dna damage induced by peroxynitrite: subsequent biological effects

NITRIC OXIDE: Biology and ChemistryVol. 1, No. 5, October, pp. 373–385 (1997)Article No. NO970143

REVIEW

DNA Damage Induced by Peroxynitrite:Subsequent Biological Effects

Csaba Szabo*,1 and Hiroshi Ohshima†

*Children’s Hospital Medical Center, Division of Critical Care, 3333 Burnet Avenue, Cincinnati, Ohio 45229;and †International Agency for Research on Cancer, Unit of Endogenous Cancer Risk Factors,150 Cours Albert-Thomas, 69372, Lyon, Cedex 08, France

Presented at the First International Conference on the Chemistry and Biology of Peroxynitrite,Ascona, Switzerland, May 11–16, 1997

or excess superoxide reduces the oxidation elicited byNitric oxide (NO) and superoxide rapidly react to peroxynitrite (4–6). The oxidant reactivity of peroxyni-

yield peroxynitrite. Peroxynitrite is a potent oxi- trite is mediated by an intermediate with the biologicaldant which reacts with proteins, lipids, and DNA. activity of the hydroxyl radical. However, this productThe present paper overviews the various DNA modi- does not appear to be the hydroxyl radical per se, butfications induced by exposure to peroxynitrite or

peroxynitrous acid (ONOOH) or its activated isomerNO and superoxide concurrently, with special refer-(ONOOH*) (3).ence to the formation of 8-nitroguanine and 8-oxo-

Peroxynitrite is produced in a variety of patho-guanine as well as the induction of DNA singlephysiological conditions. Typical examples are vari-strand breakage. In addition, we review the second-ous types of acute and chronic inflammation, andary processes that may follow the process of DNA

damage, such as activation of the nuclear enzyme, the reperfusion of ischemic organs (2, 3, 7, 8). Perox-poly(ADP-ribose) synthetase, apoptosis, and carci- ynitrite is a highly reactive species, which causesnogenesis. q 1997 Academic Press rapid oxidation of sulfhydryl groups and thioethers,

Key Words: free radical; peroxynitrite; nitric ox- as well as nitration and hydroxylation of aromaticide; superoxide; 8-nitroguanine; 8-oxoguanine; DNA compounds, such as tyrosine and tryptophan (2, 3).strand break; apoptosis; poly(ADP-ribose) synthe- Peroxynitrite also injures DNA via a number oftase; septic shock; endotoxin; inflammation; stroke; mechanisms. The present paper overviews the vari-diabetes; cancer.

ous DNA modifications induced by exposure to per-oxynitrite or NO and superoxide concurrently, withspecial reference to the formation of 8-nitroguanine

FORMATION AND REACTIVITY OF PEROXYNITRITE and 8-oxoguanine as well as the induction of DNAsingle strand breakage. In addition, we review the

Nitric oxide and superoxide rapidly combine to form secondary processes that may follow the process ofa toxic reaction product, peroxynitrite anion (ONOO0) DNA damage, such as activation of the nuclear(1–3). The ratio of superoxide to NO is important in enzyme poly(ADP-ribose) synthetase (PARS),2 anddetermining the reactivity of peroxynitrite: excess NO

2 Abbreviations used: PARS, poly(ADP-ribose) synthetase;1 To whom correspondence should be addressed. Fax: (513) 636- nox-dG, 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2 *-deoxyguanosine;

TBA, 2-thiobarbituric acid; SIN-1,3-morpholinosydnonimine.4892.

