestingly, in cancer cells, the same concentration of amitriptyline provoked an unrestorable decrease of catalase (Cordero et al., 2010). The difference of the antioxidant status observed in cancer cells, in comparison with healthy fibroblasts, may be caused by the lower antioxidant level present in the cancer cell lines used. Besides the decrease of catalase and MnSOD, amitriptyline also produces a significant decrease of CoQ level in tumor cells (Cordero et al., 2010). CoQ plays a critical protective role by either acting as an antioxidant or by the noncompetitive inhibition of the neutral sphingomyelinase of plasma membrane, preventing ceramide production (Mates et al., 1999). Most chemotherapeutic drugs do not provoke any decrement of antioxidants. Instead, they frequently induce an increase of antioxidants as a protecting mechanism against ROS generation, leading to lower cell death (Brea-Calvo et al., 2006). The fact that amitriptyline downregulates both catalase and CoQ activity is very interesting since it destroys the already decreased antioxidant defenses present in cancer cells, making the oxidative stress produced by the amitriptyline-induced ROS generation a more effective weapon.

Thus, amitriptyline promotes enhanced oxidative damage to cancer cells as this drug attacks cells by two different mechanisms: by the production of a high amount of ROS, provoking apoptosis; and by a significant decrease in antioxidant levels, seriously limiting cell reaction to oxidative stress. Therefore, amitriptyline could be used for anticancer oxidant therapy against tumors that present significant oxidative stress and/or low antioxidant defenses. For anticancer therapeutics on those tumors with a similar redox status than normal cells, a drug delivery vehicle should be used.

Fig. 3. Transmission electron microscopy showing damage and fewer mitochondria in H460 cells treated with 50 mmol/l of amitriptyline. Degenerating mitochondria (arrows) are observed in treated tumor cells. (A) Nontreated tumor cells. (B) Amitriptyline-treated tumor

In general, the increase of ROS production causes, as a response, an increase of the antioxidants activities. However, under the high input rate of ROS, enzyme inactivation prevails, which leads to the reduction of antioxidant enzymes activity and to the process of oxidative damage. Thus, tumor cells frequently possess very little antioxidative enzymes, such as catalase, SOD, and glutathione peroxidase, which are known to play a protective role against ROS in normal cells. We have observed in normal fibroblast treated with amitriptyline a decrease in protein expression of antioxidant enzymes (catalase and MnSOD) 16 h after the treatment, followed by restored levels after 24h, as a mechanism of antioxidant defense (Moreno-Fernández et al., 2008). Interestingly, in cancer cells, the same concentration of amitriptyline provoked an unrestorable decrease of catalase (Cordero et al., 2010). The difference of the antioxidant status observed in cancer cells, in comparison with healthy fibroblasts, may be caused by the lower antioxidant level present in the cancer cell lines used. Besides the decrease of catalase and MnSOD, amitriptyline also produces a significant decrease of CoQ level in tumor cells (Cordero et al., 2010). CoQ plays a critical protective role by either acting as an antioxidant or by the noncompetitive inhibition of the neutral sphingomyelinase of plasma membrane, preventing ceramide production (Mates et al., 1999). Most chemotherapeutic drugs do not provoke any decrement of antioxidants. Instead, they frequently induce an increase of antioxidants as a protecting mechanism against ROS generation, leading to lower cell death (Brea-Calvo et al., 2006). The fact that amitriptyline downregulates both catalase and CoQ activity is very interesting since it destroys the already decreased antioxidant defenses present in cancer cells, making the oxidative stress produced

by the amitriptyline-induced ROS generation a more effective weapon.

Thus, amitriptyline promotes enhanced oxidative damage to cancer cells as this drug attacks cells by two different mechanisms: by the production of a high amount of ROS, provoking apoptosis; and by a significant decrease in antioxidant levels, seriously limiting cell reaction to oxidative stress. Therefore, amitriptyline could be used for anticancer oxidant therapy against tumors that present significant oxidative stress and/or low antioxidant defenses. For anticancer therapeutics on those tumors with a similar redox status than normal cells, a drug

cells (Cordero et al., 2010).

delivery vehicle should be used.

Fig. 4. Mechanism of apoptosis induction by amitriptyline.

#### **6. Conclusion**

Evidence exists that cancer cells are under a continuous oxidative stress, facilitating ROS to act as carcinogen, as they were shown to be involved in mutagenesis, cancer promotion and progression. On the other hand, ROS may promote either cell proliferation or cell death, depending on the rate of ROS production and the activity of the antioxidant system. Accordingly, oxidative therapy against cancer arises as a ROS-generating strategy which increase oxidative status above the critical threshold required for cell survival.

In our studies, the tricyclic antidepressant amitriptyline induced high ROS generation as a result of mitochondrial dysfunction, provoking a higher level of apoptosis of tumor cells than common chemotherapeutic drugs. Moreover, it inhibits important antioxidants of the cell-defense machinery, dramatically limiting tumor cell response to ROS production. Thus, amitriptyline, as well as other tricyclic compounds, is being assayed as an anti-cancer drug in oxidative therapy.

Unfortunately, oxidative anti-cancer therapy may exert harmful effects on normal tissues, limiting the applicable dose of drugs and anti-tumor activity. So, differences in ROSinduced cell death and oxidative status between normal and neoplastic cells from different cancer types must be thoroughly investigated, taking into account the complexity of physiological and pathological pathways involved in redox balance. On the other hand, it is not strange that after ROS-generating therapies cancer cells achieve further resistance to oxidative stress. In consequence, combination therapies could be achieved in order to either

Oxidative Therapy Against Cancer 515

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**23** 

**Monensin Induced Oxidative** 

Kirsi Ketola1, Anu Vuoristo2, Matej Orešič2,

*2VTT Technical Research Centre of Finland, Espoo, 3Institute for Molecular Medicine, Finland (FIMM),* 

*1Medical Biotechnology, VTT Technical Research Centre of* 

Olli Kallioniemi3 and Kristiina Iljin1

*Finland and University of Turku, Turku,* 

*University of Helsinki,* 

 *Finland* 

**Stress Reduces Prostate Cancer Cell** 

 **Migration and Cancer Stem Cell Population**

Prostate cancer is the most common malignancy and second leading cause of cancer related death in males in developed countries (Jemal et al. 2011). Patients with localized and metastatic prostate cancer are treated with anti-androgens. Although prostate cancer cell proliferation is initially blocked or slowed down with anti-androgen therapy, eventually castration-resistant disease develops (Sharifi, Gulley & Dahut 2010). Therapeutic options for castration-resistant prostate cancer are limited and treatment responses to currently existing therapies are often unsatisfactory (Bracarda et al. 2011). For example, the cytotoxic therapy often causes severe toxicity and eventually leads also to the development of chemoresistance (Tannock et al. 2004, Berthold et al. 2008, Bracarda et al. 2011). Thus, there is an urgent need for novel agents to block the proliferation and to inhibit the progression of the primary prostate cancer cells to the advanced stage as well as to target advanced and metastatic prostate cancer. Therefore, understanding of disease progression and drug resistance mechanisms may provide valuable insights into the development of novel

We have recently performed a high-throughput cell-based screening of 4,910 known drugs and drug-like molecules in four prostate cancer cell models and two non-tumorigenic prostate epithelial cell lines to identify prostate cancer cell growth selective inhibitors (Iljin et al. 2009). Only four compounds, antibiotic ionophore monensin, aldehyde dehydrogenase (ALDH) inhibitor disulfiram, histone deacetylase inhibitor trichostatin A and fungicide thiram inhibited selectively cancer cell growth at nanomolar concentrations. The mechanistic studies indicated that monensin and disulfiram inhibited prostate cancer cell growth by inducing oxidative stress (Iljin et al. 2009, Ketola et al. 2010). In contrast to disulfiram, monensin induced apoptosis, reduced androgen receptor signalling and showed a synergistic anti-proliferative effect with anti-androgens in prostate cancer cells. Moreover,

treatment options to improve the survival of prostate cancer patients.

**1. Introduction** 


### **Monensin Induced Oxidative Stress Reduces Prostate Cancer Cell Migration and Cancer Stem Cell Population**

Kirsi Ketola1, Anu Vuoristo2, Matej Orešič2, Olli Kallioniemi3 and Kristiina Iljin1 *1Medical Biotechnology, VTT Technical Research Centre of Finland and University of Turku, Turku, 2VTT Technical Research Centre of Finland, Espoo, 3Institute for Molecular Medicine, Finland (FIMM), University of Helsinki, Finland* 

#### **1. Introduction**

520 Oxidative Stress and Diseases

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An update and review. *Biochimica et Biophysica Acta (BBA)–Bioenergetics.* 1757, (May)

Prostate cancer is the most common malignancy and second leading cause of cancer related death in males in developed countries (Jemal et al. 2011). Patients with localized and metastatic prostate cancer are treated with anti-androgens. Although prostate cancer cell proliferation is initially blocked or slowed down with anti-androgen therapy, eventually castration-resistant disease develops (Sharifi, Gulley & Dahut 2010). Therapeutic options for castration-resistant prostate cancer are limited and treatment responses to currently existing therapies are often unsatisfactory (Bracarda et al. 2011). For example, the cytotoxic therapy often causes severe toxicity and eventually leads also to the development of chemoresistance (Tannock et al. 2004, Berthold et al. 2008, Bracarda et al. 2011). Thus, there is an urgent need for novel agents to block the proliferation and to inhibit the progression of the primary prostate cancer cells to the advanced stage as well as to target advanced and metastatic prostate cancer. Therefore, understanding of disease progression and drug resistance mechanisms may provide valuable insights into the development of novel treatment options to improve the survival of prostate cancer patients.

