\*

#### **2.6 Statistical analysis**

Data are presented as mean ± SE. Differences between Wistar and SHR, young and old groups were analyzed using two-way analysis of variance (ANOVA) with the Newman-Keul's post-hoc test used for multiple comparisons among groups, considering P < 0.05 as statistically significant.

#### **3. Results**

Comparing to age-matched W rats, SBP of SHR was higher at all ages examined. The analysis of the time course of SBP showed that as early as at 40-day-old the SHR exhibited higher SBP values compared to age-matched W rats. At 4-month-old SBP increased more in comparison to the youngest rats and it remained elevated throughout the last stage studied. LVH significantly increased in SHR at 4, 11 and 19-month-old compared to age-matched W rats. Higher values were obtained at 11 and 19-month-old SHR when compared to younger SHR. An increase in LVH was also observed in W rats with aging (11 and 19-month-old) compared to younger rats (Table 1).


Table 1. Values of systolic blood pressure (SBP) and left ventricular hypertrophy (LVH) of SHR and Wistar rats of 40 days and 4, 11 and 19 months-old. \* P < 0.05 in SHR vs. Wistar; # P < 0.05 in SHR vs. 40-day-old; § P < 0.05 in Wistar vs.to 40-day-old.

Fig. 1 shows TBARS content in hearts from 4-, and 19-month-old SHR and Wistar rats. In hearts from SHR there was a significantly higher TBARS level of approximately 87% at 19 month-old compared to age-matched Wistar rats. No differences in TBARS with aging were observed in Wistar rats.

Nitrotyrosine levels from hearts of 4 and 19-month-old Wistar and SHR are depicted in Fig. 2. Immunoblotting assays showed a statistically significant increase of approximately 40 % in nitrotyrosine levels at 19-month-old SHR compared to age-matched Wistar rats. The oldest SHR and Wistar rats exhibited an increase of 200 and 120 %, respectively, in nitrotyrosine levels compared to their respective younger group.

Although there were no significant differences in NOX between SHR and Wistar hearts from young animals, an increase in aged rats (approximately 30% for Wistar and 60% for SHR) was obtained showing SHR the highest values (Fig. 3).

Similar O2–. production was obtained in hearts from Wistar rats and SHR at 4 months of age, whereas in older animals SHR showed a significantly higher O2 –. production (approximately 170%) in comparison with age matched Wistar rats (approximately 70%) (Fig. 4). Anyway, aged rats produced a higher O2–. amount that younger. The addition of the selective NOX inhibitor apocynin decreased O2–. production in hearts of aged SHR and Wistar rats. In 4 month-old SHR and Wistar rats O2–. production was lower in the presence of apocynin, but

Data are presented as mean ± SE. Differences between Wistar and SHR, young and old groups were analyzed using two-way analysis of variance (ANOVA) with the Newman-Keul's post-hoc test used for multiple comparisons among groups, considering P < 0.05 as

Comparing to age-matched W rats, SBP of SHR was higher at all ages examined. The analysis of the time course of SBP showed that as early as at 40-day-old the SHR exhibited higher SBP values compared to age-matched W rats. At 4-month-old SBP increased more in comparison to the youngest rats and it remained elevated throughout the last stage studied. LVH significantly increased in SHR at 4, 11 and 19-month-old compared to age-matched W rats. Higher values were obtained at 11 and 19-month-old SHR when compared to younger SHR. An increase in LVH was also observed in W rats with aging (11 and 19-month-old)

**40 days-old** 154 ± 5 \* 2.04 ± 0.11 115 ± 5 1.56 ± 0.15 **4 months-old** 187 ± 2 \*# 2.72 ± 0.17 \* 116 ± 3 2.05 ± 0.12 **11 months-old** 178 ± 1.5 \*# 3.18 ± 0.23 \*# 116 ± 3 2.56 ± 0.09 § **19 months-old** 191 ± 5 \*# 3.40 ± 0.26 \*# 107 ± 6 2.46 ± 0.04 § Table 1. Values of systolic blood pressure (SBP) and left ventricular hypertrophy (LVH) of SHR and Wistar rats of 40 days and 4, 11 and 19 months-old. \* P < 0.05 in SHR vs. Wistar; #

Fig. 1 shows TBARS content in hearts from 4-, and 19-month-old SHR and Wistar rats. In hearts from SHR there was a significantly higher TBARS level of approximately 87% at 19 month-old compared to age-matched Wistar rats. No differences in TBARS with aging were

Nitrotyrosine levels from hearts of 4 and 19-month-old Wistar and SHR are depicted in Fig. 2. Immunoblotting assays showed a statistically significant increase of approximately 40 % in nitrotyrosine levels at 19-month-old SHR compared to age-matched Wistar rats. The oldest SHR and Wistar rats exhibited an increase of 200 and 120 %, respectively, in

Although there were no significant differences in NOX between SHR and Wistar hearts from young animals, an increase in aged rats (approximately 30% for Wistar and 60% for

whereas in older animals SHR showed a significantly higher O2–. production (approximately 170%) in comparison with age matched Wistar rats (approximately 70%) (Fig. 4). Anyway,

inhibitor apocynin decreased O2–. production in hearts of aged SHR and Wistar rats. In 4 month-old SHR and Wistar rats O2–. production was lower in the presence of apocynin, but

–. production was obtained in hearts from Wistar rats and SHR at 4 months of age,

–. amount that younger. The addition of the selective NOX

P < 0.05 in SHR vs. 40-day-old; § P < 0.05 in Wistar vs.to 40-day-old.

nitrotyrosine levels compared to their respective younger group.

SHR) was obtained showing SHR the highest values (Fig. 3).

**SHR Wistar SBP (mmHg) LVH SBP (mmHg) LVH** 

**2.6 Statistical analysis** 

statistically significant.

compared to younger rats (Table 1).

observed in Wistar rats.

aged rats produced a higher O2

Similar O2

**3. Results** 

Fig. 1. TBARS content in nmol/mg protein, expressed in nmol/mg protein in hearts from SHR and Wistar rats at 4, and 19 months-old . \* P < 0.05 in SHR vs Wistar ; # P < 0.05 vs 4 months-old SHR.

Fig. 2. Nitrotyrosine content, expressed as percentage with respect to 4-month-old Wistar rats in hearts from SHR and Wistar rats at 4 and 19 months-old. \* P < 0.05 in SHR vs Wistar ; # P < 0.05 vs 4 months-old SHR; P < 0.05 vs 4-month-old Wistar rats.

Oxidative Damage in Cardiac Tissue from

above the background levels (Dikalov et al., 2007).

0

0

10

20

30

CAT (U/mg protein)

40

50

20

40

60

SOD (U/mg protein)

80

100

120

Normotensive and Spontaneously Hypertensive Rats: Effect of Ageing 147

the difference was not statistically significant. This may have been because the lucigenin method was unable to detect very small differences in O2–. levels that were only slightly

#

#

Fig. 5. Superoxide dismutase (SOD) activity, expressed as U/mg protein, in SHR and Wistar

**\***

4 m 19 m

Fig. 6. Catalase (CAT) activity, expressed as U/mg protein, in SHR and Wistar hearts of 4 and 19-month-old. \* P < 0.05 in SHR vs Wistar ; # P < 0.05 in 19- vs 4-month-old SHR.

4 m 19 m

hearts of 4 and 19-month-old. # P < 0.05 in 19- vs 4-month-old SHR.

60 Wistar SHR

 SHR Wistar

Fig. 3. NOX (NAD(P)H oxidase) activity, expressed as cpm/mg protein in hearts from SHR and Wistar rats at 4 and 19-month-old. \* P < 0.05 in SHR vs Wistar; # P < 0.05 in 19- vs 4 month-old SHR; P < 0.05 in 19- vs 4-month-old Wistar.

Fig. 4. Superoxide production, expressed as arbitrary units AU/mg/min, in hearts from SHR and Wistar rats at 4 and 19 months of age in the absence and presence of apocynin. \*P < 0.05 in SHR vs. Wistar rats, # P < 0.05 in 19- vs. 4-month-old SHR, § P< 0.05 in 19- vs. 4 month-old Wistar rats, ξ P < 0.05 in 19-month-old SHR and Wistar rats in the presence vs. absence of apocynin.

**\*** #

Fig. 3. NOX (NAD(P)H oxidase) activity, expressed as cpm/mg protein in hearts from SHR and Wistar rats at 4 and 19-month-old. \* P < 0.05 in SHR vs Wistar; # P < 0.05 in 19- vs 4-

4m 19m

Wistar + Apocynin **\*** #

Fig. 4. Superoxide production, expressed as arbitrary units AU/mg/min, in hearts from SHR and Wistar rats at 4 and 19 months of age in the absence and presence of apocynin. \*P < 0.05 in SHR vs. Wistar rats, # P < 0.05 in 19- vs. 4-month-old SHR, § P< 0.05 in 19- vs. 4 month-old Wistar rats, ξ P < 0.05 in 19-month-old SHR and Wistar rats in the presence vs.

4m 19m

§

P < 0.05 in 19- vs 4-month-old Wistar.

 Wistar SHR

SHR + Apocynin

 Wistar SHR

month-old SHR;

absence of apocynin.

0

50

100

150

200

Superoxide production (AU/mg/min)

250

300

350

0

5000

10000 15000

20000 25000

NOX activity (cpm/mg protein)

30000 35000

40000

the difference was not statistically significant. This may have been because the lucigenin method was unable to detect very small differences in O2–. levels that were only slightly above the background levels (Dikalov et al., 2007).

Fig. 5. Superoxide dismutase (SOD) activity, expressed as U/mg protein, in SHR and Wistar hearts of 4 and 19-month-old. # P < 0.05 in 19- vs 4-month-old SHR.

Fig. 6. Catalase (CAT) activity, expressed as U/mg protein, in SHR and Wistar hearts of 4 and 19-month-old. \* P < 0.05 in SHR vs Wistar ; # P < 0.05 in 19- vs 4-month-old SHR.

Oxidative Damage in Cardiac Tissue from

production.

Normotensive and Spontaneously Hypertensive Rats: Effect of Ageing 149

MDA (Cocco et al., 2005) or HNE (Judge et al., 2005). However, in the present study, in accordance with previously reported data (Muscari et al., 1990; Navarro-Arévalo et al., 1999; Cand & verdetti, 1989), we did not find any increase of TBARS in hearts from normotensive rats with aging. These results can be explained considering that the normal hearts have a reduced amount of substrate for the lipoperoxidation (Cand & Verdetti, 1989) or /and the end products of lipoperoxidation are readily metabolized (Muscari et al., 1990) or possess efficient antioxidant defence system . However, we detected an increase in TBARS content with aging in hearts from SHR, compared to age-matched Wistar rats. Moreover, 19-monthold SHR exhibited the highest hypertrophy index and level of lipid peroxidation suggesting that an increase of oxidative damage can be the consequence or the reason for the persistent elevated systolic blood pressure and/or increased cardiac hypertrophy in addition to aging. Nitric oxide (NO) plays pivotal roles in the maintenance of blood pressure and vascular tone (Loscalzo & Welch, 1995). Superoxide avidly reacts with NO and in the process produces highly reactive and cytotoxic products, like peroxynitrite (ONOO-). Peroxynitrite, in turn, reacts with and modifies various molecules, namely lipids, DNA, and proteins. For instance, peroxynitrite reacts with the tyrosine and cysteine residues in protein molecules to produce nitrotyrosine and nitrocysteine, leading to inactivation of important antioxidant enzymes, like SOD (Mac Millan-Crow & Cruthirds, 2001; Alvarez et al., 2004). In addition to these and other harmful biochemical reactions, the oxidation of NO by ROS inevitably results in functional NO deficiency, which can contribute to pathogenesis and maintenance of hypertension and its long-term consequences. In agreement with previous findings in the vasculature of hypertensive animals (Mc Intyre et al., 1999; Zalba et al., 2001), we detected a higher O2–. production in cardiac tissue of aged SHR compared to age-matched normotensive Wistar rats. The fact that blood pressure of SHR decreased with antioxidant therapy implies that oxidative stress is involved in the genesis and/or maintenance of hypertension (Vaziri et al., 2000). Recent investigations using hypertensive models other than SHR have shown that an increase of cellular tolerance to oxidative stress is one of the mechanisms responsible for the efficacy of anti hypertensive treatments such as calcium antagonists (Umemoto et al., 2004; Hirooka et al., 2006), angiotensin II type 1 receptor antagonists, or angiotensin-converting enzyme inhibitors (Takai et al., 2005; Tanaka et al., 2005). In our study, hearts from 4-month-old SHR and Wistar rats showed a similar nitrotyrosine content. In addition to lipid peroxidation data, this result is another demonstration that the higher LVH observed in young SHR relative to age-matched Wistar rats was not accompanied by higher nitrosative damage. Aged Wistar rats exhibited an increase in nitrotyrosilation compared with young animals. This increase was lower in Wistar in comparison to SHR, indicating that the addition of hypertrophy to aging process leads to a high degree of nitration due to an increased imbalance in myocardial production of either NO or O2–. . Although we did not measure the expression or activity of NOS, it has been reported that aged hearts exhibited increased myocardial NOS-cGMP signaling associated with an up-regulation of NOS (Zieman et al., 2001; Llorens et al., 2005). Therefore, higher levels of nitrotyrosine in aged SHR hearts would be attributed to an increase of peroxynitrite derived from an excessive production of both reactive species, NO and O2–.. Another possibility for explaining the higher oxidative and nitrosative stress of aged SHR compared to Wistar rats is a decrease in NO availability due to an increase in O2–.

The activities of antioxidant enzymes are shown in Fig. 5, 6 and 7. SOD activity significantly decreased in older hearts from SHR (approximately 17 %) while not significant differences were detected in Wistar rats with aging (Fig. 5).

Hearts from 4-month-old SHR exhibited a higher catalase activity (approximately 40%) in comparison to hearts from age-matched Wistar rats and it decreased in 19-month-old SHR. In Wistar rats CAT activity did not change with aging (Fig. 6).

Compared to younger animals, a significant decrease of GPx activity was detected in hearts from 19-month-old SHR and Wistar rats. No differences were detected between SHR and age-matched Wistar rats (Fig. 7).

Fig. 7. Glutathione peroxidase (GPx) activity, expressed as U/mg protein, in SHR and Wistar hearts of 4 and 19-month-old. # P < 0.05 in 19- vs 4-month-old SHR; P < 0.05 in 19 vs 4-month-old Wistar.

#### **4. Discussion**

The present study shows an increase of oxidative stress associated to ageing in both rat strains, showing SHR the highest values. Oxidative stress is a major contributor to the aging process (Fukagawa, 1999) and appears to be a common feature of hypertensive disorders from diverse origins (Ito et al., 1995; Dobrian et al., 2003; Vaziri & Sica, 2004; Swei et al., 1997). The damage caused by oxidative stress during aging becomes more evident when analyzing the effect of ROS on organic macromolecules, like proteins and lipids. Lipid peroxidation is a major contributor to the age-related loss of membrane fluidity, especially related to increase in the levels of two aldehydic lipid peroxidation products, malonyldialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE). Therefore, it is not surprising that lipid peroxidation is increased in the aged heart as demonstrated by higher levels of

The activities of antioxidant enzymes are shown in Fig. 5, 6 and 7. SOD activity significantly decreased in older hearts from SHR (approximately 17 %) while not significant differences

Hearts from 4-month-old SHR exhibited a higher catalase activity (approximately 40%) in comparison to hearts from age-matched Wistar rats and it decreased in 19-month-old SHR.

Compared to younger animals, a significant decrease of GPx activity was detected in hearts from 19-month-old SHR and Wistar rats. No differences were detected between SHR and

#

Fig. 7. Glutathione peroxidase (GPx) activity, expressed as U/mg protein, in SHR and

The present study shows an increase of oxidative stress associated to ageing in both rat strains, showing SHR the highest values. Oxidative stress is a major contributor to the aging process (Fukagawa, 1999) and appears to be a common feature of hypertensive disorders from diverse origins (Ito et al., 1995; Dobrian et al., 2003; Vaziri & Sica, 2004; Swei et al., 1997). The damage caused by oxidative stress during aging becomes more evident when analyzing the effect of ROS on organic macromolecules, like proteins and lipids. Lipid peroxidation is a major contributor to the age-related loss of membrane fluidity, especially related to increase in the levels of two aldehydic lipid peroxidation products, malonyldialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE). Therefore, it is not surprising that lipid peroxidation is increased in the aged heart as demonstrated by higher levels of

4 m 19 m

P < 0.05 in 19-

Wistar hearts of 4 and 19-month-old. # P < 0.05 in 19- vs 4-month-old SHR;

were detected in Wistar rats with aging (Fig. 5).

age-matched Wistar rats (Fig. 7).

vs 4-month-old Wistar.

0

50

100

150

GPx (U/mg protein)

200

250

300

**4. Discussion** 

In Wistar rats CAT activity did not change with aging (Fig. 6).

 Wistar SHR

MDA (Cocco et al., 2005) or HNE (Judge et al., 2005). However, in the present study, in accordance with previously reported data (Muscari et al., 1990; Navarro-Arévalo et al., 1999; Cand & verdetti, 1989), we did not find any increase of TBARS in hearts from normotensive rats with aging. These results can be explained considering that the normal hearts have a reduced amount of substrate for the lipoperoxidation (Cand & Verdetti, 1989) or /and the end products of lipoperoxidation are readily metabolized (Muscari et al., 1990) or possess efficient antioxidant defence system . However, we detected an increase in TBARS content with aging in hearts from SHR, compared to age-matched Wistar rats. Moreover, 19-monthold SHR exhibited the highest hypertrophy index and level of lipid peroxidation suggesting that an increase of oxidative damage can be the consequence or the reason for the persistent elevated systolic blood pressure and/or increased cardiac hypertrophy in addition to aging.

Nitric oxide (NO) plays pivotal roles in the maintenance of blood pressure and vascular tone (Loscalzo & Welch, 1995). Superoxide avidly reacts with NO and in the process produces highly reactive and cytotoxic products, like peroxynitrite (ONOO-). Peroxynitrite, in turn, reacts with and modifies various molecules, namely lipids, DNA, and proteins. For instance, peroxynitrite reacts with the tyrosine and cysteine residues in protein molecules to produce nitrotyrosine and nitrocysteine, leading to inactivation of important antioxidant enzymes, like SOD (Mac Millan-Crow & Cruthirds, 2001; Alvarez et al., 2004). In addition to these and other harmful biochemical reactions, the oxidation of NO by ROS inevitably results in functional NO deficiency, which can contribute to pathogenesis and maintenance of hypertension and its long-term consequences. In agreement with previous findings in the vasculature of hypertensive animals (Mc Intyre et al., 1999; Zalba et al., 2001), we detected a higher O2–. production in cardiac tissue of aged SHR compared to age-matched normotensive Wistar rats. The fact that blood pressure of SHR decreased with antioxidant therapy implies that oxidative stress is involved in the genesis and/or maintenance of hypertension (Vaziri et al., 2000). Recent investigations using hypertensive models other than SHR have shown that an increase of cellular tolerance to oxidative stress is one of the mechanisms responsible for the efficacy of anti hypertensive treatments such as calcium antagonists (Umemoto et al., 2004; Hirooka et al., 2006), angiotensin II type 1 receptor antagonists, or angiotensin-converting enzyme inhibitors (Takai et al., 2005; Tanaka et al., 2005). In our study, hearts from 4-month-old SHR and Wistar rats showed a similar nitrotyrosine content. In addition to lipid peroxidation data, this result is another demonstration that the higher LVH observed in young SHR relative to age-matched Wistar rats was not accompanied by higher nitrosative damage. Aged Wistar rats exhibited an increase in nitrotyrosilation compared with young animals. This increase was lower in Wistar in comparison to SHR, indicating that the addition of hypertrophy to aging process leads to a high degree of nitration due to an increased imbalance in myocardial production of either NO or O2–. . Although we did not measure the expression or activity of NOS, it has been reported that aged hearts exhibited increased myocardial NOS-cGMP signaling associated with an up-regulation of NOS (Zieman et al., 2001; Llorens et al., 2005). Therefore, higher levels of nitrotyrosine in aged SHR hearts would be attributed to an increase of peroxynitrite derived from an excessive production of both reactive species, NO and O2 –.. Another possibility for explaining the higher oxidative and nitrosative stress of aged SHR compared to Wistar rats is a decrease in NO availability due to an increase in O2–. production.

Oxidative Damage in Cardiac Tissue from

to NOX activation. The increase in O2

development in the SHR model.

**6. Acknowledgement** 

**7. References** 

6879.

**5. Conclusion** 

the increased O2

Normotensive and Spontaneously Hypertensive Rats: Effect of Ageing 151

These data are in concordance with those reported by Ito et al. (1995) and opposed to recent observations of Csonka et al. (2000). In addition, both rat strains of 19 months old showed similar antioxidant enzyme activity. Therefore, this fact could not explain the differences of oxidative damage detected between aged SHR and W rats. These differences could be attributed to a significantly higher NOX activity in aged than young SHR in accordance with

detected in young rats will be abnormal in cardiac tissue from aged SHR. In this regard, it is worth noting a previous report that an increase of SOD pharmacology potency by lecithinization is able to protect endothelial cells against alterations induced by ROS (Igarashi et al., 1992). Another explanation to the differences observed would be related to angiotensin II content, which appears involved in the genesis of oxidative stress in another tissue than heart in the SHR model (De Godoy & Rattan, 2006). This hypothesis was supported by the recent experiments performed in vascular tissue of stroke-prone SHR (Takai et al., 2005; Tanaka et al., 2005) in which the inhibition of angiotensin receptor or angiotensin-converting enzyme system produced a reduction of ROS production. Our results are also consistent with investigations showing that cardioprotective treatments are mediated by a restoration or up-regulation of antioxidant enzyme (Umemoto et al., 2004; Tanaka et al., 2005). Accumulating evidence has suggested that ROS are capable to activate directly intracellular cascades involved in the regulation of hypertrophic growth (Takano et al., 2003). It has been reported that Rho family proteins, specially Rac1, play critical roles in mechanical stress-induced hypertrophy responses and are involved in ROS-mediated activation of MAP kinases (such as p38, ERK1/2) and activation of nuclear factor-B. Moreover, Rac 1 is essential for assembly of plasma membrane NOX (Griendling et al., 2000). Thus, in our experimental conditions, sustained hemodynamic load in SHR would modulate the action of extracellular stimuli (such as angiotensin II, norepinephrine, tumor necrosis factor-, epidermal growth factor) on Rac1 activation leading

endogenous antioxidant system, activate redox-sensitive kinase cascades and transcription factors. These actions would produce an induction of immediate early genes, reexpression of fetal genes, increased mRNA content and protein synthesis thus leading to the increase in

This study shows that an increase in O2–. production in NOX dependent way and consequently higher oxidative damage appears associated to the aging process and to the increase in cardiac hypertrophy detected in hearts of SHR compared to age-matched Wistar rats. Thus, oxidative stress would be the cause and/or consequence of hypertrophy

This work was supported in part by the grant PICT 1046 from Agencia Nacional de

Aebi, H. (1984). Catalase in Vitro. *Methods in Enzymology,* Vol. 105, pp. 121-126, ISSN 0076-

myocyte cross-sectional area and fibrosis observed in aged SHR heart.

Promoción Científica y Técnica of Argentina to Dr Susana M Mosca.

–. production with aging, indicating that the compensatory mechanism

–. production by NOX would, in presence of a deficient

Mitochondria occupy a central position in the metabolism of ROS, supporting the so-called "free radical theory of aging" (Beckman & Ames, 1998; Hardman, 1956; Hardman, 1988). Other cardiovascular sources of ROS include the enzymes xanthine oxidoreductase (Berry & Hare, 2004), NOX (multisubunit membrane complexes) (Griedling et al., 2000) and eNOS uncoupling (Kuzkaya et al., 2003; Landmesser et al., 2003). This eNOS transformation takes place when its essential cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) is oxidized by ONOO- then a functional NOS is converted into a dysfunctional O2 –. generating enzyme that contributes to oxidative stress. Abnormal activation and expression of myocardial NOX have been suggested to be the mains sources of ROS in the hypertrophic and failing myocardium (Bendall et al., 2002; Li et al., 2002). A recent paper of Miyagawa et al. (2007) shows that the production of O2 –. by NOX in femoral arteries of SHR in comparison to WKY is enhanced, resulting in the inactivation of NO and impairment of endothelial modulations of vascular contractions. In our study, whereas young SHR showed a similar NOX activity as age-matched Wistar, an increase in the activity of this enzyme was detected in aged SHR, suggesting that NOX-dependent ROS production would be mediating both the hypertrophic response and aging. Apocynin is a well characterized inhibitor of NOX (Meyer & Schmitt, 2000). It acts by impeding the assembly of the p47-phox and p67-phox subunits within the membrane NOX complex (Meyer & Schmitt, 2000; Hamilton et al., 2001). Some of the effects of apocynin treatment are protection of the endothelium from the initiating events of atherosclerosis (Hamilton et al., 2001), a reduction of p22-phox mRNA expression and cardiac hypertrophy in aldosterone-infused rats (Park et al., 2004), and a prevention of hyperglycemia-induced intracellular ROS elevation and myocyte dysfunction (Privratsky et al., 2003). Aponycin has also been shown to reduce oxidative stress in stroke-prone spontaneously hypertensive rats, leading to the suppression of cardiac hypertrophy, inflammation and fibrosis (Yamamoto et al., 2006). Under our experimental conditions, apocynin blunted the O2 –. production in hearts from aged SHR and Wistar rats. Although a significant increase in NOX activity was only evident in aged SHR hearts, we suggest that NOX–dependent ROS production would mediate both the hypertrophic response and aging.

In the myocardium, as in other tissues, antioxidant enzymes protect cells by maintaining ROS at low levels, thus preventing oxidative damage to biological molecules. SOD rapidly converts O2 –. to H2O2, which is further degraded by CAT and GPx. The levels of the antioxidant enzymes are sensitive to the oxidative stress and increased or decreased levels have been reported in different pathologies in which an enhancement of ROS is cause or consequence of the disease (Navarro-Arévalo et al., 1999; Ulker et al., 2003). Our data show that SOD activity in hearts from young SHR was slightly but not significantly higher than Wistar rats. The lack of significant difference between SOD activities of hearts from both rat strains is in accordance with previous findings (Gómez-Amores et al., 2006; Wilson & Johnson, 2000; Robin et al., 2004). GPx activity was slightly but no significantly higher in hearts from young SHR compared to age-matched Wistar rats whereas CAT activity showed a significant increase. An opposite result has been recently demonstrated in thoracic aorta of SHR in which a CAT activity decreased and a concomitant increase of H2O2 were detected (Ulker et al., 2003). Although we did not have experimental evidence, the increase in CAT activity without GPx one changes detected in young SHR would indicate that CAT is acting as compensatory mechanism. This action could lead to a diminution of H2O2 amount in our preparations and could explain the similar TBARS and nitrotyrosine content obtained in young hearts from both rat strains. Aged Wistar rats did not exhibit any change in SOD and GPx activities. However, a significant diminution of antioxidant enzymes was evident in aged compared to younger SHR. These data are in concordance with those reported by Ito et al. (1995) and opposed to recent observations of Csonka et al. (2000). In addition, both rat strains of 19 months old showed similar antioxidant enzyme activity. Therefore, this fact could not explain the differences of oxidative damage detected between aged SHR and W rats. These differences could be attributed to a significantly higher NOX activity in aged than young SHR in accordance with the increased O2 –. production with aging, indicating that the compensatory mechanism detected in young rats will be abnormal in cardiac tissue from aged SHR. In this regard, it is worth noting a previous report that an increase of SOD pharmacology potency by lecithinization is able to protect endothelial cells against alterations induced by ROS (Igarashi et al., 1992). Another explanation to the differences observed would be related to angiotensin II content, which appears involved in the genesis of oxidative stress in another tissue than heart in the SHR model (De Godoy & Rattan, 2006). This hypothesis was supported by the recent experiments performed in vascular tissue of stroke-prone SHR (Takai et al., 2005; Tanaka et al., 2005) in which the inhibition of angiotensin receptor or angiotensin-converting enzyme system produced a reduction of ROS production. Our results are also consistent with investigations showing that cardioprotective treatments are mediated by a restoration or up-regulation of antioxidant enzyme (Umemoto et al., 2004; Tanaka et al., 2005). Accumulating evidence has suggested that ROS are capable to activate directly intracellular cascades involved in the regulation of hypertrophic growth (Takano et al., 2003). It has been reported that Rho family proteins, specially Rac1, play critical roles in mechanical stress-induced hypertrophy responses and are involved in ROS-mediated activation of MAP kinases (such as p38, ERK1/2) and activation of nuclear factor-B. Moreover, Rac 1 is essential for assembly of plasma membrane NOX (Griendling et al., 2000). Thus, in our experimental conditions, sustained hemodynamic load in SHR would modulate the action of extracellular stimuli (such as angiotensin II, norepinephrine, tumor necrosis factor-, epidermal growth factor) on Rac1 activation leading to NOX activation. The increase in O2 –. production by NOX would, in presence of a deficient endogenous antioxidant system, activate redox-sensitive kinase cascades and transcription factors. These actions would produce an induction of immediate early genes, reexpression of fetal genes, increased mRNA content and protein synthesis thus leading to the increase in myocyte cross-sectional area and fibrosis observed in aged SHR heart.

#### **5. Conclusion**

150 Oxidative Stress and Diseases

Mitochondria occupy a central position in the metabolism of ROS, supporting the so-called "free radical theory of aging" (Beckman & Ames, 1998; Hardman, 1956; Hardman, 1988). Other cardiovascular sources of ROS include the enzymes xanthine oxidoreductase (Berry & Hare, 2004), NOX (multisubunit membrane complexes) (Griedling et al., 2000) and eNOS uncoupling (Kuzkaya et al., 2003; Landmesser et al., 2003). This eNOS transformation takes place when its essential cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) is oxidized by

contributes to oxidative stress. Abnormal activation and expression of myocardial NOX have been suggested to be the mains sources of ROS in the hypertrophic and failing myocardium (Bendall et al., 2002; Li et al., 2002). A recent paper of Miyagawa et al. (2007) shows that the

resulting in the inactivation of NO and impairment of endothelial modulations of vascular contractions. In our study, whereas young SHR showed a similar NOX activity as age-matched Wistar, an increase in the activity of this enzyme was detected in aged SHR, suggesting that NOX-dependent ROS production would be mediating both the hypertrophic response and aging. Apocynin is a well characterized inhibitor of NOX (Meyer & Schmitt, 2000). It acts by impeding the assembly of the p47-phox and p67-phox subunits within the membrane NOX complex (Meyer & Schmitt, 2000; Hamilton et al., 2001). Some of the effects of apocynin treatment are protection of the endothelium from the initiating events of atherosclerosis (Hamilton et al., 2001), a reduction of p22-phox mRNA expression and cardiac hypertrophy in aldosterone-infused rats (Park et al., 2004), and a prevention of hyperglycemia-induced intracellular ROS elevation and myocyte dysfunction (Privratsky et al., 2003). Aponycin has also been shown to reduce oxidative stress in stroke-prone spontaneously hypertensive rats, leading to the suppression of cardiac hypertrophy, inflammation and fibrosis (Yamamoto et

from aged SHR and Wistar rats. Although a significant increase in NOX activity was only evident in aged SHR hearts, we suggest that NOX–dependent ROS production would mediate

In the myocardium, as in other tissues, antioxidant enzymes protect cells by maintaining ROS at low levels, thus preventing oxidative damage to biological molecules. SOD rapidly converts

–. to H2O2, which is further degraded by CAT and GPx. The levels of the antioxidant enzymes are sensitive to the oxidative stress and increased or decreased levels have been reported in different pathologies in which an enhancement of ROS is cause or consequence of the disease (Navarro-Arévalo et al., 1999; Ulker et al., 2003). Our data show that SOD activity in hearts from young SHR was slightly but not significantly higher than Wistar rats. The lack of significant difference between SOD activities of hearts from both rat strains is in accordance with previous findings (Gómez-Amores et al., 2006; Wilson & Johnson, 2000; Robin et al., 2004). GPx activity was slightly but no significantly higher in hearts from young SHR compared to age-matched Wistar rats whereas CAT activity showed a significant increase. An opposite result has been recently demonstrated in thoracic aorta of SHR in which a CAT activity decreased and a concomitant increase of H2O2 were detected (Ulker et al., 2003). Although we did not have experimental evidence, the increase in CAT activity without GPx one changes detected in young SHR would indicate that CAT is acting as compensatory mechanism. This action could lead to a diminution of H2O2 amount in our preparations and could explain the similar TBARS and nitrotyrosine content obtained in young hearts from both rat strains. Aged Wistar rats did not exhibit any change in SOD and GPx activities. However, a significant diminution of antioxidant enzymes was evident in aged compared to younger SHR.

–. by NOX in femoral arteries of SHR in comparison to WKY is enhanced,

–. generating enzyme that

–. production in hearts

ONOO- then a functional NOS is converted into a dysfunctional O2

al., 2006). Under our experimental conditions, apocynin blunted the O2

both the hypertrophic response and aging.

production of O2

O2

This study shows that an increase in O2–. production in NOX dependent way and consequently higher oxidative damage appears associated to the aging process and to the increase in cardiac hypertrophy detected in hearts of SHR compared to age-matched Wistar rats. Thus, oxidative stress would be the cause and/or consequence of hypertrophy development in the SHR model.

#### **6. Acknowledgement**

This work was supported in part by the grant PICT 1046 from Agencia Nacional de Promoción Científica y Técnica of Argentina to Dr Susana M Mosca.

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**8** 

*México* 

**Oxidative Stress and Mitochondrial** 

Sauri Hernández-Reséndiz, Mabel Buelna-Chontal,

Francisco Correa and Cecilia Zazueta

*Instituto Nacional de Cardiología, Ignacio Chávez,* 

*Departamento de Bioquímica,* 

 **Dysfunction in Cardiovascular Diseases** 

Reactive oxygen species (ROS) include a wide variety of molecules and free radicals derived from molecular oxygen. O2 being highly electrophilic can be reduced by one electron at a time producing relatively stable intermediates, such as superoxide anion (O2•), precursor of most ROS and a relevant mediator in many biological reactions. Dismutation of O2• produces hydrogen peroxide (H2O2), which, in turn, may be fully reduced to water or partially reduced to hydroxyl radical (•OH), one of the strongest oxidants in nature. The formation of •OH is catalyzed by reduced transition metals. On the other hand, superoxide anion is able to reduce transition metals and intensify in this way hydroxyl generation. In addition, O2• may react with other radicals, in particular, with nitric oxide (•NO) in a reaction controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is very powerful oxidant. The oxidants derived from •NO have been recently called reactive nitrogen species (RNS). Oxidative stress is an expression used to describe various deleterious processes resulting from an imbalance between the formation and elimination of ROS and/or RNS by antioxidant defenses. While small fluctuations in the steady-state concentration of these oxidants play an important role in intracellular signaling, uncontrolled increase in their concentration produces free radical-mediated chain reactions, which indiscriminately target proteins, lipids, polysaccharides, and DNA. Mitochondria are a major source of ROS, converting as much as 0.2-2% of molecular oxygen to superoxide as a by-product of the electron transfer activity (Wittenberg & Wittenberg, 1989; Alvárez et al., 2003). Other enzymatic sources of ROS include NADPH oxidases, located on the cell membrane of polymorphonuclear cells, macrophages, endothelial and other cells, and cytochrome P450-dependent oxygenases, along with the proteolytic conversion of xanthine

dehydrogenase to xanthine oxidase, which produces both O2• and H2O2.

Mitochondria's critical role in cardiomyocyte survival and death has become an exciting finding in the field of cardiac biology. Indeed, it is accepted that mitochondrial dysfunction plays a crucial role in the pathogenesis of multiple cardiac diseases, mainly due to the imbalance of the fine interplay between aerobic metabolism, calcium homeostasis, and ROS production. Reactive oxygen species generated in the mitochondria, unless adequately

**1. Introduction** 


### **Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases**

Sauri Hernández-Reséndiz, Mabel Buelna-Chontal, Francisco Correa and Cecilia Zazueta *Departamento de Bioquímica, Instituto Nacional de Cardiología, Ignacio Chávez, México* 

#### **1. Introduction**

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(2001). Oxidative stress in arterial hypertension. Role of NAD(P)H oxidase. *Hypertension, Vol.* 38, No. 6, (December 2001), pp. 1395–1399, ISSN 0194911X. Zieman, S.J.; Gerstenblith, G.; Lakatta, E.G.; Rosas, G.O.; Vandegaer, K.; Ricker, K.M. & Hare

J.M.(2001). Upregulation of the nitric oxide-cGMP pathway in aged myocardium: physiological response to l-arginine. *Circulation Research,* Vol. 88, No. 1, (January Reactive oxygen species (ROS) include a wide variety of molecules and free radicals derived from molecular oxygen. O2 being highly electrophilic can be reduced by one electron at a time producing relatively stable intermediates, such as superoxide anion (O2•), precursor of most ROS and a relevant mediator in many biological reactions. Dismutation of O2• produces hydrogen peroxide (H2O2), which, in turn, may be fully reduced to water or partially reduced to hydroxyl radical (•OH), one of the strongest oxidants in nature. The formation of •OH is catalyzed by reduced transition metals. On the other hand, superoxide anion is able to reduce transition metals and intensify in this way hydroxyl generation. In addition, O2• may react with other radicals, in particular, with nitric oxide (•NO) in a reaction controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is very powerful oxidant. The oxidants derived from •NO have been recently called reactive nitrogen species (RNS). Oxidative stress is an expression used to describe various deleterious processes resulting from an imbalance between the formation and elimination of ROS and/or RNS by antioxidant defenses. While small fluctuations in the steady-state concentration of these oxidants play an important role in intracellular signaling, uncontrolled increase in their concentration produces free radical-mediated chain reactions, which indiscriminately target proteins, lipids, polysaccharides, and DNA. Mitochondria are a major source of ROS, converting as much as 0.2-2% of molecular oxygen to superoxide as a by-product of the electron transfer activity (Wittenberg & Wittenberg, 1989; Alvárez et al., 2003). Other enzymatic sources of ROS include NADPH oxidases, located on the cell membrane of polymorphonuclear cells, macrophages, endothelial and other cells, and cytochrome P450-dependent oxygenases, along with the proteolytic conversion of xanthine dehydrogenase to xanthine oxidase, which produces both O2• and H2O2.

Mitochondria's critical role in cardiomyocyte survival and death has become an exciting finding in the field of cardiac biology. Indeed, it is accepted that mitochondrial dysfunction plays a crucial role in the pathogenesis of multiple cardiac diseases, mainly due to the imbalance of the fine interplay between aerobic metabolism, calcium homeostasis, and ROS production. Reactive oxygen species generated in the mitochondria, unless adequately

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 159

respiratory chain (State 4). As a result, the physiological steady state concentration of O2• formation increases. The formation of O2• may be further increased in the presence of certain inhibitors (for example rotenone, which inhibits Complex I, or antimycin an inhibitor of Complex III), which cause those carriers upstream from the site of inhibition to become fully reduced. In Complex I, the primary source of O2• appears to be one of the iron-sulfur clusters, whereas in Complex III, most of O2• results from ubisemiquinone auto-oxidation,

Table 1. Compartmental localization of the main mitochondrial sources of superoxide anion.

Although O2• production increases as the respiratory chain becomes more reduced, not all mitochondrial inhibitors have this effect. Most of the production of O2• by Complex III is actually inhibited if electron flow between the Rieske Fe–S protein and oxygen is blocked, for example by myxothiazol, cyanide or cytochrome *c* depletion (Turrens et al.*,* 1985). This inhibitory effect indicates that O2• must be produced as a result of the autoxidation of semiquinone (Q•), an intermediate produced in Complex III during the Q-cycle (Trumpower, 1990; Figure 2). Coenzyme Q is fully reduced and converted to ubiquinol (QH2) in the inner side of the mitochondrial membrane and then migrates to the outer side of the inner membrane carrying two protons that become part of the pool needed to sustain ADP phosphorylation. Once on the outer side of the membrane, one electron is transferred to cytochrome *c*1 (via the Rieske Fe–S protein), resulting in the formation of Q•. In a second cycle, a new QH2 transfers one of its electrons to the iron-sulphur protein (ISP) and then to cytochrome c1, whereas the second electron reduces cytochrome b566 and then cytochrome b540 (Turrens et al., 2003). This second electron reduces the Q• produced in the first cycle, yielding QH2. Despite the high efficiency of redox reactions in the Q cycle, some electrons

Another modulator of mitochondrial ROS production is the membrane potential (m), thus it has been reported that both, uncouplers and uncoupling proteins (UCPs), minimize ROS production by enhancing proton leak and providing a negative feedback loop for

Modified from Turrens, 2003.

leak and react with oxygen producing O2• (Figure 1).

both on the outer and inner sides of the inner mitochondrial membrane (Table 1).

neutralized, cause mitochondrial oxidative stress and, through reactions with polyunsaturated fatty acids, form lipid hydroperoxides and unsaturated aldehydes that propagate among cellular compartments and react with proteins and nucleic acids. In the myocardium, the oxidative stress cascade impairs several functions, like mitochondrial biogenesis, fatty acid metabolism, ionic homeostasis, and antioxidant defense mechanisms, leading to diminished cardiac energetic efficiency, altered bioenergetics, apoptosis and degradation. Besides the obvious relevance of mitochondria in energy production, new processes like mitochondrial fusion and fission are reported to be linked to ROS generation and now are included in the cast of key players in cardiac disease. In this chapter, we explore the mechanisms of mitochondrial dysfunction driven by ROS generation associated with the pathophysiology of cardiovascular diseases.

#### **2. Mitochondrial reactive oxygen species generation**

The standard reduction potential for the conversion of molecular oxygen to O2• is -0.160 V (Wood, 1987). Given the highly reducing intramitochondrial environment, various respiratory components, including complexes I and III of the mitochondrial respiratory chain (Paradies et al., 2001; Tompkins et al., 2006), flavoproteins (Prosser et al., 2011), ironsulfur clusters (Napoli et al., 2006), and ubisemiquinone (Wen & Garg, 2008) are thermodynamically capable to donate one electron to oxygen. Moreover, most steps in the respiratory chain involve single-electron reactions, further favoring the monovalent reduction of oxygen. On the other hand, the mitochondrion possesses various antioxidant defenses designed to eliminate both O2• and H2O2. As a result, the steady state concentrations of O2• and H2O2 have been estimated to be around 1 x 10-10 M and 5 x 10-9 M, respectively (Cadenas & Davies, 2000). Mitochondrial sources of O2• include several respiratory complexes and individual enzymes. Superoxide formation occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the inner mitochondrial membrane (Table 1, Figure 1). While superoxide anion generated in the matrix is mainly eliminated in that compartment, part of the O2• produced in the intermembrane space may be carried to the cytoplasm via voltage-dependent anion channels (Han et al., 2003). The relative contribution of every site to the overall O2• production varies from organ to organ and also depends on whether mitochondria are actively respiring (State 3) or if the respiratory chain is highly reduced (State 4) (Barja, 1999). Complex III appears to be responsible for most of the O2• produced in heart mitochondria (Turrens & Boveris, 1980; Turrens et al., 1982), although Complex I is thought to be the primary source of ROS in a variety of pathological scenarios ranging from ageing to Parkinson's disease (Betarbet et al., 2002; Sherer et al., 2003a). The rate of O2• formation by the respiratory chain is controlled primarily by mass action law, increasing both when electron flow slows down (the concentration of electron donors, R• is higher) and when the concentration of oxygen increases1 (Turrens et al., 1982).

$$\mathbf{d}[\mathbf{O}\_2]/\text{d} \mathbf{t} \triangleq k \, [\mathbf{O}\_2] \, [\mathbf{R}^\bullet] \tag{1}$$

The electron flow through the respiratory chain establishes an H+ gradient across the inner mitochondrial membrane, used to drive ATP synthesis through the ATP synthase complex (Complex V). In the absence of ADP, the movement of H+ through ATP synthase is ceased and the H+ gradient builds up causing slowdown of electron flow and reduction of the

neutralized, cause mitochondrial oxidative stress and, through reactions with polyunsaturated fatty acids, form lipid hydroperoxides and unsaturated aldehydes that propagate among cellular compartments and react with proteins and nucleic acids. In the myocardium, the oxidative stress cascade impairs several functions, like mitochondrial biogenesis, fatty acid metabolism, ionic homeostasis, and antioxidant defense mechanisms, leading to diminished cardiac energetic efficiency, altered bioenergetics, apoptosis and degradation. Besides the obvious relevance of mitochondria in energy production, new processes like mitochondrial fusion and fission are reported to be linked to ROS generation and now are included in the cast of key players in cardiac disease. In this chapter, we explore the mechanisms of mitochondrial dysfunction driven by ROS generation associated

The standard reduction potential for the conversion of molecular oxygen to O2• is -0.160 V (Wood, 1987). Given the highly reducing intramitochondrial environment, various respiratory components, including complexes I and III of the mitochondrial respiratory chain (Paradies et al., 2001; Tompkins et al., 2006), flavoproteins (Prosser et al., 2011), ironsulfur clusters (Napoli et al., 2006), and ubisemiquinone (Wen & Garg, 2008) are thermodynamically capable to donate one electron to oxygen. Moreover, most steps in the respiratory chain involve single-electron reactions, further favoring the monovalent reduction of oxygen. On the other hand, the mitochondrion possesses various antioxidant defenses designed to eliminate both O2• and H2O2. As a result, the steady state concentrations of O2• and H2O2 have been estimated to be around 1 x 10-10 M and 5 x 10-9 M, respectively (Cadenas & Davies, 2000). Mitochondrial sources of O2• include several respiratory complexes and individual enzymes. Superoxide formation occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the inner mitochondrial membrane (Table 1, Figure 1). While superoxide anion generated in the matrix is mainly eliminated in that compartment, part of the O2• produced in the intermembrane space may be carried to the cytoplasm via voltage-dependent anion channels (Han et al., 2003). The relative contribution of every site to the overall O2• production varies from organ to organ and also depends on whether mitochondria are actively respiring (State 3) or if the respiratory chain is highly reduced (State 4) (Barja, 1999). Complex III appears to be responsible for most of the O2• produced in heart mitochondria (Turrens & Boveris, 1980; Turrens et al., 1982), although Complex I is thought to be the primary source of ROS in a variety of pathological scenarios ranging from ageing to Parkinson's disease (Betarbet et al., 2002; Sherer et al., 2003a). The rate of O2• formation by the respiratory chain is controlled primarily by mass action law, increasing both when electron flow slows down (the concentration of electron donors, R• is higher) and when the concentration of oxygen

 d[O2]/d*t*= *k* [O2] [R•] (1) The electron flow through the respiratory chain establishes an H+ gradient across the inner mitochondrial membrane, used to drive ATP synthesis through the ATP synthase complex (Complex V). In the absence of ADP, the movement of H+ through ATP synthase is ceased and the H+ gradient builds up causing slowdown of electron flow and reduction of the

with the pathophysiology of cardiovascular diseases.

increases1 (Turrens et al., 1982).

**2. Mitochondrial reactive oxygen species generation** 

respiratory chain (State 4). As a result, the physiological steady state concentration of O2• formation increases. The formation of O2• may be further increased in the presence of certain inhibitors (for example rotenone, which inhibits Complex I, or antimycin an inhibitor of Complex III), which cause those carriers upstream from the site of inhibition to become fully reduced. In Complex I, the primary source of O2• appears to be one of the iron-sulfur clusters, whereas in Complex III, most of O2• results from ubisemiquinone auto-oxidation, both on the outer and inner sides of the inner mitochondrial membrane (Table 1).


Table 1. Compartmental localization of the main mitochondrial sources of superoxide anion. Modified from Turrens, 2003.

Although O2• production increases as the respiratory chain becomes more reduced, not all mitochondrial inhibitors have this effect. Most of the production of O2• by Complex III is actually inhibited if electron flow between the Rieske Fe–S protein and oxygen is blocked, for example by myxothiazol, cyanide or cytochrome *c* depletion (Turrens et al.*,* 1985). This inhibitory effect indicates that O2• must be produced as a result of the autoxidation of semiquinone (Q•), an intermediate produced in Complex III during the Q-cycle (Trumpower, 1990; Figure 2). Coenzyme Q is fully reduced and converted to ubiquinol (QH2) in the inner side of the mitochondrial membrane and then migrates to the outer side of the inner membrane carrying two protons that become part of the pool needed to sustain ADP phosphorylation. Once on the outer side of the membrane, one electron is transferred to cytochrome *c*1 (via the Rieske Fe–S protein), resulting in the formation of Q•. In a second cycle, a new QH2 transfers one of its electrons to the iron-sulphur protein (ISP) and then to cytochrome c1, whereas the second electron reduces cytochrome b566 and then cytochrome b540 (Turrens et al., 2003). This second electron reduces the Q• produced in the first cycle, yielding QH2. Despite the high efficiency of redox reactions in the Q cycle, some electrons leak and react with oxygen producing O2• (Figure 1).

Another modulator of mitochondrial ROS production is the membrane potential (m), thus it has been reported that both, uncouplers and uncoupling proteins (UCPs), minimize ROS production by enhancing proton leak and providing a negative feedback loop for

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 161

resulting from the convertion of arginine to citrulline, in a reaction catalyzed by a family of NADPH-dependent enzymes called nitric oxide synthases. It has been reported that the mitochondrial matrix contains a unique form of nitric oxide synthase (Alvarez et al*.,* 2003). Although its physiological role is still unclear, the formation of nitric oxide in mitochondria may have important pathological consequences, as it binds to the heme group of cytochrome oxidase, inhibiting respiration (Poderoso et al.*,* 1996). ROS are also produced within mitochondria at sites other than the inner mitochondrial membrane (Dowrakowski et al., 2008), by proteins such as monoamine oxidase (MAO) and p66Shc (reviewed in Di Lisa

Mammalian mitochondria possess a multi-leveled ROS defense network of enzymes and non-enzymatic antioxidants. Thus, constantly generated ROS, essential for normal cellular physiology and signaling process, are maintained at specific levels by intrinsic antioxidant defenses, avoiding oxidative stress. In healthy mitochondria, ROS contention is driven by manganese-dependent superoxide dismutase (MnSOD), gluthatione peroxidase (GPx), thioredoxin (TrxSH2) and thioredoxin reductase (TrxR), peroxiredoxin (Prx), and glutaredoxin (Grx), as well as water- and lipid-soluble antioxidants, i.e. vitamins C, αtocopherol (α-toc), reduced glutathione (GSH), and melatonin. It has been proposed that under certain circumstances, the mitochondrial respiratory chain can also contribute to

The tripeptide GSH is the main non-protein molecule, containing reactive thiol (-SH) groups, with scavenging properties, that provides an abundant source of reducing equivalents (Stowe & Camara, 2009). GSH reacts with hydroxyl radical (•OH), hypochlorous acid (HOCl),

thiyl radical (GS●), which potentially generates O2• among other ROS (Ježek & Hlavatá, 2005). Despite its exclusive synthesis in the cytosol, GSH is distributed in intracellular organelles, including the endoplasmic reticulum (RE), nucleus, and mitochondrion (Marí et al., 2009; Figure 2). GSH synthesis involves a two step reaction that requires ATP. Glutamate and cysteine are converted to γ-glutamyl-cysteine in a rate-limiting reaction driven by γglutamylcysteine synthetase. Then, γ-glutamylcysteine and glycine produce GSH by the action of the enzyme GSH synthetase (Figure 2). The first reaction is inhibited by GSH, a mechanism that regulates cellular GSH concentration (Marí et al., 2009). In the cytosol, GSH concentration is around 11 mM (Griffith & Meister, 1979; Mårtensson et al., 1990) and is transported into the mitochondrial matrix through a non-described high affinity carrier and a low affinity carrier, that could be the mitochondrial oxoglutarate carrier (Coll et al., 2003) or the dicarboxylate carrier (Lash et al., 2002). In mitochondria, GSH levels may fluctuate from 5 to 11 mM (Valko et al., 2007). Mitochondrial GSH plays a critical role in cell survival, as toxic cell death often correlates better with depletion of the mitochondrial GSH pool than with overall intracellular

The importance of UCPs in the control of mitochondrial ROS generation remains unclear: it is known that UCPs are inner membrane carriers that transfer protons across the mitochondrial inner membrane (MIM), by-passing ATPase (Stuart et al., 2001). A putative

●), carbon-centered radicals, and peroxynitrite anion (ONOO-

) producing

et al., 2009).

**3. Antioxidant systems in mitochondria** 

mitochondrial antioxidant defense.

**3.1 Non-enzymatic antioxidants** 

GSH depletion (Orrenius et al., 2007).

peroxyl radical (RO2

mitochondrial ROS production. A direct impact of ROS on the glutathionylation status of UCPs has been invoked to explain the activation of such proteins (Mailloux & Harper, 2011).

Fig. 1. Electron flow in the Q-cycle. Ubiquinone (Q) is reduced by electrons transferred from NADH or succinate producing ubiquinol (QH2). The reduced form undergoes a two-cycle reoxidation in which the semiquinone (Q•) is a stable intermediate that transfers electrons to oxygen via the Rieske iron-sulfur protein (ISP), cytochrome *c*1 (c1) and cytochrome *c* (c*)*. The terminal oxidase catalyzes the oxidation of reduced cytochrome c and the concomitant four-electron reduction of one O2 molecule producing water. Superoxide may be produced on both sides of the inner membrane via the autoxidation of Q, but the contribution of each pool has not yet been determined.

Special mention is worth about how Ca2+ modulates mitochondrial ROS generation. The primary role of this cation in mitochondria is the stimulation of oxidative phosphorylation by allosteric activation of pyruvate dehydrogenase, isocitrate dehydrogenase, ketoglutarate dehydrogenase, as well as other proteins of the phosphorylating machinery (Brooks et al., 2004). Thus, the stimulation of oxidative phosphorylation would enhance ROS production, as mitochondria are being forced to work faster and to consume more O2. Experimental observations are diverse, overall it appears that physiological [Ca2+]m has no direct effect on respiratory chain function or oxidation/reduction process, however pathological mitochondrial Ca2+ overload can lead to ROS increase. Possible mechanisms include Ca2+ stimulated increase of metabolic rate; Ca2+ stimulated nitric oxide production, which inhibits complex IV; Ca2+ induced cytochrome c dissociation that would inhibit the distal respiratory chain; Ca2+ induced cardiolipin peroxidation and Ca2+ induced mitochondrial permeability transition pore opening (Peng & Jou, 2010).

On the other hand, simultaneous formation of O2• and nitric oxide can produce peroxynitrite, a very strong oxidant and nitrating agent. Nitric oxide is a vasodilator resulting from the convertion of arginine to citrulline, in a reaction catalyzed by a family of NADPH-dependent enzymes called nitric oxide synthases. It has been reported that the mitochondrial matrix contains a unique form of nitric oxide synthase (Alvarez et al*.,* 2003). Although its physiological role is still unclear, the formation of nitric oxide in mitochondria may have important pathological consequences, as it binds to the heme group of cytochrome oxidase, inhibiting respiration (Poderoso et al.*,* 1996). ROS are also produced within mitochondria at sites other than the inner mitochondrial membrane (Dowrakowski et al., 2008), by proteins such as monoamine oxidase (MAO) and p66Shc (reviewed in Di Lisa et al., 2009).

#### **3. Antioxidant systems in mitochondria**

160 Oxidative Stress and Diseases

mitochondrial ROS production. A direct impact of ROS on the glutathionylation status of UCPs has been invoked to explain the activation of such proteins (Mailloux & Harper, 2011).

Fig. 1. Electron flow in the Q-cycle. Ubiquinone (Q) is reduced by electrons transferred from NADH or succinate producing ubiquinol (QH2). The reduced form undergoes a two-cycle reoxidation in which the semiquinone (Q•) is a stable intermediate that transfers electrons to oxygen via the Rieske iron-sulfur protein (ISP), cytochrome *c*1 (c1) and cytochrome *c* (c*)*. The terminal oxidase catalyzes the oxidation of reduced cytochrome c and the concomitant four-electron reduction of one O2 molecule producing water. Superoxide may be produced on both sides of the inner membrane via the autoxidation of Q, but the contribution of each

Special mention is worth about how Ca2+ modulates mitochondrial ROS generation. The primary role of this cation in mitochondria is the stimulation of oxidative phosphorylation by allosteric activation of pyruvate dehydrogenase, isocitrate dehydrogenase, ketoglutarate dehydrogenase, as well as other proteins of the phosphorylating machinery (Brooks et al., 2004). Thus, the stimulation of oxidative phosphorylation would enhance ROS production, as mitochondria are being forced to work faster and to consume more O2. Experimental observations are diverse, overall it appears that physiological [Ca2+]m has no direct effect on respiratory chain function or oxidation/reduction process, however pathological mitochondrial Ca2+ overload can lead to ROS increase. Possible mechanisms include Ca2+ stimulated increase of metabolic rate; Ca2+ stimulated nitric oxide production, which inhibits complex IV; Ca2+ induced cytochrome c dissociation that would inhibit the distal respiratory chain; Ca2+ induced cardiolipin peroxidation and Ca2+ induced

On the other hand, simultaneous formation of O2• and nitric oxide can produce peroxynitrite, a very strong oxidant and nitrating agent. Nitric oxide is a vasodilator

mitochondrial permeability transition pore opening (Peng & Jou, 2010).

pool has not yet been determined.

Mammalian mitochondria possess a multi-leveled ROS defense network of enzymes and non-enzymatic antioxidants. Thus, constantly generated ROS, essential for normal cellular physiology and signaling process, are maintained at specific levels by intrinsic antioxidant defenses, avoiding oxidative stress. In healthy mitochondria, ROS contention is driven by manganese-dependent superoxide dismutase (MnSOD), gluthatione peroxidase (GPx), thioredoxin (TrxSH2) and thioredoxin reductase (TrxR), peroxiredoxin (Prx), and glutaredoxin (Grx), as well as water- and lipid-soluble antioxidants, i.e. vitamins C, αtocopherol (α-toc), reduced glutathione (GSH), and melatonin. It has been proposed that under certain circumstances, the mitochondrial respiratory chain can also contribute to mitochondrial antioxidant defense.

#### **3.1 Non-enzymatic antioxidants**

The tripeptide GSH is the main non-protein molecule, containing reactive thiol (-SH) groups, with scavenging properties, that provides an abundant source of reducing equivalents (Stowe & Camara, 2009). GSH reacts with hydroxyl radical (•OH), hypochlorous acid (HOCl), peroxyl radical (RO2 ●), carbon-centered radicals, and peroxynitrite anion (ONOO- ) producing thiyl radical (GS●), which potentially generates O2• among other ROS (Ježek & Hlavatá, 2005). Despite its exclusive synthesis in the cytosol, GSH is distributed in intracellular organelles, including the endoplasmic reticulum (RE), nucleus, and mitochondrion (Marí et al., 2009; Figure 2). GSH synthesis involves a two step reaction that requires ATP. Glutamate and cysteine are converted to γ-glutamyl-cysteine in a rate-limiting reaction driven by γglutamylcysteine synthetase. Then, γ-glutamylcysteine and glycine produce GSH by the action of the enzyme GSH synthetase (Figure 2). The first reaction is inhibited by GSH, a mechanism that regulates cellular GSH concentration (Marí et al., 2009). In the cytosol, GSH concentration is around 11 mM (Griffith & Meister, 1979; Mårtensson et al., 1990) and is transported into the mitochondrial matrix through a non-described high affinity carrier and a low affinity carrier, that could be the mitochondrial oxoglutarate carrier (Coll et al., 2003) or the dicarboxylate carrier (Lash et al., 2002). In mitochondria, GSH levels may fluctuate from 5 to 11 mM (Valko et al., 2007). Mitochondrial GSH plays a critical role in cell survival, as toxic cell death often correlates better with depletion of the mitochondrial GSH pool than with overall intracellular GSH depletion (Orrenius et al., 2007).

The importance of UCPs in the control of mitochondrial ROS generation remains unclear: it is known that UCPs are inner membrane carriers that transfer protons across the mitochondrial inner membrane (MIM), by-passing ATPase (Stuart et al., 2001). A putative

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 163

(Rodríguez et al., 2004), helping in GSH recycling and maintaining a high GSH/GSSG ratio. Melatonin is synthesized and released to the circulation by the pineal gland, and its amphiphilic properties lead to its free access to all compartments in the cell, being concentrated especially in the nucleus and mitochondria (Escames et al., 2010). A direct role of melatonin in regulation of Complex I and IV activity and in other mitochondrial functions has been suggested. This effect, not shared by other antioxidants, would reflect redox interactions with the electron transfer chain complexes, stimulating electron flow, limiting electron leakage, and ROS generation (Escames et al., 2010). Interestingly, it has also been reported that melatonin protects mitochondria from oxidative damage by preventing

MnSOD converts superoxide anion (O2•) to hydrogen peroxide (H2O2) in the matrix side of the inner mitochondrial membrane (Liochev & Fridovich, 2010), while some O2• released into the intermembrane space is partially dismutated by copper-zinc containing superoxide dismutase (CuZnSOD) (Figure 3). Disruption of the MnSOD gene in mice has been

Fig. 3. Mitochondrial antioxidant network. Mitochondria are normally protected from oxidative damage by a network of mitochondrial antioxidant systems. See text for further details. α-toc: α-tocoferol; Cyt *c*3+ (reduced cytochrome *c*) ; CuZnSOD: copper-zinc superoxide dismutase; Gpx: glutathione peroxidase; GSH: reduced glutathione, GSSG: oxidized glutathione; L-Arg: L-arginine; mtNOS: mitochondrial nitric oxide synthase, NO: nitric oxide; Prx3-(SH)2: peroxiredoxine 3 reduced, Prx3-S2: peroxiredoxine 3 oxidized; ROS: reactive oxygen species, RNS: reactive nitrogen species; Trx2-(SH)2: tioredoxine 2 reduced,

cardiolipin oxidation (Paradies et al., 2010).

**3.2 Enzymatic antioxidants** 

Trx2-S2: tioredoxine oxidized.

Fig. 2. GSH synthesis and compartmentalization. GSH is synthesized by γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) in the cytoplasm and then redistributed to mitochondria, nucleus, and endoplasmic reticulum.

role in redox homeostasis control has been associated with their capacity to reduce mitochondrial ROS production by modulating Δm (Haines et al., 2010). In fact, UCPs reduce the damaging effects of ROS during cardiac ischemia/reperfusion injury (Barreiro et al., 2009). UCP1 isoform is involved in thermogenesis and expressed specifically in brown adipose tissue mitochondria, in which it confers a regulated proton leak across the inner membrane, whereas the physiologic functions of the other isoforms remain unclear. UCP2 and UCP3 are present in the heart and provide cardioprotection. In rat neonatal cardiomyocytes, UCP overexpression confers tolerance against oxidative stress by a mechanism related with calcium uptake (Sack, 2006b).

α-Tocopherol and melatonin scavenge lipid peroxyl radicals much faster than these radicals can react with adjacent fatty acid side-chains, so they are probably the most important inhibitors of the free-radical chain reaction of lipid peroxidation in animals (Maroz et al., 2009; Gavazza & Catalá, 2009). Aside from its various physiological functions, melatonin could acts as a scavenger of the particularly toxic •OH and carbonate radical, which is important because of their presumed role in mitochondrial damage (Srinivasan et al., 2009, Hardeland & Coto-Montes, 2010). Besides, melatonin could act as an indirect antioxidant promoting *de novo* synthesis of GSH, stimulating the activity of *γ*-glutamylcysteine synthetase and also through its effects on GPx, Grx, SOD, and CAT gene expression (Rodríguez et al., 2004), helping in GSH recycling and maintaining a high GSH/GSSG ratio. Melatonin is synthesized and released to the circulation by the pineal gland, and its amphiphilic properties lead to its free access to all compartments in the cell, being concentrated especially in the nucleus and mitochondria (Escames et al., 2010). A direct role of melatonin in regulation of Complex I and IV activity and in other mitochondrial functions has been suggested. This effect, not shared by other antioxidants, would reflect redox interactions with the electron transfer chain complexes, stimulating electron flow, limiting electron leakage, and ROS generation (Escames et al., 2010). Interestingly, it has also been reported that melatonin protects mitochondria from oxidative damage by preventing cardiolipin oxidation (Paradies et al., 2010).

#### **3.2 Enzymatic antioxidants**

162 Oxidative Stress and Diseases

Fig. 2. GSH synthesis and compartmentalization. GSH is synthesized by γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) in the cytoplasm and then redistributed

role in redox homeostasis control has been associated with their capacity to reduce mitochondrial ROS production by modulating Δm (Haines et al., 2010). In fact, UCPs reduce the damaging effects of ROS during cardiac ischemia/reperfusion injury (Barreiro et al., 2009). UCP1 isoform is involved in thermogenesis and expressed specifically in brown adipose tissue mitochondria, in which it confers a regulated proton leak across the inner membrane, whereas the physiologic functions of the other isoforms remain unclear. UCP2 and UCP3 are present in the heart and provide cardioprotection. In rat neonatal cardiomyocytes, UCP overexpression confers tolerance against oxidative stress by a

α-Tocopherol and melatonin scavenge lipid peroxyl radicals much faster than these radicals can react with adjacent fatty acid side-chains, so they are probably the most important inhibitors of the free-radical chain reaction of lipid peroxidation in animals (Maroz et al., 2009; Gavazza & Catalá, 2009). Aside from its various physiological functions, melatonin could acts as a scavenger of the particularly toxic •OH and carbonate radical, which is important because of their presumed role in mitochondrial damage (Srinivasan et al., 2009, Hardeland & Coto-Montes, 2010). Besides, melatonin could act as an indirect antioxidant promoting *de novo* synthesis of GSH, stimulating the activity of *γ*-glutamylcysteine synthetase and also through its effects on GPx, Grx, SOD, and CAT gene expression

to mitochondria, nucleus, and endoplasmic reticulum.

mechanism related with calcium uptake (Sack, 2006b).

MnSOD converts superoxide anion (O2•) to hydrogen peroxide (H2O2) in the matrix side of the inner mitochondrial membrane (Liochev & Fridovich, 2010), while some O2• released into the intermembrane space is partially dismutated by copper-zinc containing superoxide dismutase (CuZnSOD) (Figure 3). Disruption of the MnSOD gene in mice has been

Fig. 3. Mitochondrial antioxidant network. Mitochondria are normally protected from oxidative damage by a network of mitochondrial antioxidant systems. See text for further details. α-toc: α-tocoferol; Cyt *c*3+ (reduced cytochrome *c*) ; CuZnSOD: copper-zinc superoxide dismutase; Gpx: glutathione peroxidase; GSH: reduced glutathione, GSSG: oxidized glutathione; L-Arg: L-arginine; mtNOS: mitochondrial nitric oxide synthase, NO: nitric oxide; Prx3-(SH)2: peroxiredoxine 3 reduced, Prx3-S2: peroxiredoxine 3 oxidized; ROS: reactive oxygen species, RNS: reactive nitrogen species; Trx2-(SH)2: tioredoxine 2 reduced, Trx2-S2: tioredoxine oxidized.

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 165

respiratory chain or superoxide. A diminution in its concentration may inhibit the distal respiratory chain and increase ROS production. Indeed, it has been reported that upon cytochrome *c* release during apoptosis initiation, mitochondrial ROS production increases

As mentioned before, ubiquinone (Q) acts as a pro-oxidant in its semiquinone form, however when fully reduced it acts as an antioxidant. Ubiquinol (UQH2) contains phenolic hydrogen atoms that can be donated to a carbon- or oxygen centered radical, converting it to a non-radical molecule (Ježek & Hlavatá, 2005). Lipid peroxidation prevention by UQH2 has been reported (James et al., 2004). Preferential succinate oxidation can provide a tool against excessive accumulation of ROS by increasing the proportion of fully reduced ubiquinone. The antioxidant activity of UQH2 is independent from the effect of α-tocopherol (Ernster et al., 1992), which acts as a chain-breaking antioxidant, inhibiting the propagation of lipid peroxidation (Maroz et al., 2009). Even more, UQH2 can efficiently sustain the effect of αtocopherol by regenerating it from the tocopheroxyl radical, which otherwise must rely on water-soluble agents such as ascorbate (vitamin C) (Ernster & Forsmark-Andree, 1993).

The pathological role of increased mitochondrial ROS in heart disease has been established from studies in gene-modified mice with altered mitochondrial antioxidant levels. Deletion of mitochondrial thioredoxin reductase 2 is embryonically lethal in mice because of impaired hematopoiesis and impaired cardiac function (Conrad et al., 2004). Fatal dilated cardiomyopathy in mice is developed after complete deletion of mitochondrial Mn superoxide dismutase (Li et al., 1995). In contrast, attenuated left ventricular remodeling after myocardial infarct was observed in transgenic mice overexpressing mitochondrial peroxiredoxin III (Matsushima et al., 2006) or glutathione peroxidase (Shiomi et al., 2004). Also, mice with a mitochondrial-targeted overexpression of catalase have a prolonged life

 A critical role of intracellular Ca2+ overload and oxidative stress in the genesis of myocyte dysfunction is well established in cardiovascular diseases like atherosclerosis, hypertension, ischemia/reperfusion damage, cardiac hypertrophy, and heart failure (HF). In general, Ca2+ overload can be induced by direct effect of ROS on Ca2+ handling proteins or indirectly, by inducing membrane lipid peroxidation (Santos et al., 2011). Recent evidence suggests that redox modification of ryanodine receptor (RyR2) may contribute to abnormal Ca2+ handling in disease states. RyR2 dysfunction with an increase in diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) may reduce calcium transients and contribute to a reduced contractile force in the failing heart, as well as an increased likelihood of arrhythmia (González et al., 2010). In humans, increased cytosolic calcium ([Ca2+]c) has been related with augmented oxidative stress in atherosclerosis. A growing body of evidence indicates that the production of ROS is tightly linked with Angiotensin II-induced action. In this respect, a causative link between superoxide production and hypertension has been established in experiments in which SOD reduced blood pressure by 50 mm Hg in vascular smooth muscle cells (VSCM) from Angiotensin II-infused rats (Laursen et al., 1997). Much less attention has been paid to other reactions catalyzed by ROS. However, it is known that ATPase activity and inhibition of ATP-independent Ca2+ binding are severely depressed in

**4. Oxidative stress and cardiovascular diseases** 

span and improved cardiac function (Schriner et al., 2005).

(Cai & Jones, 1998).

associated with early postnatal lethality (Li et al., 1995), while MnSOD overexpression was shown to protect mitochondrial function and block apoptosis (Holley et al., 2010). The activity of MnSOD should be coordinated with H2O2-removing enzymes. Thus, H2O2 produced by MnSOD could be metabolized by Gpx, Prx, or by catalase that has been found in extremely small amounts in the mitochondrial matrix.

Besides of the importance of GSH as a direct antioxidant, it participates in multiple GSHlinked enzymatic defense systems. Among others, GSH acts as electron donor in the reduction of H2O2 and different hydroperoxides by GPx1 and GPx4 (Camara et al., 2011). Five different isoforms of GPx have been identified GPx1, GPx2, GPx3, GPx 4, and GPx 6. GPx1 is the major isoform and is localized predominantly in the cytosol, but a small proportion is also present within the mitochondrial matrix (Orrenius et al., 2007). GPx4 is a unique intracellular antioxidant enzyme that directly reduces peroxidized lipids produced in cell membranes (Nomura et al., 2000). Because GPx4 is membrane-associated, with a fraction localized in the intermembrane space of the mitochondria, possibly at the contact sites of the two membranes, and due to its small size and large hydrophobic surface it can interact with, and detoxify, membrane lipid hydroperoxides much more efficiently than the alternative pathway, phospholipase A2-GPx1 (PLA2) (Antunes et al., 1995). Hence, GPx4 is considered to be the primary enzymatic defense system against oxidative damage to cellular membranes. Accordingly, GPx4-null mice are embryonically lethal, while the heterozygotes are more sensitive to oxidants than wild type mice (Ran et al., 2003).