3731089-8603/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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374 SZABO AND OHSHIMA

FIG. 1. DNA base modifications induced by peroxynitrite. In addition to the modifications shown in this figure, increased productionof various oxidative base modifications have also been reported (see text).

apoptosis. We also overview the evidence supporting and peroxynitrite was optimal at approximately pHthe pathophysiological importance of peroxynitrite- 8 and increased dose-dependently with peroxynitriteinduced DNA modifications in various pathophysio- concentration, but was not dependent on guaninelogical conditions. concentration. The presence of Fe3//EDTA, which

has been shown to catalyze the nitration of tyrosine(10), did not affect the nitration of guanine. It wasDNA DAMAGE CAUSED BY PEROXYNITRITEproposed that either heterolytic cleavage of peroxy-

DNA Base Modification nitrite to form a nitronium ion (NO2/) or a high-

energy intermediate (ONOOH*) derived from trans-Peroxynitrite can initiate a number of DNA modi-peroxynitrite (pKa Å 7.9) (11) could be involved infications, as shown in Fig. 1. Yermilov et al. studiedthe formation of 8-nitroguanine (9).the reaction of various nucleobases and nucleosides

Reaction of nucleosides with peroxynitrite alsowith peroxynitrite in vitro (9). They found that theleads to various modifications. The reaction of 2 *-reaction of peroxynitrite with purine bases, such asdeoxyguanosine with peroxynitrite yields severalguanine and adenine, yielded a strong yellow color,compounds, two of which were identified as 4,5-dihy-whereas pyrimidine bases, such as thymine, cyto-dro-5-hydroxy-4-(nitrosooxy)-2*-deoxyguanosinesine, 5-methylcytosine, and uracil, did not. The anal-(nox-dG) and 8-nitroguanine (12). The latter com-yses of the reaction products by high-performancepound could be formed either from guanine (depuri-liquid chromatography (HPLC) and thin-layer chro-nation of dG) by peroxynitrite or depurination of 8-matography (TLC) revealed that the reaction ofnitro-2 *-deoxyguanosine generated with peroxyni-nucleobases with peroxynitrite formed several newtrite. Kinetics of the formation of nox-dG have notcompounds, indicating that peroxynitrite inducedbeen studied. The reaction of various deoxynucleo-various base modifications. The major yellow com-sides with peroxynitrite was also shown to yield 2-pound formed by the reaction between guanine andthiobarbituric acid (TBA)-reactive substances dose-peroxynitrite has been identified as 8-nitroguanine

(9). The formation of 8-nitroguanine from guanine dependently (13, 14). TLC and HPLC analyses dem-

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375PEROXYNITRITE AND DNA DAMAGE

onstrated that these TBA-responsive compounds xanthine (19). Among these, 8-nitroguanine andxanthine were formed at 100–1000 times greaterwere identical to authentic base-propenals (base-

CH|CH{CHO). The yield of base-propenals was amounts than other modified bases. Similarly, in-creased levels of 8-oxoadenine and oxazolone werehigher under acidic conditions (pH õ6) than at neu-

tral or alkaline pH. A similar pH dependence has found in calf thymus DNA (0.05 mg/ml) treated with30 mM peroxynitrite (12).been reported for malondialdehyde formation from

2 *-deoxyribose and peroxynitrite (1). Base-propenals Considerable controversy exists regarding whethersynthesized peroxynitrite reacts with guanine tohave been reported to be highly cytotoxic and easily

formed by cleavage of the deoxyribose ring with hy- form 8-oxoguanine in DNA. On one hand, Yermilovet al., Douki and Cadet, and Uppu et al. reported nodroxyl radical generated during treatment of DNA

with bleomycin (Fe2/ and oxygen) or g-irradiation significant increases in 8-oxoguanine in calf thymusDNA treated with synthesized peroxynitrite, com-(15, 16). Therefore, the mechanism for formation of

base-propenals from deoxynucleosides and peroxyni- pared to nontreated DNA or DNA treated with de-composed peroxynitrite (12, 18, 20). On the othertrite could be similar to that proposed for bleomycin;

the initial reaction could involve radical abstraction hand, Inoue and Kawanishi, Fiala et al., and Spen-cer et al. found significant increases in 8-oxoguanineof a hydrogen from the C4* position of deoxyribose

by hydroxy-radical-like intermediate(s) (ONOOH*) in calf thymus DNA treated with peroxynitrite, thelevels formed with 1 to 1.5 mM peroxynitrite beingor peroxynitrous acid (ONOOH). Base-propenals can

