Immunopharmacology and Immunotoxicology, 2009; 31(3): 388–396

R E s E a R c h a R T I c L E

Biochemical and cellular toxicology of peroxynitrite: implications in cell death and autoimmune phenomenon

Rizwan Ahmad1, Zafar Rasheed1, Haseeb Ahsan2

1Department of Biochemistry, Sardar Bhagwan Singh Post-Graduate Institute of Biomedical Sciences and Research, Balawala, Dehradun - 248161, India, and 2Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard, Hamdard University, New Delhi - 110062, India

Address for Correspondence: Haseeb Ahsan, Department of Medical Elementology, Faculty of Science, Jamia Hamdard, Hamdard University, New Delhi - 110062, India. E-mail: hahsan@rediffmail.com

(Received 01 December 2008; revised 19 December 2008; accepted 22 December 2008)

Introduction

Peroxynitrite is a member of reactive nitrogen species that also includes nitric oxide (·NO) and nitrogen diox-ide radical (NO

2·). Peroxynitrite is a reactive nitrogen

species and an anion with the formula (ONOO−). It is an unstable ‘valence isomer’ of nitrate (NO

3−), making

it an oxidant and nitrating agent. Because of its oxidiz-ing properties, peroxynitrite can damage a wide range of molecules in cells, including DNA and proteins (1). It is produced by the body in response to a variety of envi-ronmental toxins, stress, ultraviolet light and many other stimuli. It is also produced in the body due to ischemia/repurfusion injury and inflammation (2,3).

In vivo, peroxynitrite is formed from nitric oxide (·NO) and superoxide anion (O

2· –) and the production

is controlled by superoxide dismutase (SOD). Since the physiological concentration of carbon dioxide

(CO2) is high, peroxynitrite reacts nucleophilically with

CO2 to form nitrosoperoxy- carbonate (ONOOCO

2−).

Nitrosoperoxycarbonate homolyzes to form carbonate (CO

3· –) and nitrogen dioxide radicals (NO

2·). These radi-cals are believed to cause peroxynitrite related cellular damage (4).

Modification of native DNA by peroxynitrite might also lead to the generation of neoepitopes on the mol-ecule, and may be one of the factors for the induction of the immune responses as seen in an autoimmune disease like systemic lupus erythematosus (SLE).

Formation of Peroxynitrite

Free radicals are generated in biological systems both as a by-product of normal cellular processes and as a result of exposure to a number of external stimuli.

ISSN 0892-3973 print/ISSN 1532-2513 online © 2009 Informa UK LtdDOI: 10.1080/08923970802709197

abstractReactive nitrogen species include nitric oxide (·NO), peroxynitrite (ONOO−) and nitrogen dioxide radical (NO2·). Peroxynitrite is a reactive oxidant, produced from nitric oxide (·NO) and superoxide anion (O2

· –), that reacts with a variety of biological macromolecules. It is produced in the body in response to physiologi-cal stress and environmental toxins. It is a potent trigger of oxidative protein and DNA damage-including DNA strand breakage and base modification. It activates the nuclear enzyme poly-ADP ribose polymerase (PARP) resulting in energy depletion and apoptosis/necrosis of cells. Peroxynitrite generation is a crucial pathological mechanism in stroke, diabetes, inflammation, neurodegeneration, cancer, etc. Peroxynitrite modified DNA may also lead to the generation of autoantibodies in various autoimmune disorders such as systemic lupus erythematosus (SLE). In chronic inflammatory diseases, peroxynitrite formed by phago-cytic cells may cause damage to DNA, generating neoepitopes leading to the production of autoanti-bodies. Hence, understanding the pathophysiology of peroxynitrite could lead to important therapeutic interventions.