Other mitochondrial GSH-linked enzymes are glutaredoxins (Grx), which catalyze glutathione-dependent dithiol reaction, reducing protein disulfides, and monothiol reactions reducing mixed disulfides between proteins and GSH. An interesting member of this family is glutaredoxin 2 (Grx2), which was cloned and found to be present as both mitochondrial and nuclear isoforms (Lundberg et al., 2001). Modeling suggests that the GSH binding site and the hydrophobic surface of Grx2 are similar to those of Grx1 (Lundberg et al., 2001), although Grx2 lacks one of the conserved non-active site cysteine residues of Grx1 (Lundberg et al., 2001), hence it is more resistant to oxidants and oxidized glutathione (GSSG) action. Furthermore, Grx2 can be reactivated directly by thioredoxin reductase (TrxR) as well as by GSH (Johanson et al., 2004). GSSG is reduced by glutathione reductase (GR) with NADPH as a cofactor. In turn, mitochondrial NADPH can be regenerated by matrix dehydrogenases and by reaction of hydride ion transfer, which is proton motive force-dependent, utilizing intramitochondrial NADPH to reduce NADP+. Besides NADPH *per se* can serve directly as a non-enzymatic antioxidant, according to some authors (Kirsch & De Groot, 2001). Another potential source of disulfide reductase activity in mitochondria is the thioredoxin system, which includes thioredoxin 2 (Trx2) and thioredoxin reductase (TrxR2). Trx2 catalyzes the reduction of protein disulfides at much higher rates than Grx (Arner & Holmgren, 2000). This enzyme is important for life, given that disruption of the Trx2 gene in the homozygous mouse causes massive apoptosis and, finally, results in embryonic lethality (Nonn et al., 2003). Specific glutathione S-transferase (GST) isoforms: GSTα1-1, GSTα4-4, and GSTµ1-1, which neutralize reactive molecules such as 4-hydroxy-2 noneal (4-HNE), incorporating GSH to the radical molecule, have been found in mitochondria (Raza et al., 2002).

The intermembrane space of mitochondria contains ~0.7 mM cytochrome *c* (Hackenbrock et al., 1986) capable of superoxide removal. Cytochrome *c* can be alternatively reduced by the

associated with early postnatal lethality (Li et al., 1995), while MnSOD overexpression was shown to protect mitochondrial function and block apoptosis (Holley et al., 2010). The activity of MnSOD should be coordinated with H2O2-removing enzymes. Thus, H2O2 produced by MnSOD could be metabolized by Gpx, Prx, or by catalase that has been found

Besides of the importance of GSH as a direct antioxidant, it participates in multiple GSHlinked enzymatic defense systems. Among others, GSH acts as electron donor in the reduction of H2O2 and different hydroperoxides by GPx1 and GPx4 (Camara et al., 2011). Five different isoforms of GPx have been identified GPx1, GPx2, GPx3, GPx 4, and GPx 6. GPx1 is the major isoform and is localized predominantly in the cytosol, but a small proportion is also present within the mitochondrial matrix (Orrenius et al., 2007). GPx4 is a unique intracellular antioxidant enzyme that directly reduces peroxidized lipids produced in cell membranes (Nomura et al., 2000). Because GPx4 is membrane-associated, with a fraction localized in the intermembrane space of the mitochondria, possibly at the contact sites of the two membranes, and due to its small size and large hydrophobic surface it can interact with, and detoxify, membrane lipid hydroperoxides much more efficiently than the alternative pathway, phospholipase A2-GPx1 (PLA2) (Antunes et al., 1995). Hence, GPx4 is considered to be the primary enzymatic defense system against oxidative damage to cellular membranes. Accordingly, GPx4-null mice are embryonically lethal, while the heterozygotes

Other mitochondrial GSH-linked enzymes are glutaredoxins (Grx), which catalyze glutathione-dependent dithiol reaction, reducing protein disulfides, and monothiol reactions reducing mixed disulfides between proteins and GSH. An interesting member of this family is glutaredoxin 2 (Grx2), which was cloned and found to be present as both mitochondrial and nuclear isoforms (Lundberg et al., 2001). Modeling suggests that the GSH binding site and the hydrophobic surface of Grx2 are similar to those of Grx1 (Lundberg et al., 2001), although Grx2 lacks one of the conserved non-active site cysteine residues of Grx1 (Lundberg et al., 2001), hence it is more resistant to oxidants and oxidized glutathione (GSSG) action. Furthermore, Grx2 can be reactivated directly by thioredoxin reductase (TrxR) as well as by GSH (Johanson et al., 2004). GSSG is reduced by glutathione reductase (GR) with NADPH as a cofactor. In turn, mitochondrial NADPH can be regenerated by matrix dehydrogenases and by reaction of hydride ion transfer, which is proton motive force-dependent, utilizing intramitochondrial NADPH to reduce NADP+. Besides NADPH *per se* can serve directly as a non-enzymatic antioxidant, according to some authors (Kirsch & De Groot, 2001). Another potential source of disulfide reductase activity in mitochondria is the thioredoxin system, which includes thioredoxin 2 (Trx2) and thioredoxin reductase (TrxR2). Trx2 catalyzes the reduction of protein disulfides at much higher rates than Grx (Arner & Holmgren, 2000). This enzyme is important for life, given that disruption of the Trx2 gene in the homozygous mouse causes massive apoptosis and, finally, results in embryonic lethality (Nonn et al., 2003). Specific glutathione S-transferase (GST) isoforms: GSTα1-1, GSTα4-4, and GSTµ1-1, which neutralize reactive molecules such as 4-hydroxy-2 noneal (4-HNE), incorporating GSH to the radical molecule, have been found in

The intermembrane space of mitochondria contains ~0.7 mM cytochrome *c* (Hackenbrock et al., 1986) capable of superoxide removal. Cytochrome *c* can be alternatively reduced by the

in extremely small amounts in the mitochondrial matrix.

are more sensitive to oxidants than wild type mice (Ran et al., 2003).

mitochondria (Raza et al., 2002).

respiratory chain or superoxide. A diminution in its concentration may inhibit the distal respiratory chain and increase ROS production. Indeed, it has been reported that upon cytochrome *c* release during apoptosis initiation, mitochondrial ROS production increases (Cai & Jones, 1998).

As mentioned before, ubiquinone (Q) acts as a pro-oxidant in its semiquinone form, however when fully reduced it acts as an antioxidant. Ubiquinol (UQH2) contains phenolic hydrogen atoms that can be donated to a carbon- or oxygen centered radical, converting it to a non-radical molecule (Ježek & Hlavatá, 2005). Lipid peroxidation prevention by UQH2 has been reported (James et al., 2004). Preferential succinate oxidation can provide a tool against excessive accumulation of ROS by increasing the proportion of fully reduced ubiquinone. The antioxidant activity of UQH2 is independent from the effect of α-tocopherol (Ernster et al., 1992), which acts as a chain-breaking antioxidant, inhibiting the propagation of lipid peroxidation (Maroz et al., 2009). Even more, UQH2 can efficiently sustain the effect of αtocopherol by regenerating it from the tocopheroxyl radical, which otherwise must rely on water-soluble agents such as ascorbate (vitamin C) (Ernster & Forsmark-Andree, 1993).

#### **4. Oxidative stress and cardiovascular diseases**

The pathological role of increased mitochondrial ROS in heart disease has been established from studies in gene-modified mice with altered mitochondrial antioxidant levels. Deletion of mitochondrial thioredoxin reductase 2 is embryonically lethal in mice because of impaired hematopoiesis and impaired cardiac function (Conrad et al., 2004). Fatal dilated cardiomyopathy in mice is developed after complete deletion of mitochondrial Mn superoxide dismutase (Li et al., 1995). In contrast, attenuated left ventricular remodeling after myocardial infarct was observed in transgenic mice overexpressing mitochondrial peroxiredoxin III (Matsushima et al., 2006) or glutathione peroxidase (Shiomi et al., 2004). Also, mice with a mitochondrial-targeted overexpression of catalase have a prolonged life span and improved cardiac function (Schriner et al., 2005).

 A critical role of intracellular Ca2+ overload and oxidative stress in the genesis of myocyte dysfunction is well established in cardiovascular diseases like atherosclerosis, hypertension, ischemia/reperfusion damage, cardiac hypertrophy, and heart failure (HF). In general, Ca2+ overload can be induced by direct effect of ROS on Ca2+ handling proteins or indirectly, by inducing membrane lipid peroxidation (Santos et al., 2011). Recent evidence suggests that redox modification of ryanodine receptor (RyR2) may contribute to abnormal Ca2+ handling in disease states. RyR2 dysfunction with an increase in diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) may reduce calcium transients and contribute to a reduced contractile force in the failing heart, as well as an increased likelihood of arrhythmia (González et al., 2010). In humans, increased cytosolic calcium ([Ca2+]c) has been related with augmented oxidative stress in atherosclerosis. A growing body of evidence indicates that the production of ROS is tightly linked with Angiotensin II-induced action. In this respect, a causative link between superoxide production and hypertension has been established in experiments in which SOD reduced blood pressure by 50 mm Hg in vascular smooth muscle cells (VSCM) from Angiotensin II-infused rats (Laursen et al., 1997). Much less attention has been paid to other reactions catalyzed by ROS. However, it is known that ATPase activity and inhibition of ATP-independent Ca2+ binding are severely depressed in

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 167

Complex mechanisms have evolved to maintain the balance between myocardial O2 supply and O2 consumption under pathological stresses such as hypoxia, ischemia, pressure, and volume overload. These mechanisms induce changes in cardiomyocyte structure and/or function through coordinated changes in gene and protein expression and/or the activities of various proteins. Redox mechanisms are involved in the signaling pathways underlying many of these mechanisms, both via the direct effects of O2 levels in the cardiomyocyte and through the effects of ROS (Santos et al., 2011). Metabolically, the adult mammalian heart normally uses lipids as the major fuel, and mitochondria supply over 90% of the total ATP through β-oxidation of plasma fatty acids (Opie & Sack, 2002). During hypoxia, under ischemia or settings of increased cardiac workload, there is a substantial increase in glycolytic ATP generation, which may be cardioprotective during ischemia/reperfusion by ensuring an adequate ATP supply for membrane and sarcoplasmic reticulum ion pumps (Opie & Sack, 2002; Correa et al., 2008a). Recent studies suggest that a metabolic shift to glycolysis is related with the redox status in the heart. NADPH has been recognized as a critical modulator of the antioxidant defense through the regeneration of reduced pools of glutathione, while G6PDH activity was shown to be of major importance for the maintenance of redox status, Ca2+ homeostasis, and contractile function in cardiomyocytes

Mitochondrial biogenesis can be defined as a chain of events that promote growth and division of preexisting organelles. Mitochondrial biogenesis includes the synthesis, import and incorporation of proteins and lipids, as well as the replication of mitochondrial DNA (mtDNA). Replication and transcription of mtDNA are controlled by mtTFA (mitochondrial transcription factor A), and two specific transcription factors TFB1M and TFB2M (mitochondrial transcription factor B1/B2), an RNA polymerase (POLRMT), and a mitochondrial transcription termination factor (mTERF). The coordination between the expression of mitochondrial and nuclear genes is directed by nuclear respiratory factor (NRF-1 and/or NRF-2), the peroxisome proliferator-activated receptors (PPARs), estrogen receptor (ERR), and co-activators of peroxisome proliferator-activated receptor gamma (PGC-1α) (Scarpulla, 2008). PGC-1α is also involved in regulation of fatty acid oxidation

Mitochondrial biogenesis decreases in aging, obesity, insulin resistance, dyslipidemia, and hypertension, co-morbidities associated with cardiovascular diseases. Impaired activation of the renin-angiotensin-aldosterone system (RAAS) has been associated with such pathologies (Cooper et al., 2007). In fact, elevated levels of angiotensin II (Ang II) and aldosterone promote alterations in insulin metabolism, endothelial dysfunction, and loss of myocardial function (Kim et al., 2008; Sowers et al., 2009). RAAS increases the activity of NADPH oxidase and stimulates ROS generation resulting in mitochondrial damage, decreased ATP production, diminished NO availability, and attenuated mitochondrial biogenesis. Clinical and experimental observations report the loss of expression of PGC-1α, and mtTFA NRFs in hypertension (Whaley-Connell et al., 2009). In addition, downregulation of the mitochondrial biogenesis co-activator PGC-1α and its downstream nuclear

**5.1 Metabolic adaptation** 

subjected to oxidative stress (Jain et al., 2003) .

(FAO) and in co-activation of ERRα (Figure 4).

**5.2 Mitochondrial biogenesis** 

sarcolemmal membranes exposed to hydrogen peroxide and Fe2+ (Kukreja et al., 1992, Kaneko et al., 1989). In addition, augmented levels of iron pool in atherosclerotic lesions suggest that iron-catalyzed formation of free radicals may take place in the development of this pathology (Yuan & Li, 2003). High-fat diets stimulate stress response (heat shock protein 70) and signal transduction genes (Ras, MAPK1), inhibiting SOD and GPx gene expression. These effects could be prevented by scavengers of peroxides and antioxidant supplementation of the high-fat diet and caloric restriction (Rosier & Saes, 2006).

#### **5. Mitochondrial dysfunction**

Mitochondrial myopathies were described in the early 1960s, when systematic ultrastructural and histochemical studies revealed excessive proliferation of abnormally looking mitochondria in muscle of patients with weakness or exercise intolerance (Shy & Gonatas, 1964). Mitochondrial dysfunction, reflected in the structure, function and number of mitochondria within the cardiomyocyte, leads to diminished energy production, loss of myocyte contractility, altered electrical properties, and eventual cardiomyocyte cell death (Capetanaki, 2002). In addition, cardiotoxic stimuli often lead to excessive production of ROS and to Ca2+ overload in the mitochondrial matrix (Ragoni & Condolini, 2009). Evidences for a pathological role of mitochondrial ROS comes from studies in animal models of myocardial infarction, in which increased mitochondrial ROS production was observed, accompanied by decreases in mtDNA copy numbers, in mitochondrial-encoded gene transcripts, and in related enzymatic activities (complexes I, III, and IV), and from studies of genetically modified animals. Overexpression of Prx-3 (a mitochondrial antioxidant protein) improved post-myocardial infarction left ventricular function by restoring mitochondrial activity and DNA copy numbers (Matsushima et al., 2006). Other examples are studies in mice with complete deletion of mitochondrial MnSOD, which developed severe fatal dilated cardiomyopathy (Li et al., 1995). Decreased vascular SOD activities have also been associated with increased susceptibility to ischemia/reperfusion mediated damage; whereas overexpression of mitochondrial antioxidants increased cardiac tolerance to ischemia (Madamanchi, et al., 2005). Recently, the causal role of mitochondrial ROS in Angiotensin II-induced cardiomyopathy was shown by the observation that mice that overexpress catalase targeted to mitochondria, but not mice that overexpress wild-type peroxisomal catalase, are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage induced by angiotensin II (Dai et al., 2011). Monoamine oxidase (MAO) has been shown to play a prominent role in myocardial injury caused by post-ischemic reperfusion (Bianchi et al., 2005a) and to contribute to the maladaptive evolution from myocardial hypertrophy to heart failure (Kaludercic et al., 2010). MAO-mediated ROS production has been related with serotonin-induced myocyte hypertrophy *in vitro* (Bianchi et al., 2005b) and in mitogenic signaling induction by a process that may involve the activation of the metalloproteinase MMP-2, in smooth muscle cells (Coatrieux et al., 2007).

Mitochondria isolated from hearts of rabbits exposed to hypercholesterolemic diet showed significantly reduced respiration rates (state 3 and state 4) (Kojik et al., 2011), whereas increased cholesterol is related with diminution of the mitochondrial membrane potential and mitochondrial pore opening (Chávez et al., 1998; Martínez Abundis et al., 2007) and activation of apoptosis (Martínez-Abundis et al., 2009).

#### **5.1 Metabolic adaptation**

166 Oxidative Stress and Diseases

sarcolemmal membranes exposed to hydrogen peroxide and Fe2+ (Kukreja et al., 1992, Kaneko et al., 1989). In addition, augmented levels of iron pool in atherosclerotic lesions suggest that iron-catalyzed formation of free radicals may take place in the development of this pathology (Yuan & Li, 2003). High-fat diets stimulate stress response (heat shock protein 70) and signal transduction genes (Ras, MAPK1), inhibiting SOD and GPx gene expression. These effects could be prevented by scavengers of peroxides and antioxidant

Mitochondrial myopathies were described in the early 1960s, when systematic ultrastructural and histochemical studies revealed excessive proliferation of abnormally looking mitochondria in muscle of patients with weakness or exercise intolerance (Shy & Gonatas, 1964). Mitochondrial dysfunction, reflected in the structure, function and number of mitochondria within the cardiomyocyte, leads to diminished energy production, loss of myocyte contractility, altered electrical properties, and eventual cardiomyocyte cell death (Capetanaki, 2002). In addition, cardiotoxic stimuli often lead to excessive production of ROS and to Ca2+ overload in the mitochondrial matrix (Ragoni & Condolini, 2009). Evidences for a pathological role of mitochondrial ROS comes from studies in animal models of myocardial infarction, in which increased mitochondrial ROS production was observed, accompanied by decreases in mtDNA copy numbers, in mitochondrial-encoded gene transcripts, and in related enzymatic activities (complexes I, III, and IV), and from studies of genetically modified animals. Overexpression of Prx-3 (a mitochondrial antioxidant protein) improved post-myocardial infarction left ventricular function by restoring mitochondrial activity and DNA copy numbers (Matsushima et al., 2006). Other examples are studies in mice with complete deletion of mitochondrial MnSOD, which developed severe fatal dilated cardiomyopathy (Li et al., 1995). Decreased vascular SOD activities have also been associated with increased susceptibility to ischemia/reperfusion mediated damage; whereas overexpression of mitochondrial antioxidants increased cardiac tolerance to ischemia (Madamanchi, et al., 2005). Recently, the causal role of mitochondrial ROS in Angiotensin II-induced cardiomyopathy was shown by the observation that mice that overexpress catalase targeted to mitochondria, but not mice that overexpress wild-type peroxisomal catalase, are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage induced by angiotensin II (Dai et al., 2011). Monoamine oxidase (MAO) has been shown to play a prominent role in myocardial injury caused by post-ischemic reperfusion (Bianchi et al., 2005a) and to contribute to the maladaptive evolution from myocardial hypertrophy to heart failure (Kaludercic et al., 2010). MAO-mediated ROS production has been related with serotonin-induced myocyte hypertrophy *in vitro* (Bianchi et al., 2005b) and in mitogenic signaling induction by a process that may involve the activation of the

supplementation of the high-fat diet and caloric restriction (Rosier & Saes, 2006).

metalloproteinase MMP-2, in smooth muscle cells (Coatrieux et al., 2007).

activation of apoptosis (Martínez-Abundis et al., 2009).

Mitochondria isolated from hearts of rabbits exposed to hypercholesterolemic diet showed significantly reduced respiration rates (state 3 and state 4) (Kojik et al., 2011), whereas increased cholesterol is related with diminution of the mitochondrial membrane potential and mitochondrial pore opening (Chávez et al., 1998; Martínez Abundis et al., 2007) and

**5. Mitochondrial dysfunction** 

Complex mechanisms have evolved to maintain the balance between myocardial O2 supply and O2 consumption under pathological stresses such as hypoxia, ischemia, pressure, and volume overload. These mechanisms induce changes in cardiomyocyte structure and/or function through coordinated changes in gene and protein expression and/or the activities of various proteins. Redox mechanisms are involved in the signaling pathways underlying many of these mechanisms, both via the direct effects of O2 levels in the cardiomyocyte and through the effects of ROS (Santos et al., 2011). Metabolically, the adult mammalian heart normally uses lipids as the major fuel, and mitochondria supply over 90% of the total ATP through β-oxidation of plasma fatty acids (Opie & Sack, 2002). During hypoxia, under ischemia or settings of increased cardiac workload, there is a substantial increase in glycolytic ATP generation, which may be cardioprotective during ischemia/reperfusion by ensuring an adequate ATP supply for membrane and sarcoplasmic reticulum ion pumps (Opie & Sack, 2002; Correa et al., 2008a). Recent studies suggest that a metabolic shift to glycolysis is related with the redox status in the heart. NADPH has been recognized as a critical modulator of the antioxidant defense through the regeneration of reduced pools of glutathione, while G6PDH activity was shown to be of major importance for the maintenance of redox status, Ca2+ homeostasis, and contractile function in cardiomyocytes subjected to oxidative stress (Jain et al., 2003) .

#### **5.2 Mitochondrial biogenesis**

Mitochondrial biogenesis can be defined as a chain of events that promote growth and division of preexisting organelles. Mitochondrial biogenesis includes the synthesis, import and incorporation of proteins and lipids, as well as the replication of mitochondrial DNA (mtDNA). Replication and transcription of mtDNA are controlled by mtTFA (mitochondrial transcription factor A), and two specific transcription factors TFB1M and TFB2M (mitochondrial transcription factor B1/B2), an RNA polymerase (POLRMT), and a mitochondrial transcription termination factor (mTERF). The coordination between the expression of mitochondrial and nuclear genes is directed by nuclear respiratory factor (NRF-1 and/or NRF-2), the peroxisome proliferator-activated receptors (PPARs), estrogen receptor (ERR), and co-activators of peroxisome proliferator-activated receptor gamma (PGC-1α) (Scarpulla, 2008). PGC-1α is also involved in regulation of fatty acid oxidation (FAO) and in co-activation of ERRα (Figure 4).

Mitochondrial biogenesis decreases in aging, obesity, insulin resistance, dyslipidemia, and hypertension, co-morbidities associated with cardiovascular diseases. Impaired activation of the renin-angiotensin-aldosterone system (RAAS) has been associated with such pathologies (Cooper et al., 2007). In fact, elevated levels of angiotensin II (Ang II) and aldosterone promote alterations in insulin metabolism, endothelial dysfunction, and loss of myocardial function (Kim et al., 2008; Sowers et al., 2009). RAAS increases the activity of NADPH oxidase and stimulates ROS generation resulting in mitochondrial damage, decreased ATP production, diminished NO availability, and attenuated mitochondrial biogenesis. Clinical and experimental observations report the loss of expression of PGC-1α, and mtTFA NRFs in hypertension (Whaley-Connell et al., 2009). In addition, downregulation of the mitochondrial biogenesis co-activator PGC-1α and its downstream nuclear

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 169

mitochondria. Due to its central role in cell death triggering, the mPTP represents a potential therapeutic target in some cardiovascular diseases. Studies performed over 20 years have demonstrated that acute cardiac ischemia followed by reperfusion damage is associated with mPTP opening (Arteaga et al., 1992; Griffiths & Halestrap, 1993; Chipuk et al., 2006; Lucken-Ardjomande et al., 2008; Halestrap and Pasdois, 2009). Furthermore, pharmacological and conditional inhibition of mPTP formation significantly improved cardiac function reducing ischemic injury and myocardial infarction size in animal models (Argaud et a., 2005; Hausenloy & Yellon, 2003; Correa et al., 2008b) and in patients (Shanmuganathan et al., 2005; Piot et al., 2008). mPTP opening also causes cell death in isolated endothelial and vascular smooth muscle cells. Indeed, atherosclerosis is exacerbated when mitochondrial antioxidant defenses are hampered and a decrease in mitochondrial

Regulation of the mPTP by a variety of signaling molecules and cellular metabolites and ions is a complex process, and mPTP formation ultimately depends on the balance between factors favoring and inhibiting pore opening (Figure 5). The precise metabolic role of mPTP formation

Fig. 5. Metabolic inducers and inhibitors of mPTP opening in cardiac ischemia/reperfusion and tumorigenesis. ANT, adenine nucleotide translocase; CK, creatine kinase; CyP-D, cyclophilin D; HK, hexokinase; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; Pi, inorganic phosphate; PBR, peripheral benzodiazepine receptor; SOD,

under physiological conditions is still being debated; reverse pore opening with a low conductance may be attributed to regulation of mitochondrial Ca2+. Low conductive mPTinduced matrix swelling and increased mitochondrial Ca2+ can regulate ATP synthesis through the tricarboxylic acid cycle, electron transport chain, and oxidative phosphorylation. Induction of mitochondrial permeability transition (mPT) in response to pathological stresses can also be regulated with the signaling protein kinases: PKA, PKCε

superoxide dismutase; VDAC, voltage-dependent anion channel.

ROS formation reduces atherogenesis.

factors have been associated with myocardial contractile dysfunction, intracellular Ca2+ mishandling, ROS accumulation, mitochondrial damage, and loss of mitochondrial density and mtDNA content in high-fat diet–induced obesity (Dong et al., 2007).

Fig. 4. The transcriptional network that controls mitochondrial biogenesis. See text for further details.

#### **5.3 Mitochondrial ion channels**

#### **5.3.1 Permeability transition pore**

General membrane damage secondary to ROS-mediated lipid peroxidation is one mechanism by which changes in mitochondrial permeability can occur; however, severe oxidative stress accompanied by calcium overload in the mitochondrial matrix favors the formation of the pathological and non-specific mitochondrial permeability transition pore (mPTP). Opening of the mPTP induces depolarization of the mitochondrial inner membrane (MIM) leading to ATP depletion and further ROS production. The increase in MIM permeability enhances colloidal osmotic pressure in the mitochondrial matrix ultimately leading to matrix swelling and rupture of the mitochondrial outer membrane (MOM). Rupture of the MOM results in release of pro-apoptotic proteins from the mitochondrial intermembrane space to the cytoplasm initiating both caspase-dependent and caspaseindependent apoptosis. Permeabilization of the MOM may also occur due to formation of non-selective channels induced by translocation of pro-apoptotic Bcl-2 family proteins to

factors have been associated with myocardial contractile dysfunction, intracellular Ca2+ mishandling, ROS accumulation, mitochondrial damage, and loss of mitochondrial density

Fig. 4. The transcriptional network that controls mitochondrial biogenesis. See text for

General membrane damage secondary to ROS-mediated lipid peroxidation is one mechanism by which changes in mitochondrial permeability can occur; however, severe oxidative stress accompanied by calcium overload in the mitochondrial matrix favors the formation of the pathological and non-specific mitochondrial permeability transition pore (mPTP). Opening of the mPTP induces depolarization of the mitochondrial inner membrane (MIM) leading to ATP depletion and further ROS production. The increase in MIM permeability enhances colloidal osmotic pressure in the mitochondrial matrix ultimately leading to matrix swelling and rupture of the mitochondrial outer membrane (MOM). Rupture of the MOM results in release of pro-apoptotic proteins from the mitochondrial intermembrane space to the cytoplasm initiating both caspase-dependent and caspaseindependent apoptosis. Permeabilization of the MOM may also occur due to formation of non-selective channels induced by translocation of pro-apoptotic Bcl-2 family proteins to

further details.

**5.3 Mitochondrial ion channels 5.3.1 Permeability transition pore** 

and mtDNA content in high-fat diet–induced obesity (Dong et al., 2007).

mitochondria. Due to its central role in cell death triggering, the mPTP represents a potential therapeutic target in some cardiovascular diseases. Studies performed over 20 years have demonstrated that acute cardiac ischemia followed by reperfusion damage is associated with mPTP opening (Arteaga et al., 1992; Griffiths & Halestrap, 1993; Chipuk et al., 2006; Lucken-Ardjomande et al., 2008; Halestrap and Pasdois, 2009). Furthermore, pharmacological and conditional inhibition of mPTP formation significantly improved cardiac function reducing ischemic injury and myocardial infarction size in animal models (Argaud et a., 2005; Hausenloy & Yellon, 2003; Correa et al., 2008b) and in patients (Shanmuganathan et al., 2005; Piot et al., 2008). mPTP opening also causes cell death in isolated endothelial and vascular smooth muscle cells. Indeed, atherosclerosis is exacerbated when mitochondrial antioxidant defenses are hampered and a decrease in mitochondrial ROS formation reduces atherogenesis.

Regulation of the mPTP by a variety of signaling molecules and cellular metabolites and ions is a complex process, and mPTP formation ultimately depends on the balance between factors favoring and inhibiting pore opening (Figure 5). The precise metabolic role of mPTP formation

Fig. 5. Metabolic inducers and inhibitors of mPTP opening in cardiac ischemia/reperfusion and tumorigenesis. ANT, adenine nucleotide translocase; CK, creatine kinase; CyP-D, cyclophilin D; HK, hexokinase; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane; Pi, inorganic phosphate; PBR, peripheral benzodiazepine receptor; SOD, superoxide dismutase; VDAC, voltage-dependent anion channel.

under physiological conditions is still being debated; reverse pore opening with a low conductance may be attributed to regulation of mitochondrial Ca2+. Low conductive mPTinduced matrix swelling and increased mitochondrial Ca2+ can regulate ATP synthesis through the tricarboxylic acid cycle, electron transport chain, and oxidative phosphorylation. Induction of mitochondrial permeability transition (mPT) in response to pathological stresses can also be regulated with the signaling protein kinases: PKA, PKCε

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 171

Mitochondrial ATP-sensitive potassium channels (mK+ATP) were described about 20 years ago in mitoplasts obtained from rat liver mitochondria. First advances in mK+ATP research demonstrated that different drugs stimulate the opening of mK+ATP channels decreasing mitochondrial potential (Δm) and that mK+ATP channels are involved in mechanisms regulating cell volume (Garlid, 1988). Latter, it was observed that bradykinin (Yang et al., 2004), opioids (Jang et al., 2008), and adenosine (Kin H, 2005), activate signaling cascades that induce the opening of mK+ATP and provide cardioprotection against ischemia/reperfusion injury. Recently a causative link between the opening of mK+ATP and cardioprotection conferred by post-conditioning has been proposed. Garlid et al. (2008) suggested that post-conditioning induces protection via an early redox-sensitive mechanism, followed by persistent mK+ATP activation. A complex signaling system involving mitochondrial PKCε 1 and 2 may prevent the formation of the mPTP. The putative signaling cascade includes the activation of Gi protein-coupled receptors and cGMPdependent protein kinase (PKG). At mitochondrial level, this kinase binds to a hypothetic receptor, called "R1" in the mitochondrial outer membrane. This receptor may phosphorylate PKCε 1 (Jaburek et al., 2006), which, in turn, phosphorylates the mK+ATP channel favoring its opening. Once activated, the entry of K+ may produce alkalinization of the mitochondrial matrix and promote ROS production by Complex I. ROS could activate the mitochondrial matrix PKCε 2, and prevent the formation of mPTP, reducing cell death

Other proposals have been invoked to explain cardioprotection. One hypothesis is that K+ flow into the matrix may depolarize the inner membrane and reduce the driving force that sustains Ca2+ overload (Holmuhamedov et al., 1991; Murata et al., 2001). However, although Ca2+ reduction in the mitochondrial matrix may reduce mPTP opening in the post-ischemic heart, it is difficult to explain how a small depolarizing effect generated by activation of mK+ATP channels could avoid Ca2+-overload. Another assumption is that discrete mitochondrial swelling associated with K+ flow would change the architecture and respiratory control of mitochondria, creating a state of mitochondrial "super-efficiency" (Garlid, 2000). Whatever the mechanism involved in mK+ATP opening, its association with

ROS/RNS can cause cell death by non-physiological (necrotic) or regulated pathways (apoptotic) in many cardiovascular diseases such as atherosclerosis, ischemic heart disease, heart failure, stroke, hypertension, and diabetes. The mechanisms by which ROS/RNS cause or regulate apoptosis typically are caspase-dependent and include the activation of membrane receptors, Bcl-2 family proteins, and mitochondrial dysfunction. Autophagy, a caspase-independent mechanism of cell death that protects cells against oxidative damage and is involved in the degradation and recycling of oxidized proteins and damaged organelles in cells, yields amino acids for *de novo* protein synthesis or energy provision (Nishida et al., 2009). While programmed cell death participation in cardiovascular diseases is well established, insights into caspase-independent mechanisms of cell death have

**5.3.4 Mitochondrial KATP channels** 

myocardial protection is clear.

emerged recently.

**6. Programmed cell death and autophagy** 

and infarction size (Costa et al., Garlid et al., 2009) (Figure 6).

(protein kinase Cε) and GSK-3β (glycogen synthase kinase-3), which interact with the voltage-dependent anion channel (VDAC) (Bera et al., 1995; Baines et al, 2003; Javadov et al., 2009).

#### **5.3.2 Structure of the mPTP complex**

Although the crucial role of mPTPs in pathological conditions has been intensively studied in the heart, brain, and liver (Chipuk et al., 2006; Bernardi et al., 2006; Robertson et al., 2009), the actual molecular composition of the mPTP complex remains unclear. Until recently, three proteins had been accepted as key structural components of this megachannel: adenine nucleotide translocase (ANT), cyclophilin D (CyP-D), and the voltage-dependent anion channel (VDAC) located in the MIM, in the matrix and in the MOM, respectively. However, recent studies from different groups have questioned the molecular identity of the mPTP. A new model of the mPTP consisting of a phosphate carrier and ANT has been proposed where Ca2+ sensitivity of the pore is regulated by CyP-D binding to the phosphate carrier (Leung et al., 2008). Many studies have provided strong evidence that CyP-D plays a major regulatory role in mPTP formation (Baines et al., 2005; Nakagawa et al., 2005). Mitochondria isolated from CyP-D knockout mice were desensitized to the onset of the mPT, and required much higher concentrations of Ca2+ to induce pore opening compared to wild type animals, which is consistent with the role of CyP-D to regulate Ca2+-mPTP interactions (Nakagawa et al., 2005). Recent studies on transgenic mice questioned the role of ANT and VDAC as essential components of the mPTP suggesting a regulatory, rather than structural role in pore formation.

#### **5.3.3 mPTP formation in cardiac Ischemia/Reperfusion (I/R)**

mPTP opening has been examined extensively in cardiac pathological conditions, mostly in I/R (Chipuk et al., 2006; Correa et al., 2007; Halestrap & Pasdois, 2009). Acute I/R does not affect the expression of mPTP compounds due to its short duration, although it induces conformational changes in essential mPTP proteins, modifying their interactions with the pore effectors in the cytoplasm and mitochondrial matrix. Although many factors that induce pore opening are present during ischemia, including ATP depletion, Ca2+ overload, increased phosphate and ROS levels, it has been demonstrated that pore opening occurs during reperfusion rather than during ischemia (Griffiths and Halestrap, 1995). This is explained, in part, by the acidic conditions resulting from lactate and other acidic intermediates accumulation in the mitochondrial matrix. We and others demonstrated that delayed pHi recovery during reperfusion exerts beneficial effects on post-ischemic cardiac function, associated with improved mitochondrial function and inhibition of mPTP opening (Javadov et al., 2008; Correa et al., 2008a). In this regard, inhibition of the Na+/H+ exchanger 1(NHE-1) may be a promising therapeutic strategy against I/R damage (Linz & Busch, 2003; Karmazyn et al., 2001). mPTP opening causes mitochondrial uncoupling, thereby ATP is hydrolyzed rather than synthesized in the post-ischemic heart leading to myocardial death (Correa et al., 2005). mPTP opening also increases in Ca2+-induced cardiomyopathy (Nakayama et al., 2007), in diabetic cardiomyopathy (Oliveira et al., 2003), in heart failure following myocardial infarction (Javadov et al., 2005), and in intracoronary microembolization (Sharov et al., 2007).

#### **5.3.4 Mitochondrial KATP channels**

170 Oxidative Stress and Diseases

(protein kinase Cε) and GSK-3β (glycogen synthase kinase-3), which interact with the voltage-dependent anion channel (VDAC) (Bera et al., 1995; Baines et al, 2003; Javadov et al.,

Although the crucial role of mPTPs in pathological conditions has been intensively studied in the heart, brain, and liver (Chipuk et al., 2006; Bernardi et al., 2006; Robertson et al., 2009), the actual molecular composition of the mPTP complex remains unclear. Until recently, three proteins had been accepted as key structural components of this megachannel: adenine nucleotide translocase (ANT), cyclophilin D (CyP-D), and the voltage-dependent anion channel (VDAC) located in the MIM, in the matrix and in the MOM, respectively. However, recent studies from different groups have questioned the molecular identity of the mPTP. A new model of the mPTP consisting of a phosphate carrier and ANT has been proposed where Ca2+ sensitivity of the pore is regulated by CyP-D binding to the phosphate carrier (Leung et al., 2008). Many studies have provided strong evidence that CyP-D plays a major regulatory role in mPTP formation (Baines et al., 2005; Nakagawa et al., 2005). Mitochondria isolated from CyP-D knockout mice were desensitized to the onset of the mPT, and required much higher concentrations of Ca2+ to induce pore opening compared to wild type animals, which is consistent with the role of CyP-D to regulate Ca2+-mPTP interactions (Nakagawa et al., 2005). Recent studies on transgenic mice questioned the role of ANT and VDAC as essential components of the mPTP suggesting a regulatory, rather

mPTP opening has been examined extensively in cardiac pathological conditions, mostly in I/R (Chipuk et al., 2006; Correa et al., 2007; Halestrap & Pasdois, 2009). Acute I/R does not affect the expression of mPTP compounds due to its short duration, although it induces conformational changes in essential mPTP proteins, modifying their interactions with the pore effectors in the cytoplasm and mitochondrial matrix. Although many factors that induce pore opening are present during ischemia, including ATP depletion, Ca2+ overload, increased phosphate and ROS levels, it has been demonstrated that pore opening occurs during reperfusion rather than during ischemia (Griffiths and Halestrap, 1995). This is explained, in part, by the acidic conditions resulting from lactate and other acidic intermediates accumulation in the mitochondrial matrix. We and others demonstrated that delayed pHi recovery during reperfusion exerts beneficial effects on post-ischemic cardiac function, associated with improved mitochondrial function and inhibition of mPTP opening (Javadov et al., 2008; Correa et al., 2008a). In this regard, inhibition of the Na+/H+ exchanger 1(NHE-1) may be a promising therapeutic strategy against I/R damage (Linz & Busch, 2003; Karmazyn et al., 2001). mPTP opening causes mitochondrial uncoupling, thereby ATP is hydrolyzed rather than synthesized in the post-ischemic heart leading to myocardial death (Correa et al., 2005). mPTP opening also increases in Ca2+-induced cardiomyopathy (Nakayama et al., 2007), in diabetic cardiomyopathy (Oliveira et al., 2003), in heart failure following myocardial infarction (Javadov et al., 2005), and in intracoronary

2009).

**5.3.2 Structure of the mPTP complex** 

than structural role in pore formation.

microembolization (Sharov et al., 2007).

**5.3.3 mPTP formation in cardiac Ischemia/Reperfusion (I/R)** 

Mitochondrial ATP-sensitive potassium channels (mK+ATP) were described about 20 years ago in mitoplasts obtained from rat liver mitochondria. First advances in mK+ATP research demonstrated that different drugs stimulate the opening of mK+ATP channels decreasing mitochondrial potential (Δm) and that mK+ATP channels are involved in mechanisms regulating cell volume (Garlid, 1988). Latter, it was observed that bradykinin (Yang et al., 2004), opioids (Jang et al., 2008), and adenosine (Kin H, 2005), activate signaling cascades that induce the opening of mK+ATP and provide cardioprotection against ischemia/reperfusion injury. Recently a causative link between the opening of mK+ATP and cardioprotection conferred by post-conditioning has been proposed. Garlid et al. (2008) suggested that post-conditioning induces protection via an early redox-sensitive mechanism, followed by persistent mK+ATP activation. A complex signaling system involving mitochondrial PKCε 1 and 2 may prevent the formation of the mPTP. The putative signaling cascade includes the activation of Gi protein-coupled receptors and cGMPdependent protein kinase (PKG). At mitochondrial level, this kinase binds to a hypothetic receptor, called "R1" in the mitochondrial outer membrane. This receptor may phosphorylate PKCε 1 (Jaburek et al., 2006), which, in turn, phosphorylates the mK+ATP channel favoring its opening. Once activated, the entry of K+ may produce alkalinization of the mitochondrial matrix and promote ROS production by Complex I. ROS could activate the mitochondrial matrix PKCε 2, and prevent the formation of mPTP, reducing cell death and infarction size (Costa et al., Garlid et al., 2009) (Figure 6).

Other proposals have been invoked to explain cardioprotection. One hypothesis is that K+ flow into the matrix may depolarize the inner membrane and reduce the driving force that sustains Ca2+ overload (Holmuhamedov et al., 1991; Murata et al., 2001). However, although Ca2+ reduction in the mitochondrial matrix may reduce mPTP opening in the post-ischemic heart, it is difficult to explain how a small depolarizing effect generated by activation of mK+ATP channels could avoid Ca2+-overload. Another assumption is that discrete mitochondrial swelling associated with K+ flow would change the architecture and respiratory control of mitochondria, creating a state of mitochondrial "super-efficiency" (Garlid, 2000). Whatever the mechanism involved in mK+ATP opening, its association with myocardial protection is clear.

#### **6. Programmed cell death and autophagy**

ROS/RNS can cause cell death by non-physiological (necrotic) or regulated pathways (apoptotic) in many cardiovascular diseases such as atherosclerosis, ischemic heart disease, heart failure, stroke, hypertension, and diabetes. The mechanisms by which ROS/RNS cause or regulate apoptosis typically are caspase-dependent and include the activation of membrane receptors, Bcl-2 family proteins, and mitochondrial dysfunction. Autophagy, a caspase-independent mechanism of cell death that protects cells against oxidative damage and is involved in the degradation and recycling of oxidized proteins and damaged organelles in cells, yields amino acids for *de novo* protein synthesis or energy provision (Nishida et al., 2009). While programmed cell death participation in cardiovascular diseases is well established, insights into caspase-independent mechanisms of cell death have emerged recently.

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 173

Fig. 7. Simplified model of cellular apoptotic pathways. Specific ligands bind to death receptors, activating initiator and executioner caspases. In the mitochondrial pathway, several stimuli are processed favoring membrane permeabilization and the release of proapoptogenic factors as cytochrome *c*, AIF, and Smac/DIABLO to the cytosol, through membrane pore forming proteins, such as BAK/BAX or as a consequence of the mPTP opening. Once in the cytosol, cytochrome c and APAF-1 bind to caspase-9, activating caspase-3. The extrinsic pathway could activate the mitochondrial pathway, through the Bcl-

Overexpression of antioxidant proteins in several models sustains the relevance of oxidative stress in cardiomyocyte apoptosis. Catalase, glutathione peroxidase 1, metalothionein, mitochondrial glutaredoxin-2, and peroxiredoxin 2 over- expression reduce apoptosis and improve contractile dysfunction after ischemia/reperfusion injury (Shiomi et al., 2004; Nagy et al., 2008; Zhao et al., 2009). As indicated, oxidative stress may activate the intrinsic apoptotic pathway in cardiomyocytes through multiple mechanisms, such as the induction of mPTP opening, DNA damage-induced, translocation of Bax and Bad to the mitochondria, and caspase activation. However, an alternative mode of oxidative stress-induced activation of the intrinsic apoptotic pathway may also involve induction of the ER stress response, leading to caspase-12 activation and/or Ca2+-dependent opening of the mPTP (Foo et al., 2005). Catecholamines, angiotensin II, prostaglandin F2α, or endothelin-1, which interact

2 family member (Bid) that promotes BAX and BAK oligomerization.

Fig. 6. Intramitochondrial signaling pathways. The pathways leading to mK+ATP opening, ROS production, and MPT inhibition are shown.

#### **6.1 Apoptotic mitochondrial pathway**

Mitochondria contain diverse pro-apoptotic factors within their intermembrane space, such as cytochrome *c*, apoptosis-inducer factor, and Smac Diablo, which are released and propagate the death cascade. Two different pores have been described as pathways for cytochrome *c* release (reviewed in Kinally & Antonsson, 2007). The first one is the mitochondrial apoptosis-induced channel (MAC) formed by Bax and VDAC proteins (Shimizu et al., 2000). There are also reports indicating that only Bax-forming channels could account for cytochrome *c* release (Kuwana et al., 2002). In this sense, the contribution of Bax channels to cytochrome *c* release after reperfusion has been explored by Bombrun et al., 2003. A second possible pathway described for cytochrome *c* release is the mPTP. These proteins could be assembled into a continued unspecific channel, promoting mitochondrial swelling, MOM rupture, and pro-apoptotic proteins release (Figure 7).

Fig. 6. Intramitochondrial signaling pathways. The pathways leading to mK+ATP opening,

Mitochondria contain diverse pro-apoptotic factors within their intermembrane space, such as cytochrome *c*, apoptosis-inducer factor, and Smac Diablo, which are released and propagate the death cascade. Two different pores have been described as pathways for cytochrome *c* release (reviewed in Kinally & Antonsson, 2007). The first one is the mitochondrial apoptosis-induced channel (MAC) formed by Bax and VDAC proteins (Shimizu et al., 2000). There are also reports indicating that only Bax-forming channels could account for cytochrome *c* release (Kuwana et al., 2002). In this sense, the contribution of Bax channels to cytochrome *c* release after reperfusion has been explored by Bombrun et al., 2003. A second possible pathway described for cytochrome *c* release is the mPTP. These proteins could be assembled into a continued unspecific channel, promoting mitochondrial

swelling, MOM rupture, and pro-apoptotic proteins release (Figure 7).

ROS production, and MPT inhibition are shown.

**6.1 Apoptotic mitochondrial pathway** 

Fig. 7. Simplified model of cellular apoptotic pathways. Specific ligands bind to death receptors, activating initiator and executioner caspases. In the mitochondrial pathway, several stimuli are processed favoring membrane permeabilization and the release of proapoptogenic factors as cytochrome *c*, AIF, and Smac/DIABLO to the cytosol, through membrane pore forming proteins, such as BAK/BAX or as a consequence of the mPTP opening. Once in the cytosol, cytochrome c and APAF-1 bind to caspase-9, activating caspase-3. The extrinsic pathway could activate the mitochondrial pathway, through the Bcl-2 family member (Bid) that promotes BAX and BAK oligomerization.

Overexpression of antioxidant proteins in several models sustains the relevance of oxidative stress in cardiomyocyte apoptosis. Catalase, glutathione peroxidase 1, metalothionein, mitochondrial glutaredoxin-2, and peroxiredoxin 2 over- expression reduce apoptosis and improve contractile dysfunction after ischemia/reperfusion injury (Shiomi et al., 2004; Nagy et al., 2008; Zhao et al., 2009). As indicated, oxidative stress may activate the intrinsic apoptotic pathway in cardiomyocytes through multiple mechanisms, such as the induction of mPTP opening, DNA damage-induced, translocation of Bax and Bad to the mitochondria, and caspase activation. However, an alternative mode of oxidative stress-induced activation of the intrinsic apoptotic pathway may also involve induction of the ER stress response, leading to caspase-12 activation and/or Ca2+-dependent opening of the mPTP (Foo et al., 2005). Catecholamines, angiotensin II, prostaglandin F2α, or endothelin-1, which interact

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 175

Fig. 8. Signaling pathways, regulating mitochondrial function. ROS production is induced by oxidative stress, which activates a signaling cascade involving the PKCβ-dependent phosphorylation of p66 its translocation to the mitochondrial matrix. Mitochondria are also targets of ROS damage. Damaged mitochondria are removed by mitophagy, a specialized

Mitochondria are highly dynamic organelles that undergo constantly fusion and fission as part of their normal function (Detmer & Chan, 2007). These two opposite processes are accurately coordinated and necessary for proper morphology and function and are thought to play critical roles during development, cell division, and apoptosis (Cerveny et al., 2007; Chan, 2006). Disruption of mitochondrial fission and fusion has been linked to the

Mitochondrial fusion facilitates the exchange of materials between mitochondria for the maintenance of functional mitochondria, whereas mitochondrial fission contributes to the elimination of damaged mitochondrial fragments through mitophagy and contributes to the proper distribution of mitochondria in response to the local demand for ATP. Multiple proteins have been identified to mediate mitochondrial fission and fusion processes (Chan, 2006). These two opposing processes are regulated by the mitochondrial fusion proteins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2, Fzo1 in yeast) (Eura et al., 2003), and optic atrophy protein 1 (Opa1, Mgm1 in yeast) (Cipolat et al., 2004), and the mitochondrial fission proteins

form of autophagy, which is regulated by different pathways.

**7. Mitochondrial fission and fusion** 

development and progression of some diseases.

with G-protein coupled receptors and induce cardiomyocyte hypertrophy, may also induce apoptosis. A well characterized mechanism is a Gαq-mediated PKC-dependent transcriptional upregulation of the Bcl-2 family member Nix, which activates the mitochondrial death pathway (Yussman et al., 2002)

#### **6.2 Autophagy**

Autophagy is a physiological process that is necessary for cell survival and maintains stable levels of nutrients to sustain cellular homeostasis. It is also involved in various pathophysiological processes, and shows increased activity in response to extracellular and intracellular stimulation such as nutrient starvation and hypoxia. During autophagy, cytoplasmic constituents are sequestered into the autophagosome, a closed double membrane vacuole that eventually fuses with a lysosome. In the new structure, named autolysosome, the contents are degraded and recycled for protein synthesis (Cuervo, 2004). ROS act as signaling molecules in the early events of autophagy induction. If the prosurvival attempt fails, ROS cause cell death which, depending on the experimental context, involves either the autophagic or the apoptotic pathway. Mitochondria are both the major source of intracellular ROS and, at the same time, targets of ROS. An enhanced oxidative stress may activate a signaling cascade involving the PKCβ-dependent phosphorylation of p66 protein and its translocation to the mitochondrial matrix. Damaged mitochondria are degraded by a specialized form of autophagy, called mitophagy, in which mitochondrial calcium plays an active role. Normally calcium homeostasis is tightly regulated and occurs at ER-mitochondria contacts where microdomains of high calcium concentration are present. This event causes a variety of responses depending on the amount of Ca2+ increase, from stimulation of metabolism and ATP production to ROS production, mPTP opening, and apoptosis (Figure 8). In this respect, recent data have proposed a role of p66Shc in mediating the response of mitochondria to ROS-induced apoptosis or autophagy (Mammucari & Rizzuto, 2010).

Autophagy increases in response to acute myocardial ischemia (AMI), chronic myocardial ischemia, heart failure, and cardiomyopathy degeneration (Cuervo, 2004). The effect of autophagy upregulation is under debate, as some studies have demonstrated that it leads to myocyte death after ischemia/reperfusion (Valentim et al., 2006), but others indicate that autophagy has a cardioprotective effect during myocardial ischemia. Three mechanisms are invoked to explain protection: 1) generation of free amino acids and fatty acids that contributes to maintain the mitochondrial energy supply, improving cell survival (Matsui et al., 2007), 2) elimination of disordered structural proteins, harmful to cardiac myocytes, and 3) removal of damaged mitochondria. In human myocardial depression associated to endotoxemia, Hickson-Bick et al. (2008) described that mitochondrial biogenesis observed in cardiomyocytes may reflect an effort to replace the mitochondria eliminated by autophagy. In HL-1 cells subjected to oxidative stress, the induction of autophagy by rapamycin suppressed ROS production and protected cells against death (Yuan et al., 2009). These results are consistent with the notion that autophagy is a protective mechanism in this setting and limits the production of harmful ROS, either by removing damaged mitochondria or by supporting *de novo* glutathione biosynthesis through the delivery of amino acids.

with G-protein coupled receptors and induce cardiomyocyte hypertrophy, may also induce apoptosis. A well characterized mechanism is a Gαq-mediated PKC-dependent transcriptional upregulation of the Bcl-2 family member Nix, which activates the

Autophagy is a physiological process that is necessary for cell survival and maintains stable levels of nutrients to sustain cellular homeostasis. It is also involved in various pathophysiological processes, and shows increased activity in response to extracellular and intracellular stimulation such as nutrient starvation and hypoxia. During autophagy, cytoplasmic constituents are sequestered into the autophagosome, a closed double membrane vacuole that eventually fuses with a lysosome. In the new structure, named autolysosome, the contents are degraded and recycled for protein synthesis (Cuervo, 2004). ROS act as signaling molecules in the early events of autophagy induction. If the prosurvival attempt fails, ROS cause cell death which, depending on the experimental context, involves either the autophagic or the apoptotic pathway. Mitochondria are both the major source of intracellular ROS and, at the same time, targets of ROS. An enhanced oxidative stress may activate a signaling cascade involving the PKCβ-dependent phosphorylation of p66 protein and its translocation to the mitochondrial matrix. Damaged mitochondria are degraded by a specialized form of autophagy, called mitophagy, in which mitochondrial calcium plays an active role. Normally calcium homeostasis is tightly regulated and occurs at ER-mitochondria contacts where microdomains of high calcium concentration are present. This event causes a variety of responses depending on the amount of Ca2+ increase, from stimulation of metabolism and ATP production to ROS production, mPTP opening, and apoptosis (Figure 8). In this respect, recent data have proposed a role of p66Shc in mediating the response of mitochondria to ROS-induced apoptosis or autophagy

Autophagy increases in response to acute myocardial ischemia (AMI), chronic myocardial ischemia, heart failure, and cardiomyopathy degeneration (Cuervo, 2004). The effect of autophagy upregulation is under debate, as some studies have demonstrated that it leads to myocyte death after ischemia/reperfusion (Valentim et al., 2006), but others indicate that autophagy has a cardioprotective effect during myocardial ischemia. Three mechanisms are invoked to explain protection: 1) generation of free amino acids and fatty acids that contributes to maintain the mitochondrial energy supply, improving cell survival (Matsui et al., 2007), 2) elimination of disordered structural proteins, harmful to cardiac myocytes, and 3) removal of damaged mitochondria. In human myocardial depression associated to endotoxemia, Hickson-Bick et al. (2008) described that mitochondrial biogenesis observed in cardiomyocytes may reflect an effort to replace the mitochondria eliminated by autophagy. In HL-1 cells subjected to oxidative stress, the induction of autophagy by rapamycin suppressed ROS production and protected cells against death (Yuan et al., 2009). These results are consistent with the notion that autophagy is a protective mechanism in this setting and limits the production of harmful ROS, either by removing damaged mitochondria or by supporting *de novo* glutathione biosynthesis through the delivery of

mitochondrial death pathway (Yussman et al., 2002)

**6.2 Autophagy** 

(Mammucari & Rizzuto, 2010).

amino acids.

Fig. 8. Signaling pathways, regulating mitochondrial function. ROS production is induced by oxidative stress, which activates a signaling cascade involving the PKCβ-dependent phosphorylation of p66 its translocation to the mitochondrial matrix. Mitochondria are also targets of ROS damage. Damaged mitochondria are removed by mitophagy, a specialized form of autophagy, which is regulated by different pathways.

#### **7. Mitochondrial fission and fusion**

Mitochondria are highly dynamic organelles that undergo constantly fusion and fission as part of their normal function (Detmer & Chan, 2007). These two opposite processes are accurately coordinated and necessary for proper morphology and function and are thought to play critical roles during development, cell division, and apoptosis (Cerveny et al., 2007; Chan, 2006). Disruption of mitochondrial fission and fusion has been linked to the development and progression of some diseases.

Mitochondrial fusion facilitates the exchange of materials between mitochondria for the maintenance of functional mitochondria, whereas mitochondrial fission contributes to the elimination of damaged mitochondrial fragments through mitophagy and contributes to the proper distribution of mitochondria in response to the local demand for ATP. Multiple proteins have been identified to mediate mitochondrial fission and fusion processes (Chan, 2006). These two opposing processes are regulated by the mitochondrial fusion proteins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2, Fzo1 in yeast) (Eura et al., 2003), and optic atrophy protein 1 (Opa1, Mgm1 in yeast) (Cipolat et al., 2004), and the mitochondrial fission proteins

Oxidative Stress and Mitochondrial Dysfunction in Cardiovascular Diseases 177

It has been suggested that defects in mitochondrial fusion and fission processes are responsible for abnormal mitochondrial morphologies observed in many cardiac diseases. In cultured neonatal ventricular myocytes, inhibition of mitochondrial fission, by overexpressing a dominant-negative mutant form of Drp1, prevents ROS production, mitochondrial permeability transition pore opening, and subsequent cell death after ceramide treatment (Parra et al., 2008). An increase in the level of cytosolic Ca2+ induced by thapsigargin (Tg) causes cardiac mitochondrial fission associated to ROS generation in a Drp1-dependent pathway (Hom et al., 2010). Because calcium overload is a common feature in heart failure (HF), this may increase mitochondrial fission and dysfunction, thus further contributing to the decrease in the metabolic demand of the heart and increasing its injury.

Mitochondrial redox signaling is paramount to the maintenance of cardiomyocyte homeostasis. Therefore, oxidative deregulation of mitochondrial key players, like mK+ATP, mPTP, ionic transporters, metabolism enzymes, and apoptotic machinery, has enormous impact on cardiovascular function. Intense research is devoted to obtain a better understanding of the complex regulatory mechanisms ruling these systems and to enable the development of more specific therapeutic strategies for heart diseases. In addition, fascinating links are beginning to be discovered between mitochondrial function and cardiac physiology and diseases in the context of diverse signaling mechanisms. Besides, proteins with previously known function, like those driving mitochondrial fusion and fission, are now reported to have emergent functions in intracellular calcium homeostasis, apoptosis, and vascular smooth muscle cell proliferation, all, key issues in cardiac disease. These processes broaden the traditional role in energy production undertaken by mitochondria

Alvarez, S.; Valdez, L.; Zaobornyj, T. & Boveris A (2003). Oxygen dependence of

Antunes, F.; Salvador, A. & Pinto, R. (1995). PHGPx and phospholipase A2/GPx:

Argaud, L.; Gateau-Roesch, O.; Muntean, D.; Chalabreysse, L.; Loufouat, J.; Robert, D. &

Arteaga, D.; Odor, O.; Lopez, R.; Contreras, G.; Aranda, A. & Chávez, E. (1992) Impairment by cyclosporine A of reperfusion-induced arrhythmias. *Life Sciences*, 1127-1134.

mitochondria. *Free Radical Biology & Medicine.* Vol. 19, 669-77.

mitochondrial nitric oxide synthase activity. *Biochemical and Biophysical Research* 

comparative importance on the reduction of hydroperoxides in rat liver

Ovize, M. (2005). Specific inhibition of the mitochondrial permeability transition prevents lethal reperfusion injury. *Journal of Molecular Cell Cardiology.* Vol. 38, 367-374.

and provide new directions for research in cardiovascular diseases.

This work was partially supported by grant 80791-M to C. Zazueta

*Community.* Vol. 305, 771–775.

**7.2 Mitochondrial fission** 

**8. Conclusion** 

**9. Acknowledgment** 

**10. References** 

dynamin-related protein 1 (Drp1, Dnm1 in yeast) (Frank et al., 2001; Smirnova et al., 2001; Ingerman et al., 2005), and human mitochondrial fission protein 1 (hFis1) (Yoon et al., 2003). The balance between mitochondrial fusion and fission within a cell can be disrupted by a myriad of factors, including oxidative stress (Frank et al., 2001) and simulated ischemia (Brady et al., 2006), and has also been linked to aging (Kowald & Kirkwood, 2011) (Figure 9).

Fig. 9. Mitochondrial fission and fusion. Mitochondrial fission involves the action of Drp1, which can self-assemble into polymeric spirals and is recruited into the mitochondrial membrane by hFis1 and Mdv1/Caf4. Drp1 polymers wrap around the mitochondrion and constrict the membrane until fission occurs. Mitochondrial fusion involves the interaction of Mfn1 and Mfn2 proteins located in the outer mitochondrial membrane of two mitochondria until outer membranes fuse, consequently, inner mitochondrial membrane fusion occurs through interaction of Opa1 proteins. See text for further details.

#### **7.1 Mitochondrial fusion**

Data on fission and fusion proteins' role in heart diseases are scarce. Recently it has been reported that cardiac myocyte mitochondria, lacking the fusion protein Mfn2, are pleiomorphic and show enlarged morphopology. Consistent with an underlying mild mitochondrial dysfunction, Mfn2-deficient mice display modest cardiac hypertrophy accompanied by slight functional deterioration (Papanicolau et al., 2011). Expression of OPA1 is decreased in both human and rat failing hearts, which show small and fragmented mitochondria indicative of decreased fusion. OPA1 mRNA levels did not differ between failing and normal hearts, suggesting post-transcriptional control, possibly through degradation by proteases activated by ATP (Baricault et al., 2007).

#### **7.2 Mitochondrial fission**

176 Oxidative Stress and Diseases

dynamin-related protein 1 (Drp1, Dnm1 in yeast) (Frank et al., 2001; Smirnova et al., 2001; Ingerman et al., 2005), and human mitochondrial fission protein 1 (hFis1) (Yoon et al., 2003). The balance between mitochondrial fusion and fission within a cell can be disrupted by a myriad of factors, including oxidative stress (Frank et al., 2001) and simulated ischemia (Brady

et al., 2006), and has also been linked to aging (Kowald & Kirkwood, 2011) (Figure 9).

Fig. 9. Mitochondrial fission and fusion. Mitochondrial fission involves the action of Drp1, which can self-assemble into polymeric spirals and is recruited into the mitochondrial membrane by hFis1 and Mdv1/Caf4. Drp1 polymers wrap around the mitochondrion and constrict the membrane until fission occurs. Mitochondrial fusion involves the interaction of Mfn1 and Mfn2 proteins located in the outer mitochondrial membrane of two mitochondria until outer membranes fuse, consequently, inner mitochondrial membrane fusion occurs

Data on fission and fusion proteins' role in heart diseases are scarce. Recently it has been reported that cardiac myocyte mitochondria, lacking the fusion protein Mfn2, are pleiomorphic and show enlarged morphopology. Consistent with an underlying mild mitochondrial dysfunction, Mfn2-deficient mice display modest cardiac hypertrophy accompanied by slight functional deterioration (Papanicolau et al., 2011). Expression of OPA1 is decreased in both human and rat failing hearts, which show small and fragmented mitochondria indicative of decreased fusion. OPA1 mRNA levels did not differ between failing and normal hearts, suggesting post-transcriptional control, possibly through

through interaction of Opa1 proteins. See text for further details.

degradation by proteases activated by ATP (Baricault et al., 2007).

**7.1 Mitochondrial fusion** 

It has been suggested that defects in mitochondrial fusion and fission processes are responsible for abnormal mitochondrial morphologies observed in many cardiac diseases. In cultured neonatal ventricular myocytes, inhibition of mitochondrial fission, by overexpressing a dominant-negative mutant form of Drp1, prevents ROS production, mitochondrial permeability transition pore opening, and subsequent cell death after ceramide treatment (Parra et al., 2008). An increase in the level of cytosolic Ca2+ induced by thapsigargin (Tg) causes cardiac mitochondrial fission associated to ROS generation in a Drp1-dependent pathway (Hom et al., 2010). Because calcium overload is a common feature in heart failure (HF), this may increase mitochondrial fission and dysfunction, thus further contributing to the decrease in the metabolic demand of the heart and increasing its injury.

#### **8. Conclusion**

Mitochondrial redox signaling is paramount to the maintenance of cardiomyocyte homeostasis. Therefore, oxidative deregulation of mitochondrial key players, like mK+ATP, mPTP, ionic transporters, metabolism enzymes, and apoptotic machinery, has enormous impact on cardiovascular function. Intense research is devoted to obtain a better understanding of the complex regulatory mechanisms ruling these systems and to enable the development of more specific therapeutic strategies for heart diseases. In addition, fascinating links are beginning to be discovered between mitochondrial function and cardiac physiology and diseases in the context of diverse signaling mechanisms. Besides, proteins with previously known function, like those driving mitochondrial fusion and fission, are now reported to have emergent functions in intracellular calcium homeostasis, apoptosis, and vascular smooth muscle cell proliferation, all, key issues in cardiac disease. These processes broaden the traditional role in energy production undertaken by mitochondria and provide new directions for research in cardiovascular diseases.

#### **9. Acknowledgment**

This work was partially supported by grant 80791-M to C. Zazueta

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**9** 

Sarawut Kumphune

*Thailand* 

*Department of Medical Technology,* 

*Faculty of Allied Health Sciences, Naresuan University,* 

**Oxidatively Modified Biomolecules: An Early** 

**Biomarker for Acute Coronary Artery Disease** 

Cardiovascular disease is the worldwide major cause of mortality and morbidity. The 2009 annual report from World Health Organization **(**WHO**)** highlighted the mortality rate prediction of the population worldwide that, in 2030, cardiovascular disease will become the major cause of deaths, and the mortality rate will higher than other infectious diseases such as HIV, Tuberculosis, malaria infection (World Heatlh Organization ,2009). Moreover, this report also mentioned that, among cardiovascular diseases, ischemic heart disease and cerebrovascular disease, which were reported as top 2 cause of mortality in 2004, are expected to still be the major cause of death in next 20 years (World Heatlh Organization ,2009). Coronary artery disease is a sequence of pathophysiologic processes in coronary arteries, myocardial ischemia and infarction (Wudkowska et al.2010). Therefore, the early diagnostic of myocardial ischemia and infarction, will lead to the rapid and more effective of medical intervention, and safe the patients' life. The standard diagnosis of coronary artery disease focuses on clinical assessment such as history of chest pain associated with electrocardiogram (ECG) changes, and elevation of cardiac specific-biochemical markers

Determination of serum or plasma level of cardiac specific-biochemical markers is one of the most essential and effective way for diagnosing myocardial ischemia/infarction. The ideal cardiac markers should have high specificity, high sensitivity, rapidly released after the onset of the symptoms, abundant in cardiac tissue but less in other tissues, long half life in blood circulation, and capable of representing the prognosis and estimating the infarct size. It has been known that coronary artery disease, especially myocardial ischemia and ischemia-reperfusion injury is the phenomenon that related to an oxidative stress (Buja2005), which is an imbalance and inadequate production of reactive oxygen species (ROS), subsequently resulted in biochemical modifications of major principle biomolecules such as proteins, lipids, and nucleic acids (Valko et al.2007;Sbarouni et al.2008a;Sbarouni et al.2008b;Sbarouni et al.2008c;le-Donne et al.2003a;le-Donne et al.2003c). Some oxidatively modified biomolecules such as Ischemia Modified Albumin or IMA, has been approved by US Food and drug Administration (FDA) and used as a rule-out marker for acute myocardial ischemia (Apple et al.2005;Bar-Or et al.2000;Sbarouni et al.2008c;Sbarouni et

**1. Introduction** 

(Maneewong K. et al.2011).


### **Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease**

Sarawut Kumphune

*Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Thailand* 

#### **1. Introduction**

188 Oxidative Stress and Diseases

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Protection of peroxiredoxin II on oxidative stress-induced cardiomyocyte death

Cardiovascular disease is the worldwide major cause of mortality and morbidity. The 2009 annual report from World Health Organization **(**WHO**)** highlighted the mortality rate prediction of the population worldwide that, in 2030, cardiovascular disease will become the major cause of deaths, and the mortality rate will higher than other infectious diseases such as HIV, Tuberculosis, malaria infection (World Heatlh Organization ,2009). Moreover, this report also mentioned that, among cardiovascular diseases, ischemic heart disease and cerebrovascular disease, which were reported as top 2 cause of mortality in 2004, are expected to still be the major cause of death in next 20 years (World Heatlh Organization ,2009). Coronary artery disease is a sequence of pathophysiologic processes in coronary arteries, myocardial ischemia and infarction (Wudkowska et al.2010). Therefore, the early diagnostic of myocardial ischemia and infarction, will lead to the rapid and more effective of medical intervention, and safe the patients' life. The standard diagnosis of coronary artery disease focuses on clinical assessment such as history of chest pain associated with electrocardiogram (ECG) changes, and elevation of cardiac specific-biochemical markers (Maneewong K. et al.2011).

Determination of serum or plasma level of cardiac specific-biochemical markers is one of the most essential and effective way for diagnosing myocardial ischemia/infarction. The ideal cardiac markers should have high specificity, high sensitivity, rapidly released after the onset of the symptoms, abundant in cardiac tissue but less in other tissues, long half life in blood circulation, and capable of representing the prognosis and estimating the infarct size.

It has been known that coronary artery disease, especially myocardial ischemia and ischemia-reperfusion injury is the phenomenon that related to an oxidative stress (Buja2005), which is an imbalance and inadequate production of reactive oxygen species (ROS), subsequently resulted in biochemical modifications of major principle biomolecules such as proteins, lipids, and nucleic acids (Valko et al.2007;Sbarouni et al.2008a;Sbarouni et al.2008b;Sbarouni et al.2008c;le-Donne et al.2003a;le-Donne et al.2003c). Some oxidatively modified biomolecules such as Ischemia Modified Albumin or IMA, has been approved by US Food and drug Administration (FDA) and used as a rule-out marker for acute myocardial ischemia (Apple et al.2005;Bar-Or et al.2000;Sbarouni et al.2008c;Sbarouni et

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 191

polar, it can penetrate through the lipid bilayer of cell membrane or mitochondrial membrane, and destroy some biological molecules such as proteins, lipids, and nucleic acids. Cellular balancing mechanism for H2O2 is the enzyme catalase, which convert two

(Catalase)

 2 H2O2 2H2O + O2 (3) Another alternative mechanism generating ROS in the cell is the Haber-Weiss reaction, which is a chemical catalysis of superoxide and hydrogen peroxide by ferric ion (Fe3+) to

Fe3+/CU2+

In addition, superoxide can reduce ferric ion to form ferrous ion (equation 5), the reaction called "Fenton reaction". The production of ferrous ion in first Fenton reaction can react

Reactive nitrogen species (RNS) are generated from the reaction of nitric oxide (NO), which is enzymatically generated by nitric oxide synthetase (NOS). The NOS oxidized the amino acid L-arginine or L-citrulline. The member of RNS include nitric oxide (NO) and nitrogen

nitrousoxide (HNO2), and alkyl peroxynitrates (RONOO). Among these RNS molecules,

NO, and ONOO- are the most investigated species, which have significant impact in

Antioxidants are either endogenous or exogenous compounds that prevent the generation of harmful free radicals, reduce the generated radicals, inactivate their harmful reactivity, and thereby block the chain reactions of these oxidants. The primary or chain breaking antioxidants so called "scavenger" which is neutralize the free radicals by donating one of their own electrons (Kumar et al.2010). The secondary or preventative antioxidants work by sequestration of transition metal ions or removal the peroxides by catalase and glutathione peroxidase. The tertiary antioxidants defense is the repairing of damaged molecules, in

Reactive oxygen species readily attack a variety of important biomolecules, including carbohydrates, proteins, lipids, and nucleic acids. Interaction between ROS and these

and •

2), as well as non radicals nitrogen species e.g. peroxynitrite (ONOO-),

+ •

OH generation (equation 6).

Fe2+ + O2 (5)

OH (4)

OH (6)

•- + H2O2 + O2 OH-

OH) (equation 4)

•-

Fe2+ + H2O2 Fe3+ + OH- + •

attempt to avoid the accumulative damages (Kumar et al.2010).

**3. The oxidative modification of biomolecules** 

molecules of H2O2 to oxygen and water (equation 3)

with H2O2 in the second reaction and result in OH-

cardiovascular complication (Kumar et al.2010).

Fe3+ + O2

**2.2 Reactive Nitrogen Species** 

dioxide (NO•

**2.3 Antioxidants** 

•

generate hydroxyl radical (•

O2

al.2008a;Sbarouni et al.2008b;Van et al.2010). In addition, many of oxidatively modified biomolecules have been reported to correlate with the severity of coronary artery disease and possibly used as a marker for myocardial ischemia (Apple et al.2005;Bar-Or et al.2001c; Bar-Or et al.2000;le-Donne et al.2003b;Berlett & Stadtman1997;Kiyici et al.2010;Turedi et al.2010; Melanson & Tanasijevic2005;Van et al.2010;Shen et al.2010;Pantazopoulos et al.2009; Bhagavan et al.2003;Santalo et al.2003;Sinha et al.2003;Mutlu-Turkoglu et al.2005;Mocatta et al.2007; Beal2002;Docherty2010;Wudkowska et al.2010;Charpentier et al.2010;Maneewong K. et al.2011; Sbarouni et al.2008a; Sbarouni et al.2008b;Sbarouni et al.2008c;le-Donne et al.2003a; le-Donne et al.2003d).

In this chapter, studies of oxidatively modified biomolecules such as proteins, lipids, and nucleic acids, related to coronary artery diseases will be discussed. Moreover, clinical usefulness of determining these oxidatively modified biomolecules as a biomarker for coronary artery disease will also be addressed.

#### **2. Oxidative stress**

The term oxidative stress has been commonly mentioned or explained the underline pathophysiological mechanism of some diseases during the last thirty years (Hensley et al.2000). Oxidative stress is referred to an inadequate of free radicals generation and/or insufficient removal of the radicals by antioxidants, radical scavengers. Free radicals can also be defined as atoms or molecules containing one or more unpaired electrons on an open shell configuration (Lushchak2011), which generate the highly reactivity properties of the molecules. There are 2 major types of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS).

#### **2.1 Reactive Oxygen Species**

Reactive oxygen species (ROS) are generated from oxygen metabolism include superoxide anion (O2 •-), peroxyl (RO• 2), hydroperoxyl (HRO2 •-), and hydroxyl radical (• OH). In addition, ROS can also be non-radical species such as hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl). ROS can be generated from regular metabolic processes or from external sources such as X-ray exposure, air pollutants, cigarette smoking, and etc. The primary source of intracellular free radicals generated by the addition of one oxygen electron, which is resulted in superoxide (O2 •-) (equation 1).

$$\circlearrowleft \mathfrak{O}^{\bullet} \text{ + } \circlearrowleft \mathfrak{O}^{\bullet} \text{ : \tag{1}$$

Intracellular mechanism to balance the generation of superoxide is achieved by specific enzyme called superoxide dismutase (SOD), which catalyze the changing of superoxide to oxygen and hydrogen peroxide (H2O2) (equation 2).