be derived from bases with carbon atoms C1*, C2 *, 10–40 times higher than controls (19, 21, 22). Hy-droxyl radical scavengers such as ethanol and so-and C3 * of the deoxyribose molecule after cleavage

of C3 *–C4* and C1*–(ring-O) bonds (17). dium formate inhibited 8-oxoguanine formation inperoxynitrite-treated DNA (21). Epe et al. recentlyReactions of isolated DNA with authentic peroxy-

nitrite also yield various base modifications in DNA. characterized DNA damage induced by peroxyni-trite in isolated bacteriophage PM2 DNA using sev-For example, 8-nitroguanine was formed dose-de-

pendently in calf thymus DNA (1 mg/ml) incubated eral DNA repair enzymes. In this study it was foundthat peroxynitrite could induce a large number ofwith low concentrations (5–100 mM) of peroxynitrite

(18). Only peroxynitrite, but not nitrous acid, tetra- base modifications, which were sensitive to Fpg pro-tein, suggesting that 8-oxoguanine was a majornitromethane, and NO-releasing compounds, formed

8-nitroguanine. Antioxidants (urate, ascorbate, N- modification, while the numbers of oxidized pyrimi-dines (sensitive to endonuclease III) and of sites ofacetylcysteine) and desferrioxamine, but not hy-

droxyl radical scavengers (ethanol, D-mannitol, di- base loss (sensitive to exonuclease III or T4 endonu-clease V) were relatively small (23). Desferrioxa-methyl sulfoxide), inhibited the reaction (18). Bicar-

bonate (0–10 mM) caused a dose-dependent increase mine and seleno-containing compounds such as sel-enocystine and selenomethionine inhibited Fpg-of up to six-fold in the formation of 8-nitroguanine

in calf thymus DNA incubated with 0.1 mM peroxy- sensitive DNA damage induced by peroxynitrite(23). More recently Kennedy et al. also reported thatnitrite (18). 8-Nitroguanine was found to be depuri-

nated rapidly from DNA incubated at pH 7.4, 377C low doses (õ50 mM) of peroxynitrite in pUC19 plas-mid formed 8-oxoguanine dose-dependently, its(t1/2 Å Ç4 h), suggesting that 8-nitroguanine formed

in DNA is potentially mutagenic because its depuri- level being decreased with a high dose (100 mM) ofperoxynitrite (24). A similar plateau or decrease innation yields apurinic sites, which can induce G:C r

T:A transversions (18). In another investigation, calf 8-oxoguanine formation at high concentrations ofperoxynitrite was also reported (12, 18). Possiblethymus DNA (0.2 mg/ml) treated with peroxynitrite

(1 mM) has been shown to contain, in addition to 8- reasons for this controversy could be that (i) forma-tion of 8-oxoguanine is mediated by contaminantsnitroguanine, increased levels of both oxidized and

deaminated base products, including 5-hydroxyhy- (e.g., hydrogen peroxide and metal ions), the concen-trations of which may vary in different preparationsdantoin, 5-(hydroxymethyl)uracil, thymine glycol,

4,6-diamino-5-formamidepyrimidine (FAPy-adenine), of peroxynitrite; (ii) 8-oxoguanine may be formedartificially during isolation, hydrolysis, and analy-2,6-diamino-5-formamidepyrimidine (FAPy-guanine),

8-oxoadenine, 8-oxoguanine, hypoxanthine, and ses of DNA (e.g., conversion of 8-nitroguanine to 8-

