**Particularities of Oxidative Stress in Newborns**

**Particularities of Oxidative Stress in Newborns**

DOI: 10.5772/intechopen.73369

#### Melinda Mátyás and Gabriela Zaharie Melinda Mátyás and Gabriela Zaharie Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73369

#### **Abstract**

The oxidative stress at newborns is augmented by different conditions like preterm birth, asphyxia, respiratory distress, and intraventricular hemorrhage. Preterm neonates associate a more pronounced oxidative stress than healthy term newborns. Several neonatal conditions like respiratory distress (RDS), asphyxia, intraventricular hemorrhage, bronchopulmonary dysplasia, retinopathy, and necrotizing enterocolitis will increase the oxidative stress. The harmful effects of free radicals are linked to their capacity to react with polyunsaturated fatty acids of cell membranes, proteins, and nucleic acids. Free radicals will produce protein alteration with function loss and lipid peroxidation.

**Keywords:** oxidative stress, new born, prematurity

### **1. Introduction**

Oxidative stress represents all injuries caused by reactive oxygen species (ROS) on biomolecules, inducing the destruction of membranes, enzymes, receptors, as well as alteration of cell function. The consequence of oxidative stress is a disruption of the physiological balance between pro-oxidants and antioxidants [1, 2].

Newborn possesses defense mechanisms such us molecules protection, limitation of ROS production, and mechanisms for repair and adaptation to endogenous and exogenous ROS overproduction.

Reactive oxygen species are involved in physiological processes such as physical exercise, hyperbarism, regulation of vascular tone, stimulation of cell growth and proliferation, stimulation of erythropoietin secretion, the learning and memory process, as well as in pathological processes: inflammation, aging, carcinogenesis [1–3].

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In neonatal pathology, there are multiple circumstances that are associated with oxidative stress. An excess of reactive oxygen species in the context of immature, deficient antioxidant defense mechanisms may cause multisystemic diseases [2, 3].

and metabolic processes are disturbed, and morphological lesions occur. In the nerve fiber, the myelin sheath is attacked. At pulmonary level, there is an alteration of endothelial membrane permeability, with the movement of lipids from the vascular space to the extravascular

Particularities of Oxidative Stress in Newborns http://dx.doi.org/10.5772/intechopen.73369 95

Glucose and monosaccharide oxidation leads to the formation of activated molecules that may interact with other molecules, generating new compounds. By polysaccharide oxidation,

The number of oxidative attacks on DNA in humans is 10,000/cell. Thus, chromosome fragmentation occurs. Double-chain DNA is much more vulnerable than single-chain DNA. Altered fragments are eliminated as purine and pyrimidine bases by urinary excretion and they can be dosed. Oxidative DNA lesions accumulate with age. Aging and carcinogen-

The superoxide anion resulting from the xanthine/xanthine oxidase system induces DNA breaks. The hydroxyl radical through its instantaneous reaction with nucleic acids also causes DNA breaks. These breaks should be normally repaired by cell enzymes, but due to their low fidelity, the repair process is inadequate through the inclusion of inappropriate bases in the

Oxygenated water also has effects on DNA. Through its reaction with DNA-bound metal ions, oxygenated water will induce the formation of hydroxyl radical, which will immediately

The literature currently speaks about free radical disease in newborns, a term introduced by Sullivan [2], which includes the following pathogenic conditions: bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP), intra-periventricular hemorrhage (IPVH), necrotizing enterocolitis (NEC), and renal failure [1]. Premature newborns with a gestational age of less than 30 weeks and a weight of less than 1500 g, respectively, have a major risk to develop these disorders. At cerebral level, there is a predisposition to oxidative stress, due to the high amount of polyunsaturated fatty acids in the immature brain, particularly in neuronal membranes, but also due to the relatively high amount of protein-unbound iron [41]. Immaturity associated with premature birth and also oxygen therapy used for the treatment of respiratory distress significantly increase oxidative stress in premature newborns. The knowledge of oxygen toxicity mechanisms is important both for their prevention and to ensure a harmonious development of newborns in general and premature newborns in particular, given their

(interstitial) and intracellular space, and the development of pulmonary edema.

*1.1.3. Effects of oxidative stress on carbohydrates*

*1.1.4. Effects of oxidative stress on nucleic acids*

DNA structure.

fragment the DNA molecule.

**2. Oxidative stress in newborns**

low antioxidant defense capacity [1, 3].

structural changes in deoxyribonucleic acid (DNA) may occur.

esis are explained by incomplete DNA repair after oxidative attacks.

#### **1.1. Effects of reactive oxygen species on biomolecules**

Reactive oxygen species will act on molecules, inducing their deterioration as follows:


#### *1.1.1. Effects of oxidative stress on proteins and amino acids*

Reactive oxygen species (ROS) act on the side chain of amino acids. Through oxidation of amino acids in the structure of proteins, the following changes are induced: protein fragmentation, aggregation, and proteolytic degradation. Aldehydes resulting from lipid peroxidation and glycosylation will also act on proteins. The consequence will be the functional alteration of proteins, with the loss of their contractile, enzymatic, and transport function.

#### *1.1.2. Effects of oxidative stress on lipids*

The most extensively studied action on lipids is their cellular and extracellular peroxidation. Lipids and lipoproteins are involved, particularly those mainly composed of polyunsaturated fatty acids (PUFA) such as linoleic and arachidonic acids, abundantly in cholesterol esters, lecithin, and erythrocytic phospholipids. Lipoproteins can be oxidized by two pathways:


As a result of lipid peroxidation, a disorganization of the cell structure occurs, lipids being part of the membrane structure. A more rigid membrane will develop, with implications on essential membrane proteins, such as Na<sup>+</sup> -K+ -dependent ATPase. Thus, a change in the ion pump rate will occur.

Lipid peroxides alter the properties of cellular, mitochondrial, and lysosomal membranes, with the disappearance of osmotic, chemical, and electrical gradients. Thus, cell excitability and metabolic processes are disturbed, and morphological lesions occur. In the nerve fiber, the myelin sheath is attacked. At pulmonary level, there is an alteration of endothelial membrane permeability, with the movement of lipids from the vascular space to the extravascular (interstitial) and intracellular space, and the development of pulmonary edema.

#### *1.1.3. Effects of oxidative stress on carbohydrates*

In neonatal pathology, there are multiple circumstances that are associated with oxidative stress. An excess of reactive oxygen species in the context of immature, deficient antioxidant

Reactive oxygen species will act on molecules, inducing their deterioration as follows:

• DNA lesions—action on the bases in the DNA structure—thymine, cytosine, adenine, gua-

Reactive oxygen species (ROS) act on the side chain of amino acids. Through oxidation of amino acids in the structure of proteins, the following changes are induced: protein fragmentation, aggregation, and proteolytic degradation. Aldehydes resulting from lipid peroxidation and glycosylation will also act on proteins. The consequence will be the functional alteration

The most extensively studied action on lipids is their cellular and extracellular peroxidation. Lipids and lipoproteins are involved, particularly those mainly composed of polyunsaturated fatty acids (PUFA) such as linoleic and arachidonic acids, abundantly in cholesterol esters, lecithin, and erythrocytic phospholipids. Lipoproteins can be oxidized by two pathways:

• Specific enzymatic oxidation—with the formation of prostaglandins, thromboxane, prosta-

• Nonspecific enzymatic oxidation—a peroxidation process with the formation of products

As a result of lipid peroxidation, a disorganization of the cell structure occurs, lipids being part of the membrane structure. A more rigid membrane will develop, with implications on

Lipid peroxides alter the properties of cellular, mitochondrial, and lysosomal membranes, with the disappearance of osmotic, chemical, and electrical gradients. Thus, cell excitability



defense mechanisms may cause multisystemic diseases [2, 3].

nine, deoxyribose, followed by cell damage and mutations

• Glycoproteins—action on hyaluronic acid in their structure

*1.1.1. Effects of oxidative stress on proteins and amino acids*

*1.1.2. Effects of oxidative stress on lipids*

cyclin, leukotrienes, and isoprostanes

essential membrane proteins, such as Na<sup>+</sup>

with a damaging effect.

pump rate will occur.

• Alteration of NADPH—inhibition of the nucleotide coenzyme activity

• Lipid peroxidation—structural and functional changes in cell membranes

of proteins, with the loss of their contractile, enzymatic, and transport function.

• Lipids and proteins—action on the covalent bond in their structure

**1.1. Effects of reactive oxygen species on biomolecules**

94 Novel Prospects in Oxidative and Nitrosative Stress

Glucose and monosaccharide oxidation leads to the formation of activated molecules that may interact with other molecules, generating new compounds. By polysaccharide oxidation, structural changes in deoxyribonucleic acid (DNA) may occur.

#### *1.1.4. Effects of oxidative stress on nucleic acids*

The number of oxidative attacks on DNA in humans is 10,000/cell. Thus, chromosome fragmentation occurs. Double-chain DNA is much more vulnerable than single-chain DNA. Altered fragments are eliminated as purine and pyrimidine bases by urinary excretion and they can be dosed. Oxidative DNA lesions accumulate with age. Aging and carcinogenesis are explained by incomplete DNA repair after oxidative attacks.

The superoxide anion resulting from the xanthine/xanthine oxidase system induces DNA breaks. The hydroxyl radical through its instantaneous reaction with nucleic acids also causes DNA breaks. These breaks should be normally repaired by cell enzymes, but due to their low fidelity, the repair process is inadequate through the inclusion of inappropriate bases in the DNA structure.

Oxygenated water also has effects on DNA. Through its reaction with DNA-bound metal ions, oxygenated water will induce the formation of hydroxyl radical, which will immediately fragment the DNA molecule.

### **2. Oxidative stress in newborns**

The literature currently speaks about free radical disease in newborns, a term introduced by Sullivan [2], which includes the following pathogenic conditions: bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP), intra-periventricular hemorrhage (IPVH), necrotizing enterocolitis (NEC), and renal failure [1]. Premature newborns with a gestational age of less than 30 weeks and a weight of less than 1500 g, respectively, have a major risk to develop these disorders. At cerebral level, there is a predisposition to oxidative stress, due to the high amount of polyunsaturated fatty acids in the immature brain, particularly in neuronal membranes, but also due to the relatively high amount of protein-unbound iron [41]. Immaturity associated with premature birth and also oxygen therapy used for the treatment of respiratory distress significantly increase oxidative stress in premature newborns. The knowledge of oxygen toxicity mechanisms is important both for their prevention and to ensure a harmonious development of newborns in general and premature newborns in particular, given their low antioxidant defense capacity [1, 3].

#### **2.1. Implication of oxidative stress in pulmonary disorders**

Respiratory distress due to surfactant deficiency is one of the most frequent disorders in premature newborns. The higher the degree of prematurity, the higher is the incidence and severity of this disease. Due to the immature lung structure, surfactant deficiency, pulmonary fluid retention, and poorly developed bone and muscle structures, there is an increased susceptibility of the premature to pulmonary lesions. The mechanism of these pulmonary lesions is based on alveolar instability and pulmonary atelectasis.

fatty acids. During the first 3–4 days of life, pulmonary proteins are the first target of free radicals. In the context of RDS, edema develops as a result of increased membrane permeability. Due to its high protein content, its presence in the lung will make it the ideal target for the

of oxidative attack will disrupt the balance of the pulmonary protease-antiprotease system. In infants with RDS, ROS will interact with the surfactant as well as with other proteins and lipid

Patients with respiratory distress have high levels of hydrogen peroxide in the pulmonary condensate. Oxidized glutathione and the altered alpha-1 protein also show high levels in the pulmonary fluid. In addition, the antioxidant proteins catalase and ferritin are elevated, which could represent a compensatory response. Oxidative stress markers are also increased

With the increase in the survival rate of newborns with extreme prematurity, the incidence of chronic pulmonary disorders has also increased, not only as an undesired consequence of RDS, but also of mechanical ventilation used for its treatment. The most frequent chronic pulmonary disease of premature infants with RDS and a history of mechanical ventilation is bronchopulmonary dysplasia. This is a chronic lung disease developing in newborns treated with oxygen and mechanical ventilation for a primary pulmonary disease. It affects between 20 and 60% of prematures, but it may also occur in term infants with severe respiratory distress [8]. Its etiopathogeny is complex. There is the hypothesis that bronchopulmonary dysplasia might start as an acute inflammation, which subsequently turns into a chronic lung disease

Preliminary studies have shown that pulmonary lesions can be improved by administration of antioxidants such as superoxide dismutase (SOD). The function of SOD is to convert the extremely toxic superoxide radical to less toxic hydrogen peroxide and water. Superoxide dismutase is also present naturally, but not as synthetic surfactant. Genetic engineering has demonstrated that in alveolar cells, SOD survives for a longer time period. An experimental animal with an SOD gene disrupted by genetic engineering, exposed to hyperoxia, will survive for a shorter time and will have more pulmonary lesions than an animal with an intact gene. Superoxide dismutase plays a major role in the prevention of pulmonary lesions in the context of hyperoxia. If SOD activity is increased in the newborn lung, inflammatory changes

The incidence of bronchopulmonary dysplasia has increased not only with the extensive use of positive pressure ventilation for the treatment of neonatal respiratory distress, but also due to the increase of the survival rate by modern intensive care techniques in newborns with extreme prematurity. The Neonatal Research Network reported a 68% incidence of BPD in

Today, it is known that barotrauma, particularly at high inspiratory pressures, is a key factor in the development of pulmonary lesions independently of any other lesions generated by oxygen therapy. Epithelial alterations in the airways occur, as well as with an increase in capillary permeability with extravasation of protein substances. In addition to the implication


97

Particularities of Oxidative Stress in Newborns http://dx.doi.org/10.5772/intechopen.73369

initiation of oxidative attack by ROS [30, 31]. The inactivation of α<sup>1</sup>

in patients with sepsis, in those infected with HIV [40].

under the action of free radicals [2, 5].

and pulmonary lesions can be prevented [9, 20, 21].

prematures with a gestational age of 22–28 WA.

structures, delaying the normalization of pulmonary function [2, 5, 38].

Mechanical ventilation, in addition to its role of ensuring an adequate oxygen supply to the body, also has undesired effects. Thus, it interferes with inflammatory cell metabolism and pulmonary mediators. Pulmonary vascularization is supplied from the heart but is also a reservoir of neutrophils—1/3 of the total number of neutrophils outside the bone marrow is found in the lungs [38].

Animal experiments have evidenced that in the context of mechanical ventilation, the number of neutrophils and the level of mediators, platelet activating factors, thromboxane B2 in pulmonary lavage fluid, and TNF-alpha in alveolar macrophages increase [11]. In premature animals with RDS, mechanical ventilation will cause an increase in pulmonary inflammation mediators, an increase in granulocytes, and cytokine activation [8]. Ventilatory support may affect the alveolar-capillary barrier and induces a release of inflammation mediators from the alveolar space into the circulation. The translocation of endotoxins from the aerial space to the circulatory space will generate a systemic inflammatory response [8, 9].

Mechanical ventilation along with oxygen supplementation will generate oxidative stress, with protein oxidation under the action of ROS. The carbonyl group of proteins will react with 2,4-dinitrophenylhydrazine, and 2,4-dinitrophenyl-hydrazone will form, which is measured by spectrophotometry [16]. Prematures who develop bronchopulmonary dysplasia (BPD) have significantly higher levels of protein carbonyls during the first week of life [1, 16].

Newborns requiring mechanical ventilation are more exposed to free radical production as a result of oxygen therapy exposure, inflammatory response, and ischemia reperfusion [10, 49].

Experimentally, it has been demonstrated on animals that the synthesis of enzymatic and consequently, antioxidant systems occurs at the end of gestation. Hemolysis after birth associated with a low level of iron-binding antioxidants can favor protein damage [5].

Protein carbonylation in the tracheobronchial fluid is a marker of oxidative stress at pulmonary level, while the exhalation of lipid peroxidation products (ethane, pentane) is a marker of lipid peroxidation in the entire body. As a result, a high level of exhaled peroxidation products is associated with extrapulmonary morbidity [9, 10].

Enzymes protecting against oxidant agents in the lungs, such as catalase, can be the target of oxidative attack inducing their inactivation. Liposoluble free radical scavengers such as tocopherol or beta-carotene have a protective effect against oxidative attack on lipids, but not on proteins. At the end of the first week of life, a depletion of these scavengers occurs as a result of the rapid increase in ROS production under the action of pulmonary inflammatory response. Thus, lipid peroxidation products are released through oxidation of polyunsaturated fatty acids. During the first 3–4 days of life, pulmonary proteins are the first target of free radicals. In the context of RDS, edema develops as a result of increased membrane permeability. Due to its high protein content, its presence in the lung will make it the ideal target for the initiation of oxidative attack by ROS [30, 31]. The inactivation of α<sup>1</sup> -proteases under the action of oxidative attack will disrupt the balance of the pulmonary protease-antiprotease system. In infants with RDS, ROS will interact with the surfactant as well as with other proteins and lipid structures, delaying the normalization of pulmonary function [2, 5, 38].

**2.1. Implication of oxidative stress in pulmonary disorders**

96 Novel Prospects in Oxidative and Nitrosative Stress

is based on alveolar instability and pulmonary atelectasis.

found in the lungs [38].

Respiratory distress due to surfactant deficiency is one of the most frequent disorders in premature newborns. The higher the degree of prematurity, the higher is the incidence and severity of this disease. Due to the immature lung structure, surfactant deficiency, pulmonary fluid retention, and poorly developed bone and muscle structures, there is an increased susceptibility of the premature to pulmonary lesions. The mechanism of these pulmonary lesions

Mechanical ventilation, in addition to its role of ensuring an adequate oxygen supply to the body, also has undesired effects. Thus, it interferes with inflammatory cell metabolism and pulmonary mediators. Pulmonary vascularization is supplied from the heart but is also a reservoir of neutrophils—1/3 of the total number of neutrophils outside the bone marrow is

Animal experiments have evidenced that in the context of mechanical ventilation, the number of neutrophils and the level of mediators, platelet activating factors, thromboxane B2 in pulmonary lavage fluid, and TNF-alpha in alveolar macrophages increase [11]. In premature animals with RDS, mechanical ventilation will cause an increase in pulmonary inflammation mediators, an increase in granulocytes, and cytokine activation [8]. Ventilatory support may affect the alveolar-capillary barrier and induces a release of inflammation mediators from the alveolar space into the circulation. The translocation of endotoxins from the aerial space to the

Mechanical ventilation along with oxygen supplementation will generate oxidative stress, with protein oxidation under the action of ROS. The carbonyl group of proteins will react with 2,4-dinitrophenylhydrazine, and 2,4-dinitrophenyl-hydrazone will form, which is measured by spectrophotometry [16]. Prematures who develop bronchopulmonary dysplasia (BPD) have significantly higher levels of protein carbonyls during the first week of life [1, 16]. Newborns requiring mechanical ventilation are more exposed to free radical production as a result of oxygen therapy exposure, inflammatory response, and ischemia reperfusion [10, 49]. Experimentally, it has been demonstrated on animals that the synthesis of enzymatic and consequently, antioxidant systems occurs at the end of gestation. Hemolysis after birth associated

Protein carbonylation in the tracheobronchial fluid is a marker of oxidative stress at pulmonary level, while the exhalation of lipid peroxidation products (ethane, pentane) is a marker of lipid peroxidation in the entire body. As a result, a high level of exhaled peroxidation prod-

Enzymes protecting against oxidant agents in the lungs, such as catalase, can be the target of oxidative attack inducing their inactivation. Liposoluble free radical scavengers such as tocopherol or beta-carotene have a protective effect against oxidative attack on lipids, but not on proteins. At the end of the first week of life, a depletion of these scavengers occurs as a result of the rapid increase in ROS production under the action of pulmonary inflammatory response. Thus, lipid peroxidation products are released through oxidation of polyunsaturated

circulatory space will generate a systemic inflammatory response [8, 9].

with a low level of iron-binding antioxidants can favor protein damage [5].

ucts is associated with extrapulmonary morbidity [9, 10].

Patients with respiratory distress have high levels of hydrogen peroxide in the pulmonary condensate. Oxidized glutathione and the altered alpha-1 protein also show high levels in the pulmonary fluid. In addition, the antioxidant proteins catalase and ferritin are elevated, which could represent a compensatory response. Oxidative stress markers are also increased in patients with sepsis, in those infected with HIV [40].

With the increase in the survival rate of newborns with extreme prematurity, the incidence of chronic pulmonary disorders has also increased, not only as an undesired consequence of RDS, but also of mechanical ventilation used for its treatment. The most frequent chronic pulmonary disease of premature infants with RDS and a history of mechanical ventilation is bronchopulmonary dysplasia. This is a chronic lung disease developing in newborns treated with oxygen and mechanical ventilation for a primary pulmonary disease. It affects between 20 and 60% of prematures, but it may also occur in term infants with severe respiratory distress [8].

Its etiopathogeny is complex. There is the hypothesis that bronchopulmonary dysplasia might start as an acute inflammation, which subsequently turns into a chronic lung disease under the action of free radicals [2, 5].

Preliminary studies have shown that pulmonary lesions can be improved by administration of antioxidants such as superoxide dismutase (SOD). The function of SOD is to convert the extremely toxic superoxide radical to less toxic hydrogen peroxide and water. Superoxide dismutase is also present naturally, but not as synthetic surfactant. Genetic engineering has demonstrated that in alveolar cells, SOD survives for a longer time period. An experimental animal with an SOD gene disrupted by genetic engineering, exposed to hyperoxia, will survive for a shorter time and will have more pulmonary lesions than an animal with an intact gene. Superoxide dismutase plays a major role in the prevention of pulmonary lesions in the context of hyperoxia. If SOD activity is increased in the newborn lung, inflammatory changes and pulmonary lesions can be prevented [9, 20, 21].

The incidence of bronchopulmonary dysplasia has increased not only with the extensive use of positive pressure ventilation for the treatment of neonatal respiratory distress, but also due to the increase of the survival rate by modern intensive care techniques in newborns with extreme prematurity. The Neonatal Research Network reported a 68% incidence of BPD in prematures with a gestational age of 22–28 WA.

Today, it is known that barotrauma, particularly at high inspiratory pressures, is a key factor in the development of pulmonary lesions independently of any other lesions generated by oxygen therapy. Epithelial alterations in the airways occur, as well as with an increase in capillary permeability with extravasation of protein substances. In addition to the implication of high inspiratory pressure in the genesis of pulmonary lesions, increased tidal volume also plays an important role. High but also significantly decreased tidal volume may generate an accumulation of neutrophils and a release of toxic agents such as proteases and free radicals, as well as proinflammatory cytokines. An analysis of pathological anatomy data has allowed to evidence a correlation between the incidence of interstitial emphysema during the first week of life and the incidence of interstitial fibrosis or proliferation in newborns with bronchopulmonary dysplasia surviving for more than 28 days [22, 24].

A study conducted in our department, which assessed lipid peroxidation by measuring malondialdehyde (MDA) levels on the first and third days of life found that MDA values in newborns with respiratory distress increased with the increase in the severity of respiratory distress (**Table 1**).