We have recently performed a high-throughput cell-based screening of 4,910 known drugs and drug-like molecules in four prostate cancer cell models and two non-tumorigenic prostate epithelial cell lines to identify prostate cancer cell growth selective inhibitors (Iljin et al. 2009). Only four compounds, antibiotic ionophore monensin, aldehyde dehydrogenase (ALDH) inhibitor disulfiram, histone deacetylase inhibitor trichostatin A and fungicide thiram inhibited selectively cancer cell growth at nanomolar concentrations. The mechanistic studies indicated that monensin and disulfiram inhibited prostate cancer cell growth by inducing oxidative stress (Iljin et al. 2009, Ketola et al. 2010). In contrast to disulfiram, monensin induced apoptosis, reduced androgen receptor signalling and showed a synergistic anti-proliferative effect with anti-androgens in prostate cancer cells. Moreover,

Monensin Induced Oxidative Stress Reduces

cells (Iljin et al. 2009, Ketola et al., 2010).

**2. Materials and methods** 

**2.1 Cells** 

monensin on prostate cancer cell differentiation and motility.

2009, Ketola et al. 2010).

Prostate Cancer Cell Migration and Cancer Stem Cell Population

effect by inducing oxidative stress (Fang, Nakamura & Iyer 2007, Rigas, Sun 2008, Sun et al. 2011). The increased sensitivity to oxidative stress combined with dependency on antioxidative system may provide a way to selectivity inhibit cancer cell proliferation (Iljin et al.

Recently, redox control including antioxidative defence mechanisms and ROS-scavenging systems has been identified as an important regulator of cancer stem cell potential, metastasis and chemoresistance (Kobayashi, Suda 2011b, Cairns, Harris & Mak 2011, Pani, Galeotti & Chiarugi 2010). Aldehyde dehydrogenase activity is widely used as a marker for cancer stem cells and its activity has been shown to correlate with poor outcome in several cancers such as in prostate cancer (Davydov, Dobaeva & Bozhkov 2004, Burger et al. 2009, Li et al. 2010, Yu et al. 2011, Zhang et al. 2009a). Aldehyde dehydrogenases are detoxifying enzymes that are responsible for the oxidation of intracellular aldehydes (Duester 2000, Magni et al. 1996, Sophos, Vasiliou 2003, Yoshida et al. 1998). Moreover, ALDH oxidize retinol to retinoic acid (vitamin A) known to reduce oxidative stress whereas retinoic acid depletion induces oxidative stress and mitochondrial dysfunction (Ahlemeyer, Krieglstein 1998, Ahlemeyer et al. 2001, Chiu, Fischman & Hammerling 2008, Duester 2000, Chute et al. 2006). Thus, ALDH increases the antioxidative capacity in cells and protects cells from oxidative stress induction. Moreover, the inhibition of ALDH activity has recently been linked to reduced chemotherapy and radiation resistance in cancer stem cells (Croker, Allan 2011). These results suggest that not only cancer cells, but also cancer stem cells could be targeted by oxidative stress induction and/or reduction of antioxidative capacity. Interestingly, the results from mechanistic studies of prostate cancer selective compounds indicated that both disulfiram and monensin reduced the ALDH activity in prostate cancer

In this study, we explored further the molecular mechanism of monensin induced growth inhibition in cultured prostate cancer cells. Cancer pathway reporter assays and steroid profiling was performed to get insights into altered signalling and metabolite levels in monensin exposed prostate cancer cells. Since monensin reduces ALDH activity, the putative effect on cancer stem cells was evaluated. Furthermore, we studied the effect of

VCaP prostate carcinoma cells (TMPRSS2-ERG positive, received from Drs. Adrie van Bokhoven, University of Colorado Health Sciences Center, Denver, Colorado and Kenneth Pienta, University of Michigan, Michigan) were grown in Dulbecco's Modified Eagle's Medium (Korenchuk et al. 2001b, Korenchuk et al. 2001a). LNCaP prostate carcinoma cells (received from Dr. Marco Cecchini, University of Bern, Bern, Switzerland) were grown in T-Medium (Invitrogen). PC-3 prostate carcinoma cells were purchased from American Type Culture Collection (LGC Promochem AB) and grown according to provider's instructions. All cells were cultured in appropriate growth media described above including 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin

in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C.

523

monensin increased the amount of reactive oxygen species (ROS) and induced an oxidative stress signature in prostate cancer cells (VCaP and LNCaP), but not in the non-malignant prostate epithelial cells (RWPE-1, EP156T) (Ketola et al. 2010). Furthermore, antioxidant vitamin C partially rescued the monensin induced growth inhibition, indicating that oxidative stress plays a key role in the antineoplastic effect of monensin in cultured prostate cancer cells (Ketola et al. 2010).

Oxidative stress occurs in the cell when redox regulation is imbalanced. Redox balance depends on the level of intracellular free radicals and reactive oxygen species as well as on the antioxidative capacity of the cell. Figure 1 illustrates the connection between the malignant progression and increase in intracellular ROS (Fig. 1).

#### **Intracellular ROS**

Fig. 1. The balance between cell proliferation and intracellular reactive oxygen species (ROS). The malignant progression and the effects of ROS inducers and antioxidants depend on the intracellular levels of ROS. In cancer cells, intracellular ROS levels are higher than in normal cells making them more vulnerable to agents inducing ROS. Figure idea adapted from Gupte & Mumper 2009.

Cancer cells benefit from the increased mutation rate induced via oxidative stress (Shibutani, Takeshita & Grollman 1991). Therefore, cancer cells need also an active antioxidative mechanism to be able to survive under high oxidative stress. The oxidative stress level is elevated in prostate cancer cells compared to non-malignant prostate epithelial cells (Khandrika et al. 2009, Yossepowitch et al. 2007, Kumar et al. 2008). Many oncogenes are known to protect cells from oxidative stress e.g. androgen receptor (AR), ERG and MYC are known to have antioxidative properties in cancer cells (Pinthus et al. 2007, Tam et al. 2003, Benassi et al. 2006, Swanson et al. 2011, DeNicola et al. 2011). Monensin may sensitize prostate cancer cells to oxidative stress via reducing the expression of these genes (Ketola et al. 2010). In addition, many other anti-neoplastic agents such as vinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin, inostamycin, neocarzinostatin, etoposide, arsenic trioxide and nonsteroidal anti-inflammatory drugs are known to mediate their apoptotic effect by inducing oxidative stress (Fang, Nakamura & Iyer 2007, Rigas, Sun 2008, Sun et al. 2011). The increased sensitivity to oxidative stress combined with dependency on antioxidative system may provide a way to selectivity inhibit cancer cell proliferation (Iljin et al. 2009, Ketola et al. 2010).

Recently, redox control including antioxidative defence mechanisms and ROS-scavenging systems has been identified as an important regulator of cancer stem cell potential, metastasis and chemoresistance (Kobayashi, Suda 2011b, Cairns, Harris & Mak 2011, Pani, Galeotti & Chiarugi 2010). Aldehyde dehydrogenase activity is widely used as a marker for cancer stem cells and its activity has been shown to correlate with poor outcome in several cancers such as in prostate cancer (Davydov, Dobaeva & Bozhkov 2004, Burger et al. 2009, Li et al. 2010, Yu et al. 2011, Zhang et al. 2009a). Aldehyde dehydrogenases are detoxifying enzymes that are responsible for the oxidation of intracellular aldehydes (Duester 2000, Magni et al. 1996, Sophos, Vasiliou 2003, Yoshida et al. 1998). Moreover, ALDH oxidize retinol to retinoic acid (vitamin A) known to reduce oxidative stress whereas retinoic acid depletion induces oxidative stress and mitochondrial dysfunction (Ahlemeyer, Krieglstein 1998, Ahlemeyer et al. 2001, Chiu, Fischman & Hammerling 2008, Duester 2000, Chute et al. 2006). Thus, ALDH increases the antioxidative capacity in cells and protects cells from oxidative stress induction. Moreover, the inhibition of ALDH activity has recently been linked to reduced chemotherapy and radiation resistance in cancer stem cells (Croker, Allan 2011). These results suggest that not only cancer cells, but also cancer stem cells could be targeted by oxidative stress induction and/or reduction of antioxidative capacity. Interestingly, the results from mechanistic studies of prostate cancer selective compounds indicated that both disulfiram and monensin reduced the ALDH activity in prostate cancer cells (Iljin et al. 2009, Ketola et al., 2010).