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oxoguanine); and (iii) under certain conditions, es- Juedes and Wogan studied mutagenicity of perox-ynitrite in the supF gene of the pSP189 shuttle vec-pecially with high concentrations of peroxynitrite,

the 8-oxoguanine that is produced may be further tor (27). The plasmid was exposed to 2.5 mM peroxy-nitrite in vitro and then replicated in Escherichiaoxidized into the ring cleavage product by peroxyni-

trite (20, 21, 24). coli and in human AD293 cells. Mutation frequencyincreased 21-fold in pSP 189 replicated in E. coli and3-Morpholinosydnonimine (SIN-1), that simulta-

neously generates NO and superoxide, thus possibly 9-fold in plasmid replicated in human AD293 cells.In both systems, G:C r T:A transversions (Ç65%)forming peroxynitrite, has also been used to study

DNA damage. Inoue and Kawanishi reported in- were predominantly detected. In addition, G:C r

C:G transversions (Ç11%) and G:C r A:T transitionscreased production of 8-oxoguanine in DNA treatedwith SIN-1, which was inhibited by hydroxyl radical (Ç11%) as well as a number of deletions, insertions,

tandem (including CC r TT), and multiple muta-scavengers such as ethanol and formate (21). Yermi-lov et al., however, found that SIN-1 increased dose- tions were found in plasmid replicated in human

AD293 cells (27). These authors noted that the muta-dependently the level of 8-oxoguanine, but not thatof 8-nitroguanine, in DNA, in contrast with the fact tion spectra induced by peroxynitrite were similar to

those reported for singlet oxygen, but differed fromthat authentic peroxynitrite formed 8-nitroguanine,but not 8-oxoguanine, in DNA (18). One possible rea- those reported for g-irradiation or UV exposure.

G:C r T:A transversions could result from 8-oxogua-son for this observation could be that SIN-1 mayproduce other oxidants (such as the peroxynitrite nine or apurinic/apyrimidinic sites (27). On the other

hand, qualitatively very different mutation spectraradical ONOO•), which may be responsible for theformation of 8-oxoguanine (18, 20). SIN-1 has also have been reported for NO using similar test sys-

tems. Aerobic gaseous NO induced primarily A:T rbeen reported to produce malondialdehyde from de-oxyribose and benzoate, which are inhibited by su- G:C transitions (28), whereas aqueous NO, derived

from NO-releasing compounds, generated largelyperoxide dismutase, mannitol, and ethanol, indicat-ing that hydroxyl radical-like compounds are formed G:C r A:T transitions (29). Deamination of adenine

to hypoxanthine, which pairs with cytosine ratherfrom SIN-1 (25).Exposure of human skin epidermal keratinocytes than thymine in DNA, could account for A:T r G:C

transitions. G:C r A:T transitions could result fromto preformed peroxynitrite or SIN-1 led to extensiveDNA base modification (19). With both compounds, deamination of cytosine to uracil, of 5-methylcyto-

sine to thymine, and/or of guanine to xanthinelarge increases in xanthine and hypoxanthine (de-amination products of guanine and adenine, respec- (28, 29).tively) and in 8-nitroguanine were observed, whereasonly small increases in some oxidized bases includ- DNA Single Strand Breakageing 8-oxoguanine and FAPy-guanine were found (19)in the DNA from keratinocytes. A variety of DNA In 1992, King and colleagues demonstrated that

potassium peroxynitrite (ONOOK) causes DNAbase modifications have also been described in im-munostimulated macrophages which produce oxy- cleavage in solutions of end-labeled DNA restriction

fragments (30). Over the past 5 years, several groupsradicals and NO, as well as peroxynitrite. Increasedlevels of xanthine, 5-(hydroxymethyl)uracil, and 8- have independently demonstrated the induction of

single strand breakage in DNA upon exposure tooxoguanine were detected in the DNA of macro-phages activated with lipopolysaccharide and inter- peroxynitrite or to NO and superoxide concurrently

in various systems. For instance, peroxynitrite canferon-g, which were indicative of both oxidative anddeaminative DNA damage (26). Formation of both induce DNA strand breaks in vitro in plasmid DNA

such as pBR322, PM2, CMV, and pUC19, convertingxanthine and 8-oxoguanine was inhibited by an NOsynthase inhibitor, suggesting that NO plays a role the supercoiled form to relaxed or linear forms,

which can be separated by agarose gel electrophore-in both deamination and oxidation reactions (26).The authors postulated that peroxynitrite may be sis (13, 14, 23, 31–35). A significant induction of

single strand breakage was observed in pBR322one possible mediator responsible for the oxidativedamage (26). plasmid treated with as low as 1 mM peroxynitrite,