Particularities of Oxidative Stress in Newborns http://dx.doi.org/10.5772/intechopen.73369 99

MDA values in the mentioned study tended to decrease on day 3 compared to day 1, but

Also, the lipid peroxidation process was more intense in the study group-newborns with different pathologies generating oxidative stress compared with the control group of healthy

In the same study, we also monitored protein peroxidation in newborns with respiratory distress, and we found the presence of a significant correlation between protein carbonyl values on the third day of life and respiratory distress (r = 0.56; p < 0.05). For the evaluation of protein peroxidation in the mentioned study, we measured protein carbonyls on the first and third days of life using the Reznick spectrophotometric method with dinitrophenylhydrazine. The protein substrate in the lung is an optimal target for the action of ROS and the triggering of oxidation reactions of these proteins. In fact, it was demonstrated that in newborns with RDS, this protein oxidation process under the action of ROS also contributes to the pathogenesis of BPD. Under the action of reactive oxygen species, peroxidation of proteins and other lipid and protein structures occurs. Thus, the normalization of pulmonary function is delayed. Surfactant administration before the initiation of mechanical ventilation limits oxidative

The effects of ROS at endothelial level during the neonatal period are found in the following morbid conditions: septic shock, systemic inflammation, acute ischemia, inducing considerable oxidative stress [5]. Reactive oxygen species contribute to the development of ischemic and inflammatory vascular lesions by an important efflux of oxidants to the ischemic tissue and the induction of new lesions in tissues and organs in the next reperfusion stage [29].

RDS mild vs. severe MDA DOL 1 255.0 273.0 84.0 0.1159 18 14

RDS mild vs. moderate MDA DOL 1 307.5 638.5 136.5 0.0283 18 25

RDS severe vs. moderate MDA DOL 1 75.5 114.5 23.5 0.1740 6 13

MV yes vs. no MDA DOL 1 376.5 1453.5 285.5 0.7229 13 47

RDS = respiratory distress syndrome, MV = mechanical ventilation, RankSum = rank sum, p = test sign, and n = group size.

**RankSum1 RankSum2 U p n1 n2**

MDA DOL 3 281.0 247.0 110.0 0.5611 18 14

MDA DOL 3 379.0 567.0 208.0 0.6877 18 25

MDA DOL 3 79.0 111.0 20.0 0.0956 6 13

MDA DOL 3 407.5 1422.5 294.5 0.8454 13 47

term newborns, without any oxidative-stress inducing disorders (**Figures 1** and **2**).

without a statistically significant difference.

injury induced by mechanical ventilation.

**Table 1.** MDA values according to RDS severity.

**2.2. Effects of oxidative stress at endothelial level**

Another factor that plays a role in the development of BPD is inflammation. Proinflammatory cytokines were detected in the tracheal aspiration fluid during the first 1–2 weeks of life in newborns who subsequently developed BPD. Recently, it has been demonstrated that amnionitis and fetal infection are risk factors for BPD; consequently, inflammation, even prenatal, plays a role in its genesis [3].

Premature infants, due to their pulmonary immaturity, have a high risk for BPD, because they require additional oxygen supply for a longer time period in the treatment of the lung disease, their intracellular antioxidant defense capacity is affected, and they have an increased susceptibility to infections.

Studies have demonstrated the presence of a high level of lipid peroxidation products on days 1–2 of life in newborns who develop BPD. The presence of a high level of protein peroxidation products in the tracheal aspirate of infants weighing less than 1500 g compared to those with a higher weight has also been demonstrated. This confirms the fact that antioxidant defense decreases with the increase of immaturity [9, 15, 16].

There is also a close correlation between the protein oxidation level and activated neutrophils. This supports the presence of a relationship among neutrophil accumulation, oxidative stress, and the development of BPD [1].

In randomized trials for the study of STOP-ROP, some authors monitored oxygen exposure and the evolution of retrolental fibroplasia and BPD [36]. Thus, in groups with 89–94% O<sup>2</sup> Sat, compared to those with 96–99% O<sup>2</sup> Sat, the influence of saturation on the progression of retrolental fibroplasia was not significant. In contrast, in the group with higher saturation, the BPD progression rate was higher (13.2%) compared to subjects exposed to lower saturation levels (8.5%) [23, 39].

Exposure to a FiO<sup>2</sup> of 100% on the first day practically doubles the risk of BPD. Excessive oxygen administration and/or barotrauma may increase the risk of BPD. However, a high PaO<sup>2</sup> is a cofactor, not a causal agent in the development of BPD [36].

Preterm birth associates vitamin A deficiency, which is important in pulmonary epithelial lesion repair. A number of studies have evidenced a significant reduction in the incidence of bronchopulmonary dysplasia under conditions of vitamin A administration (Shenai et al.) [34, 37], but there are also studies that could not demonstrate this fact (Perason et al.) [32, 33].

Selenium deficiency can also be considered in association with low glutathione levels. Darlow et al. found a significant relationship between plasma selenium levels and the incidence of bronchopulmonary dysplasia at the age of 28 days, but could not clarify whether these were a cause or an effect of BPD.

A study conducted in our department, which assessed lipid peroxidation by measuring malondialdehyde (MDA) levels on the first and third days of life found that MDA values in newborns with respiratory distress increased with the increase in the severity of respiratory distress (**Table 1**).

MDA values in the mentioned study tended to decrease on day 3 compared to day 1, but without a statistically significant difference.

Also, the lipid peroxidation process was more intense in the study group-newborns with different pathologies generating oxidative stress compared with the control group of healthy term newborns, without any oxidative-stress inducing disorders (**Figures 1** and **2**).

In the same study, we also monitored protein peroxidation in newborns with respiratory distress, and we found the presence of a significant correlation between protein carbonyl values on the third day of life and respiratory distress (r = 0.56; p < 0.05). For the evaluation of protein peroxidation in the mentioned study, we measured protein carbonyls on the first and third days of life using the Reznick spectrophotometric method with dinitrophenylhydrazine. The protein substrate in the lung is an optimal target for the action of ROS and the triggering of oxidation reactions of these proteins. In fact, it was demonstrated that in newborns with RDS, this protein oxidation process under the action of ROS also contributes to the pathogenesis of BPD. Under the action of reactive oxygen species, peroxidation of proteins and other lipid and protein structures occurs. Thus, the normalization of pulmonary function is delayed. Surfactant administration before the initiation of mechanical ventilation limits oxidative injury induced by mechanical ventilation.

#### **2.2. Effects of oxidative stress at endothelial level**

of high inspiratory pressure in the genesis of pulmonary lesions, increased tidal volume also plays an important role. High but also significantly decreased tidal volume may generate an accumulation of neutrophils and a release of toxic agents such as proteases and free radicals, as well as proinflammatory cytokines. An analysis of pathological anatomy data has allowed to evidence a correlation between the incidence of interstitial emphysema during the first week of life and the incidence of interstitial fibrosis or proliferation in newborns with bron-

Another factor that plays a role in the development of BPD is inflammation. Proinflammatory cytokines were detected in the tracheal aspiration fluid during the first 1–2 weeks of life in newborns who subsequently developed BPD. Recently, it has been demonstrated that amnionitis and fetal infection are risk factors for BPD; consequently, inflammation, even prenatal,

Premature infants, due to their pulmonary immaturity, have a high risk for BPD, because they require additional oxygen supply for a longer time period in the treatment of the lung disease, their intracellular antioxidant defense capacity is affected, and they have an increased

Studies have demonstrated the presence of a high level of lipid peroxidation products on days 1–2 of life in newborns who develop BPD. The presence of a high level of protein peroxidation products in the tracheal aspirate of infants weighing less than 1500 g compared to those with a higher weight has also been demonstrated. This confirms the fact that antioxidant defense

There is also a close correlation between the protein oxidation level and activated neutrophils. This supports the presence of a relationship among neutrophil accumulation, oxidative stress,

In randomized trials for the study of STOP-ROP, some authors monitored oxygen exposure and the evolution of retrolental fibroplasia and BPD [36]. Thus, in groups with 89–94% O<sup>2</sup>

retrolental fibroplasia was not significant. In contrast, in the group with higher saturation, the BPD progression rate was higher (13.2%) compared to subjects exposed to lower saturation

gen administration and/or barotrauma may increase the risk of BPD. However, a high PaO<sup>2</sup>

Preterm birth associates vitamin A deficiency, which is important in pulmonary epithelial lesion repair. A number of studies have evidenced a significant reduction in the incidence of bronchopulmonary dysplasia under conditions of vitamin A administration (Shenai et al.) [34, 37], but there are also studies that could not demonstrate this fact (Perason et al.) [32, 33]. Selenium deficiency can also be considered in association with low glutathione levels. Darlow et al. found a significant relationship between plasma selenium levels and the incidence of bronchopulmonary dysplasia at the age of 28 days, but could not clarify whether these were

of 100% on the first day practically doubles the risk of BPD. Excessive oxy-

Sat, the influence of saturation on the progression of

is

chopulmonary dysplasia surviving for more than 28 days [22, 24].

plays a role in its genesis [3].

98 Novel Prospects in Oxidative and Nitrosative Stress

susceptibility to infections.

and the development of BPD [1].

levels (8.5%) [23, 39].

a cause or an effect of BPD.

Exposure to a FiO<sup>2</sup>

Sat, compared to those with 96–99% O<sup>2</sup>

decreases with the increase of immaturity [9, 15, 16].

a cofactor, not a causal agent in the development of BPD [36].

The effects of ROS at endothelial level during the neonatal period are found in the following morbid conditions: septic shock, systemic inflammation, acute ischemia, inducing considerable oxidative stress [5]. Reactive oxygen species contribute to the development of ischemic and inflammatory vascular lesions by an important efflux of oxidants to the ischemic tissue and the induction of new lesions in tissues and organs in the next reperfusion stage [29].


RDS = respiratory distress syndrome, MV = mechanical ventilation, RankSum = rank sum, p = test sign, and n = group size.

**Table 1.** MDA values according to RDS severity.

The vascular surface has the role to control critical processes for the functioning of organs. During inflammatory processes, the endothelium is exposed to oxidation [25]. It has been demonstrated that TNF-alpha stimulates the release of oxygenated water at the contact area

Particularities of Oxidative Stress in Newborns http://dx.doi.org/10.5772/intechopen.73369 101

The exposure of endothelial cells to significant concentrations of exogenous oxidants will cause specific physiological effects. Endogenous oxidants have been recognized as a physiological signal component, probably triggered by lung injury, with the release of TNF-alpha, interleukin, growth factor β, and platelet growth factors. These will stimulate tyrosine phosphorylation, with the activation of ERK, extracellular signal-regulated kinase. DNA synthesis will occur accompanied by an increase in hydrogen peroxide levels. Oxidants mediate the effects of growth factors β and insulin, which will stimulate mitogen-activated proteins [27–28].

The harmful effects of ROS at endothelial level also manifest in ROP [10, 23]. In the pathogenesis of ROP, the following factors are involved: self-regulation, which is absent in newborns, hyperoxygenation of the retina, which is frequent because antioxidant defense is reduced, particularly in the premature. Hyperoxygenation induces peroxidation of vasoactive isoprostanes. Vasoconstriction and vascular cell toxicity with ischemia and vascular proliferation occur [4, 7]. Glutathione is the most important intracellular antioxidant; however, its synthesis is reduced during intrauterine life and in prematures. In the vitreous fluid of the premature at risk for retinopathy, there is a high level of hypoxanthine with a role in the formation of free radicals [1, 12]. Papp et al. [6] found that the oxidized glutathione/reduced glutathione ratio is more than double in prematures with retinopathy compared to those without this disease. This is why the problem of using this ratio as a screening method for the detection of ocular involvement in premature infants was posed. The same authors found that in infants with active disease aged less than 3 months, reduced glutathione values were lower, and those of oxidized glutathione were higher, the greatest decrease in reduced glutathione occurring after

In ulceronecrotic enterocolitis, there are multiple pathogenic mechanisms. One of the pathogenic links is hypoxic ischemic injury. Hypoxic ischemic injury in the mesentery is followed by a cascade of events, with intestinal mucosal reperfusion injury. Cytotoxic damage of vascular endothelial cells occurs, which in turn will cause ischemia and new cytotoxic effects

The regulation of mesenteric blood flow includes a reflex self-regulation mechanism. The peripheral autonomic nervous system plays a role in the regulation of mesenteric blood flow. The initiated ischemia will induce transcapillary fluid passage and local edema. Reperfusion will exacerbate transcapillary fluid production and induce tissue destruction. This event is

between neutrophils and the endothelial cell, inducing endothelial cell retraction.

*2.2.1. Oxidative stress in retinopathy of prematurity*

the in vitro alteration of oxidative status.

through the formation of free radicals [41].

*2.2.2. Effects of oxidative stress in ulceronecrotic enterocolitis*

**Figure 1.** MDA values of case group DOL 1 vs. control group (caz = case, mar = control).

**Figure 2.** MDA values of case group DOL 3 vs. control group (caz = case, mar = control).

The vascular surface has the role to control critical processes for the functioning of organs. During inflammatory processes, the endothelium is exposed to oxidation [25]. It has been demonstrated that TNF-alpha stimulates the release of oxygenated water at the contact area between neutrophils and the endothelial cell, inducing endothelial cell retraction.

The exposure of endothelial cells to significant concentrations of exogenous oxidants will cause specific physiological effects. Endogenous oxidants have been recognized as a physiological signal component, probably triggered by lung injury, with the release of TNF-alpha, interleukin, growth factor β, and platelet growth factors. These will stimulate tyrosine phosphorylation, with the activation of ERK, extracellular signal-regulated kinase. DNA synthesis will occur accompanied by an increase in hydrogen peroxide levels. Oxidants mediate the effects of growth factors β and insulin, which will stimulate mitogen-activated proteins [27–28].

### *2.2.1. Oxidative stress in retinopathy of prematurity*

**Figure 1.** MDA values of case group DOL 1 vs. control group (caz = case, mar = control).

100 Novel Prospects in Oxidative and Nitrosative Stress

**Figure 2.** MDA values of case group DOL 3 vs. control group (caz = case, mar = control).

The harmful effects of ROS at endothelial level also manifest in ROP [10, 23]. In the pathogenesis of ROP, the following factors are involved: self-regulation, which is absent in newborns, hyperoxygenation of the retina, which is frequent because antioxidant defense is reduced, particularly in the premature. Hyperoxygenation induces peroxidation of vasoactive isoprostanes. Vasoconstriction and vascular cell toxicity with ischemia and vascular proliferation occur [4, 7].

Glutathione is the most important intracellular antioxidant; however, its synthesis is reduced during intrauterine life and in prematures. In the vitreous fluid of the premature at risk for retinopathy, there is a high level of hypoxanthine with a role in the formation of free radicals [1, 12]. Papp et al. [6] found that the oxidized glutathione/reduced glutathione ratio is more than double in prematures with retinopathy compared to those without this disease. This is why the problem of using this ratio as a screening method for the detection of ocular involvement in premature infants was posed. The same authors found that in infants with active disease aged less than 3 months, reduced glutathione values were lower, and those of oxidized glutathione were higher, the greatest decrease in reduced glutathione occurring after the in vitro alteration of oxidative status.

#### *2.2.2. Effects of oxidative stress in ulceronecrotic enterocolitis*

In ulceronecrotic enterocolitis, there are multiple pathogenic mechanisms. One of the pathogenic links is hypoxic ischemic injury. Hypoxic ischemic injury in the mesentery is followed by a cascade of events, with intestinal mucosal reperfusion injury. Cytotoxic damage of vascular endothelial cells occurs, which in turn will cause ischemia and new cytotoxic effects through the formation of free radicals [41].

The regulation of mesenteric blood flow includes a reflex self-regulation mechanism. The peripheral autonomic nervous system plays a role in the regulation of mesenteric blood flow.

The initiated ischemia will induce transcapillary fluid passage and local edema. Reperfusion will exacerbate transcapillary fluid production and induce tissue destruction. This event is mediated by factors released from the ischemic intestine, including endotoxins, histamine, prostaglandins, and superoxide anion, which result from oxygen metabolization. Superoxide will induce lipid peroxidation with the disruption of the integrity of capillaries and epithelial cells. Mucosal lesions characterized by edema, hemorrhage, ulceration, and necrosis may be induced experimentally by a combination of oxidants (hypoxanthine and xanthine oxidase) [14]. Superoxide dismutase can experimentally prevent or attenuate the described lesions. The protective effect of SOD was experimentally observed in animals with superior mesenteric artery occlusion. The sequential development of intestinal enzymes: SOD and xanthine oxidase can be a determining factor of neonatal intestinal susceptibility to ischemic mucosal injury [13, 45]. Regarding NEC prevention, many studies performed on animals show the beneficial effect of melatonin in preventing oxidative stress involved in the development of NEC lesions. Melatonin acts by reducing the level of inflammatory cytokines and by stimulating the activity of antioxidant enzymes. A decrease in TNF-α and IL-1β levels was found in animals treated with melatonin. Melatonin also counteracts the reduction of intestinal motility generated by lipopolysaccharides. Melatonin is an indolamine produced by the pineal gland, having serotonin as a precursor. It is synthesized by serotonin-rich enterochromaffin cells in the digestive tract. This synthesis takes place postprandially in the digestive tube and its level is 100 times higher than blood levels [45, 46].

Hypoxic ischemic brain injury is a long process that starts with the occurrence of the insult and continues during the recovery period, after reperfusion. This reperfusion process represents a paradoxical tissue response: the appearance of oxidative lesions in a hypoxic tissue, poorly perfused after circulatory stabilization, at the time of its perfusion with oxygenated blood. This stage of reperfusion will cause an excessive production of free radicals generating new lesions after the initial oxidative attack. In the premature, an increased risk of cerebral

Particularities of Oxidative Stress in Newborns http://dx.doi.org/10.5772/intechopen.73369 103

In the reperfusion stage, ROS with a cell damaging effect are produced, in the first place by the conversion of xanthine dehydrogenase to xanthine oxidase. This process will result in the formation of thiol groups or proteolysis by activation of proteases in energetically depressed cells. In the reoxygenation stage, hypoxanthine is converted to uric acid under the action of xanthine oxidase, and superoxide and peroxide are released through the mediation of xanthine oxidase by molecular oxygen binding. Hydroxyl radical generated through the Fenton reaction will also be released, due to the catalytic effect of metals such as iron [42]. Perinatal hypoxic stress is a frequent cause of morbidity, mortality, and neurological damage in survivors. In the perinatal hypoxic context, several factors play a role in the pathogenesis of lesions: hypoxia with initial ROS formation, followed by ischemia-reperfusion, a stage at which arachidonic acid and phagocytes will be activated under the action of inflammation mediators. Thus, a vicious circle is created. ROS will be formed, followed by tissue lesions and the genesis of new free radicals [42, 43].

At CNS level, under asphyxia conditions, encephalopathy lesions are induced by activation of leukocytes or glial cells and release of new free radicals. Hypoxic lesions will be perpetuated by release of protein-unbound iron. Endothelial lesions, hemostasis abnormalities, and inflammatory lesions occur, as well as with an increase in anaerobic metabolism, lactic acid levels, brain damage as a result of oligodendroglial vulnerability, astrocyte dysfunction,

Mitochondrial DNA damage induces changes in respiratory chain proteins, with the formation of new free radicals and subsequent cell lesions. Neonatal cerebral hypoxia stimulates activin secretion, which in turn stimulates erythropoietin, resulting in the production of

The types of lesions that occur in the brain as a result of hypoxia are variable. In the extremely immature brain, preoligodendrocytes and cell precursors of oligodendrocytes are affected. As the brain matures, the resistance of oligodendrocytes to oxidative stress increases due to an increase in antioxidant defense, as well as to the protein structure involved in programmed cell death.

After the hypoxic episode, there is an increase in the density of glutamate receptors, and mitochondrial calcium accumulation occurs, which will trigger apoptosis. Caspase 6 and, subsequently, caspase 3 are activated. NMDA receptor activation depresses the mitochondrial respiratory process and induces apoptosis—a process which is not found in adults, known as

Studies conducted by Peeters and Schulte regarding the activity of glutathione peroxidase in the cerebrospinal fluid of newborns with asphyxia and the neuron-specific enolase value evidenced a correlation between the glutathione peroxidase value and gestational age, as well

N-methyl-D-aspartate receptor abnormalities, and synaptic damage [28, 29].

nucleated red blood cells [35, 43].

the NMDA paradox [41, 48].

ischemia persists postnatally, during the first week of life [19, 26, 27].

The pathophysiological mechanism of NEC is based on a hypoxic-ischemic process similar to that found in postasphyxia brain injury. A number of studies have demonstrated that the association of melatonin with misoprostol, a gastric mucosal protective agent, is more beneficial than melatonin therapy alone. These studies on animals found that the administration of 10 mg/kg body weight melatonin for 3 days to animals with induced NEC-like lesions significantly reduced the severity of the disease by decreasing cytokine levels and stimulating antioxidant enzyme activity [45, 48].

#### *2.2.3. Effects of oxidative stress in neonatal asphyxia and hypoxic ischemic encephalopathy*

In the brain, there are some particularities that increase the vulnerability of this tissue to oxidative stress: cell membranes are rich in polyunsaturated fatty acids, the brain is poor in catalase and SOD, and there are some brain areas rich in iron. Nerve cell injury in the context of asphyxia will induce iron release [13]. Given the low antioxidant defense, the release of low molecular mass iron will allow the formation of hydroxyl radical and lipid peroxidation. By lipid peroxidation, free radicals induce molecular damage, including endothelial factor destruction. The low level of antioxidants in the serum seems to be directly involved in the genesis of cerebral hemorrhage [17]. Transferrin and ceruloplasmin levels can be indicators of the risk of cerebral hemorrhage in newborns with asphyxia at birth [17, 18]. In prematures with asphyxia, a decrease in these enzymes will precede cerebral hemorrhage. Ceruloplasmin acts as a strong ferroxidase, catalyzing iron oxidation to less reactive ions. The antioxidant defense capacity can be exceeded, making transferrin inadequate for binding, with the release of low molecular mass iron, which will subsequently induce lipid peroxidation. In case of iron overloading or severe oxidative stress, the antioxidant defense capacity is exceeded, making nontransferrin bonds available, with the release of low molecular mass iron, followed by lipid peroxidation. This may occur even if transferrin is not completely iron saturated [42].