In this study, we explored further the molecular mechanism of monensin induced growth inhibition in cultured prostate cancer cells. Cancer pathway reporter assays and steroid profiling was performed to get insights into altered signalling and metabolite levels in monensin exposed prostate cancer cells. Since monensin reduces ALDH activity, the putative effect on cancer stem cells was evaluated. Furthermore, we studied the effect of monensin on prostate cancer cell differentiation and motility.

#### **2. Materials and methods**

#### **2.1 Cells**

522 Oxidative Stress and Diseases

monensin increased the amount of reactive oxygen species (ROS) and induced an oxidative stress signature in prostate cancer cells (VCaP and LNCaP), but not in the non-malignant prostate epithelial cells (RWPE-1, EP156T) (Ketola et al. 2010). Furthermore, antioxidant vitamin C partially rescued the monensin induced growth inhibition, indicating that oxidative stress plays a key role in the antineoplastic effect of monensin in cultured prostate

Oxidative stress occurs in the cell when redox regulation is imbalanced. Redox balance depends on the level of intracellular free radicals and reactive oxygen species as well as on the antioxidative capacity of the cell. Figure 1 illustrates the connection between the

**Antioxidants**

**ROS** 

**inducers**

**Cancer cells**

**Intracellular ROS**

Fig. 1. The balance between cell proliferation and intracellular reactive oxygen species (ROS). The malignant progression and the effects of ROS inducers and antioxidants depend on the intracellular levels of ROS. In cancer cells, intracellular ROS levels are higher than in normal cells making them more vulnerable to agents inducing ROS. Figure idea adapted

Cancer cells benefit from the increased mutation rate induced via oxidative stress (Shibutani, Takeshita & Grollman 1991). Therefore, cancer cells need also an active antioxidative mechanism to be able to survive under high oxidative stress. The oxidative stress level is elevated in prostate cancer cells compared to non-malignant prostate epithelial cells (Khandrika et al. 2009, Yossepowitch et al. 2007, Kumar et al. 2008). Many oncogenes are known to protect cells from oxidative stress e.g. androgen receptor (AR), ERG and MYC are known to have antioxidative properties in cancer cells (Pinthus et al. 2007, Tam et al. 2003, Benassi et al. 2006, Swanson et al. 2011, DeNicola et al. 2011). Monensin may sensitize prostate cancer cells to oxidative stress via reducing the expression of these genes (Ketola et al. 2010). In addition, many other anti-neoplastic agents such as vinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin, inostamycin, neocarzinostatin, etoposide, arsenic trioxide and nonsteroidal anti-inflammatory drugs are known to mediate their apoptotic

malignant progression and increase in intracellular ROS (Fig. 1).

cancer cells (Ketola et al. 2010).

**Cell**

from Gupte & Mumper 2009.

**proliferation**

**Normal cells**

> VCaP prostate carcinoma cells (TMPRSS2-ERG positive, received from Drs. Adrie van Bokhoven, University of Colorado Health Sciences Center, Denver, Colorado and Kenneth Pienta, University of Michigan, Michigan) were grown in Dulbecco's Modified Eagle's Medium (Korenchuk et al. 2001b, Korenchuk et al. 2001a). LNCaP prostate carcinoma cells (received from Dr. Marco Cecchini, University of Bern, Bern, Switzerland) were grown in T-Medium (Invitrogen). PC-3 prostate carcinoma cells were purchased from American Type Culture Collection (LGC Promochem AB) and grown according to provider's instructions. All cells were cultured in appropriate growth media described above including 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C.

Monensin Induced Oxidative Stress Reduces

replicate samples were studied.

**2.8 Immuofluorescence staining** 

**2.9 Statistical analyses** 

**2.10 Steroid quantification** 

test (\*P<0.05, \*\*P<0.01, \*\*\* P<0.001).

Prostate Cancer Cell Migration and Cancer Stem Cell Population

**2.7 Fluorescence-Activated Cell Sorting analysis (FACS)** 

software (Intelligent Imaging Innovations Inc., CO, USA).


VCaP and LNCaP cells were exposed to monensin (1 µM) for 6 hours, samples were fixed with 2% paraformaldehyde, and stained with CD44 (FITC-conjugated mouse monoclonal anti-human, BD Pharmingen™ 555478) and CD24 (PE-conjugated rat monoclonal antihuman, Abcam ab25281) antibodies for 45 minutes at 4°C in the dark. Cells were washed

For immunofluorescence stainings, VCaP cells were grown on cover slip slides and exposed to monensin for 6 hours. Cells were fixed with 4 % paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS for 15 min, and blocked with 2 % BSA/PBS for 30 min. Cells were stained with E-cadherin antibody (1:100 dilution, polyclonal rabbit anti-human, Cell Signaling Technology, MA, USA) and Alexa-conjugated polyclonal donkey anti-rabbit antibody was used for secondary staining (1:300 dilution, Invitrogen, Molecular Probes, Carlsbad, CA). Cell nuclei were stained with DAPI present in Vectashield mounting medium (Vector Labs) and images were taken with Zeiss Axiovert 200M Microscope with the spinning disc confocal unit Yokogawa CSU22 and a Zeiss Plan-Neofluar 63× oil/1.4 NA objective. Z-stacks with 1 airy unit optical slices were acquired with a step size of 0.5 μm between slices, and the maximum intensity projections were created with SlideBook 4.2.0.7

Stars in the figures indicate the significance of the experiments calculated using Student's t-

VCaP cells (1x107 cells) were exposed to 1 µM monensin for 6 hours, harvested and counted. An internal standard (labeled C16:0) and chloroform:methanol (2:5) mixture were added, the samples were homogenized with Retsch system (5 min, 20 Hz), centrifuged and the supernatants were collected and evaporated. MOX (25 µl, TS-45950, Thermo Scientific, Helsinki, Finland) was added and the mixture was incubated at 45°C for 60 minutes. Next, 100 µl of MSTFA with 1% trimethylchlorosilane (Fluka, St. Louis, MO) was added and the mixture was incubated at 70°C for 60 minutes. Injection standard was added to the mixture before gas chromatography-mass spectrometry analysis (GC-MS, Agilent 6890 gas chromatograph (GC) combined with Agilent 5973 mass selective detector (MSD)). The injector (injection volume 1 µl with pulsed splitless injection) and MSD temperatures were 230°C (MS Source) and 150°C (MS Quad). The analyses were performed on Supelco 38499- 02C capillary column. Selective ion monitoring using specific masses for each target analyte was used in the detection. The following steroids were quantified: 7-ketocholesterol, aldosterone, progesterone, pregnenolone, estrone, 17B-estradiol, 4B-hydroxycholesterol,

and the fluorescence intensity was measured using Accuri C6 Flow Cytometer.

525

#### **2.2 Compounds**

Monensin was purchased from Sigma-Aldrich and diluted in ethanol.

#### **2.3 Cancer 10-pathway reporter array**

Cancer 10-pathway Reporter Luciferase Kit (Wnt (TCF/LEF), Notch (RBP-Jκ), p53/DNA damage, TGF-β (SMAD2/3/4), Cell cycle/pRb-E2F (E2F/DP1), NF-κB, Myc/Max, Hypoxia (HIF1A), MAPK/ERK (Elk-1/SRF) and MAPK/JNK (AP-1) was used to study the monensin modulated signalling (SABiosciences, Frederick, MD). The assay was performed according to manufacturer's instructions. In brief, inducible transcription factor responsive firefly luciferase reporters with constitutively expressing Renilla construct transcription factor reporters were plated onto 96-well plates with transfection reagent (siLentFect, Bio-Rad Laboratories), followed by addition of cells and incubation for 24 hours. A mixture of noninducible firefly luciferase reporter and constitutively expressing Renilla construct was used as the negative control. After 24 hours of transfection, monensin (100 nM) and control treatment were added onto the cells and plates were incubated for 18 hours. The Dual-LuciferaseReporter (DLR™) Assay System (Promega) was utilized in quantitation of reporters and results according to the manufactorer's instructions. The change in the activity of each signalling pathway was determined by comparing the normalized luciferase activity of the reporters in monensin or solvent exposed cells.

#### **2.4 Wound healing assay**

The effect of monensin (10 nM, 100 nM and 1 µM) on prostate cancer cell migration was studied using a scratch wound assay. PC-3 cells were plated on 96-well plates (Essen ImageLock, Essen Instruments, UK) and a wound was scratched with wound scratcher (Essen Instruments). Compounds and appropriate controls were added immediately after wound scratching and wound confluence was monitored with Incucyte Live-Cell Imaging System and software (Essen Instruments). Wound closure was calculated for every hour for 24 hours by comparing the mean relative wound density of three biological replicates in each experiment.