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whereas much higher concentrations of peroxyni- bility of this agent (40), or due to peroxynitrite scav-trite (ú1 mM) (13) or the presence of a catalyst such enging (41) or both (39). Strand breakage inducedas manganese porphyrin (31) were needed to induce by SIN-1 is inhibited by superoxide dismutase (23,double strand breakage. Several studies have shown 34) as well as NO-trapping agents such as oxyhemo-that NO itself dose not cause strand breakage in globin and carboxy-PTIO (34), suggesting that con-vitro in plasmid DNA (34, 35), whereas exposure of current generation of NO and superoxide is neces-plasmid DNA to preformed peroxynitrite (13, 14, 21, sary to cause strand breaks, supporting the notion23, 32–35), SIN-1 (21, 34), or NO plus superoxide that these radicals react to each other to form perox-generated from the quinone redox system (34, 36) ynitrite or other oxidant(s).can induce strand breakage. Most of the above studies were not performed in

The mechanism for DNA strand breakage by per- intact cells. DNA single strand breakage, however,oxynitrite has not been studied extensively so far. has also been reported in intact cells exposed to per-DNA cleavage caused by peroxynitrite or SIN-1 was oxynitrite (37, 42, 43), indicating that extracellularobserved at almost every nucleotide with a small peroxynitrite has the ability to enter the cells anddominance at guanine residues (21). Peroxynitrite reach the nucleus. More importantly, indirect evi-induced significantly more single strand breaks at dence supports the view that endogenously producedacidic pH than at neutral or alkaline pH, suggesting peroxynitrite can also induce DNA single strandthat hydroxy radical-like intermediate(s) (ONOOH*) breakage, During immunostimulation of variousor peroxynitrous acid (ONOOH) are responsible for cells, in addition to DNA base modifications (seethe damage (14). Salgo et al. reported that mannitol above), DNA single strand breakage also occurs (19,failed to protect DNA from damage by peroxynitrite, 44, 45). For instance, in immunostimulated macro-while benzoate and dimethyl sulfoxide amplified the phages, the generation of DNA single strand breaksbreakage, indicating that the free hydroxyl radical parallels the production of peroxynitrite (45).is not involved in the damage (32). These authors Although more detailed analyses of the productspostulated that dimethyl sulfoxide and benzoate re-

(i.e., 3 *- and 5*-DNA termini at strand breaks, low-act with peroxynitrite to form NO2, which is respon-

molecular-weight products) are needed to establishsible for the increased DNA damage observed with

a mechanism for peroxynitrite-mediated strandthese compounds, rather than allowing peroxynitrite

breakage, we propose one possible mechanism (Fig.to decompose to nitrate, an inert product (32). On2) on the basis of available data in the literature.the other hand, various compounds can inhibit per-Because peroxynitrite reacts with 2 *-deoxyriboseoxynitrite-mediated strand breakage, including sele-and deoxynucleosides to form malondialdehyde andnomethionine, selenocystine (23, 33), desferrioxa-base-propenals, respectively (1, 13, 14), the mecha-mine (23, 34), antioxidants such as ascorbate andnism for formation of strand breaks by peroxynitriteurate (34), carbon dioxide/bicarbonate (14), and car-could be similar to that proposed for bleomycin. Aboxy-PTIO (34). In theory, all of these potent peroxy-similar mechanism has also been proposed for g-nitrite scavengers are expected to reduce the extentirradiation-induced strand breaks (46). The initialof DNA single strand breakage in cells exposed toreaction could involve hydrogen abstraction and O2peroxynitrite. However, such an effect has, so far,attack at either deoxyribose C4* or C5* by hydroxy-only been reported in relation to a few agents, suchradical-like intermediate(s) (ONOOH*) or peroxyni-as the vitamin E analog Trolox (28) and melatonintrous acid (ONOOH). After the modification at C4*,(38). Although manganese porphyrins have beenthe strand breakage can be induced by either C3 *–shown to catalyze the peroxynitrite-induced DNA(phosphate-O) cleavage or C3 *–C4* plus C1*–(ring-single strand breakage in an in vitro system (31), inO) bond cleavages (17). The strand breakage couldin vivo situations, in an endotoxin-induced model ofalso be induced by cleavage between C4* and C5*,systemic inflammation, a manganese–mesoporph-following the damage at C5* (46). A similar phenom-yrin compound, MnTBAP, has been shown to reduceenon (abstraction of hydrogen atoms) has been ob-DNA single strand breakage in an ex vivo modelserved in conjunction with peroxynitrite-induced(39). In this model, however, the protection may be