Keywords: Autoimmunity; Cytotoxicity; Immunotoxicology; Nitric Oxide; Peroxynitrite

http://www.informahealthcare.com/ipi

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Toxicology of Peroxynitrite 389

Most radicals react rapidly with biological agents such as lipids, nucleic acids, carbohydrates, proteins, and these reactions have been shown to affect the biological structure, function, and subsequent cellular processing of these molecules (5,6). In vivo, peroxynitrite is formed in the macrophages, endothelial cells, platelets, leuko-cytes, neurons, etc by the reaction between O

2· – and ·NO

(7,8). Tissue inflammation and chronic infection lead to the overproduction of ·NO and O

2· –, which rapidly com-

bine to yield peroxynitrite: O2

· – + ·NO → ONO2

−.Several synthetic methods have been reported for

the preparation of peroxynitrite (9,10). Some of which include:

1. A method for the preparation of high concentration of peroxynitrite involves a displacement reaction by the hydroperoxide anion (in the aqueous phase) on isoamyl nitrite (in the organic phase). The prod-uct, peroxynitrite, formed remains in the aqueous phase (11).

2. Another method of formation of peroxynitrite involves the synergistic action of sodium nitroprus-side [Na

2Fe(CN)

5NO], a ·NO donor and pyrogallol,

a O2

· – donor, in the presence of a chelator, DTPA (diethylenetriamine pentaacetic acid). It is one of the most commonly used methods for the genera-tion of peroxynitrite in vitro (12,13).

3. A simple method for the synthesis of peroxynitrite, from nitrite (NO

2−) and hydrogen peroxide (H

2O

2),

was reported by 14. This method can generate hun-dreds of milliliters of 180 mM peroxynitrite within an hour, requires simple apparatus and is feasible in any biochemical laboratory.

Cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes mellitus or chronic smoking stimulate the production of the reactive oxygen species, O

2· –, in the vascular wall. Nicotinamide adenine

dinucleotide phosphate (NADPH) oxidases is the major source of O

2· – and has been found upregulated and acti-

vated in animal models of hypertension, diabetes and in patients with cardiovascular risk factors (15).

Endothelial ·NO synthase (eNOS) is responsible for most of the vascular ·NO produced. The eNOS oxi-dizes its substrate L-arginine to L-citrulline and ·NO. A functional eNOS requires dimerization of the enzyme, the substrate L-arginine, and an essential cofactor, BH

4

(5,6,7,8-tetrahydro-L-biopterin). The O2

· – produced can react with vascular ·NO to form peroxynitrite. Diminished levels of BH

4 promote O

2· – production by

eNOS. The transformation of eNOS from a vasoprotec-tive enzyme to a contributor to oxidative stress has been observed in several in vitro systems, animal models of cardiovascular diseases and in patients with cardiovas-cular risk factors (15). In inflammation or septic shock,

·NO is also synthesized by the inducible ·NO synthase (iNOS), an isoform that is expressed in many cells types including vascular endothelial cells, vascular smooth muscle and inflammatory cells in response to proin-flammatory cytokines.

Peroxynitrite, can be formed intravascularly in vari-ous disease conditions when there is overproduction of either ·NO or O

2· – (16). The intravascular formation

of peroxynitrite can result in oxidative modifications of plasma and vessel wall proteins including the formation of protein-3-nitrotyrosine. Protein tyrosine nitration in plasma or vessel wall proteins may be indicative of peroxynitrite formation, and constitutes a good biomar-ker of ·NO-derived oxidant production in the vascular space. Detection of 3-nitrotyrosine in vivo has attracted considerable interest not only as a biomarker of perox-ynitrite formation but also as a predictor of vascular risk (16) (Table 1).

Reactivity of Peroxynitrite

Peroxynitrite exhibits unique chemical reactivities such as protein nitration, DNA strand breakage, base modi-fication, etc., which may have cytotoxic effects and also lead to mutagenesis. It is thought to be involved in both cell death and an increased cancer risk (17–19).

The reaction of peroxynitrite with lipids leads to peroxidation (malondialdehyde and conjugated diene formation) and formation of nitrito-, nitro-, nitrosoper-oxo-, and nitrated lipid oxidation derivatives (20–22). Peroxynitrite is a particularly effective oxidant of aro-matic molecules, thioethers and organosulfur com-pounds that include free amino acids and polypeptide residues.