SOD

 2 O2 •-+ 2H+ O2 + H2O2 (2)

This hydrogen peroxide (H2O2) that is generated from equation 2 has a property of being an oxidizing agent and serve as a major source of • OH, which is one of the very harmful reactive oxygen species to the cell. According to hydrogen peroxide is non-radical and weak polar, it can penetrate through the lipid bilayer of cell membrane or mitochondrial membrane, and destroy some biological molecules such as proteins, lipids, and nucleic acids. Cellular balancing mechanism for H2O2 is the enzyme catalase, which convert two molecules of H2O2 to oxygen and water (equation 3)

$$\text{(Catalyst)}$$

$$\text{2H}\_2\text{O}\_2 \quad \xrightarrow{\text{---}} \begin{array}{c} \text{2H}\_2\text{O} + \text{O}\_2 \end{array} \tag{3}$$

Another alternative mechanism generating ROS in the cell is the Haber-Weiss reaction, which is a chemical catalysis of superoxide and hydrogen peroxide by ferric ion (Fe3+) to generate hydroxyl radical (• OH) (equation 4)

$$\text{Fe}^{3+}/\text{Cl}^{2+} $$

$$\text{O}\_2\text{"{}^{-}} + \text{H}\_2\text{O}\_2 + \text{O}\_2 \xrightarrow[]{} \xrightarrow[]{} \text{OH} + \text{'OH} \tag{4}$$

In addition, superoxide can reduce ferric ion to form ferrous ion (equation 5), the reaction called "Fenton reaction". The production of ferrous ion in first Fenton reaction can react with H2O2 in the second reaction and result in OH and • OH generation (equation 6).

$$\text{Fe}^{\ast} + \text{O}\_2^{\ast}: \begin{array}{c} \longrightarrow \longrightarrow \\ \end{array} \quad \text{Fe}^{\ast} + \text{O}\_2 \tag{5}$$

$$\text{Fe}^{2+} + \text{H}\_2\text{O}\_2 \quad \xrightarrow{\text{---}} \text{Fe}^{3+} + \text{OH} \cdot + \text{"OH} \tag{6}$$

#### **2.2 Reactive Nitrogen Species**

Reactive nitrogen species (RNS) are generated from the reaction of nitric oxide (NO), which is enzymatically generated by nitric oxide synthetase (NOS). The NOS oxidized the amino acid L-arginine or L-citrulline. The member of RNS include nitric oxide (NO) and nitrogen dioxide (NO• 2), as well as non radicals nitrogen species e.g. peroxynitrite (ONOO- ), nitrousoxide (HNO2), and alkyl peroxynitrates (RONOO). Among these RNS molecules, • NO, and ONOO- are the most investigated species, which have significant impact in cardiovascular complication (Kumar et al.2010).

#### **2.3 Antioxidants**

190 Oxidative Stress and Diseases

al.2008a;Sbarouni et al.2008b;Van et al.2010). In addition, many of oxidatively modified biomolecules have been reported to correlate with the severity of coronary artery disease and possibly used as a marker for myocardial ischemia (Apple et al.2005;Bar-Or et al.2001c; Bar-Or et al.2000;le-Donne et al.2003b;Berlett & Stadtman1997;Kiyici et al.2010;Turedi et al.2010; Melanson & Tanasijevic2005;Van et al.2010;Shen et al.2010;Pantazopoulos et al.2009; Bhagavan et al.2003;Santalo et al.2003;Sinha et al.2003;Mutlu-Turkoglu et al.2005;Mocatta et al.2007; Beal2002;Docherty2010;Wudkowska et al.2010;Charpentier et al.2010;Maneewong K. et al.2011; Sbarouni et al.2008a; Sbarouni et al.2008b;Sbarouni et al.2008c;le-Donne et

In this chapter, studies of oxidatively modified biomolecules such as proteins, lipids, and nucleic acids, related to coronary artery diseases will be discussed. Moreover, clinical usefulness of determining these oxidatively modified biomolecules as a biomarker for

The term oxidative stress has been commonly mentioned or explained the underline pathophysiological mechanism of some diseases during the last thirty years (Hensley et al.2000). Oxidative stress is referred to an inadequate of free radicals generation and/or insufficient removal of the radicals by antioxidants, radical scavengers. Free radicals can also be defined as atoms or molecules containing one or more unpaired electrons on an open shell configuration (Lushchak2011), which generate the highly reactivity properties of the molecules. There are 2 major types of free radicals such as reactive oxygen species (ROS)

Reactive oxygen species (ROS) are generated from oxygen metabolism include superoxide

addition, ROS can also be non-radical species such as hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl). ROS can be generated from regular metabolic processes or from external sources such as X-ray exposure, air pollutants, cigarette smoking, and etc. The primary source of intracellular free radicals generated by the addition of one oxygen

O2 + e- O2

Intracellular mechanism to balance the generation of superoxide is achieved by specific enzyme called superoxide dismutase (SOD), which catalyze the changing of superoxide to

SOD

This hydrogen peroxide (H2O2) that is generated from equation 2 has a property of being an

reactive oxygen species to the cell. According to hydrogen peroxide is non-radical and weak

•-) (equation 1).

•-), and hydroxyl radical (•

•-+ 2H+ O2 + H2O2 (2)

•- (1)

OH, which is one of the very harmful

OH). In

2), hydroperoxyl (HRO2

al.2003a; le-Donne et al.2003d).

**2. Oxidative stress** 

coronary artery disease will also be addressed.

and reactive nitrogen species (RNS).

•-), peroxyl (RO•

electron, which is resulted in superoxide (O2

2 O2

oxygen and hydrogen peroxide (H2O2) (equation 2).

oxidizing agent and serve as a major source of •

**2.1 Reactive Oxygen Species** 

anion (O2

Antioxidants are either endogenous or exogenous compounds that prevent the generation of harmful free radicals, reduce the generated radicals, inactivate their harmful reactivity, and thereby block the chain reactions of these oxidants. The primary or chain breaking antioxidants so called "scavenger" which is neutralize the free radicals by donating one of their own electrons (Kumar et al.2010). The secondary or preventative antioxidants work by sequestration of transition metal ions or removal the peroxides by catalase and glutathione peroxidase. The tertiary antioxidants defense is the repairing of damaged molecules, in attempt to avoid the accumulative damages (Kumar et al.2010).

#### **3. The oxidative modification of biomolecules**

Reactive oxygen species readily attack a variety of important biomolecules, including carbohydrates, proteins, lipids, and nucleic acids. Interaction between ROS and these

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 193

Lipids are the basic biomolecules found throughout the cells, such as phospholipids component of the cell membrane. Therefore, lipids can be one of an oxidative modification targets, similar to proteins. Oxidative modification of lipids are the chain reactions, lead to the degradation of lipids or so-called "lipid peroxidation", which mediated by free radicals abstract electrons from lipid molecules such as aldehyde group e.g. Malonaldehyde (MDA). Polyunsaturated fatty acids are more sensitive to the lipid peroxidation according to these lipids contain multiple double bonds in between methylene- CH2- groups that easily react with reactive hydrogen atoms (Lu et al.2010). The reactions of oxidative modified lipids consist of three major steps including; the initiation step, where is a fatty acid radicals are produced. The propagation is the direct reaction with oxygen molecules produced peroxyl fatty acid radials that react with another free fatty acid producing a different fatty acid radical and lipid peroxide or a cyclic peroxide and termination. The destruction of lipid molecules by lipid peroxidation can cause membrane permeability alteration, loss in fluidity, decreasing in electrical resistance, change in phospholipids bilayer membrane disruption, membrane-bound enzyme malfunction and loose of integrity ionic gradient, disruption or activation of enzyme function, and cellular injury (Ayres1984). Biomarkers of

Nucleic acid is one of the basic biomolecules that play many essential roles in the cell. The nucleic acids-DNA and RNA- are the principal informational molecules of the living cells.

molecules. Association and reaction of free radical to DNA can lead to DNA bases damaging, both purines and pyrimidines, and result in DNA strand break (Valavanidis et al.2009). Alteration of purines and pyrimidines play a significant role in large variety of pathological stages such as cancer (Kasai1997). Biomarkers of oxidative modified nucleic

**4. Oxidatively modified biomolecules as cardiac markers for coronary artery** 

Generation of reactive oxygen species resulted in the modification of proteins, which introduce new functional groups such as hydroxyl groups and carbonyl groups (le-Donne et al.2003b;Berlett & Stadtman1997). Among these proteins, ischemia-modified albumin (IMA) was reported as an early biomarker in many pathological disorders (Kiyici et al.2010;Montagnana et al.2006;Abboud et al.2007;Gunduz et al.2009;Sharma et al.2007).

Ischemia Modified Albumin (IMA) is serum albumin that modified at the N-terminal portion, especially at aspartate-alanine-histidine-lysine sequences, by oxidative stress generated during ischemia (Bar-Or et al.2001b). This modification reduced the ability of albumin to bind with metal ions such as cobalt, copper, and nickel (Bar-Or et al.2001a) (Figure 2). The ischemic mechanism initiated with the insufficiency of oxygen supply during ischemia, which caused cardiomyocytes cellular anaerobic metabolism. Within a few seconds after occlusion of a major coronary artery tissue oxygen content decreases and

OH can be generated and can bind to DNA

lipid peroxidation include malonaldehyde, F2-Isoprostanes, and etc.

acid include 8-hydroxy-2'-deoxyguanosine, 8-nitroguanin, and etc.

During aging processes, free radicals such as •

**3.2 Lipids** 

**3.3 Nucleic acid** 

**disease** 

**4.1 Ischemia modified albumin** 

biomolecules resulted in biochemical modifications, which alter the functions as well as the properties of these biomolecules (Figure 1).

Fig. 1. Oxidative modification of biomolecules (Becker2004). In normal physiological conditions, superoxide and hydrogen peroxide are generated. Generation of intracellular ROS may activate the intracellular signaling pathways for cardiac protection, for example in ischemic preconditioning. The generated hydrogen peroxide, especially in myocardial ischemia, reacts with ferrous ion in Fenton reaction, which results in hydroxyl radicals. Over production of the hydroxyl radical causes oxidative modification of biomolecules, such as lipids, proteins, and nucleic acids.

#### **3.1 Proteins**

It has become manifested that proteins are also concerned as a target of free radical destruction. The mechanism involved in the oxidation modification of proteins is thought to occur at the monomeric level of amino acids, especially cysteine, tyrosine, phenylalanine, tryptophan, histidine, and methionine. The process of proteins oxidation creates new functional groups such as hydroxyl groups and carbonyl groups. These added up new functional groups can be generated by different mechanisms and can also indicate the degree of oxidative modification. The outcomes of the oxidative modification of proteins cause proteins fragmentation, cross-linking and unfolding, which may activate or hinder proteolytic and proteasome-mediated turnover. The biomarkers of oxidatively modified proteins include protein carbonyl, ischemia-modified albumin, and etc. Ischemia modified albumin (IMA) and protein carbonyl (PC) are oxidatively modified proteins found in many oxidative related disorders such as myocardial ischemia, renal ischemia, (Apple et al.2005;Pantke et al.1999;Bar-Or et al.2000;Kiyici et al.2010;Turedi et al.2010;Melanson & Tanasijevic2005). According to this, many studies suggested the ability of these two oxidatively modified proteins as the early biomarkers for diagnosis of coronary artery disease.

#### **3.2 Lipids**

192 Oxidative Stress and Diseases

biomolecules resulted in biochemical modifications, which alter the functions as well as the

Fig. 1. Oxidative modification of biomolecules (Becker2004). In normal physiological conditions, superoxide and hydrogen peroxide are generated. Generation of intracellular ROS may activate the intracellular signaling pathways for cardiac protection, for example in ischemic preconditioning. The generated hydrogen peroxide, especially in myocardial ischemia, reacts with ferrous ion in Fenton reaction, which results in hydroxyl radicals. Over production of the hydroxyl radical causes oxidative modification of biomolecules, such as

It has become manifested that proteins are also concerned as a target of free radical destruction. The mechanism involved in the oxidation modification of proteins is thought to occur at the monomeric level of amino acids, especially cysteine, tyrosine, phenylalanine, tryptophan, histidine, and methionine. The process of proteins oxidation creates new functional groups such as hydroxyl groups and carbonyl groups. These added up new functional groups can be generated by different mechanisms and can also indicate the degree of oxidative modification. The outcomes of the oxidative modification of proteins cause proteins fragmentation, cross-linking and unfolding, which may activate or hinder proteolytic and proteasome-mediated turnover. The biomarkers of oxidatively modified proteins include protein carbonyl, ischemia-modified albumin, and etc. Ischemia modified albumin (IMA) and protein carbonyl (PC) are oxidatively modified proteins found in many oxidative related disorders such as myocardial ischemia, renal ischemia, (Apple et al.2005;Pantke et al.1999;Bar-Or et al.2000;Kiyici et al.2010;Turedi et al.2010;Melanson & Tanasijevic2005). According to this, many studies suggested the ability of these two oxidatively modified proteins as the early

properties of these biomolecules (Figure 1).

lipids, proteins, and nucleic acids.

biomarkers for diagnosis of coronary artery disease.

**3.1 Proteins** 

Lipids are the basic biomolecules found throughout the cells, such as phospholipids component of the cell membrane. Therefore, lipids can be one of an oxidative modification targets, similar to proteins. Oxidative modification of lipids are the chain reactions, lead to the degradation of lipids or so-called "lipid peroxidation", which mediated by free radicals abstract electrons from lipid molecules such as aldehyde group e.g. Malonaldehyde (MDA). Polyunsaturated fatty acids are more sensitive to the lipid peroxidation according to these lipids contain multiple double bonds in between methylene- CH2- groups that easily react with reactive hydrogen atoms (Lu et al.2010). The reactions of oxidative modified lipids consist of three major steps including; the initiation step, where is a fatty acid radicals are produced. The propagation is the direct reaction with oxygen molecules produced peroxyl fatty acid radials that react with another free fatty acid producing a different fatty acid radical and lipid peroxide or a cyclic peroxide and termination. The destruction of lipid molecules by lipid peroxidation can cause membrane permeability alteration, loss in fluidity, decreasing in electrical resistance, change in phospholipids bilayer membrane disruption, membrane-bound enzyme malfunction and loose of integrity ionic gradient, disruption or activation of enzyme function, and cellular injury (Ayres1984). Biomarkers of lipid peroxidation include malonaldehyde, F2-Isoprostanes, and etc.

#### **3.3 Nucleic acid**

Nucleic acid is one of the basic biomolecules that play many essential roles in the cell. The nucleic acids-DNA and RNA- are the principal informational molecules of the living cells. During aging processes, free radicals such as • OH can be generated and can bind to DNA molecules. Association and reaction of free radical to DNA can lead to DNA bases damaging, both purines and pyrimidines, and result in DNA strand break (Valavanidis et al.2009). Alteration of purines and pyrimidines play a significant role in large variety of pathological stages such as cancer (Kasai1997). Biomarkers of oxidative modified nucleic acid include 8-hydroxy-2'-deoxyguanosine, 8-nitroguanin, and etc.

#### **4. Oxidatively modified biomolecules as cardiac markers for coronary artery disease**

#### **4.1 Ischemia modified albumin**

Generation of reactive oxygen species resulted in the modification of proteins, which introduce new functional groups such as hydroxyl groups and carbonyl groups (le-Donne et al.2003b;Berlett & Stadtman1997). Among these proteins, ischemia-modified albumin (IMA) was reported as an early biomarker in many pathological disorders (Kiyici et al.2010;Montagnana et al.2006;Abboud et al.2007;Gunduz et al.2009;Sharma et al.2007).

Ischemia Modified Albumin (IMA) is serum albumin that modified at the N-terminal portion, especially at aspartate-alanine-histidine-lysine sequences, by oxidative stress generated during ischemia (Bar-Or et al.2001b). This modification reduced the ability of albumin to bind with metal ions such as cobalt, copper, and nickel (Bar-Or et al.2001a) (Figure 2). The ischemic mechanism initiated with the insufficiency of oxygen supply during ischemia, which caused cardiomyocytes cellular anaerobic metabolism. Within a few seconds after occlusion of a major coronary artery tissue oxygen content decreases and

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 195

method is the diagnostic test for acute myocardial infarction (Van et al.2010). The principle of ACB method is based on the principle of free radicals, which is generated during ischemia, alters the metal ions binding capacity of serum albumin (Sbarouni et al.2008a) (Figure 3). The test is currently called IMA test instead of the ACB test. It has been reported that IMA test could be used for screening the patients with chest pain, who suspected AMI, at the emergency department, excluded patients from the other causes of chest pain (Bar-Or et al.2000). Many studies show that IMA levels increased in patients with acute coronary syndrome and also elevated in myocardial ischemia (Sbarouni et al.2008a). The analytical sensitivity of the test is 13 u/ml, with 98% recovery (Sbarouni et al.2008a). Moreover, there were reports mentioned of non-interfering effect from bilirubin, hemoglobin, cholesterol, total proteins, and number of cardiac drugs (Govender et al.2008). It was also reported that

According to the ability to detect IMA as a result of ischemia, the event of reduce oxygen supply prior to cardiomyocytes necrosis, make IMA test good enough to be an earlier cardiac marker (Sbarouni et al.2008a). It was reported that the serum IMA level increased and can be detected within 6-10 minutes after ischemia and returned back to baseline within 6 hours (Bhagavan et al.2003). This could be an advantage of IMA, in comparison to the other conventional cardiac markers such as cardiac troponin and CK-MB, which are necrotic marker and could not be detected until 4-6 hours after onset of chest pain/ischemia (Wu et al.1999). Determination of IMA has been used triage patient who suspected from cardiac ischemia. Low level of serum IMA, would estimate low risk for a cardiac ischemic event and make a rapid consideration to exclude or discharge patient. Low level of serum IMA perhaps indirectly predicts the low level of cardiac troponin (Sbarouni et al.2008a). So, it can make a clear distinct when the negative results from IMA, troponin, and ECG can exclude the patients from ACS (Bhagavan et al.2003). The assay method can be easily and rapidly performed by spectrophotometric method. It has been shown that IMA test has high method sensitivity more than troponin as it gave positive results in 84% of patient who suspect ACS while cardiac troponin could detect only 42% (Takhshid et al.2010). Combination with triple tests including ECG, IMA, and cardiac troponin could increase the negative predictive value for ACS to 96% (Takhshid et al.2010). Furthermore, IMA test might reduce in the number of diagnostic tests such as determination of serum high sensitivity C-reactive proteins (hsCRP), NT-proBNP elevation, and cTnT release (Kazanis et al.2009), invasive imaging, which have

However, it seems like IMA test lack of specificity. High serum IMA level can also be detected in other diseases such as cancer, acute infections, end renal disease, and liver cirrhosis, (Kazanis et al.2009). Therefore, the negative results from cTnT test and IMA allow more confident to exclude the patients, who suspected AMI. However, a positive IMA alone still need further investigation. Moreover, it has been reported that serum IMA level can also be elevated in plasma from healthy subjects, 24-48 hours after exercise (Kim et al.2008). Therefore, utilization of IMA test as cardiac marker for coronary artery disease need to be

The carbonyl (CO) groups in proteins compose of aldehyde and ketone groups. Proteins Carbonyl groups (PC) is a product of oxidative modification on amino acid residues,

no biological variation of IMA regarding race and gender (Govender et al.2008).

high cost (Keating et al.2006).

**4.2 Proteins carbonyl** 

further investigated to ensure the value of the test.

mitochondrial oxidative metabolism becomes inhibited. At this point, a compensatory increase in anaerobic glycolysis for ATP production leads to accumulation of hydrogen ions and lactate, resulting in intracellular acidosis and inhibition of glycolysis (Reimer & Ideker1987). An aerobic glycolysis cannot provide sufficient ATP to meet the demand of myocardium. The depletion of ATP also causes the interruption of cellular ion-pumps and calcium influx to the cells. The excess intracellular calcium activates calcium-dependent proteases such as calpain, calmodulin, generates O2 •- and converts to H2O2. Blood consist of transition metals such as copper and iron, which can interact with O2 •- and H2O2 and form the strong oxidant •OH, which lead to cellular destruction. Proteins, predominantly albumin, are damaged by free radicals especially at amino terminus (N-terminus), resulting in the albumin N-terminal derivatives. Human serum albumin, a major protein in circulation, consists of 585 amino acid residues with half life in circulation approximately 19 days. The metal binding properties of albumin depend on the three dimensional structure binding sites, which are distributed over the molecule (Bar-Or et al.2001b;Takahashi et al.1987). The modification of albumin during ischemia is independent on cell death, and can be an early biomarker for such an early stage of ischemia.

Fig. 2. The amino acid sequences (A) and molecular structure of albumin (B). This figure is modified from figure3 of Takahashi et al. (Takahashi et al.1987). Proteins albumin is oxidatively modified at NH2 terminal of albumin.

Determination of serum or plasma IMA can be performed by Albumin Cobalt Binding (ACB) method. In 2003, US Food and drug Administration (FDA) approved that ACB method is the diagnostic test for acute myocardial infarction (Van et al.2010). The principle of ACB method is based on the principle of free radicals, which is generated during ischemia, alters the metal ions binding capacity of serum albumin (Sbarouni et al.2008a) (Figure 3). The test is currently called IMA test instead of the ACB test. It has been reported that IMA test could be used for screening the patients with chest pain, who suspected AMI, at the emergency department, excluded patients from the other causes of chest pain (Bar-Or et al.2000). Many studies show that IMA levels increased in patients with acute coronary syndrome and also elevated in myocardial ischemia (Sbarouni et al.2008a). The analytical sensitivity of the test is 13 u/ml, with 98% recovery (Sbarouni et al.2008a). Moreover, there were reports mentioned of non-interfering effect from bilirubin, hemoglobin, cholesterol, total proteins, and number of cardiac drugs (Govender et al.2008). It was also reported that no biological variation of IMA regarding race and gender (Govender et al.2008).

According to the ability to detect IMA as a result of ischemia, the event of reduce oxygen supply prior to cardiomyocytes necrosis, make IMA test good enough to be an earlier cardiac marker (Sbarouni et al.2008a). It was reported that the serum IMA level increased and can be detected within 6-10 minutes after ischemia and returned back to baseline within 6 hours (Bhagavan et al.2003). This could be an advantage of IMA, in comparison to the other conventional cardiac markers such as cardiac troponin and CK-MB, which are necrotic marker and could not be detected until 4-6 hours after onset of chest pain/ischemia (Wu et al.1999). Determination of IMA has been used triage patient who suspected from cardiac ischemia. Low level of serum IMA, would estimate low risk for a cardiac ischemic event and make a rapid consideration to exclude or discharge patient. Low level of serum IMA perhaps indirectly predicts the low level of cardiac troponin (Sbarouni et al.2008a). So, it can make a clear distinct when the negative results from IMA, troponin, and ECG can exclude the patients from ACS (Bhagavan et al.2003). The assay method can be easily and rapidly performed by spectrophotometric method. It has been shown that IMA test has high method sensitivity more than troponin as it gave positive results in 84% of patient who suspect ACS while cardiac troponin could detect only 42% (Takhshid et al.2010). Combination with triple tests including ECG, IMA, and cardiac troponin could increase the negative predictive value for ACS to 96% (Takhshid et al.2010). Furthermore, IMA test might reduce in the number of diagnostic tests such as determination of serum high sensitivity C-reactive proteins (hsCRP), NT-proBNP elevation, and cTnT release (Kazanis et al.2009), invasive imaging, which have high cost (Keating et al.2006).

However, it seems like IMA test lack of specificity. High serum IMA level can also be detected in other diseases such as cancer, acute infections, end renal disease, and liver cirrhosis, (Kazanis et al.2009). Therefore, the negative results from cTnT test and IMA allow more confident to exclude the patients, who suspected AMI. However, a positive IMA alone still need further investigation. Moreover, it has been reported that serum IMA level can also be elevated in plasma from healthy subjects, 24-48 hours after exercise (Kim et al.2008). Therefore, utilization of IMA test as cardiac marker for coronary artery disease need to be further investigated to ensure the value of the test.

#### **4.2 Proteins carbonyl**

194 Oxidative Stress and Diseases

mitochondrial oxidative metabolism becomes inhibited. At this point, a compensatory increase in anaerobic glycolysis for ATP production leads to accumulation of hydrogen ions and lactate, resulting in intracellular acidosis and inhibition of glycolysis (Reimer & Ideker1987). An aerobic glycolysis cannot provide sufficient ATP to meet the demand of myocardium. The depletion of ATP also causes the interruption of cellular ion-pumps and calcium influx to the cells. The excess intracellular calcium activates calcium-dependent proteases such as calpain, calmodulin, generates O2•- and converts to H2O2. Blood consist of transition metals such as copper and iron, which can interact with O2•- and H2O2 and form the strong oxidant •OH, which lead to cellular destruction. Proteins, predominantly albumin, are damaged by free radicals especially at amino terminus (N-terminus), resulting in the albumin N-terminal derivatives. Human serum albumin, a major protein in circulation, consists of 585 amino acid residues with half life in circulation approximately 19 days. The metal binding properties of albumin depend on the three dimensional structure binding sites, which are distributed over the molecule (Bar-Or et al.2001b;Takahashi et al.1987). The modification of albumin during ischemia is independent on cell death, and can

Fig. 2. The amino acid sequences (A) and molecular structure of albumin (B). This figure is modified from figure3 of Takahashi et al. (Takahashi et al.1987). Proteins albumin is

Determination of serum or plasma IMA can be performed by Albumin Cobalt Binding (ACB) method. In 2003, US Food and drug Administration (FDA) approved that ACB

be an early biomarker for such an early stage of ischemia.

oxidatively modified at NH2 terminal of albumin.

The carbonyl (CO) groups in proteins compose of aldehyde and ketone groups. Proteins Carbonyl groups (PC) is a product of oxidative modification on amino acid residues,

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 197

Donne et al.2003b;le-Donne et al.2006). Spectrophotometric assay for PC can be perform by 2,4-dinitrophenylhydrazine (DNPH spectrophotometric method), which based on the formation of a stable dinitrophenyl (DNP) hydrazone reacts in acidic pH solution. Spectrophotometric DNPH assay showed high sensitivity detection of carbonyl content level in purified proteins (le-Donne et al.2003b;Levine et al.1994). This method does not require any expensive or specialized equipments. It has been shown that the serum level of PC increased rapidly in blood stream and still remained at least 24 hours (Mutlu-Turkoglu et al.2005). Mutlu-Turkoglu *et al.* demonstrated that serum PC level increased in coronary artery disease, atherosclerotic lesion in human, and during ischemia-reperfusion (Mutlu-Turkoglu et al.2005). As it has also been reported that PC can be generated following the onset of myocardial infarction (Paton et al.2010) suggested the diagnostic value of PC and may be used as biomarker for coronary artery disease. The stability of this assay remained in hours and days, whereas lipid oxidation products can be removed within minutes (Mutlu-Turkoglu et al.2005). In AMI, serum PC level was significantly increased when compared with normal control (Paton et al.2010). Diagnosed value of PC in human can also be used in environmental studies, monitoring in subjects who exposed to the bunker oil (Almroth et al.2009). Although PC has been proven as a sensitive marker, but it was shown to has less specificity, similar to IMA. The increasing in serum level of PC can be detected in other human diseases such as Alzheimer' disease, cataract genesis, chronic hepatitis, diabetics, cigarette smoker, and after doing exercise (Mutlu-Turkoglu et al.2005). According to the time consuming and proteins precipitation is required in spectrophotometric method, this technique is inappropriate to determine the PC level in large number of clinical samples. Therefore, other techniques, for example ELISA, were developed. Recently, the findings from our study demonstrated that PC could be an early marker for myocardial ischemia. Serum PC level in non-ST elevation myocardial infarction (NSTEMI) was significantly higher than that in ST elevation myocardial infarction (STEMI) and healthy controls, suggesting that PC is an early marker. Moreover, combinatorial determination of PC with IMA helps to improve the diagnostic power of these two markers (Maneewong et al.2011).

As mentioned in the previous section, free radicals can attack DNA and cause molecular

the nucleobases of DNA, such as guanine, form the C8-hydroxyguanine (8-OHGua) or its nucleoside form deoxyguanosine (8-hydroxy-2'-deoxyguanine) or so called 8-OH-dG. The 8- OH-dG can be further oxidized and produced 8-oxo-7,8-dihydro-2'-deoxyguanine or 8 oxodG (Valavanidis et al.2009) (Figure 4). Although the other nucleic acids in DNA

oxidative modified nucleic acids in DNA, and known as a potential biomarker of carcinogenesis (Kasai1997). These days, the 8-OHdG can be a biomarker of oxidative stress, aging and cancer. This molecule can be measured and analyzed using high sensitivity by high performance liquid chromatography (HPLC), gas-chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry- mass spectrometry (LC-MS-MS), immunohistochemical methods, and single cell electrophoresis (Griffiths et al.2002;Halliwell & Whiteman2004;Collins et al.2004). There are many reports that the elevation of 8-OHdG related to some pathological disorders, for example, urinary 8-OHdG has been established as a marker to evaluate oxidative stress in carcinogenic exposure, environment pollutants

OH in the same manner, the 8-oxodG is the major form of

OH with

structural alterations of DNA and result in DNA strand break. The interaction of •

**4.3 8-hydroxy-2'-deoxyguanosine** 

molecules can react with •

especially proline, arginine, lysine, and threonine from free radicals reactions, forming protein carbonyl groups [53]. In addition, protein carbonyl groups can be generated from an indirect mechanism of the hydroxyl radical-mediated oxidation of lipids (Figure 4). The product of lipid peroxidation, which will be described latter in this chapter, can diffuse across cell membrane, allowing the reactive aldehyde-containing lipids, which will covalently modified proteins in the cell (Grimsrud et al.2008). Proteins oxidation changes proteins functions by changing in pattern of proteins folding, which is important for their activity, decrease catalytic activity of enzyme, and finally breakdown of proteins by proteases (Almroth et al.2009). The cleavage of proteins may occur by either the amidation pathway or by oxidation of glutamyl side chain. Redox cycling cation such as Fe2+ or Cu2+ can bind to cation binding location on proteins. Free radical attack by H2O2 or O2 • can transform side chain of amine groups into carbonyls.

Fig. 3. Principle of Albumin cobalt binding (ACB) assay. In physiological conditions, albumin capable of binding with metal ions such as cobalt, copper, and nickel, at amino terminal end of the protein. During ischemia, N-terminal portion of albumin, especially at aspartate-alanine-histidine-lysine sequences, is modified and result in the reduction in albumin-metal ions binding ability.

Laboratory measurement of PC can be performed by variety of methods, for example, spectrophotometric assay, HPLC, ELISA, and immnunoblotting (le-Donne et al.2003a;leDonne et al.2003b;le-Donne et al.2006). Spectrophotometric assay for PC can be perform by 2,4-dinitrophenylhydrazine (DNPH spectrophotometric method), which based on the formation of a stable dinitrophenyl (DNP) hydrazone reacts in acidic pH solution. Spectrophotometric DNPH assay showed high sensitivity detection of carbonyl content level in purified proteins (le-Donne et al.2003b;Levine et al.1994). This method does not require any expensive or specialized equipments. It has been shown that the serum level of PC increased rapidly in blood stream and still remained at least 24 hours (Mutlu-Turkoglu et al.2005). Mutlu-Turkoglu *et al.* demonstrated that serum PC level increased in coronary artery disease, atherosclerotic lesion in human, and during ischemia-reperfusion (Mutlu-Turkoglu et al.2005). As it has also been reported that PC can be generated following the onset of myocardial infarction (Paton et al.2010) suggested the diagnostic value of PC and may be used as biomarker for coronary artery disease. The stability of this assay remained in hours and days, whereas lipid oxidation products can be removed within minutes (Mutlu-Turkoglu et al.2005). In AMI, serum PC level was significantly increased when compared with normal control (Paton et al.2010). Diagnosed value of PC in human can also be used in environmental studies, monitoring in subjects who exposed to the bunker oil (Almroth et al.2009). Although PC has been proven as a sensitive marker, but it was shown to has less specificity, similar to IMA. The increasing in serum level of PC can be detected in other human diseases such as Alzheimer' disease, cataract genesis, chronic hepatitis, diabetics, cigarette smoker, and after doing exercise (Mutlu-Turkoglu et al.2005). According to the time consuming and proteins precipitation is required in spectrophotometric method, this technique is inappropriate to determine the PC level in large number of clinical samples. Therefore, other techniques, for example ELISA, were developed. Recently, the findings from our study demonstrated that PC could be an early marker for myocardial ischemia. Serum PC level in non-ST elevation myocardial infarction (NSTEMI) was significantly higher than that in ST elevation myocardial infarction (STEMI) and healthy controls,

### **4.3 8-hydroxy-2'-deoxyguanosine**

196 Oxidative Stress and Diseases

especially proline, arginine, lysine, and threonine from free radicals reactions, forming protein carbonyl groups [53]. In addition, protein carbonyl groups can be generated from an indirect mechanism of the hydroxyl radical-mediated oxidation of lipids (Figure 4). The product of lipid peroxidation, which will be described latter in this chapter, can diffuse across cell membrane, allowing the reactive aldehyde-containing lipids, which will covalently modified proteins in the cell (Grimsrud et al.2008). Proteins oxidation changes proteins functions by changing in pattern of proteins folding, which is important for their activity, decrease catalytic activity of enzyme, and finally breakdown of proteins by proteases (Almroth et al.2009). The cleavage of proteins may occur by either the amidation pathway or by oxidation of glutamyl side chain. Redox cycling cation such as Fe2+ or Cu2+ can bind to cation binding location on proteins. Free radical attack by H2O2 or O2

Fig. 3. Principle of Albumin cobalt binding (ACB) assay. In physiological conditions, albumin capable of binding with metal ions such as cobalt, copper, and nickel, at amino terminal end of the protein. During ischemia, N-terminal portion of albumin, especially at aspartate-alanine-histidine-lysine sequences, is modified and result in the reduction in

Laboratory measurement of PC can be performed by variety of methods, for example, spectrophotometric assay, HPLC, ELISA, and immnunoblotting (le-Donne et al.2003a;le-

albumin-metal ions binding ability.

transform side chain of amine groups into carbonyls.

• can

> As mentioned in the previous section, free radicals can attack DNA and cause molecular structural alterations of DNA and result in DNA strand break. The interaction of • OH with the nucleobases of DNA, such as guanine, form the C8-hydroxyguanine (8-OHGua) or its nucleoside form deoxyguanosine (8-hydroxy-2'-deoxyguanine) or so called 8-OH-dG. The 8- OH-dG can be further oxidized and produced 8-oxo-7,8-dihydro-2'-deoxyguanine or 8 oxodG (Valavanidis et al.2009) (Figure 4). Although the other nucleic acids in DNA molecules can react with •OH in the same manner, the 8-oxodG is the major form of oxidative modified nucleic acids in DNA, and known as a potential biomarker of carcinogenesis (Kasai1997). These days, the 8-OHdG can be a biomarker of oxidative stress, aging and cancer. This molecule can be measured and analyzed using high sensitivity by high performance liquid chromatography (HPLC), gas-chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry- mass spectrometry (LC-MS-MS), immunohistochemical methods, and single cell electrophoresis (Griffiths et al.2002;Halliwell & Whiteman2004;Collins et al.2004). There are many reports that the elevation of 8-OHdG related to some pathological disorders, for example, urinary 8-OHdG has been established as a marker to evaluate oxidative stress in carcinogenic exposure, environment pollutants

> suggesting that PC is an early marker. Moreover, combinatorial determination of PC with IMA helps to improve the diagnostic power of these two markers (Maneewong et al.2011).

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 199

samples, for example, HPLC with electrochemical detection, HPLC with a UV detector, GC-MS, and immunohistochemistry (Halliwell & Whiteman2004;Ohshima et al.2006;Sawa et al.2006). Many studies showed that 8-nitroguanine increased in inflammation, carcinogenesis, and cigarette smoke (Hiraku2010). However, there are no any evidence of the increasing in 8-nitoguanine in coronary heart diseases. Therefore, further investigation

One of the most frequently used biomarkers indicating lipid peroxidation is plasma concentration of malondialdehyde (MDA). This molecule is one of the end products of lipid peroxidation in the cell membrane or in low-density lipoproteins (LDL) (Ogino & Wang2007). Quantification of plasma MDA level can be performed by thiobarbituric acid (TBA) test (Nielsen et al.1997). TBA-reactive substances (TBARS) formed in plasma, urine, or tissue samples that need to be calibrated by sample pretreatment procedure, which forms a red adduct with 2 molecules of TBA (MDA-TBA2). The adducted compounds are separated by an HPLC method, which originally described by Wong *et. al*.(Wong et al.1987) and Carbonneau *et. al*. (Carbonneau et al.1991). The GC-MS method has been used to analyze the plasma MDA as well (Yeo et al.1994). It has been reported that the plasma from patients with coronary artery disease also had higher level of MDA than the healthy subjects, suggested that MDA could be one of the candidate biomarkers for coronary artery disease (Rajesh et al.2011;Rao & Kiran2011;Mogadam et al.2008;Pasupathi et al.2009). A recent report also suggested that serum levels of TBARS, which was determined by reverse-phase HPLC and spectrophotometric method, were a good predictive marker in patients with

Isoprostanes are a complex family of compounds generated from arachidonic acid via a free radical's catalyzed mechanism. This compound was firstly discovered in 1990 by Morrow *et. al.* who discovered prostaglandin-F2-like compounds, and termed this newly discovered compound as *F2-isoprostanes* (Morrow et al.1990). The F2 -isoprostanes can be generated by

Determination of F2-isoprostanes is similar to other techniques measuring the products from lipid peroxidation including GC-MS, which might be associated with an immunoaffinity extraction, GC-tandem MS, and LC-tandem MS (Halliwell2000). Although these techniques have high specificity, the budget cost of these techniques is the impediment of their routine use (Milne et al.2005). Determination of 15- F2t-IsoP in urine samples, by radioimmunoassay, has been validated and easier alternative to GC-MS. In addition, the new technique is developed,

F2-isoprostanes can be measured in varieties of clinical samples, for example urine, plasma, bronchoalveolar lavage fluid, bile, cerebrospinal, seminal and pericardial fluids (Iuliano et al.2001;Lindsay et al.1999;Delanty et al.1997;Cipollone et al.2000;Reilly et al.1997). In addition, F2-isoprostanes can be detected in normal tissues, including umbilical cords (Chu et al.2003). The level of F2-isoprostanes increased in cigarette smoking, similar to other oxidative modified molecules, which is known that the increasing in smoking can cause the

for example enzyme-immunoassay for detecting F2-isoprostanes (Milne et al.2005).

of 8-nitoguanine in coronary heart diseases is still need to be further investigated.

stable coronary artery disease (Walter et al.2004).

the oxidative induced peroxidation of arachidonic acid (Figure 5).

**4.5 F2-Isoprostanes** 

**4.4 MDA** 

and cigarette smoking (Kiyosawa et al.1990;Asami et al.1996). Elevation of 8-OHdG has been found in the plasma and myocardium of the patients with heart failure (Kono et al.2006). Recently, Himmetoglu *et al*. reported that the plasma level of 8-OHdG increased in patients with myocardial infarction and the level of this molecule decreased after reperfusion therapy in patients with MI, suggested that 8-OHdG could possibly be biomarker for monitoring or determining the prognosis of the patients (Himmetoglu et al.2009). In addition, Nagayoshi *et al* demonstrated that the urinary levels of 8-OHdG were significantly higher in cardiac patients when assessed the serial alteration of oxidative stress of patients with AMI (Nagayoshi et al.2005).

Fig. 4. Mechanism of ROS stimulates lipids peroxidation induced proteins carbonylation (Grimsrud et al.2008). Proteins carbonylation can be induced by the oxidative modification of polyunsaturated fatty acids (PUFA), which then undergo to lipid peroxidation reaction generating products such as ,-unsaturated aldehyde 4-HNE. These molecules act as electrophiles in the covalently modification of proteins via non-enzymatic addition reactions.

It is known that reactive nitrogen species or RNS such as nitric oxide (NO) and peroxynitrite (ONOO-) can modify the molecular structure of DNA (Ohshima et al.2006). The 8 nitroguanine is the example of nucleic acid in DNA, which can be oxidatively modified by RNS. There is overwhelm data showed that 8-nitroguanine is undetectable in normal tissues, which indicating that this molecule may be a candidate as a biomarker for DNA damage induced by RNS (Akaike et al.2003;Ma et al.2004;Horiike et al.2005;Pinlaor et al.2004). Several techniques have been developed for determining 8-nitroguanine in clinical samples, for example, HPLC with electrochemical detection, HPLC with a UV detector, GC-MS, and immunohistochemistry (Halliwell & Whiteman2004;Ohshima et al.2006;Sawa et al.2006). Many studies showed that 8-nitroguanine increased in inflammation, carcinogenesis, and cigarette smoke (Hiraku2010). However, there are no any evidence of the increasing in 8-nitoguanine in coronary heart diseases. Therefore, further investigation of 8-nitoguanine in coronary heart diseases is still need to be further investigated.

#### **4.4 MDA**

198 Oxidative Stress and Diseases

and cigarette smoking (Kiyosawa et al.1990;Asami et al.1996). Elevation of 8-OHdG has been found in the plasma and myocardium of the patients with heart failure (Kono et al.2006). Recently, Himmetoglu *et al*. reported that the plasma level of 8-OHdG increased in patients with myocardial infarction and the level of this molecule decreased after reperfusion therapy in patients with MI, suggested that 8-OHdG could possibly be biomarker for monitoring or determining the prognosis of the patients (Himmetoglu et al.2009). In addition, Nagayoshi *et al* demonstrated that the urinary levels of 8-OHdG were significantly higher in cardiac patients when assessed the serial alteration of oxidative stress of patients

Fig. 4. Mechanism of ROS stimulates lipids peroxidation induced proteins carbonylation (Grimsrud et al.2008). Proteins carbonylation can be induced by the oxidative modification of polyunsaturated fatty acids (PUFA), which then undergo to lipid peroxidation reaction generating products such as ,-unsaturated aldehyde 4-HNE. These molecules act as electrophiles in the covalently modification of proteins via non-enzymatic addition

It is known that reactive nitrogen species or RNS such as nitric oxide (NO) and peroxynitrite (ONOO-) can modify the molecular structure of DNA (Ohshima et al.2006). The 8 nitroguanine is the example of nucleic acid in DNA, which can be oxidatively modified by RNS. There is overwhelm data showed that 8-nitroguanine is undetectable in normal tissues, which indicating that this molecule may be a candidate as a biomarker for DNA damage induced by RNS (Akaike et al.2003;Ma et al.2004;Horiike et al.2005;Pinlaor et al.2004). Several techniques have been developed for determining 8-nitroguanine in clinical

with AMI (Nagayoshi et al.2005).

reactions.

One of the most frequently used biomarkers indicating lipid peroxidation is plasma concentration of malondialdehyde (MDA). This molecule is one of the end products of lipid peroxidation in the cell membrane or in low-density lipoproteins (LDL) (Ogino & Wang2007). Quantification of plasma MDA level can be performed by thiobarbituric acid (TBA) test (Nielsen et al.1997). TBA-reactive substances (TBARS) formed in plasma, urine, or tissue samples that need to be calibrated by sample pretreatment procedure, which forms a red adduct with 2 molecules of TBA (MDA-TBA2). The adducted compounds are separated by an HPLC method, which originally described by Wong *et. al*.(Wong et al.1987) and Carbonneau *et. al*. (Carbonneau et al.1991). The GC-MS method has been used to analyze the plasma MDA as well (Yeo et al.1994). It has been reported that the plasma from patients with coronary artery disease also had higher level of MDA than the healthy subjects, suggested that MDA could be one of the candidate biomarkers for coronary artery disease (Rajesh et al.2011;Rao & Kiran2011;Mogadam et al.2008;Pasupathi et al.2009). A recent report also suggested that serum levels of TBARS, which was determined by reverse-phase HPLC and spectrophotometric method, were a good predictive marker in patients with stable coronary artery disease (Walter et al.2004).

#### **4.5 F2-Isoprostanes**

Isoprostanes are a complex family of compounds generated from arachidonic acid via a free radical's catalyzed mechanism. This compound was firstly discovered in 1990 by Morrow *et. al.* who discovered prostaglandin-F2-like compounds, and termed this newly discovered compound as *F2-isoprostanes* (Morrow et al.1990). The F2 -isoprostanes can be generated by the oxidative induced peroxidation of arachidonic acid (Figure 5).

Determination of F2-isoprostanes is similar to other techniques measuring the products from lipid peroxidation including GC-MS, which might be associated with an immunoaffinity extraction, GC-tandem MS, and LC-tandem MS (Halliwell2000). Although these techniques have high specificity, the budget cost of these techniques is the impediment of their routine use (Milne et al.2005). Determination of 15- F2t-IsoP in urine samples, by radioimmunoassay, has been validated and easier alternative to GC-MS. In addition, the new technique is developed, for example enzyme-immunoassay for detecting F2-isoprostanes (Milne et al.2005).

F2-isoprostanes can be measured in varieties of clinical samples, for example urine, plasma, bronchoalveolar lavage fluid, bile, cerebrospinal, seminal and pericardial fluids (Iuliano et al.2001;Lindsay et al.1999;Delanty et al.1997;Cipollone et al.2000;Reilly et al.1997). In addition, F2-isoprostanes can be detected in normal tissues, including umbilical cords (Chu et al.2003). The level of F2-isoprostanes increased in cigarette smoking, similar to other oxidative modified molecules, which is known that the increasing in smoking can cause the

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 201

Fig. 6. Metabolic pathways of isoprostane (Cracowski & Durand2006). Free radicals interact with arachidonic acid produce arachidonyl radicals; these molecules were continue to the lipids peroxidation reaction and generated four types of prostaglandin-H2-like compounds,

Fig. 7. The Maillard reaction is chemical reaction between a reducing sugar, such as glucose, and amino acid groups. The outcome from this reaction is the formation of unstable Schiff bases that can transform to an Amadori products, which can rearrange to form advanced

which subsequently reduced to be 4 prostaglandin F2.

glycation endproducts (AGEs) (Zieman & Kass2004).

oxidative stress (Reilly et al.1996;Morrow et al.1995). The measurement of isoprostanes in biological fluids has prompted clinical investigations on the pathophysiological role of lipid peroxidation in cardiovascular diseases. In coronary artery disease, the quantified isoprostanes was mostly in 15- F2t-IsoP and 5- F2t-IsoP, which can be measured in urine samples (Haschke et al.2007). The urinary level of 15- F2t-IsoP and 5- F2t-IsoP was found to increase in, unstable angina, reperfusion following myocardial infarction and cardiopulmonary bypass, coronary angioplasty (Sakamoto et al.2002). These findings suggested that isoprostane could be biomarker for coronary artery disease.

Fig. 5. The Chemical reaction of 2'-deoxyguanosine with hydroxyl radicals. The Oxidative modification reactions of 2'-doxyguanosine cause by hydroxyl radicals. This radical adducts are oxidized to 8-hydroxy-2'-deoxyguanosine (8-OHdG), or it tautomer 8-oxo-7-hydro-2' deoxyguanosine (8-oxodG).

#### **4.6 Advanced Glycation End-Produces (AGEs)**

Advanced glycation end-produces (AGEs) are products of non-enzymatic glycation of proteins by reducing sugars (Zieman & Kass2004). AGEs was firstly discovered in early of 1900s by Louis Camille Mailard by the non-enzymatic chemical reaction between reducing sugars and amino groups on proteins to form protein-protein crosslink and complex yellowbrown pigments (Zieman & Kass2004). The Maillard reaction occurs when the reducing sugars, such as glucose, react with an amine groups, result in the formation of an unstable Shift bases (Figure 6). The produced unstable Shift bases that transform to an Amadori product, which can further rearrange to form advanced glycation endproducts (AGEs) capable of crosslinking proteins (Figure 7, 8).

oxidative stress (Reilly et al.1996;Morrow et al.1995). The measurement of isoprostanes in biological fluids has prompted clinical investigations on the pathophysiological role of lipid peroxidation in cardiovascular diseases. In coronary artery disease, the quantified isoprostanes was mostly in 15- F2t-IsoP and 5- F2t-IsoP, which can be measured in urine samples (Haschke et al.2007). The urinary level of 15- F2t-IsoP and 5- F2t-IsoP was found to increase in, unstable angina, reperfusion following myocardial infarction and cardiopulmonary bypass, coronary angioplasty (Sakamoto et al.2002). These findings

Fig. 5. The Chemical reaction of 2'-deoxyguanosine with hydroxyl radicals. The Oxidative modification reactions of 2'-doxyguanosine cause by hydroxyl radicals. This radical adducts are oxidized to 8-hydroxy-2'-deoxyguanosine (8-OHdG), or it tautomer 8-oxo-7-hydro-2'-

Advanced glycation end-produces (AGEs) are products of non-enzymatic glycation of proteins by reducing sugars (Zieman & Kass2004). AGEs was firstly discovered in early of 1900s by Louis Camille Mailard by the non-enzymatic chemical reaction between reducing sugars and amino groups on proteins to form protein-protein crosslink and complex yellowbrown pigments (Zieman & Kass2004). The Maillard reaction occurs when the reducing sugars, such as glucose, react with an amine groups, result in the formation of an unstable Shift bases (Figure 6). The produced unstable Shift bases that transform to an Amadori product, which can further rearrange to form advanced glycation endproducts (AGEs)

deoxyguanosine (8-oxodG).

**4.6 Advanced Glycation End-Produces (AGEs)** 

capable of crosslinking proteins (Figure 7, 8).

suggested that isoprostane could be biomarker for coronary artery disease.

Fig. 6. Metabolic pathways of isoprostane (Cracowski & Durand2006). Free radicals interact with arachidonic acid produce arachidonyl radicals; these molecules were continue to the lipids peroxidation reaction and generated four types of prostaglandin-H2-like compounds, which subsequently reduced to be 4 prostaglandin F2.

Fig. 7. The Maillard reaction is chemical reaction between a reducing sugar, such as glucose, and amino acid groups. The outcome from this reaction is the formation of unstable Schiff bases that can transform to an Amadori products, which can rearrange to form advanced glycation endproducts (AGEs) (Zieman & Kass2004).

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 203

following 2 major categories based on the results from electrocardiogram (ECG) including those whose ECG show ST-elevation that is diagnostic of acute ST-elevation myocardial infarction (STEMI) and those who present other patterns of ECG change, but not categorized in STEMI, called non- ST elevation ACS (NSTEACS). The latter include unstable angina (UA) and non-ST-elevation myocardial infarction (NSTEMI) (Morrow et al.2007;Wiviott & Braunwald2004). The ECG in NSTEMI can be interpreted in the way that the artery is only partially blocked, or only transiently occlusive, and results in coronary ischemia without the appearance of ST-segment elevation. The ECG is the most readily available tool for diagnosing STEMI. However, the limitation of ECG is usually occur in acute chest pain, according to the low sensitivity of the baseline ECG, which is only 60% (Panteghini2002). Undetectable of ST-elevation of ECG lead to delay in final diagnosis and affect treatment and clinical outcome. Therefore, determination of high sensitivity, specificity and early ischemic biomarker is useful for diagnosis of acute myocardial ischemia, particularly in NSTEMI patients. There are many types of conventional cardiac biomarkers such as creatine kinase- MB isoenzyme (CK-MB), cardiac troponin I or T (cTnI, cTnT). These conventional cardiac markers are known to release in blood circulation as a result of cellular necrosis, not early enough to detect the early phase of myocardial ischemia that may not excess the reference range of biochemical markers of myocardial necrosis. Determination of plasma/serum cardiac biomarkers in patients, who has arrived hospital after the onset of symptoms, may not be detected. Therefore, screening method, for measuring the early cardiac biomarkers that actually reflect the early phase of cellular injury, is extremely useful. The more rapid diagnosis, the more effective intervention and treatment, the less cost for hospital stay, secondary prevention and reduce effective budget for screening test to exclude

Creatine kinase (CK) is an enzyme responsible for transferring a phosphate group from ATP to creatine. The molecular weight of this enzyme is approximately 80,000 dalton. It is composed of M and/or B subunits build up at least 3 isoenzymes including CK-BB, CK-MB, and CK-MM or CK-1, CK-2 and CK-3, respectively. Moreover, there are one more isoenzymes that have been reported e.g. mitochondrial isoenzyme. CK-2 or CK-MB is sometime called the cardiac isoenzyme as it is predominant isoenzyme in myocardium, whereas there is only 2-5% in skeletal muscle. CK-MB can be found in large amount of infarcted myocardium and can rise up in the circulation within 3-6 hours after ischemia, peaks in 10-48 hours, and returns to normal within 72 hours (Wu et al.1999). However, an elevated serum CK-MB may occur in people with severe skeletal muscle damage (such as in muscular dystrophy, accident) and renal disease (Green et al.1986). In such cases, the ratio of CK-MB per total CK, or CK index, is very helpful. If the index is under 4%, a nonmyocardial source of high CK-MB should be concerned. One of the limitations of determining serum CK-MB is undetectable of minimal myocardial injury, late rise in the

Cardiac Troponin is a useful cardiac marker, localized in myofibrils. Troponin consists of 3 subunits including inhibitory subunit (cTnI), calcium binding subunit (cTnC), and tropomyosin binding subunit (cTnT). The troponin complex is located on the thin filaments of the contractile muscles and regulates the calcium mediated interaction of myocardial myosin and actin filaments. The specificity, sensitivity, and reliability of troponin assay for diagnosed myocardial necrosis make cardiac troponin be an ideal cardiac marker. In addition, the minimal concentration in serum cardiac troponin, from healthy people without

myocardial infarction patients (Figure 9)

setting of AMI.

Fig. 8. The formation of collagen-collagen-AGEs crosslinking, this figure was modified from Zieman et. al. AGEs from the Maillard reaction can accelerate enzymatically crosslinking reaction of collagens strands (Zieman & Kass2004).

It has been known that AGEs play important role in the pathogenesis of diabetic vascular complications, as they lead to an abnormal leakage of proteins from the circulation and a progressive constriction of the luminal area of vessel (Brownlee et al.1988;Makita et al.1991;Ono et al.1998). Moreover, AGEs have been recognized as factors in the pathogenesis of other diabetic complications, such as nephropathy and retinopathy (Makita et al.1991;Ono et al.1998). In addition, the level of serum concentration of AGEs was associated with severity of coronary arthrosclerosis and development of this pathology in type 2 diabetic patients (Kiuchi et al.2001). Interestingly, it has been reported that the serum level of AGEs were elevated and correlated significantly with oxidized LDL, especially in diabetic patients (Lopes-Virella et al.2011). A recent evidence of 18-year study showed that the serum level of AGEs could predict the mortality from cardiovascular disease and coronary heart disease in non-diabetic women (Kilhovd et al.2005).

Determination of AGEs is similar to other techniques, used in determining other oxidative modified biomarkers, such as HPLC, GC-MS, ELISA, and immunochemistry (Ogino & Wang2007). The accuracy and reproducibility of these techniques have not been well examined according to lack of universally established unit of measurement, for comparing study findings from different laboratories (Ogino & Wang2007). Furthermore, AGEs has been reported to increase in cigarette smoking, similar to the findings found in other biomarkers (Nicholl & Bucala1998).

#### **5. Early cardiac biomarkers for diagnostic acute coronary syndrome**

The biochemical markers have been routinely used to assess myocardial damage, especially in patients suspected with ACS. World health organization criteria, formulated in 1979, have classically diagnosed in ACS patients if the patient present two (probable) or three (definite) diagnostic criteria of acute coronary syndrome. The criteria including clinical history of ischemic type chest pain lasting for more than 20 minutes, changes in serial ECG tracings, and elevation of serum cardiac biomarkers. Patients with ACS are subdivided into the

Fig. 8. The formation of collagen-collagen-AGEs crosslinking, this figure was modified from Zieman et. al. AGEs from the Maillard reaction can accelerate enzymatically crosslinking

It has been known that AGEs play important role in the pathogenesis of diabetic vascular complications, as they lead to an abnormal leakage of proteins from the circulation and a progressive constriction of the luminal area of vessel (Brownlee et al.1988;Makita et al.1991;Ono et al.1998). Moreover, AGEs have been recognized as factors in the pathogenesis of other diabetic complications, such as nephropathy and retinopathy (Makita et al.1991;Ono et al.1998). In addition, the level of serum concentration of AGEs was associated with severity of coronary arthrosclerosis and development of this pathology in type 2 diabetic patients (Kiuchi et al.2001). Interestingly, it has been reported that the serum level of AGEs were elevated and correlated significantly with oxidized LDL, especially in diabetic patients (Lopes-Virella et al.2011). A recent evidence of 18-year study showed that the serum level of AGEs could predict the mortality from cardiovascular disease and coronary heart disease in

Determination of AGEs is similar to other techniques, used in determining other oxidative modified biomarkers, such as HPLC, GC-MS, ELISA, and immunochemistry (Ogino & Wang2007). The accuracy and reproducibility of these techniques have not been well examined according to lack of universally established unit of measurement, for comparing study findings from different laboratories (Ogino & Wang2007). Furthermore, AGEs has been reported to increase in cigarette smoking, similar to the findings found in other

The biochemical markers have been routinely used to assess myocardial damage, especially in patients suspected with ACS. World health organization criteria, formulated in 1979, have classically diagnosed in ACS patients if the patient present two (probable) or three (definite) diagnostic criteria of acute coronary syndrome. The criteria including clinical history of ischemic type chest pain lasting for more than 20 minutes, changes in serial ECG tracings, and elevation of serum cardiac biomarkers. Patients with ACS are subdivided into the

**5. Early cardiac biomarkers for diagnostic acute coronary syndrome** 

reaction of collagens strands (Zieman & Kass2004).

non-diabetic women (Kilhovd et al.2005).

biomarkers (Nicholl & Bucala1998).

following 2 major categories based on the results from electrocardiogram (ECG) including those whose ECG show ST-elevation that is diagnostic of acute ST-elevation myocardial infarction (STEMI) and those who present other patterns of ECG change, but not categorized in STEMI, called non- ST elevation ACS (NSTEACS). The latter include unstable angina (UA) and non-ST-elevation myocardial infarction (NSTEMI) (Morrow et al.2007;Wiviott & Braunwald2004). The ECG in NSTEMI can be interpreted in the way that the artery is only partially blocked, or only transiently occlusive, and results in coronary ischemia without the appearance of ST-segment elevation. The ECG is the most readily available tool for diagnosing STEMI. However, the limitation of ECG is usually occur in acute chest pain,

according to the low sensitivity of the baseline ECG, which is only 60% (Panteghini2002). Undetectable of ST-elevation of ECG lead to delay in final diagnosis and affect treatment and clinical outcome. Therefore, determination of high sensitivity, specificity and early ischemic biomarker is useful for diagnosis of acute myocardial ischemia, particularly in NSTEMI patients. There are many types of conventional cardiac biomarkers such as creatine kinase- MB isoenzyme (CK-MB), cardiac troponin I or T (cTnI, cTnT). These conventional cardiac markers are known to release in blood circulation as a result of cellular necrosis, not early enough to detect the early phase of myocardial ischemia that may not excess the reference range of biochemical markers of myocardial necrosis. Determination of plasma/serum cardiac biomarkers in patients, who has arrived hospital after the onset of symptoms, may not be detected. Therefore, screening method, for measuring the early cardiac biomarkers that actually reflect the early phase of cellular injury, is extremely useful. The more rapid diagnosis, the more effective intervention and treatment, the less cost for hospital stay, secondary prevention and reduce effective budget for screening test to exclude myocardial infarction patients (Figure 9)

Creatine kinase (CK) is an enzyme responsible for transferring a phosphate group from ATP to creatine. The molecular weight of this enzyme is approximately 80,000 dalton. It is composed of M and/or B subunits build up at least 3 isoenzymes including CK-BB, CK-MB, and CK-MM or CK-1, CK-2 and CK-3, respectively. Moreover, there are one more isoenzymes that have been reported e.g. mitochondrial isoenzyme. CK-2 or CK-MB is sometime called the cardiac isoenzyme as it is predominant isoenzyme in myocardium, whereas there is only 2-5% in skeletal muscle. CK-MB can be found in large amount of infarcted myocardium and can rise up in the circulation within 3-6 hours after ischemia, peaks in 10-48 hours, and returns to normal within 72 hours (Wu et al.1999). However, an elevated serum CK-MB may occur in people with severe skeletal muscle damage (such as in muscular dystrophy, accident) and renal disease (Green et al.1986). In such cases, the ratio of CK-MB per total CK, or CK index, is very helpful. If the index is under 4%, a nonmyocardial source of high CK-MB should be concerned. One of the limitations of determining serum CK-MB is undetectable of minimal myocardial injury, late rise in the setting of AMI.

Cardiac Troponin is a useful cardiac marker, localized in myofibrils. Troponin consists of 3 subunits including inhibitory subunit (cTnI), calcium binding subunit (cTnC), and tropomyosin binding subunit (cTnT). The troponin complex is located on the thin filaments of the contractile muscles and regulates the calcium mediated interaction of myocardial myosin and actin filaments. The specificity, sensitivity, and reliability of troponin assay for diagnosed myocardial necrosis make cardiac troponin be an ideal cardiac marker. In addition, the minimal concentration in serum cardiac troponin, from healthy people without

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 205

Fig. 9. The kinetics curve of conventional biomarkers. The rise and fall pattern of conventional cardiac biomarkers such as myoglobin, CK-MB, and cTnT. These markers release to circulation many hours after the onset of chest pain, post ischemia. Novel early markers, probably oxidatively modified markers, which release into blood stream, right

Early cardiac biomarkers are essential for diagnosis of acute coronary syndrome. Conventional markers might not early enough to detect the early phase of cellular injury according to ischemia. Many oxidatively modified biomolecules were studied and known to have potential as cardiac markers. Further intensive investigation of these cardiac markers, especially the diagnostic power, is very helpful and can be used in real clinical investigation

This study was supported by Naresuan University research endowment fund, and Faculty

after the onset of ischemia, will helpful.

of Allied Health Sciences, Naresuan University**.** 

**6. Conclusion** 

of coronary artery disease.

**7. Acknowledgement** 

cardiac disease, cannot be detected (Adams, III et al.1993). Among those 3 forms of cardiac troponin, troponin C cannot be used as cardiac marker according to non-specific expression in various tissues, not only the heart (Adams, III et al.1993). Cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are 2 forms that normally used as cardiac marker. It has been known that, after myocardial ischemia, elevated cTnT last for 10 days to 2 weeks. The advantages of cTnT include highly sensitive for detecting MI, cTnT level may also help to risk stratify afterward, qualitative test run in 10 minutes. In contrast, the disadvantage of using cTnT assays as cardiac markers due to non specificity of cTnT, which can be found in unstable angina, chronic renal failure (Wood et al.2003). The cTnI is also being an ideal marker for ACS, according to high sensitivity and specificity of this marker in AMI. However, these two markers could not counted as the early marker, as it need increase around 6 hours after ischemia, until the level of cTnT is significantly higher than normal level.

The oxidatively modified markers such as PC and IMA have been proven as potential cardiac marker for diagnosis of ACS. However, determining of oxidative modified markers is not specific for myocardial ischemia. For example, it has been reported that the plasma level of IMA can be increased in cerebral, gastrointestinal intestinal and skeletal muscular ischemia as well as myocardial ischemia (Matthews et al.1990;Siegel et al.1995). Therefore, it is recommended that the interpretation of a positive IMA finding should be combined with other clinical indices (Shen et al.2010). Recently, our study showed the usefulness of determining serum IMA and PC content level to identify acute myocardial infarction, particularly in STEMI. The level of both serum IMA and PC content were significantly higher in STEMI compared to healthy control and determination of serum IMA level in combination of serum PC content level improved test performance (Kumphune et al.2010). However, the results from our recent study reported that diagnosis of NSTEMI was not improved by combination of serum IMA and PC level, in contrast, individual determination of serum PC content showed a good area under ROC curve and high PPV for NSTEMI diagnosis (Maneewong et al.2011). Charpentier *et. al.* also demonstrated in a large cohort study of patients admitted to an emergency department for chest pain that IMA did not provide valuable information for ACS diagnosis (Charpentier et al.2010). The possible explanation is NSTEMI patients did not have major myocardial necrosis, unlike in patients with STEMI. Therefore, the minor myocardial damage possibly has less degree of ROS mediated proteins oxidation.

Another limitation of using oxidative modified biomarkers is the interpretation in elder patients. It has been reported that oxidative modified forms of proteins were accumulated during aging (Berlett & Stadtman1997). For example, increases in proteins carbonyls occur in rat hepatocytes, drosophila, brain, and kidney of mice and in brain tissue of gerbils (Beal2002). In humans proteins carbonyls increase with age in brain, muscle, and human eye lens (Beal2002). The carbonyl content of human fibroblasts also increases as a function of age of the donor (Beal2002).

There are some reports determining other oxidative modified molecules, such as 8-OH-dG, isoprostane, and AGEs, in ACS. However, those reports were indicated only the incidences of elevated markers in ACS, but not the efficiency of the test. Therefore, determination of analytical method efficiency of those markers is challenge and need to be further investigated.

Fig. 9. The kinetics curve of conventional biomarkers. The rise and fall pattern of conventional cardiac biomarkers such as myoglobin, CK-MB, and cTnT. These markers release to circulation many hours after the onset of chest pain, post ischemia. Novel early markers, probably oxidatively modified markers, which release into blood stream, right after the onset of ischemia, will helpful.

#### **6. Conclusion**

204 Oxidative Stress and Diseases

cardiac disease, cannot be detected (Adams, III et al.1993). Among those 3 forms of cardiac troponin, troponin C cannot be used as cardiac marker according to non-specific expression in various tissues, not only the heart (Adams, III et al.1993). Cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are 2 forms that normally used as cardiac marker. It has been known that, after myocardial ischemia, elevated cTnT last for 10 days to 2 weeks. The advantages of cTnT include highly sensitive for detecting MI, cTnT level may also help to risk stratify afterward, qualitative test run in 10 minutes. In contrast, the disadvantage of using cTnT assays as cardiac markers due to non specificity of cTnT, which can be found in unstable angina, chronic renal failure (Wood et al.2003). The cTnI is also being an ideal marker for ACS, according to high sensitivity and specificity of this marker in AMI. However, these two markers could not counted as the early marker, as it need increase around 6 hours after ischemia, until the level

The oxidatively modified markers such as PC and IMA have been proven as potential cardiac marker for diagnosis of ACS. However, determining of oxidative modified markers is not specific for myocardial ischemia. For example, it has been reported that the plasma level of IMA can be increased in cerebral, gastrointestinal intestinal and skeletal muscular ischemia as well as myocardial ischemia (Matthews et al.1990;Siegel et al.1995). Therefore, it is recommended that the interpretation of a positive IMA finding should be combined with other clinical indices (Shen et al.2010). Recently, our study showed the usefulness of determining serum IMA and PC content level to identify acute myocardial infarction, particularly in STEMI. The level of both serum IMA and PC content were significantly higher in STEMI compared to healthy control and determination of serum IMA level in combination of serum PC content level improved test performance (Kumphune et al.2010). However, the results from our recent study reported that diagnosis of NSTEMI was not improved by combination of serum IMA and PC level, in contrast, individual determination of serum PC content showed a good area under ROC curve and high PPV for NSTEMI diagnosis (Maneewong et al.2011). Charpentier *et. al.* also demonstrated in a large cohort study of patients admitted to an emergency department for chest pain that IMA did not provide valuable information for ACS diagnosis (Charpentier et al.2010). The possible explanation is NSTEMI patients did not have major myocardial necrosis, unlike in patients with STEMI. Therefore, the minor myocardial damage possibly has less degree of ROS

Another limitation of using oxidative modified biomarkers is the interpretation in elder patients. It has been reported that oxidative modified forms of proteins were accumulated during aging (Berlett & Stadtman1997). For example, increases in proteins carbonyls occur in rat hepatocytes, drosophila, brain, and kidney of mice and in brain tissue of gerbils (Beal2002). In humans proteins carbonyls increase with age in brain, muscle, and human eye lens (Beal2002). The carbonyl content of human fibroblasts also increases as a function of age

There are some reports determining other oxidative modified molecules, such as 8-OH-dG, isoprostane, and AGEs, in ACS. However, those reports were indicated only the incidences of elevated markers in ACS, but not the efficiency of the test. Therefore, determination of analytical method efficiency of those markers is challenge and need to be further

of cTnT is significantly higher than normal level.

mediated proteins oxidation.

of the donor (Beal2002).

investigated.

Early cardiac biomarkers are essential for diagnosis of acute coronary syndrome. Conventional markers might not early enough to detect the early phase of cellular injury according to ischemia. Many oxidatively modified biomolecules were studied and known to have potential as cardiac markers. Further intensive investigation of these cardiac markers, especially the diagnostic power, is very helpful and can be used in real clinical investigation of coronary artery disease.

#### **7. Acknowledgement**

This study was supported by Naresuan University research endowment fund, and Faculty of Allied Health Sciences, Naresuan University**.** 

Oxidatively Modified Biomolecules: An Early Biomarker for Acute Coronary Artery Disease 207

Brownlee, M., Cerami, A., and Vlassara, H., 1988. Advanced glycosylation end products in

Buja, L.M., 2005. Myocardial ischemia and reperfusion injury. Cardiovasc.Pathol. 14, 170-

Carbonneau, M.A., Peuchant, E., Sess, D., Canioni, P., and Clerc, M., 1991. Free and bound

Charpentier, S., Ducasse, J.L., Cournot, M., Maupas-Schwalm, F., Elbaz, M., Baixas, C.,

Chu, K.O., Wang, C.C., Rogers, M.S., and Pang, C.P., 2003. Quantifying F2-isoprostanes in

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Cracowski, J.L. and Durand, T., 2006. Cardiovascular pharmacology and physiology of the

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8-nitroguanine, a marker of nitrative nucleic acid damage, by high-performance liquid chromatography-electrochemical detection coupled with immunoaffinity purification: association with cigarette smoking. Free Radic.Biol.Med. 40, 711-720. Sbarouni, E., Georgiadou, P., Kremastinos, D.T., and Voudris, V., 2008a. Ischemia modified

albumin: is this marker of ischemia ready for prime time use? Hellenic.J Cardiol.

and Kremastinos, D.T., 2008b. Ischemia modified albumin in relation to pharmacologic stress testing in coronary artery disease. Clin Chim.Acta 396, 58-61.

Kremastinos, D.T., 2008c. The ischemia-modified albumin in relation to pacemaker

P.O., and Brecker, S.J., 2007. Evaluation of ischaemia-modified albumin as a marker of myocardial ischaemia in end-stage renal disease. Clin Sci.(Lond) 113, 25-32. Shen, X.L., Lin, C.J., Han, L.L., Lin, L., Pan, L., and Pu, X.D., 2010. Assessment of ischemia-

modified albumin levels for emergency room diagnosis of acute coronary

Normal post-race antimyosin myocardial scintigraphy in asymptomatic marathon runners with elevated serum creatine kinase MB isoenzyme and troponin T levels.

modified albumin is a sensitive marker of myocardial ischemia after percutaneous


**Section 4** 

**Diabetes Mellitus** 


## **Section 4**

**Diabetes Mellitus** 

214 Oxidative Stress and Diseases

Yeo, H.C., Helbock, H.J., Chyu, D.W., and Ames, B.N., 1994. Assay of malondialdehyde in

Zieman, S.J. and Kass, D.A., 2004. Advanced glycation endproduct crosslinking in the

391-396.

Drugs 64, 459-470.

biological fluids by gas chromatography-mass spectrometry. Anal.Biochem 220,

cardiovascular system: potential therapeutic target for cardiovascular disease.

**10** 

*Malaysia* 

**Oxidative Stress in Diabetes Mellitus:** 

The most recent statistics indicate that the global prevalence of diabetes mellitus, estimated as 366 million in 2011, will increase to 522 million by 2030 (Whiting et al., 2011). Diabetes mellitus is a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion and/or insulin action (ADA, 2011). There are basically two types of diabetes mellitus: type 1 and type 2 diabetes mellitus. Type 1 diabetes mellitus, an autoimmune disease, is characterized by the loss of pancreatic β-cells resulting in absolute insulin deficiency. It accounts for about 5-10 % of all newly diagnosed diabetes mellitus (ADA, 2011). On the other hand, type 2 diabetes mellitus is characterized by insulin resistance and β cell dysfunction. It remains the most common form of diabetes mellitus and constitutes about 90-95 % of all diabetes cases (ADA, 2011). In spite of the availability of different classes of oral hypoglycemic drugs, the incidence of microvascular complications (nephropathy, retinopathy and neuropathy) and macrovascular complications atherosclerosis, coronary artery disease, peripheral arterial disease and stroke continues to

rise unabated in diabetic patients, even with treatment (Roglic and Unwin, 2010).