related to the superoxide dismutase mimetic capa- lipid peroxidation.

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FIG. 2. A possible mechanism of DNA strand breakage induced by peroxynitrite.

BIOLOGICAL CONSEQUENCES OF THE DNA subject of debate for at least two decades, the ‘‘classi-DAMAGE CAUSED BY PEROXYNITRITE cal’’ proposition being that PARS is a DNA repair

enzyme. According to the more recent evidence,Acute Cell Injury via Activation of Poly(ADP-PARS is not effective as a DNA repair enzyme, sinceribose) Synthetase (PARS)cells from a PARS knock-out mice have normal DNA

DNA single strand breakage (but not DNA double repair characteristics (50). It has been suggestedstrand breakage or DNA base modification) is an that the physiological role of PARS is to regulateobligatory trigger for the activation of PARS (47– gene expression and cellular differentiation, trans-49). PARS is a protein-modifying and nucleotide- formation, and division and/or to slow cellular me-polymerizing enzyme which is abundantly present tabolism as an adaptive response to altered environ-in the nucleus (47–49). The activation of PARS re- mental conditions.sults in the cleavage of NAD/ into ADP-ribose and Pronounced activation of PARS can rapidly de-nicotinamide. In turn, PARS covalently attaches plete the intracellular concentration of its substrate,ADP-ribose to various nuclear proteins. PARS then NAD/, slowing the rate of glycolysis, electron trans-extends the initial ADP-ribose group into a nucleic port, and, therefore, ATP formation resulting inacid-like polymer, poly(ADP-ribose) (47–49). The acute cell dysfunction and cell death. This pathwaymajor acceptors of poly(ADP-ribose) are PARS itself of cell injury develops rapidly, and is clearly distin-(automodification domain), topoisomerase I and II, guishable from apoptosis (see below). Pharmacologi-DNA polymerases a and b, and DNA ligase 2. For cal inhibitors of PARS protect against cell death un-most of these enzymes, ADP-ribosylation results in der these conditions. This mechanism, known as thea decrease in their catalytic activities (48). It is also ‘‘PARS suicide hypothesis,’’ has first been character-noteworthy that the presence of ADP-ribose on his- ized in relation to H2O2-induced oxidant damage andtones (mainly histone H1) causes chromatin relax- radiation injury (51–56). In macrophages, smoothation (48). muscle cells, epithelial cells, and endothelial cells

exposed to authentic peroxynitrite, development ofThe physiological function of PARS has been the

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DNA strand breaks occurs, which, in turn, resultsin the activation of PARS with consequent reductionof intracellular NAD/, ATP, and mitochondrial res-piration (42–44, 49, 57, 58). The metabolic changes,but not the development of DNA strand breaks, canbe ameliorated by pharmacological inhibition ofPARS (42–44, 49, 57, 58). The finding that 3-amino-benzamide does not inhibit the development of DNAstrand breaks (43) is consistent with the proposalthat 3-aminobenzamide is not a direct scavenger ofperoxynitrite. In agreement with the findings utiliz-ing 3-aminobenzamide, cells from the PARS0/0 miceare also protected against the peroxynitrite-induced(but not nitric oxide-induced) cellular injury, whencompared to the responses in the corresponding cellsfrom the PARS/// animals (58).