The reaction of various amino acids with peroxyni-trite leads to the following products: 1) cysteine and glutathione are converted to disulfides; 2) methionine is converted to sulfoxide or is fragmented to ethylene and dimethyl disulfides. Dimethyl sulfoxide is oxidized to formaldehyde; and 3) tyrosine and tryptophan undergo one electron oxidation to radical cations, which are hydroxylated, nitrated and dimerized (23–25).

Exposure of amino acids, peptides and proteins to ionizing radiation such as gamma radiation and peroxynitrite in the presence of O

2, gives rise to

Table 1. Reaction of peroxynitrite with various substances leading to the formation of reactive products.

Reactant Product

Transition metals ·NO2

Thiols ·NO

CO2

CO3

· –

oxyHb NO3

−, metHb, O2

· –

(adapted from 15)

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390 R. Ahmad et al.

hydroperoxides. These hydroperoxides decompose to oxygen and carbon centered radicals on expo-sure to copper (Cu+) and other transition metal ions. Hydroperoxide formation on nuclear proteins results in oxidative damage to associated DNA. These hydroper-oxide-derived radicals react readily with pyrimidine DNA bases and nucleosides to form adduct species, for example 8-oxo-dG. This adduct is highly mutagenic and induces G:C to T:A transversions in human DNA after replication (26).

Purine nucleotides are vulnerable to oxidation and to adduct formation (27,28). Peroxynitrite is a mutagenic agent with a potential to produce nitration, nitrosation and deamination of DNA bases. Methylation of cytosine in DNA is important for the regulation of gene expres-sion and normal methylation patterns are altered by the carcinogenic effect of peroxynitrite (29). Prominent DNA modifications induced by peroxynitrite include the formation of 8-nitro-guanine and 8-oxyguanine, as well as the induction of single strand breaks (30,31). DNA single strand breaks generated by peroxynitrite leads to activation of the nuclear enzyme, poly (ADP-ribose) synthetase (PARS), which can trigger cellular suicidal pathway.

Single strand breaks generated by peroxynitrite can arise from two processes: 1) sugar damage, which involves abstraction of hydrogen leading to the forma-tion of sugar radical or 2) base damage, which rapidly depurinates to generate abasic sites, finally resulting in single strand breaks (30). Peroxynitrite is mutagenic in the supF gene inducing G to T transversions and dele-tions clustered at the 5/ end of the gene. The mutagenic-ity of peroxynitrite is believed to result from chemical modifications at guanine leading to miscoding (32).

Carcinogenesis is induced by altered DNA or tis-sue damage, mutations and chromosomal aberra-tions (33,34). Peroxynitrite is a mutagenic agent with the potential to produce nitration, nitrosation and deamination reactions on DNA bases. It reacts sig-nificantly only with guanine, which upon oxidation and nitration leads to mutagenicity and strand breaks, respectively (35,36). Peroxynitrite levels are elevated in inflammation and infection and play an important role in carcinogenesis. It damages tumor suppressor genes and leads to the expression of proto-oncogenes. Peroxynitrite induced DNA damage leading to muta-tions has been strongly implicated in carcinogenesis (37) (Figure 1).

Cytotoxicity of Peroxynitrite

Proteins are targets of reactive nitrogen species such as peroxynitrite and NO

2. Among the various amino acids in

proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species (38). Peroxynitrite is capable of oxidizing protein and non-protein sulfhy-dryl (-SH) groups including lipid peroxidation and reac-tivity with aromatic amino acid side chain in proteins to form nitroadducts (39).

Peroxynitrite induced tyrosine nitration may lead to dysfunction of nitrated proteins, SOD, cytoskeletal proteins, neuronal tyrosine hydroxylase, cytochrome P450 and prostacyclin synthase (40–43). Oxidation of critical -SH groups is responsible for the inhibition of mitochondrial and cytosolic aconitase and other critical enzymes in the mitochondrial respiratory chain (44). Peroxynitrite mediated nitration of myofibrillar creatine kinase activity may lead to contractile dysfunc-tion of the heart (45). Peroxynitrite-modified cellular proteins are subject to accelerated degradation via the proteosome (46).