Pharmacological agents with different mechanisms of action are often combined to achieve optimal glycemic control (Turner et al., 1999). However, despite the use of multiple drugs, a lot of diabetic patients do not achieve the optimal glycemic goal (Turner et al., 1999). The Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) have demonstrated that intensive treatment of hyperglycemia reduces risk of developing microvascular and macrovascular complications (DCCT, 1993, UKPDS, 1998). However, recent findings indicate that intensive treatment of hyperglycemia is associated with higher incidence of weight gain, hypoglycemia and mortality than conventional therapy (Ismail-Beigi et al., 2010). These findings suggest that intensive therapy is not only detrimental, but limited and may not be the best. Besides, these findings may suggest that it is high time "hyperglycemia alone" was made the "culprit" in the management of diabetes mellitus. Generally, hypoglycemic drugs are incapable to prevent

**1. Introduction** 

**Is There a Role for Hypoglycemic** 

**Drugs and/or Antioxidants?** 

Omotayo O. Erejuwa *Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia,* 

### **Oxidative Stress in Diabetes Mellitus: Is There a Role for Hypoglycemic Drugs and/or Antioxidants?**

Omotayo O. Erejuwa *Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, Malaysia* 

#### **1. Introduction**

The most recent statistics indicate that the global prevalence of diabetes mellitus, estimated as 366 million in 2011, will increase to 522 million by 2030 (Whiting et al., 2011). Diabetes mellitus is a metabolic disorder of multiple etiology characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion and/or insulin action (ADA, 2011). There are basically two types of diabetes mellitus: type 1 and type 2 diabetes mellitus. Type 1 diabetes mellitus, an autoimmune disease, is characterized by the loss of pancreatic β-cells resulting in absolute insulin deficiency. It accounts for about 5-10 % of all newly diagnosed diabetes mellitus (ADA, 2011). On the other hand, type 2 diabetes mellitus is characterized by insulin resistance and β cell dysfunction. It remains the most common form of diabetes mellitus and constitutes about 90-95 % of all diabetes cases (ADA, 2011). In spite of the availability of different classes of oral hypoglycemic drugs, the incidence of microvascular complications (nephropathy, retinopathy and neuropathy) and macrovascular complications atherosclerosis, coronary artery disease, peripheral arterial disease and stroke continues to rise unabated in diabetic patients, even with treatment (Roglic and Unwin, 2010).

Pharmacological agents with different mechanisms of action are often combined to achieve optimal glycemic control (Turner et al., 1999). However, despite the use of multiple drugs, a lot of diabetic patients do not achieve the optimal glycemic goal (Turner et al., 1999). The Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) have demonstrated that intensive treatment of hyperglycemia reduces risk of developing microvascular and macrovascular complications (DCCT, 1993, UKPDS, 1998). However, recent findings indicate that intensive treatment of hyperglycemia is associated with higher incidence of weight gain, hypoglycemia and mortality than conventional therapy (Ismail-Beigi et al., 2010). These findings suggest that intensive therapy is not only detrimental, but limited and may not be the best. Besides, these findings may suggest that it is high time "hyperglycemia alone" was made the "culprit" in the management of diabetes mellitus. Generally, hypoglycemic drugs are incapable to prevent

Oxidative Stress in Diabetes Mellitus:

Halliwell, 2011).

physiological concentrations by converting O2

concentration of H2O2 (Halliwell and Gutteridge, 2007).

**3. Oxidative stress and its sources in diabetes mellitus** 

sources of ROS and oxidative stress in diabetes mellitus include:

while less than 2% of O2 consumed is converted to O2

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 219

Gutteridge, 2007, Fukai and Ushio-Fukai, 2011). CAT metabolizes H2O2 to O2 and H2O (Halliwell and Gutteridge, 2007). CAT exerts two enzymatic activities, depending on the concentrations of its substrate (H2O2) (Scibior and Czeczot, 2006). It elicits a catalytic function at high concentrations of H2O2, whereas it produces a peroxidatic effect at lower concentrations of H2O2 (Scibior and Czeczot, 2006). GPx enzymatically reduces H2O2 to O2 and H2O using a hydrogen donor, glutathione (GSH) which is oxidized to glutathione disulfide (GSSG) (Lubos et al., 2011). Unlike CAT, GPx has a broader spectrum of substrates, detoxifying organic hydroperoxides and lipid peroxides (Lubos et al., 2011). However, CAT compared to GPx has a higher KM for H2O2 and thus can protect against a higher

The other antioxidant enzymes include glutathione reductase (GR), glutathione Stransferase (GST), peroxiredoxin, thioredoxin and thioredoxinreductase (Andreyev et al., 2005). GR scavenges O2•<sup>−</sup> and •OH non-enzymatically or by serving as an electron donor to certain enzymes involved in the metabolism of ROS (Andreyev et al., 2005, Slonchak and Obolens'ka, 2009). GR helps to regenerate GSH via reduction of oxidized glutathione (GSSG) (Slonchak and Obolens'ka, 2009). GST comprises a family of multifunctional phase II biotransformation enzymes with a broad spectrum for a variety of substrates including epoxides, carcinogens, mutagens, 4-hydroxy-2-nonenal and malondialdehyde (MDA) (Andreyev et al., 2005, Slonchak and Obolens'ka, 2009). It catalyzes the conjugation of many electrophilic compounds with GSH (Andreyev et al., 2005). These enzymes work cooperatively together in order to scavenge RS and xenobiotics and thereby protect cells against oxidative damage (Halliwell, 2011). Generally, antioxidant enzymes differ from one another in terms of structure, tissue distribution, co-factor requirement, function, substrate specificity and affinity. The uniqueness of the antioxidant defense system lies in its capability to maintain the RS at certain steady-state levels thereby create and maintain a balance between the beneficial and injurious effects of RS (Lushchak, 2010). More detailed information on oxidative stress including free radicals, ROS, RNS, antioxidant enzymes, antioxidants, antioxidant defense system, and markers of oxidative stress can be obtained from following references (Sies, 1991, Johansen et al., 2005, Halliwell and Gutteridge, 2007,

Under normal conditions and in most diseases including diabetes mellitus, mitochondria are the main source of RS and oxidative stress. Under physiological conditions, e.g. during mitochondrial oxidative metabolism, the bulk of oxygen (O2) utilized is reduced to H2O,

ROS because it may be converted to other RS including H2O2, OH and ONOO− (Andreyev et al., 2005). Within physiological conditions, the body is protected from the detrimental effects of these free radicals by a network of antioxidant defense system. However, this defense system becomes impaired in diabetes mellitus and is further exacerbated by chronic hyperglycemia which generates ROS, resulting in oxidative stress. Some of the various

•<sup>−</sup> within the

•<sup>−</sup> to H2O2, a more stable ROS (Halliwell and

•− (Brand, 2010). O2•− is an important

peroxidase (GPx) (Halliwell, 2011). SOD maintains the cellular levels of O2

pancreas degeneration, worsening of glycemic control and diabetic complications (DCCT, 1993, UKPDS, 1998, Turner et al., 1999, Ball et al., 2000). All these have been linked to increased oxidative stress in diabetes mellitus (Figueroa-Romero et al., 2008, Giacco and Brownlee, 2010). The aim of this chapter is to shed more light on the prospective of managing diabetes mellitus more effectively by targeting both "hyperglycemia and oxidative stress simultaneously". The data presented in this chapter convincingly suggest that the current management of diabetes mellitus may be improved upon by targeting hyperglycemia and oxidative stress as two potential therapeutic targets in diabetes mellitus.

#### **2. General overview of oxidative stress**

Considering the aim of this chapter, the general concept of oxidative stress will be discussed only in brief. Oxidative stress can defined as an "imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage" (Sies, 1991). According to Halliwell, "oxidative stress refers to a serious imbalance between reactive species production and antioxidant defenses" (Halliwell and Gutteridge, 2007). It occurs due to an increased generation and/or reduced elimination of reactive species (RS) by the antioxidant defense system. Oxidative stress is usually associated with oxidative damage, which is defined as "the biomolecular damage caused by attack of RS upon the constituents of living organisms" (Halliwell and Gutteridge, 2007). Most of the biologically relevant RS are either reactive oxygen species (ROS) or reactive nitrogen species (RNS). ROS include free radicals such as superoxide (O2•−) and hydroxyl (•OH), and non-free radicals such as hydrogen peroxide (H2O2). Reactive nitrogen species include free radicals such as nitric oxide (•NO) and nitrogen dioxide (NO2 •−), and non-free radicals such as peroxynitrite (OONO−) (Sies, 1991, Halliwell and Gutteridge, 2007). The generation of RS by aerobic organisms may occur as by products of metabolism (e.g. during operation of electron transfer chains), intentionally (e.g. during inflammation), or as a result of accidents of chemistry (such as the autoxidation of unstable biomolecules, e.g. dopamine) (Halliwell, 2011). Of all the RS, significant roles of O2•−, •NO, and OONO<sup>−</sup> have been implicated in diabetic cardiovascular complications (Johansen et al., 2005). In order to prevent oxidative damage, it is important that excess RS is eliminated from the cells. Oxidative damage to cellular components impairs cellular functions. Besides their toxicities, ROS are also required in certain conditions and for physiological functions. For instance, during inflammation, the phagocytes release ROS which kill invading bacteria. ROS generated during mild or moderate exercise constitute part of the mechanism of exercise- or training-induced adaptation (Sachdev and Davies, 2008).

The ability of cells or tissues to withstand oxidative stress is largely dependent on the efficiency of the overall antioxidant defense system to scavenge excess RS, without compromising the physiological roles of ROS (Halliwell, 2011). The antioxidant defense system consists of endogenously-synthesized antioxidants which include antioxidant enzymes, glutathione, vitamins, small molecules and micronutrients (Sies, 1991, Halliwell and Gutteridge, 2007). An antioxidant is defined as "any substance that delays, prevents or removes oxidative damage to a target molecule" (Halliwell and Gutteridge, 2007). Antioxidant enzymes are enzymes which scavenge or eliminate a variety of RS including those generated during biological processes (Sies, 1991, Halliwell and Gutteridge, 2007). The main antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and glutathione

pancreas degeneration, worsening of glycemic control and diabetic complications (DCCT, 1993, UKPDS, 1998, Turner et al., 1999, Ball et al., 2000). All these have been linked to increased oxidative stress in diabetes mellitus (Figueroa-Romero et al., 2008, Giacco and Brownlee, 2010). The aim of this chapter is to shed more light on the prospective of managing diabetes mellitus more effectively by targeting both "hyperglycemia and oxidative stress simultaneously". The data presented in this chapter convincingly suggest that the current management of diabetes mellitus may be improved upon by targeting hyperglycemia and oxidative stress as two potential therapeutic targets in diabetes mellitus.

Considering the aim of this chapter, the general concept of oxidative stress will be discussed only in brief. Oxidative stress can defined as an "imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage" (Sies, 1991). According to Halliwell, "oxidative stress refers to a serious imbalance between reactive species production and antioxidant defenses" (Halliwell and Gutteridge, 2007). It occurs due to an increased generation and/or reduced elimination of reactive species (RS) by the antioxidant defense system. Oxidative stress is usually associated with oxidative damage, which is defined as "the biomolecular damage caused by attack of RS upon the constituents of living organisms" (Halliwell and Gutteridge, 2007). Most of the biologically relevant RS are either reactive oxygen species (ROS) or reactive nitrogen species (RNS). ROS include free radicals

peroxide (H2O2). Reactive nitrogen species include free radicals such as nitric oxide (•NO)

1991, Halliwell and Gutteridge, 2007). The generation of RS by aerobic organisms may occur as by products of metabolism (e.g. during operation of electron transfer chains), intentionally (e.g. during inflammation), or as a result of accidents of chemistry (such as the autoxidation of unstable biomolecules, e.g. dopamine) (Halliwell, 2011). Of all the RS, significant roles of O2•−, •NO, and OONO<sup>−</sup> have been implicated in diabetic cardiovascular complications (Johansen et al., 2005). In order to prevent oxidative damage, it is important that excess RS is eliminated from the cells. Oxidative damage to cellular components impairs cellular functions. Besides their toxicities, ROS are also required in certain conditions and for physiological functions. For instance, during inflammation, the phagocytes release ROS which kill invading bacteria. ROS generated during mild or moderate exercise constitute part of the mechanism of exercise- or training-induced

The ability of cells or tissues to withstand oxidative stress is largely dependent on the efficiency of the overall antioxidant defense system to scavenge excess RS, without compromising the physiological roles of ROS (Halliwell, 2011). The antioxidant defense system consists of endogenously-synthesized antioxidants which include antioxidant enzymes, glutathione, vitamins, small molecules and micronutrients (Sies, 1991, Halliwell and Gutteridge, 2007). An antioxidant is defined as "any substance that delays, prevents or removes oxidative damage to a target molecule" (Halliwell and Gutteridge, 2007). Antioxidant enzymes are enzymes which scavenge or eliminate a variety of RS including those generated during biological processes (Sies, 1991, Halliwell and Gutteridge, 2007). The main antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and glutathione

•−) and hydroxyl (•OH), and non-free radicals such as hydrogen

•−), and non-free radicals such as peroxynitrite (OONO−) (Sies,

**2. General overview of oxidative stress** 

such as superoxide (O2

and nitrogen dioxide (NO2

adaptation (Sachdev and Davies, 2008).

peroxidase (GPx) (Halliwell, 2011). SOD maintains the cellular levels of O2 •<sup>−</sup> within the physiological concentrations by converting O2•<sup>−</sup> to H2O2, a more stable ROS (Halliwell and Gutteridge, 2007, Fukai and Ushio-Fukai, 2011). CAT metabolizes H2O2 to O2 and H2O (Halliwell and Gutteridge, 2007). CAT exerts two enzymatic activities, depending on the concentrations of its substrate (H2O2) (Scibior and Czeczot, 2006). It elicits a catalytic function at high concentrations of H2O2, whereas it produces a peroxidatic effect at lower concentrations of H2O2 (Scibior and Czeczot, 2006). GPx enzymatically reduces H2O2 to O2 and H2O using a hydrogen donor, glutathione (GSH) which is oxidized to glutathione disulfide (GSSG) (Lubos et al., 2011). Unlike CAT, GPx has a broader spectrum of substrates, detoxifying organic hydroperoxides and lipid peroxides (Lubos et al., 2011). However, CAT compared to GPx has a higher KM for H2O2 and thus can protect against a higher concentration of H2O2 (Halliwell and Gutteridge, 2007).

The other antioxidant enzymes include glutathione reductase (GR), glutathione Stransferase (GST), peroxiredoxin, thioredoxin and thioredoxinreductase (Andreyev et al., 2005). GR scavenges O2•<sup>−</sup> and •OH non-enzymatically or by serving as an electron donor to certain enzymes involved in the metabolism of ROS (Andreyev et al., 2005, Slonchak and Obolens'ka, 2009). GR helps to regenerate GSH via reduction of oxidized glutathione (GSSG) (Slonchak and Obolens'ka, 2009). GST comprises a family of multifunctional phase II biotransformation enzymes with a broad spectrum for a variety of substrates including epoxides, carcinogens, mutagens, 4-hydroxy-2-nonenal and malondialdehyde (MDA) (Andreyev et al., 2005, Slonchak and Obolens'ka, 2009). It catalyzes the conjugation of many electrophilic compounds with GSH (Andreyev et al., 2005). These enzymes work cooperatively together in order to scavenge RS and xenobiotics and thereby protect cells against oxidative damage (Halliwell, 2011). Generally, antioxidant enzymes differ from one another in terms of structure, tissue distribution, co-factor requirement, function, substrate specificity and affinity. The uniqueness of the antioxidant defense system lies in its capability to maintain the RS at certain steady-state levels thereby create and maintain a balance between the beneficial and injurious effects of RS (Lushchak, 2010). More detailed information on oxidative stress including free radicals, ROS, RNS, antioxidant enzymes, antioxidants, antioxidant defense system, and markers of oxidative stress can be obtained from following references (Sies, 1991, Johansen et al., 2005, Halliwell and Gutteridge, 2007, Halliwell, 2011).

#### **3. Oxidative stress and its sources in diabetes mellitus**

Under normal conditions and in most diseases including diabetes mellitus, mitochondria are the main source of RS and oxidative stress. Under physiological conditions, e.g. during mitochondrial oxidative metabolism, the bulk of oxygen (O2) utilized is reduced to H2O, while less than 2% of O2 consumed is converted to O2 •− (Brand, 2010). O2•− is an important ROS because it may be converted to other RS including H2O2, OH and ONOO− (Andreyev et al., 2005). Within physiological conditions, the body is protected from the detrimental effects of these free radicals by a network of antioxidant defense system. However, this defense system becomes impaired in diabetes mellitus and is further exacerbated by chronic hyperglycemia which generates ROS, resulting in oxidative stress. Some of the various sources of ROS and oxidative stress in diabetes mellitus include:

Oxidative Stress in Diabetes Mellitus:

**3.4 Hyperinsulinemia** 

**3.5 Insulin deficiency** 

2003).

upregulation and uncoupling of ROS-generating enzymes.

thereby contributes to or exacerbate oxidative stress.

**3.6 Other sources of ROS and oxidative stress in diabetes mellitus** 

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 221

the eNOS has been found to be uncoupled in diabetic blood vessels where it produces O2•− instead of •NO (Wiernsperger, 2003). Hyperglycemia might play an important role in the

Insulin resistance characterized by hyperinsulinemia is frequent in the majority of the individuals with type 2 diabetic mellitus. Insulin induces the release of H2O2 when activating its receptors (Wiernsperger, 2003). Even though H2O2 is a non-free radical, it is membrane permeable and can diffuse to other sites, different from its site of production (Sies, 1991, Halliwell and Gutteridge, 2007). Chronic hyperinsulinemia together with the impaired antioxidant defenses in diabetes will lead to inefficient scavenging of H2O2. In the presence of transition metals such as copper and iron, H2O2 undergoes Fenton reaction to generate •OH which is implicated in the initiation and propagation of lipid peroxidation. Thus, hyperinsulinemia via H2O2 formation may increase RS and contribute to oxidative stress and damage in diabetes mellitus. Besides, insulin stimulates the release of many neurotransmitters, via activation of sympathetic nervous system. Many of these neurotransmitters are known to generate ROS and induce oxidative stress (Wiernsperger,

Insulin deficiency is frequently observed in the diabetic patients. In some type 2 diabetic patients, insulin deficiency may be so severe such that the injection of exogenous insulin is required to control hyperglycemia (Turner et al., 1999, Cook et al., 2005). Insulin deficiency augments the activity of fatty acyl coenzyme A oxidase. Fatty acyl coenzyme A oxidase is an enzyme which is responsible for the oxidation of fatty acids, resulting in the increased generation of H2O2 (Schonfeld et al., 2009). H2O2 is well recognized for its role in exerting deleterious effects on cellular components such as proteins, nucleic acid and lipids including polyunsaturated fatty acids (PUFAs) (Sies, 1991, Halliwell and Gutteridge, 2007). These injurious effects of H2O2 can be mediated directly or indirectly through •OH formation or reaction with transition metals (such as copper or iron) to form toxic aldehydes, which are highly susceptible to free radical attack (Sies, 1991, Halliwell and Gutteridge, 2007). This sets up a chain reaction which further propagates the formation of more free radicals or RS and

Diabetes mellitus is characterized by lipid abnormalities such as elevated LDL and cholesterol (ADA, 2011). These abnormalities are further exacerbated by the increased oxidizing environment which enhances the formation of oxidized LDLs (oxLDLs), glycated LDL and oxysterols (formed from the oxidation of cholesterol) (Johansen et al., 2005). These oxidized lipid products bind to specific receptor proteins or activate inflammatory proteins which generate ROS (Johansen et al., 2005). The import of oxLDLs in the vascular wall is the main mechanism by which ROS and oxidative stress induce atherosclerosis (Sies, 1991, Wiernsperger, 2003, Halliwell and Gutteridge, 2007). Evidence indicates that the levels of certain pro-oxidants such as ferritin and homocysteine are elevated in diabetes (Penckofer et

#### **3.1 Hyperglycemia**

Chronic hyperglycemia is the hallmark of diabetes mellitus. Evidence implicates mitochondrial generation of O2 •<sup>−</sup> as a significant source of ROS (Andreyev et al., 2005). With persistent hyperglycemia, disproportionate amounts of glucose are delivered to the cells. This results in enhanced glucose flux through glycolysis and the tricarboxylic acid (TCA) cycle (Drews et al., 2010). This leads to an overdrive of the mitochondrial electron transport chain, which generates greater amounts of O2•<sup>−</sup> more than mitochondrial SOD can dismutase (Wiernsperger, 2003, Brand, 2010). This tilts the normal delicate balance between mitochondrial ROS production and mitochondrial ROS degradation in favor of mitochondrial ROS generation, and oxidative stress ensues (Brand, 2010). Evidence indicates that hyperglycemia-induced excessive mitochondrial O2•<sup>−</sup> production plays an important role in generating other RS in diabetes mellitus (Nishikawa et al., 2000b). Furthermore, glucose autooxidation produces ROS in the presence of transition metal ions (Johansen et al., 2005). Elevated glucose in diabetes may also react with lipids, resulting in the generation of RS (Johansen et al., 2005). Through non-enzymatic glycation reaction, glucose can react with proteins to produce several intermediate products such as Amadori and Sciff base products before generating advanced glycosylation endproducts (AGEs) (Johansen et al., 2005). Evidence indicates ROS are generated at each step of these reactions. Moreover, with excessive glucose in diabetes, it has been shown that glucose is diverted to other pathways such as sorbitol and hexosamine pathways where glucose is metabolized and ROS are generated (Johansen et al., 2005, Figueroa-Romero et al., 2008, Giacco and Brownlee, 2010).

#### **3.2 Impaired antioxidant defense system**

Impaired antioxidant defense system, such as reduced levels of endogenous antioxidants, reduced/enhanced antioxidant enzyme activities and increased levels of oxidative stress markers such as MDA, is very common in diabetes mellitus (Maritim et al., 2003, Johansen et al., 2005, Rahimi et al., 2005, Erejuwa et al., 2010a). The insufficient scavenging of RS as a result of impaired antioxidant defense system in diabetes may contribute to increased oxidative damage. The mechanisms for the impaired antioxidant defenses in diabetes mellitus remain poorly understood. It may be due to non-enzymatic glycation of these enzymes by hyperglycemia and thereby impairs their individual functions. Furthermore, evidence indicates that the antioxidant enzymes produce optimal protection when they function together (Michiels et al., 1994). Thus, glycation of any of these antioxidant enzymes may impair the efficiency of the entire antioxidant defense system or network. For instance, if SOD activity is impaired, this may result in O2 •<sup>−</sup> build-up. On the other hand, if SOD activity is up-regulated, this may result in increased levels of H2O2. In either case, this may affect the activity of CAT or GPx. Similarly, if GR activity is impaired, there may be increased steady-state GSSG levels which will prevent regeneration of GSH, an endogenous antioxidant.

#### **3.3 Increased activity of ROS-generating enzymes**

The activities of many ROS-generating enzymes such as cyclooxygenase, xanthine oxidase, lipoxygenases, myeloperoxidase, NADPH oxidases and eNOS are augmented in diabetes (Wiernsperger, 2003). Besides, evidence implicates a role of endothelial NO synthase (eNOS). The eNOS produces NO which scavenges O2•− in non-diabetic subjects. However, the eNOS has been found to be uncoupled in diabetic blood vessels where it produces O2•− instead of •NO (Wiernsperger, 2003). Hyperglycemia might play an important role in the upregulation and uncoupling of ROS-generating enzymes.

#### **3.4 Hyperinsulinemia**

220 Oxidative Stress and Diseases

Chronic hyperglycemia is the hallmark of diabetes mellitus. Evidence implicates

persistent hyperglycemia, disproportionate amounts of glucose are delivered to the cells. This results in enhanced glucose flux through glycolysis and the tricarboxylic acid (TCA) cycle (Drews et al., 2010). This leads to an overdrive of the mitochondrial electron transport chain, which generates greater amounts of O2•<sup>−</sup> more than mitochondrial SOD can dismutase (Wiernsperger, 2003, Brand, 2010). This tilts the normal delicate balance between mitochondrial ROS production and mitochondrial ROS degradation in favor of mitochondrial ROS generation, and oxidative stress ensues (Brand, 2010). Evidence indicates that hyperglycemia-induced excessive mitochondrial O2•<sup>−</sup> production plays an important role in generating other RS in diabetes mellitus (Nishikawa et al., 2000b). Furthermore, glucose autooxidation produces ROS in the presence of transition metal ions (Johansen et al., 2005). Elevated glucose in diabetes may also react with lipids, resulting in the generation of RS (Johansen et al., 2005). Through non-enzymatic glycation reaction, glucose can react with proteins to produce several intermediate products such as Amadori and Sciff base products before generating advanced glycosylation endproducts (AGEs) (Johansen et al., 2005). Evidence indicates ROS are generated at each step of these reactions. Moreover, with excessive glucose in diabetes, it has been shown that glucose is diverted to other pathways such as sorbitol and hexosamine pathways where glucose is metabolized and ROS are generated (Johansen et al., 2005, Figueroa-Romero et al., 2008, Giacco and Brownlee, 2010).

Impaired antioxidant defense system, such as reduced levels of endogenous antioxidants, reduced/enhanced antioxidant enzyme activities and increased levels of oxidative stress markers such as MDA, is very common in diabetes mellitus (Maritim et al., 2003, Johansen et al., 2005, Rahimi et al., 2005, Erejuwa et al., 2010a). The insufficient scavenging of RS as a result of impaired antioxidant defense system in diabetes may contribute to increased oxidative damage. The mechanisms for the impaired antioxidant defenses in diabetes mellitus remain poorly understood. It may be due to non-enzymatic glycation of these enzymes by hyperglycemia and thereby impairs their individual functions. Furthermore, evidence indicates that the antioxidant enzymes produce optimal protection when they function together (Michiels et al., 1994). Thus, glycation of any of these antioxidant enzymes may impair the efficiency of the entire antioxidant defense system or network. For instance, if SOD activity is impaired, this may result in O2•<sup>−</sup> build-up. On the other hand, if SOD activity is up-regulated, this may result in increased levels of H2O2. In either case, this may affect the activity of CAT or GPx. Similarly, if GR activity is impaired, there may be increased steady-state GSSG levels which will prevent regeneration of GSH, an endogenous

The activities of many ROS-generating enzymes such as cyclooxygenase, xanthine oxidase, lipoxygenases, myeloperoxidase, NADPH oxidases and eNOS are augmented in diabetes (Wiernsperger, 2003). Besides, evidence implicates a role of endothelial NO synthase (eNOS). The eNOS produces NO which scavenges O2•− in non-diabetic subjects. However,

•<sup>−</sup> as a significant source of ROS (Andreyev et al., 2005). With

**3.1 Hyperglycemia** 

mitochondrial generation of O2

**3.2 Impaired antioxidant defense system** 

**3.3 Increased activity of ROS-generating enzymes** 

antioxidant.

Insulin resistance characterized by hyperinsulinemia is frequent in the majority of the individuals with type 2 diabetic mellitus. Insulin induces the release of H2O2 when activating its receptors (Wiernsperger, 2003). Even though H2O2 is a non-free radical, it is membrane permeable and can diffuse to other sites, different from its site of production (Sies, 1991, Halliwell and Gutteridge, 2007). Chronic hyperinsulinemia together with the impaired antioxidant defenses in diabetes will lead to inefficient scavenging of H2O2. In the presence of transition metals such as copper and iron, H2O2 undergoes Fenton reaction to generate •OH which is implicated in the initiation and propagation of lipid peroxidation. Thus, hyperinsulinemia via H2O2 formation may increase RS and contribute to oxidative stress and damage in diabetes mellitus. Besides, insulin stimulates the release of many neurotransmitters, via activation of sympathetic nervous system. Many of these neurotransmitters are known to generate ROS and induce oxidative stress (Wiernsperger, 2003).

#### **3.5 Insulin deficiency**

Insulin deficiency is frequently observed in the diabetic patients. In some type 2 diabetic patients, insulin deficiency may be so severe such that the injection of exogenous insulin is required to control hyperglycemia (Turner et al., 1999, Cook et al., 2005). Insulin deficiency augments the activity of fatty acyl coenzyme A oxidase. Fatty acyl coenzyme A oxidase is an enzyme which is responsible for the oxidation of fatty acids, resulting in the increased generation of H2O2 (Schonfeld et al., 2009). H2O2 is well recognized for its role in exerting deleterious effects on cellular components such as proteins, nucleic acid and lipids including polyunsaturated fatty acids (PUFAs) (Sies, 1991, Halliwell and Gutteridge, 2007). These injurious effects of H2O2 can be mediated directly or indirectly through •OH formation or reaction with transition metals (such as copper or iron) to form toxic aldehydes, which are highly susceptible to free radical attack (Sies, 1991, Halliwell and Gutteridge, 2007). This sets up a chain reaction which further propagates the formation of more free radicals or RS and thereby contributes to or exacerbate oxidative stress.

#### **3.6 Other sources of ROS and oxidative stress in diabetes mellitus**

Diabetes mellitus is characterized by lipid abnormalities such as elevated LDL and cholesterol (ADA, 2011). These abnormalities are further exacerbated by the increased oxidizing environment which enhances the formation of oxidized LDLs (oxLDLs), glycated LDL and oxysterols (formed from the oxidation of cholesterol) (Johansen et al., 2005). These oxidized lipid products bind to specific receptor proteins or activate inflammatory proteins which generate ROS (Johansen et al., 2005). The import of oxLDLs in the vascular wall is the main mechanism by which ROS and oxidative stress induce atherosclerosis (Sies, 1991, Wiernsperger, 2003, Halliwell and Gutteridge, 2007). Evidence indicates that the levels of certain pro-oxidants such as ferritin and homocysteine are elevated in diabetes (Penckofer et

Oxidative Stress in Diabetes Mellitus:

type 2 diabetes (Drews et al., 2010).

**4.2 Role of oxidative stress in insulin resistance** 

transferred to O2 leading to production of O2

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 223

2010). Research within the last decade has recognized the role of glucotoxicity as the main causal determinant of β-cell dysfunction (Drews et al., 2010). The role of glucotoxicity in βcell dysfunction is demonstrated by studies which indicate increased glucose concentrations impair insulin release in non-diabetic subjects (Marchetti et al., 2008). In contrast, improved glycemic control results in improved insulin secretion in patients with type 2 diabetes (Marchetti et al., 2008). The fact that antioxidants reduce or prevent the toxicities of elevated glucose on the expression of insulin mRNA, insulin content and secretion lends support to the role of oxidative stress in mediating the toxic effects of glucotoxicity (Tanaka et al., 1999). Besides glucotoxicity, lipotoxicity may also play a role in impaired β-cell function. Free fatty acids have been shown to uncouple mitochondrial oxidative phosphorylation and increase ROS formation in rat pancreatic islets (Carlsson et al., 1999), deplete pancreatic β-cell insulin content (Bollheimer et al., 1998) and inhibit glucose-induced insulin secretion and biosynthesis (Zhou and Grill, 1994). Both glucotoxicity and lipotoxicity may be involved in β-cell dysfunction in diabetes mellitus (Marchetti et al., 2008). The role of oxidative stress is also implicated in β-cell deficit, as well as increased apoptosis observed in humans with

Insulin resistance precedes the onset of diabetes mellitus and is influenced by factors such as genetic make-up and environmental factors including increased calorie intake, sedentary lifestyle, obesity, pregnancy and abnormally elevated levels of certain hormones (Evans et al., 2003). The role of oxidative stress is implicated in the pathogenesis of insulin resistance (Kim et al., 2006). Both pyruvate and fatty acids can serve as energy substrate in muscle and adipose tissue. Pyruvate is derived from glucose and other sugars, while fatty acids originate from fats. Once transported across the inner mitochondrial membrane, these fuel substrates are converted to acetyl CoA by mitochondrial enzymes. During citric acid cycle, oxidation of the carbon atoms of the acetyl groups in acetyl CoA generates CO2. Besides CO2 formation, the citric acid cycle generates high-energy electrons which are carried by NADH and FADH2. The increased uptake of energy substrate in the muscle and adipose tissue enhances citric acid cycle activity. This in turn generates mitochondrial NADH more than required. This leads to an overdrive of the oxidative phosphorylation which increases the mitochondrial transmembrane proton gradient. These high-energy electrons are then

This sets the stage for oxidative stress (Talior et al., 2003). A recent study showed that the skeletal muscle of high-fat diet-induced insulin-resistant rats liberated more mitochondrial H2O2 and had impaired ability to maintain normal redox balance compared to the data obtained in the skeletal muscle of the insulin-sensitive control rats (Anderson et al., 2009). Similar findings were observed in insulin-resistant, morbidly obese human subjects (Anderson et al., 2009). This study provides strong evidence in support of a role of mitochondrial ROS production and oxidative stress in the pathogenesis of insulin resistance. Various mechanisms by which oxidative stress contributes to insulin resistance have been identified. These include oxidative stress-impaired insulin-induced GLUT4 translocation in adipocytes (Rudich et al., 1998), oxidative stress-induced impairment in insulin stimulation of protein kinase B and glucose transport in adipocytes (Rudich et al., 1999), oxidative stress-induced interactions between the PI3-kinase-dependent signaling pathway and

•− which is further converted to other ROS.

al., 2002). Free iron can increase ROS generation and the oxidation of LDL cholesterol. Similarly, homocysteine can generate ROS in the presence of transition metals which may enhance the oxidation of LDL cholesterol (Penckofer et al., 2002). Another source of ROS in diabetes mellitus is leptin. Elevated levels of leptin are associated with insulin resistance and diabetes mellitus. Evidence implicates a role of leptin in inducing ROS and oxidative stress in aortic endothelial cells in a dose-dependent manner while it produces additive effects with those of glucose (Wiernsperger, 2003). Other potential sources of ROS include aging, menopause, diet and physical activity (Penckofer et al., 2002). Increased ROS is reported in older people. Menopause may also enhance ROS production in older women. The levels of oestrogen, an antioxidant which decreases the oxidation of LDL cholesterol, usually decline during menopause (Penckofer et al., 2002). Aging and menopause may exacerbate oxidative stress since a large number of type 2 diabetics are older men and women. Consumption of foods rich in carbohydrates and fats, as opposed to antioxidant-enriched diets, may also enhance ROS formation. Sedentary lifestyle may predispose to increased ROS generation. This is corroborated by evidence which indicates mild or moderate, but not strenuous, exercise induces antioxidant defenses (Sachdev and Davies, 2008).

#### **4. Role of oxidative stress in the pathogenesis of diabetes mellitus**

Evidence implicates the role of oxidative stress in the different stages of the development of diabetes mellitus, starting from the pre-diabetes state, impaired glucose tolerance, postprandial hyperglycemia, mild diabetes and finally to overt diabetes mellitus (Ceriello et al., 1998). Loss of β-cell function, resulting from impaired secretory capacity and increased apoptosis, is a main occurrence in the pathogenesis of both types of diabetes (Drews et al., 2010). Besides β-cell dysfunction, insulin resistance is also a major characteristic feature of type 2 diabetes mellitus (Evans et al., 2003). Oxidative stress plays an important role in the pathogenesis of both β-cell dysfunction and insulin resistance (Evans et al., 2003, Drews et al., 2010).

#### **4.1 Role of oxidative stress in β-cell dysfunction**

The β-cells express low level of antioxidant enzymes such as SOD, CAT and GPx and thereby increase their susceptibility to oxidative stress (Tiedge et al., 1997). Increased mitochondrial ROS production in the β-cells results from enhanced glucose or fatty acid flux through glycolysis and the TCA cycle (Drews et al., 2010). This generates excess O2•− which gives rise to other ROS and RNS. The insufficiency of antioxidant enzymes to scavenge these ROS leads to oxidative stress. Besides mitochondria, NADPH oxidases, nitric oxide synthases, and phagocytes are other key sources of ROS in the β-cells (Drews et al., 2010). In type 1 diabetes mellitus, evidence implicates the role of ROS in impaired -cell function caused by autoimmune reactions, cytokines and inflammatory proteins (Drews et al., 2010). Similarly, in type 2 diabetes mellitus, the role of ROS is implicated in the β-cell dysfunction as well as insulin resistance (Drews et al., 2010). The pancreas is highly susceptible to oxidative stress as evidenced by studies which show that H2O2 impairs insulin secretion in pancreatic -cells (Maechler et al., 1999) and products of oxidative stress inhibit glucosestimulated insulin secretion (Miwa et al., 2000). Other evidence shows that overexpression of antioxidant enzymes in islets or transgenic mice and antioxidants such as N-acetyl-Lcysteine (NAC) protect against ROS-induced -cell toxicity (Tiedge et al., 1998, Drews et al.,

al., 2002). Free iron can increase ROS generation and the oxidation of LDL cholesterol. Similarly, homocysteine can generate ROS in the presence of transition metals which may enhance the oxidation of LDL cholesterol (Penckofer et al., 2002). Another source of ROS in diabetes mellitus is leptin. Elevated levels of leptin are associated with insulin resistance and diabetes mellitus. Evidence implicates a role of leptin in inducing ROS and oxidative stress in aortic endothelial cells in a dose-dependent manner while it produces additive effects with those of glucose (Wiernsperger, 2003). Other potential sources of ROS include aging, menopause, diet and physical activity (Penckofer et al., 2002). Increased ROS is reported in older people. Menopause may also enhance ROS production in older women. The levels of oestrogen, an antioxidant which decreases the oxidation of LDL cholesterol, usually decline during menopause (Penckofer et al., 2002). Aging and menopause may exacerbate oxidative stress since a large number of type 2 diabetics are older men and women. Consumption of foods rich in carbohydrates and fats, as opposed to antioxidant-enriched diets, may also enhance ROS formation. Sedentary lifestyle may predispose to increased ROS generation. This is corroborated by evidence which indicates mild or moderate, but not strenuous,

exercise induces antioxidant defenses (Sachdev and Davies, 2008).

**4.1 Role of oxidative stress in β-cell dysfunction** 

al., 2010).

**4. Role of oxidative stress in the pathogenesis of diabetes mellitus** 

Evidence implicates the role of oxidative stress in the different stages of the development of diabetes mellitus, starting from the pre-diabetes state, impaired glucose tolerance, postprandial hyperglycemia, mild diabetes and finally to overt diabetes mellitus (Ceriello et al., 1998). Loss of β-cell function, resulting from impaired secretory capacity and increased apoptosis, is a main occurrence in the pathogenesis of both types of diabetes (Drews et al., 2010). Besides β-cell dysfunction, insulin resistance is also a major characteristic feature of type 2 diabetes mellitus (Evans et al., 2003). Oxidative stress plays an important role in the pathogenesis of both β-cell dysfunction and insulin resistance (Evans et al., 2003, Drews et

The β-cells express low level of antioxidant enzymes such as SOD, CAT and GPx and thereby increase their susceptibility to oxidative stress (Tiedge et al., 1997). Increased mitochondrial ROS production in the β-cells results from enhanced glucose or fatty acid flux through glycolysis and the TCA cycle (Drews et al., 2010). This generates excess O2•− which gives rise to other ROS and RNS. The insufficiency of antioxidant enzymes to scavenge these ROS leads to oxidative stress. Besides mitochondria, NADPH oxidases, nitric oxide synthases, and phagocytes are other key sources of ROS in the β-cells (Drews et al., 2010). In type 1 diabetes mellitus, evidence implicates the role of ROS in impaired -cell function caused by autoimmune reactions, cytokines and inflammatory proteins (Drews et al., 2010). Similarly, in type 2 diabetes mellitus, the role of ROS is implicated in the β-cell dysfunction as well as insulin resistance (Drews et al., 2010). The pancreas is highly susceptible to oxidative stress as evidenced by studies which show that H2O2 impairs insulin secretion in pancreatic -cells (Maechler et al., 1999) and products of oxidative stress inhibit glucosestimulated insulin secretion (Miwa et al., 2000). Other evidence shows that overexpression of antioxidant enzymes in islets or transgenic mice and antioxidants such as N-acetyl-Lcysteine (NAC) protect against ROS-induced -cell toxicity (Tiedge et al., 1998, Drews et al., 2010). Research within the last decade has recognized the role of glucotoxicity as the main causal determinant of β-cell dysfunction (Drews et al., 2010). The role of glucotoxicity in βcell dysfunction is demonstrated by studies which indicate increased glucose concentrations impair insulin release in non-diabetic subjects (Marchetti et al., 2008). In contrast, improved glycemic control results in improved insulin secretion in patients with type 2 diabetes (Marchetti et al., 2008). The fact that antioxidants reduce or prevent the toxicities of elevated glucose on the expression of insulin mRNA, insulin content and secretion lends support to the role of oxidative stress in mediating the toxic effects of glucotoxicity (Tanaka et al., 1999). Besides glucotoxicity, lipotoxicity may also play a role in impaired β-cell function. Free fatty acids have been shown to uncouple mitochondrial oxidative phosphorylation and increase ROS formation in rat pancreatic islets (Carlsson et al., 1999), deplete pancreatic β-cell insulin content (Bollheimer et al., 1998) and inhibit glucose-induced insulin secretion and biosynthesis (Zhou and Grill, 1994). Both glucotoxicity and lipotoxicity may be involved in β-cell dysfunction in diabetes mellitus (Marchetti et al., 2008). The role of oxidative stress is also implicated in β-cell deficit, as well as increased apoptosis observed in humans with type 2 diabetes (Drews et al., 2010).

#### **4.2 Role of oxidative stress in insulin resistance**

Insulin resistance precedes the onset of diabetes mellitus and is influenced by factors such as genetic make-up and environmental factors including increased calorie intake, sedentary lifestyle, obesity, pregnancy and abnormally elevated levels of certain hormones (Evans et al., 2003). The role of oxidative stress is implicated in the pathogenesis of insulin resistance (Kim et al., 2006). Both pyruvate and fatty acids can serve as energy substrate in muscle and adipose tissue. Pyruvate is derived from glucose and other sugars, while fatty acids originate from fats. Once transported across the inner mitochondrial membrane, these fuel substrates are converted to acetyl CoA by mitochondrial enzymes. During citric acid cycle, oxidation of the carbon atoms of the acetyl groups in acetyl CoA generates CO2. Besides CO2 formation, the citric acid cycle generates high-energy electrons which are carried by NADH and FADH2. The increased uptake of energy substrate in the muscle and adipose tissue enhances citric acid cycle activity. This in turn generates mitochondrial NADH more than required. This leads to an overdrive of the oxidative phosphorylation which increases the mitochondrial transmembrane proton gradient. These high-energy electrons are then transferred to O2 leading to production of O2•− which is further converted to other ROS. This sets the stage for oxidative stress (Talior et al., 2003). A recent study showed that the skeletal muscle of high-fat diet-induced insulin-resistant rats liberated more mitochondrial H2O2 and had impaired ability to maintain normal redox balance compared to the data obtained in the skeletal muscle of the insulin-sensitive control rats (Anderson et al., 2009). Similar findings were observed in insulin-resistant, morbidly obese human subjects (Anderson et al., 2009). This study provides strong evidence in support of a role of mitochondrial ROS production and oxidative stress in the pathogenesis of insulin resistance.

Various mechanisms by which oxidative stress contributes to insulin resistance have been identified. These include oxidative stress-impaired insulin-induced GLUT4 translocation in adipocytes (Rudich et al., 1998), oxidative stress-induced impairment in insulin stimulation of protein kinase B and glucose transport in adipocytes (Rudich et al., 1999), oxidative stress-induced interactions between the PI3-kinase-dependent signaling pathway and

Oxidative Stress in Diabetes Mellitus:

causes diabetic complications.

diabetes mellitus.

**5.1 Hyperglycemia-enhanced polyol pathway** 

healthcare providers.

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 225

on the need for adherence to dietary regimens and compliance to prescribed medications. These recommendations could also be an important component of training received by

There is strong evidence implicating a role of oxidative stress in diabetic nephropathy, retinopathy and neuropathy which constitute the microvascular complications (Figueroa-Romero et al., 2008, Giacco and Brownlee, 2010, Brownlee, 2005). Similarly, a role of oxidative stress is implicated in the macrovascular complications (coronary artery disease, peripheral arterial disease and cerebrovascular disease) (Giacco and Brownlee, 2010, Brownlee, 2005). This section highlights the different mechanisms by which hyperglycemia

The polyol pathway comprises two enzymes: aldose reductase (AR) and sorbitol dehydrogenase (SDH) (Brownlee, 2005). AR reduces a broad spectrum of substrates such as glucose, galactose, methylglyoxal, glucosone, deoxyglucosone and lipid-derived aldehydes (Petrash, 2004). Under euglycemic conditions, glucose is not reduced by AR. The bulk of glucose is normally phosphorylated by hexokinase to produce glucose-6-phosphate, a substrate for glycolysis and pentose phosphate pathway. However, under hyperglycemic conditions, AR reduces glucose to sorbitol (Brownlee, 2005). Sorbitol is oxidized to fructose by SDH (Giacco and Brownlee, 2010). With chronic hyperglycemia, the AR pathway becomes enhanced leading to increased formation of sorbitol. As a result of activated AR pathway, there is increased consumption of NADPH (as an obligate co-factor) by AR (Figueroa-Romero et al., 2008). The GR also requires NADPH, as co-factor, for the regeneration of GSH, an endogenous scavenger of ROS. Therefore, increased utilization of NADPH caused by enhanced activity of AR reduces intracellular concentration of GSH. Reduced levels of GSH will impair the activity of GPx which utilizes GSH as a hydrogen donor (Halliwell and Gutteridge, 2007). Taken together, decreased NADPH impairs GR activity, reduces GSH level and impairs GPx activity. This impairs the antioxidant defense network and increases cellular susceptibility to oxidative stress. NO• has been shown to inhibit AR activity in diabetes (Chandra et al., 2002). In view of evidence which indicates ROS can decrease NO• bioavailability, therefore, increased oxidative stress in diabetes will further augment AR activity. Thus, hyperglycemia-enhanced polyol pathway will exacerbate oxidative stress in

Besides, the increased levels of sorbitol caused by enhanced AR activity increase the osmolality of intracellular milieu. Sorbitol, myoinositol, glycerophosphorylcholine, betaine and taurine are physiological osmolytes which help to maintain homeostasis in cells such as renal medullary cells (Yancey and Burg, 1989). Thus, as a compensatory mechanism, this leads to efflux of intracellular osmolytes (Figueroa-Romero et al., 2008). Some of these osmolytes e.g. myoinositol play a vital role in signaling transduction, while others such as taurine are endogenous antioxidants (Figueroa-Romero et al., 2008). Hence, increased sorbitol formation reduces endogenous antioxidants and other important osmolytes. This further impairs cellular function and increases intracellular susceptibility to oxidative stress. In contrast, the SDH pathway generates fructose from sorbitol. Similar to AR pathway,

**5. Role of oxidative stress in the complications of diabetes mellitus** 

activation of p38 MAPK (Kim et al., 2006), and interruption of insulin-induced cellular redistribution of insulin receptor substrate-1 and phosphatidylinositol 3-kinase in adipocytes (Tirosh et al., 1999). Other studies indicate that oxidative stress can directly induce considerable insulin resistance in skeletal muscle by interfering with insulin signaling, glucose uptake and glycogen synthesis (Dokken et al., 2008). This oxidative stressinduced insulin resistance is mediated in part through reduced insulin-modulated suppression of glycogen synthase kinase-3 (GSK-3beta) (Dokken *et al.*, 2008; Henriksen, 2010). Evidence has also implicated a role of inducible nitric oxide synthase and •NO donor in the degradation of insulin receptor substrate-1. Increased S-nitrosylation of certain molecules or pathways involved in insulin signaling such as insulin receptor, insulin receptor substrate-1, and protein kinase B/Akt in skeletal muscle have also been reported to play an important role in insulin resistance (Carvalho-Filho et al., 2009).

In order to protect against increased glucose-induced oxidative stress (Talior et al., 2003), cells tend to respond by limiting more energy fuel or substrates from gaining access into the cells. Cells may utilize various mechanisms, including inhibition of α-ketoglutarate dehydrogenase, to limit the amount of NADH available for the oxidative phosphorylation. Therefore, to limit insulin-dependent nutrient uptake into the cells, the insulin receptors become less sensitive to the action of insulin. This marks the onset of insulin resistance - a phenomenon whereby normal amounts of insulin can no longer activate the glucose transport system in insulin-sensitive tissues such as skeletal muscle and adipose tissue. This results in enhanced steady-state glucose levels in the blood. It is suggested that, in this setting, insulin resistance may be a compensatory mechanism employed by the cells to prevent further uptake of insulin-stimulated glucose and fatty acids (Hoehn et al., 2009). This compensatory mechanism may result in reduced formation and accumulation of RS leading to reduced oxidative stress and damage. With persistently increased blood glucose levels, hyperglycemia ensues leading to overt type 2 diabetes mellitus.

#### **4.3 Could antioxidants play a role in preventing diabetes mellitus and/or its progression?**

In view of evidence which implicates a role of oxidative stress in β-cell dysfunction and insulin resistance, the question that arises is: could antioxidants play a role in preventing diabetes mellitus and/or its progression? As discussed earlier, there is a possibility for such a role of antioxidants. Antioxidants such as vitamin C, vitamin E, β-carotene, α-lipoic acids and honey have been shown to ameliorate hyperglycemia through increased β-cell mass and insulin secretion. However, at the moment, the evidence to recommend or prescribe antioxidants for the prevention of diabetes mellitus is very weak. Instead, efforts should be made to create and increase the awareness on (1) the role of ROS and oxidative stress in the pathogenesis and/or progression of diabetes mellitus and (2) the importance of increased consumption of antioxidant-enriched diets including fruits and vegetables, as opposed to increased calorie intake. The recommendation for increased consumption of fruits and vegetables is very important because even in the developed countries, majority of the population do not meet these requirements. The importance of exercise should also not be left out. These could be achieved by incorporating into educational curricula - at all levels of education. In addition, those who are already diabetic should be enlightened on the importance of maintaining good glycemic control in order to prevent or delay the progression or complications of diabetes mellitus. Diabetic patients also need to be informed

activation of p38 MAPK (Kim et al., 2006), and interruption of insulin-induced cellular redistribution of insulin receptor substrate-1 and phosphatidylinositol 3-kinase in adipocytes (Tirosh et al., 1999). Other studies indicate that oxidative stress can directly induce considerable insulin resistance in skeletal muscle by interfering with insulin signaling, glucose uptake and glycogen synthesis (Dokken et al., 2008). This oxidative stressinduced insulin resistance is mediated in part through reduced insulin-modulated suppression of glycogen synthase kinase-3 (GSK-3beta) (Dokken *et al.*, 2008; Henriksen, 2010). Evidence has also implicated a role of inducible nitric oxide synthase and •NO donor in the degradation of insulin receptor substrate-1. Increased S-nitrosylation of certain molecules or pathways involved in insulin signaling such as insulin receptor, insulin receptor substrate-1, and protein kinase B/Akt in skeletal muscle have also been reported to

In order to protect against increased glucose-induced oxidative stress (Talior et al., 2003), cells tend to respond by limiting more energy fuel or substrates from gaining access into the cells. Cells may utilize various mechanisms, including inhibition of α-ketoglutarate dehydrogenase, to limit the amount of NADH available for the oxidative phosphorylation. Therefore, to limit insulin-dependent nutrient uptake into the cells, the insulin receptors become less sensitive to the action of insulin. This marks the onset of insulin resistance - a phenomenon whereby normal amounts of insulin can no longer activate the glucose transport system in insulin-sensitive tissues such as skeletal muscle and adipose tissue. This results in enhanced steady-state glucose levels in the blood. It is suggested that, in this setting, insulin resistance may be a compensatory mechanism employed by the cells to prevent further uptake of insulin-stimulated glucose and fatty acids (Hoehn et al., 2009). This compensatory mechanism may result in reduced formation and accumulation of RS leading to reduced oxidative stress and damage. With persistently increased blood glucose

play an important role in insulin resistance (Carvalho-Filho et al., 2009).

levels, hyperglycemia ensues leading to overt type 2 diabetes mellitus.

**progression?** 

**4.3 Could antioxidants play a role in preventing diabetes mellitus and/or its** 

In view of evidence which implicates a role of oxidative stress in β-cell dysfunction and insulin resistance, the question that arises is: could antioxidants play a role in preventing diabetes mellitus and/or its progression? As discussed earlier, there is a possibility for such a role of antioxidants. Antioxidants such as vitamin C, vitamin E, β-carotene, α-lipoic acids and honey have been shown to ameliorate hyperglycemia through increased β-cell mass and insulin secretion. However, at the moment, the evidence to recommend or prescribe antioxidants for the prevention of diabetes mellitus is very weak. Instead, efforts should be made to create and increase the awareness on (1) the role of ROS and oxidative stress in the pathogenesis and/or progression of diabetes mellitus and (2) the importance of increased consumption of antioxidant-enriched diets including fruits and vegetables, as opposed to increased calorie intake. The recommendation for increased consumption of fruits and vegetables is very important because even in the developed countries, majority of the population do not meet these requirements. The importance of exercise should also not be left out. These could be achieved by incorporating into educational curricula - at all levels of education. In addition, those who are already diabetic should be enlightened on the importance of maintaining good glycemic control in order to prevent or delay the progression or complications of diabetes mellitus. Diabetic patients also need to be informed on the need for adherence to dietary regimens and compliance to prescribed medications. These recommendations could also be an important component of training received by healthcare providers.

#### **5. Role of oxidative stress in the complications of diabetes mellitus**

There is strong evidence implicating a role of oxidative stress in diabetic nephropathy, retinopathy and neuropathy which constitute the microvascular complications (Figueroa-Romero et al., 2008, Giacco and Brownlee, 2010, Brownlee, 2005). Similarly, a role of oxidative stress is implicated in the macrovascular complications (coronary artery disease, peripheral arterial disease and cerebrovascular disease) (Giacco and Brownlee, 2010, Brownlee, 2005). This section highlights the different mechanisms by which hyperglycemia causes diabetic complications.

#### **5.1 Hyperglycemia-enhanced polyol pathway**

The polyol pathway comprises two enzymes: aldose reductase (AR) and sorbitol dehydrogenase (SDH) (Brownlee, 2005). AR reduces a broad spectrum of substrates such as glucose, galactose, methylglyoxal, glucosone, deoxyglucosone and lipid-derived aldehydes (Petrash, 2004). Under euglycemic conditions, glucose is not reduced by AR. The bulk of glucose is normally phosphorylated by hexokinase to produce glucose-6-phosphate, a substrate for glycolysis and pentose phosphate pathway. However, under hyperglycemic conditions, AR reduces glucose to sorbitol (Brownlee, 2005). Sorbitol is oxidized to fructose by SDH (Giacco and Brownlee, 2010). With chronic hyperglycemia, the AR pathway becomes enhanced leading to increased formation of sorbitol. As a result of activated AR pathway, there is increased consumption of NADPH (as an obligate co-factor) by AR (Figueroa-Romero et al., 2008). The GR also requires NADPH, as co-factor, for the regeneration of GSH, an endogenous scavenger of ROS. Therefore, increased utilization of NADPH caused by enhanced activity of AR reduces intracellular concentration of GSH. Reduced levels of GSH will impair the activity of GPx which utilizes GSH as a hydrogen donor (Halliwell and Gutteridge, 2007). Taken together, decreased NADPH impairs GR activity, reduces GSH level and impairs GPx activity. This impairs the antioxidant defense network and increases cellular susceptibility to oxidative stress. NO• has been shown to inhibit AR activity in diabetes (Chandra et al., 2002). In view of evidence which indicates ROS can decrease NO• bioavailability, therefore, increased oxidative stress in diabetes will further augment AR activity. Thus, hyperglycemia-enhanced polyol pathway will exacerbate oxidative stress in diabetes mellitus.

Besides, the increased levels of sorbitol caused by enhanced AR activity increase the osmolality of intracellular milieu. Sorbitol, myoinositol, glycerophosphorylcholine, betaine and taurine are physiological osmolytes which help to maintain homeostasis in cells such as renal medullary cells (Yancey and Burg, 1989). Thus, as a compensatory mechanism, this leads to efflux of intracellular osmolytes (Figueroa-Romero et al., 2008). Some of these osmolytes e.g. myoinositol play a vital role in signaling transduction, while others such as taurine are endogenous antioxidants (Figueroa-Romero et al., 2008). Hence, increased sorbitol formation reduces endogenous antioxidants and other important osmolytes. This further impairs cellular function and increases intracellular susceptibility to oxidative stress. In contrast, the SDH pathway generates fructose from sorbitol. Similar to AR pathway,

Oxidative Stress in Diabetes Mellitus:

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 227

dehydrogenase (GAPDH) by increased ROS (Giacco and Brownlee, 2010). Besides DAG, evidence indicates that AGEs can also activate PKC pathway to increase the expression of vascular endothelial growth factor (VEGF) (Ahmed, 2005). Similarly, increased ROS levels in vascular endothelial cells may also enhance PKC pathway (Nishikawa et al., 2000a). Enhanced PKC activity induces several cytokines and protein signals including plasminogen activator inhibitor (PAI-1), NF-κB, NAD(P)H oxidases, endothelin-1, transforming growth factor β (TGF-β) and extracellular matrix (ECM) (Nishikawa et al., 2000a). These pathological alterations have been implicated in basement membrane thickening, vasoconstriction, altered capillary permeability, hypoxia and activation of

Evidence has implicated the role of hexosamine pathway in the toxic or adverse effects of hyperglycemia in diabetes mellitus (Schleicher and Weigert, 2000). Under physiological conditions, a small quantity of fructose-6 phosphate derived from glycolysis is diverted to the hexosamine pathway. Glutamine: fructose-6 phosphate amidotransferase (GFAT) then converts fructose-6 phosphate to glucosamine-6 phosphate, which is converted to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) (Schleicher and Weigert, 2000). The enzyme O-GlcNAc transferase then utilizes UDP-GlcNAc as a substrate, fixing O-GlcNAC to protein residues of transcription factors such as Sp1 and thus modify their expression (Figueroa-Romero et al., 2008). Similar to the other pathways, with chronic hyperglycemia, the hexosamine pathway becomes enhanced (Figueroa-Romero et al., 2008, Schleicher and Weigert, 2000). This leads to increased formation of UDP-GlcNAc and increased activity of O-GlcNAc transferase, with consequent alterations in gene expression (Figueroa-Romero et al., 2008). This pathway is implicated in the hyperglycemia-mediated increases in the transcription of TGF-α and TGF-β1. Over-expression of these transcription factors such as TGF-β1 is known to activate the proliferation of collagen matrix, basement membrane thickening and inhibition of mesangial cell mitogenesis, thus contributing to microvascular

angiogenesis (Nishikawa et al., 2000a, Figueroa-Romero et al., 2008).

complications such as nephropathy (Schleicher and Weigert, 2000).

**5.5 Hyperglycemia-activated Poly-ADP Ribose Polymerase (PARP) pathway** 

Poly(ADP-ribose) polymerase (PARP) is a family of enzymes that detect single- and doublestranded DNA and repair damaged DNA (Virag and Szabo, 2002). Activation of PARP is a direct cellular response to metabolic- or chemical-induced DNA damage. Once PARP senses and identifies a damaged DNA, it binds to the DNA and forms homodimers and catalyzes the cleavage of nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADPribose (Virag and Szabo, 2002). It then uses ADP-ribose to synthesize a poly(ADP-ribose) chain (PAR) which serves as a signal for several DNA repairing enzymes such as DNA ligase III and DNA polymerase beta (polβ). In hyperglycemic environment, there is increased ROS formation leading to oxidative damage which activates PARP. As a result of NAD+ utilization, this depletes cellular NAD+ stores and induces a progressive ATP depletion. This further increases the vulnerability of cells to oxidative stress and damage as well as cell death (Figueroa-Romero et al., 2008). Available evidence suggests that PARP's catalytic activity may cause altered gene expression, increased oxidative stress and diversion of glycolytic intermediates to other pathogenic pathways (Figueroa-Romero et al.,

**5.4 Hyperglycemia-enhanced hexosamine pathway** 

chronic hyperglycemia increases the activity of SDH which results in the formation of high amounts of fructose (Figueroa-Romero et al., 2008). Increased SDH activity result in an increased NADH:NAD+ ratio, which may inhibit oxidation of triose phosphates (Nishikawa et al., 2000a). Accumulation of triose phosphates increases *de novo* synthesis of diacylglycerols (DAG) which activate protein kinase C. Increased levels of triose phosphate may also increase generation of methylglyoxal, a potent AGE-precursor. Besides, increased fructose levels may enhance glycation and further contribute to reduced levels of NADPH, impaired antioxidant defenses and formation of AGEs.

#### **5.2 Hyperglycemia-enhanced formation of advanced glycation end products**

Advanced glycation end products (AGEs) are adducts formed non-enzymatically by the reaction between reducing carbohydrates and proteins, DNA, or lipids (Ahmed, 2005). AGEs also include products that are formed non-enzymatically from the reaction between AGE precursors, glycated sugars or oxidized products of fatty acid (in arterial endothelial cells) and protein (Giacco and Brownlee, 2010). They are produced through three major pathways: (1) convertion of glucose to glyoxal; (2) degradation of Amadori products to 3 deoxyglucosone; and (3) conversion of glyceraldehyde-3-phosphate to methylglyoxal (Nishikawa et al., 2000a, Ahmed, 2005, Figueroa-Romero et al., 2008). In diabetes mellitus, the levels of AGEs become elevated as a result of chronic hyperglycemia (Duran-Jimenez et al., 2009). The formation of AGEs occurs in different stages. During these stages of AGE formation, a number of highly reactive intermediates and cross-linkers, which enhance the binding affinity of AGEs to proteins, are also formed (Ahmed, 2005). AGEs can inhibit the antiproliferative effects of nitric oxide (Maritim et al., 2003). AGEs bind to and modify intracellular proteins thereby altering their functions (Giacco and Brownlee, 2010). In the vasculature, AGEs interact with cell surface protein or extracellular matrix components resulting in the formation of cross-linked proteins which enhance stiffening within the arterial vessel (Ahmed, 2005). AGEs can also modify plasma proteins which in turn activate receptor for advanced glycation end products (RAGE) on cells such as macrophages, vascular endothelial and smooth muscle cells (Giacco and Brownlee, 2010). AGEs can bind directly to and activate the receptors for AGEs.(Griesmacher *et al.*, 1995, Ahmed, 2005).

The binding of AGEs or AGE-modified plasma proteins to RAGE induces the release of ROS (Nishikawa et al., 2000a; Ahmed, 2005; Giacco and Brownlee, 2010). The ROS activate the expression of several genes and proteins that are involved in inflammatory cascade and implicated in the pathogenesis of diabetic cardiovascular disease. These genes and proteins include nuclear factor kappa , tumor necrosis factor α, interleukin-1 and granulocytemacrophage colony-stimulating factor (Nishikawa et al., 2000a, Ahmed, 2005, Giacco and Brownlee, 2010).

#### **5.3 Hyperglycemia-activated protein kinase C pathway**

Protein kinase C (PKC) is an enzyme that modulates the functions of other proteins through their phosphorylation. PKC is activated by the elevated level of DAG, derived from enhanced formation of triose phosphate via hyperglycemia (Giacco and Brownlee, 2010). Hyperglycemia may also increase DAG content through phosphatidylcholine hydrolysis (Nishikawa et al., 2000a). In diabetes, elevated levels of triose phosphate occur through increased *de novo* synthesis due to inhibition of glycolytic enzyme glyceraldehyde phosphate

chronic hyperglycemia increases the activity of SDH which results in the formation of high amounts of fructose (Figueroa-Romero et al., 2008). Increased SDH activity result in an increased NADH:NAD+ ratio, which may inhibit oxidation of triose phosphates (Nishikawa et al., 2000a). Accumulation of triose phosphates increases *de novo* synthesis of diacylglycerols (DAG) which activate protein kinase C. Increased levels of triose phosphate may also increase generation of methylglyoxal, a potent AGE-precursor. Besides, increased fructose levels may enhance glycation and further contribute to reduced levels of NADPH,

Advanced glycation end products (AGEs) are adducts formed non-enzymatically by the reaction between reducing carbohydrates and proteins, DNA, or lipids (Ahmed, 2005). AGEs also include products that are formed non-enzymatically from the reaction between AGE precursors, glycated sugars or oxidized products of fatty acid (in arterial endothelial cells) and protein (Giacco and Brownlee, 2010). They are produced through three major pathways: (1) convertion of glucose to glyoxal; (2) degradation of Amadori products to 3 deoxyglucosone; and (3) conversion of glyceraldehyde-3-phosphate to methylglyoxal (Nishikawa et al., 2000a, Ahmed, 2005, Figueroa-Romero et al., 2008). In diabetes mellitus, the levels of AGEs become elevated as a result of chronic hyperglycemia (Duran-Jimenez et al., 2009). The formation of AGEs occurs in different stages. During these stages of AGE formation, a number of highly reactive intermediates and cross-linkers, which enhance the binding affinity of AGEs to proteins, are also formed (Ahmed, 2005). AGEs can inhibit the antiproliferative effects of nitric oxide (Maritim et al., 2003). AGEs bind to and modify intracellular proteins thereby altering their functions (Giacco and Brownlee, 2010). In the vasculature, AGEs interact with cell surface protein or extracellular matrix components resulting in the formation of cross-linked proteins which enhance stiffening within the arterial vessel (Ahmed, 2005). AGEs can also modify plasma proteins which in turn activate receptor for advanced glycation end products (RAGE) on cells such as macrophages, vascular endothelial and smooth muscle cells (Giacco and Brownlee, 2010). AGEs can bind directly to and activate the receptors for AGEs.(Griesmacher *et al.*, 1995, Ahmed, 2005).

The binding of AGEs or AGE-modified plasma proteins to RAGE induces the release of ROS (Nishikawa et al., 2000a; Ahmed, 2005; Giacco and Brownlee, 2010). The ROS activate the expression of several genes and proteins that are involved in inflammatory cascade and implicated in the pathogenesis of diabetic cardiovascular disease. These genes and proteins include nuclear factor kappa , tumor necrosis factor α, interleukin-1 and granulocytemacrophage colony-stimulating factor (Nishikawa et al., 2000a, Ahmed, 2005, Giacco and

Protein kinase C (PKC) is an enzyme that modulates the functions of other proteins through their phosphorylation. PKC is activated by the elevated level of DAG, derived from enhanced formation of triose phosphate via hyperglycemia (Giacco and Brownlee, 2010). Hyperglycemia may also increase DAG content through phosphatidylcholine hydrolysis (Nishikawa et al., 2000a). In diabetes, elevated levels of triose phosphate occur through increased *de novo* synthesis due to inhibition of glycolytic enzyme glyceraldehyde phosphate

**5.2 Hyperglycemia-enhanced formation of advanced glycation end products** 

impaired antioxidant defenses and formation of AGEs.

**5.3 Hyperglycemia-activated protein kinase C pathway** 

Brownlee, 2010).

dehydrogenase (GAPDH) by increased ROS (Giacco and Brownlee, 2010). Besides DAG, evidence indicates that AGEs can also activate PKC pathway to increase the expression of vascular endothelial growth factor (VEGF) (Ahmed, 2005). Similarly, increased ROS levels in vascular endothelial cells may also enhance PKC pathway (Nishikawa et al., 2000a). Enhanced PKC activity induces several cytokines and protein signals including plasminogen activator inhibitor (PAI-1), NF-κB, NAD(P)H oxidases, endothelin-1, transforming growth factor β (TGF-β) and extracellular matrix (ECM) (Nishikawa et al., 2000a). These pathological alterations have been implicated in basement membrane thickening, vasoconstriction, altered capillary permeability, hypoxia and activation of angiogenesis (Nishikawa et al., 2000a, Figueroa-Romero et al., 2008).

#### **5.4 Hyperglycemia-enhanced hexosamine pathway**

Evidence has implicated the role of hexosamine pathway in the toxic or adverse effects of hyperglycemia in diabetes mellitus (Schleicher and Weigert, 2000). Under physiological conditions, a small quantity of fructose-6 phosphate derived from glycolysis is diverted to the hexosamine pathway. Glutamine: fructose-6 phosphate amidotransferase (GFAT) then converts fructose-6 phosphate to glucosamine-6 phosphate, which is converted to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) (Schleicher and Weigert, 2000). The enzyme O-GlcNAc transferase then utilizes UDP-GlcNAc as a substrate, fixing O-GlcNAC to protein residues of transcription factors such as Sp1 and thus modify their expression (Figueroa-Romero et al., 2008). Similar to the other pathways, with chronic hyperglycemia, the hexosamine pathway becomes enhanced (Figueroa-Romero et al., 2008, Schleicher and Weigert, 2000). This leads to increased formation of UDP-GlcNAc and increased activity of O-GlcNAc transferase, with consequent alterations in gene expression (Figueroa-Romero et al., 2008). This pathway is implicated in the hyperglycemia-mediated increases in the transcription of TGF-α and TGF-β1. Over-expression of these transcription factors such as TGF-β1 is known to activate the proliferation of collagen matrix, basement membrane thickening and inhibition of mesangial cell mitogenesis, thus contributing to microvascular complications such as nephropathy (Schleicher and Weigert, 2000).

#### **5.5 Hyperglycemia-activated Poly-ADP Ribose Polymerase (PARP) pathway**

Poly(ADP-ribose) polymerase (PARP) is a family of enzymes that detect single- and doublestranded DNA and repair damaged DNA (Virag and Szabo, 2002). Activation of PARP is a direct cellular response to metabolic- or chemical-induced DNA damage. Once PARP senses and identifies a damaged DNA, it binds to the DNA and forms homodimers and catalyzes the cleavage of nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADPribose (Virag and Szabo, 2002). It then uses ADP-ribose to synthesize a poly(ADP-ribose) chain (PAR) which serves as a signal for several DNA repairing enzymes such as DNA ligase III and DNA polymerase beta (polβ). In hyperglycemic environment, there is increased ROS formation leading to oxidative damage which activates PARP. As a result of NAD+ utilization, this depletes cellular NAD+ stores and induces a progressive ATP depletion. This further increases the vulnerability of cells to oxidative stress and damage as well as cell death (Figueroa-Romero et al., 2008). Available evidence suggests that PARP's catalytic activity may cause altered gene expression, increased oxidative stress and diversion of glycolytic intermediates to other pathogenic pathways (Figueroa-Romero et al.,

Oxidative Stress in Diabetes Mellitus:

of oxidative stress?

**ameliorate oxidative stress?** 

management of diabetic complications?

complications?

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 229

effects of reduced hyperglycemia or glycemic control on the risk of developing diabetic complications (DCCT, 1993, UKPDS, 1998). Nevertheless, recent findings suggest that treatment of chronic hyperglycemia to achieve optimal glycemic goal in diabetic patients is limited and detrimental (Ismail-Beigi et al., 2010). It is recommended that the consequences (higher mortality rate, hypoglycemia and weight gain) should be weighed against the benefits of intensive therapy (Ismail-Beigi et al., 2010). Besides, available evidence indicates that achieving and/or maintaining optimal glycemic control in diabetic patients is difficult (Turner et al., 1999, Cook et al., 2005). The difficulty in maintaining optimal glycemic control is attributed to deterioration of pancreatic β-cell function, which is linked to hyperglycemiainduced oxidative stress (Drews et al., 2010). Interestingly, even in diabetic patients given pancreatic transplants, diabetic complications such as nephropathy continued to deteriorate at least five years after their diabetes had been cured (Fioretto et al., 1998). This contradicts evidence that links hyperglycemia to diabetic complications. Therefore, this section attempts to explain the interrelation among glycemic control, oxidative stress and diabetic complications and possible role of hypoglycemic drugs (and/or insulin) and antioxidants in

the management of diabetic complications by answering the following questions:

1. Does reduced or intensive therapy of hyperglycemia prevent induction or development

2. Does reduced or intensive therapy of hyperglycemia prevent diabetic complications? 3. Why does reduced or intensive therapy of hyperglycemia not prevent diabetic

4. Could there be a role of antioxidants in the management of diabetic complications? 5. Could there be a role of hypoglycemic drugs (and/or insulin) and antioxidants in the

**6.1 Does reduced or intensive therapy of hyperglycemia completely restore or** 

status (Rahimi et al., 2005, Erejuwa et al., 2010a, Erejuwa et al., 2011b).

Hyperglycemia induces oxidative stress in diabetes mellitus. However, does evidence indicate that reduced or intensive treatment of hyperglycemia completely prevent development of oxidative stress? This section aims to answer this important question by presenting both experimental and clinical data. In diabetic rats, after two months of poor glycemic control, reinstitution of good glycemic control for seven additional months partially reduced the elevated caspase-3 activity, levels of NF-k, lipid peroxides and nitric oxides with no beneficial effect on nitrotyrosine formation. In contrast, after six months of poor glycemic control, re-institution of good glycemic control for seven additional months demonstrated no significant effects on the elevated caspase-3 activity, NF-k, and oxidative stress parameters (Kowluru, 2003, Kowluru *et al.*, 2004). In another follow up study, after six months of poor glycemic control, normalization of hyperglycemia for another 6 months also had no significant effect on retinal nitrotyrosine levels, neither did oxidative stress parameters improve (Kowluru et al., 2007). Other studies have also shown that reduced hyperglycemia does not completely restore redox

Evidence suggests that proteins (collagen) are likely to be glycated irrespective of blood glucose levels (Monnier et al., 1999). In patients with type 2 diabetes mellitus, insulin treatment only partially improved oxidative stress parameters (Seghrouchni et al., 2002). This is evidenced by the elevated levels of thiobarbituric acid reactive substances and reduced erythrocyte GSH (Seghrouchni et al., 2002). In type 2 diabetic patients, treatment

2008). A recent study demonstrated the beneficial effects of PARP inhibition in diabetic complications (Lupachyk et al., 2011).