#### **2.5 Cell viability assay**

Cell viability was determined with CellTiter-Glo (CTG) cell viability assay (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, 2,000 cells per well were plated in 35 μl of their respective growth media and left to attach overnight. Monensin (100 nM) was added to the cells and incubated for 12 or 24 hours. CTG reagent was added and the signals were quantified using Envision Multilabel Plate Reader (Perkin-Elmer, Massachusetts, MA).

#### **2.6 RNA extraction and quantitative real-time PCR**

VCaP cells were exposed to monensin for 6 hours, total RNA was extracted and quantitative real-time PCR was done as previously described (Ketola et al. 2010). TaqMan gene expression probes and primers from the Universal Probe Library (Roche Diagnostics, Espoo, Finland) were used to study E-cadherin (5´-cccgggacaacgtttattac-3´ and 5´-gctggctcaagtcaaagtcc-3´) and -actin (5´-ccaaccgcgagaagatga -3´ and 5´-ccagaggcgtacagggatag -3´) mRNA expression. Three replicate samples were studied.

#### **2.7 Fluorescence-Activated Cell Sorting analysis (FACS)**

VCaP and LNCaP cells were exposed to monensin (1 µM) for 6 hours, samples were fixed with 2% paraformaldehyde, and stained with CD44 (FITC-conjugated mouse monoclonal anti-human, BD Pharmingen™ 555478) and CD24 (PE-conjugated rat monoclonal antihuman, Abcam ab25281) antibodies for 45 minutes at 4°C in the dark. Cells were washed and the fluorescence intensity was measured using Accuri C6 Flow Cytometer.

#### **2.8 Immuofluorescence staining**

524 Oxidative Stress and Diseases

Cancer 10-pathway Reporter Luciferase Kit (Wnt (TCF/LEF), Notch (RBP-Jκ), p53/DNA damage, TGF-β (SMAD2/3/4), Cell cycle/pRb-E2F (E2F/DP1), NF-κB, Myc/Max, Hypoxia (HIF1A), MAPK/ERK (Elk-1/SRF) and MAPK/JNK (AP-1) was used to study the monensin modulated signalling (SABiosciences, Frederick, MD). The assay was performed according to manufacturer's instructions. In brief, inducible transcription factor responsive firefly luciferase reporters with constitutively expressing Renilla construct transcription factor reporters were plated onto 96-well plates with transfection reagent (siLentFect, Bio-Rad Laboratories), followed by addition of cells and incubation for 24 hours. A mixture of noninducible firefly luciferase reporter and constitutively expressing Renilla construct was used as the negative control. After 24 hours of transfection, monensin (100 nM) and control treatment were added onto the cells and plates were incubated for 18 hours. The Dual-LuciferaseReporter (DLR™) Assay System (Promega) was utilized in quantitation of reporters and results according to the manufactorer's instructions. The change in the activity of each signalling pathway was determined by comparing the normalized luciferase activity

The effect of monensin (10 nM, 100 nM and 1 µM) on prostate cancer cell migration was studied using a scratch wound assay. PC-3 cells were plated on 96-well plates (Essen ImageLock, Essen Instruments, UK) and a wound was scratched with wound scratcher (Essen Instruments). Compounds and appropriate controls were added immediately after wound scratching and wound confluence was monitored with Incucyte Live-Cell Imaging System and software (Essen Instruments). Wound closure was calculated for every hour for 24 hours by comparing the mean relative wound density of three biological replicates in

Cell viability was determined with CellTiter-Glo (CTG) cell viability assay (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, 2,000 cells per well were plated in 35 μl of their respective growth media and left to attach overnight. Monensin (100 nM) was added to the cells and incubated for 12 or 24 hours. CTG reagent was added and the signals were quantified using Envision Multilabel Plate Reader (Perkin-Elmer,

VCaP cells were exposed to monensin for 6 hours, total RNA was extracted and quantitative real-time PCR was done as previously described (Ketola et al. 2010). TaqMan gene expression probes and primers from the Universal Probe Library (Roche Diagnostics, Espoo, Finland) were used to study E-cadherin (5´-cccgggacaacgtttattac-3´ and 5´-gctggctcaagtcaaagtcc-3´) and

Monensin was purchased from Sigma-Aldrich and diluted in ethanol.

**2.2 Compounds** 

**2.3 Cancer 10-pathway reporter array** 

of the reporters in monensin or solvent exposed cells.

**2.6 RNA extraction and quantitative real-time PCR** 

**2.4 Wound healing assay** 

each experiment.

**2.5 Cell viability assay** 

Massachusetts, MA).

For immunofluorescence stainings, VCaP cells were grown on cover slip slides and exposed to monensin for 6 hours. Cells were fixed with 4 % paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS for 15 min, and blocked with 2 % BSA/PBS for 30 min. Cells were stained with E-cadherin antibody (1:100 dilution, polyclonal rabbit anti-human, Cell Signaling Technology, MA, USA) and Alexa-conjugated polyclonal donkey anti-rabbit antibody was used for secondary staining (1:300 dilution, Invitrogen, Molecular Probes, Carlsbad, CA). Cell nuclei were stained with DAPI present in Vectashield mounting medium (Vector Labs) and images were taken with Zeiss Axiovert 200M Microscope with the spinning disc confocal unit Yokogawa CSU22 and a Zeiss Plan-Neofluar 63× oil/1.4 NA objective. Z-stacks with 1 airy unit optical slices were acquired with a step size of 0.5 μm between slices, and the maximum intensity projections were created with SlideBook 4.2.0.7 software (Intelligent Imaging Innovations Inc., CO, USA).

#### **2.9 Statistical analyses**

Stars in the figures indicate the significance of the experiments calculated using Student's ttest (\*P<0.05, \*\*P<0.01, \*\*\* P<0.001).

#### **2.10 Steroid quantification**

VCaP cells (1x107 cells) were exposed to 1 µM monensin for 6 hours, harvested and counted. An internal standard (labeled C16:0) and chloroform:methanol (2:5) mixture were added, the samples were homogenized with Retsch system (5 min, 20 Hz), centrifuged and the supernatants were collected and evaporated. MOX (25 µl, TS-45950, Thermo Scientific, Helsinki, Finland) was added and the mixture was incubated at 45°C for 60 minutes. Next, 100 µl of MSTFA with 1% trimethylchlorosilane (Fluka, St. Louis, MO) was added and the mixture was incubated at 70°C for 60 minutes. Injection standard was added to the mixture before gas chromatography-mass spectrometry analysis (GC-MS, Agilent 6890 gas chromatograph (GC) combined with Agilent 5973 mass selective detector (MSD)). The injector (injection volume 1 µl with pulsed splitless injection) and MSD temperatures were 230°C (MS Source) and 150°C (MS Quad). The analyses were performed on Supelco 38499- 02C capillary column. Selective ion monitoring using specific masses for each target analyte was used in the detection. The following steroids were quantified: 7-ketocholesterol, aldosterone, progesterone, pregnenolone, estrone, 17B-estradiol, 4B-hydroxycholesterol,

Monensin Induced Oxidative Stress Reduces

al. 2006, Koh et al. 2010).

cell cultures.

Prostate Cancer Cell Migration and Cancer Stem Cell Population

the overall functionality of the assay (van Bokhoven et al. 2003).

clearly the most active pathway (fold change FC > 300, compared to the negative control). In addition, TGF-ß (FC = 5), cell cycle (FC = 12), Myc/Max (FC = 7) and hypoxia (FC = 5) were active in VCaP cells. In LNCaP cells, MAPK/JNK was the most active pathway (FC = 30 compared to the negative control), followed by NF-κB (FC = 23), p53 (FC = 10), Myc/Max (FC = 7) and hypoxia (FC = 6). Interestingly, NF-κB pathway was 14 times more active in TMPRSS2-ERG fusion positive VCaP cells than in ERG negative LNCaP cells. These results are in agreement with previous data indicating that ERG induces NF-κB activity in prostate cells *in vitro* and *in vivo* (Wang et al. 2010). The results with p53 pathway are also in accordance with the previous literature since VCaP cells are known to have an inactivating mutation in p53 (Trp-248) whereas LNCaP cells express the active form, supporting further

Monensin exposure inhibited NF-κB activity in both VCaP (by 88%) and LNCaP (by 83%) cells. NF-κB is a transcription factor that regulates various cellular processes such as cellular antioxidant defence capacity (Gloire, Legrand-Poels & Piette 2006). These results suggest that monensin induced oxidative stress may result from reduced NF-κB signalling. In addition, monensin reduced TGF-ß (by 75%), cell cycle (by 85%) and Myc/Max (by 71%) activities in VCaP cells and p53 (by 94%), hypoxia (by 82%) and Myc/Max (by 90%) activities in LNCaP cells. The reduced Myc/Max signalling is supported by reduced MYC mRNA expression seen in monensin exposed VCaP and LNCaP cells (Ketola et al. 2010). Taken together, monensin reduced the activities of multiple signalling pathways such as NF-κB, TGF-ß, hypoxia and Myc/Max, all associated with cancer cell survival, oxidative stress, stem cell potential and metastasis (Jones, Pu & Kyprianou 2009, Blum et al. 2009, Mimeault, M. & Batra, S.K. 2011, Benassi et