Hypoxic ischemic brain injury is a long process that starts with the occurrence of the insult and continues during the recovery period, after reperfusion. This reperfusion process represents a paradoxical tissue response: the appearance of oxidative lesions in a hypoxic tissue, poorly perfused after circulatory stabilization, at the time of its perfusion with oxygenated blood. This stage of reperfusion will cause an excessive production of free radicals generating new lesions after the initial oxidative attack. In the premature, an increased risk of cerebral ischemia persists postnatally, during the first week of life [19, 26, 27].

mediated by factors released from the ischemic intestine, including endotoxins, histamine, prostaglandins, and superoxide anion, which result from oxygen metabolization. Superoxide will induce lipid peroxidation with the disruption of the integrity of capillaries and epithelial cells. Mucosal lesions characterized by edema, hemorrhage, ulceration, and necrosis may be induced experimentally by a combination of oxidants (hypoxanthine and xanthine oxidase) [14]. Superoxide dismutase can experimentally prevent or attenuate the described lesions. The protective effect of SOD was experimentally observed in animals with superior mesenteric artery occlusion. The sequential development of intestinal enzymes: SOD and xanthine oxidase can be a determining factor of neonatal intestinal susceptibility to ischemic mucosal injury [13, 45]. Regarding NEC prevention, many studies performed on animals show the beneficial effect of melatonin in preventing oxidative stress involved in the development of NEC lesions. Melatonin acts by reducing the level of inflammatory cytokines and by stimulating the activity of antioxidant enzymes. A decrease in TNF-α and IL-1β levels was found in animals treated with melatonin. Melatonin also counteracts the reduction of intestinal motility generated by lipopolysaccharides. Melatonin is an indolamine produced by the pineal gland, having serotonin as a precursor. It is synthesized by serotonin-rich enterochromaffin cells in the digestive tract. This synthesis takes place postprandially in the digestive tube and

The pathophysiological mechanism of NEC is based on a hypoxic-ischemic process similar to that found in postasphyxia brain injury. A number of studies have demonstrated that the association of melatonin with misoprostol, a gastric mucosal protective agent, is more beneficial than melatonin therapy alone. These studies on animals found that the administration of 10 mg/kg body weight melatonin for 3 days to animals with induced NEC-like lesions significantly reduced the severity of the disease by decreasing cytokine levels and stimulating

In the brain, there are some particularities that increase the vulnerability of this tissue to oxidative stress: cell membranes are rich in polyunsaturated fatty acids, the brain is poor in catalase and SOD, and there are some brain areas rich in iron. Nerve cell injury in the context of asphyxia will induce iron release [13]. Given the low antioxidant defense, the release of low molecular mass iron will allow the formation of hydroxyl radical and lipid peroxidation. By lipid peroxidation, free radicals induce molecular damage, including endothelial factor destruction. The low level of antioxidants in the serum seems to be directly involved in the genesis of cerebral hemorrhage [17]. Transferrin and ceruloplasmin levels can be indicators of the risk of cerebral hemorrhage in newborns with asphyxia at birth [17, 18]. In prematures with asphyxia, a decrease in these enzymes will precede cerebral hemorrhage. Ceruloplasmin acts as a strong ferroxidase, catalyzing iron oxidation to less reactive ions. The antioxidant defense capacity can be exceeded, making transferrin inadequate for binding, with the release of low molecular mass iron, which will subsequently induce lipid peroxidation. In case of iron overloading or severe oxidative stress, the antioxidant defense capacity is exceeded, making nontransferrin bonds available, with the release of low molecular mass iron, followed by lipid

*2.2.3. Effects of oxidative stress in neonatal asphyxia and hypoxic ischemic encephalopathy*

peroxidation. This may occur even if transferrin is not completely iron saturated [42].

its level is 100 times higher than blood levels [45, 46].

antioxidant enzyme activity [45, 48].

102 Novel Prospects in Oxidative and Nitrosative Stress

In the reperfusion stage, ROS with a cell damaging effect are produced, in the first place by the conversion of xanthine dehydrogenase to xanthine oxidase. This process will result in the formation of thiol groups or proteolysis by activation of proteases in energetically depressed cells. In the reoxygenation stage, hypoxanthine is converted to uric acid under the action of xanthine oxidase, and superoxide and peroxide are released through the mediation of xanthine oxidase by molecular oxygen binding. Hydroxyl radical generated through the Fenton reaction will also be released, due to the catalytic effect of metals such as iron [42]. Perinatal hypoxic stress is a frequent cause of morbidity, mortality, and neurological damage in survivors. In the perinatal hypoxic context, several factors play a role in the pathogenesis of lesions: hypoxia with initial ROS formation, followed by ischemia-reperfusion, a stage at which arachidonic acid and phagocytes will be activated under the action of inflammation mediators. Thus, a vicious circle is created. ROS will be formed, followed by tissue lesions and the genesis of new free radicals [42, 43].

At CNS level, under asphyxia conditions, encephalopathy lesions are induced by activation of leukocytes or glial cells and release of new free radicals. Hypoxic lesions will be perpetuated by release of protein-unbound iron. Endothelial lesions, hemostasis abnormalities, and inflammatory lesions occur, as well as with an increase in anaerobic metabolism, lactic acid levels, brain damage as a result of oligodendroglial vulnerability, astrocyte dysfunction, N-methyl-D-aspartate receptor abnormalities, and synaptic damage [28, 29].

Mitochondrial DNA damage induces changes in respiratory chain proteins, with the formation of new free radicals and subsequent cell lesions. Neonatal cerebral hypoxia stimulates activin secretion, which in turn stimulates erythropoietin, resulting in the production of nucleated red blood cells [35, 43].

The types of lesions that occur in the brain as a result of hypoxia are variable. In the extremely immature brain, preoligodendrocytes and cell precursors of oligodendrocytes are affected. As the brain matures, the resistance of oligodendrocytes to oxidative stress increases due to an increase in antioxidant defense, as well as to the protein structure involved in programmed cell death.

After the hypoxic episode, there is an increase in the density of glutamate receptors, and mitochondrial calcium accumulation occurs, which will trigger apoptosis. Caspase 6 and, subsequently, caspase 3 are activated. NMDA receptor activation depresses the mitochondrial respiratory process and induces apoptosis—a process which is not found in adults, known as the NMDA paradox [41, 48].

Studies conducted by Peeters and Schulte regarding the activity of glutathione peroxidase in the cerebrospinal fluid of newborns with asphyxia and the neuron-specific enolase value evidenced a correlation between the glutathione peroxidase value and gestational age, as well as between the neuronal enolase value and the neurological evolution of perinatal asphyxia cases. The influence of the genetic factor on postischemic evolution was also demonstrated, as well as on the presence of a correlation with patient's sex, males being more predisposed to develop lesions compared to females.

should attempt to identify free radical scavengers that can be administered to newborns and could be useful in limiting oxidative stress, particularly in prematures, in whom antioxidant

Particularities of Oxidative Stress in Newborns http://dx.doi.org/10.5772/intechopen.73369 105

Oxidative stress at newborns has role in pathogenesis of different neonatal diseases. The oxidative stress is more severe in preterm than in term neonates. The antioxidant defense of preterm less developed than in term neonates, mainly the enzymatic antioxidant defense.

In the near future, studies are needed which should attempt to identify free radical scavengers that can be administered to newborns and could be useful in limiting oxidative stress, particularly in prematures, in whom antioxidant defense is impaired due to the end of pregnancy

Department of Neonatology, University of Medicine and Pharmacy, Cluj-Napoca, Romania

[1] Saugstad OD. Update on oxygen radical disease in neonatology. Current Opinion in

[2] Sullivan JL. Iron, plasma antioxidants, and the oxygen radical disease of prematurity.

[3] Saugstad OD. Chronic lung disease: The role of oxidative stress. Biologia Neonatorum.

[4] Raju TN, Langenberg P, Bhutani V, Quinn GE. Vitamin E prophylaxis to reduce rethinopaty of prematurity; a reappraisal of published trials. Journal of Pediatrics. 1997;

[5] Luukkainen P, Aejmelaeus R, Alho H, et al. Plasma chain–breaking antioxidants in preterm infants with good and poor short term outcome. Free Radical Research. 1999;

[6] Papp A, Nemeth I, Karg E, Papp E. Gluthatione status in retinopathy of prematurity.

defense is impaired due to the end of pregnancy before term [44, 47].

**3. Conclusion**

before term.

**Author details**

**References**

1998;**74**:21-28

**131**:844850

(3):189-197

Melinda Mátyás\* and Gabriela Zaharie

\*Address all correspondence to: melimatyas@yahoo.com

Obstetrics & Gynecology. 2001;**13**:147-153

Free Radical Biology & Medicine. 1999;**27**:738-743

American Journal of Diseases of Children. 1988;**142**:1341-1344

With the stabilization of newborns with asphyxia at birth, in the reoxygenation stage, the exposure of the diseased cell to a new oxidative attack follows. The use of 100% oxygen in resuscitation is an important source for the formation of ROS. Hyperoxia causes an increase in the activity of antioxidant enzymes such as catalase or superoxide dismutase, as well as an activation of enzymes in the glutathione reductase cycle: GSH-reductase and GSH-Stransferase. Some studies demonstrated that the urinary level of N-acetyl-glucosaminidase is correlated with the value of oxidized glutathione and is higher in newborns exposed to a FiO<sup>2</sup> of 100%. Based on these findings, resuscitation guidelines were changed in 2010, indicating to start neonatal resuscitation with atmospheric air, followed by an increase by titration in the FiO<sup>2</sup> value depending on the improvement of blood oxygen saturation [1, 3, 43].

In the study conducted in our unit, we found that lipid peroxidation in newborns with asphyxia was maintained at a high level on the third day compared to the first day of life, without a statistically significant difference. This high plateau value on day 3 was attributed to perinatal hypoxic stress and to the reoxygenation-reperfusion process. None of the newborns with asphyxia received hypothermia treatment, because at the time of the study, this therapeutic method was not available in our unit. The analysis of lipid peroxidation after neonatal asphyxia by gestational age groups evidenced more intense peroxidation in premature infants compared to term infants with asphyxia. We explained this process by the association of neonatal asphyxia with respiratory distress in the case of prematures, which involved oxygen therapy and respiratory support for its treatment, factors that increased oxidative stress and implicitly, malondialdehyde values. Regarding protein peroxidation, its markers are found in high amounts in the plasma of prematures with asphyxia. Their high values are maintained until the seventh day of life [43]. In newborns with hypoxic ischemic encephalopathy in our study, protein peroxidation was more intense compared to the other newborns. Hypoxic injury is aggravated by protein-unbound iron release. In its presence, as part of the Fenton reaction, hydroxyl radicals are released, which will exert a strong toxic effect on the brain. ROS toxicity in newborns is enhanced by the increased production of ROS, their rapid tissue growth, and impaired antioxidant defense. In the developing brain, endothelial lesions, hemostasis abnormalities, inflammatory reaction, an increase of anaerobic metabolism occur, which are followed by lactic acid accumulation. Oligodendroglial injury, astrocyte dysfunction, and synaptic abnormalities will result [43, 44].

Oxidative stress markers have an important predictive value for neuronal injury in newborns at risk. The correlation between the values of oxidative stress markers and imaging electrophysiological brain changes, near-infrared spectroscopy (NIRS) aspects, and the degree of neurological impairment is not currently described in the literature. This is why further studies are required in this respect. Also, research on therapies protecting against cerebral oxidative stress after perinatal asphyxia has not reported a clearly beneficial medication at this stage of knowledge. The only therapy currently applied with positive, beneficial results in perinatal asphyxia is controlled hypothermia. In the near future, studies are needed which should attempt to identify free radical scavengers that can be administered to newborns and could be useful in limiting oxidative stress, particularly in prematures, in whom antioxidant defense is impaired due to the end of pregnancy before term [44, 47].

### **3. Conclusion**

as between the neuronal enolase value and the neurological evolution of perinatal asphyxia cases. The influence of the genetic factor on postischemic evolution was also demonstrated, as well as on the presence of a correlation with patient's sex, males being more predisposed to

With the stabilization of newborns with asphyxia at birth, in the reoxygenation stage, the exposure of the diseased cell to a new oxidative attack follows. The use of 100% oxygen in resuscitation is an important source for the formation of ROS. Hyperoxia causes an increase in the activity of antioxidant enzymes such as catalase or superoxide dismutase, as well as an activation of enzymes in the glutathione reductase cycle: GSH-reductase and GSH-Stransferase. Some studies demonstrated that the urinary level of N-acetyl-glucosaminidase is correlated with the value of oxidized glutathione and is higher in newborns exposed to a FiO<sup>2</sup> of 100%. Based on these findings, resuscitation guidelines were changed in 2010, indicating to start neonatal resuscitation with atmospheric air, followed by an increase by titration in the

value depending on the improvement of blood oxygen saturation [1, 3, 43].

In the study conducted in our unit, we found that lipid peroxidation in newborns with asphyxia was maintained at a high level on the third day compared to the first day of life, without a statistically significant difference. This high plateau value on day 3 was attributed to perinatal hypoxic stress and to the reoxygenation-reperfusion process. None of the newborns with asphyxia received hypothermia treatment, because at the time of the study, this therapeutic method was not available in our unit. The analysis of lipid peroxidation after neonatal asphyxia by gestational age groups evidenced more intense peroxidation in premature infants compared to term infants with asphyxia. We explained this process by the association of neonatal asphyxia with respiratory distress in the case of prematures, which involved oxygen therapy and respiratory support for its treatment, factors that increased oxidative stress and implicitly, malondialdehyde values. Regarding protein peroxidation, its markers are found in high amounts in the plasma of prematures with asphyxia. Their high values are maintained until the seventh day of life [43]. In newborns with hypoxic ischemic encephalopathy in our study, protein peroxidation was more intense compared to the other newborns. Hypoxic injury is aggravated by protein-unbound iron release. In its presence, as part of the Fenton reaction, hydroxyl radicals are released, which will exert a strong toxic effect on the brain. ROS toxicity in newborns is enhanced by the increased production of ROS, their rapid tissue growth, and impaired antioxidant defense. In the developing brain, endothelial lesions, hemostasis abnormalities, inflammatory reaction, an increase of anaerobic metabolism occur, which are followed by lactic acid accumulation. Oligodendroglial injury, astrocyte dysfunc-

Oxidative stress markers have an important predictive value for neuronal injury in newborns at risk. The correlation between the values of oxidative stress markers and imaging electrophysiological brain changes, near-infrared spectroscopy (NIRS) aspects, and the degree of neurological impairment is not currently described in the literature. This is why further studies are required in this respect. Also, research on therapies protecting against cerebral oxidative stress after perinatal asphyxia has not reported a clearly beneficial medication at this stage of knowledge. The only therapy currently applied with positive, beneficial results in perinatal asphyxia is controlled hypothermia. In the near future, studies are needed which

develop lesions compared to females.

104 Novel Prospects in Oxidative and Nitrosative Stress

tion, and synaptic abnormalities will result [43, 44].

FiO<sup>2</sup>

Oxidative stress at newborns has role in pathogenesis of different neonatal diseases. The oxidative stress is more severe in preterm than in term neonates. The antioxidant defense of preterm less developed than in term neonates, mainly the enzymatic antioxidant defense.

In the near future, studies are needed which should attempt to identify free radical scavengers that can be administered to newborns and could be useful in limiting oxidative stress, particularly in prematures, in whom antioxidant defense is impaired due to the end of pregnancy before term.

### **Author details**

Melinda Mátyás\* and Gabriela Zaharie

\*Address all correspondence to: melimatyas@yahoo.com

Department of Neonatology, University of Medicine and Pharmacy, Cluj-Napoca, Romania

### **References**


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[11] Yu XQ, Medbo S, Saugstad OD. Sodium nitroprusside prevents oxygen-free-radicalincluded pulmonary vasoconstriction in newborn piglets. Biology of the Neonate. 1999;

[12] Hardy P, Dumont I, Bhattacharya M, et al. Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: A basis for ischemic retinopathy. Cardiovascular

[13] Saugstad OD. Hypoxanthine as a measurement of hypoxia. Pediatric Research. 1975;

[14] Supnet MC, David-Cu R, Walther FJ. Plasma xanthine oxidase and lipid hydroperoxide

[15] Rogers S, Witz G, Anwar M, et al. Antioxidant capacity and oxygen radical disease in the preterm newborn. Archives of Pediatrics & Adolescent Medicine. 2000;**154**:544-548

[16] Buonocore G, Perrone S, Longini M, et al. Total hydroperoxide and advanced oxidation protein products in preterm hypoxidic babies. Pediatric Research. 2000;**47**:221-224 [17] Domergues MA, Gallego J, Evrard P, Gressens P. Iron supplementation aggravates periventricular cystic white matter lesions in newborn mice. European Journal of Pediatric

[18] Palmer C, Menzies SL, Roberts RL, et al. Changes in iron histochemistry after hypoxicischemic brain injury in the neonatal rat. Journal of Neuroscience Research. 1999;**56**:60-71

[19] Vento M, Garcia-Sala F, Vina J, et al. The use of room air ventilation enhances the establishment of a sustained spontaneous respiratory pattern after perinatal asphyxia.

[20] Rosenfeld WN, Davis JM, Parton L, Richter SE, Price A, Flaster E, Kassem N. Safety and pharmacokinetics of human recombinant superoxide dismutase administered intratracheally to premature neonates with respiratory distress syndrome. Pediatrics.

levels in the preterm infants. Pediatric Research. 1994;**36**:283-287

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Pediatric Research. 2000;**47**:640-645

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mentation. Pediatric Research. 2000;**48**:84-90


[34] Saugstad OD. Bronchopulmonary dysplasia – Oxidative stress and antioxidants. Seminars in Neonatology. 2003;**8**:39-49

**Chapter 6**

**Provisional chapter**

**Oxidative Stress in Hadrontherapy**

**Oxidative Stress in Hadrontherapy**

DOI: 10.5772/intechopen.73238

Conventional radiotherapy has shown its efficiency since decades with large progresses during the 1990s. However, for 15–20% of treated patients, there is no prognosis improvement either due to tumor radiation resistance and/or to side effects on normal tissues representing the limiting dose given during a radiotherapy protocol. A new modality of radiation therapy has emerged representing a technological breakthrough: hadrontherapy. This regroups mainly proton and carbon ion therapy. Dose deposit is in favor of hadrons compared to photons as it occurs at a precise depth in human body sparing upstream and downstream normal tissues. Mechanisms of action of photons and hadrons are different. When photons mainly act by water radiolysis—producing e− aq, H●, ●OH, H2

transfer of ion energy to biological macromolecules. Moreover, efficiency of carbon ions is considered threefold higher (1.1 for protons) than X-rays in killing tumor cells, whereas it is considered lower for normal cells. These findings suggest strong advantages of hadrontherapy compared to conventional radiotherapy. However, some recent studies tend to show a stronger increase in oxidative stress in normal cells after protons or carbon

**Keywords:** hadrontherapy, oxidative stress, carbon ions, protons, DNA damage, tumor killing efficiency, normal tissue toxicity, senescence, inflammation

●−…, carbon ions and protons mainly act by direct effects, i.e. by direct

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Oxidative stress is of major interest in killing tumor cells. In this way, radiation therapy is one of the most used modality for cancer treatment (**Figure 1**). Ionizing radiations lead to the production of deleterious reactive oxygen species that overcome antioxidant systems resulting in tumor cell death. On the 14 million of new cancer cases each year in the world, about half of

http://dx.doi.org/10.5772/intechopen.73238

Carine Laurent

**Abstract**

O2 , O2

ions than X-rays.

them will benefit from this treatment [1].

**1. Introduction**

Carine Laurent


### **Chapter 6**

**Provisional chapter**

## **Oxidative Stress in Hadrontherapy**

**Oxidative Stress in Hadrontherapy**

#### Carine Laurent Carine Laurent Additional information is available at the end of the chapter

[34] Saugstad OD. Bronchopulmonary dysplasia – Oxidative stress and antioxidants. Semi-

[35] Szabo M, Vasarhelyi B, Balla G, Szabo T, Machay T, Tulassay T. Acute postnatal increase of extracellular antioxidant defence of neonates: The role of iron metabolism. Acta Pae-

[36] The STOP-ROP Multicenter Study Group. Supplemental therapeutic oxygen for Prethreshold retinopathy of prematurity (STOP-ROP), a randomized, controlled trial. I:

[37] Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radical Biology and

[38] Gitto E, Reiter RJ, Xian-Tan D, Barberi I. Respiratory distress syndrome in the newborn:

[39] Grobben DJH, Lindeman E, Houdkamp RM, Moison JT, Berger WHM. Markers of oxidative stress and antioxidant activity in plasma and erythrocytes in neonates with respi-

[40] Sola A, Rodigo MR, Deulofeut R. Oxygen as a neonatal health hazard: Call for détente in

[41] Perrone S, Tataranno ML, Negro S, Longini M, Marzocchi B, Proietti F, Iacoponi F, Capitani S, Buonocore G. Early identification of the risk for free radical-related diseases

[42] Torres-Cuevasa I, Parra-Llorcaa A, S-Illanaa A, Nuñez-Ramiroa A. Oxygen and oxida-

[43] Buonocore G, Groenendaal F. Anti-oxidant strategies. Seminars in Fetal & Neonatal

[44] Tataranno ML, Perrone S, Buonocore G. Plasma biomarkers of oxidative stress in neona-

[45] Marseglia et al. Oxidative stress and necrotizing Enterocolitis. American Journal of Peri-

[46] Thor PJ, Krolczyk G, Gil K, Zurowski D, Nowak L. Melatonin and serotonin effects on gastrointestinal motility. Journal of Physiology and Pharmacology. 2007;**58**(Suppl 6):

[47] Guven A, Uysal B, Gundogdu G, Oztas E, Ozturk H, Korkmaz A. Melatonin ameliorates necrotizing enterocolitis in a neonatal ratmodel. Journal of Pediatric Surgery. 2011;

[48] Negi R, Pande D, Karki R, Kumar A, Khanna RS, Khanna HD. A Novel approach to study oxidative stress in neonatal respiratory distress syndrome. BBA Clinical, 2015;**3**:

Role of oxidative stress. Intensive Care Medicine. 2001;**27**:1116-1123

ratory distress syndrome. Acta Paediatrica. 1997;**86**(12):1356-1362

in preterm newborns. Early Human Development. 2010;**86**:241-244

tive stress in the perinatal period. Redox Biology. 2017;**12**:674-681

tal brain injury. Clinics in Perinatology. 2015 sept;**42**:529-539

clinical practice. Acta Paediatrica. 2007 jun;**96**(6):801-812

nars in Neonatology. 2003;**8**:39-49

Primary outcomes. Pediatrics. 2000;**105**:295-310

diatrica. 2001;**90**:1167-1170

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Medicine. 2000;**28**:463-499

Medicine. 2007;**12**:287-295

natology 2015;**32**:905-909

97-103

65-69

**46**(11):2101-2107

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73238

#### **Abstract**

Conventional radiotherapy has shown its efficiency since decades with large progresses during the 1990s. However, for 15–20% of treated patients, there is no prognosis improvement either due to tumor radiation resistance and/or to side effects on normal tissues representing the limiting dose given during a radiotherapy protocol. A new modality of radiation therapy has emerged representing a technological breakthrough: hadrontherapy. This regroups mainly proton and carbon ion therapy. Dose deposit is in favor of hadrons compared to photons as it occurs at a precise depth in human body sparing upstream and downstream normal tissues. Mechanisms of action of photons and hadrons are different. When photons mainly act by water radiolysis—producing e− aq, H●, ●OH, H2 O2 , O2 ●−…, carbon ions and protons mainly act by direct effects, i.e. by direct transfer of ion energy to biological macromolecules. Moreover, efficiency of carbon ions is considered threefold higher (1.1 for protons) than X-rays in killing tumor cells, whereas it is considered lower for normal cells. These findings suggest strong advantages of hadrontherapy compared to conventional radiotherapy. However, some recent studies tend to show a stronger increase in oxidative stress in normal cells after protons or carbon ions than X-rays.

DOI: 10.5772/intechopen.73238

**Keywords:** hadrontherapy, oxidative stress, carbon ions, protons, DNA damage, tumor killing efficiency, normal tissue toxicity, senescence, inflammation

#### **1. Introduction**

Oxidative stress is of major interest in killing tumor cells. In this way, radiation therapy is one of the most used modality for cancer treatment (**Figure 1**). Ionizing radiations lead to the production of deleterious reactive oxygen species that overcome antioxidant systems resulting in tumor cell death. On the 14 million of new cancer cases each year in the world, about half of them will benefit from this treatment [1].