#### **5.6 Hyperglycemia-induced mitochondrial O2•<sup>−</sup> overproduction**

Recent data indicates that hyperglycemia-induced mitochondrial O2•− overproduction is the sole underlying mechanism (directly or indirectly) by which hyperglycemia induces cellular damage (Giacco and Brownlee, 2010). During mitochondrial oxidative phosphorylation, O2 •− is generated due to leakage of electrons from electron transport chain (ETC) on molecular oxygen. In euglycemic environment, about 0.2–2% of O2 utilized by the mitochondria is reduced to O2•− (Bashan et al., 2009, Brand, 2010). The antioxidant defense network maintains the mitochondrial level of ROS within physiological concentrations (Andreyev et al., 2005, Brand, 2010). However, in hyperglycemic environment, enhanced glucose flux through glycolysis and TCA causes an overdrive of the mitochondrial ETC resulting in mitochondrial dysfunction and increased ROS formation (Bashan *et al.*, 2009; Brand, 2010). Elevated levels of ROS lead to oxidative stress and damage. Oxidative damage to DNA can activate Poly(ADP-ribose) polymerase (PARP) pathway (Wei, 1998).

Besides this pathway, evidence suggests that hyperglycemia, via O2 •<sup>−</sup> overproduction , inhibits G6PDH. G6PDH is the rate-limiting enzyme of the pentose phosphate pathway necessary for generating reducing equivalents to the antioxidant defense system (Brand, 2010). Inhibition of G6PDH leads to increased levels of glycolytic intermediates resulting in increased flux into these pathways. For instance, glyceraldehyde 3-phosphate can nonenzymatically be converted to methylglyoxal which activates AGEs pathway (Giacco and Brownlee, 2010). Similarly, glyceraldehyde 3-phosphate is a precursor of DAG which can activate PKC pathway. Increased levels of fructose 6-phosphate will increase flux through the hexosamine pathway (Giacco and Brownlee, 2010). Furthermore, reduced levels of G6PDH result in increased glucose concentrations which further enhances flux through the polyol pathway. The aldose reductase pathway becomes intensified leading to increased formation of sorbitol. This sets up a chain reaction that continuously activates one pathway or the other generating ROS which may cause DNA damage. This causes activation of PARP pathway. Thus, virtually all these pathways, namely polyol pathway, formation of AGEs pathway, PKC pathway, hexosamine pathway, and poly-ADP ribose polymerase (PARP) pathway, can be activated by hyperglycemia-induced mitochondrial O2•<sup>−</sup> overproduction.

#### **6. Interrelation among glycemic control, oxidative stress and diabetic complications**

So far, this chapter has identified the various sources of oxidative stress in diabetes mellitus. It has also presented evidence that indicates hyperglycemia enhances formation of ROS in diabetes mellitus. It has also shown that hyperglycemia-induced oxidative stress plays an important role in the activation of several pathogenic pathways implicated in the pathogenesis of diabetic complications. Generally, the mechanisms by which hyperglycemia causes cellular damage can be classified into two groups (Nishikawa et al., 2000a). The first group of mechanisms entails constant acute fluctuations in cellular metabolism which are reversible following restoration of euglycemia. The second group of mechanisms involves cumulative changes in long-lived macromolecules which are irreversible even after euglycemia is restored (Nishikawa et al., 2000a). Studies have demonstrated the beneficial

2008). A recent study demonstrated the beneficial effects of PARP inhibition in diabetic

Recent data indicates that hyperglycemia-induced mitochondrial O2•− overproduction is the sole underlying mechanism (directly or indirectly) by which hyperglycemia induces cellular damage (Giacco and Brownlee, 2010). During mitochondrial oxidative phosphorylation, O2•− is generated due to leakage of electrons from electron transport chain (ETC) on molecular oxygen. In euglycemic environment, about 0.2–2% of O2 utilized by the mitochondria is reduced to O2•− (Bashan et al., 2009, Brand, 2010). The antioxidant defense network maintains the mitochondrial level of ROS within physiological concentrations (Andreyev et al., 2005, Brand, 2010). However, in hyperglycemic environment, enhanced glucose flux through glycolysis and TCA causes an overdrive of the mitochondrial ETC resulting in mitochondrial dysfunction and increased ROS formation (Bashan *et al.*, 2009; Brand, 2010). Elevated levels of ROS lead to oxidative stress and damage. Oxidative damage

to DNA can activate Poly(ADP-ribose) polymerase (PARP) pathway (Wei, 1998).

**6. Interrelation among glycemic control, oxidative stress and diabetic** 

So far, this chapter has identified the various sources of oxidative stress in diabetes mellitus. It has also presented evidence that indicates hyperglycemia enhances formation of ROS in diabetes mellitus. It has also shown that hyperglycemia-induced oxidative stress plays an important role in the activation of several pathogenic pathways implicated in the pathogenesis of diabetic complications. Generally, the mechanisms by which hyperglycemia causes cellular damage can be classified into two groups (Nishikawa et al., 2000a). The first group of mechanisms entails constant acute fluctuations in cellular metabolism which are reversible following restoration of euglycemia. The second group of mechanisms involves cumulative changes in long-lived macromolecules which are irreversible even after euglycemia is restored (Nishikawa et al., 2000a). Studies have demonstrated the beneficial

Besides this pathway, evidence suggests that hyperglycemia, via O2•<sup>−</sup> overproduction , inhibits G6PDH. G6PDH is the rate-limiting enzyme of the pentose phosphate pathway necessary for generating reducing equivalents to the antioxidant defense system (Brand, 2010). Inhibition of G6PDH leads to increased levels of glycolytic intermediates resulting in increased flux into these pathways. For instance, glyceraldehyde 3-phosphate can nonenzymatically be converted to methylglyoxal which activates AGEs pathway (Giacco and Brownlee, 2010). Similarly, glyceraldehyde 3-phosphate is a precursor of DAG which can activate PKC pathway. Increased levels of fructose 6-phosphate will increase flux through the hexosamine pathway (Giacco and Brownlee, 2010). Furthermore, reduced levels of G6PDH result in increased glucose concentrations which further enhances flux through the polyol pathway. The aldose reductase pathway becomes intensified leading to increased formation of sorbitol. This sets up a chain reaction that continuously activates one pathway or the other generating ROS which may cause DNA damage. This causes activation of PARP pathway. Thus, virtually all these pathways, namely polyol pathway, formation of AGEs pathway, PKC pathway, hexosamine pathway, and poly-ADP ribose polymerase (PARP) pathway, can be activated by hyperglycemia-induced mitochondrial O2•<sup>−</sup> overproduction.

**overproduction** 

complications (Lupachyk et al., 2011).

**complications** 

**5.6 Hyperglycemia-induced mitochondrial O2•<sup>−</sup>**

effects of reduced hyperglycemia or glycemic control on the risk of developing diabetic complications (DCCT, 1993, UKPDS, 1998). Nevertheless, recent findings suggest that treatment of chronic hyperglycemia to achieve optimal glycemic goal in diabetic patients is limited and detrimental (Ismail-Beigi et al., 2010). It is recommended that the consequences (higher mortality rate, hypoglycemia and weight gain) should be weighed against the benefits of intensive therapy (Ismail-Beigi et al., 2010). Besides, available evidence indicates that achieving and/or maintaining optimal glycemic control in diabetic patients is difficult (Turner et al., 1999, Cook et al., 2005). The difficulty in maintaining optimal glycemic control is attributed to deterioration of pancreatic β-cell function, which is linked to hyperglycemiainduced oxidative stress (Drews et al., 2010). Interestingly, even in diabetic patients given pancreatic transplants, diabetic complications such as nephropathy continued to deteriorate at least five years after their diabetes had been cured (Fioretto et al., 1998). This contradicts evidence that links hyperglycemia to diabetic complications. Therefore, this section attempts to explain the interrelation among glycemic control, oxidative stress and diabetic complications and possible role of hypoglycemic drugs (and/or insulin) and antioxidants in the management of diabetic complications by answering the following questions:


#### **6.1 Does reduced or intensive therapy of hyperglycemia completely restore or ameliorate oxidative stress?**

Hyperglycemia induces oxidative stress in diabetes mellitus. However, does evidence indicate that reduced or intensive treatment of hyperglycemia completely prevent development of oxidative stress? This section aims to answer this important question by presenting both experimental and clinical data. In diabetic rats, after two months of poor glycemic control, reinstitution of good glycemic control for seven additional months partially reduced the elevated caspase-3 activity, levels of NF-k, lipid peroxides and nitric oxides with no beneficial effect on nitrotyrosine formation. In contrast, after six months of poor glycemic control, re-institution of good glycemic control for seven additional months demonstrated no significant effects on the elevated caspase-3 activity, NF-k, and oxidative stress parameters (Kowluru, 2003, Kowluru *et al.*, 2004). In another follow up study, after six months of poor glycemic control, normalization of hyperglycemia for another 6 months also had no significant effect on retinal nitrotyrosine levels, neither did oxidative stress parameters improve (Kowluru et al., 2007). Other studies have also shown that reduced hyperglycemia does not completely restore redox status (Rahimi et al., 2005, Erejuwa et al., 2010a, Erejuwa et al., 2011b).

Evidence suggests that proteins (collagen) are likely to be glycated irrespective of blood glucose levels (Monnier et al., 1999). In patients with type 2 diabetes mellitus, insulin treatment only partially improved oxidative stress parameters (Seghrouchni et al., 2002). This is evidenced by the elevated levels of thiobarbituric acid reactive substances and reduced erythrocyte GSH (Seghrouchni et al., 2002). In type 2 diabetic patients, treatment

Oxidative Stress in Diabetes Mellitus:

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 231

In type I diabetic patients whose diabetes had been cured (through pancreas transplantation) but still had diabetic nephropathy, the thickness of glomerular and tubular basement membranes was still similar at 5 years versus baseline values. In contrast, it was significantly reduced only by the 10th year versus baseline values. The study further showed that while mesangial fractional volume had increased by the 5th year, it significantly decreased by the 10th year (Fioretto et al., 1998). In the DCCT, due to the considerable benefits of intensive therapy, patients in the conventional therapy group were transitioned to intensive therapy group (DCCT, 1993). A long-term follow-up study under the EDIC Trial (DCCT/EDIC, 2002, DCCT/EDIC, 2003, Pop-Busui et al., 2009) showed that patients who were formerly in the intensive therapy group had lower incidence of diabetic microvascular complications than the patients who were originally in the conventional treatment group, despite 8 years of similar and normalized glycemic control in the EDIC trial (DCCT/EDIC, 2002). It was further reported that the conventionally treated-DCCT patients who were transitioned to intensive therapy group in the EDIC trial still had greater incidence of macrovascular pathology or complications (Nathan et al., 2003, Nathan et al., 2005, Cleary et al., 2006, Patel et al., 2008). Evidence from clinical trials also suggests that the incidence or development of diabetic complications is more likely to depend on the intensity of oxidative stress than on the level of glycemic control (Monnier et al., 1999, Genuth et al., 2005). Besides, evidence indicates that the degree of insulin resistance correlates with the onset of diabetic complications, independently of glycemic levels (Chillaron et al., 2009). In type 2 diabetic patients, it was reported that gliclazide treatment delayed the progression of diabetic nephropathy only, whereas it produced no significant effect on the development or progression of retinopathy or macrovascular complications (Patel et al., 2008). These findings are very important because gliclazide is one of the few hypoglycemic agents with an antioxidant effect (Fava et al., 2002). It is evident from the different aforementioned hyperglycemia-induced mechanistic pathways and the data (both experimental and clinical) presented in this section that the pathogenesis of diabetic complications is a complex process. It involves oxidative stress and ROS-activated pathways including several inflammatory proteins, cytokines and growth factors. It is clear that hyperglycemia exerts long-term injurious effects in patients with type 1 and type 2 diabetes mellitus. Similar long-lasting detrimental effects of hyperglycemia also occur in animals (dogs). In both animals and humans with diabetes, glycemic control only delays the development or progression of diabetic complications. It does not prevent or completely restore diabetic complications. This is understandable in view of the fact that these diabetic complications are not primary effects of hyperglycemia but are its sequelae (secondary effects). In fact, in few cases in which normalization of hyperglycemia or cure of diabetes mellitus do prevent diabetic complications, it has to be initiated at a very early stage of the disease and maintained for several years. Overall, these findings clearly show that intensive therapy or

normalization of hyperglycemia does not prevent diabetic complications.

**complications?** 

**6.3 Why does reduced or intensive therapy of hyperglycemia not prevent diabetic** 

Investigators have introduced different phrases to describe this concept or observation in which reduced or intensive therapy of hyperglycemia does not prevent diabetic complications. Some of these phrases include ''glycemic memory", "hyperglycemic memory", "metabolic memory'' or "lasting memory". Glycemic memory is a term that refers to the development of diabetic complications during post-hyperglycemic normoglycemia

with gliclazide for 12 weeks ameliorated oxidative stress better than did glibenclamide (Fava et al., 2002). Available data from DCCT and Epidemiology of Diabetic Complications and Interventions (EDIC) Trial indicated that type 1 diabetic patients in the intensive therapy group still had increased levels of protein glycation products and AGEs despite intensive treatment (Genuth et al., 2005). However, protein glycation and AGE formation were less compared with those in the conventional treatment group (Genuth et al., 2005).

The data highlighted in this section reveal that reduced or intensive therapy of hyperglycemia does not completely prevent induction of oxidative stress. In other words, once hyperglycemia (either via mitochondrial O2 •− overproduction or other mechanisms) activates any of these mechanistic pathways (especially the polyol pathway), intensive therapy or even normalization of hyperglycemia (which is even difficult with the current hypoglycemic drugs) would have limited effects on oxidative stress. Once oxidative stress is induced in diabetes mellitus, it can enhance the generation of ROS and initiate redox-chain reactions which may activate various inflammatory proteins and signaling pathways including all the abovementioned mechanistic pathways. Even if normal glucose level is restored, these inflammatory proteins and signaling pathways on their own could cause oxidative stress and/or sustain the oxidative stress originally or previously induced by hyperglycemia. Thus, with or without hyperglycemia, the resulting ROS and oxidative stress secondary to hyperglycemia would suffice to activate some of these pathways and set the stage for oxidative stress or exacerbate the already developed or existing oxidative stress. Besides, evidence implicates a role of oxidative stress in many neurodegenerative disorders such as hypertension, cancer and Alzheimer's disease. All of these disorders are characterized by euglycemia. Therefore, it means or suggests that oxidative stress can be an entirely independent phenomenon which can exist even with normalization of hyperglycemia.

#### **6.2 Does reduced or intensive therapy of hyperglycemia prevent diabetic complications?**

Data from animal and human studies indicate that intensive therapy may delay the progression of diabetic complications, but does not prevent diabetic complications. A study that investigated the effect of improved glycemic control on the progression of retinopathy in diabetic dogs found that diabetic dogs with 5-year poor glycemic control developed diabetic retinopathy while those with 5-year good glycemic control had no diabetic retinopathy (Engerman and Kern, 1987). The third group comprised diabetic dogs with 2.5 year poor glycemic control. It was observed that these diabetic dogs did not develop diabetic retinopathy. However, the dogs later developed diabetic retinopathy despite 2.5 year good glycemic control (Engerman and Kern, 1987). The study further revealed that the extent of pathology of diabetic retinopathy in the third group (with 2.5-year poor glycemic control + 2.5-year good glycemic control) was similar to that of the dogs with 5-year poor glycemic control (Engerman and Kern, 1987). Similarly, in sucrose-fed diabetic rats, cure of diabetes via islet transplantation at 12 weeks (but only at 6 weeks after the confirmation of diabetes) did not prevent lesions or progression of diabetic retinopathy (Hammes et al., 1993). Kowluru and colleagues (2007) also reported that after 6 months of poor glycemic control in rats, normalization of hyperglycemia for 6 months had no effect on the lesions or pathology of diabetic retinopathy.

with gliclazide for 12 weeks ameliorated oxidative stress better than did glibenclamide (Fava et al., 2002). Available data from DCCT and Epidemiology of Diabetic Complications and Interventions (EDIC) Trial indicated that type 1 diabetic patients in the intensive therapy group still had increased levels of protein glycation products and AGEs despite intensive treatment (Genuth et al., 2005). However, protein glycation and AGE formation were less compared with those in the conventional treatment group (Genuth et al., 2005).

The data highlighted in this section reveal that reduced or intensive therapy of hyperglycemia does not completely prevent induction of oxidative stress. In other words, once hyperglycemia (either via mitochondrial O2•− overproduction or other mechanisms) activates any of these mechanistic pathways (especially the polyol pathway), intensive therapy or even normalization of hyperglycemia (which is even difficult with the current hypoglycemic drugs) would have limited effects on oxidative stress. Once oxidative stress is induced in diabetes mellitus, it can enhance the generation of ROS and initiate redox-chain reactions which may activate various inflammatory proteins and signaling pathways including all the abovementioned mechanistic pathways. Even if normal glucose level is restored, these inflammatory proteins and signaling pathways on their own could cause oxidative stress and/or sustain the oxidative stress originally or previously induced by hyperglycemia. Thus, with or without hyperglycemia, the resulting ROS and oxidative stress secondary to hyperglycemia would suffice to activate some of these pathways and set the stage for oxidative stress or exacerbate the already developed or existing oxidative stress. Besides, evidence implicates a role of oxidative stress in many neurodegenerative disorders such as hypertension, cancer and Alzheimer's disease. All of these disorders are characterized by euglycemia. Therefore, it means or suggests that oxidative stress can be an entirely independent phenomenon which can exist even with normalization of

**6.2 Does reduced or intensive therapy of hyperglycemia prevent diabetic** 

Data from animal and human studies indicate that intensive therapy may delay the progression of diabetic complications, but does not prevent diabetic complications. A study that investigated the effect of improved glycemic control on the progression of retinopathy in diabetic dogs found that diabetic dogs with 5-year poor glycemic control developed diabetic retinopathy while those with 5-year good glycemic control had no diabetic retinopathy (Engerman and Kern, 1987). The third group comprised diabetic dogs with 2.5 year poor glycemic control. It was observed that these diabetic dogs did not develop diabetic retinopathy. However, the dogs later developed diabetic retinopathy despite 2.5 year good glycemic control (Engerman and Kern, 1987). The study further revealed that the extent of pathology of diabetic retinopathy in the third group (with 2.5-year poor glycemic control + 2.5-year good glycemic control) was similar to that of the dogs with 5-year poor glycemic control (Engerman and Kern, 1987). Similarly, in sucrose-fed diabetic rats, cure of diabetes via islet transplantation at 12 weeks (but only at 6 weeks after the confirmation of diabetes) did not prevent lesions or progression of diabetic retinopathy (Hammes et al., 1993). Kowluru and colleagues (2007) also reported that after 6 months of poor glycemic control in rats, normalization of hyperglycemia for 6 months had no effect on the lesions or

hyperglycemia.

**complications?** 

pathology of diabetic retinopathy.

In type I diabetic patients whose diabetes had been cured (through pancreas transplantation) but still had diabetic nephropathy, the thickness of glomerular and tubular basement membranes was still similar at 5 years versus baseline values. In contrast, it was significantly reduced only by the 10th year versus baseline values. The study further showed that while mesangial fractional volume had increased by the 5th year, it significantly decreased by the 10th year (Fioretto et al., 1998). In the DCCT, due to the considerable benefits of intensive therapy, patients in the conventional therapy group were transitioned to intensive therapy group (DCCT, 1993). A long-term follow-up study under the EDIC Trial (DCCT/EDIC, 2002, DCCT/EDIC, 2003, Pop-Busui et al., 2009) showed that patients who were formerly in the intensive therapy group had lower incidence of diabetic microvascular complications than the patients who were originally in the conventional treatment group, despite 8 years of similar and normalized glycemic control in the EDIC trial (DCCT/EDIC, 2002). It was further reported that the conventionally treated-DCCT patients who were transitioned to intensive therapy group in the EDIC trial still had greater incidence of macrovascular pathology or complications (Nathan et al., 2003, Nathan et al., 2005, Cleary et al., 2006, Patel et al., 2008). Evidence from clinical trials also suggests that the incidence or development of diabetic complications is more likely to depend on the intensity of oxidative stress than on the level of glycemic control (Monnier et al., 1999, Genuth et al., 2005). Besides, evidence indicates that the degree of insulin resistance correlates with the onset of diabetic complications, independently of glycemic levels (Chillaron et al., 2009). In type 2 diabetic patients, it was reported that gliclazide treatment delayed the progression of diabetic nephropathy only, whereas it produced no significant effect on the development or progression of retinopathy or macrovascular complications (Patel et al., 2008). These findings are very important because gliclazide is one of the few hypoglycemic agents with an antioxidant effect (Fava et al., 2002).

It is evident from the different aforementioned hyperglycemia-induced mechanistic pathways and the data (both experimental and clinical) presented in this section that the pathogenesis of diabetic complications is a complex process. It involves oxidative stress and ROS-activated pathways including several inflammatory proteins, cytokines and growth factors. It is clear that hyperglycemia exerts long-term injurious effects in patients with type 1 and type 2 diabetes mellitus. Similar long-lasting detrimental effects of hyperglycemia also occur in animals (dogs). In both animals and humans with diabetes, glycemic control only delays the development or progression of diabetic complications. It does not prevent or completely restore diabetic complications. This is understandable in view of the fact that these diabetic complications are not primary effects of hyperglycemia but are its sequelae (secondary effects). In fact, in few cases in which normalization of hyperglycemia or cure of diabetes mellitus do prevent diabetic complications, it has to be initiated at a very early stage of the disease and maintained for several years. Overall, these findings clearly show that intensive therapy or normalization of hyperglycemia does not prevent diabetic complications.

#### **6.3 Why does reduced or intensive therapy of hyperglycemia not prevent diabetic complications?**

Investigators have introduced different phrases to describe this concept or observation in which reduced or intensive therapy of hyperglycemia does not prevent diabetic complications. Some of these phrases include ''glycemic memory", "hyperglycemic memory", "metabolic memory'' or "lasting memory". Glycemic memory is a term that refers to the development of diabetic complications during post-hyperglycemic normoglycemia

Oxidative Stress in Diabetes Mellitus:

complications).

**complications?** 

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 233

demonstrated by Ceriello and colleagues (2008), using a euinsulinemic hyperglycemic clamp, glucose at 5, 10, and 15 mmol/l was given in rising steps as a single "spike", and oscillating between basal and high levels over 24-hour in non-diabetic subjects and type 2 diabetic patients. The authors reported that glucose at 10 and 15 mmol/l led to a concentration-dependent fasting blood glucose-independent impaired endothelial function and increased intensity of oxidative stress in both non-diabetic subjects and type 2 diabetic patients (Ceriello et al., 2008). The study also revealed that oscillating glucose concentrations between 5 and 15 mmol/l every 6 hour for 24 hours caused further significant impairments in endothelial function and induced oxidative stress compared with continuous 10 or 15 mmol/l glucose (Ceriello et al., 2008). These findings are very relevant because the current management of diabetes mellitus (including intensive therapy) does not control postprandial hyperglycemia. Besides, both endothelial function and oxidative stress are implicated in the pathogenesis of diabetic micro- and macrovascular complications. Oxidative stress also plays an important role in endothelial dysfunction (Sies, 1991, Halliwell and Gutteridge, 2007). This study is also remarkable because it shows that oscillating levels of glucose result in endothelial dysfunction and induced oxidative stress even in non-diabetic subjects (euglycemia). These postprandial hyperglycemia fluctuations may contribute to the continuous deterioration of diabetic complications, despite restoration of euglycemia. Therefore, postprandial hyperglycemia-induced endothelial dysfunction and

oxidative stress may explain the mechanism of glycemic or hyperglycemic memory.

**6.4 Could there be a role of antioxidants in the management of diabetic** 

The findings indicate that ROS-induced mutated DNA mitochondria may continuously generate RS (Nishikawa et al., 2000a). The data also reveal that AGEs can modify or glycate protein content of mitochondrial TCA cycle and the electron respiratory chain (Ceriello, 2009, Ceriello et al., 2009). Irrespective of the level of glycemic control, all these will generate RS and trigger cellular injury. Hence, it does suggest that oxidative stress in diabetes mellitus can exist as an independent entity. As explained previously, oxidative stress is also implicated in other disorders characterized by euglycemia. Since hyperglycemia enhances the pathogenic pathways of diabetic complications via oxidative stress, therefore, whether hyperglycemia is normalized or the recommended optimal glycemic goal ≤ 6.5% HbA1c is achieved and/or maintained, the already existing oxidative stress can activate these same mechanistic pathways and thus propagate glycemic memory (induce diabetic

Since oxidative stress is implicated in the pathogenesis of diabetes and its complications, there ought to be a place for antioxidants in the treatment of diabetes mellitus. However, previous clinical trials using antioxidants have yielded both promise and inconsistent results. Even though the data from the large scale clinical trials are inconclusive, it is noteworthy that many of those clinical trials were characterized by inappropriate study designs or several limitations (Johansen et al., 2005, Penckofer et al., 2002, Wierzba, 2005, Robinson et al., 2006, Willcox et al., 2008). These include: trials did not address specific diabetic populations; some studies included both healthy and unhealthy subjects; no data establishing the occurrence of oxidative stress in the patients before treatment and comparing such data with those obtained after treatment; the short duration of treatment; most of the trials were performed with vitamins A, C and E without consideration for other

(Nishikawa et al., 2000a). It is a phenomenon whereby early glycemic milieu or environment is remembered in many target organs such as heart, eye, nerve and kidney. Evidence suggests that oxidative stress may contribute to the inability of reduced or intensive treatment of hyperglycemia to prevent diabetic complications (Ceriello et al., 2009). It is proposed that glycemic memory could involve two stages: induction and perpetuation (Nishikawa et al., 2000a). During induction, hyperglycemia generates increased ROS levels. This may result from increased production of reducing equivalents formed from overdrive of the mitochondrial ETC. As a result of increased ROS, cellular dysfunction and mutations in mitochondrial DNA may occur (Wei, 1998). On the other hand, perpetuation, which is glycemic memory itself, could occur because ROS-induced mutated mitochondrial DNA would encode defective ETC subunits (Nishikawa et al., 2000a). This hypothesis of defective mitochondrial DNA subunits seems valid. Two years after this hypothesis, a study demonstrated that methylglyoxal modified mitochondrial proteins causing disturbances in mitochondria in kidney of rats (Rosca *et al.*, 2002). The study further showed that methylglyoxal produced an inhibitory effect on the tricarboxylic acid (TCA) cycle and the electron respiratory chain in kidney of rats. In another related study, it was shown that methylglyoxal-modified mitochondria considerably augmented O2 •− production,, independently of the level of hyperglycemia, and were characterized by oxidative damage (Rosca *et al.*, 2005).

Furthermore, Ihnat and colleagues (2007) also provided evidence in support of the role of oxidative stress in mediating the hyperglycemic memory (Ihnat et al., 2007). The authors showed that in human endothelial and ARPE-19 retinal cells, the levels of protein kinase Cbeta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, ffibronectin and poly (ADP-ribose) remained elevated for 1 week after the levels of glucose had normalized. The study showed that inhibition of ROS production using antioxidant alpha-lipoic acid prevented the induction of these glucose-induced oxidative stress markers (Ihnat et al., 2007). Similar findings were also reported in aortic endothelial cells both *in vitro* and in non-diabetic mice (El-Osta et al., 2008). The study showed that short-term hyperglycemic spikes produced long-lasting effects on vascular cells (El-Osta et al., 2008). This suggests that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications (El-Osta et al., 2008). A recent study also indicated that elevated glucose levels caused continual changes in cell viability and apoptosis-related gene expressions even after recovery of normoglycemia (Wei et al., 2011). The changes were associated with increased ROS production (Wei et al., 2011). Besides, evidence implicates the role of protein glycation and formation of AGEs in metabolic memory (Genuth et al., 2005, Ceriello et al., 2009). AGEs may reduce NADPH and impair antioxidant system which in turn can generate more ROS (Ahmed, 2005). Data from the DCCT suggest that the tendency of collagen to be glycated is less dependent on the level of blood glucose (Monnier et al., 1999). It was also demonstrated that both conventional and intensive therapies did not prevent protein glycation and formation of AGEs (Genuth et al., 2005). The data further revealed that the incidences of retinopathy and nephropathy were significantly associated with the levels of protein glycation and AGEs (Genuth et al., 2005). This phenomenon termed "glycemic memory" has also been corroborated in several other studies (Nathan et al., 2005, Holman et al., 2008).

Recent findings indicate that elevated oscillating glucose concentrations may contribute to increased risk for cardiovascular diabetic complications (Ceriello et al., 2008). As

(Nishikawa et al., 2000a). It is a phenomenon whereby early glycemic milieu or environment is remembered in many target organs such as heart, eye, nerve and kidney. Evidence suggests that oxidative stress may contribute to the inability of reduced or intensive treatment of hyperglycemia to prevent diabetic complications (Ceriello et al., 2009). It is proposed that glycemic memory could involve two stages: induction and perpetuation (Nishikawa et al., 2000a). During induction, hyperglycemia generates increased ROS levels. This may result from increased production of reducing equivalents formed from overdrive of the mitochondrial ETC. As a result of increased ROS, cellular dysfunction and mutations in mitochondrial DNA may occur (Wei, 1998). On the other hand, perpetuation, which is glycemic memory itself, could occur because ROS-induced mutated mitochondrial DNA would encode defective ETC subunits (Nishikawa et al., 2000a). This hypothesis of defective mitochondrial DNA subunits seems valid. Two years after this hypothesis, a study demonstrated that methylglyoxal modified mitochondrial proteins causing disturbances in mitochondria in kidney of rats (Rosca *et al.*, 2002). The study further showed that methylglyoxal produced an inhibitory effect on the tricarboxylic acid (TCA) cycle and the electron respiratory chain in kidney of rats. In another related study, it was shown that methylglyoxal-modified mitochondria considerably augmented O2•− production,, independently of the level of hyperglycemia, and were characterized by oxidative damage

Furthermore, Ihnat and colleagues (2007) also provided evidence in support of the role of oxidative stress in mediating the hyperglycemic memory (Ihnat et al., 2007). The authors showed that in human endothelial and ARPE-19 retinal cells, the levels of protein kinase Cbeta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, ffibronectin and poly (ADP-ribose) remained elevated for 1 week after the levels of glucose had normalized. The study showed that inhibition of ROS production using antioxidant alpha-lipoic acid prevented the induction of these glucose-induced oxidative stress markers (Ihnat et al., 2007). Similar findings were also reported in aortic endothelial cells both *in vitro* and in non-diabetic mice (El-Osta et al., 2008). The study showed that short-term hyperglycemic spikes produced long-lasting effects on vascular cells (El-Osta et al., 2008). This suggests that transient spikes of hyperglycemia may be an HbA1c-independent risk factor for diabetic complications (El-Osta et al., 2008). A recent study also indicated that elevated glucose levels caused continual changes in cell viability and apoptosis-related gene expressions even after recovery of normoglycemia (Wei et al., 2011). The changes were associated with increased ROS production (Wei et al., 2011). Besides, evidence implicates the role of protein glycation and formation of AGEs in metabolic memory (Genuth et al., 2005, Ceriello et al., 2009). AGEs may reduce NADPH and impair antioxidant system which in turn can generate more ROS (Ahmed, 2005). Data from the DCCT suggest that the tendency of collagen to be glycated is less dependent on the level of blood glucose (Monnier et al., 1999). It was also demonstrated that both conventional and intensive therapies did not prevent protein glycation and formation of AGEs (Genuth et al., 2005). The data further revealed that the incidences of retinopathy and nephropathy were significantly associated with the levels of protein glycation and AGEs (Genuth et al., 2005). This phenomenon termed "glycemic memory" has also been corroborated in several other studies (Nathan et

Recent findings indicate that elevated oscillating glucose concentrations may contribute to increased risk for cardiovascular diabetic complications (Ceriello et al., 2008). As

(Rosca *et al.*, 2005).

al., 2005, Holman et al., 2008).

demonstrated by Ceriello and colleagues (2008), using a euinsulinemic hyperglycemic clamp, glucose at 5, 10, and 15 mmol/l was given in rising steps as a single "spike", and oscillating between basal and high levels over 24-hour in non-diabetic subjects and type 2 diabetic patients. The authors reported that glucose at 10 and 15 mmol/l led to a concentration-dependent fasting blood glucose-independent impaired endothelial function and increased intensity of oxidative stress in both non-diabetic subjects and type 2 diabetic patients (Ceriello et al., 2008). The study also revealed that oscillating glucose concentrations between 5 and 15 mmol/l every 6 hour for 24 hours caused further significant impairments in endothelial function and induced oxidative stress compared with continuous 10 or 15 mmol/l glucose (Ceriello et al., 2008). These findings are very relevant because the current management of diabetes mellitus (including intensive therapy) does not control postprandial hyperglycemia. Besides, both endothelial function and oxidative stress are implicated in the pathogenesis of diabetic micro- and macrovascular complications. Oxidative stress also plays an important role in endothelial dysfunction (Sies, 1991, Halliwell and Gutteridge, 2007). This study is also remarkable because it shows that oscillating levels of glucose result in endothelial dysfunction and induced oxidative stress even in non-diabetic subjects (euglycemia). These postprandial hyperglycemia fluctuations may contribute to the continuous deterioration of diabetic complications, despite restoration of euglycemia. Therefore, postprandial hyperglycemia-induced endothelial dysfunction and oxidative stress may explain the mechanism of glycemic or hyperglycemic memory.

The findings indicate that ROS-induced mutated DNA mitochondria may continuously generate RS (Nishikawa et al., 2000a). The data also reveal that AGEs can modify or glycate protein content of mitochondrial TCA cycle and the electron respiratory chain (Ceriello, 2009, Ceriello et al., 2009). Irrespective of the level of glycemic control, all these will generate RS and trigger cellular injury. Hence, it does suggest that oxidative stress in diabetes mellitus can exist as an independent entity. As explained previously, oxidative stress is also implicated in other disorders characterized by euglycemia. Since hyperglycemia enhances the pathogenic pathways of diabetic complications via oxidative stress, therefore, whether hyperglycemia is normalized or the recommended optimal glycemic goal ≤ 6.5% HbA1c is achieved and/or maintained, the already existing oxidative stress can activate these same mechanistic pathways and thus propagate glycemic memory (induce diabetic complications).

#### **6.4 Could there be a role of antioxidants in the management of diabetic complications?**

Since oxidative stress is implicated in the pathogenesis of diabetes and its complications, there ought to be a place for antioxidants in the treatment of diabetes mellitus. However, previous clinical trials using antioxidants have yielded both promise and inconsistent results. Even though the data from the large scale clinical trials are inconclusive, it is noteworthy that many of those clinical trials were characterized by inappropriate study designs or several limitations (Johansen et al., 2005, Penckofer et al., 2002, Wierzba, 2005, Robinson et al., 2006, Willcox et al., 2008). These include: trials did not address specific diabetic populations; some studies included both healthy and unhealthy subjects; no data establishing the occurrence of oxidative stress in the patients before treatment and comparing such data with those obtained after treatment; the short duration of treatment; most of the trials were performed with vitamins A, C and E without consideration for other

Oxidative Stress in Diabetes Mellitus:

failure of antioxidants in clinical trials.

**the management of diabetic complications?** 

including oxidative stress parameters (Sindhu et al., 2004).

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 235

This is corroborated by findings of Gupta and co-workers (2011). That could also explain the

**6.5 Could there be a role of hypoglycemic drugs (and/or insulin) and antioxidants in** 

A closer look at the various mechanistic pathways implicated in the pathogenesis of diabetic complications indicates that hyperglycemia-enhanced polyol pathway (via depletion of intracellular NADPH) and O2•− inhibition of G6PDH (via hyperglycemia-enhanced O2•<sup>−</sup> overproduction) would play a major role in impairing antioxidant defenses. This will increase intracellular susceptibility to oxidative stress during diabetes. As regards evidence, very few studies have investigated the effects of combined hypoglycemic agents and antioxidants in diabetic rodents or patients. Interestingly, all these studies found beneficial effects of combination of these two agents in both animals and human with diabetes mellitus. A study that investigated the effects of 4-week insulin and/or antioxidant (vitamin E and C) supplementation in diabetic rats found that the antioxidant treatment improved some of the oxidative stress parameters whereas insulin treatment prevented weight loss and ameliorated the activities and expression of antioxidant enzymes. In contrast, the combination of insulin and antioxidants resulted in normalization of all measurements

Similarly, comparison of the effects of glibenclamide alone or combined with honey in pancreas of diabetic rats indicated that, even though glibenclamide reduced hyperglycemia, it only partially ameliorated oxidative stress parameters (most of the data were insignificant) (Erejuwa et al., 2011b). However, the combination of glibenclamide and honey significantly reduced hyperglycemia and ameliorated oxidative stress parameters in pancreas of diabetic rats (Erejuwa et al., 2011b). A similar study also showed that a combination of glibenclamide and metformin produced a limited antioxidant effect compared to when they were combined with honey in pancreas of diabetic rats (Erejuwa et al., 2010b). In the kidney of diabetic rats treated with metformin and/or glibenclamide, impaired antioxidant defenses were reported. In contrast, metformin and/or glibenclamide combined with honey significantly ameliorated oxidative stress parameters and restored the activities of antioxidant enzymes in the kidney of diabetic rats (Erejuwa et al., 2011a).

In type 1 and type 2 diabetic patients, a study found that while optimal glycemic control reduced the levels of MDA and increased the levels of GSH and vitamin E, it did not normalize the oxidative stress parameters (Chugh et al., 1999). However, after 4 weeks of vitamin E supplementation, the levels of oxidative stress markers were further reduced while those of endogenous antioxidants were increased compared to the optimal control values (without antioxidant treatment) (Chugh et al., 1999). A similar beneficial effect of vitamin E supplementation and optimal glycemic control was also reported in type 2 diabetic patients (Sharma et al., 2000). A number of other studies have also shown that antioxidants reduce glucose levels, improve insulin secretion and insulin resistance during diabetes (Penckofer et al., 2002). Moreover, a study found that in type 1 diabetic patients, normalization of glucose levels did not ameliorate hyperglycemia-induced endothelial dysfunction (Ceriello et al., 2007). The authors reported that, neither insulin nor vitamin C was able to ameliorate oxidative stress or normalize endothelial dysfunction (Ceriello et al., 2007). On the contrary, combination of insulin and vitamin C significantly decrease the

antioxidants; the use of vitamin E supplementation without the concurrent use of vitamin C (Johansen et al., 2005, Penckofer et al., 2002, Wierzba, 2005, Robinson et al., 2006, Willcox et al., 2008). Other limitations include: some trials are gender-specific (comprising either men or women); lack of pharmacokinetic data of these antioxidants, before and after treatment, so as to ascertain if these antioxidants reached the target cells/tissues in adequate concentrations; no data to show that effects of different doses of each antioxidant were investigated so as to obtain and select optimal dose; the suppression of gamma-tocopherol by alpha-tocopherol; vitamins inappropriately administered relative to meal ingestion; and poor patient compliance are some of the issues that may contribute to the failure of antioxidants in clinical studies (Johansen et al., 2005, Penckofer et al., 2002, Wierzba, 2005, Robinson et al., 2006, Willcox et al., 2008). Some endpoints that were not directly related to oxidative stress such as mortality were used in some trials (Johansen et al., 2005). Hence, limited research and findings are available on the effects of antioxidants in diabetic patients. However, available evidences in small or medium sample-sized diabetic studies, both experimental and clinical, suggest antioxidants might play a role in the management of diabetes mellitus.

In diabetic rats with neuropathy, α-lipoic acid supplementation ameliorated oxidative stress parameters and improved lesions of diabetic neuropathy such as conduction velocity of the digital nerve, deficits in nerve conduction and nerve blood flow (Coppey et al., 2001). Evidence suggests that antioxidants can inhibit some of the pathways of diabetic complications such as protein kinase C-signaled increases in TGF- in mesangial cells (Scott and King, 2004). Similarly, antioxidant treatment prevented the elevation in the levels of protein kinase C-beta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3 nitrotyrosine, fibronectin and poly (ADP) ribose in the retina of diabetic rats (Ihnat et al., 2007). In the kidney of diabetic rats, honey supplementation considerably reduced hyperglycemia, attenuated antioxidant enzymes, ameliorated oxidative stress markers and reduced mesangial matrix expansion and glomerular basement membrane thickness (Erejuwa et al., 2010a, Erejuwa et al., 2010c).

In patients with type 1 diabetes, supplementation with vitamins E and/or C combination ameliorated oxidative stress and improved endothelium-dependent vasorelaxation (Johansen et al., 2005, Rahimi et al., 2005). A study found that supplementation with combined chromium (Cr) and vitamins C and E ameliorated oxidative stress, reduced fasting blood glucose, HbA1c and insulin resistance in type 2 diabetes (Lai, 2008). Similarly, a recent study reported that vitamin E supplementation significantly reduced malondialdehyde (MDA) levels and increased the concentrations of GSH and vitamin E in type 1 diabetic patients (Gupta et al., 2011). The study also found a negative correlation between oxidative stress marker (MDA) and antioxidants (vitamin E and GSH) and a positive correlation between exogenously administered antioxidant (vitamin E) and endogenous antioxidant (GSH) (Gupta et al., 2011). However, the study showed that vitamin E supplementation in type 1 diabetic patients did not produce significant effects in metabolic parameters (Gupta et al., 2011). In patients with type 1 diabetes mellitus, vitamins C and E supplementation ameliorated oxidative stress markers, improved vascular dysfunction, retinal blood flow and creatinine clearance (Scott and King, 2004). These studies indicate that antioxidants could play a role in the management of diabetes mellitus. However, considering that diabetes mellitus is a disorder with multiple etiology and metabolic derangements, antioxidant supplementation alone is likely to be less effective.

antioxidants; the use of vitamin E supplementation without the concurrent use of vitamin C (Johansen et al., 2005, Penckofer et al., 2002, Wierzba, 2005, Robinson et al., 2006, Willcox et al., 2008). Other limitations include: some trials are gender-specific (comprising either men or women); lack of pharmacokinetic data of these antioxidants, before and after treatment, so as to ascertain if these antioxidants reached the target cells/tissues in adequate concentrations; no data to show that effects of different doses of each antioxidant were investigated so as to obtain and select optimal dose; the suppression of gamma-tocopherol by alpha-tocopherol; vitamins inappropriately administered relative to meal ingestion; and poor patient compliance are some of the issues that may contribute to the failure of antioxidants in clinical studies (Johansen et al., 2005, Penckofer et al., 2002, Wierzba, 2005, Robinson et al., 2006, Willcox et al., 2008). Some endpoints that were not directly related to oxidative stress such as mortality were used in some trials (Johansen et al., 2005). Hence, limited research and findings are available on the effects of antioxidants in diabetic patients. However, available evidences in small or medium sample-sized diabetic studies, both experimental and clinical, suggest antioxidants might play a role in the management of

In diabetic rats with neuropathy, α-lipoic acid supplementation ameliorated oxidative stress parameters and improved lesions of diabetic neuropathy such as conduction velocity of the digital nerve, deficits in nerve conduction and nerve blood flow (Coppey et al., 2001). Evidence suggests that antioxidants can inhibit some of the pathways of diabetic complications such as protein kinase C-signaled increases in TGF- in mesangial cells (Scott and King, 2004). Similarly, antioxidant treatment prevented the elevation in the levels of protein kinase C-beta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3 nitrotyrosine, fibronectin and poly (ADP) ribose in the retina of diabetic rats (Ihnat et al., 2007). In the kidney of diabetic rats, honey supplementation considerably reduced hyperglycemia, attenuated antioxidant enzymes, ameliorated oxidative stress markers and reduced mesangial matrix expansion and glomerular basement membrane thickness

In patients with type 1 diabetes, supplementation with vitamins E and/or C combination ameliorated oxidative stress and improved endothelium-dependent vasorelaxation (Johansen et al., 2005, Rahimi et al., 2005). A study found that supplementation with combined chromium (Cr) and vitamins C and E ameliorated oxidative stress, reduced fasting blood glucose, HbA1c and insulin resistance in type 2 diabetes (Lai, 2008). Similarly, a recent study reported that vitamin E supplementation significantly reduced malondialdehyde (MDA) levels and increased the concentrations of GSH and vitamin E in type 1 diabetic patients (Gupta et al., 2011). The study also found a negative correlation between oxidative stress marker (MDA) and antioxidants (vitamin E and GSH) and a positive correlation between exogenously administered antioxidant (vitamin E) and endogenous antioxidant (GSH) (Gupta et al., 2011). However, the study showed that vitamin E supplementation in type 1 diabetic patients did not produce significant effects in metabolic parameters (Gupta et al., 2011). In patients with type 1 diabetes mellitus, vitamins C and E supplementation ameliorated oxidative stress markers, improved vascular dysfunction, retinal blood flow and creatinine clearance (Scott and King, 2004). These studies indicate that antioxidants could play a role in the management of diabetes mellitus. However, considering that diabetes mellitus is a disorder with multiple etiology and metabolic derangements, antioxidant supplementation alone is likely to be less effective.

diabetes mellitus.

(Erejuwa et al., 2010a, Erejuwa et al., 2010c).

This is corroborated by findings of Gupta and co-workers (2011). That could also explain the failure of antioxidants in clinical trials.

#### **6.5 Could there be a role of hypoglycemic drugs (and/or insulin) and antioxidants in the management of diabetic complications?**

A closer look at the various mechanistic pathways implicated in the pathogenesis of diabetic complications indicates that hyperglycemia-enhanced polyol pathway (via depletion of intracellular NADPH) and O2•− inhibition of G6PDH (via hyperglycemia-enhanced O2•<sup>−</sup> overproduction) would play a major role in impairing antioxidant defenses. This will increase intracellular susceptibility to oxidative stress during diabetes. As regards evidence, very few studies have investigated the effects of combined hypoglycemic agents and antioxidants in diabetic rodents or patients. Interestingly, all these studies found beneficial effects of combination of these two agents in both animals and human with diabetes mellitus. A study that investigated the effects of 4-week insulin and/or antioxidant (vitamin E and C) supplementation in diabetic rats found that the antioxidant treatment improved some of the oxidative stress parameters whereas insulin treatment prevented weight loss and ameliorated the activities and expression of antioxidant enzymes. In contrast, the combination of insulin and antioxidants resulted in normalization of all measurements including oxidative stress parameters (Sindhu et al., 2004).

Similarly, comparison of the effects of glibenclamide alone or combined with honey in pancreas of diabetic rats indicated that, even though glibenclamide reduced hyperglycemia, it only partially ameliorated oxidative stress parameters (most of the data were insignificant) (Erejuwa et al., 2011b). However, the combination of glibenclamide and honey significantly reduced hyperglycemia and ameliorated oxidative stress parameters in pancreas of diabetic rats (Erejuwa et al., 2011b). A similar study also showed that a combination of glibenclamide and metformin produced a limited antioxidant effect compared to when they were combined with honey in pancreas of diabetic rats (Erejuwa et al., 2010b). In the kidney of diabetic rats treated with metformin and/or glibenclamide, impaired antioxidant defenses were reported. In contrast, metformin and/or glibenclamide combined with honey significantly ameliorated oxidative stress parameters and restored the activities of antioxidant enzymes in the kidney of diabetic rats (Erejuwa et al., 2011a).

In type 1 and type 2 diabetic patients, a study found that while optimal glycemic control reduced the levels of MDA and increased the levels of GSH and vitamin E, it did not normalize the oxidative stress parameters (Chugh et al., 1999). However, after 4 weeks of vitamin E supplementation, the levels of oxidative stress markers were further reduced while those of endogenous antioxidants were increased compared to the optimal control values (without antioxidant treatment) (Chugh et al., 1999). A similar beneficial effect of vitamin E supplementation and optimal glycemic control was also reported in type 2 diabetic patients (Sharma et al., 2000). A number of other studies have also shown that antioxidants reduce glucose levels, improve insulin secretion and insulin resistance during diabetes (Penckofer et al., 2002). Moreover, a study found that in type 1 diabetic patients, normalization of glucose levels did not ameliorate hyperglycemia-induced endothelial dysfunction (Ceriello et al., 2007). The authors reported that, neither insulin nor vitamin C was able to ameliorate oxidative stress or normalize endothelial dysfunction (Ceriello et al., 2007). On the contrary, combination of insulin and vitamin C significantly decrease the

Oxidative Stress in Diabetes Mellitus:

adverse effects of these drugs.

**7. Conclusions** 

diabetes mellitus.

Is There a Role for Hypoglycemic Drugs and/or Antioxidants? 237

reported (Bhor and Sivakami, 2003). Similarly, increased protein glycation and lipid peroxidation might exacerbate diabetes-related alterations in BBM fluidity (Watala and Winocour, 1992). Therefore, antioxidants may ameliorate intestinal oxidative stress and improve BBM fluidity, thereby promote healing and enhance gastrointestinal tract health in diabetes mellitus. This might impact positively on glycemic control. Antioxidants may also augment bioavailability of essential macronutrients or co-administered hypoglycemic drugs (Faria et al., 2009). Furthermore, antioxidants may help to ameliorate liver damage and hepatic oxidative stress which are common in diabetes mellitus (Dias et al., 2005, Erejuwa et al., 2012b). Considering the role of liver in glucose homeostasis and the fact that some hypoglycemic agents (e.g. glibenclamide) mediate their effects via liver, these hepatic effects of antioxidants combined with those of hypoglycemic drugs may enhance liver functions and contribute to improved glycemic control. Moreover, evidence suggests that the use of antioxidants is associated with reduced weight gain (Razquin et al., 2009, Erejuwa et al., 2012a), therefore, co-administration of antioxidants and hypoglycemic agents, especially glibenclamide, may be beneficial in type 2 diabetic patients, majority of whom are obese. Majority of diabetic patients end up developing hypertension which further increases the risk of developing diabetic complications including cardiovascular events. Interestingly, oxidative stress is also implicated in the pathogenesis and/or complications of hypertension. Therefore, a combination of hypoglycemic drugs and antioxidants, via improved glycemic control and amelioration of oxidative stress, may help to prevent or delay the development of hypertension and diabetic complications (Erejuwa et al., 2011c, 2012a). Besides, the combination of both agents may help to minimize the adverse effects or toxicities of hypoglycemic agents. The use of antioxidants may necessitate lower doses of hypoglycemic agents to achieve the same therapeutic effect, thereby limiting the side or

These studies indicate that hyperglycemia exerts long-term injurious effects in patients with type 1 and type 2 diabetes mellitus. Similar long-lasting detrimental effects of hyperglycemia also occur in diabetic animals. In both animals and humans with diabetes, glycemic control only delays the development or progression of diabetic complications. It does not prevent or completely restore diabetic complications. In few cases in which good glycemic control or cure of diabetes does prevent diabetic complications, it has to be initiated at a very early stage of the disease and maintained for several years. This is probably impracticable in the larger diabetic population. Evidence indicates it is not easy to achieve and/or maintain glycemic control in many diabetic patients close to the physiological range commonly observed in their healthy counterparts. Together with recent findings which demonstrate the deleterious effect of intensive therapy of hyperglycemia, it can be inferred that any therapeutic option that target hyperglycemia alone is not only limited and ineffective but may also be detrimental in diabetic patients. In view of the alarming rate of global prevalence of diabetes mellitus and the associated complications, morbidity and mortality, there is an urgent need for a better or new therapeutic management. While efforts are being made by researchers and scientists to unravel the main cause(s) of diabetes mellitus, it is high time clinicians, physicians and diabetologists began to look for an alternative and/or a complementary therapy to the current management of

intensity of oxidative stress and normalized endothelial dysfunction in type 1 diabetic patients (Ceriello et al., 2007).

A critical analysis of the mechanistic pathways of hyperglycemia-induced diabetic complications indicates that they are all characterized by increased formation of ROS and impaired antioxidant defense network, which would further exacerbate oxidative stress and damage. In other words, these pathways begin and end with oxidative stress. Besides, evidence indicates that postprandial hyperglycemic fluctuations can cause endothelial dysfunction and induce oxidative stress in diabetic subjects and even in euglycemic subjects. Hence, findings from these studies clearly indicate that it is oxidative stress all over in diabetes mellitus and its complications. Moreover, the data from experimental and clinical studies indicate that there is a role for co-administration of hypoglycemic drugs or insulin and antioxidants in diabetes mellitus. It is possible that normalization of hyperglycemia may be achieved with hypoglycemic drugs, insulin, their combinations or even via pancreatic transplant. However, in patients with diabetic complications, whether euglycemia is achieved and/or maintained, oxidative stress (and oxidative stress-induced sequelae) becomes an independent entity. Thus, oxidative stress, as a possible independent entity, in diabetes mellitus necessitates antioxidant therapy. On account of these observations, findings and data, there seems little doubt that antioxidant therapy or other therapeutic intervention of oxidative stress in combination with hypoglycemic drugs or insulin should result in better management of diabetes mellitus. This should also prevent or reduce ROSlinked diabetic complications.

#### **6.6 Other potential beneficial effects of hypoglycemic drugs (and/or insulin) and antioxidants in diabetes mellitus**

Diabetes mellitus is characterized by impairments in renal and hepatic function as well as impaired metabolism of glucose, lipid and protein. Lipid abnormalities and induction of oxidative stress enhance the oxidation and glycation of low-density lipoproteins (LDLs), thereby exacerbate endothelial dysfunction (Penckofer et al., 2002). Studies have shown that antioxidants and some hypoglycemic drugs can prevent oxidation of LDL (Fava et al., 2002, Maritim et al., 2003, Rahimi et al., 2005). Besides, evidence indicates that antioxidants can ameliorate lipid abnormalities (Rahimi et al., 2005, Erejuwa et al., 2011d). The beneficial effects of antioxidants on glycemic control (blood glucose, fructosamine and glycosylated hemoglobin) in diabetes have also been documented (Rahimi et al., 2005, Lai, 2008, Erejuwa et al., 2010a; 2011d). Furthermore, antioxidants improve C-peptide and insulin levels as well as insulin resistance in diabetes mellitus (Rahimi et al., 2005, Lai, 2008, Erejuwa et al., 2011d). Co-administration of hypoglycemic drugs (glibenclamide or metformin) and antioxidant (honey) considerably improved glycemic control and lipid parameters in diabetic rats more than the effects produced by individual hypoglycemic drug (Erejuwa et al., 2011d). It is worth mentioning that other non-antioxidant constituents of honey, such as fructose and oligosaccharides, might contribute to this improved glycemic control and lipid parameters. In addition, antioxidants ameliorated and improved impaired renal function which has been documented in diabetes mellitus (Slyvka et al., 2009, Erejuwa et al., 2011d) or in combination with hypoglycemic drugs may produce synergism (Erejuwa et al., 2011d).

In the duodenum and jejunum of diabetic rats, a number of alterations in the brush border membrane (BBM) fluidity, non-enzymatic glycation, oxidative stress and damage have been reported (Bhor and Sivakami, 2003). Similarly, increased protein glycation and lipid peroxidation might exacerbate diabetes-related alterations in BBM fluidity (Watala and Winocour, 1992). Therefore, antioxidants may ameliorate intestinal oxidative stress and improve BBM fluidity, thereby promote healing and enhance gastrointestinal tract health in diabetes mellitus. This might impact positively on glycemic control. Antioxidants may also augment bioavailability of essential macronutrients or co-administered hypoglycemic drugs (Faria et al., 2009). Furthermore, antioxidants may help to ameliorate liver damage and hepatic oxidative stress which are common in diabetes mellitus (Dias et al., 2005, Erejuwa et al., 2012b). Considering the role of liver in glucose homeostasis and the fact that some hypoglycemic agents (e.g. glibenclamide) mediate their effects via liver, these hepatic effects of antioxidants combined with those of hypoglycemic drugs may enhance liver functions and contribute to improved glycemic control. Moreover, evidence suggests that the use of antioxidants is associated with reduced weight gain (Razquin et al., 2009, Erejuwa et al., 2012a), therefore, co-administration of antioxidants and hypoglycemic agents, especially glibenclamide, may be beneficial in type 2 diabetic patients, majority of whom are obese. Majority of diabetic patients end up developing hypertension which further increases the risk of developing diabetic complications including cardiovascular events. Interestingly, oxidative stress is also implicated in the pathogenesis and/or complications of hypertension. Therefore, a combination of hypoglycemic drugs and antioxidants, via improved glycemic control and amelioration of oxidative stress, may help to prevent or

delay the development of hypertension and diabetic complications (Erejuwa et al., 2011c, 2012a). Besides, the combination of both agents may help to minimize the adverse effects or toxicities of hypoglycemic agents. The use of antioxidants may necessitate lower doses of hypoglycemic agents to achieve the same therapeutic effect, thereby limiting the side or adverse effects of these drugs.

#### **7. Conclusions**

236 Oxidative Stress and Diseases

intensity of oxidative stress and normalized endothelial dysfunction in type 1 diabetic

A critical analysis of the mechanistic pathways of hyperglycemia-induced diabetic complications indicates that they are all characterized by increased formation of ROS and impaired antioxidant defense network, which would further exacerbate oxidative stress and damage. In other words, these pathways begin and end with oxidative stress. Besides, evidence indicates that postprandial hyperglycemic fluctuations can cause endothelial dysfunction and induce oxidative stress in diabetic subjects and even in euglycemic subjects. Hence, findings from these studies clearly indicate that it is oxidative stress all over in diabetes mellitus and its complications. Moreover, the data from experimental and clinical studies indicate that there is a role for co-administration of hypoglycemic drugs or insulin and antioxidants in diabetes mellitus. It is possible that normalization of hyperglycemia may be achieved with hypoglycemic drugs, insulin, their combinations or even via pancreatic transplant. However, in patients with diabetic complications, whether euglycemia is achieved and/or maintained, oxidative stress (and oxidative stress-induced sequelae) becomes an independent entity. Thus, oxidative stress, as a possible independent entity, in diabetes mellitus necessitates antioxidant therapy. On account of these observations, findings and data, there seems little doubt that antioxidant therapy or other therapeutic intervention of oxidative stress in combination with hypoglycemic drugs or insulin should result in better management of diabetes mellitus. This should also prevent or reduce ROS-

**6.6 Other potential beneficial effects of hypoglycemic drugs (and/or insulin) and** 

Diabetes mellitus is characterized by impairments in renal and hepatic function as well as impaired metabolism of glucose, lipid and protein. Lipid abnormalities and induction of oxidative stress enhance the oxidation and glycation of low-density lipoproteins (LDLs), thereby exacerbate endothelial dysfunction (Penckofer et al., 2002). Studies have shown that antioxidants and some hypoglycemic drugs can prevent oxidation of LDL (Fava et al., 2002, Maritim et al., 2003, Rahimi et al., 2005). Besides, evidence indicates that antioxidants can ameliorate lipid abnormalities (Rahimi et al., 2005, Erejuwa et al., 2011d). The beneficial effects of antioxidants on glycemic control (blood glucose, fructosamine and glycosylated hemoglobin) in diabetes have also been documented (Rahimi et al., 2005, Lai, 2008, Erejuwa et al., 2010a; 2011d). Furthermore, antioxidants improve C-peptide and insulin levels as well as insulin resistance in diabetes mellitus (Rahimi et al., 2005, Lai, 2008, Erejuwa et al., 2011d). Co-administration of hypoglycemic drugs (glibenclamide or metformin) and antioxidant (honey) considerably improved glycemic control and lipid parameters in diabetic rats more than the effects produced by individual hypoglycemic drug (Erejuwa et al., 2011d). It is worth mentioning that other non-antioxidant constituents of honey, such as fructose and oligosaccharides, might contribute to this improved glycemic control and lipid parameters. In addition, antioxidants ameliorated and improved impaired renal function which has been documented in diabetes mellitus (Slyvka et al., 2009, Erejuwa et al., 2011d) or in combination with hypoglycemic drugs may produce synergism (Erejuwa et al., 2011d). In the duodenum and jejunum of diabetic rats, a number of alterations in the brush border membrane (BBM) fluidity, non-enzymatic glycation, oxidative stress and damage have been

patients (Ceriello et al., 2007).

linked diabetic complications.

**antioxidants in diabetes mellitus** 

These studies indicate that hyperglycemia exerts long-term injurious effects in patients with type 1 and type 2 diabetes mellitus. Similar long-lasting detrimental effects of hyperglycemia also occur in diabetic animals. In both animals and humans with diabetes, glycemic control only delays the development or progression of diabetic complications. It does not prevent or completely restore diabetic complications. In few cases in which good glycemic control or cure of diabetes does prevent diabetic complications, it has to be initiated at a very early stage of the disease and maintained for several years. This is probably impracticable in the larger diabetic population. Evidence indicates it is not easy to achieve and/or maintain glycemic control in many diabetic patients close to the physiological range commonly observed in their healthy counterparts. Together with recent findings which demonstrate the deleterious effect of intensive therapy of hyperglycemia, it can be inferred that any therapeutic option that target hyperglycemia alone is not only limited and ineffective but may also be detrimental in diabetic patients. In view of the alarming rate of global prevalence of diabetes mellitus and the associated complications, morbidity and mortality, there is an urgent need for a better or new therapeutic management. While efforts are being made by researchers and scientists to unravel the main cause(s) of diabetes mellitus, it is high time clinicians, physicians and diabetologists began to look for an alternative and/or a complementary therapy to the current management of diabetes mellitus.

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At the moment, one of such options or alternatives is the prospective of managing diabetes mellitus by targeting both hyperglycemia and oxidative stress simultaneously. As the data presented in this chapter have revealed, co-administration of oral hypoglycemic drugs (and/or insulin) and antioxidants might prove to be a better therapy in the management of diabetes mellitus. This is because, with the combination, hypoglycemic drugs (and/or insulin) will target hyperglycemia to improve glycemic control and reduce hyperglycemiaenhanced ROS production. In addition, administration of antioxidants will help to scavenge or eliminate RS including those generated in the various pathways highlighted in this chapter. Besides, many antioxidants have hypoglycemic effect which may also contribute to improved glycemic control. The co-administration of these two agents will help to minimize the level of oxidative stress in the vasculature and other targets of diabetic complications such as kidney (reducing diabetic nephropathy), retina (reducing diabetic retinopathy), nerves (reducing diabetic neuropathy) and heart (reducing diabetic cardiomyopathy). Moreover, evidence has shown that the pancreas and the liver are also target of oxidative stress in diabetes mellitus. Hence, co-administration of antioxidants will help to ameliorate oxidative stress in these tissues and organs (pancreas and liver) which play key roles in glucose homeostasis in diabetes mellitus.

In addition to improved glycemic control and amelioration of oxidative stress, evidence suggests that co-administration of hypoglycemic drugs and antioxidants may exert other beneficial effects on gastrointestinal tract, lipid profile, renal function and others which will contribute to better management of diabetic patients. Considering the limitations with antioxidants, coupled with the latest advances in our understanding of the various mechanisms involved in ROS formation, other interventions such as inhibition of the mitochondrial ROS overproduction, developing mitochondria-targeted antioxidants, blockage of hyperglycemia-induced mechanistic pathways are viable therapeutic options. This chapter has shown that the prospective of managing diabetes mellitus more effectively by targeting both hyperglycemia and oxidative stress simultaneously holds much promise. This new therapeutic option is worth investigating in patients with diabetes mellitus. Hence, both small and large, well designed, randomized clinical trials that examine the effect of combination of hypoglycemic drugs (and/or insulin) and specific antioxidants in patients with type 1 or type 2 diabetes mellitus are recommended. This may revolutionize the management of diabetes mellitus, at least in the interim, while attempts are being made to discover its main cause(s) and develop more potent and effective antidiabetic drugs.

#### **8. Dedication**

This chapter is dedicated to the memory of my dad, Educator Sephaniah Adeyemi Erejuwa, who battled diabetes mellitus and later succumbed to its complications. It is also dedicated to millions of people globally who are suffering from this disorder and its complications

#### **9. References**

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At the moment, one of such options or alternatives is the prospective of managing diabetes mellitus by targeting both hyperglycemia and oxidative stress simultaneously. As the data presented in this chapter have revealed, co-administration of oral hypoglycemic drugs (and/or insulin) and antioxidants might prove to be a better therapy in the management of diabetes mellitus. This is because, with the combination, hypoglycemic drugs (and/or insulin) will target hyperglycemia to improve glycemic control and reduce hyperglycemiaenhanced ROS production. In addition, administration of antioxidants will help to scavenge or eliminate RS including those generated in the various pathways highlighted in this chapter. Besides, many antioxidants have hypoglycemic effect which may also contribute to improved glycemic control. The co-administration of these two agents will help to minimize the level of oxidative stress in the vasculature and other targets of diabetic complications such as kidney (reducing diabetic nephropathy), retina (reducing diabetic retinopathy), nerves (reducing diabetic neuropathy) and heart (reducing diabetic cardiomyopathy). Moreover, evidence has shown that the pancreas and the liver are also target of oxidative stress in diabetes mellitus. Hence, co-administration of antioxidants will help to ameliorate oxidative stress in these tissues and organs (pancreas and liver) which play key roles in

In addition to improved glycemic control and amelioration of oxidative stress, evidence suggests that co-administration of hypoglycemic drugs and antioxidants may exert other beneficial effects on gastrointestinal tract, lipid profile, renal function and others which will contribute to better management of diabetic patients. Considering the limitations with antioxidants, coupled with the latest advances in our understanding of the various mechanisms involved in ROS formation, other interventions such as inhibition of the mitochondrial ROS overproduction, developing mitochondria-targeted antioxidants, blockage of hyperglycemia-induced mechanistic pathways are viable therapeutic options. This chapter has shown that the prospective of managing diabetes mellitus more effectively by targeting both hyperglycemia and oxidative stress simultaneously holds much promise. This new therapeutic option is worth investigating in patients with diabetes mellitus. Hence, both small and large, well designed, randomized clinical trials that examine the effect of combination of hypoglycemic drugs (and/or insulin) and specific antioxidants in patients with type 1 or type 2 diabetes mellitus are recommended. This may revolutionize the management of diabetes mellitus, at least in the interim, while attempts are being made to

discover its main cause(s) and develop more potent and effective antidiabetic drugs.

This chapter is dedicated to the memory of my dad, Educator Sephaniah Adeyemi Erejuwa, who battled diabetes mellitus and later succumbed to its complications. It is also dedicated to millions of people globally who are suffering from this disorder and its complications

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**8. Dedication** 

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**11** 

*Australia* 

**Oxidative Stress and Novel Antioxidant** 

Sih Min Tan, Arpeeta Sharma and Judy B. de Haan *Oxidative Stress Laboratory, Diabetic Complications Division,* 

*Baker IDI Heart and Diabetes Institute, Melbourne,* 

**Approaches to Reduce Diabetic Complications** 

In 1995, the International Diabetes Federation estimated the prevalence of diabetes to be approximately 135 million patients worldwide. More recently in 2010, it was estimated that around 285 million people were diabetic and this number is predicted to reach 438 million by 2030, accounting for 7.7% of the population aged 20-79 (Shaw et al 2010). However, despite greater knowledge of the disease, approximately one-third of people with diabetes remain undiagnosed. Although intensive blood glucose and blood pressure control have reduced the risk of diabetes-associated microvascular (nephropathy, retinopathy, neuropathy) and macrovascular complications (atherosclerosis), diabetes remains a major risk factor for cardiovascular complications, cardiomyopathy, end-stage renal disease (ESRD), blindness and neuropathy. There is therefore an urgent need to develop more effective therapeutic strategies to prevent and/or halt the progression of diabetic

Accumulating evidence suggests that oxidative stress plays a pivotal role in the aetiology of diabetic complications. Many biochemical pathways associated with hyperglycaemia increase the production of free radicals leading to oxidative stress, including glucose autooxidation, the polyol pathway, prostanoids synthesis, protein glycation and the protein kinase C (PKC) pathway (Giugliano et al 1996). Hyperglycaemia alters reactive oxygen species (ROS) production, particularly in the mitochondria, leading to increased intracellular ROS and activated stress-sensitive pathways such as nuclear factor κB (NFκB), p38 mitogen-activated protein kinase (MAPK), and the c-Jun NH2-terminal kinase/stressactivated protein kinase (JNK/SAPK) pathways (Johansen et al 2005). Subsequently, PKC activity, advanced glycation end-products (AGE) and sorbitol levels increase and this can lead to more ROS generation in a positive regulatory feedback loop to chronically stimulate stress-sensitive pathways. ROS can also inflict direct damage upon cellular macromolecules

Under physiological conditions, reactive oxygen and reactive nitrogen species (RNS) are produced and maintained at steady-state levels within a cell (Lushchak 2011). On the other hand, oxidative stress arises when an imbalance occurs between the production of ROS/RNS and the antioxidant defences that neutralise them, shifting the balance in favour

which, in turn, result in further oxidative stress (Evans et al 2002).

**1. Introduction** 

complications.


### **Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications**

Sih Min Tan, Arpeeta Sharma and Judy B. de Haan *Oxidative Stress Laboratory, Diabetic Complications Division, Baker IDI Heart and Diabetes Institute, Melbourne, Australia* 

#### **1. Introduction**

246 Oxidative Stress and Diseases

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inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty

In 1995, the International Diabetes Federation estimated the prevalence of diabetes to be approximately 135 million patients worldwide. More recently in 2010, it was estimated that around 285 million people were diabetic and this number is predicted to reach 438 million by 2030, accounting for 7.7% of the population aged 20-79 (Shaw et al 2010). However, despite greater knowledge of the disease, approximately one-third of people with diabetes remain undiagnosed. Although intensive blood glucose and blood pressure control have reduced the risk of diabetes-associated microvascular (nephropathy, retinopathy, neuropathy) and macrovascular complications (atherosclerosis), diabetes remains a major risk factor for cardiovascular complications, cardiomyopathy, end-stage renal disease (ESRD), blindness and neuropathy. There is therefore an urgent need to develop more effective therapeutic strategies to prevent and/or halt the progression of diabetic complications.

Accumulating evidence suggests that oxidative stress plays a pivotal role in the aetiology of diabetic complications. Many biochemical pathways associated with hyperglycaemia increase the production of free radicals leading to oxidative stress, including glucose autooxidation, the polyol pathway, prostanoids synthesis, protein glycation and the protein kinase C (PKC) pathway (Giugliano et al 1996). Hyperglycaemia alters reactive oxygen species (ROS) production, particularly in the mitochondria, leading to increased intracellular ROS and activated stress-sensitive pathways such as nuclear factor κB (NFκB), p38 mitogen-activated protein kinase (MAPK), and the c-Jun NH2-terminal kinase/stressactivated protein kinase (JNK/SAPK) pathways (Johansen et al 2005). Subsequently, PKC activity, advanced glycation end-products (AGE) and sorbitol levels increase and this can lead to more ROS generation in a positive regulatory feedback loop to chronically stimulate stress-sensitive pathways. ROS can also inflict direct damage upon cellular macromolecules which, in turn, result in further oxidative stress (Evans et al 2002).