**3.2 Monensin reduces the cancer stem cell population in prostate cancer cell cultures**  Monensin exposure reduced the activities of multiple pathways maintaining antioxidative capacity and promoting the growth and survival of cancer stem cells such as NF-κB, HIF1A, MYC and ALDH, in cultured prostate cancer cells. Therefore, the effect of monensin on prostate cancer stem cell population was studied. Prostate cancer stem cells can be identified by high expression of CD44 and low expression of CD24 antigens (Klarmann et al. 2009). Interestingly, CD44 cell surface glycoprotein has recently been shown to increase antioxidative capacity in cancer cells, indicating that cancer initiating cells could be targeted by impairing oxidative stress defence mechanisms (Ishimoto et al. 2011). Thus, we analyzed the effect of monensin exposure to the fraction of CD44+/CD24 cells in cultured prostate cancer cells. VCaP and LNCaP cells exposed to monensin (1 µM) for 6 hours were stained with CD44 and CD24 recognizing antibodies and the samples were analyzed using FACS. The results showed that monensin reduced the fraction of CD44+/CD24- cells in both VCaP (from 3 to 1.3%) and LNCaP (3.1 to 2.6%) cells (Fig. 3). In agreement with these results, genome-wide gene expression analysis indicated that monensin decreased CD44 (by 20%) and induced CD24 (by 30%) mRNAs compared to the levels in ethanol control at 6-hour time point (Ketola et al. 2010). Taken together, these results indicate that monensin reduces the fraction of cancer stem cells in prostate cancer

527

25-hydroxycholesterol, 5a,6a-epoxycholesterol (Mono-TMS), dihydrotestosterone and testosterone (the standards were from Steraloids, Newport, RI)).

#### **3. Results**

#### **3.1 Monensin reduces NF-κB pathway activity in prostate cancer cells**

Here, we studied the effect of monensin exposure on the activities of ten cancer signalling pathways using Cancer pathway Reporter Array in prostate cancer cells. Inducible transcription factor responsive firefly luciferase reporters were transfected to VCaP and LNCaP cells with constitutively active Renilla reporters and incubated for 24 hours. Monensin (100 nM) or solvent control was added onto the transfected cells for 18 hours followed by measure of luciferase activities. The results are presented in Fig. 2. Comparison of the basal pathway activities in VCaP prostate cancer cells indicated that NF-κB was

Fig. 2. Cancer pathway activities in VCaP and LNCaP prostate cancer cells. A) VCaP and B) LNCaP cells were exposed to ethanol or 100 nM monensin for 24 hours and pathway activities were measured as described in the text. Results with negative control measuring the background firefly luciferase activity (non-inducible firefly luciferase reporter and constitutively expressing Renilla construct) are indicated and used to determine the basal pathway activities in VCaP and LNCaP cells. The y-axis was set to 50 to allow direct comparison of relative luciferase units (RLU) in VCaP and LNCaP cells, although the basal NK-κB activity in VCaP cells extended RLU 324. Statistical significance of monensin induced changes are shown for the active pathways \*P<0.05, \*\*P<0.01, \*\*\*P<0.001.

25-hydroxycholesterol, 5a,6a-epoxycholesterol (Mono-TMS), dihydrotestosterone and

Here, we studied the effect of monensin exposure on the activities of ten cancer signalling pathways using Cancer pathway Reporter Array in prostate cancer cells. Inducible transcription factor responsive firefly luciferase reporters were transfected to VCaP and LNCaP cells with constitutively active Renilla reporters and incubated for 24 hours. Monensin (100 nM) or solvent control was added onto the transfected cells for 18 hours followed by measure of luciferase activities. The results are presented in Fig. 2. Comparison of the basal pathway activities in VCaP prostate cancer cells indicated that NF-κB was

**VCaP**

**cycle**

**\* \*\*\***

**cycle**

changes are shown for the active pathways \*P<0.05, \*\*P<0.01, \*\*\*P<0.001.

**LNCaP**

**\*\*\* \*\* \*\* \***

Fig. 2. Cancer pathway activities in VCaP and LNCaP prostate cancer cells. A) VCaP and B) LNCaP cells were exposed to ethanol or 100 nM monensin for 24 hours and pathway activities were measured as described in the text. Results with negative control measuring the background firefly luciferase activity (non-inducible firefly luciferase reporter and constitutively expressing Renilla construct) are indicated and used to determine the basal pathway activities in VCaP and LNCaP cells. The y-axis was set to 50 to allow direct comparison of relative luciferase units (RLU) in VCaP and LNCaP cells, although the basal NK-κB activity in VCaP cells extended RLU 324. Statistical significance of monensin induced

**NF-κB Myc**

**\*\*\***

**NF-κB Myc**

**/Max**

**/Max**

**\***

**Hypoxia MAPK /ERK**

**Hypoxia MAPK /ERK**

**MAPK /JNK**

**MAPK /JNK**

**Negative ctrl**

**Negative ctrl**

testosterone (the standards were from Steraloids, Newport, RI)).

**Wnt Notch p53 TGFß Cell**

**Wnt Notch p53 TGFß Cell**

**3.1 Monensin reduces NF-κB pathway activity in prostate cancer cells** 

**3. Results** 

**A**

**RLU**

**ctrl monensin**

**50**

**<sup>50</sup> ctrl**

**monensin**

**RLU**

**B**

clearly the most active pathway (fold change FC > 300, compared to the negative control). In addition, TGF-ß (FC = 5), cell cycle (FC = 12), Myc/Max (FC = 7) and hypoxia (FC = 5) were active in VCaP cells. In LNCaP cells, MAPK/JNK was the most active pathway (FC = 30 compared to the negative control), followed by NF-κB (FC = 23), p53 (FC = 10), Myc/Max (FC = 7) and hypoxia (FC = 6). Interestingly, NF-κB pathway was 14 times more active in TMPRSS2-ERG fusion positive VCaP cells than in ERG negative LNCaP cells. These results are in agreement with previous data indicating that ERG induces NF-κB activity in prostate cells *in vitro* and *in vivo* (Wang et al. 2010). The results with p53 pathway are also in accordance with the previous literature since VCaP cells are known to have an inactivating mutation in p53 (Trp-248) whereas LNCaP cells express the active form, supporting further the overall functionality of the assay (van Bokhoven et al. 2003).

Monensin exposure inhibited NF-κB activity in both VCaP (by 88%) and LNCaP (by 83%) cells. NF-κB is a transcription factor that regulates various cellular processes such as cellular antioxidant defence capacity (Gloire, Legrand-Poels & Piette 2006). These results suggest that monensin induced oxidative stress may result from reduced NF-κB signalling. In addition, monensin reduced TGF-ß (by 75%), cell cycle (by 85%) and Myc/Max (by 71%) activities in VCaP cells and p53 (by 94%), hypoxia (by 82%) and Myc/Max (by 90%) activities in LNCaP cells. The reduced Myc/Max signalling is supported by reduced MYC mRNA expression seen in monensin exposed VCaP and LNCaP cells (Ketola et al. 2010). Taken together, monensin reduced the activities of multiple signalling pathways such as NF-κB, TGF-ß, hypoxia and Myc/Max, all associated with cancer cell survival, oxidative stress, stem cell potential and metastasis (Jones, Pu & Kyprianou 2009, Blum et al. 2009, Mimeault, M. & Batra, S.K. 2011, Benassi et al. 2006, Koh et al. 2010).

#### **3.2 Monensin reduces the cancer stem cell population in prostate cancer cell cultures**

Monensin exposure reduced the activities of multiple pathways maintaining antioxidative capacity and promoting the growth and survival of cancer stem cells such as NF-κB, HIF1A, MYC and ALDH, in cultured prostate cancer cells. Therefore, the effect of monensin on prostate cancer stem cell population was studied. Prostate cancer stem cells can be identified by high expression of CD44 and low expression of CD24 antigens (Klarmann et al. 2009). Interestingly, CD44 cell surface glycoprotein has recently been shown to increase antioxidative capacity in cancer cells, indicating that cancer initiating cells could be targeted by impairing oxidative stress defence mechanisms (Ishimoto et al. 2011). Thus, we analyzed the effect of monensin exposure to the fraction of CD44+/CD24 cells in cultured prostate cancer cells. VCaP and LNCaP cells exposed to monensin (1 µM) for 6 hours were stained with CD44 and CD24 recognizing antibodies and the samples were analyzed using FACS. The results showed that monensin reduced the fraction of CD44+/CD24- cells in both VCaP (from 3 to 1.3%) and LNCaP (3.1 to 2.6%) cells (Fig. 3). In agreement with these results, genome-wide gene expression analysis indicated that monensin decreased CD44 (by 20%) and induced CD24 (by 30%) mRNAs compared to the levels in ethanol control at 6-hour time point (Ketola et al. 2010). Taken together, these results indicate that monensin reduces the fraction of cancer stem cells in prostate cancer cell cultures.