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

**Figure 1.** Cancer treatment: focus on radiotherapy.

Conventional radiotherapy—by photons (γ- or X-rays)—has known a revolution since the 1990s, mainly thanks to progresses in imagery, computer sciences and robotics. In this way, new modalities of radiation therapy occurred: intensity-modulated radiotherapy (IMXRT), image-guided radiation therapy (IGRT) and respiratory-gated radiotherapy: the 4D radiotherapy. These new kinds of treatments allowed to overcome the main difficulties encountered in conventional radiotherapy: the exponential dose deposit which leads to an overdose in normal tissues upstream and downstream from the tumor (**Figure 2**, left panel) [2].

tails. In this way, normal surrounding tissues received a percentage of dose that could be non-

**Figure 2.** Dose deposit after X-rays in comparison to hadrons. Left panel: Dose deposit of X-rays according to depth in human body. Right panel: Dose deposit of hadrons according to depth in human body. Bragg peak: Continuous black

Radiations lead to a wide range of oxidative damage to DNA, lipids and proteins. Effects of photons were widely studied *in vitro* and *in vivo* since decades. When photons mainly act

carbon ions and protons mainly act by direct effects, i.e. by direct transfer of ion energy to

We propose to develop involvements of oxidative stress in: (i) tumor cell killing efficiency of hadrontherapy and (ii) side effects of hadrontherapy—secondary tumors and normal tissue

The main advantages of the use of hadrons in comparison with photons are their superior dose localization, their efficiency against radioresistant and hypoxic tumors and the ability to

Due to their high charge, heavy ions lead to concentrate ionizations when they cross matter. These concentrate ionizations result in concentrate oxidative damage. On the contrary, when photons (low LET) encounter matter, they produce low ionization densities. Tumor cell killing is more efficient with hadrons as, for example, clusters of DNA damage are produced leading to difficult DNA repair in comparison with photons producing more easily repaired SSB (single-strand breaks). Efficiency—RBE for Relative Biological Efficiency—of carbon ions

**2. Oxidative stress and tumor cell killing efficiency of hadrontherapy**

aq, H●, ●OH, H2

, H2 O2 , H<sup>+</sup> , OH− , O2 ●−…,

Oxidative Stress in Hadrontherapy

111

http://dx.doi.org/10.5772/intechopen.73238

negligible according to the tumor size and localization.

by indirect effects, i.e. water radiolysis—producing e−

**2.1. Interest of protons and carbon ions in clinics**

biological macromolecules.

line. SOBP: Dotted line.

shorten treatment planning.

injury.

In parallel, therapy by accelerated hadrons was developed since the 1950s (Berkeley, United States). Hadronic particles regroup neutrons, protons, pions, antiprotons, helium, lithium, boron, carbon and oxygen ions. The major interest of protons and heavy ions (mass greater than helium) lies in the profile of dose deposit: the Bragg peak (**Figure 2**, right panel). Contrary to conventional radiations, dose distribution is in favor of normal surrounding tissues as the maximum of dose is deposited at a precise depth in the matter with a larger peak for protons than for carbon ions. However, a plateau phase does exist upstream from the peak, resulting in a small proportion of dose deposition in normal tissues preceding the tumor, as well as a fragmentation tail downstream from the peak (except for protons which cannot fragment in smaller particles). Moreover, in the case of heavy ions, their fragmentation when encountering matter lead to secondary particles, which properties are different in terms of LET (linear transfer energy) and biological effects. In addition, to treat the whole tumor volume, hadron beam energy and direction are modified to spread the peak: SOBP, Spread Out Bragg Peak (**Figure 2**, right panel). This leads to an addition of plateau phases as well as fragmentation

**Figure 2.** Dose deposit after X-rays in comparison to hadrons. Left panel: Dose deposit of X-rays according to depth in human body. Right panel: Dose deposit of hadrons according to depth in human body. Bragg peak: Continuous black line. SOBP: Dotted line.

tails. In this way, normal surrounding tissues received a percentage of dose that could be nonnegligible according to the tumor size and localization.

Radiations lead to a wide range of oxidative damage to DNA, lipids and proteins. Effects of photons were widely studied *in vitro* and *in vivo* since decades. When photons mainly act by indirect effects, i.e. water radiolysis—producing e− aq, H●, ●OH, H2 , H2 O2 , H<sup>+</sup> , OH− , O2 ●−…, carbon ions and protons mainly act by direct effects, i.e. by direct transfer of ion energy to biological macromolecules.

We propose to develop involvements of oxidative stress in: (i) tumor cell killing efficiency of hadrontherapy and (ii) side effects of hadrontherapy—secondary tumors and normal tissue injury.

### **2. Oxidative stress and tumor cell killing efficiency of hadrontherapy**

The main advantages of the use of hadrons in comparison with photons are their superior dose localization, their efficiency against radioresistant and hypoxic tumors and the ability to shorten treatment planning.

#### **2.1. Interest of protons and carbon ions in clinics**

Conventional radiotherapy—by photons (γ- or X-rays)—has known a revolution since the 1990s, mainly thanks to progresses in imagery, computer sciences and robotics. In this way, new modalities of radiation therapy occurred: intensity-modulated radiotherapy (IMXRT), image-guided radiation therapy (IGRT) and respiratory-gated radiotherapy: the 4D radiotherapy. These new kinds of treatments allowed to overcome the main difficulties encountered in conventional radiotherapy: the exponential dose deposit which leads to an overdose

In parallel, therapy by accelerated hadrons was developed since the 1950s (Berkeley, United States). Hadronic particles regroup neutrons, protons, pions, antiprotons, helium, lithium, boron, carbon and oxygen ions. The major interest of protons and heavy ions (mass greater than helium) lies in the profile of dose deposit: the Bragg peak (**Figure 2**, right panel). Contrary to conventional radiations, dose distribution is in favor of normal surrounding tissues as the maximum of dose is deposited at a precise depth in the matter with a larger peak for protons than for carbon ions. However, a plateau phase does exist upstream from the peak, resulting in a small proportion of dose deposition in normal tissues preceding the tumor, as well as a fragmentation tail downstream from the peak (except for protons which cannot fragment in smaller particles). Moreover, in the case of heavy ions, their fragmentation when encountering matter lead to secondary particles, which properties are different in terms of LET (linear transfer energy) and biological effects. In addition, to treat the whole tumor volume, hadron beam energy and direction are modified to spread the peak: SOBP, Spread Out Bragg Peak (**Figure 2**, right panel). This leads to an addition of plateau phases as well as fragmentation

in normal tissues upstream and downstream from the tumor (**Figure 2**, left panel) [2].

**Figure 1.** Cancer treatment: focus on radiotherapy.

110 Novel Prospects in Oxidative and Nitrosative Stress

Due to their high charge, heavy ions lead to concentrate ionizations when they cross matter. These concentrate ionizations result in concentrate oxidative damage. On the contrary, when photons (low LET) encounter matter, they produce low ionization densities. Tumor cell killing is more efficient with hadrons as, for example, clusters of DNA damage are produced leading to difficult DNA repair in comparison with photons producing more easily repaired SSB (single-strand breaks). Efficiency—RBE for Relative Biological Efficiency—of carbon ions is considered twofold to threefold higher (1.1 for protons) than X-rays in killing tumor cells. These RBE are calculated for a percentage of clonogenic survival of 10%. However, experiments leading to these values were performed under a broad range of conditions, among other things: LET or cell cycle phase—cell irradiation at confluence stage or during exponential phase. In this way, higher RBE than 3 were found—up to 5, for example, 3.3 for normal human skin fibroblasts exposed at confluence stage to mimic skin physiology to carbon ions in the plateau phase before Bragg peak as it would be the case during radiotherapy [3]. Biological interactions of protons and carbon ions being a lot more complex than photons, and to improve hadrontherapy, there is a need of a better knowledge of biological effects, at early and late times, of hadrons according to LET, fractionation, cell type, oxygenation, cell cycle phase, etc. (for review [4]). Due to the favorable dose deposit profile, this kind of therapy is recommended for unresectable and radioresistant tumors. Until now, more than 110,000 patients have been treated by proton therapy and 15,000 patients by carbon ion therapy.

X-ray-irradiated cells. Moreover, Weyrather et al. [33] highlighted that carbon ion RBE was related to cell repair capacity. The role of HR (homologous recombination) was highlighted after proton irradiation as deficiency in this pathway leads to a sensitization of cells to protons [34]. DNA repair by the Ku-dependent NHEJ (non-homologous end joining) pathway was shown as inhibited by high-LET irradiation [35]: the yield of DSB should be the same after low- or high-LET irradiation but high-LET induced smaller fragments inhibiting the efficient binding of Ku to DSB fragment. However, there are reports of a primordial role of NHEJ after carbon ions as inhibition of DNA-PKcs led to a sensitization of cancer cells to carbon ions [36]. Recent report pointed out another response to DNA damage after carbon ion irradiation: mitotic catastrophe. Kobayashi et al. [37] demonstrated that mitotic catastrophe phenomenon was induced in a larger manner after carbon ions than after X-rays in 20 human cancer cell lines, whereas apoptosis and senescence were unchanged between both radiation types.

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http://dx.doi.org/10.5772/intechopen.73238

Hypoxic tumor cells are reoxygenated during radiotherapy treatment, and this reoxygenation plays an important role on treatment efficacy [38]. Hypoxic tumors resist to X-rays, whereas carbon ion exposure remains efficient [39]. The same study showed that this could come from a faster reoxygenation of tumors after carbon ion irradiation compared to X-rays. This was confirmed by Oya et al. [40] and also by Fukawa et al. [41] by pO2 measurements in mouse fibrosarcomas. Recently, Wozny et al. [42] have shown that hypoxia-induced factor HIF-1α, whose role was demonstrated in radioresistance to conventional radiotherapy, is expressed earlier in carbon irradiated cancer stem cells—subpopulation of head and neck squamous cell carcinoma—localized in tumor hypoxic areas. In the presence of oxygen, ROS quantity increases leading possibly to a major oxidative stress and so to a stronger attack of biological macromolecules. Oxygen effect could play a major role in the difference observed between carbon ion and photon responses as hypoxia leads to a decrease in DSB repair capacity [43]. Hirayama et al. [44] observed a decrease in DNA damage after hypoxia but this was much less significant after carbon ion than after X-ray irradiation. Moreover, the same study demonstrated that repaired DSB percentage was unchanged after carbon ion irradiation in hypoxic conditions, which is not the case for X-rays. The authors concluded that DSB repair plays an important role in oxygen effect as this effect was decreased after carbon ion irradiation compared to X-rays. This could be related to a stable effect of oxygen on DSB during the time after

Studies are controversial concerning protons. The use of edaravone, a radical scavenger, did not decrease DNA DSB formation in MOLT-4 tumor cells after protons as it was the case for X-rays leading to conclude that radical-induced indirect DNA damage was lower with protons than with X-rays [45]. However, Baran et al. [46] showed that proton irradiation led to a disruption of the electron flow in the complex I of the mitochondrial respiratory chain in human leukemia Jurkat T cell, and the use of antioxidants in HeLa cancer cell line allowed an

carbon ion irradiation, whereas it decreases after X-ray irradiation.

attenuation of the enhancement of radiation-activated gene expression [47].

**2.4. Role of oxidative stress in hadrontherapy efficiency**

**2.3. Oxygen effect and carbon ions**

Concerning protons, numbers of pediatric tumors were treated by protons as the dose deposit should be favorable for normal surrounding tissues: medulloblastoma [5, 6], rhabdomyosarcoma [7, 8], craniopharyngioma [9], etc. There is a trend to extend the indications for proton therapy from already treated skull base [10, 11] and brain [12–14] tumors to prostate [15–17], lung [18–20], head and neck (for review, [21]), gastrointestinal (for review, [22]) and breast [23] cancers. Compared to conventional radiotherapy, proton therapy obtained the same results in terms of tumor local control (for review, [24]). The superiority of protons is still discussed, except in large ocular melanomas, chordomas and chondrosarcomas [25].

The main experienced facilities providing carbon ion beams and treating a big number of patients are: NIRS (Japan) and GSI and then HIT (Germany). The main indications were, as for protons, not only pediatric cancers but also bone and soft tissue sarcomas; head and neck cancers; pancreas, prostate and cervix cancers; hepatocellular carcinomas and glioblastoma (for review, [26–28]). Carbon ion therapy present significant advantages, but, due to a lack of available data in the literature, clinical evidences are still lacking.

#### **2.2. DNA damage and repair, mitotic catastrophe**

Hadrons are considered as acting mainly by direct effects. Carbon ions are particularly deleterious in terms of cell survival, viability and apoptosis, even on very radioresistant tumor cell lines [29, 30]. This efficiency to kill tumor cells could come from the type of damage produced by carbon ions: DSB (double-strand breaks) and clustered DNA damage considered as difficult to repair. Clusters of damage could be a criterion explaining ion irradiation efficiency as it was shown that cluster number increases with LET. However, Hada et al. [31] have shown that DNA damage (DSB, abasic sites, oxidized bases) number decreased in genomic DNA irradiated at high LET and DSB was more frequent than other damage after charged particles, even low-LET protons, than after X-rays. In the same manner, Heilmann et al. [32] demonstrated that carbon ion irradiation (LET from 14 to 400 keV/μm) did not generate more DSB than X-rays (kV) with a maximum of about 38 DSB/Gy/cell. A possible explanation for the strong RBE of carbon ions could be related to DNA damage repair. Moertel et al. [29] showed that residual DSB were more numerous in ion-irradiated human glioblastoma cells than in X-ray-irradiated cells. Moreover, Weyrather et al. [33] highlighted that carbon ion RBE was related to cell repair capacity. The role of HR (homologous recombination) was highlighted after proton irradiation as deficiency in this pathway leads to a sensitization of cells to protons [34]. DNA repair by the Ku-dependent NHEJ (non-homologous end joining) pathway was shown as inhibited by high-LET irradiation [35]: the yield of DSB should be the same after low- or high-LET irradiation but high-LET induced smaller fragments inhibiting the efficient binding of Ku to DSB fragment. However, there are reports of a primordial role of NHEJ after carbon ions as inhibition of DNA-PKcs led to a sensitization of cancer cells to carbon ions [36]. Recent report pointed out another response to DNA damage after carbon ion irradiation: mitotic catastrophe. Kobayashi et al. [37] demonstrated that mitotic catastrophe phenomenon was induced in a larger manner after carbon ions than after X-rays in 20 human cancer cell lines, whereas apoptosis and senescence were unchanged between both radiation types.

### **2.3. Oxygen effect and carbon ions**

is considered twofold to threefold higher (1.1 for protons) than X-rays in killing tumor cells. These RBE are calculated for a percentage of clonogenic survival of 10%. However, experiments leading to these values were performed under a broad range of conditions, among other things: LET or cell cycle phase—cell irradiation at confluence stage or during exponential phase. In this way, higher RBE than 3 were found—up to 5, for example, 3.3 for normal human skin fibroblasts exposed at confluence stage to mimic skin physiology to carbon ions in the plateau phase before Bragg peak as it would be the case during radiotherapy [3]. Biological interactions of protons and carbon ions being a lot more complex than photons, and to improve hadrontherapy, there is a need of a better knowledge of biological effects, at early and late times, of hadrons according to LET, fractionation, cell type, oxygenation, cell cycle phase, etc. (for review [4]). Due to the favorable dose deposit profile, this kind of therapy is recommended for unresectable and radioresistant tumors. Until now, more than 110,000 patients have been treated by proton therapy and 15,000 patients by carbon ion therapy.

Concerning protons, numbers of pediatric tumors were treated by protons as the dose deposit should be favorable for normal surrounding tissues: medulloblastoma [5, 6], rhabdomyosarcoma [7, 8], craniopharyngioma [9], etc. There is a trend to extend the indications for proton therapy from already treated skull base [10, 11] and brain [12–14] tumors to prostate [15–17], lung [18–20], head and neck (for review, [21]), gastrointestinal (for review, [22]) and breast [23] cancers. Compared to conventional radiotherapy, proton therapy obtained the same results in terms of tumor local control (for review, [24]). The superiority of protons is still

The main experienced facilities providing carbon ion beams and treating a big number of patients are: NIRS (Japan) and GSI and then HIT (Germany). The main indications were, as for protons, not only pediatric cancers but also bone and soft tissue sarcomas; head and neck cancers; pancreas, prostate and cervix cancers; hepatocellular carcinomas and glioblastoma (for review, [26–28]). Carbon ion therapy present significant advantages, but, due to a lack of

Hadrons are considered as acting mainly by direct effects. Carbon ions are particularly deleterious in terms of cell survival, viability and apoptosis, even on very radioresistant tumor cell lines [29, 30]. This efficiency to kill tumor cells could come from the type of damage produced by carbon ions: DSB (double-strand breaks) and clustered DNA damage considered as difficult to repair. Clusters of damage could be a criterion explaining ion irradiation efficiency as it was shown that cluster number increases with LET. However, Hada et al. [31] have shown that DNA damage (DSB, abasic sites, oxidized bases) number decreased in genomic DNA irradiated at high LET and DSB was more frequent than other damage after charged particles, even low-LET protons, than after X-rays. In the same manner, Heilmann et al. [32] demonstrated that carbon ion irradiation (LET from 14 to 400 keV/μm) did not generate more DSB than X-rays (kV) with a maximum of about 38 DSB/Gy/cell. A possible explanation for the strong RBE of carbon ions could be related to DNA damage repair. Moertel et al. [29] showed that residual DSB were more numerous in ion-irradiated human glioblastoma cells than in

discussed, except in large ocular melanomas, chordomas and chondrosarcomas [25].

available data in the literature, clinical evidences are still lacking.

**2.2. DNA damage and repair, mitotic catastrophe**

112 Novel Prospects in Oxidative and Nitrosative Stress

Hypoxic tumor cells are reoxygenated during radiotherapy treatment, and this reoxygenation plays an important role on treatment efficacy [38]. Hypoxic tumors resist to X-rays, whereas carbon ion exposure remains efficient [39]. The same study showed that this could come from a faster reoxygenation of tumors after carbon ion irradiation compared to X-rays. This was confirmed by Oya et al. [40] and also by Fukawa et al. [41] by pO2 measurements in mouse fibrosarcomas. Recently, Wozny et al. [42] have shown that hypoxia-induced factor HIF-1α, whose role was demonstrated in radioresistance to conventional radiotherapy, is expressed earlier in carbon irradiated cancer stem cells—subpopulation of head and neck squamous cell carcinoma—localized in tumor hypoxic areas. In the presence of oxygen, ROS quantity increases leading possibly to a major oxidative stress and so to a stronger attack of biological macromolecules. Oxygen effect could play a major role in the difference observed between carbon ion and photon responses as hypoxia leads to a decrease in DSB repair capacity [43]. Hirayama et al. [44] observed a decrease in DNA damage after hypoxia but this was much less significant after carbon ion than after X-ray irradiation. Moreover, the same study demonstrated that repaired DSB percentage was unchanged after carbon ion irradiation in hypoxic conditions, which is not the case for X-rays. The authors concluded that DSB repair plays an important role in oxygen effect as this effect was decreased after carbon ion irradiation compared to X-rays. This could be related to a stable effect of oxygen on DSB during the time after carbon ion irradiation, whereas it decreases after X-ray irradiation.

#### **2.4. Role of oxidative stress in hadrontherapy efficiency**

Studies are controversial concerning protons. The use of edaravone, a radical scavenger, did not decrease DNA DSB formation in MOLT-4 tumor cells after protons as it was the case for X-rays leading to conclude that radical-induced indirect DNA damage was lower with protons than with X-rays [45]. However, Baran et al. [46] showed that proton irradiation led to a disruption of the electron flow in the complex I of the mitochondrial respiratory chain in human leukemia Jurkat T cell, and the use of antioxidants in HeLa cancer cell line allowed an attenuation of the enhancement of radiation-activated gene expression [47].

Concerning carbon ions, studies performed at high radiation doses (30 Gy) on murine squamous cell carcinoma and fibrosarcoma transplanted in mouse allowed to provide evidence of a strong upregulation of stress-responsive and cell communication genes after carbon ion irradiation compared to γ-rays [48]. Moreover, glutathione depletion in human squamous cell carcinoma cell lines potentiates the effects of carbon ion irradiation [49]. In this way, heavy ions do not act only by direct interaction with biological macromolecules but also by an induction of oxidative phenomena.

of carbon ion therapy versus conventional radiotherapy are still missing. Concerning bone and soft tissue sarcomas, toxicities were mostly decreased compared to conventional radiotherapy. A report showed that, on 78 patients treated by carbon ion therapy for unresectable osteosarcomas, grade 3 acute and late skin reactions were seen in 3 and 4 patients, respectively, and grade 4 skin and soft tissue reaction occurred in 3 patients [54]. However, for an escalation dose protocol, toxicities were considerably increased: 34 on 35 patients present acute skin reactions and 26 on 27 patients late skin reactions up to grade 4 [55]. For unresectable sarcomas: on 47 patients treated for non-sacral spinal sarcomas, 1 patient presented grades 3 and 4 late skin reaction and 1 patient grade 3 spinal cord reaction [56]; 6 patients and 2 patients on 188 patients with sacral chordomas presented grade 3 peripheral nerve and grade 4 skin toxicity, respectively [57]; and 4 patients on 75 patients treated for non-skull base chondrosarcomas report grade 3 or 4 late skin and soft tissue reactions [58]. Except bone and soft tissue sarcomas, most of toxicity was encountered for cervical cancers: a dose escalation protocol led to 18% of major gastrointestinal toxicity [59], and in another study, 8 patients on 29 developed bladder complications and 4 patients presented grade 4 rectal toxicities [60]. Clinical trials are in progress to register toxicities in the different facili-

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Induction of secondary tumors was also reported. Concerning protons, Chung et al. [61] studied 558 patients treated by protons and 558 treated by photons: second malignancies occurred in 5.2% of proton patients compared to 7.5% of photons. They concluded that proton therapy was not associated with a significantly increased risk of secondary malignancies compared with photon therapy, but the follow-up of these patients was only around 6 years after radiation therapy. This reduced risk of secondary malignancies due to proton therapy was confirmed by Sethi et al. [62], whereas there are no enough long-term reports after carbon therapy. Indeed, concerning carbon ions, literature on secondary tumors is still poor but a study pointed out that 30% of patients treated for cervical cancers developed distant metastases [63]; a case was reported of a brain tumor induced by heavy particle radiotherapy [64]. Preclinical studies, recently performed on mice exposed to carbon ions in comparison to photons, revealed that interstitial chromosome deletions were more increased in secondary cancers induced by carbon exposure [65]. They contradict previous results of Ando et al. [66] showing the same

Production of clusters of DNA damage can lead to mitotic catastrophe in fast or slow renewal normal tissues then leading to early or late toxicities. A lower immediate increase in DNA damage measured by alkaline comet assay was observed in confluent primary cultures of skin fibroblasts after carbon ion versus X-ray irradiation but a late increase in DNA damage was observed only after carbon ions whereas it was not the case after X-rays [3]. The lower immediate increase could be explained by the production of smaller fragments after carbon ions compared to X-rays whereas the late production of DNA damage after carbon ions could come from DNA repair. Indeed, micronucleus frequency—described as a result of impaired repair of DNA double-strand breaks—was 1.7-fold increased 24 hours after carbon irradiation compared to X-rays (unpublished results) and this increase persisted 2 weeks after irradiation (unpublished results) where a late wave of oxidative damage was observed [3].

induction in carbon locally irradiated mice of secondary tumors after γ-rays.