Similar to the results observed with authentic per-oxynitrite, immunostimulation of J774 cells and vas-cular smooth muscle cells in culture results in DNAstrand breakage and PARS activation, and thesechanges parallel the onset of NO and peroxynitriteproduction (44, 45). Pharmacological inhibitors of

FIG. 3. Proposed scheme of PARS-dependent and PARS-inde-PARS activity, as well as pharmacological inhibitionpendent cytotoxic pathways involving nitric oxide (NO•), hydroxylof NO biosynthesis inhibit the depression of mito-radical (OH•), and peroxynitrite (ONOO0). Proinflammatory con-chondrial respiration in these cells (44, 45). Takenditions induce the expression of the inducible NO synthase

together, these data suggest that activation of PARS, (iNOS), whereas NMDA receptor ligands activate the constitutivecaused by DNA strand breakage due to endoge- neuronal NOS (bNOS). NO, in turn, combines with superoxide to

yield peroxynitrite. During reperfusion injury, NO derived fromnously produced peroxynitrite, contributes to acutethe constitutive endothelial NO synthase (ecNOS) combines withcell damage (Fig. 3).superoxide to produce peroxynitrite. Hydroxyl radical and peroxy-Peroxynitrite-induced PARS activation has im-nitrite or peroxynitrous acid induce the development of DNA sin-

portant functional consequences in various in vitro gle strand breakage, with consequent activation of PARS. Deple-and in vivo experimental systems, and has been tion of the cellular NAD/ leads to inhibition of cellular ATP-

generating pathways, leading to cellular dysfunction. NO aloneshown to mediate pulmonary epithelial and intesti-does not induce DNA single strand breakage, but may combinenal epithelial hyperpermeability (42, 57), vascularwith superoxide (produced from the mitochondrial chain or fromsmooth muscle hypocontractility in endotoxic shockother cellular sources) to yield peroxynitrite. Under conditions of(44), and the development of endothelial dysfunctionlow cellular L-arginine, NOS may produce both superoxide and

(reduced relaxant responsiveness to endothelium- NO, which then combine to form peroxynitrite. There are PARS-dependent, but not to endothelium-independent va- independent, parallel pathways of cellular metabolic inhibition,

and these pathways can be activated by NO, hydroxyl radical,sorelaxants) in endotoxic shock (58). The importancesuperoxide, and by peroxynitrite (alone, or in combination). Theof the peroxynitrite–PARS pathway in mediatingrelative importance of the PARS-dependent and PARS-indepen-vascular hypocontractility, endothelial dysfunctiondent component of oxidant cytotoxicity is cell-type dependent.

and intestinal hyperpermeability has also been con-firmed in vivo, in splanchnic artery ischemia/reper-fusion (59). Furthermore, PARS activation, most data implicating the role of PARS activation by a NO

or a NO-related species (most likely peroxynitrite) inlikely due to peroxynitrite formation, has beenshown to play an important role in the injury of zymosan-induced multiple organ failure (63), in the

pancreatic islet cell injury and the development ofcardiac myoblasts upon hypoxia and reoxygenation(60) and in the reperfusion injury associated with diabetes (64–67) and in stroke and reperfusion in-

jury in the brain (68–74). The primary trigger oftransient coronary occlusion (61, 62). There are also

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DNA single strand breakage, in these conditions, pharmacological inhibition of PARS. These findingsare consistent with the results of a recent studyappears to be peroxynitrite, rather than NO per se

(43, 49). where the effects of peroxynitrite were studiedin primary neuronal cultures obtained from thePARS0/0 mice: these cells are not protected againstPeroxynitrite-Induced Apoptosisapoptosis when compared to the response in wild-type controls (89).Several reports demonstrated that NO and perox-

ynitrite either cause acute cell death (necrosis) ordelayed cell death (apoptosis) in a variety of cell