Monensin Induced Oxidative Stress Reduces

**ctrl monensin**

in VCaP cells.

Prostate Cancer Cell Migration and Cancer Stem Cell Population

**A B**

Fig. 4. Monensin induces E-cadherin expression in VCaP prostate cancer cells. A)

**3.4 Monensin reduces migration in cultured prostate cancer cells** 

comparison to control are shown at 12 and 24 hour time points in Fig. 5C.

**level of androgen precursor and antioxidative steroid** 

**3.5 Monensin increases oxidative stress inducing steroids as well as reduces the** 

Our previous results indicated that monensin induced oxidative stress and altered the expression of genes involved in cholesterol and steroid biosynthesis (Ketola et al. 2010). Moreover, monensin reduced androgen receptor (AR) signalling and showed synergistic growth inhibitory effects with anti-androgens in prostate cancer cells. To validate the monensin induced changes in cellular steroid levels, steroid profiling was performed in VCaP prostate cancer cells. Cells were exposed to ethanol or monensin (1 µM) for six hours and steroid profiles were studied using gas chromatocraphy - mass spectrometry (GC-MS). The results presented as a heat-map in Fig. 6 show that the most prominent changes in response to monensin exposure were the induction of 7-ketocholesterol and aldosterone

Immunofluorescence staining of E-cadherin (red) in response to 1 µM monensin or ethanol (ctrl) for 6-hour in VCaP cells. Nuclei are stained with DAPI (blue). B) Relative mRNA expression of E-cadherin in response to 1 µM monensin or ethanol (ctrl) exposure for 6-hour

Epithelial-to-mesenchymal transition is a perquisite for cancer cell migration (Baum, Settleman & Quinlan 2008). Moreover, in addition to the role in promoting cancer stem cell growth and survival, NF-κB and ALDH activities as well as high CD44 and low CD24 expressions are known to enhance prostate cancer cell migration (van den Hoogen et al. 2010, Klarmann et al. 2009). Since monensin induced cell differentiation, the effect of monensin exposure on prostate cancer cell migration was studied. VCaP and LNCaP cells do not migrate and therefore PC-3 prostate cancer cells were used as a model in migration assay. The results are presented in Fig. 5. Interestingly, already at 10 nM concentration, monensin significantly reduced cell migration. The anti-migratorial effect was stronger at higher concentrations (100 nM and 1 µM) (Fig. 5A). Thus, monensin was able to reduce cell migration at nanomolar concentrations in PC-3 cells although the same concentrations did not significantly decrease cell viability in these cells even at 24 hour time point (Fig. 5B). Pictures showing the decrease in wound closure in monensin exposed PC-3 cells in

529

\*

**ctrl monensin**

**1.0 0.8 0.6 0.4 0.2 0**

**Relative mRNA expression** **1.2 1.4 1.6 1.8**

Fig. 3. FACS analysis of CD44 and CD24 immunostained VCaP and LNCaP prostate cancer cells. Monensin reduces the fraction of CD44+/CD24- cells in cultured prostate cancer cells. VCaP and LNCaP cells were stained with CD44 and CD24 antibodies in response to six hour monensin or ethanol exposure. Representative images from one out of four replicates are shown.

#### **3.3 Monensin induces cell differentiation in prostate cancer cells**

Cancer stem cells have been hypothesized to arise intrinsically through oncogenic transformation of normal tissue stem or progenitor cells in the early stage of the tumorigenesis or through induction of epithelial-to-mesenchymal transition (EMT) at later stages (Chaffer, Weinberg 2011). The expression of E-cadherin is considered as a marker of epithelial differentiation and it is lost during EMT. Interestingly, ERG knock-down in VCaP cells has been shown to induce E-cadherin expression (Gupta et al. 2010). Moreover, our previous results indicated that monensin reduced ERG expression in VCaP cells (Ketola et al. 2010). Thus, to study whether monensin affects prostate cancer cell differentiation, E-cadherin expression was analysed in monensin exposed VCaP cells. The results from immunochemical staining and quantitative RT-PCR showed that monensin induced E-cadherin expression in VCaP cells (Fig. 4A and B), indicating that monensin promotes cell differentiation in cultured prostate cancer cells.

**VCaP ctrl VCaP monensin**

**CD44**

**101 102**

**CD24 CD24**

**CD24 CD24**

Fig. 3. FACS analysis of CD44 and CD24 immunostained VCaP and LNCaP prostate cancer cells. Monensin reduces the fraction of CD44+/CD24- cells in cultured prostate cancer cells. VCaP and LNCaP cells were stained with CD44 and CD24 antibodies in response to six hour monensin or ethanol exposure. Representative images from one out of four replicates are

Cancer stem cells have been hypothesized to arise intrinsically through oncogenic transformation of normal tissue stem or progenitor cells in the early stage of the tumorigenesis or through induction of epithelial-to-mesenchymal transition (EMT) at later stages (Chaffer, Weinberg 2011). The expression of E-cadherin is considered as a marker of epithelial differentiation and it is lost during EMT. Interestingly, ERG knock-down in VCaP cells has been shown to induce E-cadherin expression (Gupta et al. 2010). Moreover, our previous results indicated that monensin reduced ERG expression in VCaP cells (Ketola et al. 2010). Thus, to study whether monensin affects prostate cancer cell differentiation, E-cadherin expression was analysed in monensin exposed VCaP cells. The results from immunochemical staining and quantitative RT-PCR showed that monensin induced E-cadherin expression in VCaP cells (Fig. 4A and B), indicating that monensin promotes cell

**LNCaP ctrl LNCaP monensin**

**CD44**

**101 102**

**101 102 103 104 105 106 107**

**101 102 103 104 105 106 107**

**101 102 103 104 105 106 107**

**101 102 103 104 105 106 107**

differentiation in cultured prostate cancer cells.

**3.3 Monensin induces cell differentiation in prostate cancer cells** 

**CD44**

**CD44**

shown.

**101 102**

**101 102**

Fig. 4. Monensin induces E-cadherin expression in VCaP prostate cancer cells. A) Immunofluorescence staining of E-cadherin (red) in response to 1 µM monensin or ethanol (ctrl) for 6-hour in VCaP cells. Nuclei are stained with DAPI (blue). B) Relative mRNA expression of E-cadherin in response to 1 µM monensin or ethanol (ctrl) exposure for 6-hour in VCaP cells.

#### **3.4 Monensin reduces migration in cultured prostate cancer cells**

Epithelial-to-mesenchymal transition is a perquisite for cancer cell migration (Baum, Settleman & Quinlan 2008). Moreover, in addition to the role in promoting cancer stem cell growth and survival, NF-κB and ALDH activities as well as high CD44 and low CD24 expressions are known to enhance prostate cancer cell migration (van den Hoogen et al. 2010, Klarmann et al. 2009). Since monensin induced cell differentiation, the effect of monensin exposure on prostate cancer cell migration was studied. VCaP and LNCaP cells do not migrate and therefore PC-3 prostate cancer cells were used as a model in migration assay. The results are presented in Fig. 5. Interestingly, already at 10 nM concentration, monensin significantly reduced cell migration. The anti-migratorial effect was stronger at higher concentrations (100 nM and 1 µM) (Fig. 5A). Thus, monensin was able to reduce cell migration at nanomolar concentrations in PC-3 cells although the same concentrations did not significantly decrease cell viability in these cells even at 24 hour time point (Fig. 5B). Pictures showing the decrease in wound closure in monensin exposed PC-3 cells in comparison to control are shown at 12 and 24 hour time points in Fig. 5C.

#### **3.5 Monensin increases oxidative stress inducing steroids as well as reduces the level of androgen precursor and antioxidative steroid**

Our previous results indicated that monensin induced oxidative stress and altered the expression of genes involved in cholesterol and steroid biosynthesis (Ketola et al. 2010). Moreover, monensin reduced androgen receptor (AR) signalling and showed synergistic growth inhibitory effects with anti-androgens in prostate cancer cells. To validate the monensin induced changes in cellular steroid levels, steroid profiling was performed in VCaP prostate cancer cells. Cells were exposed to ethanol or monensin (1 µM) for six hours and steroid profiles were studied using gas chromatocraphy - mass spectrometry (GC-MS). The results presented as a heat-map in Fig. 6 show that the most prominent changes in response to monensin exposure were the induction of 7-ketocholesterol and aldosterone

Monensin Induced Oxidative Stress Reduces

progesterone and pregnenolone levels.