Peroxynitrite inhibits SOD, glutaredoxin and other antioxidant enzymes. Peroxynitrite mediated deple-tion of one of the key cellular antioxidants—glutath-ione (GSH) leads to intracellular oxidation and the generation and exacerbation of cellular injury (47,48). Peroxynitrite formation in human body fluids causes antioxidant depletion and oxidative damage to pro-teins and lipids in plasma. It decreases the total per-oxyl trapping capacity of plasma, which leads to rapid oxidation of ascorbic acid, uric acid and plasma -SH groups (49).

Peroxynitrite inhibits a variety of ion-pumps includ-ing calcium pumps, calcium-activated potassium chan-nels and membrane Na+/K+ ATPase activity resulting in

Mutagenesis

Modification ofDNA bases

Change in DNA conformation

ReplicationDefects

MismatchBase Pairing

MutationErrors

Peroxynitrite

StrandBreaks

DNA adducts

Figure 1. Peroxynitrite mediated biochemical toxicology leading to DNA damage and mutagenesis. Peroxynitrite may cause modi-fication of DNA bases or change in DNA conformation through modified bases, strand breaks or DNA adducts ultimately leading to mutations.

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Toxicology of Peroxynitrite 391

global dysregulation of ion-balance (50). Peroxynitrite reacts with NADH resulting in the forma tion of NAD, O

2· – and H

2O

2. Peroxynitrite mediated oxidation of cat-

echolamines contribute to a variety of central nervous system and cardiovascular pathologies (51). It induces an imbalance in cellular pyrimidine nucleotide levels and causes cytotoxic oxidant generation (52).

Peroxynitrite mediates both oxidative and deami-native DNA injury (53). DNA single strand breakage, initiated by endogenous or exogenous peroxynitrite, is a potent trigger of poly-ADP ribose polymerase (PARP) activation, which is a major contributor of programmed cell death and necrosis under conditions of severe oxi-dative stress (54–57). Peroxynitrite generation and con-sequent PARP activation is known to play an important role in many diseases such as diabetes, ischemia/reper-fusion, etc. (Table 2). In motor neurons, both axotomy and peroxynitrite exposure leads to accumulation of DNA single strand breaks (86).

Nitric oxide and peroxynitrite react with mitochon-dria leading to various responses such as modulation of mitochondrial respiration, mitochondrial dysfunc-tion, and apoptotic cell death (87). Nitric oxide prima-rily interacts with cytochrome c oxidase, which affect calcium uptake and may regulate processes such as mitochondrial transition pore (MTP) opening and the release of pro-apoptotic proteins. (INDENT)Large or persistent levels of ·NO in mitochondria promote mitochondrial oxidant formation. Peroxynitrite formed either extra- or intra-mitochondrially leads to oxida-tive damage to ATPase, aconitase and Mn-superoxide dismutase. Mitochondrial scavenging systems for per-oxynitrite and peroxynitrite-derived radicals such as carbonate (CO

3· –) and nitrogen dioxide radicals (·NO

2)

include cytochrome c oxidase, glutathione and ubiqui-nol and serve to partially attenuate the reactions of these oxidants with critical mitochondrial targets (88).

Peroxynitrite affects signaling pathways by nitra-tion as well as by oxidation: while nitration of tyrosine residues by peroxynitrite modulates signaling processes relying on tyrosine phosphorylation and dephospho-rylation, oxidation of phosphotyrosine phosphatases may lead to an alteration in the tyrosine phosphoryla-tion/dephosphorylation balance (89). Peroxynitrite, being a terminal mediator of cell injury, also enhances

and triggers a variety of pro-inflammatory processes. It mediates the cytokine induced (interleukin-8) IL-8 expression in human leukocytes. These alterations can culminate in a dysregulation of cellular signal transduc-tion pathways (90,91).