**3.2. DNA damage and repair, mitotic catastrophe**

ties providing carbon ion therapy [26].

### **3. Oxidative stress and side effects of hadrontherapy**

Radiotherapy aims to destroy cancer cells by the use of photons, protons or heavy ions. But this is a double-edged sword as it can also kill normal cells. Two types of side effects can appear: deterministic (pneumonitis, gastrointestinal or cutaneous syndrome, etc.) or stochastic (carcinogenesis and genetic effects). Indeed, dose deposit is exponential for photons so that the maximum of the dose is given at the entrance in the body before reaching tumor. In this way, normal tissues—present upstream and downstream from the tumor—receive ionizing radiations leading to ROS (reactive oxygen species) production. When normal cells are unable to detoxify these ROS, there is an imbalance leading to oxidative stress. Signaling pathways leading to inflammation maintain this process, therefore participating to side effects on normal tissues. It is considered that 5–10% of the general population exhibit acute or late adverse effects after radiotherapy. For example, pneumonitis is observed in 5–15% of patients irradiated for breast, lung and mediastinal tumors [50]. By the use of hadrons, organs at risk present around the tumor could be spared, and biological efficiency is considered higher in tumors than in normal tissues. In this way, treatment time could be shortened by hypofractionation of the total radiotherapy dose: 3 weeks compared to 6–7 weeks.

#### **3.1. Toxicity encountered in patients after proton or carbon ion therapy**

Toxicities of radiation therapy can not only occur at skin level (dermatitis, telangiectasia, etc.), cardiovascular and pulmonary level (pneumonitis, cardiovascular disease, etc.), gastrointestinal level (xerostomia, mucositis, esophagitis, enteritis, proctitis, emesis) and genitourinary level (cystitis, erectile dysfunction, vaginal dryness and stenosis, infertility and teratogenicity), but also at psychological level with fatigue and depression (for review [51]).

Proton therapy studies reported approximately the same proportion of early toxicities than photon therapy. However, comparative studies to photons are still necessary when possible. Recent reports tend to show a decrease in early and late toxicity: Romessser et al. [52] reported that proton therapy for head and neck cancers had significantly lower rates of early grade 2 (grade represents the degree of gravity of toxicity) or greater acute dysgeusia (5.6 vs. 65.2%), mucositis (16.7 vs. 52.2%) and nausea (11.1 vs. 56.5%). Yock et al. [53] reported ototoxicity and neuroendocrine deficit, but no cardiac, pulmonary or gastrointestinal late effects after treatment of medulloblastomas by protons, with a median follow-up of 7 years.

First studies of patients undergoing carbon ion therapy and presenting side effects were reported during the end of the 2000s (for review, [26]). Comparative studies on toxicities of carbon ion therapy versus conventional radiotherapy are still missing. Concerning bone and soft tissue sarcomas, toxicities were mostly decreased compared to conventional radiotherapy. A report showed that, on 78 patients treated by carbon ion therapy for unresectable osteosarcomas, grade 3 acute and late skin reactions were seen in 3 and 4 patients, respectively, and grade 4 skin and soft tissue reaction occurred in 3 patients [54]. However, for an escalation dose protocol, toxicities were considerably increased: 34 on 35 patients present acute skin reactions and 26 on 27 patients late skin reactions up to grade 4 [55]. For unresectable sarcomas: on 47 patients treated for non-sacral spinal sarcomas, 1 patient presented grades 3 and 4 late skin reaction and 1 patient grade 3 spinal cord reaction [56]; 6 patients and 2 patients on 188 patients with sacral chordomas presented grade 3 peripheral nerve and grade 4 skin toxicity, respectively [57]; and 4 patients on 75 patients treated for non-skull base chondrosarcomas report grade 3 or 4 late skin and soft tissue reactions [58]. Except bone and soft tissue sarcomas, most of toxicity was encountered for cervical cancers: a dose escalation protocol led to 18% of major gastrointestinal toxicity [59], and in another study, 8 patients on 29 developed bladder complications and 4 patients presented grade 4 rectal toxicities [60]. Clinical trials are in progress to register toxicities in the different facilities providing carbon ion therapy [26].

Induction of secondary tumors was also reported. Concerning protons, Chung et al. [61] studied 558 patients treated by protons and 558 treated by photons: second malignancies occurred in 5.2% of proton patients compared to 7.5% of photons. They concluded that proton therapy was not associated with a significantly increased risk of secondary malignancies compared with photon therapy, but the follow-up of these patients was only around 6 years after radiation therapy. This reduced risk of secondary malignancies due to proton therapy was confirmed by Sethi et al. [62], whereas there are no enough long-term reports after carbon therapy. Indeed, concerning carbon ions, literature on secondary tumors is still poor but a study pointed out that 30% of patients treated for cervical cancers developed distant metastases [63]; a case was reported of a brain tumor induced by heavy particle radiotherapy [64]. Preclinical studies, recently performed on mice exposed to carbon ions in comparison to photons, revealed that interstitial chromosome deletions were more increased in secondary cancers induced by carbon exposure [65]. They contradict previous results of Ando et al. [66] showing the same induction in carbon locally irradiated mice of secondary tumors after γ-rays.

#### **3.2. DNA damage and repair, mitotic catastrophe**

Concerning carbon ions, studies performed at high radiation doses (30 Gy) on murine squamous cell carcinoma and fibrosarcoma transplanted in mouse allowed to provide evidence of a strong upregulation of stress-responsive and cell communication genes after carbon ion irradiation compared to γ-rays [48]. Moreover, glutathione depletion in human squamous cell carcinoma cell lines potentiates the effects of carbon ion irradiation [49]. In this way, heavy ions do not act only by direct interaction with biological macromolecules but also by

Radiotherapy aims to destroy cancer cells by the use of photons, protons or heavy ions. But this is a double-edged sword as it can also kill normal cells. Two types of side effects can appear: deterministic (pneumonitis, gastrointestinal or cutaneous syndrome, etc.) or stochastic (carcinogenesis and genetic effects). Indeed, dose deposit is exponential for photons so that the maximum of the dose is given at the entrance in the body before reaching tumor. In this way, normal tissues—present upstream and downstream from the tumor—receive ionizing radiations leading to ROS (reactive oxygen species) production. When normal cells are unable to detoxify these ROS, there is an imbalance leading to oxidative stress. Signaling pathways leading to inflammation maintain this process, therefore participating to side effects on normal tissues. It is considered that 5–10% of the general population exhibit acute or late adverse effects after radiotherapy. For example, pneumonitis is observed in 5–15% of patients irradiated for breast, lung and mediastinal tumors [50]. By the use of hadrons, organs at risk present around the tumor could be spared, and biological efficiency is considered higher in tumors than in normal tissues. In this way, treatment time could be shortened by hypofractionation

Toxicities of radiation therapy can not only occur at skin level (dermatitis, telangiectasia, etc.), cardiovascular and pulmonary level (pneumonitis, cardiovascular disease, etc.), gastrointestinal level (xerostomia, mucositis, esophagitis, enteritis, proctitis, emesis) and genitourinary level (cystitis, erectile dysfunction, vaginal dryness and stenosis, infertility and teratogenic-

Proton therapy studies reported approximately the same proportion of early toxicities than photon therapy. However, comparative studies to photons are still necessary when possible. Recent reports tend to show a decrease in early and late toxicity: Romessser et al. [52] reported that proton therapy for head and neck cancers had significantly lower rates of early grade 2 (grade represents the degree of gravity of toxicity) or greater acute dysgeusia (5.6 vs. 65.2%), mucositis (16.7 vs. 52.2%) and nausea (11.1 vs. 56.5%). Yock et al. [53] reported ototoxicity and neuroendocrine deficit, but no cardiac, pulmonary or gastrointestinal late effects after treat-

First studies of patients undergoing carbon ion therapy and presenting side effects were reported during the end of the 2000s (for review, [26]). Comparative studies on toxicities

an induction of oxidative phenomena.

114 Novel Prospects in Oxidative and Nitrosative Stress

**3. Oxidative stress and side effects of hadrontherapy**

of the total radiotherapy dose: 3 weeks compared to 6–7 weeks.

**3.1. Toxicity encountered in patients after proton or carbon ion therapy**

ity), but also at psychological level with fatigue and depression (for review [51]).

ment of medulloblastomas by protons, with a median follow-up of 7 years.

Production of clusters of DNA damage can lead to mitotic catastrophe in fast or slow renewal normal tissues then leading to early or late toxicities. A lower immediate increase in DNA damage measured by alkaline comet assay was observed in confluent primary cultures of skin fibroblasts after carbon ion versus X-ray irradiation but a late increase in DNA damage was observed only after carbon ions whereas it was not the case after X-rays [3]. The lower immediate increase could be explained by the production of smaller fragments after carbon ions compared to X-rays whereas the late production of DNA damage after carbon ions could come from DNA repair. Indeed, micronucleus frequency—described as a result of impaired repair of DNA double-strand breaks—was 1.7-fold increased 24 hours after carbon irradiation compared to X-rays (unpublished results) and this increase persisted 2 weeks after irradiation (unpublished results) where a late wave of oxidative damage was observed [3]. Results obtained by Antonelli et al. [67] on quantification of γ-H2AX foci after carbon ion vs. X-ray irradiation in lung fibroblasts showed a longer persistence of γ-H2AX foci after carbon ion irradiation which is in agreement with a more difficult repair of DNA complex damage. Moreover, Gustafsson et al. [68] studied, in normal human skin fibroblasts, clustered DSB and non-DSB lesions which convert into DSB during preparation for pulsed-field gel electrophoresis and their results showed a similar increase after carbon ion or low-LET irradiation. It was recently shown that clustered DSB perturb normal human fibroblast DNA repair after high LET irradiation [69]. In confluent normal fibroblasts, accumulations of p53 at early times and p21 at late times were 2–3 times higher after carbon ions than after X-rays [70]. DNA repair proteins (hMRE11, p21, PCNA) were accumulated along ion trajectory in normal human fibroblasts and this was dependent of chromatin compaction [71].

and NAD(P)H dehydrogenase-quinone 1 expression [85]. *In vivo*, mouse whole-body carbon irradiation was shown to decrease glutathione level and to increase MDA content in testis one week after irradiation [86]. At longer term - 2 months after exposure - and in comparison to gamma-rays, mouse whole body irradiation led, in intestine and colon, to: (i) a persistent increase in ROS, mitochondrial cardiolipin oxidation and lipid damage; (ii) a late decrease in antioxidant enzyme activities [87]. The use of other antioxidants indirectly pointed out an important role of oxidative phenomena. Indeed, some antioxidants allowed to decrease effects of carbon ions in normal cells or tissues: curcumin ameliorates cognitive deficits in carbon-irradiated mice via SOD increase, MDA decrease and upregulation of important genes in oxidative stress pathways like heme oxygenase-1 and NAD(P)H quinine oxidoreductase 1 [88]; melatonin reduced carbon-induced apoptosis in mouse carbon-irradiated testes [89] and brain [90] via a decrease in carbonyl and MDA content and an increase in SOD and catalase activities; Dragon's blood decreased hydrogen peroxide and MDA levels and increased SOD activity and glutathione content in carbon-irradiated rat brain [91]. These last experiments

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provide indirect proofs of the major role of oxidative stress in hadrontherapy toxicity.

In normal human fibroblasts, radiation exposure lead to a G1 cell cycle arrest evolving in quiescence or senescence [92]. Premature senescence, or SIPS (stress-induced premature senescence), differs from replicative senescence. SIPS phenomenon was generally observed in fibroblasts exposed to prolonged or repeated stresses [93, 94] and was also shown after X-ray exposure [95, 96]. Naka et al. [96] showed, by the use of ATM mutated fibroblasts, that pathway leading to premature senescence in fibroblasts after oxidative stress or X-ray exposure could also be ATM-dependent and could act via p38MAPK and p16INK4A. After carbon ion exposure, a higher accumulation of p21 in carbon-irradiated confluent normal fibroblasts was observed at late times compared to X-rays [70]. In normal human lung fibroblasts, carbon ion irradiation led to a faster senescence than γ-rays [97] However, this phenomenon of premature senescence was observed in the same proportion as for X-rays in the progeny of human fibroblasts after an immediate cell cycle arrest and senescence reappeared and persisted after 5 months after exposure [98]. Our experiments on confluent primary cultures of normal human skin fibroblasts showed a lower proportion of senescence-associated β-galactosidase cells 3 weeks after carbon ion exposure compared to X-rays (unpublished results) (**Figure 3**).

Schematically, acute side effects in normal tissues would be generally related to a loss of fast renewal cells, whereas late effects would appear due to several more complex phenomena as the loss of low renewal cells, progressive ischemia due to the loss of microvascularization endothelial cells and the development of late fibrosis, mainly due to inflammatory processes [99, 100]. After irradiation, it is known that cytokines, which are important mediators of late radiation-induced effects, are not only secreted at early times after irradiation but also at later times-months or years after exposure. Normal tissues monocytes and macrophages produce proinflammatory cytokines like IL-1, IL-6 and TNF-α, which attract macrophages and

**3.4. Stress-induced premature senescence**

**3.5. Inflammation and late toxicity**

#### **3.3. Role of oxidative stress in hadrontherapy toxicity**

Highest toxicity of carbon ions, and in a lower extent of protons, could come from indirect effects of irradiation, i.e. due to a stronger concentration of reactive oxygen and nitrogen species that cells would not be able to detoxify. However, only few studies were interested in oxidative phenomena occurring after carbon ion or proton irradiation.

Wan et al. [72] showed that ROS production in human epithelial cells occurred in the same proportion after proton or X-ray irradiation. Whole body proton irradiation of mice also led to an early differential modulation of oxidative stress gene expression in liver: only proton irradiation led to an increase in Prdx6 and Sod3, mainly, whereas other genes were common to photon irradiation [73]. Chang et al. [74] demonstrated that whole body proton irradiation of C57BL/6 J mice leads to a late increase in ROS production, NOX4 transcription and DNA damage in hematopoietic stem cells from irradiated mice. Proton irradiation of rat eye led to an upregulation of oxidative stress and apoptosis gene expression [75]. Baluchamy et al. [76] concluded that, after proton irradiation, mouse brain presented modifications in expression of genes related to oxidative stress which could lead to programmed cell death. Moreover, the use of antioxidants allowed to protect against biological effects of protons not only *in vitro* [77] but also *in vivo* [78], which tends to demonstrate the importance of oxidative stress. Transgenic mice overexpressing human mitochondrial catalase presented protective effects on low-dose proton-induced brain injury [79]. In the same manner, neuroprotective effects of reducing mitochondrial ROS were also shown by Liao et al. [80] in proton irradiated mice not only at low dose but also at a higher dose of 2 Gy. SOD mimetic was also shown efficient in reducing oxidative damage in retinal cells from proton eye-irradiated rats [81] and in ameliorating acute and chronic proctitis in focal proton irradiated rat rectum [82].

After carbon ion irradiation, an increase in oxidative stress was observed in confluent irradiated primary cultures of normal human skin fibroblasts with an increase in biological macromolecule damage and a decrease in antioxidant enzyme activities in comparison with X-rays [3, 83]. This trend was confirmed by Dettmering et al. [84]: an increase in superoxide anion production was measured in normal human fibroblasts and the maximum level was obtained at a lower dose after carbon irradiation than after X-rays. In human hematopoietic stem/ progenitor cells (HSPCs), carbon irradiation led to a strong increase in heme oxygenase-1 and NAD(P)H dehydrogenase-quinone 1 expression [85]. *In vivo*, mouse whole-body carbon irradiation was shown to decrease glutathione level and to increase MDA content in testis one week after irradiation [86]. At longer term - 2 months after exposure - and in comparison to gamma-rays, mouse whole body irradiation led, in intestine and colon, to: (i) a persistent increase in ROS, mitochondrial cardiolipin oxidation and lipid damage; (ii) a late decrease in antioxidant enzyme activities [87]. The use of other antioxidants indirectly pointed out an important role of oxidative phenomena. Indeed, some antioxidants allowed to decrease effects of carbon ions in normal cells or tissues: curcumin ameliorates cognitive deficits in carbon-irradiated mice via SOD increase, MDA decrease and upregulation of important genes in oxidative stress pathways like heme oxygenase-1 and NAD(P)H quinine oxidoreductase 1 [88]; melatonin reduced carbon-induced apoptosis in mouse carbon-irradiated testes [89] and brain [90] via a decrease in carbonyl and MDA content and an increase in SOD and catalase activities; Dragon's blood decreased hydrogen peroxide and MDA levels and increased SOD activity and glutathione content in carbon-irradiated rat brain [91]. These last experiments provide indirect proofs of the major role of oxidative stress in hadrontherapy toxicity.

#### **3.4. Stress-induced premature senescence**

Results obtained by Antonelli et al. [67] on quantification of γ-H2AX foci after carbon ion vs. X-ray irradiation in lung fibroblasts showed a longer persistence of γ-H2AX foci after carbon ion irradiation which is in agreement with a more difficult repair of DNA complex damage. Moreover, Gustafsson et al. [68] studied, in normal human skin fibroblasts, clustered DSB and non-DSB lesions which convert into DSB during preparation for pulsed-field gel electrophoresis and their results showed a similar increase after carbon ion or low-LET irradiation. It was recently shown that clustered DSB perturb normal human fibroblast DNA repair after high LET irradiation [69]. In confluent normal fibroblasts, accumulations of p53 at early times and p21 at late times were 2–3 times higher after carbon ions than after X-rays [70]. DNA repair proteins (hMRE11, p21, PCNA) were accumulated along ion trajectory in normal

Highest toxicity of carbon ions, and in a lower extent of protons, could come from indirect effects of irradiation, i.e. due to a stronger concentration of reactive oxygen and nitrogen species that cells would not be able to detoxify. However, only few studies were interested in

Wan et al. [72] showed that ROS production in human epithelial cells occurred in the same proportion after proton or X-ray irradiation. Whole body proton irradiation of mice also led to an early differential modulation of oxidative stress gene expression in liver: only proton irradiation led to an increase in Prdx6 and Sod3, mainly, whereas other genes were common to photon irradiation [73]. Chang et al. [74] demonstrated that whole body proton irradiation of C57BL/6 J mice leads to a late increase in ROS production, NOX4 transcription and DNA damage in hematopoietic stem cells from irradiated mice. Proton irradiation of rat eye led to an upregulation of oxidative stress and apoptosis gene expression [75]. Baluchamy et al. [76] concluded that, after proton irradiation, mouse brain presented modifications in expression of genes related to oxidative stress which could lead to programmed cell death. Moreover, the use of antioxidants allowed to protect against biological effects of protons not only *in vitro* [77] but also *in vivo* [78], which tends to demonstrate the importance of oxidative stress. Transgenic mice overexpressing human mitochondrial catalase presented protective effects on low-dose proton-induced brain injury [79]. In the same manner, neuroprotective effects of reducing mitochondrial ROS were also shown by Liao et al. [80] in proton irradiated mice not only at low dose but also at a higher dose of 2 Gy. SOD mimetic was also shown efficient in reducing oxidative damage in retinal cells from proton eye-irradiated rats [81] and in amelio-

human fibroblasts and this was dependent of chromatin compaction [71].

oxidative phenomena occurring after carbon ion or proton irradiation.

rating acute and chronic proctitis in focal proton irradiated rat rectum [82].

After carbon ion irradiation, an increase in oxidative stress was observed in confluent irradiated primary cultures of normal human skin fibroblasts with an increase in biological macromolecule damage and a decrease in antioxidant enzyme activities in comparison with X-rays [3, 83]. This trend was confirmed by Dettmering et al. [84]: an increase in superoxide anion production was measured in normal human fibroblasts and the maximum level was obtained at a lower dose after carbon irradiation than after X-rays. In human hematopoietic stem/ progenitor cells (HSPCs), carbon irradiation led to a strong increase in heme oxygenase-1

**3.3. Role of oxidative stress in hadrontherapy toxicity**

116 Novel Prospects in Oxidative and Nitrosative Stress

In normal human fibroblasts, radiation exposure lead to a G1 cell cycle arrest evolving in quiescence or senescence [92]. Premature senescence, or SIPS (stress-induced premature senescence), differs from replicative senescence. SIPS phenomenon was generally observed in fibroblasts exposed to prolonged or repeated stresses [93, 94] and was also shown after X-ray exposure [95, 96]. Naka et al. [96] showed, by the use of ATM mutated fibroblasts, that pathway leading to premature senescence in fibroblasts after oxidative stress or X-ray exposure could also be ATM-dependent and could act via p38MAPK and p16INK4A. After carbon ion exposure, a higher accumulation of p21 in carbon-irradiated confluent normal fibroblasts was observed at late times compared to X-rays [70]. In normal human lung fibroblasts, carbon ion irradiation led to a faster senescence than γ-rays [97] However, this phenomenon of premature senescence was observed in the same proportion as for X-rays in the progeny of human fibroblasts after an immediate cell cycle arrest and senescence reappeared and persisted after 5 months after exposure [98]. Our experiments on confluent primary cultures of normal human skin fibroblasts showed a lower proportion of senescence-associated β-galactosidase cells 3 weeks after carbon ion exposure compared to X-rays (unpublished results) (**Figure 3**).

#### **3.5. Inflammation and late toxicity**

Schematically, acute side effects in normal tissues would be generally related to a loss of fast renewal cells, whereas late effects would appear due to several more complex phenomena as the loss of low renewal cells, progressive ischemia due to the loss of microvascularization endothelial cells and the development of late fibrosis, mainly due to inflammatory processes [99, 100]. After irradiation, it is known that cytokines, which are important mediators of late radiation-induced effects, are not only secreted at early times after irradiation but also at later times-months or years after exposure. Normal tissues monocytes and macrophages produce proinflammatory cytokines like IL-1, IL-6 and TNF-α, which attract macrophages and

**Figure 3.** Premature senescence in normal human skin fibroblasts exposed to carbon ions or X-rays at an isosurvival dose (unpublished results). Cells were irradiated at confluence and kept until 14 or 21 days post irradiation. After fixation, SA-β-galactosidase staining was performed (citric acid/sodium phosphate 40 mM, NaCl 150 mM, MgCl2 2 mM, potassium ferrocyanide 5 mM, potassium ferricyanide 5 mM and X-gal 1 mg/mL). SA-β-galactosidase activity was determined by counting blue cells using a microscope. Data represent mean percentage of β-gal positive cells ± SEM.

led to less damage than a fractionated dose. However, after 20 population doublings, there were more damage on cells irradiated in one time than in several fractions. Dose hypofractionation, which is presented as a major advantage of carbon therapy, could therefore engender more late effects to bystander normal tissues. Inflammatory pathways playing an important role in oxidative stress persistence in normal tissues after irradiation thus leading to normal tissue injury, the study of bystander effects on the secretion of inflammatory cytokines is of major interest. Carbon microbeam irradiation of a low proportion (0.45%) of immune cells led to decreased cytokine levels [107]. Oxidized extracellular DNA could also be a signaling factor in bystander effects. 8-oxodG is the main oxidatively generated DNA lesion and is formed either by direct oxidation or can be incorporated in DNA from oxidized nucleotide pool by DNA polymerase. Its extracellular presence can be due to DNA repair, cell death, mitochondrial turnover, cellular uptake or salvage of DNA damage products. Carbon-irradiated confluent skin fibroblasts exhibited a 1.5-fold increase in extracellular 8-oxodG 24 hours and 2 weeks after C-ion beam exposure compared to X-rays (see **Table 1**, personal unpublished data). In this way, the role of bystander effects in carbon or proton therapy remains unclear and needs

**Table 1.** 8-oxodG concentration in normal human skin fibroblast culture supernatants exposed to carbon ions or X-rays

**8-oxodG concentration (nM)**

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**Time after irradiation 24 hours 14 days** X-ray control 0.775 +/- 0.140 0.817 +/- 0.036 X-ray irradiated 1.980 +/- 0.064 2.175 +/- 0.073 C-ion control 0.822 +/- 0.103 0.830 +/- 0.084 C-ion irradiated 3.103 +/- 0.296 3.153 +/- 0.262

Cell culture supernatants were purified by solid phase extraction, and samples were adjusted for the standard addition method in order to correct for the matrix effects contributed by the culture medium constituents as reported previously [108]. An optimized method for the quantification of 8-oxodG has been applied. HPLC-ECD signals were recorded in the culture supernatants spiked with the external standard. Data represent mean 8-oxodG concentration ± SEM.