**4. Conclusion** 

Prostate Cancer Cell Migration and Cancer Stem Cell Population

levels as well as decrease in progesterone and pregnenolone levels. Aldosterone and 7 ketocholesterol are known oxidative stress inducers whereas progesterone has antioxidant properties (Leonarduzzi et al., 2006, Gramajo et al., 2010, Lee et al., 2009, Calo et al., 2010, Queisser et al., 2011, Ozacmak et al., 2009). Moreover, progesterone and pregnenolone are androgen precursors which have been suggested to play a major role in prostate cancer cell survival (Locke et al. 2008). Interestingly, progesterone has also been shown to promote mammary stem cell expansion (Joshi et al. 2010). The steroid profiling validates also the previous Connectivity Map results which indicated that monensin has opposite effects to progesterone and pregnenolone (Ketola et al. 2010). Taken together, monensin induced alterations in cellular steroid profile indicated that monensin induced oxidative stress results from increased aldosterone and 7-ketocholesterol levels as well as decreased

In this study, we explored the molecular consequences of monensin exposure in cultured prostate cancer cells. Our previous study indicated that monensin inhibited selectively prostate cancer cell viability at nanomolar concentrations by inducing oxidative stress. Cancer cells are constantly under pro-oxidative state (Szatrowski, Nathan 1991, Toyokuni et al. 1995). Long-term oxidative stress stimulates cell growth and proliferation, contributes to metastatic process and promotes cancer cell invasiveness and migration (Mori, Shibanuma & Nose 2004, Sung et al. 2006). Therefore, cancer cells need strong antioxidant mechanisms to survive and profit from these oxidative stress induced changes. Interfering redox balance has been suggested as a potential mean to selectively target cancer cells for example by increasing the cellular ROS level or reducing the expression of antioxidative enzymes (Pelicano, Carney & Huang 2004). Our results with monensin support this hypothesis.

Our previous Connectivity Map results indicated that monensin has agonistic effects to NF-κB inactivator and oxidative stress inducer niclosamide supporting monensin as a potent NF-κB inhibitor. Here, we showed that although monensin reduced the activities of several pathways known to play a role in tumourigenesis, the strongest reduction was seen in NF κB signalling. NF-κB activity promotes cell viability, tumorigenesis and metastasis as well as correlates with poor prognosis in prostate cancer patients (Blum et al. 2009, Sarkar et al. 2008). Importantly, NF-κB regulates the expression of genes responsible for antioxidant defence capacity and its inhibition induces oxidative stress as well as reduces tumourigenesis, metastasis and cancer stem cell potential (Gloire, Legrand-Poels & Piette 2006, Sarkar et al. 2008, Gluschnaider et al. 2010). NF-κB inhibitors are known to decrease AR signalling *in vitro* and reduce the growth of androgen deprivation-resistant prostate cancer xenografts *in vivo* (Jin et al. 2008, Zhang et al. 2009b). However, at present no specific NF-κB inhibitors have reached the stage of clinical trials for prostate cancer treatment (Mahon et al. 2011). Our results support NF-κB as the main mediator of monensin induced oxidative stress, which may also contribute to the reduced androgen signalling and

Recently, cancer stem cell targeting has raised a lot of interest as a prominent way to target cancer drug resistance and metastasic growth (Mimeault, Batra 2011, Clayton, Mousa 2011). In comparison to cancer cells, ROS levels in cancer stem cells are lower due to controlled

induction of apoptosis in monensin exposed prostate cancer cells.

531

Fig. 5. The effect of monensin exposure on PC-3 prostate cancer cell migration. A) Relative wound confluency in response to monensin (10 nM, 100 nM and 1 µM) or control (ethanol) was monitored for 24 hours. B) Results from cell viability assay in response to monensin exposure (100 nM) for 12 and 24 hours in PC-3 cells. C) Pictures of scratch-wounded wells in response to 100 nM monensin or ethanol exposure at 12- and 24-hour time points. The wound margin in the beginning of the experiment is coloured in dark grey.

Fig. 6. Steroid profiling heat-map of monensin exposed VCaP prostate cancer cells. The cells were exposed to monensin (1 µM) for 6 hours and the steroid profile was measured with gas chromatography-mass spectrometry (GC-MS). The levels of individual steroids in monensin exposed cells were compared to ethanol exposed samples (presented as fold change, red: induction, blue: reduction).

levels as well as decrease in progesterone and pregnenolone levels. Aldosterone and 7 ketocholesterol are known oxidative stress inducers whereas progesterone has antioxidant properties (Leonarduzzi et al., 2006, Gramajo et al., 2010, Lee et al., 2009, Calo et al., 2010, Queisser et al., 2011, Ozacmak et al., 2009). Moreover, progesterone and pregnenolone are androgen precursors which have been suggested to play a major role in prostate cancer cell survival (Locke et al. 2008). Interestingly, progesterone has also been shown to promote mammary stem cell expansion (Joshi et al. 2010). The steroid profiling validates also the previous Connectivity Map results which indicated that monensin has opposite effects to progesterone and pregnenolone (Ketola et al. 2010). Taken together, monensin induced alterations in cellular steroid profile indicated that monensin induced oxidative stress results from increased aldosterone and 7-ketocholesterol levels as well as decreased progesterone and pregnenolone levels.

#### **4. Conclusion**

530 Oxidative Stress and Diseases

**120 140**

**ctrl 12 h monensin 12 h ctrl 24 h monensin 24 h**

Fig. 5. The effect of monensin exposure on PC-3 prostate cancer cell migration. A) Relative wound confluency in response to monensin (10 nM, 100 nM and 1 µM) or control (ethanol) was monitored for 24 hours. B) Results from cell viability assay in response to monensin exposure (100 nM) for 12 and 24 hours in PC-3 cells. C) Pictures of scratch-wounded wells in response to 100 nM monensin or ethanol exposure at 12- and 24-hour time points. The

Fig. 6. Steroid profiling heat-map of monensin exposed VCaP prostate cancer cells. The cells were exposed to monensin (1 µM) for 6 hours and the steroid profile was measured with gas chromatography-mass spectrometry (GC-MS). The levels of individual steroids in monensin exposed cells were compared to ethanol exposed samples (presented as fold change, red:

wound margin in the beginning of the experiment is coloured in dark grey.

**Time (h) 0 4 8 12 16 20 24**

**Wound confluence (%)**

**C**

**ctrl**

induction, blue: reduction).

**monensin 10 nM monensin 100 nM monensin 1 µM**

**A B**

**12h 24h**

**12h 24h**

**100 nM**

**ctrl monensin**

**7-Ketocholesterol 17ß-Estradiol Aldosterone Testosterone**

**25-Hydroxycholesterol**

**Dihydrotestosterone 5a,6a-Epoxycholesterol 4B-Hydroxycholesterol**

**Pregnenolone Progesterone**

**Estrone**

In this study, we explored the molecular consequences of monensin exposure in cultured prostate cancer cells. Our previous study indicated that monensin inhibited selectively prostate cancer cell viability at nanomolar concentrations by inducing oxidative stress. Cancer cells are constantly under pro-oxidative state (Szatrowski, Nathan 1991, Toyokuni et al. 1995). Long-term oxidative stress stimulates cell growth and proliferation, contributes to metastatic process and promotes cancer cell invasiveness and migration (Mori, Shibanuma & Nose 2004, Sung et al. 2006). Therefore, cancer cells need strong antioxidant mechanisms to survive and profit from these oxidative stress induced changes. Interfering redox balance has been suggested as a potential mean to selectively target cancer cells for example by increasing the cellular ROS level or reducing the expression of antioxidative enzymes (Pelicano, Carney & Huang 2004). Our results with monensin support this hypothesis.

Our previous Connectivity Map results indicated that monensin has agonistic effects to NF-κB inactivator and oxidative stress inducer niclosamide supporting monensin as a potent NF-κB inhibitor. Here, we showed that although monensin reduced the activities of several pathways known to play a role in tumourigenesis, the strongest reduction was seen in NF κB signalling. NF-κB activity promotes cell viability, tumorigenesis and metastasis as well as correlates with poor prognosis in prostate cancer patients (Blum et al. 2009, Sarkar et al. 2008). Importantly, NF-κB regulates the expression of genes responsible for antioxidant defence capacity and its inhibition induces oxidative stress as well as reduces tumourigenesis, metastasis and cancer stem cell potential (Gloire, Legrand-Poels & Piette 2006, Sarkar et al. 2008, Gluschnaider et al. 2010). NF-κB inhibitors are known to decrease AR signalling *in vitro* and reduce the growth of androgen deprivation-resistant prostate cancer xenografts *in vivo* (Jin et al. 2008, Zhang et al. 2009b). However, at present no specific NF-κB inhibitors have reached the stage of clinical trials for prostate cancer treatment (Mahon et al. 2011). Our results support NF-κB as the main mediator of monensin induced oxidative stress, which may also contribute to the reduced androgen signalling and induction of apoptosis in monensin exposed prostate cancer cells.