Peroxynitrite induced cell death

The mechanism involved in induction of cell death by peroxynitrite is not fully understood. However, the pic-ture is becoming clearer after a better understanding of its toxicology, physiology and biochemistry (2,3,92). It was shown for the first time by Whiteman et al., (87) that peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calcium-dependent cysteine proteases (calpains). The mode of cell death induced by peroxynitrite can be necrosis, apoptosis or mixed types of cell death, depends on the peroxynitrite concentration, duration of peroxyni-trite exposure, and the intracellular ATP levels (93–95. Activation of poly(ADP-ribose) polymerase by damaged DNA is suggested to be a major cause of peroxynitrite-induced necrosis (1).

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders and the mechanism involved remains a critical area of investiga-tion (96). Peroxynitrite caused DNA fragmentation and apoptosis was first identified in several cell types such as thymocytes, HL-60 cells, cultured cortical neurons, and PC12 cells. Peroxynitrite induces apoptosis in a number of cell types in culture, including pheochromocytoma-derived PC12 cells, cortical neurons, HL-60 cells and rat thymocytes.

Shacka et al. (97) have reported that peroxynitrite activation of the intrinsic or mitochondrial apoptotic pathway in PC12 cells is preceded by phosphorylation of p38 and c-Jun-N-terminal Kinase (JNK), mitogen activated protein kinases (MAPK), and translocation of phosphorylated JNK to the mitochondria. Furthermore, they show that the inactivation of the phosphatidyl-inositol 3-kinase/Protein Kinase B (PI3K/Akt) pathway is important in peroxynitrite-induced apoptosis. The results reveal an important link between peroxynitrite-induced upstream signaling events and downstream activation of the intrinsic apoptotic pathway, suggesting that induction of apoptosis by peroxynitrite is not a con-sequence of direct effects on the mitochondria.

When isolated mitochondria are treated with per-oxynitrite the permeability transition pore (MTP) is activated and cytochrome c is released, suggesting that peroxynitrite-induced apoptosis may be the result of direct oxidative damage to the mitochondria. Treatment

Table 2. Pathophysiological conditions in which peroxynitrite generation leads to subsequent PARP activation.

Diseases References

1. Ischemia/Reperfusion 58–64

2. Cardiac Failure 58,65–74

3. Shock 59,63,64,75–78

4. Diabetes 68, 73,79–85

5. Inflammation 62,77

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392 R. Ahmad et al.

with peroxynitrite also decreases the activity of the PI3K pathway, while stimulating the activity of p38 and JNK mitogen-activated protein kinase pathways. Both the inactivation of PI3K and the activation of p38 and JNK/MAPK have been shown to promote apoptosis. Furthermore, activation of PI3K is important in prevent-ing apoptotic signaling in PC12 cells. These observations suggest that peroxynitrite-induced cell death might result from the activation of specific apoptotic signaling pathways and not merely from nonspecific oxidative damage (97).

Peroxynitrite induces ER stress in human vascular endothelial cells resulting in apoptosis. Peroxynitrite treatment leads to the following cellular responses: depletion of ER–Ca2+, phosphorylation of eIF2 via activation of the phosphorylated extracellular signal regulated protein kinase (pERK) pathway and pro-grammed cell death. Peroxynitrite-induced apoptosis is prevented by the overexpression of GRP78, an ER marker (98).

Immunotoxicology of Peroxynitrite

DNA is a non-immunogenic entity, but any significant unrepaired alteration in its basic structure could render it “foreign,” leading to the activation of immune path-ways. A change in the structure of DNA could either be due to radiation or interaction with different free radicals. NO and its derivatives are among the radicals known to interact with DNA and are primarily involved in deamination of DNA bases.