Under physiological conditions, reactive oxygen and reactive nitrogen species (RNS) are produced and maintained at steady-state levels within a cell (Lushchak 2011). On the other hand, oxidative stress arises when an imbalance occurs between the production of ROS/RNS and the antioxidant defences that neutralise them, shifting the balance in favour

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 249

Due to its vasorelaxation, anti-inflammatory and anti-proliferative properties, EDNO is often viewed as vasculoprotective. Diabetes is associated with an attenuation of EDNO bioavailability, which is lowered by either decreased formation or enhanced removal of

NO. One way in which hyperglycaemia attenuates the level of EDNO is by blocking the function of endothelial NOS (eNOS) synthase in endothelial and vascular smooth muscle cells (De Vriese et al 2000). However, evidence now points most strongly at the prevention of EDNO reaching its molecular target, rather than its attenuated production as being critical for the loss of EDNO bioavailability in diabetes. An increase in ROS, such as •

within the endothelium is one of the most significant factors known to decrease EDNO (de

functional EDNO causes impaired relaxation of the vessel wall and inhibition of the

damage via lipid peroxidation, and the inactivation of enzymes and structural proteins by oxidation and nitration. Furthermore, ONOO- can activate matrix metalloproteinases (MMPs), trigger the release of pro-apoptic factors such as cytochrome c and induce DNA damage (Pacher & Szabo 2006). ONOO- is also involved in oxidising tetrahydrobiopterin (BH4), an important cofactor of eNOS, thereby uncoupling eNOS which then produces •

angiotensin-converting enzyme (ACE) activity and the generation of Ang II and •

(Schulman et al 2006). While EDNO inhibits the production of endothelin-1 (ET-1) which is a vasoconstricting peptide (Boulanger & Luscher 1990), increased Ang II can stimulate the endothelial cell to synthesise and release ET-1, thereby contributing to vascular smooth muscle dysfunction (Sasser et al 2002). A disruption in vascular smooth muscle function may lead to plaque destabilisation and rupture, with often fatal consequences (Beckman et al 2002). Since EDNO also limits inflammation by reducing leukocyte adhesion and migration (Chen et al 1998), lowering EDNO will also promote atherogenesis via accelerated pro-inflammatory pathways. Additionally, EDNO is also involved in the inhibition of platelet activation (Loscalzo 2001). A reduction in the bioavailability of EDNO in diabetes therefore potentiates platelet activation, adhesion and aggregate formation, leading to

O2-

O2 -

effects of EDNO in the vasculature leading to increased inflammation, thrombosis, plaque

shown to activate the nuclear enzyme poly(ADP-ribose) polymerase (PARP) which in turn leads to metabolic alterations that activate NFκB, AGE/receptor for AGE (RAGE) and the polyol pathways (Pacher & Szabo 2006). Upregulation of these pathways results in more •

production, while NFκB activation increases the expression of many proinflammatory mediators, leading to endothelial dysfunction. Furthermore, it is well accepted that ROS such

 are involved in the oxidation of low-density lipoprotein (ox-LDL), which is not recognised by the LDL receptor and is preferentially taken up by scavenger receptors on macrophages, leading to foam cell formation and atherosclerotic plaques (Boullier et al 2001).

numerous and mostly detrimental to the cells of the vasculature. For example, •

NO, producing the highly reactive oxidant, ONOO- (Beckman 1996). Loss of

can reduce EDNO bioavailability due to its propensity

NO, as evident in diabetes, stimulates endothelial

, as seen in diabetes, limits the protective

levels in the mitochondria are

O2 -

has been

O2 -

O2 -

O2-

O2-

can induce cell

**2.1.1 The role of ROS in diabetes-associated atherosclerosis** 

O2 -

proliferative effects of EDNO (Maritim et al 2003a). In addition, ONOO-

•

Haan & Cooper 2011). Increased •

instead of EDNO (Maritim et al 2003a). It is also known that a reduction in •

thrombosis. Hence, the increased presence of •

The effects of hyperglycaemia-mediated increases in •

destabilisation and plaque rupture (Fig.1).

to react with •

as • O2 -

of enhanced ROS levels. The consequence of this shift is cellular damage to biologically important molecules and organelles (Sies 1997). Elevations in ROS/RNS levels are mainly caused by an imbalance between the activity of endogenous pro-oxidant enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase or the mitochondrial respiratory chain, and the antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), heme oxygenase (HO), thioredoxin (Trx) peroxidase/peroxiredoxin, catalase and paraoxonase (Forstermann 2008). ROS include the superoxide anion (• O2 - ) and the hydroxyl radical (• OH), as well as non-radical species such as hydrogen peroxide (H2O2). RNS include the free radicals nitric oxide (• NO) and nonradical species such as peroxynitrite (ONOO- ) and nitrogen dioxide (NO2)(Johansen et al 2005). In a hyperglycaemic milieu, • O2 - increases through the enhanced activity of enzymatic sources, including NADPH oxidase and xanthine oxidase, and non-enzymatic sources such as the mitochondrial respiratory chain, glucose autoxidation, AGE formation and activation of the polyol pathway. In addition, antioxidant defences are known to decrease in a hyperglycaemic milieu, shifting the balance away from steady-state levels of ROS towards an environment of oxidative stress.

#### **2. Oxidative stress and diabetic complications**

#### **2.1 Diabetes-associated atherosclerosis**

Atherosclerosis is a major cause of mortality and morbidity in patients with diabetes (Beckman et al 2002). The buildup of fat and cholesterol along the walls of arteries is progressive; it thickens and hardens, forming calcium deposits, and may eventually block the arteries. Blockage of the arteries and/or rupture of vulnerable plaques is a common cause of heart attack and stroke. Diabetes has been shown to accelerate the clinical course of atherosclerosis in the coronary arteries (coronary artery disease, including myocardial infarction), lower extremities (peripheral arterial disease) and extracranial carotid arteries (cerebrovascular disease, including stroke) (Beckman et al 2002).

An understanding of the underlying mechanisms that accelerate diabetes-associated atherosclerosis is important in the search for treatments to protect against or retard the progression of this disease. The abnormal metabolic state associated with diabetes, which includes chronic hyperglycaemia, dyslipidemia and insulin resistance can alter the function of multiple cell types including endothelial cells, smooth muscle cells and platelets. The single layer of endothelial cells that line the vessels of the circulatory system, provide a metabolically active interface between the blood and the underlying tissue to facilitate blood flow and nutrient delivery. Disruption of the integrity of the endothelium leads to inflammation, activation of platelets, coagulation, and thrombosis (Cines et al 1998). To protect against this, the endothelium synthesises important bioactive substances such as endothelial-derived NO (EDNO), prostaglandins, endothelin (ET) and angiotensin II (Ang II) that regulate blood vessel function and structure (Beckman et al 2002). It is now known that hyperglycaemia-mediated dysregulation of these vasoprotective agents either enhances the intensity of oxidative stress directly or is affected by oxidative stress. The consequences of hyperglycaemia-driven enhanced ROS/RNS on the vasculature will be discussed below.

#### **2.1.1 The role of ROS in diabetes-associated atherosclerosis**

248 Oxidative Stress and Diseases

of enhanced ROS levels. The consequence of this shift is cellular damage to biologically important molecules and organelles (Sies 1997). Elevations in ROS/RNS levels are mainly caused by an imbalance between the activity of endogenous pro-oxidant enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase or the mitochondrial respiratory chain, and the antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), heme oxygenase (HO), thioredoxin (Trx) peroxidase/peroxiredoxin, catalase and paraoxonase (Forstermann 2008). ROS include the

sources, including NADPH oxidase and xanthine oxidase, and non-enzymatic sources such as the mitochondrial respiratory chain, glucose autoxidation, AGE formation and activation of the polyol pathway. In addition, antioxidant defences are known to decrease in a hyperglycaemic milieu, shifting the balance away from steady-state levels of ROS towards

Atherosclerosis is a major cause of mortality and morbidity in patients with diabetes (Beckman et al 2002). The buildup of fat and cholesterol along the walls of arteries is progressive; it thickens and hardens, forming calcium deposits, and may eventually block the arteries. Blockage of the arteries and/or rupture of vulnerable plaques is a common cause of heart attack and stroke. Diabetes has been shown to accelerate the clinical course of atherosclerosis in the coronary arteries (coronary artery disease, including myocardial infarction), lower extremities (peripheral arterial disease) and extracranial carotid arteries

An understanding of the underlying mechanisms that accelerate diabetes-associated atherosclerosis is important in the search for treatments to protect against or retard the progression of this disease. The abnormal metabolic state associated with diabetes, which includes chronic hyperglycaemia, dyslipidemia and insulin resistance can alter the function of multiple cell types including endothelial cells, smooth muscle cells and platelets. The single layer of endothelial cells that line the vessels of the circulatory system, provide a metabolically active interface between the blood and the underlying tissue to facilitate blood flow and nutrient delivery. Disruption of the integrity of the endothelium leads to inflammation, activation of platelets, coagulation, and thrombosis (Cines et al 1998). To protect against this, the endothelium synthesises important bioactive substances such as endothelial-derived NO (EDNO), prostaglandins, endothelin (ET) and angiotensin II (Ang II) that regulate blood vessel function and structure (Beckman et al 2002). It is now known that hyperglycaemia-mediated dysregulation of these vasoprotective agents either enhances the intensity of oxidative stress directly or is affected by oxidative stress. The consequences of hyperglycaemia-driven enhanced

OH), as well as non-radical species such

) and nitrogen dioxide (NO2)(Johansen et al

O2- increases through the enhanced activity of enzymatic

NO) and non-

) and the hydroxyl radical (•

as hydrogen peroxide (H2O2). RNS include the free radicals nitric oxide (•

superoxide anion (•

O2 -

2005). In a hyperglycaemic milieu, •

an environment of oxidative stress.

**2.1 Diabetes-associated atherosclerosis** 

radical species such as peroxynitrite (ONOO-

**2. Oxidative stress and diabetic complications** 

(cerebrovascular disease, including stroke) (Beckman et al 2002).

ROS/RNS on the vasculature will be discussed below.

Due to its vasorelaxation, anti-inflammatory and anti-proliferative properties, EDNO is often viewed as vasculoprotective. Diabetes is associated with an attenuation of EDNO bioavailability, which is lowered by either decreased formation or enhanced removal of • NO. One way in which hyperglycaemia attenuates the level of EDNO is by blocking the function of endothelial NOS (eNOS) synthase in endothelial and vascular smooth muscle cells (De Vriese et al 2000). However, evidence now points most strongly at the prevention of EDNO reaching its molecular target, rather than its attenuated production as being critical for the loss of EDNO bioavailability in diabetes. An increase in ROS, such as • O2 within the endothelium is one of the most significant factors known to decrease EDNO (de Haan & Cooper 2011). Increased • O2 can reduce EDNO bioavailability due to its propensity to react with • NO, producing the highly reactive oxidant, ONOO- (Beckman 1996). Loss of functional EDNO causes impaired relaxation of the vessel wall and inhibition of the proliferative effects of EDNO (Maritim et al 2003a). In addition, ONOO can induce cell damage via lipid peroxidation, and the inactivation of enzymes and structural proteins by oxidation and nitration. Furthermore, ONOO- can activate matrix metalloproteinases (MMPs), trigger the release of pro-apoptic factors such as cytochrome c and induce DNA damage (Pacher & Szabo 2006). ONOO- is also involved in oxidising tetrahydrobiopterin (BH4), an important cofactor of eNOS, thereby uncoupling eNOS which then produces • O2 instead of EDNO (Maritim et al 2003a).

It is also known that a reduction in • NO, as evident in diabetes, stimulates endothelial angiotensin-converting enzyme (ACE) activity and the generation of Ang II and • O2- (Schulman et al 2006). While EDNO inhibits the production of endothelin-1 (ET-1) which is a vasoconstricting peptide (Boulanger & Luscher 1990), increased Ang II can stimulate the endothelial cell to synthesise and release ET-1, thereby contributing to vascular smooth muscle dysfunction (Sasser et al 2002). A disruption in vascular smooth muscle function may lead to plaque destabilisation and rupture, with often fatal consequences (Beckman et al 2002). Since EDNO also limits inflammation by reducing leukocyte adhesion and migration (Chen et al 1998), lowering EDNO will also promote atherogenesis via accelerated pro-inflammatory pathways. Additionally, EDNO is also involved in the inhibition of platelet activation (Loscalzo 2001). A reduction in the bioavailability of EDNO in diabetes therefore potentiates platelet activation, adhesion and aggregate formation, leading to thrombosis. Hence, the increased presence of • O2-, as seen in diabetes, limits the protective effects of EDNO in the vasculature leading to increased inflammation, thrombosis, plaque destabilisation and plaque rupture (Fig.1).

The effects of hyperglycaemia-mediated increases in • O2 levels in the mitochondria are numerous and mostly detrimental to the cells of the vasculature. For example, • O2 has been shown to activate the nuclear enzyme poly(ADP-ribose) polymerase (PARP) which in turn leads to metabolic alterations that activate NFκB, AGE/receptor for AGE (RAGE) and the polyol pathways (Pacher & Szabo 2006). Upregulation of these pathways results in more • O2 production, while NFκB activation increases the expression of many proinflammatory mediators, leading to endothelial dysfunction. Furthermore, it is well accepted that ROS such as • O2 are involved in the oxidation of low-density lipoprotein (ox-LDL), which is not recognised by the LDL receptor and is preferentially taken up by scavenger receptors on macrophages, leading to foam cell formation and atherosclerotic plaques (Boullier et al 2001).

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 251

dysfunction in STZ-induced murine models of diabetes. Treatment with FP15 prevented loss of endothelium-dependent relaxant ability of blood vessels and improved both diastolic and systolic dysfunction of the diabetic heart, supporting the concept that neutralisation of ONOO- can be of significant therapeutic benefit. Furthermore, hyperglycaemia-mediated oxidative stress has been shown to cause abnormal gene expression, altered signal transduction, and the activation of pathways leading to myocyte apoptosis (Cai & Kang 2001). Myocyte death causes a loss of contractile units, compensatory hypertrophy of myocytes and reparative fibrosis, all characteristics of diabetic cardiomyopathy (Kang 2001). In a study by Frustaci et al. (2000), increased levels of nitrotyrosine were associated with apoptosis and necrosis of cardiac cells including myocytes, endothelial cells and fibroblasts in patients with diabetes, as well as in STZ-induced diabetic mice (Cai et al 2005). However, the mechanisms underlying hyperglycaemia-induced myocyte loss are still poorly

Recently, it was shown that hyperglycaemia-induced oxidative stress results in the activation of the hexosamine biosynthetic pathway (HBP), leading to increased *O*-GlcNAcylation (endproduct of HBP) of the pro-apoptotic peptide, BAD. In this manner oxidative stress is linked to increased cardiac myocyte death (Rajamani & Essop 2010). Other known mediators of hyperglycaemia-induced cardiac cell death include AGEs (Li et al 2007), apoptosis signal-regulating kinase 1 (ASK1) (Thandavarayan et al 2008) and mitochondrial dysfunction (Duncan 2011). All of these pathways appear to induce myocyte apoptosis via an increase in oxidative stress in the diabetic heart. Myocyte loss and myocyte injury are believed to contribute to systolic dysfunction due to the impairment in the ability of the myocardium to develop force, resulting in reduced contractility, decreased pump

important regulators of myocardial function, it is not surprising that hemodynamic studies have indicated that endothelium-dependent vasodilatory responses are impaired in the diabetic milieu (Farhangkhoee et al 2006). Indeed, the bioavailability of vasodilatory •

was found to be reduced with the progression of diabetic cardiomyopathy (Joffe et al 1999). In the heart, both eNOS and inducible NOS (iNOS) are the principal producers of •

relaxation and decrease oxygen consumption in cardiac myocytes, whereas high levels of

NO produced by iNOS decrease the contraction of cardiac myocytes and induce apoptosis (Khullar et al 2010). Under pathological conditions, such as diabetes, both enzymes can

dysfunction, resulting in increased permeability of the vessel wall and reduced blood flow through the myocardium causing tissue ischemia. In response, endothelial cells release growth factors, such as transforming growth factor-β (TGF-β), resulting in increased basement membrane thickening, extracellular matrix (ECM) deposition and interstitial

Diabetes-mediated increases in ROS are also known to affect structural proteins pertinent to the integrity of the myocardium, as well as proteins that affect its function. ROS have been shown to cause alterations in the function of regulatory and contractile proteins such as the

to form within the diabetic myocardium, as detailed above, •

NO. Since •

and increase oxidative stress and inflammation (Razavi et al

NO bioavailability in diabetes causes endothelial cell

NO produced by eNOS increase diastolic

O2 -

NO

NO.

NO is one of the most

function, and decreased ejection fraction (Fang et al 2004).

must interact with and reduce the bioavailability of •

Under physiological conditions, low levels of •

O2 -

understood.

For destructive ONOO-

produce highly reactive •

2005). Additionally, loss of •

fibrosis (Farhangkhoee et al 2006).

•

Finally, it is important to highlight that increased • O2 via enzymatic antioxidant conversion (see Fig.2) gives rise to increased levels of H2O2, an important ROS implicated in proinflammatory processes that are further amplified as diabetes develops (Nicolls et al 2007). H2O2 has also been shown to upregulate vascular cell adhesion molecule-1 (VCAM-1), an important adhesion molecule that aids in the migration of leukocytes from the blood into the tissue (Cook-Mills 2006). It is therefore clear that elevations in hyperglycaemia-mediated ROS lead to cellular, molecular and functional vascular alterations that initiate, mediate and ultimately hasten cardiovascular complications associated with diabetes.

#### **2.2 Diabetic cardiomyopathy**

Diabetic cardiomyopathy is considered a distinct clinical entity first recognised by Rubler et al. (1972) in 4 diabetic patients with congestive heart failure without evidence of hypertension, coronary artery disease or congenital heart disease. The Framingham study showed that diabetic men and women had a 2- and 5-fold greater incidence of heart failure respectively, even after taking into account other common risk factors such as coronary artery disease, age, blood pressure, weight and cholesterol (Kannel et al 1974). Diabetic cardiomyopathy is characterised by early diastolic dysfunction and late systolic impairment, and is accompanied by a wide range of structural abnormalities and pathophysiological impairments (Hayat et al 2004). Despite intensive investigations, the aetiology of diabetic cardiomyopathy remains elusive.

#### **2.2.1 The role of ROS in diabetic cardiomyopathy**

Hyperglycaemia is known to upregulate the production of Ang II, which is the overt hormone of the renin-angiotensin system (RAS). This has a profound effect on the myocardium given that most of the cellular components of the RAS including angiotensinogen, renin and the angiotensin II type I (AT1) receptor are found in myocytes (Fiordaliso et al 2000). Although Ang II is known to contribute to the development of diabetic cardiomyopathy through its hemodynamic vasoconstrictor effects and its ability to act as a proinflammatory mediator, it is now becoming increasingly clear that Ang II mediates its effects on the myocardium via its ability to enhance production of • O2- (Cooper 2004). Indeed, Privratsky et al. (2003) found that hyperglycaemia induces cardiac myopathies via the AT1 receptor, with activation of NADPH oxidase and increased ROS generation. Furthermore, • O2- levels were attenuated with an ACE inhibitor (Fiordaliso et al 2006). However, other sources of ROS generation are known to contribute to the oxidative stress that accompanies diabetic cardiomyopathy. For example, treatment of diabetic mice with allopurinol, the xanthine oxidase inhibitor, improved type 2 diabetes-induced cardiac dysfunction by decreasing oxidative/nitrosative stress and fibrosis (Rajesh et al 2009).

The biochemical pathways described earlier for diabetes-associated atherosclerosis, including those leading to endothelial dysfunction and inflammation due to the overproduction of ROS, appear to play a causal role in the pathogenesis of diabetic cardiomyopathy (Fig.1). Several lines of evidence suggest that nitrosative stress and peroxynitrite-induced damage contribute to the pathogenesis of diabetic cardiomyopathies. In particular, Szabo et al. (2002) showed that a metalloporphyrin peroxynitrite decomposition catalyst, FP15, which neutralises ONOO-, ameliorates cardiovascular

(see Fig.2) gives rise to increased levels of H2O2, an important ROS implicated in proinflammatory processes that are further amplified as diabetes develops (Nicolls et al 2007). H2O2 has also been shown to upregulate vascular cell adhesion molecule-1 (VCAM-1), an important adhesion molecule that aids in the migration of leukocytes from the blood into the tissue (Cook-Mills 2006). It is therefore clear that elevations in hyperglycaemia-mediated ROS lead to cellular, molecular and functional vascular alterations that initiate, mediate and

Diabetic cardiomyopathy is considered a distinct clinical entity first recognised by Rubler et al. (1972) in 4 diabetic patients with congestive heart failure without evidence of hypertension, coronary artery disease or congenital heart disease. The Framingham study showed that diabetic men and women had a 2- and 5-fold greater incidence of heart failure respectively, even after taking into account other common risk factors such as coronary artery disease, age, blood pressure, weight and cholesterol (Kannel et al 1974). Diabetic cardiomyopathy is characterised by early diastolic dysfunction and late systolic impairment, and is accompanied by a wide range of structural abnormalities and pathophysiological impairments (Hayat et al 2004). Despite intensive investigations, the aetiology of diabetic

Hyperglycaemia is known to upregulate the production of Ang II, which is the overt hormone of the renin-angiotensin system (RAS). This has a profound effect on the myocardium given that most of the cellular components of the RAS including angiotensinogen, renin and the angiotensin II type I (AT1) receptor are found in myocytes (Fiordaliso et al 2000). Although Ang II is known to contribute to the development of diabetic cardiomyopathy through its hemodynamic vasoconstrictor effects and its ability to act as a proinflammatory mediator, it is now becoming increasingly clear that Ang II

2004). Indeed, Privratsky et al. (2003) found that hyperglycaemia induces cardiac myopathies via the AT1 receptor, with activation of NADPH oxidase and increased ROS

2006). However, other sources of ROS generation are known to contribute to the oxidative stress that accompanies diabetic cardiomyopathy. For example, treatment of diabetic mice with allopurinol, the xanthine oxidase inhibitor, improved type 2 diabetes-induced cardiac dysfunction by decreasing oxidative/nitrosative stress and fibrosis (Rajesh et al 2009).

The biochemical pathways described earlier for diabetes-associated atherosclerosis, including those leading to endothelial dysfunction and inflammation due to the overproduction of ROS, appear to play a causal role in the pathogenesis of diabetic cardiomyopathy (Fig.1). Several lines of evidence suggest that nitrosative stress and peroxynitrite-induced damage contribute to the pathogenesis of diabetic cardiomyopathies. In particular, Szabo et al. (2002) showed that a metalloporphyrin peroxynitrite decomposition catalyst, FP15, which neutralises ONOO-, ameliorates cardiovascular

O2- levels were attenuated with an ACE inhibitor (Fiordaliso et al

mediates its effects on the myocardium via its ability to enhance production of •

ultimately hasten cardiovascular complications associated with diabetes.

O2 -

via enzymatic antioxidant conversion

O2- (Cooper

Finally, it is important to highlight that increased •

**2.2 Diabetic cardiomyopathy** 

cardiomyopathy remains elusive.

generation. Furthermore, •

**2.2.1 The role of ROS in diabetic cardiomyopathy** 

dysfunction in STZ-induced murine models of diabetes. Treatment with FP15 prevented loss of endothelium-dependent relaxant ability of blood vessels and improved both diastolic and systolic dysfunction of the diabetic heart, supporting the concept that neutralisation of ONOO- can be of significant therapeutic benefit. Furthermore, hyperglycaemia-mediated oxidative stress has been shown to cause abnormal gene expression, altered signal transduction, and the activation of pathways leading to myocyte apoptosis (Cai & Kang 2001). Myocyte death causes a loss of contractile units, compensatory hypertrophy of myocytes and reparative fibrosis, all characteristics of diabetic cardiomyopathy (Kang 2001). In a study by Frustaci et al. (2000), increased levels of nitrotyrosine were associated with apoptosis and necrosis of cardiac cells including myocytes, endothelial cells and fibroblasts in patients with diabetes, as well as in STZ-induced diabetic mice (Cai et al 2005). However, the mechanisms underlying hyperglycaemia-induced myocyte loss are still poorly understood.

Recently, it was shown that hyperglycaemia-induced oxidative stress results in the activation of the hexosamine biosynthetic pathway (HBP), leading to increased *O*-GlcNAcylation (endproduct of HBP) of the pro-apoptotic peptide, BAD. In this manner oxidative stress is linked to increased cardiac myocyte death (Rajamani & Essop 2010). Other known mediators of hyperglycaemia-induced cardiac cell death include AGEs (Li et al 2007), apoptosis signal-regulating kinase 1 (ASK1) (Thandavarayan et al 2008) and mitochondrial dysfunction (Duncan 2011). All of these pathways appear to induce myocyte apoptosis via an increase in oxidative stress in the diabetic heart. Myocyte loss and myocyte injury are believed to contribute to systolic dysfunction due to the impairment in the ability of the myocardium to develop force, resulting in reduced contractility, decreased pump function, and decreased ejection fraction (Fang et al 2004).

For destructive ONOO- to form within the diabetic myocardium, as detailed above, • O2 must interact with and reduce the bioavailability of • NO. Since • NO is one of the most important regulators of myocardial function, it is not surprising that hemodynamic studies have indicated that endothelium-dependent vasodilatory responses are impaired in the diabetic milieu (Farhangkhoee et al 2006). Indeed, the bioavailability of vasodilatory • NO was found to be reduced with the progression of diabetic cardiomyopathy (Joffe et al 1999). In the heart, both eNOS and inducible NOS (iNOS) are the principal producers of • NO. Under physiological conditions, low levels of • NO produced by eNOS increase diastolic relaxation and decrease oxygen consumption in cardiac myocytes, whereas high levels of • NO produced by iNOS decrease the contraction of cardiac myocytes and induce apoptosis (Khullar et al 2010). Under pathological conditions, such as diabetes, both enzymes can produce highly reactive • O2 - and increase oxidative stress and inflammation (Razavi et al 2005). Additionally, loss of • NO bioavailability in diabetes causes endothelial cell dysfunction, resulting in increased permeability of the vessel wall and reduced blood flow through the myocardium causing tissue ischemia. In response, endothelial cells release growth factors, such as transforming growth factor-β (TGF-β), resulting in increased basement membrane thickening, extracellular matrix (ECM) deposition and interstitial fibrosis (Farhangkhoee et al 2006).

Diabetes-mediated increases in ROS are also known to affect structural proteins pertinent to the integrity of the myocardium, as well as proteins that affect its function. ROS have been shown to cause alterations in the function of regulatory and contractile proteins such as the

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 253

filtration surface of the glomerulus, a process known as glomerulosclerosis (Kalant 1978). Furthermore, there is now compelling evidence to suggest that disruption of the tubulointerstitial architecture is as important, if not more important in contributing to

Despite intensive glucose control and blockade of the RAS (Barit & Cooper 2008; Keane et al 2006), DN continues to progress in a significant proportion of patients and often leads to organ failure and the need for dialysis and/or kidney transplantation. Therefore, the development of novel targeted therapeutics is warranted to reduce or eliminate kidney

An upregulation of ROS in diabetes has been implicated in the pathogenesis of kidney injury (Forbes et al 2008). ROS activate a number of signalling pathways including PKC, p38 MAPK, p42/p44 MAPK and the transcription factor NF-κB, which leads to the increased activation of growth factors such as TGF-β that contribute to the pathogenesis of DN. In the diabetic kidney, enhanced glucose uptake occurs in many of the cell populations including glomerular epithelial cells, mesangial cells and proximal tubular epithelial cells, leading to the excessive production of intracellular ROS, making these cells particularly susceptible to

Sufficient evidence exists, from both clinical and pre-clinical studies, to suggest that oxidative stress accompanies the progression of diabetic nephropathy. Hyperglycaemia has been shown to increase 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative mitochondrial DNA damage in diabetic rat kidneys (Kakimoto et al 2002). In this study, intervention by insulin treatment normalised renal 8-OHdG level in diabetic rats, clearly linking the diabetic milieu and increased oxidative stress in this pre-clinical model (Kakimoto et al 2002). In type 2 diabetic patients, it was found that urinary 8-OHdG excretion was significantly higher than in healthy controls and furthermore, that this increase was proportional to the severity of the tubulointerstitial lesion observed in the kidneys of these patients (Kanauchi et al 2002). In addition, it was reported that the 24-hour urinary content of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a product of oxidative DNA damage, strongly predicted the progression of DN in type 2 diabetic patients in a 5-

Several studies have examined the pathways through which increased ROS may mediate its damaging effects on glomerular and tubular injury in the diabetic kidney. One study showed that ROS mediates high glucose-induced activation of PKC in mesangial cells, leading to an increase in TGF-β expression (Studer et al 1997). Ha et al. (2002) have demonstrated that by inhibiting ROS with a series of antioxidants, high glucose-induced activation of NFκB and NFκB-dependent monocyte chemoattractant protein-1 (MCP-1) expression was inhibited in mesangial cells. Furthermore, increased ROS led to accelerated glomerulosclerosis through TGF-β-mediated plasminogen activator inhibitor-1 (PAI-1) upregulation in mesangial cells (Jiang et al 2003b). Similarly, it was proposed that ROS mediate kidney fibrosis in renal cells through the upregulation of the transcription factors NFκB and activator protein-1 (AP-1), that in turn increase MCP-1, TGF-β and PAI-1, resulting in the increased accumulation of ECM (Lee et al 2003). Using several disparate antioxidants, another study found that the TGF-βinduced cellular ROS, the phosphorylation of Smad2, p38 MAPK and extracellular signal-

kidney injury as glomerular damage (Nangaku 2004).

**2.3.1 The role of ROS in diabetic nephropathy** 

the diabetic milieu (Forbes et al 2008).

year follow up study (Hinokio et al 2002).

disease in diabetic patients.

sarcoplasmic reticulum Ca2+-ATPase and Na+-Ca2+ exchanger in the heart (Fang et al., 2004). This leads to diminished calcium sensitivity of proteins involved in the regulation of the cardiac actomyosin system, a reduction in the sarcoplasmic reticulum Ca2+-ATPase and a decrease in the sarcoplasmic reticulum calcium (SERCA2a) pump protein (Abe et al., 2002). These deficits may all contribute to impaired LV function. Indeed, abnormal diastolic and systolic function was normalised in streptozotocin-induced diabetic rat hearts when SERCA2a was overexpressed (Trost et al., 2002).

Fig. 1. Roles of reactive oxygen species (ROS) in the pathophysiology of diabetic macrovascular complications, namely atherosclerosis and diabetic cardiomyopathy.

#### **2.3 Diabetic nephropathy**

Diabetic nephropathy (DN) has become a worldwide epidemic, accounting for approximately one third of all cases of ESRD (Rossing 2006). DN is classically defined as the increase in protein excretion in the urine. The early stage of DN is characterised by a small increase in urinary albumin excretion (microalbuminuria), while overt diabetic nephropathy is defined as the presence of macroalbuminuria or proteinuria (Zelmanovitz et al 2009).

The earliest structural changes associated with diabetic nephropathy are the expansion of glomerular mesangial area, mesangial cell hypertrophy and thickening of the glomerular basement membrane (Gilbert & Cooper 1999), leading to a progressive reduction in the filtration surface of the glomerulus, a process known as glomerulosclerosis (Kalant 1978). Furthermore, there is now compelling evidence to suggest that disruption of the tubulointerstitial architecture is as important, if not more important in contributing to kidney injury as glomerular damage (Nangaku 2004).

Despite intensive glucose control and blockade of the RAS (Barit & Cooper 2008; Keane et al 2006), DN continues to progress in a significant proportion of patients and often leads to organ failure and the need for dialysis and/or kidney transplantation. Therefore, the development of novel targeted therapeutics is warranted to reduce or eliminate kidney disease in diabetic patients.

#### **2.3.1 The role of ROS in diabetic nephropathy**

252 Oxidative Stress and Diseases

sarcoplasmic reticulum Ca2+-ATPase and Na+-Ca2+ exchanger in the heart (Fang et al., 2004). This leads to diminished calcium sensitivity of proteins involved in the regulation of the cardiac actomyosin system, a reduction in the sarcoplasmic reticulum Ca2+-ATPase and a decrease in the sarcoplasmic reticulum calcium (SERCA2a) pump protein (Abe et al., 2002). These deficits may all contribute to impaired LV function. Indeed, abnormal diastolic and systolic function was normalised in streptozotocin-induced diabetic rat hearts when

Fig. 1. Roles of reactive oxygen species (ROS) in the pathophysiology of diabetic macrovascular complications, namely atherosclerosis and diabetic cardiomyopathy.

Diabetic nephropathy (DN) has become a worldwide epidemic, accounting for approximately one third of all cases of ESRD (Rossing 2006). DN is classically defined as the increase in protein excretion in the urine. The early stage of DN is characterised by a small increase in urinary albumin excretion (microalbuminuria), while overt diabetic nephropathy is defined as the presence of macroalbuminuria or proteinuria (Zelmanovitz et al 2009).

The earliest structural changes associated with diabetic nephropathy are the expansion of glomerular mesangial area, mesangial cell hypertrophy and thickening of the glomerular basement membrane (Gilbert & Cooper 1999), leading to a progressive reduction in the

SERCA2a was overexpressed (Trost et al., 2002).

**2.3 Diabetic nephropathy** 

An upregulation of ROS in diabetes has been implicated in the pathogenesis of kidney injury (Forbes et al 2008). ROS activate a number of signalling pathways including PKC, p38 MAPK, p42/p44 MAPK and the transcription factor NF-κB, which leads to the increased activation of growth factors such as TGF-β that contribute to the pathogenesis of DN. In the diabetic kidney, enhanced glucose uptake occurs in many of the cell populations including glomerular epithelial cells, mesangial cells and proximal tubular epithelial cells, leading to the excessive production of intracellular ROS, making these cells particularly susceptible to the diabetic milieu (Forbes et al 2008).

Sufficient evidence exists, from both clinical and pre-clinical studies, to suggest that oxidative stress accompanies the progression of diabetic nephropathy. Hyperglycaemia has been shown to increase 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative mitochondrial DNA damage in diabetic rat kidneys (Kakimoto et al 2002). In this study, intervention by insulin treatment normalised renal 8-OHdG level in diabetic rats, clearly linking the diabetic milieu and increased oxidative stress in this pre-clinical model (Kakimoto et al 2002). In type 2 diabetic patients, it was found that urinary 8-OHdG excretion was significantly higher than in healthy controls and furthermore, that this increase was proportional to the severity of the tubulointerstitial lesion observed in the kidneys of these patients (Kanauchi et al 2002). In addition, it was reported that the 24-hour urinary content of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a product of oxidative DNA damage, strongly predicted the progression of DN in type 2 diabetic patients in a 5 year follow up study (Hinokio et al 2002).

Several studies have examined the pathways through which increased ROS may mediate its damaging effects on glomerular and tubular injury in the diabetic kidney. One study showed that ROS mediates high glucose-induced activation of PKC in mesangial cells, leading to an increase in TGF-β expression (Studer et al 1997). Ha et al. (2002) have demonstrated that by inhibiting ROS with a series of antioxidants, high glucose-induced activation of NFκB and NFκB-dependent monocyte chemoattractant protein-1 (MCP-1) expression was inhibited in mesangial cells. Furthermore, increased ROS led to accelerated glomerulosclerosis through TGF-β-mediated plasminogen activator inhibitor-1 (PAI-1) upregulation in mesangial cells (Jiang et al 2003b). Similarly, it was proposed that ROS mediate kidney fibrosis in renal cells through the upregulation of the transcription factors NFκB and activator protein-1 (AP-1), that in turn increase MCP-1, TGF-β and PAI-1, resulting in the increased accumulation of ECM (Lee et al 2003). Using several disparate antioxidants, another study found that the TGF-βinduced cellular ROS, the phosphorylation of Smad2, p38 MAPK and extracellular signal-

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 255

Fig. 2. Removal of reactive oxygen species (ROS) by antioxidant defence systems.

oxidase, xanthine oxidase and a dysfunctional mitochondrial respiratory chain. •

production is greatly enhanced under pathological situations via enzymes such as NADPH

neutralised to water via a two-step process involving superoxide dismutase (SOD) in the first step, and glutathione peroxidase (GPx) or catalase in a second step. Increased

the intermediate hydrogen peroxide (H2O2). H2O2 forms the toxic oxygen species hydroxyl

forming lipid hydroperoxides (LOOH). The functional importance of GPx resides in its ability to remove H2O2 and LOOH and neutralise these to water and lipid alcohol,

O2

GPx activity is decreased in patients with type 1 diabetes, as well as in experimentallyinduced diabetic rats (Chiu et al 2005; Dominguez et al 1998), although some studies have shown opposite results (Maritim et al 2003b). The decrease in GPx activity may contribute to the progression of diabetic complications due to the build-up of ROS such as H2O2 and

) is generated in low levels under physiological states but its

O2- and/or impairment of antioxidant defence systems lead to a build-up of


NO). GPx also functions to

OH) via Fenton biochemistry, which is highly reactive and causes lipid peroxidation

O2 is

Superoxide radical (•

production of •

neutralise ONOO-

anion (•

ONOO-

O2-

respectively. Additionally, the increase in •

.

(ONOO-) which reduces the bioavailability of nitric oxide (•

, leading to lipid peroxidation and oxidative injury.

**3.1.2 GPx in diabetes-associated complications** 

regulated kinase (ERK), as well as endothelial-mesenchymal transition (EMT) were inhibited, further suggesting an important role for ROS in TGF-β-dependent pathways in renal tubular epithelial cells (Rhyu et al 2005).

#### **3. A role for antioxidant defence in diabetic complications**

Evidence suggests that glucose alters antioxidant defences in endothelial cells (Ceriello et al 1996) and in patients with diabetic complications such as DN (Ceriello et al 2000; Hodgkinson et al 2003). Fibroblasts derived from type 1 diabetic patients susceptible to microvascular complications were unable to upregulate their protective antioxidative defences after exposure to high glucose compared with skin fibroblasts from normal subjects, suggesting a failure of antioxidant defences in diabetic patients with nephropathy (Ceriello et al 1996). In addition, the concentration of the antioxidant glutathione (GSH) is found to decrease in a range of organs including the liver, kidney, pancreas, plasma, and red blood cells of chemically induced diabetic animals (Maritim et al 2003b). Given that reduced GSH functions as a direct free-radical scavenger and a cosubstrate for GPx activity, as well as a cofactor for many enzymes, reductions in this antioxidant induced by the hyperglycaemic environment is likely to impact on the progression of diabetic complications. Thus, these findings suggest that increased ROS in diabetes is not only the result of their increased production, as detailed in section 2, but also a consequence of impaired antioxidant defences.

#### **3.1 Glutathione peroxidase**

Pre-clinical and clinical evidence are now mounting in support of an important role for GPx in the protection against diseases such as atherosclerosis, both in a non-diabetic and a diabetic setting. The selenocysteine-containing GPx family of antioxidant enzymes attenuates oxidative stress by utilising GSH to reduce hydrogen and lipid peroxides to water and their corresponding alcohol (Fig.2). Additionally, GPx also functions to remove harmful ONOO-. Thus the major role for GPx in the protection against pathogenesis may reside in the fact that it is the only antioxidant enzyme that metabolises three major ROS, H2O2, lipid peroxide (LOOH) and ONOO- (Fig.2). Several isoforms of GPx have been identified and they are each encoded by separate genes, which vary in cellular location, substrate specificity and tissue-specific functions (Brigelius-Flohé 1999).

#### **3.1.1 Different isoforms of GPx**

GPx1, also known as cellular GPx, was first identified as an erythrocyte enzyme that protects haemoglobin from oxidative injury (Mills 1957). Its ubiquitous expression in almost all tissues, together with its abundant expression in organs such as the kidney and liver have meant that this isoform is one of the most well-characterised of the GPx family (Lei 2001). GPx2 is most prominent in the gastrointestinal tract and its role is mainly to protect intestinal epithelium from oxidative stress (Chu et al 1997; Esworthy et al 1998). GPx3 is secreted by the kidney and is the main source of plasma GPx; however GPx3 is also expressed in other tissues, for example in the heart (Reeves & Hoffmann 2009). GPx4 reduces phospholipid hydroperoxides (Conrad et al 2007; Thomas et al 1990) and is thought to play a protective role in oxidative stress-induced apoptosis, possibly through the mitochondrial death pathway (Nomura et al 1999; Seiler et al 2008).

regulated kinase (ERK), as well as endothelial-mesenchymal transition (EMT) were inhibited, further suggesting an important role for ROS in TGF-β-dependent pathways in renal tubular

Evidence suggests that glucose alters antioxidant defences in endothelial cells (Ceriello et al 1996) and in patients with diabetic complications such as DN (Ceriello et al 2000; Hodgkinson et al 2003). Fibroblasts derived from type 1 diabetic patients susceptible to microvascular complications were unable to upregulate their protective antioxidative defences after exposure to high glucose compared with skin fibroblasts from normal subjects, suggesting a failure of antioxidant defences in diabetic patients with nephropathy (Ceriello et al 1996). In addition, the concentration of the antioxidant glutathione (GSH) is found to decrease in a range of organs including the liver, kidney, pancreas, plasma, and red blood cells of chemically induced diabetic animals (Maritim et al 2003b). Given that reduced GSH functions as a direct free-radical scavenger and a cosubstrate for GPx activity, as well as a cofactor for many enzymes, reductions in this antioxidant induced by the hyperglycaemic environment is likely to impact on the progression of diabetic complications. Thus, these findings suggest that increased ROS in diabetes is not only the result of their increased production, as detailed in section 2, but also a consequence of

Pre-clinical and clinical evidence are now mounting in support of an important role for GPx in the protection against diseases such as atherosclerosis, both in a non-diabetic and a diabetic setting. The selenocysteine-containing GPx family of antioxidant enzymes attenuates oxidative stress by utilising GSH to reduce hydrogen and lipid peroxides to water and their corresponding alcohol (Fig.2). Additionally, GPx also functions to remove harmful ONOO-. Thus the major role for GPx in the protection against pathogenesis may reside in the fact that it is the only antioxidant enzyme that metabolises three major ROS, H2O2, lipid peroxide (LOOH) and ONOO- (Fig.2). Several isoforms of GPx have been identified and they are each encoded by separate genes, which vary in cellular location,

GPx1, also known as cellular GPx, was first identified as an erythrocyte enzyme that protects haemoglobin from oxidative injury (Mills 1957). Its ubiquitous expression in almost all tissues, together with its abundant expression in organs such as the kidney and liver have meant that this isoform is one of the most well-characterised of the GPx family (Lei 2001). GPx2 is most prominent in the gastrointestinal tract and its role is mainly to protect intestinal epithelium from oxidative stress (Chu et al 1997; Esworthy et al 1998). GPx3 is secreted by the kidney and is the main source of plasma GPx; however GPx3 is also expressed in other tissues, for example in the heart (Reeves & Hoffmann 2009). GPx4 reduces phospholipid hydroperoxides (Conrad et al 2007; Thomas et al 1990) and is thought to play a protective role in oxidative stress-induced apoptosis, possibly through the

substrate specificity and tissue-specific functions (Brigelius-Flohé 1999).

mitochondrial death pathway (Nomura et al 1999; Seiler et al 2008).

**3. A role for antioxidant defence in diabetic complications** 

epithelial cells (Rhyu et al 2005).

impaired antioxidant defences.

**3.1 Glutathione peroxidase** 

**3.1.1 Different isoforms of GPx** 

Fig. 2. Removal of reactive oxygen species (ROS) by antioxidant defence systems. Superoxide radical (• O2- ) is generated in low levels under physiological states but its production is greatly enhanced under pathological situations via enzymes such as NADPH oxidase, xanthine oxidase and a dysfunctional mitochondrial respiratory chain. • O2 is neutralised to water via a two-step process involving superoxide dismutase (SOD) in the first step, and glutathione peroxidase (GPx) or catalase in a second step. Increased production of • O2- and/or impairment of antioxidant defence systems lead to a build-up of the intermediate hydrogen peroxide (H2O2). H2O2 forms the toxic oxygen species hydroxyl anion (• OH) via Fenton biochemistry, which is highly reactive and causes lipid peroxidation forming lipid hydroperoxides (LOOH). The functional importance of GPx resides in its ability to remove H2O2 and LOOH and neutralise these to water and lipid alcohol, respectively. Additionally, the increase in • O2 - also favours the formation of peroxynitrite (ONOO-) which reduces the bioavailability of nitric oxide (• NO). GPx also functions to neutralise ONOO-.

#### **3.1.2 GPx in diabetes-associated complications**

GPx activity is decreased in patients with type 1 diabetes, as well as in experimentallyinduced diabetic rats (Chiu et al 2005; Dominguez et al 1998), although some studies have shown opposite results (Maritim et al 2003b). The decrease in GPx activity may contribute to the progression of diabetic complications due to the build-up of ROS such as H2O2 and ONOO- , leading to lipid peroxidation and oxidative injury.

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 257

all selenium-dependent enzymes (Reddi & Bollineni 2001; Rosenblat & Aviram 1998). Furthermore, studying GPx1 knockout mice allows us to draw meaningful conclusions about the protective role of this isoform of the GPx family, since standard assays do not discriminate between different isoforms (Lewis et al 2007). This specific knockout model also facilitates the distinction between the contribution of GPx1, catalase (a peroxisomal H2O2 metabolising enzyme) and thioredoxin reductase in the peroxidation of H2O2 to water

Forgione et al. first reported vascular functional changes in mice associated with both a heterozygous (Forgione et al 2002a) and homozygous deficiency of GPx1 compared to wildtype (WT) mice (Forgione et al 2002b). Mesenteric arterioles of GPx+/- and GPx-/- mice demonstrated paradoxical vasoconstriction to endothelium-dependent vasodilatory compounds such as acetylcholine and bradykinin, whereas WT arterioles showed dosedependent vasodilation. Superfusion of GPx-/- vessels with an endothelium-independent vasodilator, sodium nitroprusside (SNP) resulted in dose-dependent arteriolar vasodilation that was similar in GPx-/- and WT vessels. These results suggest that GPx-/- mice may have a depletion of bioavailable EDNO. Furthermore, the observed endothelial dysfunction was accompanied by increased nitrotyrosine levels in the endothelial layer of the vessel wall, as well as elevated plasma isoprostanes, indicative that lack of GPx1 leads to oxidative stress in this tissue. By increasing intracellular thiol pools (GSH, cysteine) in the vascular tissue of GPx-/- mice using (L)-2-Oxothiazolidine carboxylic acid (OTC), endothelial dysfunction and oxidant stress was attenuated, further indicating the importance of GPx1 in maintaining normal endothelial function, as well as protecting the blood vessels from oxidative injury

Several isoforms of GPx are present in kidney, however GPx1 is the major isoform expressed in normal kidney, and accounts for >96% of the renal GPx activity (de Haan et al 1998). Protection against oxidative stress is therefore most likely due to lipid and H2O2 quenching effects of the GPx1 isoform in the kidney. Until recently, no study has directly linked GPx1 to the protection against DN (Chew et al 2010). Our initial studies using diabetic C57Bl/6J GPx-/- mice, surprisingly failed to show accelerated kidney injury compared with diabetic WT mice (de Haan et al 2005). Furthermore, an assessment of atherosclerosis, which is only possible in the aortic sinus after feeding mice diets rich in fats, cholesterol and choline, failed to reveal a protective role for GPx1 in G57Bl/J6 GPx-/- mice (de Haan et al 2006). As detailed below, the importance of GPx1 in limiting diabetes-associated atherosclerosis and diabetic nephropathy became evident in ApoE/GPx1 double knockout (dKO) mice, a murine model that encompasses three important risk factors, namely hyperglycaemia,

hyperlipidemia and enhanced intensity of oxidative stress, seen in diabetic patients.

**associated atherosclerosis and diabetic nephropathy** 

**3.1.4 Diabetic ApoE/GPx1 double knockout mouse: a model of accelerated diabetes-**

Since the lack of GPx1 plays a major role in endothelial dysfunction (Forgione et al 2002b), which is known to be an important mediator of hyperglycaemia-induced atherosclerosis (Nakagami et al 2005), we hypothesised that a lack of GPx1 would accelerate atherosclerosis in a diabetic setting. Because rodents are more resilient than humans to the development of diabetic atherosclerosis, in order to generate a mouse model with similar disease progression and aetiology, we crossed our GPx1-/- mice with ApoE-deficient mice that

(de Haan & Cooper 2011).

(Forgione et al 2002b).

Clinical evidence has shown that diabetic patients with cardiovascular complications have significantly lower enzymatic antioxidant defences, including an impairment in GPx activity, with this defect being more pronounced in younger patients (Čolak et al 2005). GPx activity is also found to decrease in diabetic rats in the heart, kidney and brain, leading to enhanced oxidative stress and secondary organ damage (Aliciguzel et al 2003). In particular, glomerular expression of GPx is significantly reduced in both human and rats with diabetes (Chiu et al 2005). Furthermore, diabetic rats with reduced glomerular GPx expression were found to have more severe glomerulosclerosis and mesangial expansion (Chiu et al 2005). Moreover, patients with type 1 diabetes together with DN displayed a defective GPx defence mechanism, in contrast to patients with type 1 diabetes but without DN (Ceriello et al 2000). Several studies have linked selenium deficiency to a reduction in GPx mRNA expression and activity in the kidney together with elevated plasma glucose, albuminuria and glomerulosclerosis (Fujieda et al 2007; Reddi & Bollineni 2001). These changes may be mediated by the profibrotic growth factor, TGF-β, since inhibition of TGF-β with a TGF-β neutralising antibody, abrogated the reduction in GPx activity, as well as the increase in lipid peroxidation, albuminuria and glomerular injury in rats fed with a selenium-deficient diet (Reddi & Bollineni 2001).

Several clinical studies have linked reduced GPx1 levels with diabetes-associated atherogenesis (Čolak et al 2005; Hamanishi et al 2004). Polymorphisms identified within the GPx1 gene resulting in reduced GPx1 activity have been linked with increased intima-media thickness of carotid arteries and an increased risk of cardiovascular and peripheral vascular disease in type 2 diabetic patients (Hamanishi et al 2004). Moreover, a reduction in red blood cell GPx1 activity has been associated with an increased risk of cardiovascular events in a prospective cohort study assessing the extent of atherosclerosis (Espinola-Klein et al 2007; Winter et al 2003), while atherosclerotic plaques of patients with carotid artery disease have reduced GPx1 activity (Lapenna et al 1998). These evidence, although correlative, suggest that GPx1 is a key enzyme for the protection of vessels against oxidative stress and atherogenesis, particularly in the highly pro-oxidant diabetic environment (de Haan & Cooper 2011).

The roles of GPx in mediating diabetes-associated heart injury are less well understood. Current evidence suggest that the compensatory upregulation of antioxidant enzymes, including GPx1 is impaired in the heart of severely hyperglycaemic mice (Fujita et al 2005). More recently, GPx3 is reported to be upregulated in the heart of STZ-diabetic mice, suggesting that GPx3 is the major antioxidative enzyme of the heart in the cellular defence against oxidative stress under hyperglycaemia (Iwata et al 2006). However, these were short term studies of only 4 to 10 weeks of diabetes, therefore further studies are required to investigate whether diabetes has long term effects on the expression and/or activity of GPx3 in the heart.

#### **3.1.3 The GPx1 knockout mouse: a model of enhanced intensity of oxidative stress**

GPx1 knockout (-/-) mice, generated in our laboratory (de Haan et al 1998) and by others (Cheng et al 1997; Esposito et al 2000; Yoshida et al 1997) have become an important research tool to specifically study the protective role of GPx1 in the ROS-mediated progression and promotion of oxidative stress-mediated injury. Most studies investigating the role of GPx do so by limiting selenium intake which results in non-specific reductions of

Clinical evidence has shown that diabetic patients with cardiovascular complications have significantly lower enzymatic antioxidant defences, including an impairment in GPx activity, with this defect being more pronounced in younger patients (Čolak et al 2005). GPx activity is also found to decrease in diabetic rats in the heart, kidney and brain, leading to enhanced oxidative stress and secondary organ damage (Aliciguzel et al 2003). In particular, glomerular expression of GPx is significantly reduced in both human and rats with diabetes (Chiu et al 2005). Furthermore, diabetic rats with reduced glomerular GPx expression were found to have more severe glomerulosclerosis and mesangial expansion (Chiu et al 2005). Moreover, patients with type 1 diabetes together with DN displayed a defective GPx defence mechanism, in contrast to patients with type 1 diabetes but without DN (Ceriello et al 2000). Several studies have linked selenium deficiency to a reduction in GPx mRNA expression and activity in the kidney together with elevated plasma glucose, albuminuria and glomerulosclerosis (Fujieda et al 2007; Reddi & Bollineni 2001). These changes may be mediated by the profibrotic growth factor, TGF-β, since inhibition of TGF-β with a TGF-β neutralising antibody, abrogated the reduction in GPx activity, as well as the increase in lipid peroxidation, albuminuria and glomerular injury in rats fed with a selenium-deficient

Several clinical studies have linked reduced GPx1 levels with diabetes-associated atherogenesis (Čolak et al 2005; Hamanishi et al 2004). Polymorphisms identified within the GPx1 gene resulting in reduced GPx1 activity have been linked with increased intima-media thickness of carotid arteries and an increased risk of cardiovascular and peripheral vascular disease in type 2 diabetic patients (Hamanishi et al 2004). Moreover, a reduction in red blood cell GPx1 activity has been associated with an increased risk of cardiovascular events in a prospective cohort study assessing the extent of atherosclerosis (Espinola-Klein et al 2007; Winter et al 2003), while atherosclerotic plaques of patients with carotid artery disease have reduced GPx1 activity (Lapenna et al 1998). These evidence, although correlative, suggest that GPx1 is a key enzyme for the protection of vessels against oxidative stress and atherogenesis, particularly in the highly pro-oxidant diabetic environment (de Haan &

The roles of GPx in mediating diabetes-associated heart injury are less well understood. Current evidence suggest that the compensatory upregulation of antioxidant enzymes, including GPx1 is impaired in the heart of severely hyperglycaemic mice (Fujita et al 2005). More recently, GPx3 is reported to be upregulated in the heart of STZ-diabetic mice, suggesting that GPx3 is the major antioxidative enzyme of the heart in the cellular defence against oxidative stress under hyperglycaemia (Iwata et al 2006). However, these were short term studies of only 4 to 10 weeks of diabetes, therefore further studies are required to investigate whether diabetes has long term effects on the expression and/or activity of GPx3

**3.1.3 The GPx1 knockout mouse: a model of enhanced intensity of oxidative stress**  GPx1 knockout (-/-) mice, generated in our laboratory (de Haan et al 1998) and by others (Cheng et al 1997; Esposito et al 2000; Yoshida et al 1997) have become an important research tool to specifically study the protective role of GPx1 in the ROS-mediated progression and promotion of oxidative stress-mediated injury. Most studies investigating the role of GPx do so by limiting selenium intake which results in non-specific reductions of

diet (Reddi & Bollineni 2001).

Cooper 2011).

in the heart.

all selenium-dependent enzymes (Reddi & Bollineni 2001; Rosenblat & Aviram 1998). Furthermore, studying GPx1 knockout mice allows us to draw meaningful conclusions about the protective role of this isoform of the GPx family, since standard assays do not discriminate between different isoforms (Lewis et al 2007). This specific knockout model also facilitates the distinction between the contribution of GPx1, catalase (a peroxisomal H2O2 metabolising enzyme) and thioredoxin reductase in the peroxidation of H2O2 to water (de Haan & Cooper 2011).

Forgione et al. first reported vascular functional changes in mice associated with both a heterozygous (Forgione et al 2002a) and homozygous deficiency of GPx1 compared to wildtype (WT) mice (Forgione et al 2002b). Mesenteric arterioles of GPx+/- and GPx-/- mice demonstrated paradoxical vasoconstriction to endothelium-dependent vasodilatory compounds such as acetylcholine and bradykinin, whereas WT arterioles showed dosedependent vasodilation. Superfusion of GPx-/- vessels with an endothelium-independent vasodilator, sodium nitroprusside (SNP) resulted in dose-dependent arteriolar vasodilation that was similar in GPx-/- and WT vessels. These results suggest that GPx-/- mice may have a depletion of bioavailable EDNO. Furthermore, the observed endothelial dysfunction was accompanied by increased nitrotyrosine levels in the endothelial layer of the vessel wall, as well as elevated plasma isoprostanes, indicative that lack of GPx1 leads to oxidative stress in this tissue. By increasing intracellular thiol pools (GSH, cysteine) in the vascular tissue of GPx-/- mice using (L)-2-Oxothiazolidine carboxylic acid (OTC), endothelial dysfunction and oxidant stress was attenuated, further indicating the importance of GPx1 in maintaining normal endothelial function, as well as protecting the blood vessels from oxidative injury (Forgione et al 2002b).

Several isoforms of GPx are present in kidney, however GPx1 is the major isoform expressed in normal kidney, and accounts for >96% of the renal GPx activity (de Haan et al 1998). Protection against oxidative stress is therefore most likely due to lipid and H2O2 quenching effects of the GPx1 isoform in the kidney. Until recently, no study has directly linked GPx1 to the protection against DN (Chew et al 2010). Our initial studies using diabetic C57Bl/6J GPx-/- mice, surprisingly failed to show accelerated kidney injury compared with diabetic WT mice (de Haan et al 2005). Furthermore, an assessment of atherosclerosis, which is only possible in the aortic sinus after feeding mice diets rich in fats, cholesterol and choline, failed to reveal a protective role for GPx1 in G57Bl/J6 GPx-/- mice (de Haan et al 2006). As detailed below, the importance of GPx1 in limiting diabetes-associated atherosclerosis and diabetic nephropathy became evident in ApoE/GPx1 double knockout (dKO) mice, a murine model that encompasses three important risk factors, namely hyperglycaemia, hyperlipidemia and enhanced intensity of oxidative stress, seen in diabetic patients.

#### **3.1.4 Diabetic ApoE/GPx1 double knockout mouse: a model of accelerated diabetesassociated atherosclerosis and diabetic nephropathy**

Since the lack of GPx1 plays a major role in endothelial dysfunction (Forgione et al 2002b), which is known to be an important mediator of hyperglycaemia-induced atherosclerosis (Nakagami et al 2005), we hypothesised that a lack of GPx1 would accelerate atherosclerosis in a diabetic setting. Because rodents are more resilient than humans to the development of diabetic atherosclerosis, in order to generate a mouse model with similar disease progression and aetiology, we crossed our GPx1-/- mice with ApoE-deficient mice that

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 259

Diabetes is associated with a decrease in SOD activity in most animal studies (Brocca et al 2008; Fujita et al 2009; Fukuda et al 2010). Lowered SOD levels are reported in serum and urine of STZ-treated Sprague-Dawley rats (Luo et al 2010), and decreased SOD1 and SOD3 levels are suggested to play a key role in the pathogenesis of diabetic nephropathy (Fujita et al 2009). Manipulations of SOD, through the use of SOD knockout mice or SOD overexpressing mice, have shown the importance of these enzymes in the protection against diabetic complications. Indeed, SOD1 knockout mice have clearly shown the importance of protection by SOD1 against superoxide-mediated DN. These mice demonstrated significant mesangial matrix expansion, renal cortical malondialdehyde content and severe tubulointerstitial injury compared with diabetic controls (DeRubertis et al 2007). Importantly, these pathological changes were attenuated in the presence of the SODmimetic, tempol (discussed in more detail in section 4.3.2). The significance of SOD2 was revealed in eMnSOD-Tg mice where the overexpression of mitochondrial-specific SOD targeted to the endothelium prevented diabetic retinopathy (Goto et al 2008). Furthermore, the targeted overexpression of Mn-SOD significantly attenuated morphological changes in diabetic hearts and improved contractility in diabetic cardiomyocytes (Shen et al 2006).

Catalase is present mainly in the peroxisomes of mammalian cells as a tetrameric enzyme of four identically arranged subunits; each containing a heme group and NADPH at its active centre (MatÉs et al 1999). Similar to GPx1, the enzyme neutralise H2O2 to water and oxygen (Fig.2). A role for catalase in the protection against atherosclerosis comes from the analysis of mice overexpressing catalase. In these experiments, overexpression of catalase significantly reduced the severity of lesions in ApoE-deficient mice (Yang et al 2004). However, the role of catalase in diabetes is debatable; studies have shown that onset and progression of diabetes is accompanied by reductions in catalase activity (Ali & Agha 2009; Kamboj et al 2010; Pari et al 2010; Patel et al 2009), while others report an increase in the

More recently, mutations within the catalase gene have been suggested to contribute to the increased risk of diabetes (Góth & Eaton 2000). However, other studies report no such association with catalase gene polymorphisms and the development of diabetic complications (Letonja et al 2011; Panduru et al 2010). For example, one study reported that blood catalase activity was lowered due to the downregulation of catalase synthesis, rather than specific catalase gene mutations in type 2 diabetic patients. This was also associated

The mammalian Trx system is ubiquitously expressed and consists of Trx, Trx reductase and NADPH. The antioxidant properties of Trx is exerted mostly through the antioxidant enzymes, Trx peroxidase (also known as peroxiredoxin), which uses sulfhydryl (SH) groups as reducing equivalents (Chae et al., 1994). Trx reduces oxidised peroxiredoxin, which then scavenges H2O2 to produce water (Kang et al., 1998), thus attenuating oxidative stress in cells. The role of the Trx system in diabetic complications has gained considerable interest

with increased H2O2 levels and dysfunctional insulin receptor signalling (Góth 2008).

O2-, leads to improved outcomes

Collectively, these studies show that targeted removal of •

activity of catalase (Kakkar et al 1996; Kesavulu et al 2000).

in diabetic nephropathy and retinopathy.

**3.2.2 Catalase** 

**3.2.3 Thioredoxin system** 

were also on a C57/BL6 background (Lewis et al 2007). We then compared aortic lesion formation and atherogenic pathways in ApoE-deficient and ApoE/GPx1 dKO mice after these mice were rendered diabetic using the diabetogenic agent, streptozotocin (STZ). STZ destroys the pancreatic β-islet cells, thus providing a robust model of insulin deficient diabetes (Wilson & Leiter 1990).

In our study, we demonstrated that atherosclerotic lesions within the aortic sinus region, as well as lesions within the arch, thoracic and abdominal region were significantly increased in diabetic ApoE/GPx1 dKO aortas compared with diabetic ApoE-/- aortas (Lewis et al 2007). This increase in aortic lesions was accompanied by an increase in macrophages, α-smooth muscle actin (α-SMA), RAGE and various proinflammatory (VCAM-1, MCP-1) and profibrotic mediators (vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF)). Gene expression analyses also revealed a concomitant increase in RAGE, VCAM-1, VEGF and CTGF in diabetic dKO aortas compared with diabetic controls. Furthermore, the oxidative stress marker nitrotyrosine was also significantly increased in the diabetic dKO aortas. These findings were observed despite upregulation of other antioxidants, suggesting that a lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE-/- mice. Similar results were reported in a separate study using ApoE/GPx1-/- dKO mice, but in this instance atherosclerosis was induced by high fat diet (Torzewski et al 2007). In particular, these authors demonstrated increased atherosclerosis in their dKO mice, which was accompanied by increased cellularity in atherosclerotic lesions, as well as increased nitrotyrosine levels in the aortic wall and a lower level of bioactive • NO (Torzewski et al 2007).

As previously discussed, diabetic GPx1-deficient mice on a C57Bl/J6 background did not show accelerated kidney injury. However, on examination of the kidneys of diabetic dKO mice, we observed increased albuminuria and renal pathological changes which included mesangial expansion of the glomeruli and upregulation of profibrotic (collagen I and III, fibronectin, TGF-β) and proinflammatory mediators (VCAM-1, MCP-1) (Chew et al 2010). Thus, we believe that in the diabetic C57Bl/J6 GPx1-/- mice, the significance of a lack of GPx1 may not have been properly revealed since lipid levels were unaffected in this model (de Haan et al 2005). Elevated lipid levels have been shown to be critical in accelerating DN since clinical observation suggests that hyperlipidemia is an important contributory factor to the progression of diabetic renal disease (Jenkins et al 2003; Tolonen et al 2009). Importantly, we show enhanced staining for nitrotyrosine, which is a marker of ONOO damage in diabetic ApoE/Gpx1 dKO glomeruli and tubules of the kidney compared to diabetic ApoE- /- controls. We have therefore established a role for GPx1 in limiting and/or preventing DN in the pathophysiologically relevant milieu of increased lipids known to accompany diabetes (Chew et al 2010).

#### **3.2 Other antioxidant defence systems**

#### **3.2.1 Superoxide Dismutase (SOD)**

The SOD family of enzymes catalyse the conversion of • O2 - into H2O2 and oxygen in the first step of the antioxidant pathway (Fig.2), thereby performing an important role in the removal of • O2 -. Three isoforms exists in humans, Cu/Zn-SOD (also known as SOD1), Mn-SOD (also known as SOD2) and SOD3 with distinct cellular localisation, namely cytosolic, mitochondrial and extracellular, respectively.

Diabetes is associated with a decrease in SOD activity in most animal studies (Brocca et al 2008; Fujita et al 2009; Fukuda et al 2010). Lowered SOD levels are reported in serum and urine of STZ-treated Sprague-Dawley rats (Luo et al 2010), and decreased SOD1 and SOD3 levels are suggested to play a key role in the pathogenesis of diabetic nephropathy (Fujita et al 2009). Manipulations of SOD, through the use of SOD knockout mice or SOD overexpressing mice, have shown the importance of these enzymes in the protection against diabetic complications. Indeed, SOD1 knockout mice have clearly shown the importance of protection by SOD1 against superoxide-mediated DN. These mice demonstrated significant mesangial matrix expansion, renal cortical malondialdehyde content and severe tubulointerstitial injury compared with diabetic controls (DeRubertis et al 2007). Importantly, these pathological changes were attenuated in the presence of the SODmimetic, tempol (discussed in more detail in section 4.3.2). The significance of SOD2 was revealed in eMnSOD-Tg mice where the overexpression of mitochondrial-specific SOD targeted to the endothelium prevented diabetic retinopathy (Goto et al 2008). Furthermore, the targeted overexpression of Mn-SOD significantly attenuated morphological changes in diabetic hearts and improved contractility in diabetic cardiomyocytes (Shen et al 2006). Collectively, these studies show that targeted removal of • O2-, leads to improved outcomes in diabetic nephropathy and retinopathy.

#### **3.2.2 Catalase**

258 Oxidative Stress and Diseases

were also on a C57/BL6 background (Lewis et al 2007). We then compared aortic lesion formation and atherogenic pathways in ApoE-deficient and ApoE/GPx1 dKO mice after these mice were rendered diabetic using the diabetogenic agent, streptozotocin (STZ). STZ destroys the pancreatic β-islet cells, thus providing a robust model of insulin deficient

In our study, we demonstrated that atherosclerotic lesions within the aortic sinus region, as well as lesions within the arch, thoracic and abdominal region were significantly increased in diabetic ApoE/GPx1 dKO aortas compared with diabetic ApoE-/- aortas (Lewis et al 2007). This increase in aortic lesions was accompanied by an increase in macrophages, α-smooth muscle actin (α-SMA), RAGE and various proinflammatory (VCAM-1, MCP-1) and profibrotic mediators (vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF)). Gene expression analyses also revealed a concomitant increase in RAGE, VCAM-1, VEGF and CTGF in diabetic dKO aortas compared with diabetic controls. Furthermore, the oxidative stress marker nitrotyrosine was also significantly increased in the diabetic dKO aortas. These findings were observed despite upregulation of other antioxidants, suggesting that a lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE-/- mice. Similar results were reported in a separate study using ApoE/GPx1-/- dKO mice, but in this instance atherosclerosis was induced by high fat diet (Torzewski et al 2007). In particular, these authors demonstrated increased atherosclerosis in their dKO mice, which was accompanied by increased cellularity in atherosclerotic lesions, as well as increased nitrotyrosine levels in the aortic wall and a lower

As previously discussed, diabetic GPx1-deficient mice on a C57Bl/J6 background did not show accelerated kidney injury. However, on examination of the kidneys of diabetic dKO mice, we observed increased albuminuria and renal pathological changes which included mesangial expansion of the glomeruli and upregulation of profibrotic (collagen I and III, fibronectin, TGF-β) and proinflammatory mediators (VCAM-1, MCP-1) (Chew et al 2010). Thus, we believe that in the diabetic C57Bl/J6 GPx1-/- mice, the significance of a lack of GPx1 may not have been properly revealed since lipid levels were unaffected in this model (de Haan et al 2005). Elevated lipid levels have been shown to be critical in accelerating DN since clinical observation suggests that hyperlipidemia is an important contributory factor to the progression of diabetic renal disease (Jenkins et al 2003; Tolonen et al 2009). Importantly,

diabetic ApoE/Gpx1 dKO glomeruli and tubules of the kidney compared to diabetic ApoE- /- controls. We have therefore established a role for GPx1 in limiting and/or preventing DN in the pathophysiologically relevant milieu of increased lipids known to accompany

step of the antioxidant pathway (Fig.2), thereby performing an important role in the

SOD (also known as SOD2) and SOD3 with distinct cellular localisation, namely cytosolic,

O2


damage in


we show enhanced staining for nitrotyrosine, which is a marker of ONOO-

diabetes (Wilson & Leiter 1990).

level of bioactive •

diabetes (Chew et al 2010).

O2

removal of •

**3.2 Other antioxidant defence systems 3.2.1 Superoxide Dismutase (SOD)** 

mitochondrial and extracellular, respectively.

The SOD family of enzymes catalyse the conversion of •

NO (Torzewski et al 2007).

Catalase is present mainly in the peroxisomes of mammalian cells as a tetrameric enzyme of four identically arranged subunits; each containing a heme group and NADPH at its active centre (MatÉs et al 1999). Similar to GPx1, the enzyme neutralise H2O2 to water and oxygen (Fig.2). A role for catalase in the protection against atherosclerosis comes from the analysis of mice overexpressing catalase. In these experiments, overexpression of catalase significantly reduced the severity of lesions in ApoE-deficient mice (Yang et al 2004). However, the role of catalase in diabetes is debatable; studies have shown that onset and progression of diabetes is accompanied by reductions in catalase activity (Ali & Agha 2009; Kamboj et al 2010; Pari et al 2010; Patel et al 2009), while others report an increase in the activity of catalase (Kakkar et al 1996; Kesavulu et al 2000).

More recently, mutations within the catalase gene have been suggested to contribute to the increased risk of diabetes (Góth & Eaton 2000). However, other studies report no such association with catalase gene polymorphisms and the development of diabetic complications (Letonja et al 2011; Panduru et al 2010). For example, one study reported that blood catalase activity was lowered due to the downregulation of catalase synthesis, rather than specific catalase gene mutations in type 2 diabetic patients. This was also associated with increased H2O2 levels and dysfunctional insulin receptor signalling (Góth 2008).

#### **3.2.3 Thioredoxin system**

The mammalian Trx system is ubiquitously expressed and consists of Trx, Trx reductase and NADPH. The antioxidant properties of Trx is exerted mostly through the antioxidant enzymes, Trx peroxidase (also known as peroxiredoxin), which uses sulfhydryl (SH) groups as reducing equivalents (Chae et al., 1994). Trx reduces oxidised peroxiredoxin, which then scavenges H2O2 to produce water (Kang et al., 1998), thus attenuating oxidative stress in cells. The role of the Trx system in diabetic complications has gained considerable interest

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 261

We hypothesised that ebselen, in its capacity to act as a mimetic of GPx, would attenuate oxidative stress and lessen diabetes-associated atherosclerosis (Chew et al 2009). Eight week-old male C57Bl/J6 ApoE-/- mice were rendered diabetic with STZ and assigned to ebselen-gavaged and non-gavaged groups. Ebselen was administered twice daily at 10mg/kg/day body weight starting at 10 weeks of age and continued for 20 weeks. Our analysis showed that ebselen reduced lesion formation in most regions of the aorta including the arch, thoracic and abdominal regions of diabetic ApoE-/- mice. In addition, ebselen attenuated aortic nitrotyrosine levels and the expression of the Nox2 subunit of NADPH oxidase. The cellularity of the aorta associated with a pro-atherosclerotic phenotype (increased α-SMA-positive cells and increased macrophage infiltration) was also decreased by ebselen, together with a reduction in the aortic expression of the proatherosclerotic mediators, RAGE and VEGF (Chew et al 2009). These data support the notion of Blankenberg et al. (2003) that bolstering GPx-like activity reduces atherosclerosis. Furthermore, similar results were observed in our diabetic ApoE/GPx1 dKO mice where ebselen significantly reduced total aortic plaque, as well as regional plaque (arch, thoracic and abdominal) (Chew et al 2010). These reductions in plaque were also accompanied by a decrease in vascular oxidative stress (aortic 4-hydroxynonenal (HNE), nitrotyrosine, the Nox2 subunit of NADPH oxidase), as well as reductions in plasma hydroperoxides and urinary 8-isoprostanes. Additionally, the pro-inflammatory and pro-atherogenic mediators VCAM-1, MCP-1, CTGF and VEGF were attenuated by ebselen in diabetic ApoE/GPx1 deficient aortas. These data suggest that ebselen is able to replenish GPx1 activity in this

As mentioned in section 3.1.4, diabetic ApoE/GPx1 dKO mice demonstrated significant renal injury with an increase in albuminuria, hyperfiltration, mesangial expansion, oxidative stress (nitrotyrosine), pro-fibrotic (TGF-β, CTGF, collagen I, III and IV, fibronectin) and proinflammatory mediators (MCP-1, VCAM-1 and TNF-α) (Chew et al 2010). Ebselen significantly attenuated all of these parameters in diabetic dKO kidneys (Chew et al 2010). Our results are in agreement with Chander et al. (2004) where ebselen improved renal function and attenuated structural defects such as glomerulosclerosis, tubulointerstitial fibrosis and vasculopathy in the Zucker diabetic fat rat. Moreover, ebselen prevented the accumulation of lipid peroxidation products and 3-nitrotyrosine-modified proteins, and

Ebselen is well-characterised for its ability to act as a GPx mimic and can be catalytically maintained at the expense of GSH (Muller et al 1984; Sies & Masumoto 1997). Ebselen has been shown to reduce oxidative stress in several *in vivo* models; however this protective effect is unlikely to be solely due to the direct pro-oxidant interception by ebselen. Recently, ebselen has been reported to be an inducer of NF-E2-Related Factor 2- (Nrf-2)-dependent gene activation (Tamasi et al 2004). Nrf-2 is a member of the Cap'n'Collar family of basic region-leucine zipper (bZIP) transcription factors (Chui et al 1995). Genes upregulated by

**4.1.1 Ebselen in experimental models of diabetes associated atherosclerosis and** 

**nephropathy** 

model, thereby reducing atherosclerosis.

restored renal tissue levels of GSH (Chander et al 2004).