Mainly due to the cost of hadrontherapy facilities, there are too few studies dealing with biological effects of protons, carbon ions or other particles on tumors and normal tissues. In addition, a large proportion of these works did not compare carbon ion effects to X-ray effects. Advantages of hadrons, mostly on tumors, are often highlighted but particular attention should be paid on side effects of hadrons, especially hypofractionation which could lead to major injuries in normal tissues. Killing efficiency of carbon ions is often considered lower for normal cells than for tumor cells. However, some recent studies tend to show a strong increase in oxidative stress in normal cells after protons [74, 79] or carbon ions [3, 84].

further investigations.

at an isosurvival dose (unpublished results).

**4. Conclusion**

lymphocytes. Activated macrophages and stimulated stromal cells synthetize fibrogenic cytokines such as TGF-β and PDGF modulating fibroblast proliferation-differentiation balance and protein synthesis and degradation via metalloproteinases (MMP) and their inhibitors (TIMP) (for review, [99, 101]). In this way, specificity of proton or carbon ion irradiation concerning these pathways is of main interest to modulate late effects of hadrontherapy. Fournier et al. [102] showed an accumulation of fibrocytes and extracellular matrix proteins in normal human foreskin fibroblasts exposed to carbon ions. However, a lowered increase in IL-6 was observed in normal human skin fibroblasts exposed to carbon ion compared to X-ray irradiation [3]. The use of Dragon's blood, which presents antioxidant and anti-inflammatory properties, did not allow to reduce TNF-α, IFN-γ and IL-6 levels in carbon-irradiated rat brain as it was the case for γ-rays [91]. A recent report on carbon-irradiated normal human skin models showed similar inflammatory processes than after the same dose of X-rays [103].

#### **3.6. Bystander effects**

Bystander effect, i.e. biological effects to cells which were not irradiated via signals coming from irradiated cells, could be at the origin of normal surrounding tissue injury and to, for example, abscopal effects. Oxidative stress signal pathways could play an important role in these effects.

Indeed, confluent human skin fibroblasts were shown to present a persistent oxidative stress after exposure of 0.036–0.4% of them to proton or X-ray microbeam but this was not the case for carbon ions [104]. However, when normal cell cultures exposed to low-LET protons were co-cultured with unirradiated cells and after 20 population doublings, no changes in survival, chromosomal damage, protein oxidation and lipid peroxidation were observed [105]. This was not the case for higher LET (iron and silicon ions) for which a higher level of oxidative damage, a decrease in antioxidant enzyme activities and an alteration of mitochondiral proteins - encoded by mitochondiral DNA - were observed [105].

Recently, Autsavapromporn et al. [106] have shown that glioblastoma cell carbon irradiation led to damage in unirradiated normal fibroblasts. Moreover, a single dose of carbon irradiation


**Table 1.** 8-oxodG concentration in normal human skin fibroblast culture supernatants exposed to carbon ions or X-rays at an isosurvival dose (unpublished results).

led to less damage than a fractionated dose. However, after 20 population doublings, there were more damage on cells irradiated in one time than in several fractions. Dose hypofractionation, which is presented as a major advantage of carbon therapy, could therefore engender more late effects to bystander normal tissues. Inflammatory pathways playing an important role in oxidative stress persistence in normal tissues after irradiation thus leading to normal tissue injury, the study of bystander effects on the secretion of inflammatory cytokines is of major interest. Carbon microbeam irradiation of a low proportion (0.45%) of immune cells led to decreased cytokine levels [107]. Oxidized extracellular DNA could also be a signaling factor in bystander effects. 8-oxodG is the main oxidatively generated DNA lesion and is formed either by direct oxidation or can be incorporated in DNA from oxidized nucleotide pool by DNA polymerase. Its extracellular presence can be due to DNA repair, cell death, mitochondrial turnover, cellular uptake or salvage of DNA damage products. Carbon-irradiated confluent skin fibroblasts exhibited a 1.5-fold increase in extracellular 8-oxodG 24 hours and 2 weeks after C-ion beam exposure compared to X-rays (see **Table 1**, personal unpublished data). In this way, the role of bystander effects in carbon or proton therapy remains unclear and needs further investigations.

Cell culture supernatants were purified by solid phase extraction, and samples were adjusted for the standard addition method in order to correct for the matrix effects contributed by the culture medium constituents as reported previously [108]. An optimized method for the quantification of 8-oxodG has been applied. HPLC-ECD signals were recorded in the culture supernatants spiked with the external standard. Data represent mean 8-oxodG concentration ± SEM.

### **4. Conclusion**

lymphocytes. Activated macrophages and stimulated stromal cells synthetize fibrogenic cytokines such as TGF-β and PDGF modulating fibroblast proliferation-differentiation balance and protein synthesis and degradation via metalloproteinases (MMP) and their inhibitors (TIMP) (for review, [99, 101]). In this way, specificity of proton or carbon ion irradiation concerning these pathways is of main interest to modulate late effects of hadrontherapy. Fournier et al. [102] showed an accumulation of fibrocytes and extracellular matrix proteins in normal human foreskin fibroblasts exposed to carbon ions. However, a lowered increase in IL-6 was observed in normal human skin fibroblasts exposed to carbon ion compared to X-ray irradiation [3]. The use of Dragon's blood, which presents antioxidant and anti-inflammatory properties, did not allow to reduce TNF-α, IFN-γ and IL-6 levels in carbon-irradiated rat brain as it was the case for γ-rays [91]. A recent report on carbon-irradiated normal human skin models

**Figure 3.** Premature senescence in normal human skin fibroblasts exposed to carbon ions or X-rays at an isosurvival dose (unpublished results). Cells were irradiated at confluence and kept until 14 or 21 days post irradiation. After fixation, SA-β-galactosidase staining was performed (citric acid/sodium phosphate 40 mM, NaCl 150 mM, MgCl2

potassium ferrocyanide 5 mM, potassium ferricyanide 5 mM and X-gal 1 mg/mL). SA-β-galactosidase activity was determined by counting blue cells using a microscope. Data represent mean percentage of β-gal positive cells ± SEM.

2 mM,

showed similar inflammatory processes than after the same dose of X-rays [103].

teins - encoded by mitochondiral DNA - were observed [105].

Bystander effect, i.e. biological effects to cells which were not irradiated via signals coming from irradiated cells, could be at the origin of normal surrounding tissue injury and to, for example, abscopal effects. Oxidative stress signal pathways could play an important role in

Indeed, confluent human skin fibroblasts were shown to present a persistent oxidative stress after exposure of 0.036–0.4% of them to proton or X-ray microbeam but this was not the case for carbon ions [104]. However, when normal cell cultures exposed to low-LET protons were co-cultured with unirradiated cells and after 20 population doublings, no changes in survival, chromosomal damage, protein oxidation and lipid peroxidation were observed [105]. This was not the case for higher LET (iron and silicon ions) for which a higher level of oxidative damage, a decrease in antioxidant enzyme activities and an alteration of mitochondiral pro-

Recently, Autsavapromporn et al. [106] have shown that glioblastoma cell carbon irradiation led to damage in unirradiated normal fibroblasts. Moreover, a single dose of carbon irradiation

**3.6. Bystander effects**

118 Novel Prospects in Oxidative and Nitrosative Stress

these effects.

Mainly due to the cost of hadrontherapy facilities, there are too few studies dealing with biological effects of protons, carbon ions or other particles on tumors and normal tissues. In addition, a large proportion of these works did not compare carbon ion effects to X-ray effects. Advantages of hadrons, mostly on tumors, are often highlighted but particular attention should be paid on side effects of hadrons, especially hypofractionation which could lead to major injuries in normal tissues. Killing efficiency of carbon ions is often considered lower for normal cells than for tumor cells. However, some recent studies tend to show a strong increase in oxidative stress in normal cells after protons [74, 79] or carbon ions [3, 84].

[2] Bragg S. On the ionization of various gases by the alpha particles of radium. Proceedings

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[3] Laurent C, Leduc A, Pottier I, Prévost V, Sichel F, Lefaix JL. Dramatic increase in oxidative stress in carbon-irradiated normal human skin fibroblasts. PLoS One. 2013;**8**(12):e85158

[4] Mohan R, Grosshans D. Proton therapy – present and future. Advanced Drug Delivery

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[6] Jimenez RB, Sethi R, Depauw N, Pulsifer MB, Adams J, McBride SM, et al. Proton radiation therapy for pediatric medulloblastoma and supratentorial primitive neuroectodermal tumors: Outcomes for very young children treated with upfront chemotherapy. International Journal of Radiation Oncology, Biology, Physics. 2013;**87**(1):120-126

[7] Ladra MM, Szymonifka JD, Mahajan A, Friedmann AM, Yong Yeap B, Goebel CP, et al. Preliminary results of a phase II trial of proton radiotherapy for pediatric rhabdomyo-

[8] McGovern SL, Okcu MF, Munsell MF, Kumbalasseriyil N, Grosshans DR, McAleer MF, et al. Outcomes and acute toxicities of proton therapy for pediatric atypical teratoid/rhabdoid tumor of the central nervous system. International Journal of Radiation

[9] Bishop AJ, Greenfield B, Mahajan A, Paulino AC, Okcu MF, Allen PK, et al. Proton beam therapy versus conformal photon radiation therapy for childhood craniopharyngioma: Multi-institutional analysis of outcomes, cyst dynamics, and toxicity. International

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[11] Grosshans DR, Zhu XR, Melancon A, Allen PK, Poenisch F, Palmer M, et al. Spot scanning proton therapy for malignancies of the base of skull: Treatment planning, acute toxicities, and preliminary clinical outcomes. International Journal of Radiation Oncology,

[12] Weber DC, Schneider R, Goitein G, Koch T, Ares C, Geismar JH, et al. Spot scanningbased proton therapy for intracranial meningioma: Long-term results from the Paul Scherrer Institute. International Journal of Radiation Oncology, Biology, Physics. 2012;

[13] Hauswald H, Rieken S, Ecker S, Kessel KA, Herfarth K, Debus J, et al. First experiences in treatment of low-grade glioma grade I and II with proton therapy. Radiation Oncology.

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**Figure 4.** Proposed schema of biological effects of hadron irradiation leading to tumor killing and normal tissue toxicity.

According to the literature, **Figure 4** proposed a schema of biological effects leading to tumor cell death or to normal cell toxicity.

Further investigations are needed to better understand toxicity of protons and carbon ions. Prediction of side effects for each patient should be of major interest in order to adapt radiotherapy protocol and/or to prevent deleterious effects due to normal tissue irradiation or to bystander phenomena. The use of antioxidants, which were demonstrated as efficient in reducing late effects of protons and carbon ions, could be of major interest in preserving normal tissue during proton or carbon ion therapy. Another guideline for reflection is related to the drawbacks of protons and carbon ions: they could lead to an interest of other ions as helium ions which should lead to less toxicity in normal tissues but are also less efficient on cancer cells and which do not present the same interest as carbon ions in killing tumor cells in hypoxic conditions. In conclusion, due to complex effects of hadrons when encountering normal tissues and tumors, there is a strong need in preclinical studies—at early and late times post-irradiation and in comparison to photons—to determine biological effects of SOBP, ion fragmentation, LET distribution in depth, hypofractionation, beam scanning, etc.

### **Author details**

Carine Laurent

Address all correspondence to: carine.laurent@unicaen.fr

Saphyn-Archade (Advanced Resource Centre for HADrontherapy in Europe), University of Caen, Caen, France

### **References**

[1] Jaffray DA, Gospodarowicz MK. Radiation therapy for cancer. In: Gelband H, Jha P, HSankaranarayanan R, Horton S, Editors. Cancer: Disease Control Priorities. 3rd ed. Vol. 3. Washington (DC): The International Bank for Reconstruction and Development/The World Bank; 2015. p. 239-248


According to the literature, **Figure 4** proposed a schema of biological effects leading to tumor

**Figure 4.** Proposed schema of biological effects of hadron irradiation leading to tumor killing and normal tissue toxicity.

Further investigations are needed to better understand toxicity of protons and carbon ions. Prediction of side effects for each patient should be of major interest in order to adapt radiotherapy protocol and/or to prevent deleterious effects due to normal tissue irradiation or to bystander phenomena. The use of antioxidants, which were demonstrated as efficient in reducing late effects of protons and carbon ions, could be of major interest in preserving normal tissue during proton or carbon ion therapy. Another guideline for reflection is related to the drawbacks of protons and carbon ions: they could lead to an interest of other ions as helium ions which should lead to less toxicity in normal tissues but are also less efficient on cancer cells and which do not present the same interest as carbon ions in killing tumor cells in hypoxic conditions. In conclusion, due to complex effects of hadrons when encountering normal tissues and tumors, there is a strong need in preclinical studies—at early and late times post-irradiation and in comparison to photons—to determine biological effects of SOBP, ion

fragmentation, LET distribution in depth, hypofractionation, beam scanning, etc.

Saphyn-Archade (Advanced Resource Centre for HADrontherapy in Europe), University of

[1] Jaffray DA, Gospodarowicz MK. Radiation therapy for cancer. In: Gelband H, Jha P, HSankaranarayanan R, Horton S, Editors. Cancer: Disease Control Priorities. 3rd ed. Vol. 3. Washington (DC): The International Bank for Reconstruction and Development/The

Address all correspondence to: carine.laurent@unicaen.fr

cell death or to normal cell toxicity.

120 Novel Prospects in Oxidative and Nitrosative Stress

**Author details**

Carine Laurent

Caen, Caen, France

World Bank; 2015. p. 239-248

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sure to H2


**Chapter 7**

**Provisional chapter**

**Erythrocyte Nitric Oxide**

**Erythrocyte Nitric Oxide**

http://dx.doi.org/10.5772/intechopen.75931

**Abstract**

pathophysiology

**1. Introduction**

Carlota Saldanha and Ana Silva-Herdade

Carlota Saldanha and Ana Silva-Herdade

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

DOI: 10.5772/intechopen.75931

Nitric oxide (NO) is a vasoactive molecule that, by stimulated and functional vascular endothelial cells, is released to the lumen of the vessel and into the surrounding smooth muscle cells. Once in the lumen, NO is captured by red blood cells and scavenged inside through hemoglobin and derived as NO metabolites. The delivery ability of erythrocytes allowing the NO efflux also occurs. Manipulation of NO levels inside the erythrocyte through different external (acetylcholine, acetylcholinesterase inhibitors, fibrinogen and CD47 4N1K peptide) and internal (redox and protein phosphorylation levels) stimuli will be described. The values of NO efflux from the erythrocytes and its association with the data quantified in the hemorheology properties and in clinical parameters obtained from patients with vascular diseases will also be present. The in vivo animal experimental studies highlighting the ability of NO efflux (delivered) from the erythrocytes where is scavenged and its influence in inflammatory and hemorheological responses will be addressed. So, the aim of this chapter is to present the knowledge obtained about the NO signal transduction mechanism in erythrocytes and the association between erythrocyte availability in NO with clinical biomarkers obtained in inflammatory vascular diseases. A final question is raised—namely, could NO be considered a hemorheological parameter?

**Keywords:** erythrocyte, nitric oxide, deformability, intravital microscopy,

Vascular endothelium cells behave like "meeting points" between white blood cells and mediator factor participants in the steps of inflammatory response allowing "cross-talk" with blood, red blood cells (RBCs), platelets, fibrinogen, lipoproteins and other blood biomolecule components [1]. Endothelial cells (ECs) under influence of mechanical, physical and chemical stimuli are prone to secrete vasoactive molecules into the lumen vessels and smooth muscle cells [2].

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

#### **Chapter 7 Provisional chapter**

#### **Erythrocyte Nitric Oxide Erythrocyte Nitric Oxide**

Carlota Saldanha and Ana Silva-Herdade Carlota Saldanha and Ana Silva-Herdade

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75931

#### **Abstract**

Nitric oxide (NO) is a vasoactive molecule that, by stimulated and functional vascular endothelial cells, is released to the lumen of the vessel and into the surrounding smooth muscle cells. Once in the lumen, NO is captured by red blood cells and scavenged inside through hemoglobin and derived as NO metabolites. The delivery ability of erythrocytes allowing the NO efflux also occurs. Manipulation of NO levels inside the erythrocyte through different external (acetylcholine, acetylcholinesterase inhibitors, fibrinogen and CD47 4N1K peptide) and internal (redox and protein phosphorylation levels) stimuli will be described. The values of NO efflux from the erythrocytes and its association with the data quantified in the hemorheology properties and in clinical parameters obtained from patients with vascular diseases will also be present. The in vivo animal experimental studies highlighting the ability of NO efflux (delivered) from the erythrocytes where is scavenged and its influence in inflammatory and hemorheological responses will be addressed. So, the aim of this chapter is to present the knowledge obtained about the NO signal transduction mechanism in erythrocytes and the association between erythrocyte availability in NO with clinical biomarkers obtained in inflammatory vascular diseases. A final question is raised—namely, could NO be considered a hemorheological parameter?

DOI: 10.5772/intechopen.75931

**Keywords:** erythrocyte, nitric oxide, deformability, intravital microscopy, pathophysiology

#### **1. Introduction**

Vascular endothelium cells behave like "meeting points" between white blood cells and mediator factor participants in the steps of inflammatory response allowing "cross-talk" with blood, red blood cells (RBCs), platelets, fibrinogen, lipoproteins and other blood biomolecule components [1]. Endothelial cells (ECs) under influence of mechanical, physical and chemical stimuli are prone to secrete vasoactive molecules into the lumen vessels and smooth muscle cells [2].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The endothelium-derived factor secreted by vascular endothelial cells, known by its vasodilator property, was further identified as nitric oxide (NO) by Robert F. Furchgott, Louis J. Ignarro and Ferid Murad who received the Nobel Prize in 1998 [3, 4].

Endothelial cells and lymphocytes are able, by the participation of choline acetyltransferase, to synthesize ACh which is released into the plasma through vesicular acetylcholine trans-

Erythrocyte Nitric Oxide

133

http://dx.doi.org/10.5772/intechopen.75931

Depending on the degree of endothelium integrity the circulating ACh induced vasodilation or vasoconstriction according to the amount of nitric oxide synthesized and released [3]. The NO released from endothelial cells and platelets is scavenged by erythrocyte and blood cellfree hemoglobin [25]. In order to identify the signal transduction mechanism of the erythrocyte ability to scavenge or deliver NO, in vitro studies mimicking normal and inflammatory conditions were performed and are presented in the next section. This section also includes ex vivo studies showing the quantification of the NO efflux values, performed with RBCs obtained from blood samples of healthy donors and patients with vascular inflammatory diseases and their association with clinical biomarkers. Also, *in vivo* studies' conduct in animal models of hypertension and inflammation are included in the next section to show erythrocyte NO availability, contribution and association with inflammatory vascular diseases. From all data obtained and herein described we are able to conclude the erythrocyte NO transloca-

Erythrocyte membrane acetylcholinesterase (AChE) is a hydrolytic enzyme with a rare kinetic profile with an optimum substrate concentration (So) from on AChE activity decrease with the augment of its substrate ACh [26, 27]. At lower or higher So values, the AChE-ACh enzyme

Based on the fact that SNO-Hb and GSNO have been considered reservoirs of NO and ACh is an endogenous compound with vasoactive properties, present in blood circulation, we raised three questions, whether ACh induces changes on erythrocyte deformability, if there is NO inside erythrocyte and whether it could be mobilized to the outside. In order to answer these, human erythrocyte suspensions, in the presence of ACh, were loaded with the permeable nonfluorescent probe diamine fluorescein-2 diacetate (DAF-2 Da). Intra-erythrocyte fluorescence intensity of triazolofluorescein (DAF-2 T) was visualized, by fluorescence microscopy, as a result of the reaction between NO and the 4, 5- diamine fluorescein [28]. So, inside the erythrocyte, there is NO when stimulated with ACh and also there is an increase in the levels of NO2− and NO3− [28]. When erythrocytes are in the presence of ACh, erythrocyte deformability, during the impairment of oxygen hemoglobin affinity and of erythrocyte aggregation (EA), has been verified [29]. The presence of an active complex (AChE-ACh) in red blood cells is able to trigger band 3 protein phosphorylation when PTP is inhibited, with a higher mobilization of NO-derived metabolites [30]. This complex is unable to induce band 3 phosphorylation upon p53/56lyn and p72syk inhibition, providing a lower degree of NO efflux and NOx mobilization [30]. This mobilization is enhanced with phosphorylated but not a dephosphorylated band 3 protein. The maximum translocation of NO efflux from RBC achieved upon acetylcholine stimulation and band 3 phosphorylation was related to the higher levels of the methemoglobin,

tion across the erythrocyte membrane as a hemorheological parameter.

complex forms are active or the less active ones, respectively [26, 27].

**2. Erythrocyte nitric oxide studies**

porter [22–24].

**2.1. In vitro**

Ignarro's spectral analysis of hemoglobin (Hb) evidenced that when this biomolecule was exposed to endothelial cells' stimulate by acetylcholine (ACh), NO is liberated and a shift of the Hb absorption curve occurs, establishing for the first time the NO binding to Hb and an indirect link between red blood cells (RBCs) and NO [4].