Peroxynitrite, on the other hand, leads to more exten-sive damage than that caused by an equivalent dose of ·NO. Formation occurs both intracellularly inside mac-rophages and extracellularly, and causes DNA strand breaks and modification of guanine (99). The two main products identified from the reaction of deoxyguanosine with peroxynitrite are 8-oxodeoxyguanine and 8-nitro-guanine. The former has long been regarded as a reliable biomarker for monitoring DNA damage in studies with various oxidizing agents. The peroxynitrite-modified DNA has been shown to acquire immunogenicity and was suspected to be one of the causes for generation of autoantibodies in cancer and autoimmune disorders (12,100,101) (Figure 2).

The peroxynitrite modified DNA is a potent immuniz-ing stimulus, inducing high-titer immunogen-specific antibodies in rabbits. Peroxynitrite modification might have generated potential neoepitopes against which antibodies are raised. The analysis of cross-reactivity indicates that anti-peroxynitrite-DNA IgG is immuno-gen-specific, showing various extents of cross-reactivity attributable to sharing of common antigenic determi-nants. The common antigenic determinants between peroxynitrite-DNA and nDNA could possibly be the sugar-phosphate backbone, since peroxynitrite attacks DNA and causes single strand breaks through sugar fragmentation. Induced antibodies also recognized syn-thetic polynucleotides, representing A/B conformations, with a preference for the B-form (13).

Elevated levels of ·NO in systemic lupus erythemato-sus (SLE) patients suggest a role for ·NO in the pathogen-esis of the disease. Murine models of SLE demonstrate

Peroxynitrite

Protein DamageLipid Peroxidation

DNA Damage

damage to tumor suppressor genes and

proto-oncogenes

Carcinogenesis

ProliferationCell Death

Generation of neo-epitopes

anti-nucleic acid antibodies

DNA Replication and Repair Errors

autoimmunity

Signal Transduction

ApoptosisNecrosis

Figure 2. Peroxynitrite mediated cell and molecular toxicology leading to various pathophysiological changes. Peroxynitrite may damage biolog-ical macromolecules including key proteins and genes leading to important pathological conditions such as autoimmunity, carcinogenesis, etc.

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Toxicology of Peroxynitrite 393

abnormally high levels of ·NO compared with normal mice, whereas systemic blockade of ·NO production reduces disease activity. Elevated serum nitrate levels correlate with indices of disease activity and, along with serum titers of anti-(ds DNA) antibodies, serve as indi-cators of SLE (102,103). Auto-antibody production in SLE has been attributed to either selective stimulation of autoreactive B-cells by self-antigens or antigens cross-reactive with self.

The persistence of anti-DNA antibodies in SLE patients, despite systems to suppress self-recognition, suggests that the response is driven by an antigen resembling nDNA. The DNA damage by peroxynitrite is far more lethal than that caused by ·NO alone, lead-ing to the perturbations in nDNA that render it immu-nogenic. This modified DNA might therefore play a role in the induction of circulating anti-DNA autoan-tibodies in various autoimmune disorders including SLE (12).

Conclusion

Evidence suggests that most of the cytotoxicity attrib-uted to nitric oxide is due to peroxynitrite, produced from the reaction between the free radical species, ·NO and O

2· –. Peroxynitrite interacts with lipids, DNA

and proteins causing oxidative damage and other free radical induced chain reactions. These reactions trig-ger cellular responses such as cell signaling, oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathological mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, inflammation, neurodegenerative disorders and can-cer. Even though nucleic acid antigens are by them-selves poorly immunogenic, their antigenicity can be enhanced by modification through different free radicals.

A number of studies support the role of free radi-cals in the initiation and progression of autoimmune response. Therefore, in chronic inflammatory diseases, peroxynitrite generated by phagocytic cells may cause damage to DNA, generating neoepitopes that lead to the production of antibodies cross-reacting with nDNA.

Hence, further research on understanding the biochemical toxicology, cellular physiology and molecular pathology of peroxynitrite could lead to important therapeutic interventions for the design of various pharmacological agents against this increas-ingly important and physiologically relevant reactive nitrogen species.

Acknowledgments

This review article is dedicated to our mentors and supervisors at the Nucleic Acid Immunology Laboratory, Jawaharlal Nehru Medical College, Aligarh.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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