**4.1.2 Mechanistic understanding of the actions of ebselen** 

since thioredoxin-interacting protein (Txnip), which is an inhibitor of Trx activity, was discovered to be a highly upregulated hyperglycaemia-induced gene in both human and animal studies (Kobayashi et al 2003; Qi et al 2007; Shalev et al 2002). Txnip directly binds to the catalytic active site of Trx, thus inhibiting the reducing activity of Trx (Nishiyama et al 1999).

*In vitro* studies have shown Txnip expression to be significantly upregulated by glucose in mesangial cells (Kobayashi et al 2003), proximal tubule cells (Qi et al 2007) and distal tubule/collecting duct cells (Advani et al 2009; Kobayashi et al 2003). Overexpression of Txnip in STZ-treated diabetic rats was associated with an increase in ROS, as well as ECM accumulation in the kidney (Kobayashi et al 2003; Tan et al 2011). By reducing Txnip gene transcription with siRNA in kidney cells, high glucose-mediated increases in ROS production and ECM accumulation were attenuated, together with a restoration of Trx activity (Advani et al 2009). These results clearly delineate a role for Trx in maintaining redox homeostasis and highlight the importance of Txnip dysregulation in diabetic complications.

Txnip also plays an important role as a biomechanical effector of atherosclerosis (World et al 2006). In the endothelium of intact rabbit aorta, exposure to physiological fluid shear stress decreased Txnip expression and increased Trx activity, leading to a reduction in proinflammatory events mediated by the tumour necrosis factor (TNF)-ASK-1-JNK/p38 pathway, in association with a decrease in TNF-mediated VCAM1 expression (Yamawaki et al 2005). Furthermore, a calcium channel blocker promoted cardiac myocyte survival and improved cardiac function by reducing cardiac Txnip expression, suggesting a role for Txnip in mediating cardiac myocyte apoptosis in diabetic cardiomyopathy (Chen et al 2009).

#### **4. Novel antioxidants to limit diabetic micro- and macrovascular disease**

#### **4.1 GPx1-mimetic ebselen**

Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) is a synthetic, lipid soluble, non-toxic seleno-organic compound (Sies & Masumoto 1997) with anti-inflammatory and antioxidant activities (Muller et al 1984). Ebselen eliminates hydroperoxides, including H2O2 and lipid peroxides due to its GPx mimetic activity (Maiorino et al 1988; Parnham et al 1987; Safayhi et al 1985). Additionally, ebselen also scavenges other ROS such as peroxyl radicals and ONOO- (Sies & Masumoto 1997). Several enzymes involved in inflammatory processes, including 5-lipoxygenases, NOS, NADPH oxidase, PKC and ATPase are also inhibited by ebselen (Sies & Masumoto 1997). Importantly, ebselen has shown tremendous potential in reducing injury in various experimental models. Ebselen conferred protection against endothelial dysfunction in stroke-prone hypertensive rats (Sui et al 2005) and improved cardiac function in a model of chronic iron overload (Davis & Bartfay 2004). Furthermore, ebselen partially restored endothelial dysfunction in Zucker diabetic rats (Brodsky et al 2004). Additionally, arterial lesions were reduced by ebselen in a superoxide-driven noninflammatory transgenic murine model, suggesting a role for ebselen in reducing atherosclerosis (Khatri et al 2004). Importantly, in a clinical trial, ebselen has shown improved neurological outcomes in patients after cerebral infarction (Yamaguchi et al 1998) and a safety and pharmacokinetic profile of ebselen has been established in normal volunteers in a Phase I trial to determine whether ebselen is protective against noiseinduced hearing loss (Lynch & Kil 2009).

since thioredoxin-interacting protein (Txnip), which is an inhibitor of Trx activity, was discovered to be a highly upregulated hyperglycaemia-induced gene in both human and animal studies (Kobayashi et al 2003; Qi et al 2007; Shalev et al 2002). Txnip directly binds to the catalytic active site of Trx, thus inhibiting the reducing activity of Trx (Nishiyama et al

*In vitro* studies have shown Txnip expression to be significantly upregulated by glucose in mesangial cells (Kobayashi et al 2003), proximal tubule cells (Qi et al 2007) and distal tubule/collecting duct cells (Advani et al 2009; Kobayashi et al 2003). Overexpression of Txnip in STZ-treated diabetic rats was associated with an increase in ROS, as well as ECM accumulation in the kidney (Kobayashi et al 2003; Tan et al 2011). By reducing Txnip gene transcription with siRNA in kidney cells, high glucose-mediated increases in ROS production and ECM accumulation were attenuated, together with a restoration of Trx activity (Advani et al 2009). These results clearly delineate a role for Trx in maintaining redox homeostasis and

Txnip also plays an important role as a biomechanical effector of atherosclerosis (World et al 2006). In the endothelium of intact rabbit aorta, exposure to physiological fluid shear stress decreased Txnip expression and increased Trx activity, leading to a reduction in proinflammatory events mediated by the tumour necrosis factor (TNF)-ASK-1-JNK/p38 pathway, in association with a decrease in TNF-mediated VCAM1 expression (Yamawaki et al 2005). Furthermore, a calcium channel blocker promoted cardiac myocyte survival and improved cardiac function by reducing cardiac Txnip expression, suggesting a role for Txnip in mediating cardiac myocyte apoptosis in diabetic cardiomyopathy (Chen et al 2009).

**4. Novel antioxidants to limit diabetic micro- and macrovascular disease** 

Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) is a synthetic, lipid soluble, non-toxic seleno-organic compound (Sies & Masumoto 1997) with anti-inflammatory and antioxidant activities (Muller et al 1984). Ebselen eliminates hydroperoxides, including H2O2 and lipid peroxides due to its GPx mimetic activity (Maiorino et al 1988; Parnham et al 1987; Safayhi et al 1985). Additionally, ebselen also scavenges other ROS such as peroxyl radicals and ONOO- (Sies & Masumoto 1997). Several enzymes involved in inflammatory processes, including 5-lipoxygenases, NOS, NADPH oxidase, PKC and ATPase are also inhibited by ebselen (Sies & Masumoto 1997). Importantly, ebselen has shown tremendous potential in reducing injury in various experimental models. Ebselen conferred protection against endothelial dysfunction in stroke-prone hypertensive rats (Sui et al 2005) and improved cardiac function in a model of chronic iron overload (Davis & Bartfay 2004). Furthermore, ebselen partially restored endothelial dysfunction in Zucker diabetic rats (Brodsky et al 2004). Additionally, arterial lesions were reduced by ebselen in a superoxide-driven noninflammatory transgenic murine model, suggesting a role for ebselen in reducing atherosclerosis (Khatri et al 2004). Importantly, in a clinical trial, ebselen has shown improved neurological outcomes in patients after cerebral infarction (Yamaguchi et al 1998) and a safety and pharmacokinetic profile of ebselen has been established in normal volunteers in a Phase I trial to determine whether ebselen is protective against noise-

highlight the importance of Txnip dysregulation in diabetic complications.

1999).

**4.1 GPx1-mimetic ebselen** 

induced hearing loss (Lynch & Kil 2009).

#### **4.1.1 Ebselen in experimental models of diabetes associated atherosclerosis and nephropathy**

We hypothesised that ebselen, in its capacity to act as a mimetic of GPx, would attenuate oxidative stress and lessen diabetes-associated atherosclerosis (Chew et al 2009). Eight week-old male C57Bl/J6 ApoE-/- mice were rendered diabetic with STZ and assigned to ebselen-gavaged and non-gavaged groups. Ebselen was administered twice daily at 10mg/kg/day body weight starting at 10 weeks of age and continued for 20 weeks. Our analysis showed that ebselen reduced lesion formation in most regions of the aorta including the arch, thoracic and abdominal regions of diabetic ApoE-/- mice. In addition, ebselen attenuated aortic nitrotyrosine levels and the expression of the Nox2 subunit of NADPH oxidase. The cellularity of the aorta associated with a pro-atherosclerotic phenotype (increased α-SMA-positive cells and increased macrophage infiltration) was also decreased by ebselen, together with a reduction in the aortic expression of the proatherosclerotic mediators, RAGE and VEGF (Chew et al 2009). These data support the notion of Blankenberg et al. (2003) that bolstering GPx-like activity reduces atherosclerosis.

Furthermore, similar results were observed in our diabetic ApoE/GPx1 dKO mice where ebselen significantly reduced total aortic plaque, as well as regional plaque (arch, thoracic and abdominal) (Chew et al 2010). These reductions in plaque were also accompanied by a decrease in vascular oxidative stress (aortic 4-hydroxynonenal (HNE), nitrotyrosine, the Nox2 subunit of NADPH oxidase), as well as reductions in plasma hydroperoxides and urinary 8-isoprostanes. Additionally, the pro-inflammatory and pro-atherogenic mediators VCAM-1, MCP-1, CTGF and VEGF were attenuated by ebselen in diabetic ApoE/GPx1 deficient aortas. These data suggest that ebselen is able to replenish GPx1 activity in this model, thereby reducing atherosclerosis.

As mentioned in section 3.1.4, diabetic ApoE/GPx1 dKO mice demonstrated significant renal injury with an increase in albuminuria, hyperfiltration, mesangial expansion, oxidative stress (nitrotyrosine), pro-fibrotic (TGF-β, CTGF, collagen I, III and IV, fibronectin) and proinflammatory mediators (MCP-1, VCAM-1 and TNF-α) (Chew et al 2010). Ebselen significantly attenuated all of these parameters in diabetic dKO kidneys (Chew et al 2010). Our results are in agreement with Chander et al. (2004) where ebselen improved renal function and attenuated structural defects such as glomerulosclerosis, tubulointerstitial fibrosis and vasculopathy in the Zucker diabetic fat rat. Moreover, ebselen prevented the accumulation of lipid peroxidation products and 3-nitrotyrosine-modified proteins, and restored renal tissue levels of GSH (Chander et al 2004).

#### **4.1.2 Mechanistic understanding of the actions of ebselen**

Ebselen is well-characterised for its ability to act as a GPx mimic and can be catalytically maintained at the expense of GSH (Muller et al 1984; Sies & Masumoto 1997). Ebselen has been shown to reduce oxidative stress in several *in vivo* models; however this protective effect is unlikely to be solely due to the direct pro-oxidant interception by ebselen. Recently, ebselen has been reported to be an inducer of NF-E2-Related Factor 2- (Nrf-2)-dependent gene activation (Tamasi et al 2004). Nrf-2 is a member of the Cap'n'Collar family of basic region-leucine zipper (bZIP) transcription factors (Chui et al 1995). Genes upregulated by

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 263

small selenium compounds as functional mimics of GPx, either by modifying the basic structure of ebselen or by incorporating some structural features of the native enzyme (Alberto et al 2010; Back 2009; Bhabak & Mugesh 2010). These synthetic mimics can be classified into three major categories: (i) cyclic selenenyl amides having a Se-N bond, (ii) diaryl diselenides, and (iii) aromatic or aliphatic monoselenides (Bhabak & Mugesh 2010).

In addition to their GPx-like activity, the antioxidant activity of diphenyl diselenides may also be attributed to their capacity to be a substrate for mammalian Trx reductase (De Freitas et al 2010). In a study where acidosis was used to mediate oxidative stress in rat kidney homogenates, diphenyl diselenide significantly protected against lipid peroxidation, whilst the protection afforded by ebselen was only minor (Hassan et al 2009). Furthermore, diphenyl diselenide was also effective in protecting against acute renal failure induced by glycerol in rats (Brandão et al 2009). In cholesterol-fed rabbits, where animals exhibited hypercholesterolaemia and oxidative stress, diphenyl diselenide significantly reduced both of these risk factors of coronary artery disease in these animals (De Bem et al 2009). More recently, diphenyl diselenide has also been reported to attenuate atherosclerotic lesions in

The clinical benefits of ebselen and/or these novel GPx-mimetics in attenuating diabetesassociated complications are yet to be reported. Our pre-clinical assessments, as well as those of others, have highlighted the attractiveness of this class of therapeutic for their clinical use to prevent or attenuate both diabetes-associated atherosclerosis and DN, two co-

Increased NADPH oxidase (NOX) activity, which catalyses the production of ROS, has been implicated in the pathogenesis of diabetic complications (Kakehi & Yabe-Nishimura 2008). NOX4 has been shown to not only mediate the increase in ROS, but also activate profibrotic pathways in type 2 DN (Sedeek et al 2010). Additionally, inhibition of NOX1 suppresses neointimal formation in the prevention of vascular complications associated with diabetes

Several small molecule and peptide inhibitors of the NOX enzymes have been developed and are showing promise in experimental studies, but issues of specificity, potency and toxicity militate against any of the existing compounds as candidates for drug development (Kim et al 2011; Williams & Griendling 2007). In a recent study, apocynin, a proven NADPH oxidase inhibitor, attenuated albuminuria, improved kidney structure (glomerular and mesangial expansion) and reduced oxidative stress (urinary 8-OHdG and malondialdehyde) in aged Otsuka Long Evans Tokushima Fatty (OLETF) rats (Nam et al 2009). However, apocynin is considered to be non-selective in its mode of action as it also targets other

One of the most specific NOX inhibitors developed so far is gp91 (ds-tat), an 18-amino acid peptide which interferes with NOX assembly and activation (Rey et al 2001). This peptide functions by mimicking the binding region of NOX2, and possibly NOX1, which interacts

The most widely studied among the novel GPx-mimetics is diphenyl selenide.

LDLr-/- mice by lowering oxidative stress and inflammation (Hort et al 2011).

morbidities often present in diabetic patients.

enzymes such as Rho-kinase (Heumüller et al 2008).

**4.3 Other novel antioxidants** 

**4.3.1 NOX inhibitors** 

(Lee et al 2009).

Nrf-2 can be broadly classified into three separate classes; those that are involved in GSH synthesis (cystine membrane transporters), detoxication enzymes (rat glutathione Stransferase A2, a non-selenium-dependent glutathione peroxidase, and NAD(P)H:quinine oxidoreductase), and those directly involved in the amelioration of oxidative stress (heme oxygenase-1 (HO-1), peroxiredoxin MSP23, and Trx reductase) (Tamasi et al 2004). Tamasi et al. (2004) showed that ebselen can directly induce Nrf-2-dependent gene transcription, including the increase of intracellular GSH production, which acts as a substrate for GPx activity. These data showed that the activation of Nrf-2-dependent signalling by ebselen could indirectly augment cellular defences, independent of the direct interception of ROS by ebselen.

To further elucidate the mechanistic actions of ebselen observed in our diabetic experimental models, we examined the effect of ebselen on various signalling pathways implicated in atherosclerosis and nephropathy in human aortic endothelial cells (HAEC) (Chew et al 2009) and normal rat kidney (NRK) cells (Chew et al 2010). Pre-treatment of HAEC with 0.03 µM ebselen prior to exposure to 100 µM H2O2 reduced the H2O2-mediated increase in IkB-kinase (IKK) phosphorylation on critical activatory residues. Since IKK is a key regulator of NK-κB activation (Schmid & Birbach 2008), it is anticipated that by reducing IKK phosphorylation, ebselen anchors NF-κB in the cytoplasm thereby preventing the activation of proinflammatory genes. We further showed that ebselen reduced the H2O2-mediated increase in Nox2 expression; Nox2 is known to be regulated by NK-κB (Anrather et al 2006), further confirming the view that ebselen affects downstream targets of NF-κB.

The cytokine TNF-α is an important diabetes-associated pro-inflammatory mediator and is involved in the activation of NF-κB (Hacker & Karin 2006). Our *in vitro* data showed that H2O2-induced upregulation of TNF-α was reduced by ebselen. Our results support previous findings that ebselen inhibits TNF-α-induced pro-inflammatory responses in endothelial cells (Yoshizumi et al 2004) and other cell types (Sharma et al 2008; Tewari et al 2009). We also investigated the effects of ebselen on H2O2-mediated phosphorylation of JNK, a kinase involved in the activation of the transcription factor, AP-1. Our *in vitro* analysis showed that ebselen effectively attenuated H2O2-mediated phosphorylation of JNK, an important finding since phosphorylated JNK has been implicated in TNF-α-mediated endothelial activation (Min & Pober 1997) in particular through the interaction of AP-1 and NF-κB (Read et al 1997). Collectively, our results with ebselen have implications not only for inflammatory genes known to be regulated by these pathways, but also on the proatherosclerotic pathway itself, since inflammatory events are integrally linked with the development and progression of atherosclerosis.

Our *in vitro* studies in NRK cells further strengthen the notion that ebselen downregulates proinflammatory pathways in renal cells (Chew et al 2010). Similar to what we observed in HAEC, ebselen attenuated the phosphorylation of the pro-inflammatory mediator, IKK. Furthermore, stress-response kinases such as JNK and p38 MAPK phosphorylation were also attenuated by ebselen in NRK cells (Chew et al 2010).

#### **4.2 Novel GPx1-mimetics**

Novel GPx-mimetics, synthesised for their greater solubility and efficacy than ebselen are now available. Several laboratories have reported the synthesis and characterisation of novel small selenium compounds as functional mimics of GPx, either by modifying the basic structure of ebselen or by incorporating some structural features of the native enzyme (Alberto et al 2010; Back 2009; Bhabak & Mugesh 2010). These synthetic mimics can be classified into three major categories: (i) cyclic selenenyl amides having a Se-N bond, (ii) diaryl diselenides, and (iii) aromatic or aliphatic monoselenides (Bhabak & Mugesh 2010). The most widely studied among the novel GPx-mimetics is diphenyl selenide.

In addition to their GPx-like activity, the antioxidant activity of diphenyl diselenides may also be attributed to their capacity to be a substrate for mammalian Trx reductase (De Freitas et al 2010). In a study where acidosis was used to mediate oxidative stress in rat kidney homogenates, diphenyl diselenide significantly protected against lipid peroxidation, whilst the protection afforded by ebselen was only minor (Hassan et al 2009). Furthermore, diphenyl diselenide was also effective in protecting against acute renal failure induced by glycerol in rats (Brandão et al 2009). In cholesterol-fed rabbits, where animals exhibited hypercholesterolaemia and oxidative stress, diphenyl diselenide significantly reduced both of these risk factors of coronary artery disease in these animals (De Bem et al 2009). More recently, diphenyl diselenide has also been reported to attenuate atherosclerotic lesions in LDLr-/- mice by lowering oxidative stress and inflammation (Hort et al 2011).

The clinical benefits of ebselen and/or these novel GPx-mimetics in attenuating diabetesassociated complications are yet to be reported. Our pre-clinical assessments, as well as those of others, have highlighted the attractiveness of this class of therapeutic for their clinical use to prevent or attenuate both diabetes-associated atherosclerosis and DN, two comorbidities often present in diabetic patients.

#### **4.3 Other novel antioxidants**

#### **4.3.1 NOX inhibitors**

262 Oxidative Stress and Diseases

Nrf-2 can be broadly classified into three separate classes; those that are involved in GSH synthesis (cystine membrane transporters), detoxication enzymes (rat glutathione Stransferase A2, a non-selenium-dependent glutathione peroxidase, and NAD(P)H:quinine oxidoreductase), and those directly involved in the amelioration of oxidative stress (heme oxygenase-1 (HO-1), peroxiredoxin MSP23, and Trx reductase) (Tamasi et al 2004). Tamasi et al. (2004) showed that ebselen can directly induce Nrf-2-dependent gene transcription, including the increase of intracellular GSH production, which acts as a substrate for GPx activity. These data showed that the activation of Nrf-2-dependent signalling by ebselen could indirectly augment cellular defences, independent of the direct interception of ROS by

To further elucidate the mechanistic actions of ebselen observed in our diabetic experimental models, we examined the effect of ebselen on various signalling pathways implicated in atherosclerosis and nephropathy in human aortic endothelial cells (HAEC) (Chew et al 2009) and normal rat kidney (NRK) cells (Chew et al 2010). Pre-treatment of HAEC with 0.03 µM ebselen prior to exposure to 100 µM H2O2 reduced the H2O2-mediated increase in IkB-kinase (IKK) phosphorylation on critical activatory residues. Since IKK is a key regulator of NK-κB activation (Schmid & Birbach 2008), it is anticipated that by reducing IKK phosphorylation, ebselen anchors NF-κB in the cytoplasm thereby preventing the activation of proinflammatory genes. We further showed that ebselen reduced the H2O2-mediated increase in Nox2 expression; Nox2 is known to be regulated by NK-κB (Anrather et al 2006), further

The cytokine TNF-α is an important diabetes-associated pro-inflammatory mediator and is involved in the activation of NF-κB (Hacker & Karin 2006). Our *in vitro* data showed that H2O2-induced upregulation of TNF-α was reduced by ebselen. Our results support previous findings that ebselen inhibits TNF-α-induced pro-inflammatory responses in endothelial cells (Yoshizumi et al 2004) and other cell types (Sharma et al 2008; Tewari et al 2009). We also investigated the effects of ebselen on H2O2-mediated phosphorylation of JNK, a kinase involved in the activation of the transcription factor, AP-1. Our *in vitro* analysis showed that ebselen effectively attenuated H2O2-mediated phosphorylation of JNK, an important finding since phosphorylated JNK has been implicated in TNF-α-mediated endothelial activation (Min & Pober 1997) in particular through the interaction of AP-1 and NF-κB (Read et al 1997). Collectively, our results with ebselen have implications not only for inflammatory genes known to be regulated by these pathways, but also on the proatherosclerotic pathway itself, since inflammatory events are integrally linked with the development and

Our *in vitro* studies in NRK cells further strengthen the notion that ebselen downregulates proinflammatory pathways in renal cells (Chew et al 2010). Similar to what we observed in HAEC, ebselen attenuated the phosphorylation of the pro-inflammatory mediator, IKK. Furthermore, stress-response kinases such as JNK and p38 MAPK phosphorylation were

Novel GPx-mimetics, synthesised for their greater solubility and efficacy than ebselen are now available. Several laboratories have reported the synthesis and characterisation of novel

confirming the view that ebselen affects downstream targets of NF-κB.

ebselen.

progression of atherosclerosis.

**4.2 Novel GPx1-mimetics** 

also attenuated by ebselen in NRK cells (Chew et al 2010).

Increased NADPH oxidase (NOX) activity, which catalyses the production of ROS, has been implicated in the pathogenesis of diabetic complications (Kakehi & Yabe-Nishimura 2008). NOX4 has been shown to not only mediate the increase in ROS, but also activate profibrotic pathways in type 2 DN (Sedeek et al 2010). Additionally, inhibition of NOX1 suppresses neointimal formation in the prevention of vascular complications associated with diabetes (Lee et al 2009).

Several small molecule and peptide inhibitors of the NOX enzymes have been developed and are showing promise in experimental studies, but issues of specificity, potency and toxicity militate against any of the existing compounds as candidates for drug development (Kim et al 2011; Williams & Griendling 2007). In a recent study, apocynin, a proven NADPH oxidase inhibitor, attenuated albuminuria, improved kidney structure (glomerular and mesangial expansion) and reduced oxidative stress (urinary 8-OHdG and malondialdehyde) in aged Otsuka Long Evans Tokushima Fatty (OLETF) rats (Nam et al 2009). However, apocynin is considered to be non-selective in its mode of action as it also targets other enzymes such as Rho-kinase (Heumüller et al 2008).

One of the most specific NOX inhibitors developed so far is gp91 (ds-tat), an 18-amino acid peptide which interferes with NOX assembly and activation (Rey et al 2001). This peptide functions by mimicking the binding region of NOX2, and possibly NOX1, which interacts

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 265

Manganese(Il) complex with a bis(cyclohexylpyridine)-substituted macrocyclic ligand (M40403) is another SOD-mimetic which has high catalytic SOD activity and is chemically and biologically stable *in vivo*. Injection of M40403 into rat models of inflammation and ischemia-reperfusion injury protected the animals against tissue damage, possibly by

(Salvemini et al 1999). M40403 also reversed endothelial dysfunction in ApoE-/- aortas *ex* 

The targeted delivery of antioxidants to the mitochondria is an attractive approach to effectively remove or attenuate pathogenic ROS produced by the mitochondria. Animal studies using transgenic mice over-expressing mitochondrially-targeted SOD and catalase have already shown the potential of this strategy, since these transgenic mice were associated with significant reductions in cardiac mitochondrial oxidative stress and improvements in left ventricular function after antiretroviral-induced cardiomyopathy (Kohler et al 2009). Indeed, the development of antioxidants that specifically target the matrix-facing inner surface of the mitochondrial membrane is hypothesised to protect against mitochondrial oxidative damage (Ross et al 2005). Because of the high negative membrane potential of the inner mitochondrial membrane, antioxidants conjugated with a lipophilic triphenylphosphonium (TPP) cation such as mitoquinone, mitovitamin E and mitophenyltertbutyline accumulate in the mitochondrial matrix at concentrations severalfold greater than cytosolic non-mitochondrially targeted antioxidants (Subramanian et al 2010; Victor et al 2009). In particular, MitoQ, which has a TPP modification added to coenzyme Q is 100 times more potent than idebenone, a coenzymes Q derivative. Furthermore, MitoVitE (vitamin E attached to a TPP cation) is 350 times more potent than Trolox, a water soluble version of vitamin E, in fibroblasts from patients (Victor et al 2009). *In vitro* experiments using both mitoquinone and mitovitamin E have shown promising reductions in peroxide-mediated oxidant stress and apoptosis whilst maintaining proteasomal function in bovine aortic endothelial cells (BAEC) (Dhanasekaran et al 2004). Moreover, Mito-carboxy proxyl (Mito-CP), a mitochondrial-targeted SOD, significantly diminished glucose/glucose oxidase-induced formation of intracellular ROS and apoptosis in BAEC, while the "untargeted" carboxy proxyl (CP) nitroxide probe did not (Dhanasekaran et al 2005). Furthermore, a mitochondrial-targeted form of the Gpx1 mimetic, MitoPeroxidase, which contains an ebselen moiety covalently linked to a TPP cation, decreases glucose and H2O2-mediated apoptosis in a rat basophilic leukemia cell line (RBL-2H3) (Filipovska et al 2005). Several studies have shown the accumulation of these compounds in the mitochondria of various tissues including brain, heart, liver and kidney, in mice fed mitrochondrially-targeted antioxidant compounds for several weeks (Smith et al 1999). However, to date, their therapeutic potential in militating against diabetic

O2

, as well as assisting in the direct removal of •


O2 -

preventing the formation of ONOO-

complications has not been explored.

**4.3.4 Bolstering antioxidant defences via the transcription factor Nrf2** 

Nrf2 is a redox sensitive transcription factor which regulates the expression of important cytoprotective enzymes (Kensler et al 2007). Nrf2 plays an important role in endogenous defence against sustained oxidative stress by upregulating important detoxifying phase II

*vivo* by decreasing NADPH oxidase-dependent •

**4.3.3 Mitochondrially-targeted antioxidants** 

with p47phox. In doing so, it inhibits subunit assembly, resulting in the specific inhibition of • O2- production from NOX and not from other oxidases such as xanthine oxidase. Gp91 (dstat) has shown promise in reducing vascular ROS associated with Ang II-mediated hypertension in mice (Rey et al 2001), as well as reducing endothelial dysfunction and vascular ROS in the Dahl salt-sensitive rat model (Zhou et al 2006). However, limited bioavailability of this peptide has hampered its usefulness as a therapeutic agent; at present, it can only be administered via intravenous injection (Williams & Griendling 2007). VAS2870 is a fairly new NOX inhibitor discovered via high-throughput screening and is specific for NADPH oxidase activity (ten Freyhaus et al 2006). It has been shown to attenuate platelet-derived growth factor (PDGF)-dependent smooth muscle cell chemotaxis *in vitro* via a mechanism that includes the complete abolition of NOX activation and ROS production (ten Freyhaus et al 2006).

Only limited *in vitro* and *in vivo* data are available for these novel compounds. Further testing in pre-clinical models is necessary to determine if these approaches represent feasible therapeutic strategies for diabetic complications.

#### **4.3.2 SOD mimetics**

As discussed in section 3.2.1, SOD is the first line of defence against both physiological and pathological ROS, by catalysing the dismutation of • O2 - to H2O2 (Fig.2). Protective and beneficial effects of SOD enzymes have been demonstrated in a broad range of superoxidedriven diseases, both pre-clinically and clinically (Muscoli et al 2003). When tested in humans, Orgotein (a bovine CuZn-SOD mimetic) showed promising results in acute and chronic conditions associated with inflammation; however, because of its non-human origin, the use of bovine native enzyme in the human context caused a variety of immunological disorders (Muscoli et al 2003). Since then, several synthetic, low-molecular weight mimetics of SOD have been produced with promising results in pre-clinical models.

The most well-characterised of the SOD-mimetics is tempol (4-hydroxy-2,2,6,6, tetramethylpiperidine-*N*-oxyl). Tempol has been reported to protect animals and mammalian cells from cytotoxicity induced by oxygen radicals such as H2O2 and • O2 - (Mitchell et al 1990). One attractive attribute of tempol is its ability to penetrate cell membranes and hence react with ROS both intracellularly and extracellularly, as well as within important organelles such as the mitochondria (Simonsen et al 2009). Tempol has been shown to improve acetylcholineand arachidonic acid-induced relaxation in skeletal muscle arteries and in coronary arteries from diabetic animals (Gao et al 2007; Xiang et al 2008). Furthermore, in an *in vivo* one-kidney one-clip hypertensive rat model, tempol improved endothelial function in small arteries exposed to high blood pressure (Christensen et al 2007). However, the use of tempol may be limited in some instances by its propensity to increase H2O2, thereby exacerbating the progression of disease. For instance, in an experimental model of glomerulonephritis, tempol increased proteinuria and crescentric glomerulonephritis with leukocyte infiltration, as well as accelerating mortality in the treated group (Lu et al 2010). Moreover, tempol upregulated p65- NFκB and osteopontin in the kidney and increased H2O2 levels in the urine. In another study, tempol could not prevent the development of hypertension in a hypertensive rat model induced by inhibiting renal medullary SOD with diethyldithiocarbamic acid (Chen et al 2003). These results may be due to the increased formation of H2O2, as a result of the dismutation of • O2 by tempol, causing constriction of the medullary vessels, and counteracting the vasodilatory actions of tempol.

Manganese(Il) complex with a bis(cyclohexylpyridine)-substituted macrocyclic ligand (M40403) is another SOD-mimetic which has high catalytic SOD activity and is chemically and biologically stable *in vivo*. Injection of M40403 into rat models of inflammation and ischemia-reperfusion injury protected the animals against tissue damage, possibly by preventing the formation of ONOO- , as well as assisting in the direct removal of • O2 - (Salvemini et al 1999). M40403 also reversed endothelial dysfunction in ApoE-/- aortas *ex vivo* by decreasing NADPH oxidase-dependent • O2 - levels (Jiang et al 2003a).

#### **4.3.3 Mitochondrially-targeted antioxidants**

264 Oxidative Stress and Diseases

with p47phox. In doing so, it inhibits subunit assembly, resulting in the specific inhibition of • O2- production from NOX and not from other oxidases such as xanthine oxidase. Gp91 (dstat) has shown promise in reducing vascular ROS associated with Ang II-mediated hypertension in mice (Rey et al 2001), as well as reducing endothelial dysfunction and vascular ROS in the Dahl salt-sensitive rat model (Zhou et al 2006). However, limited bioavailability of this peptide has hampered its usefulness as a therapeutic agent; at present, it can only be administered via intravenous injection (Williams & Griendling 2007). VAS2870 is a fairly new NOX inhibitor discovered via high-throughput screening and is specific for NADPH oxidase activity (ten Freyhaus et al 2006). It has been shown to attenuate platelet-derived growth factor (PDGF)-dependent smooth muscle cell chemotaxis *in vitro* via a mechanism that includes the complete abolition of NOX activation and ROS

Only limited *in vitro* and *in vivo* data are available for these novel compounds. Further testing in pre-clinical models is necessary to determine if these approaches represent feasible

As discussed in section 3.2.1, SOD is the first line of defence against both physiological and

beneficial effects of SOD enzymes have been demonstrated in a broad range of superoxidedriven diseases, both pre-clinically and clinically (Muscoli et al 2003). When tested in humans, Orgotein (a bovine CuZn-SOD mimetic) showed promising results in acute and chronic conditions associated with inflammation; however, because of its non-human origin, the use of bovine native enzyme in the human context caused a variety of immunological disorders (Muscoli et al 2003). Since then, several synthetic, low-molecular weight mimetics

The most well-characterised of the SOD-mimetics is tempol (4-hydroxy-2,2,6,6, tetramethylpiperidine-*N*-oxyl). Tempol has been reported to protect animals and mammalian

One attractive attribute of tempol is its ability to penetrate cell membranes and hence react with ROS both intracellularly and extracellularly, as well as within important organelles such as the mitochondria (Simonsen et al 2009). Tempol has been shown to improve acetylcholineand arachidonic acid-induced relaxation in skeletal muscle arteries and in coronary arteries from diabetic animals (Gao et al 2007; Xiang et al 2008). Furthermore, in an *in vivo* one-kidney one-clip hypertensive rat model, tempol improved endothelial function in small arteries exposed to high blood pressure (Christensen et al 2007). However, the use of tempol may be limited in some instances by its propensity to increase H2O2, thereby exacerbating the progression of disease. For instance, in an experimental model of glomerulonephritis, tempol increased proteinuria and crescentric glomerulonephritis with leukocyte infiltration, as well as accelerating mortality in the treated group (Lu et al 2010). Moreover, tempol upregulated p65- NFκB and osteopontin in the kidney and increased H2O2 levels in the urine. In another study, tempol could not prevent the development of hypertension in a hypertensive rat model induced by inhibiting renal medullary SOD with diethyldithiocarbamic acid (Chen et al 2003). These results may be due to the increased formation of H2O2, as a result of the dismutation of •

by tempol, causing constriction of the medullary vessels, and counteracting the

of SOD have been produced with promising results in pre-clinical models.

cells from cytotoxicity induced by oxygen radicals such as H2O2 and •

O2 -

to H2O2 (Fig.2). Protective and

O2 -

(Mitchell et al 1990).

production (ten Freyhaus et al 2006).

**4.3.2 SOD mimetics** 

O2 -

vasodilatory actions of tempol.

therapeutic strategies for diabetic complications.

pathological ROS, by catalysing the dismutation of •

The targeted delivery of antioxidants to the mitochondria is an attractive approach to effectively remove or attenuate pathogenic ROS produced by the mitochondria. Animal studies using transgenic mice over-expressing mitochondrially-targeted SOD and catalase have already shown the potential of this strategy, since these transgenic mice were associated with significant reductions in cardiac mitochondrial oxidative stress and improvements in left ventricular function after antiretroviral-induced cardiomyopathy (Kohler et al 2009). Indeed, the development of antioxidants that specifically target the matrix-facing inner surface of the mitochondrial membrane is hypothesised to protect against mitochondrial oxidative damage (Ross et al 2005). Because of the high negative membrane potential of the inner mitochondrial membrane, antioxidants conjugated with a lipophilic triphenylphosphonium (TPP) cation such as mitoquinone, mitovitamin E and mitophenyltertbutyline accumulate in the mitochondrial matrix at concentrations severalfold greater than cytosolic non-mitochondrially targeted antioxidants (Subramanian et al 2010; Victor et al 2009). In particular, MitoQ, which has a TPP modification added to coenzyme Q is 100 times more potent than idebenone, a coenzymes Q derivative. Furthermore, MitoVitE (vitamin E attached to a TPP cation) is 350 times more potent than Trolox, a water soluble version of vitamin E, in fibroblasts from patients (Victor et al 2009). *In vitro* experiments using both mitoquinone and mitovitamin E have shown promising reductions in peroxide-mediated oxidant stress and apoptosis whilst maintaining proteasomal function in bovine aortic endothelial cells (BAEC) (Dhanasekaran et al 2004). Moreover, Mito-carboxy proxyl (Mito-CP), a mitochondrial-targeted SOD, significantly diminished glucose/glucose oxidase-induced formation of intracellular ROS and apoptosis in BAEC, while the "untargeted" carboxy proxyl (CP) nitroxide probe did not (Dhanasekaran et al 2005). Furthermore, a mitochondrial-targeted form of the Gpx1 mimetic, MitoPeroxidase, which contains an ebselen moiety covalently linked to a TPP cation, decreases glucose and H2O2-mediated apoptosis in a rat basophilic leukemia cell line (RBL-2H3) (Filipovska et al 2005). Several studies have shown the accumulation of these compounds in the mitochondria of various tissues including brain, heart, liver and kidney, in mice fed mitrochondrially-targeted antioxidant compounds for several weeks (Smith et al 1999). However, to date, their therapeutic potential in militating against diabetic complications has not been explored.

#### **4.3.4 Bolstering antioxidant defences via the transcription factor Nrf2**

Nrf2 is a redox sensitive transcription factor which regulates the expression of important cytoprotective enzymes (Kensler et al 2007). Nrf2 plays an important role in endogenous defence against sustained oxidative stress by upregulating important detoxifying phase II

Oxidative Stress and Novel Antioxidant Approaches to Reduce Diabetic Complications 267

than 1 ml/min/1.73 m2 per year, therefore (Brenner et al 2001), improvements with bardoxolone methyl of between 5-10 ml/min/1.73 m2 are seen as a major advance over standard therapies. These results may lead to further pre-clinical and clinical activity to identify additional Nrf2 activators with possibly even greater efficacy. Indeed, the strategy of bolstering antioxidant defences by manipulating Nrf2 may represent a new class of therapy with potentially major advances over conventional therapy in the treatment of

Fig. 3. Novel antioxidant strategies to attenuate increased cellular ROS production and/or increase the activity of endogenous antioxidant defence systems in diabetes-associated

Increasing evidence has implicated a role for oxidative stress in mediating diabetesassociated complications. Despite this, very few therapies are currently available in clinical practice to effectively target oxidative stress and lessen the burden of diabetic complications. The use of vitamins in clinical trials have been mostly disappointing, showing no overall benefit for major cardiovascular events and in some instances, even increasing cardiovascular mortality (McQueen et al 2005). Failure of vitamins in the clinic may be due to their lack of specificity in not correctly targeting the ROS responsible for pathogenesis; conversely, total ablation of ROS could be detrimental as ROS are essential for basic cell signalling and homeostasis. Thus, the challenge for developing an effective antioxidant therapy for diabetes-associated complications would be to target either the production of specific ROS involved in diabetes-mediated injury or to eliminate ROS now appreciated to contribute to diabetes-associated-atherosclerosis such as hydrogen peroxide. A further

diabetic complications such as diabetic nephropathy.

complications.

**5. Conclusion** 

enzymes, such as NAD(P)H:quinine oxidoreductase (NQO1) and antioxidant proteins, such as HO-1, through an antioxidant response element (ARE)-dependent pathway. The protective role of Nrf2 in diabetes-mediated kidney injury has gained considerable attention recently. Diabetic Nrf2 knockout mice demonstrated increased glomerular ROS production and greater oxidative DNA damage and renal injury compared to control mice (Jiang et al 2010). In addition, in human renal mesangial (Jiang et al 2010) and coronary arterial endothelial cells (Ungvari et al 2011), high glucose induced ROS production and the enhanced expression of Nrf2 and its downstream genes, such as NQO1, glutathione Stransferase (GST), glutamate-cysteine ligase catalytic (GCLC) and HO-1. These effects of high glucose were significantly attenuated by silencing Nrf2 expression using siRNA or overexpression of kelch-like ECH-associated protein (Keap-1), which is an inhibitor of Nrf2 (Ungvari et al 2011). Furthermore, overexpression of Nrf2 inhibited the promoter activity of TGF-β1 in a dose-dependent manner, whereas knockdown of Nrf2 by siRNA enhanced TGF-β1 transcription and fibronectin production, suggesting that Nrf2 plays a protective role in attenuating diabetic nephropathy (Jiang et al 2010).

Hyperglycaemia is associated with the increased formation of AGE and enhanced oxidative stress, leading to the progression of diabetic cardiovascular disease (Thomas et al 2005). It was recently reported that Nrf2 is activated by AGE in BAEC, resulting in the induction of the antioxidant genes HO-1 and NQO1, thus confirming a protective role of Nrf2 against oxidative stress in diabetes (He et al 2011). Furthermore, to test the protective effects of Nrf2 under metabolic stress, which often occurs concurrently with diabetes, Nrf2-/- mice were subjected to high fat diet (Ungvari et al 2011). These mice failed to show significant increases in the gene expression of the Nrf2 downstream targets GCLC and HO-1. In addition, increased ROS and endothelial dysfunction was attenuated in Nrf2-/- aortas, in contrast to Nrf2+/+ controls, further confirming that an adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions (Ungvari et al 2011).

Nonetheless, very few studies have directly demonstrated the therapeutic potential of increasing Nrf2 in pre-clinical models. Oltipraz, an Nrf2 activator, significantly prevented the development of insulin resistance and obesity in high fat diet (HFD)-induced C57BL/6J mice (Yu et al 2011). Control mice fed with HFD demonstrated reduced nuclear content of Nrf2 in adipose tissue, which was associated with increased Keap-1 mRNA expression and reduced HO-1 and NQO1; all of which were attenuated with oltipraz. Moreover, resveratrol, which is a polyphenolic phytoalexin that occurs naturally in many plant species, including grapevines and berries, was shown to attenuate STZ-induced diabetic nephropathy in rats, through the preservation of Nrf2 mRNA and protein expression, with an inhibitory effect on diabetes-induced upregulation of Keap-1 in diabetic kidneys (Palsamy & Subramanian 2011). As mentioned earlier, ebselen can directly upregulate the expression of Nrf2-dependent genes, in addition to its ability to quench H2O2 (Tamasi et al 2004). Furthermore, ebselen has been shown to modify Keap-1, thereby relieving the inhibitory effect of Keap-1 on Nrf2 (Sakurai et al 2006).

A recent clinical trial using the Nrf2 activator, bardoxolone methyl, (Pergola et al 2011) has generated particular interest in this type of drug to lessen the burden of DN. In this doubleblind, randomized, placebo-controlled trial, bardoxolone significantly improved the estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes and impaired renal function. Current interventions appear to slow the decline in renal function by less than 1 ml/min/1.73 m2 per year, therefore (Brenner et al 2001), improvements with bardoxolone methyl of between 5-10 ml/min/1.73 m2 are seen as a major advance over standard therapies. These results may lead to further pre-clinical and clinical activity to identify additional Nrf2 activators with possibly even greater efficacy. Indeed, the strategy of bolstering antioxidant defences by manipulating Nrf2 may represent a new class of therapy with potentially major advances over conventional therapy in the treatment of diabetic complications such as diabetic nephropathy.

Fig. 3. Novel antioxidant strategies to attenuate increased cellular ROS production and/or increase the activity of endogenous antioxidant defence systems in diabetes-associated complications.

#### **5. Conclusion**

266 Oxidative Stress and Diseases

enzymes, such as NAD(P)H:quinine oxidoreductase (NQO1) and antioxidant proteins, such as HO-1, through an antioxidant response element (ARE)-dependent pathway. The protective role of Nrf2 in diabetes-mediated kidney injury has gained considerable attention recently. Diabetic Nrf2 knockout mice demonstrated increased glomerular ROS production and greater oxidative DNA damage and renal injury compared to control mice (Jiang et al 2010). In addition, in human renal mesangial (Jiang et al 2010) and coronary arterial endothelial cells (Ungvari et al 2011), high glucose induced ROS production and the enhanced expression of Nrf2 and its downstream genes, such as NQO1, glutathione Stransferase (GST), glutamate-cysteine ligase catalytic (GCLC) and HO-1. These effects of high glucose were significantly attenuated by silencing Nrf2 expression using siRNA or overexpression of kelch-like ECH-associated protein (Keap-1), which is an inhibitor of Nrf2 (Ungvari et al 2011). Furthermore, overexpression of Nrf2 inhibited the promoter activity of TGF-β1 in a dose-dependent manner, whereas knockdown of Nrf2 by siRNA enhanced TGF-β1 transcription and fibronectin production, suggesting that Nrf2 plays a protective

Hyperglycaemia is associated with the increased formation of AGE and enhanced oxidative stress, leading to the progression of diabetic cardiovascular disease (Thomas et al 2005). It was recently reported that Nrf2 is activated by AGE in BAEC, resulting in the induction of the antioxidant genes HO-1 and NQO1, thus confirming a protective role of Nrf2 against oxidative stress in diabetes (He et al 2011). Furthermore, to test the protective effects of Nrf2 under metabolic stress, which often occurs concurrently with diabetes, Nrf2-/- mice were subjected to high fat diet (Ungvari et al 2011). These mice failed to show significant increases in the gene expression of the Nrf2 downstream targets GCLC and HO-1. In addition, increased ROS and endothelial dysfunction was attenuated in Nrf2-/- aortas, in contrast to Nrf2+/+ controls, further confirming that an adaptive activation of the Nrf2/ARE pathway

Nonetheless, very few studies have directly demonstrated the therapeutic potential of increasing Nrf2 in pre-clinical models. Oltipraz, an Nrf2 activator, significantly prevented the development of insulin resistance and obesity in high fat diet (HFD)-induced C57BL/6J mice (Yu et al 2011). Control mice fed with HFD demonstrated reduced nuclear content of Nrf2 in adipose tissue, which was associated with increased Keap-1 mRNA expression and reduced HO-1 and NQO1; all of which were attenuated with oltipraz. Moreover, resveratrol, which is a polyphenolic phytoalexin that occurs naturally in many plant species, including grapevines and berries, was shown to attenuate STZ-induced diabetic nephropathy in rats, through the preservation of Nrf2 mRNA and protein expression, with an inhibitory effect on diabetes-induced upregulation of Keap-1 in diabetic kidneys (Palsamy & Subramanian 2011). As mentioned earlier, ebselen can directly upregulate the expression of Nrf2-dependent genes, in addition to its ability to quench H2O2 (Tamasi et al 2004). Furthermore, ebselen has been shown to modify Keap-1, thereby relieving the

A recent clinical trial using the Nrf2 activator, bardoxolone methyl, (Pergola et al 2011) has generated particular interest in this type of drug to lessen the burden of DN. In this doubleblind, randomized, placebo-controlled trial, bardoxolone significantly improved the estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes and impaired renal function. Current interventions appear to slow the decline in renal function by less

confers endothelial protection under diabetic conditions (Ungvari et al 2011).

role in attenuating diabetic nephropathy (Jiang et al 2010).

inhibitory effect of Keap-1 on Nrf2 (Sakurai et al 2006).

Increasing evidence has implicated a role for oxidative stress in mediating diabetesassociated complications. Despite this, very few therapies are currently available in clinical practice to effectively target oxidative stress and lessen the burden of diabetic complications. The use of vitamins in clinical trials have been mostly disappointing, showing no overall benefit for major cardiovascular events and in some instances, even increasing cardiovascular mortality (McQueen et al 2005). Failure of vitamins in the clinic may be due to their lack of specificity in not correctly targeting the ROS responsible for pathogenesis; conversely, total ablation of ROS could be detrimental as ROS are essential for basic cell signalling and homeostasis. Thus, the challenge for developing an effective antioxidant therapy for diabetes-associated complications would be to target either the production of specific ROS involved in diabetes-mediated injury or to eliminate ROS now appreciated to contribute to diabetes-associated-atherosclerosis such as hydrogen peroxide. A further

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Smith RAJ, Porteous CM, Coulter CV, Murphy MP. 1999. Selective targeting of an antioxidant to mitochondria. *European Journal of Biochemistry* 263:709-16 Studer RK, Craven PA, DeRubertis FR. 1997. Antioxidant inhibition of protein kinase C-

Subramanian S, Kalyanaraman B, Migrino RQ. 2010. Mitochondrially targeted antioxidants

Sui H, Wang W, Wang PH, Liu LS. 2005. Effect of glutathione peroxidase mimic ebselen

Szabó C, Mabley JG, Moeller SM, Shimanovich R, Pacher P, et al. 2002. Part I: Pathogenetic

Sies H. 1997. Oxidative stress: oxidants and antioxidants. *Experimental Physiology* 82:291-5 Sies H, Masumoto H. 1997. Ebselen as a glutathione peroxidase mimic and as a scavenger of

peroxynitrite. *Advances in pharmacology (San Diego, Calif.)* 38:229-46

signaling complex. *International Journal of Cancer* 123:2204-12

2010 and 2030. *Diabetes research and clinical practice* 87:4-14

vasodilatation. *Pharmacological Reports* 61:105-15

*Metabolism: Clinical and Experimental* 46:918-25

hypertensive rats. *Blood Pressure* 14:366-72

*Discovery* 5:54-65

AIF-Mediated Cell Death. *Cell Metabolism* 8:237-48

Signaling Pathway. *Endocrinology* 143:3695-

*Physiology* 283:R243-R8

*Physiology* 299:F1348-F58

24:S45-S50

hypertension. *American Journal of Physiology - Regulatory Integrative and Comparative* 

the endothelium: Role in atherosclerosis and hypertension. *Journal of Hypertension*

based NADPH oxidase in glucose-induced oxidative stress in the kidney: Implications in type 2 diabetic nephropathy. *American Journal of Physiology - Renal* 

Senses and Translates Oxidative Stress into 12/15-Lipoxygenase Dependent- and

Oligonucleotide Microarray Analysis of Intact Human Pancreatic Islets: Identification of Glucose-Responsive Genes and a Highly Regulated TGF-β

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Overexpression of MnSOD Reduces Diabetic Cardiomyopathy. *Diabetes* 55:798-805

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signaled increases in transforming growth factor-beta in mesangial cells.

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(PZ51) on endothelium and vascular structure of stroke‐prone spontaneously

role of peroxynitrite in the development of diabetes and diabetic vascular

complications: Studies with FP15, a novel potent peroxynitrite decomposition catalyst. *Molecular Medicine* 8:571-80


**12** 

*Romania* 

**Evaluation of Oxidative Stress** 

**and the Efficacy of Antioxidant** 

**Treatment in Diabetes Mellitus** 

*University of Medicine and Pharmacy, Tîrgu-Mureş, Medical Biochemistry Department,* 

Studies on the efficacy of antioxidant treatment in type 1 diabetes mellitus is an interesting, actual research subject. Reactive oxygen species (ROS) are continuously produced and eliminated by living organisms normally maintaining ROS at certain steady-state levels. Under some circumstances, the balance between ROS generation and elimination is disturbed leading to enhanced ROS level causing oxidative stress Lushchak, 2011. Oxidative stress is involved in the development of several important diseases (cancer, ulcer, atherosclerosis, autoimmune

Oxidative stress due to increased production of reactive oxygen species and/or impaired antioxidant capacity of the body plays a special, important role in the development of type 1 diabetes mellitus and its complications Baynes, 1991, Giugliano et al., 1995, Krippeit-Drews et al., 1994, Nakazaki et al., 1995, Nomikos et al., 1989, Raes et al, 1995, Shinn, 1998.

Under normal conditions, oxidative tissue damage is prevented by enzymatic and nonenzymatic antioxidants. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) are three enzymes involved in detoxification of reactive oxygen species (superoxide radical, hydroxyl radical, and hydrogen peroxide). Pancreatic cells have a poor antioxidant defence system, so they are very vulnerable to oxidative stress, especially to

Evaluation of oxidative status can be made by various methods, most of them very sophisticated, requiring laboratories with modern equipment, and are used almost

V. Balogh-Sămărghiţan1, Elena Cristina Crăciun2, R. Morar3, Dana Liana Pusta3, Fazakas Zita1,

<sup>1</sup>*University of Medicine and Pharmacy, Tîrgu-Mureş, Medical Biochemistry Department, Romania 2 University of Medicine and Pharmacy "Iuliu Haţieganu", Cluj-Napoca, Pharmaceutical Biochemistry and* 

*3 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine,* 

*4 II. Clinical Hospital of Pediatrics, Tîrgu-Mureş, Romania 5 Santa Medical Unit, Tîrgu-Mureş, Romania, 6 University of Medicine and Pharmacy, Tîrgu-Mureş, Clinical Biochemistry Department, Romania*

Szőcs-Molnár Terézia4, Dunca Iulia4, Sánta Dóra5 and Minodora Dobreanu6

diseases, ischaemia-reperfusion injury, emphysema, inflammation, etc.).

superoxide mediated radical damage Dejica, 2000.

exclusively for research purposes.

*Clinical Laboratory Department, Romania* 

*Cluj-Napoca, Romania* 

 \*

**1. Introduction** 

Nemes-Nagy Enikő et al.\*


### **Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus**

Nemes-Nagy Enikő et al.\* *University of Medicine and Pharmacy, Tîrgu-Mureş, Medical Biochemistry Department, Romania* 

#### **1. Introduction**

280 Oxidative Stress and Diseases

Xiang L, Dearman J, Abram SR, Carter C, Hester RL. 2008. Insulin resistance and impaired

Yamaguchi T, Sano K, Takakura K, Saito I, Shinohara Y, et al. 1998. Ebselen in Acute Ischemic Stroke : A Placebo-Controlled, Double-blind Clinical Trial. *Stroke* 29:12-7 Yamawaki H, Pan S, Lee RT, Berk BC. 2005. Fluid shear stress inhibits vascular

Yang H, Roberts LJ, Ming JS, Li CZ, Ballard BR, et al. 2004. Retardation of atherosclerosis by

Yoshida T, Maulik N, Engelman RM, Ho YS, Magnenat JL, et al. 1997. Glutathione

Yoshizumi M, Fujita Y, Izawa Y, Suzaki Y, Kyaw M, et al. 2004. Ebselen inhibits tumor

molecule expression in endothelial cells. *Experimental Cell Research* 292:1-10 Yu Z, Shao W, Chiang Y, Foltz W, Zhang Z, et al. 2011. Oltipraz upregulates the nuclear

Zelmanovitz T, Gerchman F, Balthazar A, Thomazelli F, Matos J, Canani L. 2009. Diabetic

Zhou M-S, Schulman IH, Pagano PJ, Jaimes EA, Raij L. 2006. Reduced NAD(P)H Oxidase in

mice lacking apolipoprotein E. *Circulation Research* 95:1075-81

nephropathy. *Diabetology & Metabolic Syndrome* 1:10-26

Low Renin Hypertension. *Hypertension* 47:81-6

*and Circulatory Physiology* 294:H1658-H66

*Clin Invest* 115:733-8

54:922-34

injury. *Circulation* 96:II216-II20

functional vasodilation in obese Zucker rats. *American Journal of Physiology - Heart* 

inflammation by decreasing thioredoxin-interacting protein in endothelial cells. *J* 

overexpression of catalase or both Cu/Zn-superoxide dismutase and catalase in

peroxidase knockout mice are susceptible to myocardial ischemia reperfusion

necrosis factor-[alpha]-induced c-Jun N-terminal kinase activation and adhesion

respiratory factor 2 alpha subunit (NRF2) antioxidant system and prevents insulin resistance and obesity induced by a high-fat diet in C57BL/6J mice. *Diabetologia*

Studies on the efficacy of antioxidant treatment in type 1 diabetes mellitus is an interesting, actual research subject. Reactive oxygen species (ROS) are continuously produced and eliminated by living organisms normally maintaining ROS at certain steady-state levels. Under some circumstances, the balance between ROS generation and elimination is disturbed leading to enhanced ROS level causing oxidative stress Lushchak, 2011. Oxidative stress is involved in the development of several important diseases (cancer, ulcer, atherosclerosis, autoimmune diseases, ischaemia-reperfusion injury, emphysema, inflammation, etc.).

Oxidative stress due to increased production of reactive oxygen species and/or impaired antioxidant capacity of the body plays a special, important role in the development of type 1 diabetes mellitus and its complications Baynes, 1991, Giugliano et al., 1995, Krippeit-Drews et al., 1994, Nakazaki et al., 1995, Nomikos et al., 1989, Raes et al, 1995, Shinn, 1998.

Under normal conditions, oxidative tissue damage is prevented by enzymatic and nonenzymatic antioxidants. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) are three enzymes involved in detoxification of reactive oxygen species (superoxide radical, hydroxyl radical, and hydrogen peroxide). Pancreatic cells have a poor antioxidant defence system, so they are very vulnerable to oxidative stress, especially to superoxide mediated radical damage Dejica, 2000.

Evaluation of oxidative status can be made by various methods, most of them very sophisticated, requiring laboratories with modern equipment, and are used almost exclusively for research purposes.

<sup>\*</sup> V. Balogh-Sămărghiţan1, Elena Cristina Crăciun2, R. Morar3, Dana Liana Pusta3, Fazakas Zita1, Szőcs-Molnár Terézia4, Dunca Iulia4, Sánta Dóra5 and Minodora Dobreanu6

<sup>1</sup>*University of Medicine and Pharmacy, Tîrgu-Mureş, Medical Biochemistry Department, Romania 2 University of Medicine and Pharmacy "Iuliu Haţieganu", Cluj-Napoca, Pharmaceutical Biochemistry and Clinical Laboratory Department, Romania* 

*<sup>3</sup> Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania* 

*<sup>4</sup> II. Clinical Hospital of Pediatrics, Tîrgu-Mureş, Romania 5 Santa Medical Unit, Tîrgu-Mureş, Romania, 6 University of Medicine and Pharmacy, Tîrgu-Mureş, Clinical Biochemistry Department, Romania*

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 283

every prooxidant is necessary a toxic compound. Antioxidants are substances that, in low concentrations compared to an oxidizable substrate, prevent or delay oxidation initiated by

Several vitamins are well known for their antioxidant properties: vitamin C exhibits its effect in hydrophilic environment, while vitamin A and E protect especially the cell membranes, and act in hydrophobic phase. Many other, more complex antioxidant

**3. Implication of oxidative stress and glycation process in diabetes mellitus**  In type 2 diabetic patients, besides the relative insulin deficiency, there is a certain grade of insulin resistance. The relationship between reactive oxygen species and the effect of insulin has been studied, and the results showed that in elderly people, presenting intense exposure to oxidative stress, the ratio between GSH/GSSG is reduced, leading to intensified lipoperoxidation. This phenomenon might exhibit a negative influence on the integrity of plasma membranes, leading to their disfunction, regarding for instance the transmembrane glucose transport. There is a high probability that the periferal action of insulin is disturbed by the negative effect of reactive oxygen species on membrane ATP-ase activity Dejica, 2000.

Peroxidation of lipids (especially LDL) plays an important role in inducing macrovascular lesions found in both diabetes and in atherosclerotic disease. Susceptibility to oxidation of lipoproteins seems to be a key element in the initiation and propagation of the atherogenic process Dobreanu M., Módy, 1998. In diabetic patients, lipid oxidation affects circulating lipids, and also those present in cell membranes or myelin layers. Hyperglycemia, excessive autooxidation and decreased antioxidant capacity registered in diabetic patients are

The speed of non-enzymatic glycation is proportional to the blood sugar level. Glucose (and fructose and galactose) are attached to the extreme N-terminal peptide chain, initially forming a Schiff base (aldimine), which is unstable, then by Amadori rearrangement a stable cetoimine is formed, and advanced glycation end products, leading to alterations in

Amadori-products can also be oxidized, by reactions catalyzed by transition metals, releasing eritronic acid and forming carboxymethylated lysine. Its level is double in the collagen of the skin of diabetic patients compared to non-diabetic subjects, and it is positively correlated with the presence of retino- and nephropathy in diabetic patients

All structural proteins and those circulating in the body can suffer non-enzymatic glycation processes. They can alter protein structure and function of vessels, nerves, liver, skin and other organs. Glycosylation of LDL lipoprotein particles decreases their catabolism and accelerates HDL catabolism, disorders that may explain in part the modifications present in macroangiopathy. Glycosylated proteins are more susceptible to attack by reactive oxygen

Enzymatic glycosylation of proteins is also important in development of chronic diabetic complications. The collagen molecules are thus glycosylated, glycoproteins and

configuration, of the electronegative charge and molecular recognition of proteins.

a prooxidant Dejica, 2001.

substances are found in plants.

responsible for intensified oxidative processes.

Wolff, 1993.

species.

Exploring the body oxidative status can be made through the following ways:

	- lipid compounds conjugated dienes, hydroperoxides, aldehydes (malondialdehyde, hydroxynonenal)
	- ethane and pentane measurements in expired gases
	- DNA beta-hydroxydeoxyguanosine
	- protein derivatives (carbonyl or thiol groups)
	- amino acids methionine sulfoxide, ortho-tyrosine, dityrosine, nitrotyrosine, chlorotyrosine.

#### **2. Antioxidants**

Determination of trace elements is also of interest because some are in the active center of antioxidant enzymes: for example Cu, Zn and Mn are found in the structure of SOD, and Se is present in the active center of GPX, but also possesses antioxidant effect independent of this enzyme Olinescu, 1994.

The variety of antioxidant substances in the body, the difficulty of measuring their individual level and the interactions between them require methods for measuring total antioxidant capacity (TAOC) in different biological samples Cadenas & Packer, 2002.

By measuring TAOC one can assess not only the interaction effects of antioxidants known, but also the antioxidant action of unidentified components present in human plasma. In most methods uric acid is the major contributor to the TAOC of plasma, so increasing plasma levels of uric acid can mask the depletion of ascorbate or other antioxidants in some pathological conditions if only TAOC measurement is carried out.

TAOC are methods used to measure indirect inhibition involving a prooxidant (typically a free radical) and an oxidizable substrate. The prooxidant induces oxidative deterioration of the substrate, which is inhibited in the presence of the antioxidant.

Antioxidant capacity can be defined as the ability of a compound to reduce prooxidants. In biological systems, prooxidants are usually defined as toxic substances causing oxidative damage to lipids, proteins, nucleic acids, leading to a variety of pathological events, but not

1. Free radical measurement by absorption spectroscopy with electronic spin resonance

2. Measurement of chemical uptake (chemical trapping) by quantitative determination of the elimination of specific derivatives of salicylic acid, hydroxylated or nitrosylated

3. Measuring the antioxidant capacity of each antioxidant in part or total plasma

4. Determination of antioxidant enzyme activites (SOD, CAT, GPX) and non-enzymatic antioxidants (tocopherols and tocotrienols, vitamin C, ubiquinone, glutathione, carotenoids and vitamin A, bilirubin, melatonin, uric acid, ceruloplasmin, vitamin K,



6. Measurement of the antioxidants / oxidizing substances ratio (eg. ascorbic acid / dehydroascorbic acid and reduced glutathione / oxidized glutathione) Prior & Cao,

Determination of trace elements is also of interest because some are in the active center of antioxidant enzymes: for example Cu, Zn and Mn are found in the structure of SOD, and Se is present in the active center of GPX, but also possesses antioxidant effect independent of

The variety of antioxidant substances in the body, the difficulty of measuring their individual level and the interactions between them require methods for measuring total antioxidant capacity (TAOC) in different biological samples Cadenas & Packer, 2002.

By measuring TAOC one can assess not only the interaction effects of antioxidants known, but also the antioxidant action of unidentified components present in human plasma. In most methods uric acid is the major contributor to the TAOC of plasma, so increasing plasma levels of uric acid can mask the depletion of ascorbate or other antioxidants in some

TAOC are methods used to measure indirect inhibition involving a prooxidant (typically a free radical) and an oxidizable substrate. The prooxidant induces oxidative deterioration of

Antioxidant capacity can be defined as the ability of a compound to reduce prooxidants. In biological systems, prooxidants are usually defined as toxic substances causing oxidative damage to lipids, proteins, nucleic acids, leading to a variety of pathological events, but not

5. The measurement of biological compounds resulting from oxidative processes:


pathological conditions if only TAOC measurement is carried out.

the substrate, which is inhibited in the presence of the antioxidant.


Exploring the body oxidative status can be made through the following ways:

(ESR) and electronic paramagnetic resonance (EPR) Olinescu, 1994.

compounds.

antioxidant capacity.

lipoic acid) Dejica, 2000, 2001.


hydroxynonenal)

chlorotyrosine.

this enzyme Olinescu, 1994.

1999.

**2. Antioxidants** 

every prooxidant is necessary a toxic compound. Antioxidants are substances that, in low concentrations compared to an oxidizable substrate, prevent or delay oxidation initiated by a prooxidant Dejica, 2001.

Several vitamins are well known for their antioxidant properties: vitamin C exhibits its effect in hydrophilic environment, while vitamin A and E protect especially the cell membranes, and act in hydrophobic phase. Many other, more complex antioxidant substances are found in plants.

#### **3. Implication of oxidative stress and glycation process in diabetes mellitus**

In type 2 diabetic patients, besides the relative insulin deficiency, there is a certain grade of insulin resistance. The relationship between reactive oxygen species and the effect of insulin has been studied, and the results showed that in elderly people, presenting intense exposure to oxidative stress, the ratio between GSH/GSSG is reduced, leading to intensified lipoperoxidation. This phenomenon might exhibit a negative influence on the integrity of plasma membranes, leading to their disfunction, regarding for instance the transmembrane glucose transport. There is a high probability that the periferal action of insulin is disturbed by the negative effect of reactive oxygen species on membrane ATP-ase activity Dejica, 2000.

Peroxidation of lipids (especially LDL) plays an important role in inducing macrovascular lesions found in both diabetes and in atherosclerotic disease. Susceptibility to oxidation of lipoproteins seems to be a key element in the initiation and propagation of the atherogenic process Dobreanu M., Módy, 1998. In diabetic patients, lipid oxidation affects circulating lipids, and also those present in cell membranes or myelin layers. Hyperglycemia, excessive autooxidation and decreased antioxidant capacity registered in diabetic patients are responsible for intensified oxidative processes.

The speed of non-enzymatic glycation is proportional to the blood sugar level. Glucose (and fructose and galactose) are attached to the extreme N-terminal peptide chain, initially forming a Schiff base (aldimine), which is unstable, then by Amadori rearrangement a stable cetoimine is formed, and advanced glycation end products, leading to alterations in configuration, of the electronegative charge and molecular recognition of proteins.

Amadori-products can also be oxidized, by reactions catalyzed by transition metals, releasing eritronic acid and forming carboxymethylated lysine. Its level is double in the collagen of the skin of diabetic patients compared to non-diabetic subjects, and it is positively correlated with the presence of retino- and nephropathy in diabetic patients Wolff, 1993.

All structural proteins and those circulating in the body can suffer non-enzymatic glycation processes. They can alter protein structure and function of vessels, nerves, liver, skin and other organs. Glycosylation of LDL lipoprotein particles decreases their catabolism and accelerates HDL catabolism, disorders that may explain in part the modifications present in macroangiopathy. Glycosylated proteins are more susceptible to attack by reactive oxygen species.

Enzymatic glycosylation of proteins is also important in development of chronic diabetic complications. The collagen molecules are thus glycosylated, glycoproteins and

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 285

0.5 g/group/day), the 4th group was treated with Eridiarom® (1.2 g/group/day), and the fifth group was given Diavit® (1.2 g/group/day). Healthy, normally fed rats formed the

After 5 days, and than each month glycaemia was measured, and after 100 days histopathologic examination (pancreas, liver, kidney, heart, muscles, eyes) were performed

During the experiment one animal in each treated group died, and in the group treated with

The dynamics of glycaemia increases at 7 days after the induction of subclinical diabetes (176.8 – 185.5 mg/dl +/- 3.0 – 5.6 SD) in each of the streptozotocin-treated groups compared

After 30 days the glycaemia is close to normal (120.1 – 127.8 mg/dl +/- 2.0 – 2.1 SD) in groups 4 and treated with the phytotherapeutic products Eridiarom® and Diavit®5 (14.6- 22.6% higher than the initial values), and it is high (151.6 mg/dl +/- 4.0 – 4.1 SD) in groups 2 and 3 treated with the antidiabetic sulphamides Siofor® and Meguan® (45.46% higher than the initial values), but lower compared to the first, witness group (164.6 mg/dl +/- 4.2 SD). After 90 days the glycaemia is practically normalized in groups 4 and 5 (102.8 – 114.3 mg/dl +/- 1.5 -2.3 SD) (the final value 1.37% lower, and 9.6% higher than the initial values) and it is slightly increased in the first 3 groups (135.0 – 133.3 mg/dl +/- 3.3 – 4.0 mg/dl) (29,1-27.9%

The histopathologic examination of the pancreas (Trichromic stain) revealed a strong

In the first witness group we can observe pancreatic cytonecrosis, atrophy with hypofunction and disturbing of the ratio between and secretory cells after administering streptozotocin. 100 days later the Langerhans islets of the rats in the first, witness group present partial regeneration and recovery of pseudolobules with cells, general interstitial oedema, atrophy

In group 2, treated with Siofor®, 100 days after the beginning of the experiment partial recovery can be observed in the Langerhans islets, with cells during the pseudolobules' reconstruction, and partial recovery of the exocrine secretor function by acinary and lobular

In group 3 treated with Meguan®, 100 days after the initiation of the experiment regenerated Langerhans islets can be seen with and cells' reorganisation and the formation of sinusoidal capillary, and acinus-lobulary hyperfunction with the hypertrophy

In group 4, treated with Eridiarom®, 100 days after the beginning of the experiment, Langerhans islets with regenerated and cells can be observed, with increased mitotic index and complete recovery, and generalised acinus-lobulary functional hypertrophy.

In group 5, treated with Diavit®, 100 days after the initiation of the experiment, Langerhans islets with complete recovery of the cell-architectonics can be observed, specific for normal

destructive action of streptozotocin against the endocrine and exocrine pancreas.

of the acini's level with the significant reducing of the cellular secretory pole.

on 2 animals of each group, and the rest of the rats were kept under observation.

6th, non streptozotocin-treated witness group.

to the initial values (103.2 – 105.3 mg/dl +/- 1.6 – 2.2 SD).

Siofor two animals died.

higher than the initial values).

of the secretor pole in pancreatic acini.

hyperfunction.

proteoglycans suffer similar processes. At the level of nerves, lens, livers or other organs, glycosylation of proteins is involved in the occurrence of diabetic neuropathy, cataracts, dysmetabolic hepatopathy Gherasim, 1998, Kovács, 2001.

Based on these evidences, antioxidant treatment is a promising approach for complementary therapy in diabetes mellitus. Several phytotherapeutical products contain a complexity of free radical scavengers and exhibit no side effects on long term treatment.

#### **4. Evaluation of oxidative stress and the efficacy of antioxidant treatment in diabetes mellitus**

#### **4.1. Studies on animals**

Some of the plants known for their high antioxidant power are: Allium sativa, Ricinus communis, Securinega virosa, Viscum album, pomegranates, berries (including strawberries, blueberries, and raspberries), walnuts, sunflower seeds, ginger and several plants of Indian traditional medicine: Emblica officinalis L., Curcuma longa L., Mangifera indica L., Momordica charantia L., Santalum album L, Swertia chirata Buch-Ham, Withania somnifera and Cassia auriculata Capasso et al, 2003, Dejica, 2001, Moshi & Mbwambo, 2003, Pari & Latha, 2003, Pietta, 1998, Scartezzini & Speroni, 2000, Varga et al., 2001.

Our studies carried out in streptozotocin diabetic rats demonstrated that treatment with the blueberry (Eridiarom®) or blueberry and sea buckthorn concentrate (Diavit®) for two months had regenerative effect on pancreatic beta cells Crăciun et al., 2007, Morar et al., 2004.

The therapeutic properties of blueberry are attributed to its anthocyanosides which belong to a class of substances known as plant bioflavonoids. Pharmacologically, anthocyanosides are thought to decrease vascular permeability and improve microcirculation. They are also thought to have antioxidant activity.

Diavit® is a dietary supplement with a more complex composition compared to Eridiarom®, it contains quinolizidine alkaloids, anthocyanosides, sugars, carotenoids, vitamins (C, E, PP, B1, B2, folic acid), minerals (K, Ca, P, S, Mg, Cl, Mn, Fe), organic acids, flavonoids, etc..

Carotenoids are best recognized for their antioxidant capacity, they are considered the most potent biological quenchers of singlet oxygen Morar, 2003, Paiva & Russell, 1999, Pizzorno & Murray, 2003, Rombi, 1998, Slosse & Hootele, 1981, Timberlake & Henry, 1988, Verette, 1984, Zeb, 2004.

The experiment was carried out on 5 groups of male Wistar rats, the first five group received Streptozotocin i.v. 4 mg/100 g body weight to induce diabetes.

The toxicity of Streptozotocin can be counteracted by desferioxamine, suggesting that oxidative reactions catalyzed by transition metals could be responsible for the toxicity of this substance. Streptozotocin, being one of the glico-nitrosureas, could exhibit its diabetesinducing effect also by inadequate NO release Giugliano et al., 1995.