Other authors evidenced the ability of RBCs to rescue NO liberated from endothelium cells and the need to liberate it according the tissues oxygen partial pressure [5, 6]. Experiments conducted in vitro using RBCs under normoxia conditions submitting to low oxygen tension (hypoxia) showed liberation of oxygen and NO binding to deoxygenated hemoglobin demonstrating consequently allosteric structure transitions of the Hb molecule [5]. The role of erythrocyte membrane band 3 protein into NO through erythrocyte was studied [5]. The bioavailability of RBCs in NO may be the trigger or the consequence of the involvement of the RBC's hemoglobin as the oxygen sensor [5, 6]. NO binds to oxygenated Hb in its thiol group of cysteine β93 at high-tissue oxygen pressure (PaO<sup>2</sup> ) originating as S-nitrosohemoglobin (SNO-HbO2 ), while at low PaO2, NO binds to the iron ion of the hemoglobin; heme group generates nitrosylhemoglobin molecules [7, 8]. Regarding the efflux of NO from erythrocytes, the transfer of NO between SNO-HbO2 and the thiol group of band 3 protein was verified [9–11]. The transnitrosation reaction could occur with the thiol group of other biomolecules [9–11]. Using inhibitors of protein tyrosine kinase (PTK) p72 syk, Src Lyn and of SHP-2 protein tyrosine phosphatase (PTP), in vitro studies have evidenced, respectively, band 3 protein phosphorylation and dephosphorylation at tyrosine residues [12]. The dephosphorylation of band 3 is associated with oxyhemoglobin and glycolytic enzymes binding which, upon band 3 phosphorylation, delivers oxygen and glyceraldehyde dehydrogenase, aldolase and phosphofructokinase closer to the cytosol [13]. Glutathione (GSH) is a redox biomolecule resulting from the reaction between the three peptides glycine, glutamic and cysteine showing its thiol group that can bind directly NO transferred from SNO-HbO2, originating from nitrosothiol such as S-nitrosoglutathione (GSNO) [14]. The GSNO is a transient reservoir of NO because it is essential to be in its reduced state for the regeneration of NADPH to NADP levels of the erythrocyte. However, the inactivation of glutathione reductase induced by oxidative stress influences the concentration of GSH which is needed for regeneration of oxidized proteins [15]. For instance, dithiothreitol (DTT) is a thiolreducing agent capable of regenerating disulfide-containing proteins and establishing an interchangeable thiol-disulfide reaction with glutathione [16]. Beyond that, DTT's presence induces changes on enzyme activity states, for example, of the PTP and PTK [17].

If auto-oxidation of hemoglobin occurs the peroxide anion will be produced, which generates peroxynitrite after reaction with NO [18]. The decomposition of peroxynitrite molecules leads to nitrite (NO2−) and nitrate (NO3−) which are designated NO derivative molecules (NOx ) whose concentrations are changed by external RBC-binding biomolecules as shown [19, 20]. It was evidenced that NO release from SNOHb could bind to thiol groups and be exported from RBCs as nitrosothiol or may be so as oxidation generates nitrate [19]. The NO in the presence of oxyhemoglobin molecules induces methemoglobin and nitrate formation [20]. The hemoglobin reductase with the NADH produced in the glycolytic pathway maintains the methemoglobin concentration [21].

Endothelial cells and lymphocytes are able, by the participation of choline acetyltransferase, to synthesize ACh which is released into the plasma through vesicular acetylcholine transporter [22–24].

Depending on the degree of endothelium integrity the circulating ACh induced vasodilation or vasoconstriction according to the amount of nitric oxide synthesized and released [3]. The NO released from endothelial cells and platelets is scavenged by erythrocyte and blood cellfree hemoglobin [25]. In order to identify the signal transduction mechanism of the erythrocyte ability to scavenge or deliver NO, in vitro studies mimicking normal and inflammatory conditions were performed and are presented in the next section. This section also includes ex vivo studies showing the quantification of the NO efflux values, performed with RBCs obtained from blood samples of healthy donors and patients with vascular inflammatory diseases and their association with clinical biomarkers. Also, *in vivo* studies' conduct in animal models of hypertension and inflammation are included in the next section to show erythrocyte NO availability, contribution and association with inflammatory vascular diseases. From all data obtained and herein described we are able to conclude the erythrocyte NO translocation across the erythrocyte membrane as a hemorheological parameter.

### **2. Erythrocyte nitric oxide studies**

#### **2.1. In vitro**

The endothelium-derived factor secreted by vascular endothelial cells, known by its vasodilator property, was further identified as nitric oxide (NO) by Robert F. Furchgott, Louis

Ignarro's spectral analysis of hemoglobin (Hb) evidenced that when this biomolecule was exposed to endothelial cells' stimulate by acetylcholine (ACh), NO is liberated and a shift of the Hb absorption curve occurs, establishing for the first time the NO binding to Hb and an

Other authors evidenced the ability of RBCs to rescue NO liberated from endothelium cells and the need to liberate it according the tissues oxygen partial pressure [5, 6]. Experiments conducted in vitro using RBCs under normoxia conditions submitting to low oxygen tension (hypoxia) showed liberation of oxygen and NO binding to deoxygenated hemoglobin demonstrating consequently allosteric structure transitions of the Hb molecule [5]. The role of erythrocyte membrane band 3 protein into NO through erythrocyte was studied [5]. The bioavailability of RBCs in NO may be the trigger or the consequence of the involvement of the RBC's hemoglobin as the oxygen sensor [5, 6]. NO binds to oxygenated Hb in its thiol group of cysteine β93 at

low PaO2, NO binds to the iron ion of the hemoglobin; heme group generates nitrosylhemoglobin molecules [7, 8]. Regarding the efflux of NO from erythrocytes, the transfer of NO between

tion could occur with the thiol group of other biomolecules [9–11]. Using inhibitors of protein tyrosine kinase (PTK) p72 syk, Src Lyn and of SHP-2 protein tyrosine phosphatase (PTP), in vitro studies have evidenced, respectively, band 3 protein phosphorylation and dephosphorylation at tyrosine residues [12]. The dephosphorylation of band 3 is associated with oxyhemoglobin and glycolytic enzymes binding which, upon band 3 phosphorylation, delivers oxygen and glyceraldehyde dehydrogenase, aldolase and phosphofructokinase closer to the cytosol [13]. Glutathione (GSH) is a redox biomolecule resulting from the reaction between the three peptides glycine, glutamic and cysteine showing its thiol group that can bind directly NO transferred from SNO-HbO2, originating from nitrosothiol such as S-nitrosoglutathione (GSNO) [14]. The GSNO is a transient reservoir of NO because it is essential to be in its reduced state for the regeneration of NADPH to NADP levels of the erythrocyte. However, the inactivation of glutathione reductase induced by oxidative stress influences the concentration of GSH which is needed for regeneration of oxidized proteins [15]. For instance, dithiothreitol (DTT) is a thiolreducing agent capable of regenerating disulfide-containing proteins and establishing an interchangeable thiol-disulfide reaction with glutathione [16]. Beyond that, DTT's presence induces

If auto-oxidation of hemoglobin occurs the peroxide anion will be produced, which generates peroxynitrite after reaction with NO [18]. The decomposition of peroxynitrite molecules leads to nitrite (NO2−) and nitrate (NO3−) which are designated NO derivative molecules (NOx

whose concentrations are changed by external RBC-binding biomolecules as shown [19, 20]. It was evidenced that NO release from SNOHb could bind to thiol groups and be exported from RBCs as nitrosothiol or may be so as oxidation generates nitrate [19]. The NO in the presence of oxyhemoglobin molecules induces methemoglobin and nitrate formation [20]. The hemoglobin reductase with the NADH produced in the glycolytic pathway maintains the

changes on enzyme activity states, for example, of the PTP and PTK [17].

and the thiol group of band 3 protein was verified [9–11]. The transnitrosation reac-

) originating as S-nitrosohemoglobin (SNO-HbO2

), while at

)

J. Ignarro and Ferid Murad who received the Nobel Prize in 1998 [3, 4].

indirect link between red blood cells (RBCs) and NO [4].

high-tissue oxygen pressure (PaO<sup>2</sup>

132 Novel Prospects in Oxidative and Nitrosative Stress

methemoglobin concentration [21].

SNO-HbO2

Erythrocyte membrane acetylcholinesterase (AChE) is a hydrolytic enzyme with a rare kinetic profile with an optimum substrate concentration (So) from on AChE activity decrease with the augment of its substrate ACh [26, 27]. At lower or higher So values, the AChE-ACh enzyme complex forms are active or the less active ones, respectively [26, 27].

Based on the fact that SNO-Hb and GSNO have been considered reservoirs of NO and ACh is an endogenous compound with vasoactive properties, present in blood circulation, we raised three questions, whether ACh induces changes on erythrocyte deformability, if there is NO inside erythrocyte and whether it could be mobilized to the outside. In order to answer these, human erythrocyte suspensions, in the presence of ACh, were loaded with the permeable nonfluorescent probe diamine fluorescein-2 diacetate (DAF-2 Da). Intra-erythrocyte fluorescence intensity of triazolofluorescein (DAF-2 T) was visualized, by fluorescence microscopy, as a result of the reaction between NO and the 4, 5- diamine fluorescein [28]. So, inside the erythrocyte, there is NO when stimulated with ACh and also there is an increase in the levels of NO2− and NO3− [28]. When erythrocytes are in the presence of ACh, erythrocyte deformability, during the impairment of oxygen hemoglobin affinity and of erythrocyte aggregation (EA), has been verified [29]. The presence of an active complex (AChE-ACh) in red blood cells is able to trigger band 3 protein phosphorylation when PTP is inhibited, with a higher mobilization of NO-derived metabolites [30]. This complex is unable to induce band 3 phosphorylation upon p53/56lyn and p72syk inhibition, providing a lower degree of NO efflux and NOx mobilization [30]. This mobilization is enhanced with phosphorylated but not a dephosphorylated band 3 protein. The maximum translocation of NO efflux from RBC achieved upon acetylcholine stimulation and band 3 phosphorylation was related to the higher levels of the methemoglobin, [L-lactate], concentration ratio between cyclic guanylyl cyclase (cGMP) and cyclic adenosine monophosphate (cAMP) and lower oxygen affinity to hemoglobin value and of oxyhemoglobin concentration [30]. At variance, the effect of the AChE inhibitor velnacrine maleate (VM) induced a higher degree of [NO] efflux/[NOx] mobilization through the AChE-VM inhibitor complex in the presence of p53/56lyn and p72syk inhibitors [30]. When in the case of erythrocyte membrane band 3 protein dephosphorylated state, the inactive complex form of the AChE promotes higher NO efflux than the AChE active complex form [30]. But the opposite was observed with erythrocyte membrane band 3 protein phosphorylation [30]. When experiments were done with the AChE strong inhibitor, VM, an almost inactive complex, results and induces lower NO efflux from erythrocytes and higher GSNO and peroxynitrite concentration values than those obtained with the active complex form AChE-ACh [30].

century was CD47 established as a binding target in the erythrocyte membrane for the soluble

Erythrocyte Nitric Oxide

135

http://dx.doi.org/10.5772/intechopen.75931

It was shown that for soluble Fib, in physiological concentrations, the NO efflux from erythrocytes decreased with increased GSNO, nitrite and nitrate levels [44]. The scavenging NO RBC ability to reduce efflux was surpassed showing normal values when both 4N1K (the CD47 peptide analog of thrombospondin binding site) and high fibrinogen levels are present or when 4N1K is absent [45]. These data show the dependence of lower cyclic adenosine monophosphate (cAMP) associated with adenylate cyclase (AC) inhibition by CD47Gαi [45]. When phosphorylation of the erythrocyte membrane protein band 3 is induced in the presence of high fibrinogen concentration and in the absence or presence of 4N1K, the NO efflux increases [46, 47]. The NO efflux from erythrocytes at high fibrinogen concentration is dependent on band 3 protein phosphorylation which was confirmed in the experiments where the erythrocyte casein kinase 2 (a cytosol protein that phosphorylates the band 3 protein) inhibitor was

During inflammation high levels of both acetylcholine and fibrinogen are presented and normal values of NO efflux from erythrocytes have been observed in vitro [39, 49]. Besides, a higher NO efflux from RBC will be expected resulting of the presence of ACh and high fibrinogen concentration, normal values were obtained; the AChE-ACh molecular conformational state activates PKC which inhibits PDE 3 with increase of cAMP concentration that normalize the lower levels of cAMP derived from the inhibition of AC [35, 38, 45, 49]. Interestingly, the presence of forskolin (activator of the enzyme AC) in an in vitro model of hyperfibrinogenemia did not change the levels of NO efflux from erythrocytes, because the PDE3 is functional to hydrolase cAMP [27, 48]. When stimulating the erythrocyte redox thiol status by loading dithiothreitol (DTT), there is a decreased NO efflux concomitant with increased levels of nitrite, nitrate and GSNO [50]. It is well known that dithiothreitol induces band 3 dephosphorylation and a dephosphorylated

used, showing unchanged levels of NO efflux in relation to its absence [27, 48].

state accounts for the AChE inhibitors and fibrinogen effects on red cells [17, 30, 49].

High concentrations of oxidized LDL when in the presence of blood samples of healthy human erythrocytes increase its ability to scavenge NO [51]. The same behavior was obtained in another study conducted with blood samples taken from healthy humans and exposed or not (control aliquot) to two different concentrations of LDL/HDL; no changes in NO efflux values from the erythrocyte, no alterations on intra-erythrocyte peroxynitrite concentrations and an unaltered deformability profile, at all shear stresses, were observed. At variance the levels of intra-erythrocyte NO derivative molecules nitrite, nitrate and GSNO showed significantly increased values when compared with control aliquots. The unchanged deformability values obtained at lower and high shear stresses for all treated blood sample aliquots with LDL/HDL are indicative of membrane stability, internal viscosity maintenance and normal interactions of membrane peripheral and cytoskeleton [52]. The absence of erythrocyte membrane instability obtained in blood sample aliquots under LDL/HDL addition is confirmed by the unchanged nitrogen reactive species concentration of peroxynitrite, as evidence by the normal levels obtained for peroxynitrite is an index of auto-oxidation of oxyhemoglobin [52, 53]. The addition of different concentrations of the lipoprotein sub-fractions' LDL/HDL seems not to favor hemoglobin auto-oxidation. Superoxide anion will be formed from the

form of fibrinogen [43].

Additional studies were performed taking into account the identification of the type of a G-protein involved in the erythrocyte ACh/NO signaling pathway. It was evidenced that at the N-terminal band 3 protein domain only Gαi1/2 binds. Gαi1/2 and the G<sup>β</sup> are associated with band 3 protein at the C-terminal site domain independently of the band 3 phosphorylation degree [31]. This chapter confirmed our previous hypothesis of the potential involvement of a heterotrimeric G protein in signal events mediated by the erythrocyte membrane AChE-ACh complex or AChE-inhibitor complex band 3 protein interactions with the participation of adenylyl cyclase inhibition [20, 31].

The quantification of NO efflux from the erythrocyte was assessed, by the first time for us, using the amino-IV sensor by the amperometric method which is described [32, 33]. The nitric oxide release from RBC in presence of ACh is sense by an electrode which oxidize NO at the working platinum electrode, resulting on electric current. The redox current is proportional to the NO concentration outside the membrane and it was continuously monitored with a computer. The AChE-ACh active complex activates PKC which phosphorylates PTP and PTK switching them to inactive and activate enzymes states, resulting in band-3 protein phosphorylation by PTK active form without with consequently NO release [32–34].

Beyond the AChE's strong inhibitor velnacrine, the moderate AChE inhibitor timolol was used, forming a less active AChE-timolol complex, and a lower erythrocyte efflux from NO was quantified in relation to those values obtained with AChE-ACh [35–37].

For the first time we evidenced that when erythrocytes were in the presence of ACh or timolol, the efflux of GSNO was lower with AChE-timolol than with AChE-ACh, both values being higher than in their absence [38].

It was evidenced by those in vitro studies that AChE's active and less active molecular conformations induce increased or decreased NO efflux from erythrocytes, respectively [38].

In the presence of SpermineNONOate, one among other NO donors, there is an increase in erythrocyte deformability and oxygen hemoglobin affinity (29).

The plasma levels of ACh increase in inflammatory pathologies like fibrinogen (Fib), a plasma molecule predominantly produced by the liver [39, 40]. From many years, it was recognized that Fib behavior in vascular domains, where blood circulation is under low shear stress, acts as the most influent molecule in erythrocyte aggregation (EA) [41]. The association between Fib and EA has been verified in several pathological conditions [42]. Only in this twenty-first century was CD47 established as a binding target in the erythrocyte membrane for the soluble form of fibrinogen [43].

[L-lactate], concentration ratio between cyclic guanylyl cyclase (cGMP) and cyclic adenosine monophosphate (cAMP) and lower oxygen affinity to hemoglobin value and of oxyhemoglobin concentration [30]. At variance, the effect of the AChE inhibitor velnacrine maleate (VM) induced a higher degree of [NO] efflux/[NOx] mobilization through the AChE-VM inhibitor complex in the presence of p53/56lyn and p72syk inhibitors [30]. When in the case of erythrocyte membrane band 3 protein dephosphorylated state, the inactive complex form of the AChE promotes higher NO efflux than the AChE active complex form [30]. But the opposite was observed with erythrocyte membrane band 3 protein phosphorylation [30]. When experiments were done with the AChE strong inhibitor, VM, an almost inactive complex, results and induces lower NO efflux from erythrocytes and higher GSNO and peroxynitrite concentration

Additional studies were performed taking into account the identification of the type of a G-protein involved in the erythrocyte ACh/NO signaling pathway. It was evidenced that at the N-terminal

C-terminal site domain independently of the band 3 phosphorylation degree [31]. This chapter confirmed our previous hypothesis of the potential involvement of a heterotrimeric G protein in signal events mediated by the erythrocyte membrane AChE-ACh complex or AChE-inhibitor complex band 3 protein interactions with the participation of adenylyl cyclase inhibition [20, 31]. The quantification of NO efflux from the erythrocyte was assessed, by the first time for us, using the amino-IV sensor by the amperometric method which is described [32, 33]. The nitric oxide release from RBC in presence of ACh is sense by an electrode which oxidize NO at the working platinum electrode, resulting on electric current. The redox current is proportional to the NO concentration outside the membrane and it was continuously monitored with a computer. The AChE-ACh active complex activates PKC which phosphorylates PTP and PTK switching them to inactive and activate enzymes states, resulting in band-3 protein phos-

are associated with band 3 protein at the

values than those obtained with the active complex form AChE-ACh [30].

phorylation by PTK active form without with consequently NO release [32–34].

was quantified in relation to those values obtained with AChE-ACh [35–37].

erythrocyte deformability and oxygen hemoglobin affinity (29).

Beyond the AChE's strong inhibitor velnacrine, the moderate AChE inhibitor timolol was used, forming a less active AChE-timolol complex, and a lower erythrocyte efflux from NO

For the first time we evidenced that when erythrocytes were in the presence of ACh or timolol, the efflux of GSNO was lower with AChE-timolol than with AChE-ACh, both values being

It was evidenced by those in vitro studies that AChE's active and less active molecular conformations induce increased or decreased NO efflux from erythrocytes, respectively [38].

In the presence of SpermineNONOate, one among other NO donors, there is an increase in

The plasma levels of ACh increase in inflammatory pathologies like fibrinogen (Fib), a plasma molecule predominantly produced by the liver [39, 40]. From many years, it was recognized that Fib behavior in vascular domains, where blood circulation is under low shear stress, acts as the most influent molecule in erythrocyte aggregation (EA) [41]. The association between Fib and EA has been verified in several pathological conditions [42]. Only in this twenty-first

band 3 protein domain only Gαi1/2 binds. Gαi1/2 and the G<sup>β</sup>

134 Novel Prospects in Oxidative and Nitrosative Stress

higher than in their absence [38].

It was shown that for soluble Fib, in physiological concentrations, the NO efflux from erythrocytes decreased with increased GSNO, nitrite and nitrate levels [44]. The scavenging NO RBC ability to reduce efflux was surpassed showing normal values when both 4N1K (the CD47 peptide analog of thrombospondin binding site) and high fibrinogen levels are present or when 4N1K is absent [45]. These data show the dependence of lower cyclic adenosine monophosphate (cAMP) associated with adenylate cyclase (AC) inhibition by CD47Gαi [45]. When phosphorylation of the erythrocyte membrane protein band 3 is induced in the presence of high fibrinogen concentration and in the absence or presence of 4N1K, the NO efflux increases [46, 47]. The NO efflux from erythrocytes at high fibrinogen concentration is dependent on band 3 protein phosphorylation which was confirmed in the experiments where the erythrocyte casein kinase 2 (a cytosol protein that phosphorylates the band 3 protein) inhibitor was used, showing unchanged levels of NO efflux in relation to its absence [27, 48].

During inflammation high levels of both acetylcholine and fibrinogen are presented and normal values of NO efflux from erythrocytes have been observed in vitro [39, 49]. Besides, a higher NO efflux from RBC will be expected resulting of the presence of ACh and high fibrinogen concentration, normal values were obtained; the AChE-ACh molecular conformational state activates PKC which inhibits PDE 3 with increase of cAMP concentration that normalize the lower levels of cAMP derived from the inhibition of AC [35, 38, 45, 49]. Interestingly, the presence of forskolin (activator of the enzyme AC) in an in vitro model of hyperfibrinogenemia did not change the levels of NO efflux from erythrocytes, because the PDE3 is functional to hydrolase cAMP [27, 48].

When stimulating the erythrocyte redox thiol status by loading dithiothreitol (DTT), there is a decreased NO efflux concomitant with increased levels of nitrite, nitrate and GSNO [50]. It is well known that dithiothreitol induces band 3 dephosphorylation and a dephosphorylated state accounts for the AChE inhibitors and fibrinogen effects on red cells [17, 30, 49].

High concentrations of oxidized LDL when in the presence of blood samples of healthy human erythrocytes increase its ability to scavenge NO [51]. The same behavior was obtained in another study conducted with blood samples taken from healthy humans and exposed or not (control aliquot) to two different concentrations of LDL/HDL; no changes in NO efflux values from the erythrocyte, no alterations on intra-erythrocyte peroxynitrite concentrations and an unaltered deformability profile, at all shear stresses, were observed. At variance the levels of intra-erythrocyte NO derivative molecules nitrite, nitrate and GSNO showed significantly increased values when compared with control aliquots. The unchanged deformability values obtained at lower and high shear stresses for all treated blood sample aliquots with LDL/HDL are indicative of membrane stability, internal viscosity maintenance and normal interactions of membrane peripheral and cytoskeleton [52]. The absence of erythrocyte membrane instability obtained in blood sample aliquots under LDL/HDL addition is confirmed by the unchanged nitrogen reactive species concentration of peroxynitrite, as evidence by the normal levels obtained for peroxynitrite is an index of auto-oxidation of oxyhemoglobin [52, 53]. The addition of different concentrations of the lipoprotein sub-fractions' LDL/HDL seems not to favor hemoglobin auto-oxidation. Superoxide anion will be formed from the auto-oxidation of hemoglobin, but without its generation plus unchanged values of peroxynitrite concentrations it was evidenced that when the thiol status of erythrocyte was maintained in normal range, no alterations were verified in erythrocyte deformability [50].

So, a study was performed to evaluate the associations between hemorheology parameters including the erythrocyte NO rescue ability of RBCs and the cardiovascular risk factors, inflammatory parameters and subclinical atherosclerosis. Erythrocyte NO efflux was significantly associated with both carotid intima-media thickness (cIMT) and the presence of plaques (negative association) and was an independent predictor of cIMT [67]. Erythrocyte NO production can be looked at as a compensatory mechanism [67]. As mentioned above, under low tissue oxygen partial tension, erythrocytes release NO bound to hemoglobin, promoting vasodilation [61, 62]. Besides, NO could represent a protective factor against atherosclerosis; it could be produced in large amounts by the inducible nitric oxide synthase (iNOS) which is characteristic of the dysfunctional endothelium which combining with superoxide anion generates peroxynitrite molecules that have oxidant properties; this NO derivative

Erythrocyte Nitric Oxide

137

http://dx.doi.org/10.5772/intechopen.75931

worse the dysfunctional endothelial wall hindering it to return to be functional [68].

disease in RA and SLA patients [67].

further potential therapeutic targets [70].

index quantified in sub lingual microcirculation [72].