Recently, cancer stem cell targeting has raised a lot of interest as a prominent way to target cancer drug resistance and metastasic growth (Mimeault, Batra 2011, Clayton, Mousa 2011). In comparison to cancer cells, ROS levels in cancer stem cells are lower due to controlled

Monensin Induced Oxidative Stress Reduces

7-ketocholesterol aldosterone

**Pro-oxidants**

**5. Acknowledgement** 

study.

**6. References** 

ROS

Prostate Cancer Cell Migration and Cancer Stem Cell Population

adapted from Cairns, R.A., Harris, I.S. & Mak, T.W. 2011.

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Fig. 7. Overview of monensin induced changes in prostate cancer cells. The figure idea

We thank Mika Hilvo, Tuulia Hyötyläinen, Anna-Liisa Ruskeepää (VTT Technical Research Centre of Finland, Espoo, Finland) for their contribution in steroid profiling part of this

Abidi, P., Zhang, H., Zaidi, S.M., Shen, W.J., Leers-Sucheta, S., Cortez, Y., Han, J. & Azhar, S.

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CD44+/CD24-

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**Antioxidative capacity**

AR signalling, MYC, ERG

redox balance system such as high ALDH and CD44 expression protecting cancer stem cells from oxidative stress (Kobayashi, Suda 2011a, Croker, Allan 2011, Ishimoto et al. 2011). Interestingly, NF-κB inhibition induces apoptosis in prostate cancer stem cells and thus NF-κB is considered as an attractive chemotherapeutic target also against cancer stem cells (Jin et al. 2008, Birnie et al. 2008). Since monensin reduced ALDH and NF-κB activities, we studied the fraction of CD44+/CD24- cells in prostate cancer cell cultures in response to monensin exposure. The results confirmed that monensin reduced the amount of prostate cancer stem cells. Moreover, monensin induced epithelial cell differentiation and reduced motility in cultured prostate cancer cells, suggesting that monensin inhibits prostate tumorigenesis by multiple ways. Cancer stem cell inhibitor and cell differentiation inducer salinomycin shares a similar structure as monensin, supporting the functional similarities between these two compounds (Gupta et al. 2009).

Steroidogenic enzymes as well as stem cell markers are induced in castration-resistant prostate cancer both *in vitro* and *in vivo* (Blum et al. 2009, Pfeiffer et al. 2011). Several studies have shown that steroidogenesis is inhibited by ROS (Tsai et al. 2003, Stocco, Wells & Clark 1993, Kodaman, Aten & Behrman 1994, Lee et al. 2009, Abidi et al. 2008). Our previous results indicated that monensin reduced androgen receptor signalling. Here, we showed that monensin increases the levels of oxidative stress inducing steroids, 7 ketocholesterol and aldosterone, and reduces androgen precursor and antioxidative steroid progesterone in cultured prostate cancer cells. Interestingly, 7-ketocholesterol is a ligand for aryl hydrocarbon receptor (AhR) and acts as AhR antagonist (Savouret et al. 2001). AhR expression is elevated in malignant prostate cells and its signalling is activated in prostate cancer stem cells (Blum et al. 2009, Gluschnaider et al. 2010). AhR pathway increases the expression of ALDH proteins and protects cells against oxidative stress and foreign chemicals (Lindros et al. 1998, Vrzal, Ulrichova & Dvorak 2004, Nebert et al. 2000, Kohle, Bock 2007). Interestingly, AhR binds to NF-κB, induces MYC activation and reduces E-cadherin expression in breast cancer cells (Kim et al. 2000, Dietrich, Kaina 2010). Moreover, AhR can form a complex with androgen receptor and protect prostate cancer cells during androgen ablation (Ohtake, Fujii-Kuriyama & Kato 2009, Gluschnaider et al. 2010). Thus, our results indicate that monensin induced oxidative stress is potentially transmitted via reduced AhR signalling. This hypothesis is further supported by our previous gene expression results indicating that although AhR itself was not altered, the expression of AhR target gene mRNAs were decreased in response to monensin exposure (Ketola et al. 2010).

Taken together, we hypothesize that monensin induced anti-neoplastic effects result mainly due to increase in oxidative stress. The overview figure 7 illustrates the various changes occurring in prostate cancer cells in response to monensin exposure. The cancer selectiveness could be explained by increased intracellular ROS due to reduced antioxidative capacity sensitizing prostate cancer cells to oxidative stress. Since normal prostate epithelial cells are not under intensive oxidative stress and therefore, are less dependent on the function of antioxidative genes, they are not as sensitive to monensin exposure as cancer cells. In conclusion, our results support the idea that impairing the redox control, which has a crucial role in cancer cells enabling survival in high intracellular ROS, is a potent way to target prostate cancer cells.

Fig. 7. Overview of monensin induced changes in prostate cancer cells. The figure idea adapted from Cairns, R.A., Harris, I.S. & Mak, T.W. 2011.

#### **5. Acknowledgement**

We thank Mika Hilvo, Tuulia Hyötyläinen, Anna-Liisa Ruskeepää (VTT Technical Research Centre of Finland, Espoo, Finland) for their contribution in steroid profiling part of this study.

#### **6. References**

532 Oxidative Stress and Diseases

redox balance system such as high ALDH and CD44 expression protecting cancer stem cells from oxidative stress (Kobayashi, Suda 2011a, Croker, Allan 2011, Ishimoto et al. 2011). Interestingly, NF-κB inhibition induces apoptosis in prostate cancer stem cells and thus NF-κB is considered as an attractive chemotherapeutic target also against cancer stem cells (Jin et al. 2008, Birnie et al. 2008). Since monensin reduced ALDH and NF-κB activities, we studied the fraction of CD44+/CD24- cells in prostate cancer cell cultures in response to monensin exposure. The results confirmed that monensin reduced the amount of prostate cancer stem cells. Moreover, monensin induced epithelial cell differentiation and reduced motility in cultured prostate cancer cells, suggesting that monensin inhibits prostate tumorigenesis by multiple ways. Cancer stem cell inhibitor and cell differentiation inducer salinomycin shares a similar structure as monensin, supporting the functional similarities

Steroidogenic enzymes as well as stem cell markers are induced in castration-resistant prostate cancer both *in vitro* and *in vivo* (Blum et al. 2009, Pfeiffer et al. 2011). Several studies have shown that steroidogenesis is inhibited by ROS (Tsai et al. 2003, Stocco, Wells & Clark 1993, Kodaman, Aten & Behrman 1994, Lee et al. 2009, Abidi et al. 2008). Our previous results indicated that monensin reduced androgen receptor signalling. Here, we showed that monensin increases the levels of oxidative stress inducing steroids, 7 ketocholesterol and aldosterone, and reduces androgen precursor and antioxidative steroid progesterone in cultured prostate cancer cells. Interestingly, 7-ketocholesterol is a ligand for aryl hydrocarbon receptor (AhR) and acts as AhR antagonist (Savouret et al. 2001). AhR expression is elevated in malignant prostate cells and its signalling is activated in prostate cancer stem cells (Blum et al. 2009, Gluschnaider et al. 2010). AhR pathway increases the expression of ALDH proteins and protects cells against oxidative stress and foreign chemicals (Lindros et al. 1998, Vrzal, Ulrichova & Dvorak 2004, Nebert et al. 2000, Kohle, Bock 2007). Interestingly, AhR binds to NF-κB, induces MYC activation and reduces E-cadherin expression in breast cancer cells (Kim et al. 2000, Dietrich, Kaina 2010). Moreover, AhR can form a complex with androgen receptor and protect prostate cancer cells during androgen ablation (Ohtake, Fujii-Kuriyama & Kato 2009, Gluschnaider et al. 2010). Thus, our results indicate that monensin induced oxidative stress is potentially transmitted via reduced AhR signalling. This hypothesis is further supported by our previous gene expression results indicating that although AhR itself was not altered, the expression of AhR target gene mRNAs were decreased in response to monensin exposure

Taken together, we hypothesize that monensin induced anti-neoplastic effects result mainly due to increase in oxidative stress. The overview figure 7 illustrates the various changes occurring in prostate cancer cells in response to monensin exposure. The cancer selectiveness could be explained by increased intracellular ROS due to reduced antioxidative capacity sensitizing prostate cancer cells to oxidative stress. Since normal prostate epithelial cells are not under intensive oxidative stress and therefore, are less dependent on the function of antioxidative genes, they are not as sensitive to monensin exposure as cancer cells. In conclusion, our results support the idea that impairing the redox control, which has a crucial role in cancer cells enabling survival in high intracellular ROS, is

between these two compounds (Gupta et al. 2009).

(Ketola et al. 2010).

a potent way to target prostate cancer cells.


Monensin Induced Oxidative Stress Reduces

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**Section 7** 

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