The first group served as a diabetic witness, the second group was treated with Siofor® ½ tablet/day/group (equivalent to 12 mg/kg), the third group received Meguan® (2 tablets of

proteoglycans suffer similar processes. At the level of nerves, lens, livers or other organs, glycosylation of proteins is involved in the occurrence of diabetic neuropathy, cataracts,

Based on these evidences, antioxidant treatment is a promising approach for complementary therapy in diabetes mellitus. Several phytotherapeutical products contain a complexity of free radical scavengers and exhibit no side effects on long term treatment.

**4. Evaluation of oxidative stress and the efficacy of antioxidant treatment in** 

Some of the plants known for their high antioxidant power are: Allium sativa, Ricinus communis, Securinega virosa, Viscum album, pomegranates, berries (including strawberries, blueberries, and raspberries), walnuts, sunflower seeds, ginger and several plants of Indian traditional medicine: Emblica officinalis L., Curcuma longa L., Mangifera indica L., Momordica charantia L., Santalum album L, Swertia chirata Buch-Ham, Withania somnifera and Cassia auriculata Capasso et al, 2003, Dejica, 2001, Moshi & Mbwambo, 2003, Pari & Latha, 2003, Pietta, 1998, Scartezzini & Speroni, 2000, Varga et al., 2001. Our studies carried out in streptozotocin diabetic rats demonstrated that treatment with the blueberry (Eridiarom®) or blueberry and sea buckthorn concentrate (Diavit®) for two months had regenerative effect on pancreatic beta cells Crăciun et al., 2007, Morar et al.,

The therapeutic properties of blueberry are attributed to its anthocyanosides which belong to a class of substances known as plant bioflavonoids. Pharmacologically, anthocyanosides are thought to decrease vascular permeability and improve microcirculation. They are also

Diavit® is a dietary supplement with a more complex composition compared to Eridiarom®, it contains quinolizidine alkaloids, anthocyanosides, sugars, carotenoids, vitamins (C, E, PP, B1, B2, folic acid), minerals (K, Ca, P, S, Mg, Cl, Mn, Fe), organic acids,

Carotenoids are best recognized for their antioxidant capacity, they are considered the most potent biological quenchers of singlet oxygen Morar, 2003, Paiva & Russell, 1999, Pizzorno & Murray, 2003, Rombi, 1998, Slosse & Hootele, 1981, Timberlake & Henry,

The experiment was carried out on 5 groups of male Wistar rats, the first five group received

The toxicity of Streptozotocin can be counteracted by desferioxamine, suggesting that oxidative reactions catalyzed by transition metals could be responsible for the toxicity of this substance. Streptozotocin, being one of the glico-nitrosureas, could exhibit its diabetes-

The first group served as a diabetic witness, the second group was treated with Siofor® ½ tablet/day/group (equivalent to 12 mg/kg), the third group received Meguan® (2 tablets of

Streptozotocin i.v. 4 mg/100 g body weight to induce diabetes.

inducing effect also by inadequate NO release Giugliano et al., 1995.

dysmetabolic hepatopathy Gherasim, 1998, Kovács, 2001.

**diabetes mellitus** 

2004.

flavonoids, etc..

**4.1. Studies on animals** 

thought to have antioxidant activity.

1988, Verette, 1984, Zeb, 2004.

0.5 g/group/day), the 4th group was treated with Eridiarom® (1.2 g/group/day), and the fifth group was given Diavit® (1.2 g/group/day). Healthy, normally fed rats formed the 6th, non streptozotocin-treated witness group.

After 5 days, and than each month glycaemia was measured, and after 100 days histopathologic examination (pancreas, liver, kidney, heart, muscles, eyes) were performed on 2 animals of each group, and the rest of the rats were kept under observation.

During the experiment one animal in each treated group died, and in the group treated with Siofor two animals died.

The dynamics of glycaemia increases at 7 days after the induction of subclinical diabetes (176.8 – 185.5 mg/dl +/- 3.0 – 5.6 SD) in each of the streptozotocin-treated groups compared to the initial values (103.2 – 105.3 mg/dl +/- 1.6 – 2.2 SD).

After 30 days the glycaemia is close to normal (120.1 – 127.8 mg/dl +/- 2.0 – 2.1 SD) in groups 4 and treated with the phytotherapeutic products Eridiarom® and Diavit®5 (14.6- 22.6% higher than the initial values), and it is high (151.6 mg/dl +/- 4.0 – 4.1 SD) in groups 2 and 3 treated with the antidiabetic sulphamides Siofor® and Meguan® (45.46% higher than the initial values), but lower compared to the first, witness group (164.6 mg/dl +/- 4.2 SD). After 90 days the glycaemia is practically normalized in groups 4 and 5 (102.8 – 114.3 mg/dl +/- 1.5 -2.3 SD) (the final value 1.37% lower, and 9.6% higher than the initial values) and it is slightly increased in the first 3 groups (135.0 – 133.3 mg/dl +/- 3.3 – 4.0 mg/dl) (29,1-27.9% higher than the initial values).

The histopathologic examination of the pancreas (Trichromic stain) revealed a strong destructive action of streptozotocin against the endocrine and exocrine pancreas.

In the first witness group we can observe pancreatic cytonecrosis, atrophy with hypofunction and disturbing of the ratio between and secretory cells after administering streptozotocin. 100 days later the Langerhans islets of the rats in the first, witness group present partial regeneration and recovery of pseudolobules with cells, general interstitial oedema, atrophy of the acini's level with the significant reducing of the cellular secretory pole.

In group 2, treated with Siofor®, 100 days after the beginning of the experiment partial recovery can be observed in the Langerhans islets, with cells during the pseudolobules' reconstruction, and partial recovery of the exocrine secretor function by acinary and lobular hyperfunction.

In group 3 treated with Meguan®, 100 days after the initiation of the experiment regenerated Langerhans islets can be seen with and cells' reorganisation and the formation of sinusoidal capillary, and acinus-lobulary hyperfunction with the hypertrophy of the secretor pole in pancreatic acini.

In group 4, treated with Eridiarom®, 100 days after the beginning of the experiment, Langerhans islets with regenerated and cells can be observed, with increased mitotic index and complete recovery, and generalised acinus-lobulary functional hypertrophy.

In group 5, treated with Diavit®, 100 days after the initiation of the experiment, Langerhans islets with complete recovery of the cell-architectonics can be observed, specific for normal

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 287

We obtained good correlation of the results provided by the three methods: r = 0.8087, P = 0.0151 comparing LPO-586 method with Fogelman procedure, r = 0.6580, P = 0.0007 comparing Fogelman method with the procedure described by Kei Satoh, and r = 0.9085, p = 0.0007 comparing the LPO-586 method with Kei Satoh procedure Nemes-Nagy, 2004a.

In the diabetic children's group the average value of HbA1c was 10.0% +/- 1.9 (SD) (range 6.7 – 15.4 %), and the average malondialdehyde concentration was 3.7 nmol/ml +/- 1.04 (SD) (range 1.7 – 8.1 nmol/ml), and the MDA concentration was significantly higher (P0.001) compared to the average value of children presenting glucose intolerance (2.6

HbA1c level highly correlated with malondialdehyde concentration (r = 0.814, P<0.05) in the

HbA1c was determined by chromatographic method using venous blood samples collected

Fig. 2. Correlation between glycated hemoglobin level and serum malondialdehyde

We also determined serum malondialdehyde concentration in three different groups of type 2 diabetic patients: subjects presenting cardiovascular (CV) diseases, patients having a high risk to develop such diseases, and those who had no other cardiovascular risk factor besides

nmol/ml +/- 1.1 SD).

diabetic children's group (60 patients).

concentration in type 1 diabetic children

suffering from diabetes mellitus.

on EDTA-K2 as anticoagulant.

functioning Langerhans cells, and hyperthrophy with general acinus-lobulary moderate secretion.

Based on these experimental data we can conclude that the two phytotherapeutic products exhibited a powerful regenerative effect on the pancreatic cells, presenting a better efficacy compared to the widely used antidiabetic sulphamides Crăciun et al., 2007, Morar et al., 2004.

#### **4.2 Studies on human subjects**

In our research in humans we evaluated oxidative stress using several methods, measuring lipid peroxidation products in type 1 diabetic patients compared to non-diabetic subjects of the same age-group. We used the LPO 586 (R&D Systems) kit and two methods based on the reaction between malondialdehyde and thiobarbituric acid Nemes-Nagy et al., 2004a, Satoh, 1978.

After the incubation period, malondialdehyde concentration was determined by photometric dosage, in case of some methods comparing the results obtained for the samples with those for the reference series with known concentrations, previously prepared.

We revealed that oxidative stress was more intense in diabetic children (78 patients, aged 12.8 years +/- 4.2 SD) compared to healthy subjects of their age group (P=0.0022).

Fig. 1. Malondialdehyde concentration in diabetic and non-diabetic patients by Kei Satoh method

functioning Langerhans cells, and hyperthrophy with general acinus-lobulary moderate

Based on these experimental data we can conclude that the two phytotherapeutic products exhibited a powerful regenerative effect on the pancreatic cells, presenting a better efficacy compared to the widely used antidiabetic sulphamides Crăciun et al., 2007, Morar et al.,

In our research in humans we evaluated oxidative stress using several methods, measuring lipid peroxidation products in type 1 diabetic patients compared to non-diabetic subjects of the same age-group. We used the LPO 586 (R&D Systems) kit and two methods based on the reaction between malondialdehyde and thiobarbituric acid Nemes-Nagy et al., 2004a,

After the incubation period, malondialdehyde concentration was determined by photometric dosage, in case of some methods comparing the results obtained for the samples with those for the reference series with known concentrations, previously prepared. We revealed that oxidative stress was more intense in diabetic children (78 patients, aged

12.8 years +/- 4.2 SD) compared to healthy subjects of their age group (P=0.0022).

Fig. 1. Malondialdehyde concentration in diabetic and non-diabetic patients by Kei Satoh

secretion.

2004.

Satoh, 1978.

method

**4.2 Studies on human subjects** 

We obtained good correlation of the results provided by the three methods: r = 0.8087, P = 0.0151 comparing LPO-586 method with Fogelman procedure, r = 0.6580, P = 0.0007 comparing Fogelman method with the procedure described by Kei Satoh, and r = 0.9085, p = 0.0007 comparing the LPO-586 method with Kei Satoh procedure Nemes-Nagy, 2004a.

In the diabetic children's group the average value of HbA1c was 10.0% +/- 1.9 (SD) (range 6.7 – 15.4 %), and the average malondialdehyde concentration was 3.7 nmol/ml +/- 1.04 (SD) (range 1.7 – 8.1 nmol/ml), and the MDA concentration was significantly higher (P0.001) compared to the average value of children presenting glucose intolerance (2.6 nmol/ml +/- 1.1 SD).

HbA1c level highly correlated with malondialdehyde concentration (r = 0.814, P<0.05) in the diabetic children's group (60 patients).

HbA1c was determined by chromatographic method using venous blood samples collected on EDTA-K2 as anticoagulant.

Fig. 2. Correlation between glycated hemoglobin level and serum malondialdehyde concentration in type 1 diabetic children

We also determined serum malondialdehyde concentration in three different groups of type 2 diabetic patients: subjects presenting cardiovascular (CV) diseases, patients having a high risk to develop such diseases, and those who had no other cardiovascular risk factor besides suffering from diabetes mellitus.

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 289

Our research team demonstrated the powerful antioxidant and hypoglycemic effect of a blueberry (*Vaccinium myrtillus*) concentrate (Eridiarom®) in diabetic children (initially this product was used for treatment of diarrhoea in humans). We selected 29 infants presenting

Fig. 4. Erythrocyte Cu-Zn superoxide dismutase activity in type 1 diabetic children

Fig. 5. Whole blood glutathione peroxidase dismutase activity in type 1 diabetic children

poor carbohydrate metabolic balance to participate to the study.

We obtained significant differences between the first and third (P = 0.0015), and the second and the third group (P = 0.0018) concerning malondialdehyde concentration, the patients in the third group having lower levels Moldován, 2003 compared to those from the first two groups.

Fig. 3. Average values of serum malondialdehyde concentration in different cardiovascular risk groups of type 2 diabetic patients

We compared Cu/Zn SOD and GPX activities in diabetic children and a non-diabetic infant group of similar age. In diabetic infants we found significantly lower SOD activities (1200.8 U/gHb 101.4 SD) compared to the control group (1404.9 U/gHb +/- 125.4 SD), the difference is significant (P<0.005). No notable differences could be observed regarding GPX activity in the 2 studied groups of patients (P>0.05).

According to the literature normal values of erythrocyte SOD are considered between 1102- 1601 U/gHb. In our diabetic group only 16,7% of the patients presented values lower than normal, the minimum SOD activity value was 1014.2 U/gHb and the maximum was 1386 U/gHb.

Physiological values of GPX activity are between 27.5-73.6 U/gHb, only one of the studied diabetic children exhibited a value slightly lower than normal Jákó et al, 2009.

We obtained significant differences between the first and third (P = 0.0015), and the second and the third group (P = 0.0018) concerning malondialdehyde concentration, the patients in the third group having lower levels Moldován, 2003 compared to those from the first two

Fig. 3. Average values of serum malondialdehyde concentration in different cardiovascular

We compared Cu/Zn SOD and GPX activities in diabetic children and a non-diabetic infant group of similar age. In diabetic infants we found significantly lower SOD activities (1200.8 U/gHb 101.4 SD) compared to the control group (1404.9 U/gHb +/- 125.4 SD), the difference is significant (P<0.005). No notable differences could be observed regarding GPX

According to the literature normal values of erythrocyte SOD are considered between 1102- 1601 U/gHb. In our diabetic group only 16,7% of the patients presented values lower than normal, the minimum SOD activity value was 1014.2 U/gHb and the maximum was 1386

Physiological values of GPX activity are between 27.5-73.6 U/gHb, only one of the studied

diabetic children exhibited a value slightly lower than normal Jákó et al, 2009.

risk groups of type 2 diabetic patients

U/gHb.

activity in the 2 studied groups of patients (P>0.05).

groups.

Our research team demonstrated the powerful antioxidant and hypoglycemic effect of a blueberry (*Vaccinium myrtillus*) concentrate (Eridiarom®) in diabetic children (initially this product was used for treatment of diarrhoea in humans). We selected 29 infants presenting poor carbohydrate metabolic balance to participate to the study.

Fig. 4. Erythrocyte Cu-Zn superoxide dismutase activity in type 1 diabetic children

Fig. 5. Whole blood glutathione peroxidase dismutase activity in type 1 diabetic children

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 291

The average MDA concentration before the study was 6.7 nmol/ml 0.7 (SD), after 3 months of treatment the average value was 4.5 nmol/ml 0.8 (SD), the difference is

Prior to the study the serum magnesium and calcium concentrations were under the normal range in 43% of the patients. The average magnesium level at the beginning of the study was 1.7 mg/dl, while after 3 months of Eridiarom® treatment in case of all these patients the magnesium level turned to normal, the average value being 2.2 mg/dl; the difference is

significant (P0.0001) Balogh-Sămărghiţan et al., 2004, Nemes-Nagy et al., 2006.

Fig. 7. Dynamics of serum magnesium concentration under blueberry containing

Several studies were made on the implications of magnesium in diabetes mellitus. Certain key enzymes of carbohydrate metabolism (glucokinase, hexokinase, glucose-6-phosphate dehydrogenase) and lipid metabolism (mevalonate kinase, lecithin-cholesterol acyl-

Insulin, along with catecholamines, has major effects on intracellular homeostasis of magnesium, being, besides vitamin D and taurine, an important magnesium-linking substance. Insulin receptors exhibit a magnesium-dependent kinase activity Vereşiu, 2000. Studies evaluating magnesium levels in diabetic patients compared to healthy controls showed decreases in case of diabetic subjects, and other studies have shown an increased risk for this disease in patients with magnesium deficiency. Possible correlation of Mg with chronic diabetes complications have been the subject of other studies, that have found lower values in patients with retinopathy De Valk, 1999 and neuropathy DeLourdes, 1998.

significant (P0.0001).

Eridiarom® treatment

transferase) are magnesium-dependent.

The average glycemic level at the beginning of the study was 179.4 mg/dl 39.2 (SD), after 3 months of Eridiarom® treatment the value decreased to 159.1 mg/dl +/- 40.7 (SD), the difference is significant (P<0.005).

Insulin doses (UI/kg) could be lowered in 78.6% of the patients. Initially the average dose was 0.98 UI/kg 0.2 (SD) and after 3 months of treatment 0.91 UI/kg 0.2 (SD), the difference is significant (P<0.05). This result can be explained by the regenerative effect of this phytotherapeutic product on the pancreatic beta cells, improving their insulin secretion.

We observed that the longer the treatment with this dietary supplement, better the results are.

After 3 months of Eridiarom® treatment HbA1c values presented decrease in 71.43% of the patients. Using the Student paired t test, we obtained significant differences (P<0.05) regarding the HbA1c values at the beginning of the study (9.6% 1.6 SD) and 8.5% 1.5 (SD) after 3 months of Eridiarom® treatment.

After 3 months of Eridiarom® treatment MDA concentration decreased in 92.9% of the patients, 7.1% showed practically no modification of the MDA level.

Fig. 6. Serum MDA concentration before and after Eridiarom® treatment

The average glycemic level at the beginning of the study was 179.4 mg/dl 39.2 (SD), after 3 months of Eridiarom® treatment the value decreased to 159.1 mg/dl +/- 40.7 (SD), the

Insulin doses (UI/kg) could be lowered in 78.6% of the patients. Initially the average dose was 0.98 UI/kg 0.2 (SD) and after 3 months of treatment 0.91 UI/kg 0.2 (SD), the difference is significant (P<0.05). This result can be explained by the regenerative effect of this phytotherapeutic product on the pancreatic beta cells, improving their insulin secretion. We observed that the longer the treatment with this dietary supplement, better the results

After 3 months of Eridiarom® treatment HbA1c values presented decrease in 71.43% of the patients. Using the Student paired t test, we obtained significant differences (P<0.05) regarding the HbA1c values at the beginning of the study (9.6% 1.6 SD) and 8.5% 1.5

After 3 months of Eridiarom® treatment MDA concentration decreased in 92.9% of the

patients, 7.1% showed practically no modification of the MDA level.

Fig. 6. Serum MDA concentration before and after Eridiarom® treatment

difference is significant (P<0.005).

(SD) after 3 months of Eridiarom® treatment.

are.

The average MDA concentration before the study was 6.7 nmol/ml 0.7 (SD), after 3 months of treatment the average value was 4.5 nmol/ml 0.8 (SD), the difference is significant (P0.0001).

Prior to the study the serum magnesium and calcium concentrations were under the normal range in 43% of the patients. The average magnesium level at the beginning of the study was 1.7 mg/dl, while after 3 months of Eridiarom® treatment in case of all these patients the magnesium level turned to normal, the average value being 2.2 mg/dl; the difference is significant (P0.0001) Balogh-Sămărghiţan et al., 2004, Nemes-Nagy et al., 2006.

Several studies were made on the implications of magnesium in diabetes mellitus. Certain key enzymes of carbohydrate metabolism (glucokinase, hexokinase, glucose-6-phosphate dehydrogenase) and lipid metabolism (mevalonate kinase, lecithin-cholesterol acyltransferase) are magnesium-dependent.

Insulin, along with catecholamines, has major effects on intracellular homeostasis of magnesium, being, besides vitamin D and taurine, an important magnesium-linking substance. Insulin receptors exhibit a magnesium-dependent kinase activity Vereşiu, 2000.

Studies evaluating magnesium levels in diabetic patients compared to healthy controls showed decreases in case of diabetic subjects, and other studies have shown an increased risk for this disease in patients with magnesium deficiency. Possible correlation of Mg with chronic diabetes complications have been the subject of other studies, that have found lower values in patients with retinopathy De Valk, 1999 and neuropathy DeLourdes, 1998.

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 293

Hydrogen peroxide, formed by the reaction catalyzed by SOD, is decomposed by two enzymes: GPX and CAT. Dosage of blood catalase activity could have helped to have a better picture on the enzymatic antioxidant equipment of these diabetic patients under the

HbA1C levels were significantly lower after treatment with the dietary supplement (9.2 1.6 % versus 4the initial 10.2 2.3 %; P<0.05) and C-peptide average value increased significantly (P<0.05) after 2 month of treatment with this dietary supplement (0.2 ng/ml 0.1 SD) compared to the initial average value (0.04 ng/ml 0.02 SD). Insulin requirement reduced significantly from the average of 0.96 0.27 IU/kg bodyweight to 0.89 0.28 IU/kg after 2 months of treatment with the product (P<0.05), insulin doses were reduced in 66.7%

Lower blood glucose and HbA1c values may be due to a regenerative effect that the product has on pancreatic beta cells. Significantly higher C-peptide levels after 2 months of treatment

A scientific team in Harvard University, Howard Hughes Medical Institute, under the leadership of Prof. Douglas Melton, published their findings about the capacity of pancreatic beta cells to regenerate by self-duplication due to some latent embrional cell remains or adult stem cells Zhou et al., 2008, and this regenerative process might explain our results with the product studied. We suppose that better results could be obtained if the treatment with the concentrate begins soon after the diabetic disease is diagnosed, maybe because long term insulin treatment causes the atrophy of pancreatic beta-cells, similar to corticosteroid-caused hypofunction of corticosuprarenals. This hypothesis should be verified in latter studies. Based on our experimental data it seems that the longer the

Hyperglycemia in diabetes mellitus produces increased oxidative stress via non-enzymatic glycation, glucose autooxidation, and alteration in polyol pathway activity. This is characterized by increased lipid peroxide production and decreased antioxidative defence (e.g. inactivation of SOD by glycation) which affects the entire body. Two months using Diavit® lead to a significant increase in SOD which may have occurred as a result of its antioxidant and hypoglycemic effects. The antioxidant effect of this product might be partially due to anthocyanosides, known as scavengers of superoxide anions, inhibitors of lipid peroxidation. Lower glycaemic levels during the study might cause lower superoxide radical production and decrease the inactivation rate of this antioxidant enzyme, leading to

Protection of free radical scavengers might help to maintain higher levels of antioxidants under treatment with this concentrate, and several components of the product (carotenoids, vitamin E, C) show important protective role against oxidative stress. According to recent studies, it might be a link between GPX and ascorbate: intracellular vitamin C cooperates in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C is recycled through a mechanism involving

Several data suggest that oxygen metabolits are involved in the pathogenesis of autoimmune destruction of pancreatic beta cells, involving inflammatory process, and especially superoxide

the simultaneous neutralization of oxidant species Montecinos et al., 2007.

with the extract support this hypothesis Nemes-Nagy et al., 2008.

treatment with these dietary supplements, the better the results are.

treatment with this dietary supplement.

of the patients.

higher SOD levels than before.

Effects of magnesium administration in patients with diabetes to improve glycemic control and prevention of chronic complications are conflicting and awaiting further confirmation.

A few years later we studied the effect of another dietary supplement (Diavit®), containing blueberry (*Vaccinum myrtillus*) and sea buckthorn (*Hippophae rhamnoides*) concentrate with a complex composition previously presented.

We compared glycaemic profile, glycated hemoglobin (HbA1C) after 2 and 3 months of treatment, C peptide level and changes in antioxidant enzyme activity (Cu/Zn superoxide dismutase and glutation peroxidase) after two months of treatment with the Diavit® dietary supplement versus after placebo treatment.

Values for activity of erythrocyte Cu/Zn SOD, a scavenger of superoxide radicals, were significantly higher (P<0.05) in diabetic patients after two months of treatment with the concentrate (1260.9 66.9 U/g Hb) compared to those obtained before treatment (1201.6 105.6 U/g Hb).

There was no significant difference in GPX activity before (40.8 9.2 U/g Hb) and after the study (43.9 13.9 U/g Hb), only a slight, not significant increase could be observed (P>0.05) Capasso et al, 2003, Nemes-Nagy et al., 2007, 2008, 2010, Paglia & Valentine, 1967, Szőcs-Molnár et al., 2006.

Fig. 8. Dynamics of blood SOD activity under blueberry and sea buckthorn containing Diavit® treatment

Effects of magnesium administration in patients with diabetes to improve glycemic control and prevention of chronic complications are conflicting and awaiting further confirmation. A few years later we studied the effect of another dietary supplement (Diavit®), containing blueberry (*Vaccinum myrtillus*) and sea buckthorn (*Hippophae rhamnoides*) concentrate with a

We compared glycaemic profile, glycated hemoglobin (HbA1C) after 2 and 3 months of treatment, C peptide level and changes in antioxidant enzyme activity (Cu/Zn superoxide dismutase and glutation peroxidase) after two months of treatment with the Diavit® dietary

Values for activity of erythrocyte Cu/Zn SOD, a scavenger of superoxide radicals, were significantly higher (P<0.05) in diabetic patients after two months of treatment with the concentrate (1260.9 66.9 U/g Hb) compared to those obtained before treatment (1201.6

There was no significant difference in GPX activity before (40.8 9.2 U/g Hb) and after the study (43.9 13.9 U/g Hb), only a slight, not significant increase could be observed (P>0.05) Capasso et al, 2003, Nemes-Nagy et al., 2007, 2008, 2010, Paglia & Valentine, 1967,

Fig. 8. Dynamics of blood SOD activity under blueberry and sea buckthorn containing

complex composition previously presented.

supplement versus after placebo treatment.

105.6 U/g Hb).

Szőcs-Molnár et al., 2006.

Diavit® treatment

Hydrogen peroxide, formed by the reaction catalyzed by SOD, is decomposed by two enzymes: GPX and CAT. Dosage of blood catalase activity could have helped to have a better picture on the enzymatic antioxidant equipment of these diabetic patients under the treatment with this dietary supplement.

HbA1C levels were significantly lower after treatment with the dietary supplement (9.2 1.6 % versus 4the initial 10.2 2.3 %; P<0.05) and C-peptide average value increased significantly (P<0.05) after 2 month of treatment with this dietary supplement (0.2 ng/ml 0.1 SD) compared to the initial average value (0.04 ng/ml 0.02 SD). Insulin requirement reduced significantly from the average of 0.96 0.27 IU/kg bodyweight to 0.89 0.28 IU/kg after 2 months of treatment with the product (P<0.05), insulin doses were reduced in 66.7% of the patients.

Lower blood glucose and HbA1c values may be due to a regenerative effect that the product has on pancreatic beta cells. Significantly higher C-peptide levels after 2 months of treatment with the extract support this hypothesis Nemes-Nagy et al., 2008.

A scientific team in Harvard University, Howard Hughes Medical Institute, under the leadership of Prof. Douglas Melton, published their findings about the capacity of pancreatic beta cells to regenerate by self-duplication due to some latent embrional cell remains or adult stem cells Zhou et al., 2008, and this regenerative process might explain our results with the product studied. We suppose that better results could be obtained if the treatment with the concentrate begins soon after the diabetic disease is diagnosed, maybe because long term insulin treatment causes the atrophy of pancreatic beta-cells, similar to corticosteroid-caused hypofunction of corticosuprarenals. This hypothesis should be verified in latter studies. Based on our experimental data it seems that the longer the treatment with these dietary supplements, the better the results are.

Hyperglycemia in diabetes mellitus produces increased oxidative stress via non-enzymatic glycation, glucose autooxidation, and alteration in polyol pathway activity. This is characterized by increased lipid peroxide production and decreased antioxidative defence (e.g. inactivation of SOD by glycation) which affects the entire body. Two months using Diavit® lead to a significant increase in SOD which may have occurred as a result of its antioxidant and hypoglycemic effects. The antioxidant effect of this product might be partially due to anthocyanosides, known as scavengers of superoxide anions, inhibitors of lipid peroxidation. Lower glycaemic levels during the study might cause lower superoxide radical production and decrease the inactivation rate of this antioxidant enzyme, leading to higher SOD levels than before.

Protection of free radical scavengers might help to maintain higher levels of antioxidants under treatment with this concentrate, and several components of the product (carotenoids, vitamin E, C) show important protective role against oxidative stress. According to recent studies, it might be a link between GPX and ascorbate: intracellular vitamin C cooperates in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C is recycled through a mechanism involving the simultaneous neutralization of oxidant species Montecinos et al., 2007.

Several data suggest that oxygen metabolits are involved in the pathogenesis of autoimmune destruction of pancreatic beta cells, involving inflammatory process, and especially superoxide

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 295

folium, and the smallest amount of flavonoids was found in Phaseoli tegumen. The results

The highest concentration of malondialdehyde was found in Centauri herba (149 mmol/100g), smaller amounts were found in Myrtilli folium (146 mmol/100g), Mori folium (115 mmol/100g), Crataegi summitas (100 mmol/100g), Urticae herba (97 mmol/100g), Menthae piperitae (97 mmol/100g), Visci albae stipes (94 mmol/100g), Juglandis folium (89 mmol/100g), Millefolii flos (79 mmol/100g) and Phaseoli tegumen (73 mmol/100g). The antioxidant capacity of these plants is inversely proportional to their MDA contents, a measure of ROS-induced damage to lipids Nemes-Nagy et al., 2004,

**Medicinal teas Flavonoid content (g% hyperoside)** 

We measured, by photometric diclorphenol-indophenol method the vitamin C concentration in freshly squeezed juices and in those preserved, available in stores. As a result we found that lemon, orange and grapefruit contain the biggest ascorbate quantity, especially freshly squeezed (22.0 – 22.2 mg/dl) the same volume of juice contains higher

In case of orange juices, we compared the vitamin C content of 3 samples from juices sold in boxes from different firms (the results obtained were 20.5 mg/dl, 18.2 mg/dl and 15.0 mg/dl), so we concluded that the concentration of ascorbic acid was 7.7%, 18.0% and 32.0%

ascorbate concentration compared to those sold in boxes (15.0 mg/dl – 21.9 mg/dl).

**Freshly squeezed juices Vitamin C content (mg/dl)** 

lower compared to the result obtained from the freshly squeezed juices.

Orange 22.2 Lemon 22.2 Grapefruit 22.0 Grapes 17.2 Tomato 17.0

Table 2. Vitamin C contents of freshly squeezed juices

Juglandis folium 1.79 Crataegi summitas 0.75 Menthae piperitae 0.70 Urticae herba 0.68 Mori folium 0.59 Myrtilli folium 0.31 Millefolii flos 0.24 Centauri herba 0.24 Visci albae stipes 0.18 Phaseoli tegumen 0.03

Table 1. Flavonoid content of medicinal teas

were calculated in hyperoside units.

Lushchak Vl., 2011.

radical is required for the expression of the disease. Pancreatic beta cells are particularly sensitive to superoxide mediated radical damage, having a poor antioxidant defence system. Superoxide itself or derivative radicals may be the direct cause of cell damage. Radical generation leads to breakage of cell DNA, which initiates the repair process resulting in depletion of cellular NADH + H+ levels, leading to an inhibition of pro-insulin synthesis and renders the cell more sensitive to radical damage because NAD is involved in the electrontransport process required for radical scavenging by the cell. Oxygen radicals are also involved in the production of the cytokines (IL-1, TNF) by the cells of the inflammatory focus that could be involved in the cell damage Nemes-Nagy et al., 2008.

Another important component of Diavit® is the phytoestrogen called resveratrol, which is a natural polyphenolic compound found largely in the skin of red grapes, but also in blueberries.

Growing evidence suggests that resveratrol may play an important role in the prevention of many human diseases. Many of the biological actions of this polyphenol have been attributed to its antioxidant properties, it exhibits anticoagulant, vasodilator, antiinflammatory effects, inhibits oxidation of LDL-cholesterol particles, thus preventing atherosclerosis, and also increases sensitivity to insulin.

Certain studies evaluated the effect of resveratrol on intracellular reduced glutathione (GSH) and membrane sulphydryl groups in erythrocytes subjected to oxidative stress in vitro to test the efficacy of the antioxidant effect of resveratrol on human erythrocytes. In one of these studies subjecting erythrocytes to oxidative stress (in vitro) by incubating them with t-BHP (10 micromolar) caused a significant decrease in the intracellular GSH level and membrane -SH content compared with basal values.

Incubation of erythrocytes/membranes with resveratrol (1-100 micromolar final concentration) resulted in significant protection against the t-BHP-induced oxidative stress as evidenced by the increase in GSH level and membrane -SH content. It was observed that the effect of resveratrol is dose/concentration and time-dependent, it protects erythrocytes experimentally exposed to oxidative stress. Since resveratrol is naturally present in many fruits and vegetables, a diet rich in resveratrol, or dietary supplements containing this substance may provide protection against degenerative diseases and prevent diabetes complications [Pandey & Rizvi, 2010].

A new promising study on humans demonstrated the effect of regular consumption of a resveratrol supplement and the health of patients with impaired glucose tolerance. Resveratrol has been tested in relation to diabetes before, but only in animal subjects or on cell lines. Those studies have repeatedly shown promising effects on insulin secretion, insulin sensitivity and glucose tolerance, leading to the initiation of this first-of-its-kind pilot clinical study on humans [Crandall, 2010].

#### **5. Studies on antioxidant components of medicinal teas, fruit and vegetable juices**

We also determined the flavonoid content and the antioxidant capacity of several medicinal teas used by diabetic patients. We found the highest amount of flavonoids in Juglandis folium, and the smallest amount of flavonoids was found in Phaseoli tegumen. The results were calculated in hyperoside units.

The highest concentration of malondialdehyde was found in Centauri herba (149 mmol/100g), smaller amounts were found in Myrtilli folium (146 mmol/100g), Mori folium (115 mmol/100g), Crataegi summitas (100 mmol/100g), Urticae herba (97 mmol/100g), Menthae piperitae (97 mmol/100g), Visci albae stipes (94 mmol/100g), Juglandis folium (89 mmol/100g), Millefolii flos (79 mmol/100g) and Phaseoli tegumen (73 mmol/100g). The antioxidant capacity of these plants is inversely proportional to their MDA contents, a measure of ROS-induced damage to lipids Nemes-Nagy et al., 2004, Lushchak Vl., 2011.


Table 1. Flavonoid content of medicinal teas

294 Oxidative Stress and Diseases

radical is required for the expression of the disease. Pancreatic beta cells are particularly sensitive to superoxide mediated radical damage, having a poor antioxidant defence system. Superoxide itself or derivative radicals may be the direct cause of cell damage. Radical generation leads to breakage of cell DNA, which initiates the repair process resulting in depletion of cellular NADH + H+ levels, leading to an inhibition of pro-insulin synthesis and renders the cell more sensitive to radical damage because NAD is involved in the electrontransport process required for radical scavenging by the cell. Oxygen radicals are also involved in the production of the cytokines (IL-1, TNF) by the cells of the inflammatory focus that

Another important component of Diavit® is the phytoestrogen called resveratrol, which is a natural polyphenolic compound found largely in the skin of red grapes, but also in

Growing evidence suggests that resveratrol may play an important role in the prevention of many human diseases. Many of the biological actions of this polyphenol have been attributed to its antioxidant properties, it exhibits anticoagulant, vasodilator, antiinflammatory effects, inhibits oxidation of LDL-cholesterol particles, thus preventing

Certain studies evaluated the effect of resveratrol on intracellular reduced glutathione (GSH) and membrane sulphydryl groups in erythrocytes subjected to oxidative stress in vitro to test the efficacy of the antioxidant effect of resveratrol on human erythrocytes. In one of these studies subjecting erythrocytes to oxidative stress (in vitro) by incubating them with t-BHP (10 micromolar) caused a significant decrease in the intracellular GSH level and

Incubation of erythrocytes/membranes with resveratrol (1-100 micromolar final concentration) resulted in significant protection against the t-BHP-induced oxidative stress as evidenced by the increase in GSH level and membrane -SH content. It was observed that the effect of resveratrol is dose/concentration and time-dependent, it protects erythrocytes experimentally exposed to oxidative stress. Since resveratrol is naturally present in many fruits and vegetables, a diet rich in resveratrol, or dietary supplements containing this substance may provide protection against degenerative diseases and prevent diabetes

A new promising study on humans demonstrated the effect of regular consumption of a resveratrol supplement and the health of patients with impaired glucose tolerance. Resveratrol has been tested in relation to diabetes before, but only in animal subjects or on cell lines. Those studies have repeatedly shown promising effects on insulin secretion, insulin sensitivity and glucose tolerance, leading to the initiation of this first-of-its-kind pilot

**5. Studies on antioxidant components of medicinal teas, fruit and vegetable** 

We also determined the flavonoid content and the antioxidant capacity of several medicinal teas used by diabetic patients. We found the highest amount of flavonoids in Juglandis

could be involved in the cell damage Nemes-Nagy et al., 2008.

atherosclerosis, and also increases sensitivity to insulin.

membrane -SH content compared with basal values.

complications [Pandey & Rizvi, 2010].

clinical study on humans [Crandall, 2010].

**juices** 

blueberries.

We measured, by photometric diclorphenol-indophenol method the vitamin C concentration in freshly squeezed juices and in those preserved, available in stores. As a result we found that lemon, orange and grapefruit contain the biggest ascorbate quantity, especially freshly squeezed (22.0 – 22.2 mg/dl) the same volume of juice contains higher ascorbate concentration compared to those sold in boxes (15.0 mg/dl – 21.9 mg/dl).

In case of orange juices, we compared the vitamin C content of 3 samples from juices sold in boxes from different firms (the results obtained were 20.5 mg/dl, 18.2 mg/dl and 15.0 mg/dl), so we concluded that the concentration of ascorbic acid was 7.7%, 18.0% and 32.0% lower compared to the result obtained from the freshly squeezed juices.


Table 2. Vitamin C contents of freshly squeezed juices

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 297

Raman scattering spectroscopy is a highly specific method for skin carotenoid determination. This method is able to discern carotenoids from other potentially interfering compounds present in the skin due to its ability to identify molecules with long conjugated double-bond structures. Raman spectroscopy involves a blue, low-energy laser light source of 470 – 490 nm, directed onto the surface of the skin, where Raman resonance light scattering events cause the

carotenoids to emit a green signal at 510 – 530 nm, which is detected and quantified.

Fig. 10. Comparision of skin carotenoid score in diabetic and non-diabetic patients

concentration being found in the palm.

gender, age or skin pigmentation.

result.

Studies showed that the Raman spectroscopic method accurately reflects the presence of carotenoids in the human skin with high reproducibility. Significant differences in carotenoid concentrations were found between five different skin sites, the highest

An epidemiological study performed on a large number of healthy volunteers determined that the Raman spectroscopic method of measuring skin carotenoids is not really affected by

Raman intensity measurements were positively related to the amount of fruit and vegetable intake and use of carotenoid-containing dietary supplements, and inversely related to body fat content and smoking. This study confirmed that skin carotenoids follow similar dietary

Before the measurement, every patient has to fill in a special questionnaire containing questions regarding its lifestyle, dietary habits and other aspects that could influence the

and demographic patterns as in case of serum or plasma carotenoid measurements.

#### **6. Vitamin C dynamics in human milk**

We also followed the dynamics of ascorbate in human milk after juice and vitamin C tablet ingestion. The highest ascorbate level in milk was 1 hour after juice ingestion and half an hour after taking 1000 mg vitamin C containing tablets, the assimilation from natural sources being better. It is a close relationship between mothers' diet and the quality of their milk. We can recommend to consume juices one hour before brestfeeding, to offer the infant the highest amount of ascorbate Jákó et al., 2008.

Fig. 9. The dynamics of ascorbic acid in human milk after ingestion of freshly squeezed juices and C vitamin tablets

#### **7. Determination of carotenoid score in diabetic patients**

Another study was made on adult subjects, we used Raman spectroscopy (a method that can identify carotene molecules in the skin) to determine the carotenoide score, which gives information on the antioxidant capacity of the body. Significantly lower average value was obtained in adult diabetic compared to the control non-diabetic group of similar age; the difference is significant (P0.05) Jákó et al, 2009.

Food carotenoids are transported into the skin, protecting the tegument against ultraviolet radiation. The skin carotenoid concentration undergoes smaller variation compared to that in the blood depending on the dietary intake. Skin carotenoid determination by HPLC method using bioptic material is an invasive procedure and cannot be introduced in the every day practice. A very good correlation can be found between the serum carotenoid concentration and that in the palm skin (r = 0.78, P<0.001) determined by Raman spectroscopy, and the biophotonic scanner operates based on this observation Jákó et al, 2009, [Peng et al., 1995], Smidt & Shieh, 2003. Thus, skin carotenoid measurement appears to be a valuable biomarker of carotenoid nutritional status.

We also followed the dynamics of ascorbate in human milk after juice and vitamin C tablet ingestion. The highest ascorbate level in milk was 1 hour after juice ingestion and half an hour after taking 1000 mg vitamin C containing tablets, the assimilation from natural sources being better. It is a close relationship between mothers' diet and the quality of their milk. We can recommend to consume juices one hour before brestfeeding, to offer the infant

Fig. 9. The dynamics of ascorbic acid in human milk after ingestion of freshly squeezed

Another study was made on adult subjects, we used Raman spectroscopy (a method that can identify carotene molecules in the skin) to determine the carotenoide score, which gives information on the antioxidant capacity of the body. Significantly lower average value was obtained in adult diabetic compared to the control non-diabetic group of similar age; the

Food carotenoids are transported into the skin, protecting the tegument against ultraviolet radiation. The skin carotenoid concentration undergoes smaller variation compared to that in the blood depending on the dietary intake. Skin carotenoid determination by HPLC method using bioptic material is an invasive procedure and cannot be introduced in the every day practice. A very good correlation can be found between the serum carotenoid concentration and that in the palm skin (r = 0.78, P<0.001) determined by Raman spectroscopy, and the biophotonic scanner operates based on this observation Jákó et al, 2009, [Peng et al., 1995], Smidt & Shieh, 2003. Thus, skin carotenoid measurement appears

**7. Determination of carotenoid score in diabetic patients** 

difference is significant (P0.05) Jákó et al, 2009.

to be a valuable biomarker of carotenoid nutritional status.

**6. Vitamin C dynamics in human milk** 

the highest amount of ascorbate Jákó et al., 2008.

juices and C vitamin tablets

Raman scattering spectroscopy is a highly specific method for skin carotenoid determination. This method is able to discern carotenoids from other potentially interfering compounds present in the skin due to its ability to identify molecules with long conjugated double-bond structures. Raman spectroscopy involves a blue, low-energy laser light source of 470 – 490 nm, directed onto the surface of the skin, where Raman resonance light scattering events cause the carotenoids to emit a green signal at 510 – 530 nm, which is detected and quantified.

Fig. 10. Comparision of skin carotenoid score in diabetic and non-diabetic patients

Studies showed that the Raman spectroscopic method accurately reflects the presence of carotenoids in the human skin with high reproducibility. Significant differences in carotenoid concentrations were found between five different skin sites, the highest concentration being found in the palm.

An epidemiological study performed on a large number of healthy volunteers determined that the Raman spectroscopic method of measuring skin carotenoids is not really affected by gender, age or skin pigmentation.

Raman intensity measurements were positively related to the amount of fruit and vegetable intake and use of carotenoid-containing dietary supplements, and inversely related to body fat content and smoking. This study confirmed that skin carotenoids follow similar dietary and demographic patterns as in case of serum or plasma carotenoid measurements.

Before the measurement, every patient has to fill in a special questionnaire containing questions regarding its lifestyle, dietary habits and other aspects that could influence the result.

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 299

Regarding proper evaluation of antioxidant status in the human body, measuring lipoperoxidation products is a valuable tool because its high value positively correlates with the intensified oxidative stress. Interpretation of antioxidant enzyme activities could be sometimes difficult because it can decrease in case of intense consumption when free radical production is intensified, and in other situations even higher values can be observed due to

We can conclude that proper nutrition including adequate fresh fruit and vegetable intake are important sources of natural antioxidants. Patients suffering from diseases like diabetes mellitus which involves high oxidative stress should take dietary supplements containing antioxidant vitamins, phytoterapeutical products and oligoelements, these could help diabetic patients to achieve a better metabolic balance and to prevent several complications

It would be interesting to perform a placebo-controlled double-blind study on the effect of the dietary supplement Diavit®, containing blueberry and sea buckthorn concentrate, on a large group of type 2 diabetic patients followed for several years, to observe the long term effect of this phytotherapeutic product, or other, more complex dietary supplements could be used in similar studies. This could be a possibility to reduce the incidence of complications in diabetic patients, and to help them to achieve a proper metabolic balance without taking antidiabetic drugs, or at least lower the doses of their usual medication,

Balogh-Sămărghiţan V., Nemes-Nagy E., I. Dunca, Szőcs T., Pap Z., Máthé J. & Şt. Hobai:

Baynes J.W.: Role of oxidative stress in development of complications in diabetes, Diabetes,

Capasso F., Gaginella TS., Grandolini G. & Izzo AA.: Phytotherapy A Quick Reference to

Cadenas E., Packer L. (eds): *Handbook of Antioxidants*, Ed. Marcel Dekker Inc., New York,

Crăciun E. C., Szőcs-Molnár T., Nemes-Nagy E., Dunca I., Balogh-Sămărghiţan V., Hobai Şt.,

Oxygen Biology and Medicine, Paris, 11-13 April (2007), (abstract p. 31) Crandall J.: Promising Results of First-Ever Human Clinical Trial of a Resveratrol Supplement's Impact on Pre-diabetes, ADA's 70th Annual Meeting, 2010 Dejica D.: Antioxidanţi şi terapie antioxidantă (Antioxidants and antioxidant therapy), Ed.

Dejica D.: Stresul oxidativ (Oxidative stress), Ed. Casa Cărţii de Ştiinţă, Cluj-Napoca, 2000 DeLourdes M., Cruz T., Pousada J., et al: *The effect of Magnezium Supplementation in Increasing Doses on the Control of Type 2 Diabetes*, Diabetes Care, 1998, 21(5), p. 682-686

Pusta D. & Morar R.: Effect of dietary supplement Diavit on antioxidant capacity in diabetes mellitus disease (poster), 2nd Symposium International - Nutrition,

Modificări ionice la copii cu diabet zaharat de tip 1 sub tratament cu Eridiarom (Ionic modifications in type 1 diabetic children under Eridiarom treatment), in Diabetul - alternative fitoterapeutice, sub redacţia Roman Morar, Dana Liana Pusta,

compensatory mechanisms, being a way of adaptation to intensified oxidative stress.

**8. Conclusion, perspectives** 

decreasing the risk of developing side effects.

1991, 40, 405-412

2002, p. 47-55

Ed. Todesco, Cluj-Napoca, 2004, p. 105-115

Herbal Medicine, Ed. Springer, 2003

Casa Cărţii de Ştiinţă, Cluj-Napoca, 2001

of this disease.

**9. References** 

Fig. 11. Biophotonic scanner showing the carotenoid score of a non-diabetic patient

Fig. 12. Low carotenoid score of an old diabetic patient

#### **8. Conclusion, perspectives**

298 Oxidative Stress and Diseases

Fig. 11. Biophotonic scanner showing the carotenoid score of a non-diabetic patient

Fig. 12. Low carotenoid score of an old diabetic patient

Regarding proper evaluation of antioxidant status in the human body, measuring lipoperoxidation products is a valuable tool because its high value positively correlates with the intensified oxidative stress. Interpretation of antioxidant enzyme activities could be sometimes difficult because it can decrease in case of intense consumption when free radical production is intensified, and in other situations even higher values can be observed due to compensatory mechanisms, being a way of adaptation to intensified oxidative stress.

We can conclude that proper nutrition including adequate fresh fruit and vegetable intake are important sources of natural antioxidants. Patients suffering from diseases like diabetes mellitus which involves high oxidative stress should take dietary supplements containing antioxidant vitamins, phytoterapeutical products and oligoelements, these could help diabetic patients to achieve a better metabolic balance and to prevent several complications of this disease.

It would be interesting to perform a placebo-controlled double-blind study on the effect of the dietary supplement Diavit®, containing blueberry and sea buckthorn concentrate, on a large group of type 2 diabetic patients followed for several years, to observe the long term effect of this phytotherapeutic product, or other, more complex dietary supplements could be used in similar studies. This could be a possibility to reduce the incidence of complications in diabetic patients, and to help them to achieve a proper metabolic balance without taking antidiabetic drugs, or at least lower the doses of their usual medication, decreasing the risk of developing side effects.

#### **9. References**


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DeLourdes M., Cruz T., Pousada J., et al: *The effect of Magnezium Supplementation in Increasing Doses on the Control of Type 2 Diabetes*, Diabetes Care, 1998, 21(5), p. 682-686

Evaluation of Oxidative Stress and the Efficacy of Antioxidant Treatment in Diabetes Mellitus 301

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**13** 

*UAE* 

Natheer H. Al-Rawi

**Diabetes, Oxidative Stress,** 

*Dept. Oral & Craniofacial Health Science, College of Dentistry, University of Sharjah,* 

**Antioxidants and Saliva: A Review** 

Diabetes mellitus is a devastating disease throughout the world. It has been estimated that the number of peoples affected with diabetes in the world will increase to 300 million by 2025 (1). Diabetes is associated with several mechanisms, one of which is oxidative stress. Increased oxidative stress is a widely accepted participant in the development and progression of diabetes and its complications (2,3). Oxidative stress is a general term used to describe the imbalance between the production and manifestation of reactive oxygen species and a biological system's ability to ready detoxify the reactive intermediates or to repair the resulting damage (4). Oxidative stress occurs when free radical production exceeds the body's ability to neutralize them. The imbalance may be due to either: decrease production of antioxidants; or excessive production of free radicals. In diabetes, free radicals are formed disproportionately by glucose oxidation, non-enzymatic glycation of proteins and the subsequent oxidative degradation of glycated proteins (5). Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation and development of insulin resistance (6). These consequences of oxidative stress can promote the

There are multiple sources of oxidative stress in diabetes including non enzymatic,

Non enzymatic sources of oxidative stress originate from the oxidative biochemistry of glucose. Hyperglycemia can directly cause increased Reactive Oxygen Species (ROS) generation. Glucose can undergo autooxidation and generate hydroxyl \*OH- radicals (7). In addition, glucose reacts with proteins in a non enzymatic manner leading to the formation of advanced glycation end products (AGEs). ROS is generated at multiple steps during this process. In hyperglycemia, there is enhanced metabolism of glucose through the polyol

Enzymatic sources of augmented generation of reactive species in diabetes include Nitrous Oxide Species (NOS), NAD(P)H oxidase and xanthine oxidase (8-10). The mitochondrial

(sorbitol) pathway, which also results in enhanced production of superoxides (\*O2-) .

**1. Introduction** 

development of complications of diabetes mellitus.

**2. Sources of oxidative stress in diabetes** 

enzymatic and mitochondrial pathway.


### **Diabetes, Oxidative Stress, Antioxidants and Saliva: A Review**

Natheer H. Al-Rawi

*Dept. Oral & Craniofacial Health Science, College of Dentistry, University of Sharjah, UAE* 

#### **1. Introduction**

302 Oxidative Stress and Diseases

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Diabetes mellitus is a devastating disease throughout the world. It has been estimated that the number of peoples affected with diabetes in the world will increase to 300 million by 2025 (1). Diabetes is associated with several mechanisms, one of which is oxidative stress. Increased oxidative stress is a widely accepted participant in the development and progression of diabetes and its complications (2,3). Oxidative stress is a general term used to describe the imbalance between the production and manifestation of reactive oxygen species and a biological system's ability to ready detoxify the reactive intermediates or to repair the resulting damage (4). Oxidative stress occurs when free radical production exceeds the body's ability to neutralize them. The imbalance may be due to either: decrease production of antioxidants; or excessive production of free radicals. In diabetes, free radicals are formed disproportionately by glucose oxidation, non-enzymatic glycation of proteins and the subsequent oxidative degradation of glycated proteins (5). Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation and development of insulin resistance (6). These consequences of oxidative stress can promote the development of complications of diabetes mellitus.

#### **2. Sources of oxidative stress in diabetes**

There are multiple sources of oxidative stress in diabetes including non enzymatic, enzymatic and mitochondrial pathway.

Non enzymatic sources of oxidative stress originate from the oxidative biochemistry of glucose. Hyperglycemia can directly cause increased Reactive Oxygen Species (ROS) generation. Glucose can undergo autooxidation and generate hydroxyl \*OH- radicals (7). In addition, glucose reacts with proteins in a non enzymatic manner leading to the formation of advanced glycation end products (AGEs). ROS is generated at multiple steps during this process. In hyperglycemia, there is enhanced metabolism of glucose through the polyol (sorbitol) pathway, which also results in enhanced production of superoxides (\*O2-) .

Enzymatic sources of augmented generation of reactive species in diabetes include Nitrous Oxide Species (NOS), NAD(P)H oxidase and xanthine oxidase (8-10). The mitochondrial

Diabetes, Oxidative Stress, Antioxidants and Saliva: A Review 305

Reduced glutathione detoxify reactive oxygen species such as hydrogen peroxide and lipid peroxide directly or in a glutathione peroxidase (GPX) catalyzed mechanism. Glutathione reductase (GRD) catalyzes the NAD(P)H dependent reduction of oxidized glutathione,

Blood GSH was significantly decreased in different phases of type 2 diabetes mellitus such

Measurement of salivary GPX and GRD activities and GSSG/GSH ratio, provide a noninvasive method to assess the degree of oxidative stress in pathophysiologic status, such as

The decrease in salivary reduced-glutathione levels in patients with type 1 DM may have a role in periodontal tissue destruction by predisposing tissues to oxidative stress.(23). Our previous study (24) identified GSH activity in serum and saliva of patients with type 2 diabetes which was significantly low when compared with control group. This finding was explained on the basis that oxidative stress may consumes some naturally occurring local antioxidants such as reduced glutathione and this reflects the overwhelming adaptive response to the challenge of oxidative stress in the diabetic state with or without

SOD and catalase are also major antioxidant enzymes, SOD exists in 3 different isoforms; Cu,Zn-SOD is mostly in the cytosol and dismutate superoxide to hydrogen peroxide, Extracellular SOD is found in the plasma and extracellular space and Mn-SOD is located in mitochondria. Catalase is H2O2 decomposing enzyme mainly localized to peroxicomes or microperoxicomes. Superoxide may react with other reactive oxygen species such as Nitric

The major reason for the decreased SOD activity is the glycosylation of Cu,Zn-SOD which has been shown to lead to enzyme inactivation both in vivo and in vitro (26). Salivary SOD was measured in saliva (27) .Belce et al suggested that the main reason for the decrease of salivary SOD activity may be increased glycation of the enzyme and/or deleterious effect of increased free oxygen radicals by glycated proteins on SOD activity in diabetes which could lead to oral complications in diabetic patients.However; Al-Rawi study (24) demonstrated an increase in the level of SOD in serum and saliva of diabetic patients, this increase could be due to the existance or increased free radicals production which could enhance the

The polyol pathway consists of two enzymes. The first enzyme, aldose reductase (AR), reduces glucose to sorbitol with the aid of its co-factor NADPH, and the second enzyme, sorbitol dehydrogenase (SDH), with its co-factor NAD+, converts sorbitol to fructose. In animal models, treatment with AR inhibitors (ARI) was shown to be effective in preventing the development of various diabetic complications, including cataract, neuropathy, and nephropathy (28). The possibility of determination of sorbitol and fructosamine in saliva has been studied in healthy volunteers and patients with diabetes. It was concluded that saliva

antioxidant defense system that couter-balance the pro-oxidant environment.

serving to maintain intercellular glutathione stores and a favorable redox status (19).

as: glucose intolerance and early hyperglycemia (20) and poor glycemic control (21)

2. Alteration in glutathione metabolism:

3. Impairment of SOD and catalase activity:

Oxide to form highly toxic species such as peroxynitrite (25).

diabetes (22).

complications

4. Polyol Pathway:

respiratory chain is another source of non enzymatic generation of reactive species. Hyperglycemia-induced generation of \*O2- at the mitochondrial level is the initial trigger of vicious cycle of oxidative stress in diabetes (11,12).

#### **3. Saliva as diagnostic fluid**

Saliva in humans is a mouth fluid possessing several functions involved in oral health and homeostasis, with an active protective role in maintaining oral health. It plays a role in the preliminary digestion of food, fascilitates taste perception, maintains teeth enamel mineralization, buffers the acid components of food, and antimicrobial functions. The assay of saliva is an increasing area of research with implications of basic and clinical purposes. Recently, the use of saliva has provided a substantial addition to the diagnostic armamentarium as an investigative tool for disease processes and disorders. In addition to its oral indications, the analysis of saliva provides important information about the functioning of various organs within the body. Saliva analyses have been used mainly in dentistry and for studies in oral disease to help assess the risk of caries, by measuring buffering capacity and bacterial contents (13). Oral fluid is mainly utilized for research and diagnostic purposes concerning systemic diseases such as diabetes.

The determination of the oxidative stress and antioxidants require sometimes invasive techniques such as venepuncture. Whole saliva is an important physiologic fluid that contains a highly complex mixture of substances. Variable amounts of blood, serum markers that accurately reflect the redox status of the body can be determined in saliva and may have great clinical interest. The assay of salivary oxidative stress parameters has brought substantial insight into the pathogenesis and evolution of many systemic diseases including diabetes.

#### **4. Mechanisms for increased oxidative stress in diabetes**

1. Advanced glycation end products (AGEs):

AGEs are products of glycation and oxidation (glycol-oxidation), which are increased with age, and at accelerated rate in diabetes (14,15). The formation of AGEs is an important biochemical abnormality that accompanies diabetes mellitus. AGEs initiate oxidative reactions that promote the formation of oxidized LDL. Interaction of AGEs with endothelial cells as well as other cells accumulating within the atherosclerotic plaque, such as mononuclear phagocytes and smooth muscle cells provides a mechanism to augment vascular dysfunction (16)

Nuclear magnetic resonance spectra of AGEs were determined in saliva of 52 consecutive patients with diabetes mellitus and 47 age-matched healthy control subjects. Resonance spectra showed specific peaks at 2.3, 7.3, and 8.4 ppm in saliva from patients with diabetes mellitus, indicating the presence of advanced glycation endproducts which was associated with approximal plaque index. (17).

In a study of Garay-Sevilla et al (18) who measured AGEs in skin, serum and saliva of diabetic patients with complications they concluded that the AGEs measurement in saliva is useful to evaluate diabetes complications.

#### 2. Alteration in glutathione metabolism:

304 Oxidative Stress and Diseases

respiratory chain is another source of non enzymatic generation of reactive species. Hyperglycemia-induced generation of \*O2- at the mitochondrial level is the initial trigger of

Saliva in humans is a mouth fluid possessing several functions involved in oral health and homeostasis, with an active protective role in maintaining oral health. It plays a role in the preliminary digestion of food, fascilitates taste perception, maintains teeth enamel mineralization, buffers the acid components of food, and antimicrobial functions. The assay of saliva is an increasing area of research with implications of basic and clinical purposes. Recently, the use of saliva has provided a substantial addition to the diagnostic armamentarium as an investigative tool for disease processes and disorders. In addition to its oral indications, the analysis of saliva provides important information about the functioning of various organs within the body. Saliva analyses have been used mainly in dentistry and for studies in oral disease to help assess the risk of caries, by measuring buffering capacity and bacterial contents (13). Oral fluid is mainly utilized for research and

The determination of the oxidative stress and antioxidants require sometimes invasive techniques such as venepuncture. Whole saliva is an important physiologic fluid that contains a highly complex mixture of substances. Variable amounts of blood, serum markers that accurately reflect the redox status of the body can be determined in saliva and may have great clinical interest. The assay of salivary oxidative stress parameters has brought substantial insight into the pathogenesis and evolution of many systemic diseases including

AGEs are products of glycation and oxidation (glycol-oxidation), which are increased with age, and at accelerated rate in diabetes (14,15). The formation of AGEs is an important biochemical abnormality that accompanies diabetes mellitus. AGEs initiate oxidative reactions that promote the formation of oxidized LDL. Interaction of AGEs with endothelial cells as well as other cells accumulating within the atherosclerotic plaque, such as mononuclear phagocytes and smooth muscle cells provides a mechanism to augment

Nuclear magnetic resonance spectra of AGEs were determined in saliva of 52 consecutive patients with diabetes mellitus and 47 age-matched healthy control subjects. Resonance spectra showed specific peaks at 2.3, 7.3, and 8.4 ppm in saliva from patients with diabetes mellitus, indicating the presence of advanced glycation endproducts which was associated

In a study of Garay-Sevilla et al (18) who measured AGEs in skin, serum and saliva of diabetic patients with complications they concluded that the AGEs measurement in saliva is

vicious cycle of oxidative stress in diabetes (11,12).

diagnostic purposes concerning systemic diseases such as diabetes.

**4. Mechanisms for increased oxidative stress in diabetes** 

1. Advanced glycation end products (AGEs):

**3. Saliva as diagnostic fluid** 

diabetes.

vascular dysfunction (16)

with approximal plaque index. (17).

useful to evaluate diabetes complications.

Reduced glutathione detoxify reactive oxygen species such as hydrogen peroxide and lipid peroxide directly or in a glutathione peroxidase (GPX) catalyzed mechanism. Glutathione reductase (GRD) catalyzes the NAD(P)H dependent reduction of oxidized glutathione, serving to maintain intercellular glutathione stores and a favorable redox status (19).

Blood GSH was significantly decreased in different phases of type 2 diabetes mellitus such as: glucose intolerance and early hyperglycemia (20) and poor glycemic control (21)

Measurement of salivary GPX and GRD activities and GSSG/GSH ratio, provide a noninvasive method to assess the degree of oxidative stress in pathophysiologic status, such as diabetes (22).

The decrease in salivary reduced-glutathione levels in patients with type 1 DM may have a role in periodontal tissue destruction by predisposing tissues to oxidative stress.(23). Our previous study (24) identified GSH activity in serum and saliva of patients with type 2 diabetes which was significantly low when compared with control group. This finding was explained on the basis that oxidative stress may consumes some naturally occurring local antioxidants such as reduced glutathione and this reflects the overwhelming adaptive response to the challenge of oxidative stress in the diabetic state with or without complications

#### 3. Impairment of SOD and catalase activity:

SOD and catalase are also major antioxidant enzymes, SOD exists in 3 different isoforms; Cu,Zn-SOD is mostly in the cytosol and dismutate superoxide to hydrogen peroxide, Extracellular SOD is found in the plasma and extracellular space and Mn-SOD is located in mitochondria. Catalase is H2O2 decomposing enzyme mainly localized to peroxicomes or microperoxicomes. Superoxide may react with other reactive oxygen species such as Nitric Oxide to form highly toxic species such as peroxynitrite (25).

The major reason for the decreased SOD activity is the glycosylation of Cu,Zn-SOD which has been shown to lead to enzyme inactivation both in vivo and in vitro (26). Salivary SOD was measured in saliva (27) .Belce et al suggested that the main reason for the decrease of salivary SOD activity may be increased glycation of the enzyme and/or deleterious effect of increased free oxygen radicals by glycated proteins on SOD activity in diabetes which could lead to oral complications in diabetic patients.However; Al-Rawi study (24) demonstrated an increase in the level of SOD in serum and saliva of diabetic patients, this increase could be due to the existance or increased free radicals production which could enhance the antioxidant defense system that couter-balance the pro-oxidant environment.

#### 4. Polyol Pathway:

The polyol pathway consists of two enzymes. The first enzyme, aldose reductase (AR), reduces glucose to sorbitol with the aid of its co-factor NADPH, and the second enzyme, sorbitol dehydrogenase (SDH), with its co-factor NAD+, converts sorbitol to fructose. In animal models, treatment with AR inhibitors (ARI) was shown to be effective in preventing the development of various diabetic complications, including cataract, neuropathy, and nephropathy (28). The possibility of determination of sorbitol and fructosamine in saliva has been studied in healthy volunteers and patients with diabetes. It was concluded that saliva

Diabetes, Oxidative Stress, Antioxidants and Saliva: A Review 307

damage produced by nitric oxide and other free radicals. The estimation of vitamin levels and other antioxidants in saliva could provide a good insight about the body function

The saliva matrix is an upcoming area of research for basic and clinical application purposes, with considerable potential for growth and progress. Nevertheless, to date salivary assays are still little used compared with plasma assays, even it is possible to have a

[2] Cerielo A. Oxidative stress and glycemic regulation. Metabolism 2000;49 (2)suppl 1:27-29 [3] Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new

[4] Maritim AC, Sanders RA, Watkins JB 3rd . Diabetes, oxidative stress, and antioxidants: A

[5] Atalay M, Laaksonen. Diabetes, Oxidative stress and physical exercise. J Sports Science

[6] Johansen JS, Harris AK, Rychly DJ, Ergul A. Oxidative stress and the use of antioxidants

[7] Turko IV, Marcondes S, Murad F. Diabetes-associated nitration of tyrosine and

[8] Cuzik TJ, West NE, Black E, McDonald D, Ratnatunga C, Pillai R, Channon KM.

[9] Cuzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM.

[10] Aliciguzel Y, Ozen I, Aslan M, Karayalcin U. Activities of xanthine oxireductase and

[11] Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA,

[12] Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature

[13] Van Nieuw Amerongen AV, Bolscher JG, Veerman ECI. Salivary proteins: protective

and diagnostic value in cariology: Caries Res 2004;38:247-53.

dysfunction and clinical risk factors. Circ Res 2000;86(9):E85-90.

in diabetes: Linking basic science to clinical practice. Cardiovascular Diabetology

inactivation of succinyl-CoA:3-oxoacid CoA- transferase. Am J Physiol Heart Circul

Vascular superoxide production by NAD(P)H oxidase:association with endothelial

Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase.

antioxidant enzymes in different tissues of diabetic rats. J Lab Clin Med

Beebe D, Oates PJ, Hammes HP, et al. Normalizing mitochondrial superoxide production block three pathways of hyperglycemic damage. Nature

quantitative estimate of oxidative stress markers and antioxidants in saliva.

[1] Current Diabetes scenario in India. Br. J Diabetes Vasc Dis 2007; 7(1): 12-16

perspective on the old paradigm. Diabetes 1999;48:1-9.

review. J Biochem Mol Toxicol 2003;17(1):24-38.

against oxidative stress and it can be used to monitor therapy.

**7. Conclusion** 

**8. References** 

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Physiol 2001;281(6):H2289-94.

Circulation 2002;105(14):1656-62.

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2001;414(6865):813-20.

2005;4:5.

sorbitol and fructosamine levels measurements may be used as diagnostic tests in diabetes and serve as indicators of efficacy of therapy in diabetes (29).

#### **5. Lipid peroxidation and protein oxidation in diabetes**

*Lipid peroxidation:* Lipid peroxidation end-products very commonly detected by the measurement of thiobarbituric acid reactive substance (TBARS). The use of TBARS as an index of lipid peroxidation has been increased in plasma of diabetic patients (30-35). Thiobarbituric acid reacting substances (TBARS) are produced during lipoperoxidationoxidative stress-induced damage of lipids and are, thus, a widely used marker of oxidative stress (36,37). However, they represent a heterogeneous group of compounds – best known is malondialdehyde (MDA). TBARS is associated with parodontopathies when measured directly in the injured gingival tissue (38). In previous studies we have shown that TBARS can be found in measurable concentrations in saliva and that these levels are higher in patients with parodontopathies and their origin is unlikely to be plasma (39,40). Whether the difference in patients is caused by a rise of MDA or instead of and which other factors influence salivary TBARS levels is unknown.(40) thus, assume, that salivary TBARS may reflect the local oral oxidative stress, although the producer is still hidden (41). Salivary MDA levels are directly affected by sytemic oxidative stress, since MDA levels were also elevated in saliva of diabtetic patients without parodontopathies (24). Astaneie et al (42) have reported no difference in salivary versus serum MDA levels and presence of high Antioxidant activity (AOA) in type 1 diabetics. Studies conducted on diabetic rats have reported an increase in salivary and serum MDA with variable antioxidant activity (43). Celec et al (44) have found an increase in MDA levels in non diabetics which was attributed to age, altered periodontal status and smoking. Hodosy et al (45) suggest that MDA levels depend on the time of sampling and also are affected by factors like tooth brushing and antioxidative therapy received by the patients. Studies by Reznick et al (46)and Astaneie et al (42) have shown both salivary and serum antioxidants to increase depending on HbA1C levels and severity of diabetes. The AOA levels of both the groups did not show notable correlation with Fasting Plasma Glucosa (FPG) but a significant correlation existed between salivary MDA and FPG levels in the diabetic group.

#### **6. Diabetes and antioxidants**

Antioxidants are substances that inhibit the destructive eefects of oxidation. Some of the general antioxidants that are known are glutathione effects, glutathione peroxidase, vitamins A,C,E, catalase and SOD. The decreased efficiency of antioxidant defenses (both enzymatic and non-enzymatic) seems to correlate with the severity of pathological tissue changes in type 1 diabetes (47).

Administration of the antioxidants, for example, the vitamin C and free amino acids, get a better reaction to insulin and can supply extra benefit to the proposed reduction of oxidative stress in tissues (48,49). Experimental study on diabetic rats suggested that nutritional vitamin E supplementation helps fatty acids metabolism and lower lipid peroxidation in rat tissues (50). Oral vitamin C and vitamin E has the ability to lower the oxidative stress in eye (51) and the vascular endothelia function get better in type1 and not type 2 diabetes (52). Vitamin C and Vitamin E, probably have an important role in reducing the oxidative damage produced by nitric oxide and other free radicals. The estimation of vitamin levels and other antioxidants in saliva could provide a good insight about the body function against oxidative stress and it can be used to monitor therapy.

#### **7. Conclusion**

306 Oxidative Stress and Diseases

sorbitol and fructosamine levels measurements may be used as diagnostic tests in diabetes

*Lipid peroxidation:* Lipid peroxidation end-products very commonly detected by the measurement of thiobarbituric acid reactive substance (TBARS). The use of TBARS as an index of lipid peroxidation has been increased in plasma of diabetic patients (30-35). Thiobarbituric acid reacting substances (TBARS) are produced during lipoperoxidationoxidative stress-induced damage of lipids and are, thus, a widely used marker of oxidative stress (36,37). However, they represent a heterogeneous group of compounds – best known is malondialdehyde (MDA). TBARS is associated with parodontopathies when measured directly in the injured gingival tissue (38). In previous studies we have shown that TBARS can be found in measurable concentrations in saliva and that these levels are higher in patients with parodontopathies and their origin is unlikely to be plasma (39,40). Whether the difference in patients is caused by a rise of MDA or instead of and which other factors influence salivary TBARS levels is unknown.(40) thus, assume, that salivary TBARS may reflect the local oral oxidative stress, although the producer is still hidden (41). Salivary MDA levels are directly affected by sytemic oxidative stress, since MDA levels were also elevated in saliva of diabtetic patients without parodontopathies (24). Astaneie et al (42) have reported no difference in salivary versus serum MDA levels and presence of high Antioxidant activity (AOA) in type 1 diabetics. Studies conducted on diabetic rats have reported an increase in salivary and serum MDA with variable antioxidant activity (43). Celec et al (44) have found an increase in MDA levels in non diabetics which was attributed to age, altered periodontal status and smoking. Hodosy et al (45) suggest that MDA levels depend on the time of sampling and also are affected by factors like tooth brushing and antioxidative therapy received by the patients. Studies by Reznick et al (46)and Astaneie et al (42) have shown both salivary and serum antioxidants to increase depending on HbA1C levels and severity of diabetes. The AOA levels of both the groups did not show notable correlation with Fasting Plasma Glucosa (FPG) but a significant correlation existed between

Antioxidants are substances that inhibit the destructive eefects of oxidation. Some of the general antioxidants that are known are glutathione effects, glutathione peroxidase, vitamins A,C,E, catalase and SOD. The decreased efficiency of antioxidant defenses (both enzymatic and non-enzymatic) seems to correlate with the severity of pathological tissue

Administration of the antioxidants, for example, the vitamin C and free amino acids, get a better reaction to insulin and can supply extra benefit to the proposed reduction of oxidative stress in tissues (48,49). Experimental study on diabetic rats suggested that nutritional vitamin E supplementation helps fatty acids metabolism and lower lipid peroxidation in rat tissues (50). Oral vitamin C and vitamin E has the ability to lower the oxidative stress in eye (51) and the vascular endothelia function get better in type1 and not type 2 diabetes (52). Vitamin C and Vitamin E, probably have an important role in reducing the oxidative

and serve as indicators of efficacy of therapy in diabetes (29).

salivary MDA and FPG levels in the diabetic group.

**6. Diabetes and antioxidants** 

changes in type 1 diabetes (47).

**5. Lipid peroxidation and protein oxidation in diabetes** 

The saliva matrix is an upcoming area of research for basic and clinical application purposes, with considerable potential for growth and progress. Nevertheless, to date salivary assays are still little used compared with plasma assays, even it is possible to have a quantitative estimate of oxidative stress markers and antioxidants in saliva.

#### **8. References**


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sialic acid level and Cu-Zn superoxide dismutase activity in type 1 diabetes


**Section 5** 

**Systemic, Neuronal and Hormonal Pathologies** 