The data of the hemorheological and inflammatory evaluations performed ex vivo in blood samples of women with SLE suggested greater risk of arterial thrombosis and prediction of higher mortality than humans with normal blood viscosity and fibrinogen values [67, 69]. Both SLE and RA patients showed high erythrocyte aggregation independent of the medication undertaken by SLE patients. This ex vivo study shows that hemorheological parameters are independently associated with the early stages of atherosclerosis in SLE and RA patients. Additionally, it documents disturbed and unfavorable hemorheological features in association with disease activity and with traditional CV risk factors contributing to atherogenesis in inflammatory rheumatic diseases. So, the evaluation of NO and also of hemorheological parameters must be done in order to predict the development course of the autoimmune

In patients with amyotrophic lateral sclerosis (ALS) that is a neurodegenerative disease of the motor system, our aim was to assess RBCs' biochemical and hemorheological parameters and identify novel biomarkers of one of the most painful and fast mortal disease after diagnosis [70]. The erythrocyte deformability and AChE activity of blood samples were increased in patients with ALS in comparison to healthy donors [70]. This ex vivo study conducted with blood samples of ALS patients showed lower values of NO efflux from RBCs and nitrites than those obtained in healthy humans [70]. Due to variability between the duration of this disease until death, the higher NO quartile values are associated with the worse respiratory function [70]. A positive relation of quartiles values were obtained between AChE enzyme activity and nitrites levels [70]. Both erythrocyte NO and AChE were suggested as biomarkers of ALS and

Another very sad complex situation with high mortality covering healthy humans from all ages and under a variety of situations from a simple infection or travel accident to a postsurgery complication is sepsis [71]. Several pathophysiological mechanisms from unbalanced pro−/anti -inflammatory, hypo and hyper-insulinemia, to hypo- and hyper-glycemia are simultaneously deregulated in intensive care units (ICUs). Follow-up studies carried out in ICU are urgently needed [71]. We have verified that in septic shock patients before dead, at 24 in IUC showed higher efflux of NO from erythrocytes and worse blood circulation observed by hemodynamic parameters namely high unequal blood flow and high microvascular flow

The NO efflux from erythrocytes is gender independent [54] at variance with higher women's RBC AChE enzyme activity than men as previously evidenced [54]. Timolol maleate is a topically therapeutic drug used in glaucoma patients that, when incubated with blood samples of patients with this nerve optical disease, they did not induce significant differences in NO efflux from erythrocytes and nor in GSNO concentration values inside RBCs when compared to the absence of timolol in the blood aliquots of those erythrocytes [55]. Both NO efflux and GSNO values obtained were significantly higher than those quantified in blood samples of healthy persons [55]. Erythrocytes' NO metabolism in glaucoma patients are not affected by timolol treatment [55].

The same amperometric NO sensor, mentioned above, was used in confluent human umbilical endothelial cells (HUVECs) in which we have demonstrated the existence of membranebound acetylcholinesterase and higher NO production in the presence of ACh in relation to velnacrine [56–58]. The activation of the signal transduction mechanism induced by the AChE-ACh active complex revealed high values of [cAMP] and [cGMP] which are lowered by the AChE-VM inactive complex [58].

### **2.2. Ex vivo**

Hemorheology is the science which studies blood deformation and its components' interaction with vessel walls, occurring inside blood vessels of macro and microcirculation. In the past, the longitudinal and follow-up clinical studies done, ex vivo, have as an objective the characterization of the intravascular profile of different diseases according to hemorheological parameters and inflammatory factors [41, 48, 59]. To accomplish this aim, blood samples were taken from patients with acute myocardial infarction, glaucoma, Bechet, renal diseases whether submitted or not to chronic hemodialysis or kidney transplant and diabetic retinopathy degree, and an association between the laboratorial data and the clinical parameter values was observed [41, 48, 59].

Erythrocytes scavenge and liberate oxygen and NO at high and low local tissue oxygen partial pressure, respectively [60, 61]. Erythrocyte deformability is a biorheological influent factor on blood viscosity, cellular oxygenation and a biomarker of acute and chronic inflammation [62].

Patients with hypercholesterolemia, hypertension and renal transplant present lower ability to reversible change its shape (decrease erythrocyte deformability values) and higher values of NO efflux from erythrocytes when stimulated with ACh [63]. In the same study, an inverse relationship between erythrocyte deformability values and NO efflux concentrations from erythrocytes obtained from blood samples of those patients, was evidenced [63]. In all samples lower values of NO efflux were verified in relation to those of healthy persons—what could be considered as a compensatory mechanism to avoid more wall vessel damage [63]. We will present in vivo studies later in this section.

Disturbed blood rheology in patients with systemic lupus erythematosus (SLE) and patients with rheumatoid arthritis (RA) that could contribute to atherosclerosis is described [64–66]. So, a study was performed to evaluate the associations between hemorheology parameters including the erythrocyte NO rescue ability of RBCs and the cardiovascular risk factors, inflammatory parameters and subclinical atherosclerosis. Erythrocyte NO efflux was significantly associated with both carotid intima-media thickness (cIMT) and the presence of plaques (negative association) and was an independent predictor of cIMT [67]. Erythrocyte NO production can be looked at as a compensatory mechanism [67]. As mentioned above, under low tissue oxygen partial tension, erythrocytes release NO bound to hemoglobin, promoting vasodilation [61, 62]. Besides, NO could represent a protective factor against atherosclerosis; it could be produced in large amounts by the inducible nitric oxide synthase (iNOS) which is characteristic of the dysfunctional endothelium which combining with superoxide anion generates peroxynitrite molecules that have oxidant properties; this NO derivative worse the dysfunctional endothelial wall hindering it to return to be functional [68].

auto-oxidation of hemoglobin, but without its generation plus unchanged values of peroxynitrite concentrations it was evidenced that when the thiol status of erythrocyte was maintained

The NO efflux from erythrocytes is gender independent [54] at variance with higher women's RBC AChE enzyme activity than men as previously evidenced [54]. Timolol maleate is a topically therapeutic drug used in glaucoma patients that, when incubated with blood samples of patients with this nerve optical disease, they did not induce significant differences in NO efflux from erythrocytes and nor in GSNO concentration values inside RBCs when compared to the absence of timolol in the blood aliquots of those erythrocytes [55]. Both NO efflux and GSNO values obtained were significantly higher than those quantified in blood samples of healthy persons [55]. Erythrocytes' NO metabolism in glaucoma patients are not affected by

The same amperometric NO sensor, mentioned above, was used in confluent human umbilical endothelial cells (HUVECs) in which we have demonstrated the existence of membranebound acetylcholinesterase and higher NO production in the presence of ACh in relation to velnacrine [56–58]. The activation of the signal transduction mechanism induced by the AChE-ACh active complex revealed high values of [cAMP] and [cGMP] which are lowered

Hemorheology is the science which studies blood deformation and its components' interaction with vessel walls, occurring inside blood vessels of macro and microcirculation. In the past, the longitudinal and follow-up clinical studies done, ex vivo, have as an objective the characterization of the intravascular profile of different diseases according to hemorheological parameters and inflammatory factors [41, 48, 59]. To accomplish this aim, blood samples were taken from patients with acute myocardial infarction, glaucoma, Bechet, renal diseases whether submitted or not to chronic hemodialysis or kidney transplant and diabetic retinopathy degree, and an association between the laboratorial data and the clinical parameter values was observed [41, 48, 59].

Erythrocytes scavenge and liberate oxygen and NO at high and low local tissue oxygen partial pressure, respectively [60, 61]. Erythrocyte deformability is a biorheological influent factor on blood viscosity, cellular oxygenation and a biomarker of acute and chronic inflammation [62].

Patients with hypercholesterolemia, hypertension and renal transplant present lower ability to reversible change its shape (decrease erythrocyte deformability values) and higher values of NO efflux from erythrocytes when stimulated with ACh [63]. In the same study, an inverse relationship between erythrocyte deformability values and NO efflux concentrations from erythrocytes obtained from blood samples of those patients, was evidenced [63]. In all samples lower values of NO efflux were verified in relation to those of healthy persons—what could be considered as a compensatory mechanism to avoid more wall vessel damage [63].

Disturbed blood rheology in patients with systemic lupus erythematosus (SLE) and patients with rheumatoid arthritis (RA) that could contribute to atherosclerosis is described [64–66].

in normal range, no alterations were verified in erythrocyte deformability [50].

timolol treatment [55].

136 Novel Prospects in Oxidative and Nitrosative Stress

**2.2. Ex vivo**

by the AChE-VM inactive complex [58].

We will present in vivo studies later in this section.

The data of the hemorheological and inflammatory evaluations performed ex vivo in blood samples of women with SLE suggested greater risk of arterial thrombosis and prediction of higher mortality than humans with normal blood viscosity and fibrinogen values [67, 69]. Both SLE and RA patients showed high erythrocyte aggregation independent of the medication undertaken by SLE patients. This ex vivo study shows that hemorheological parameters are independently associated with the early stages of atherosclerosis in SLE and RA patients. Additionally, it documents disturbed and unfavorable hemorheological features in association with disease activity and with traditional CV risk factors contributing to atherogenesis in inflammatory rheumatic diseases. So, the evaluation of NO and also of hemorheological parameters must be done in order to predict the development course of the autoimmune disease in RA and SLA patients [67].

In patients with amyotrophic lateral sclerosis (ALS) that is a neurodegenerative disease of the motor system, our aim was to assess RBCs' biochemical and hemorheological parameters and identify novel biomarkers of one of the most painful and fast mortal disease after diagnosis [70]. The erythrocyte deformability and AChE activity of blood samples were increased in patients with ALS in comparison to healthy donors [70]. This ex vivo study conducted with blood samples of ALS patients showed lower values of NO efflux from RBCs and nitrites than those obtained in healthy humans [70]. Due to variability between the duration of this disease until death, the higher NO quartile values are associated with the worse respiratory function [70]. A positive relation of quartiles values were obtained between AChE enzyme activity and nitrites levels [70]. Both erythrocyte NO and AChE were suggested as biomarkers of ALS and further potential therapeutic targets [70].

Another very sad complex situation with high mortality covering healthy humans from all ages and under a variety of situations from a simple infection or travel accident to a postsurgery complication is sepsis [71]. Several pathophysiological mechanisms from unbalanced pro−/anti -inflammatory, hypo and hyper-insulinemia, to hypo- and hyper-glycemia are simultaneously deregulated in intensive care units (ICUs). Follow-up studies carried out in ICU are urgently needed [71]. We have verified that in septic shock patients before dead, at 24 in IUC showed higher efflux of NO from erythrocytes and worse blood circulation observed by hemodynamic parameters namely high unequal blood flow and high microvascular flow index quantified in sub lingual microcirculation [72].

#### **2.3. In vivo studies**

The ACh molecule has a ubiquity property with an anti-inflammatory effect, decreasing leukocytes' adherence and plasma TNF-α concentration evidenced in an in vivo animal model of lipopolysaccharide-induced inflammation [73, 74].

phosphorylation. At variance, higher NO mobilization inside the erythrocytes happens under

Erythrocyte Nitric Oxide

139

http://dx.doi.org/10.5772/intechopen.75931

At normal acetylcholine plasma levels, the erythrocyte NO efflux increases by a signal pathway dependent of membrane band 3 protein phosphorylation, Giαβ protein, AC, acetylcholin-

The erythrocyte aggregation tendency is impaired by the presence of the AChE-ACh complex and is reinforced by higher thiol redox status inside the erythrocyte. The erythrocyte aggregation is impaired by changes on band 3 phosphorylation/dephosphorylation equilibrium; besides, higher values are associated with a higher phosphorylation degree. On the contrary,

The ability of RBC to scavenge NO may be considered as a compensatory mechanism acting against the overproduced NO by the endothelial-inducible NO synthase when the vascular

Under unstimulated erythrocytes, GSNO efflux did not occur, and it is regarded as a poten-

It is possible to modulate erythrocyte availability in NO by plasma fibrinogen in a nonlinear, dose-dependent manner in human erythrocytes. Lower intra-erythrocyte cAMP is an influent condition to the NO efflux in an in vitro model of hyperfibrinogenemia. These results may be considered a useful therapeutic approach for the storage of blood that is used in transfusions. Fibrinogen-C47-triggered erythrocyte GSNO and decreased NO efflux may, if verified in vivo, be associated with coagulopathy and hypotension under acute phase states. These effects show on NO-derived molecules, allowing intra-erythrocyte NO scavenging as a protector under inflammatory conditions as we evidenced in an animal model of acute inflammation and verified ex vivo in a vascular inflammatory disease with low-grade or chronic inflammation. An anti-reactive nitrogen role can be attributed to ox-LDL for its contribution in the erythrocyte-scavenged ability for nitric oxide. From all studies reviewed here, we can suggest NO efflux or influx from or into RBCs and the internal mobilization between the NOx as a hemorheological parameter participating in the erythrocyte deformability in dependence of the structural protein conformations or phosphorylation degree components of the membrane or the internal RBC compartment.

This work was funded by Fundação para a Ciência e Tecnologia: LISBOA-01-0145-FEDER-007391, and the project was co-funded by FEDER, through POR Lisboa 2020—Programa Operacional Regional de Lisboa, PORTUGAL 2020. The authors thank Teresa Freitas and Emilia

Alves for their technical and type-writing support, respectively.

simultaneous influence of AChE-velnacrine and band 3 protein dephosphorylation.

esterase enzyme activity and its molecular conformations, PKC and PD3.

ED increases in the presence of the AChE-ACh complex.

tially therapeutical agent, acting as a store or donor of NO.

endothelium is dysfunctional [50].

**Acknowledgements**

**Conflict of interest**

The authors declare no conflict of interest.

The influence of NO on the hemoglobin affinity to oxygen and on biorheology properties of the erythrocytes was shown in healthy and in ill humans as described earlier. NO influx into erythrocytes induced by spermineNONOate or the efflux stimulates by ACh increased the reversible discocyte shape, or erythrocyte deformability, under shear stress values characteristic of the microcirculation network as shown [29].

The angiotensin II AT1-receptor antagonist, valsartan, is able to restore, in hypertensive Sprague–Dawley rats which are LNAME dependent, their systolic blood pressure (SBP) to the physiological values, as well as normalize the whole blood viscosity (WBV) values increased during the hypertensive time [75]. The NO efflux from erythrocytes decreases in parallel to WBV and SBP returning to normal values after valsartan application [75]. Regarding erythrocyte deformability values that decreased during the hypertensive state of the animals, lower or normal values at lower and higher shear stress, respectively, were maintained after systolic blood pressure recovery by valsartan [75]. This in vivo animal experimental model of hypertension demonstrated the relation between the endothelial cells and the erythrocytes' availability of NO, beavering as a compensatory mechanism vascular disturbance [75]. This could be considered as an antagonist effect to the occurrence of reactive nitrogen species (RNS) and to the amplification of oxidative reactive species (ORS).

The in vivo mice model of acute inflammation induced by intra-scrotal injection of the platelet-activator factor (PAF, a phospholipid mediator of inflammation) showed that the NO efflux from erythrocytes decreases with acute-phase response development [76] The end of the acute inflammatory response visualized by intravital microscopy showed normalization of the number of labeled neutrophils rolling and adherent, rolling velocity and vessel diameter values [76]. NO normal values rewound with inflammation recover, besides the maintenance of decreased RBCs' deformability [76]. There are PA receptors (PAF-R) constitutively present on platelets, leukocytes and endothelial cells but are absent in red blood cells; besides, PAF stimulates the breakdown of sphingomyelin on RBCs in isotonic conditions [77]. Therefore, PAF may cause changes in the physicochemical structure of the erythrocyte membrane, which in turn may cause changes in RBC deformability maintained after the recovery of the initial phase of inflammatory response [77]. The NO efflux from erythrocyte behavior in this model of acute inflammatory response during 6 h in mice reinforces the idea of the NO rescue inside erythrocyte as a compensatory mechanism in low-grade or chronic inflammation of those diseases reported earlier [63, 77].

### **3. Conclusions**

The nitric oxide mobilization inside or outside the erythrocyte is possible under either the action of the non-neuronal cholinergic components, acetylcholinesterase and acetylcholine, or the manipulation of band 3 protein phosphorylation degree. Higher NO efflux occurs under the influence of the AChE-ACh complex as well as, simultaneously, with the band 3 protein phosphorylation. At variance, higher NO mobilization inside the erythrocytes happens under simultaneous influence of AChE-velnacrine and band 3 protein dephosphorylation.

At normal acetylcholine plasma levels, the erythrocyte NO efflux increases by a signal pathway dependent of membrane band 3 protein phosphorylation, Giαβ protein, AC, acetylcholinesterase enzyme activity and its molecular conformations, PKC and PD3.

The erythrocyte aggregation tendency is impaired by the presence of the AChE-ACh complex and is reinforced by higher thiol redox status inside the erythrocyte. The erythrocyte aggregation is impaired by changes on band 3 phosphorylation/dephosphorylation equilibrium; besides, higher values are associated with a higher phosphorylation degree. On the contrary, ED increases in the presence of the AChE-ACh complex.

The ability of RBC to scavenge NO may be considered as a compensatory mechanism acting against the overproduced NO by the endothelial-inducible NO synthase when the vascular endothelium is dysfunctional [50].

Under unstimulated erythrocytes, GSNO efflux did not occur, and it is regarded as a potentially therapeutical agent, acting as a store or donor of NO.

It is possible to modulate erythrocyte availability in NO by plasma fibrinogen in a nonlinear, dose-dependent manner in human erythrocytes. Lower intra-erythrocyte cAMP is an influent condition to the NO efflux in an in vitro model of hyperfibrinogenemia. These results may be considered a useful therapeutic approach for the storage of blood that is used in transfusions. Fibrinogen-C47-triggered erythrocyte GSNO and decreased NO efflux may, if verified in vivo, be associated with coagulopathy and hypotension under acute phase states. These effects show on NO-derived molecules, allowing intra-erythrocyte NO scavenging as a protector under inflammatory conditions as we evidenced in an animal model of acute inflammation and verified ex vivo in a vascular inflammatory disease with low-grade or chronic inflammation. An anti-reactive nitrogen role can be attributed to ox-LDL for its contribution in the erythrocyte-scavenged ability for nitric oxide. From all studies reviewed here, we can suggest NO efflux or influx from or into RBCs and the internal mobilization between the NOx as a hemorheological parameter participating in the erythrocyte deformability in dependence of the structural protein conformations or phosphorylation degree components of the membrane or the internal RBC compartment.

### **Acknowledgements**

**2.3. In vivo studies**

138 Novel Prospects in Oxidative and Nitrosative Stress

**3. Conclusions**

lipopolysaccharide-induced inflammation [73, 74].

istic of the microcirculation network as shown [29].

to the amplification of oxidative reactive species (ORS).

The ACh molecule has a ubiquity property with an anti-inflammatory effect, decreasing leukocytes' adherence and plasma TNF-α concentration evidenced in an in vivo animal model of

The influence of NO on the hemoglobin affinity to oxygen and on biorheology properties of the erythrocytes was shown in healthy and in ill humans as described earlier. NO influx into erythrocytes induced by spermineNONOate or the efflux stimulates by ACh increased the reversible discocyte shape, or erythrocyte deformability, under shear stress values character-

The angiotensin II AT1-receptor antagonist, valsartan, is able to restore, in hypertensive Sprague–Dawley rats which are LNAME dependent, their systolic blood pressure (SBP) to the physiological values, as well as normalize the whole blood viscosity (WBV) values increased during the hypertensive time [75]. The NO efflux from erythrocytes decreases in parallel to WBV and SBP returning to normal values after valsartan application [75]. Regarding erythrocyte deformability values that decreased during the hypertensive state of the animals, lower or normal values at lower and higher shear stress, respectively, were maintained after systolic blood pressure recovery by valsartan [75]. This in vivo animal experimental model of hypertension demonstrated the relation between the endothelial cells and the erythrocytes' availability of NO, beavering as a compensatory mechanism vascular disturbance [75]. This could be considered as an antagonist effect to the occurrence of reactive nitrogen species (RNS) and

The in vivo mice model of acute inflammation induced by intra-scrotal injection of the platelet-activator factor (PAF, a phospholipid mediator of inflammation) showed that the NO efflux from erythrocytes decreases with acute-phase response development [76] The end of the acute inflammatory response visualized by intravital microscopy showed normalization of the number of labeled neutrophils rolling and adherent, rolling velocity and vessel diameter values [76]. NO normal values rewound with inflammation recover, besides the maintenance of decreased RBCs' deformability [76]. There are PA receptors (PAF-R) constitutively present on platelets, leukocytes and endothelial cells but are absent in red blood cells; besides, PAF stimulates the breakdown of sphingomyelin on RBCs in isotonic conditions [77]. Therefore, PAF may cause changes in the physicochemical structure of the erythrocyte membrane, which in turn may cause changes in RBC deformability maintained after the recovery of the initial phase of inflammatory response [77]. The NO efflux from erythrocyte behavior in this model of acute inflammatory response during 6 h in mice reinforces the idea of the NO rescue inside erythrocyte as a compensatory mechanism in low-grade or chronic inflammation of those diseases reported earlier [63, 77].

The nitric oxide mobilization inside or outside the erythrocyte is possible under either the action of the non-neuronal cholinergic components, acetylcholinesterase and acetylcholine, or the manipulation of band 3 protein phosphorylation degree. Higher NO efflux occurs under the influence of the AChE-ACh complex as well as, simultaneously, with the band 3 protein This work was funded by Fundação para a Ciência e Tecnologia: LISBOA-01-0145-FEDER-007391, and the project was co-funded by FEDER, through POR Lisboa 2020—Programa Operacional Regional de Lisboa, PORTUGAL 2020. The authors thank Teresa Freitas and Emilia Alves for their technical and type-writing support, respectively.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Carlota Saldanha\* and Ana Silva-Herdade

\*Address all correspondence to: carlotasaldanha@medicina.ulisboa.pt

Faculty of Medicine, Institute of Biochemistry, Institute of Molecular Medicine, University of Lisbon, Lisbon, Portugal

[12] Bordin L, Brunati AM, Donella-Deana A, Baggio B, Toninello A, Clari G. Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes.

Erythrocyte Nitric Oxide

141

http://dx.doi.org/10.5772/intechopen.75931

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**Author details**

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## *Edited by Pinar Atukeren*

Oxidative stress plays a crucial role in the pathophysiology of various diseases when there is a disruption of the intracellular redox balance and the homeostatic balance between cellular oxidants and antioxidants. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) react with molecular targets including proteins, lipids, and nucleic acids contributing to mitochondrial injury and cellular dysfunction. This book intends to provide the readers with an extensive overview of the novel approaches and prospects based on oxidative and nitrosative stress in the pathophysiology of various diseases and in the current treatment strategies with antioxidants.

Published in London, UK © 2018 IntechOpen © Holly Chisholm / unsplash

Novel Prospects in Oxidative and Nitrosative Stress

Novel Prospects in Oxidative

and Nitrosative Stress