Role of Peroxynitrite-Mediated DNA Damage intypes (75–79). It appears that sustained exposure Inflammation-Related Canceror low levels of NO or peroxynitrite cause apoptosis,whereas sudden exposure to high concentrations of Chronic infection by bacteria, parasites, or viruses

and tissue inflammation such as gastritis, hepatitis,peroxynitrite or NO results in cell necrosis. Peroxy-nitrite-related apoptosis has been proposed to play and colitis are recognized risk factors for human can-

cers at various sites (90, 91). Nitric oxide and otheran important role in inflammation.The cellular mechanisms of peroxynitrite-induced oxygen radicals produced in infected and inflamed

tissues have been proposed as contributing to theapoptosis have not been clearly elucidated. As dem-onstrated in CHO-Em9 cells defective in their ability multistage process of carcinogenesis by damaging

DNA and tissues. Since peroxynitrite is now consid-to repair DNA single strand breaks, unrepaired sin-gle strand breaks lead to the formation of double ered to be a major compound responsible for tissue

damage caused by inflammation, it may also play anstrand breaks, with eventual cell death (35). Thedetermination of the fate of the cells after DNA in- important role in carcinogenesis. As described

above, peroxynitrite causes various DNA modifica-jury (apoptosis vs repair) is a subject of extensiveinvestigations. In relation to NO- or peroxynitrite- tions and induces strand breakage. Predominant

mutations detected in the supF gene of the pSP189induced DNA damage, it appears that a number ofregulatory factors such as p53 and CPP32 determine shuttle vector, exposed in vitro to peroxynitrite, fol-

lowed by replication in E. coli and in human AD293the fate of the cell (80, 81). It is also important thatdifferent types of DNA injuries can cause cell cycle cells, were G:C r T:A transversions. This type of

mutation is very common in a variety of genes fromarrest in different phases, which, in turn, can affectthe fate of the cell (80, 81). Although a role for PARS all types of human cancers (92). In particular,

G:C r T:A transversions account for 30% of all p53in the development of apoptosis has previously beenproposed in a variety of cell types (82, 83), the role gene mutations in lung cancer, and are also high in

liver and breast cancer. It has been proposed thatof PARS in the mediation of NO- or peroxynitrite-induced apoptosis is unclear. In human leukemia G:C r T:A transversions are predominantly caused

by polycyclic aromatic hydrocarbons such as benzo-cells, the PARS inhibitors 3-aminobenzamide andnicotinamide reduced NO-induced apoptosis (84), (a)pyrene present in tobacco smoke. However, the

same G:C r T:A transversions can also be inducedwhereas these inhibitors were ineffective in blockingapoptosis in RAW murine macrophages (85). It is by peroxynitrite. There is now increasing evidence

which suggests that peroxynitrite could be formedclear that the NO-induced apoptosis, which is associ-ated with proteolytic cleavage of PARS (86), involves in cigarette smoke, which contains high concentra-

tions of NO in the gas phase and superoxide-generat-mechanisms that are different from the apoptosistriggered by potent DNA single strand breaking ing quinone/hydroquinone redox system in tar (30,

93, 94). NO can also be formed endogenously by aagents, such as peroxynitrite and hydroxyl radical.Indeed, it appears that the hydrogen peroxide-in- constitutive and inducible NO synthase in the lung

(95, 96). NO and superoxide may react to form perox-duced apoptosis in intestinal epithelial cells (87), theperoxynitrite-induced apoptosis in human umbilical ynitrite in the lungs of smokers, and play an im-

portant role in smoking-related diseases includingvein endothelial cells (58), and the peroxynitrite- orimmunostimulation-induced apoptosis in cultured lung cancer. Similarly, the reaction between cate-

chol–estrogens and nitric oxide can produce oxi-rat aortic smooth muscle cells (88) is not affected by

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dants which are similar to peroxynitrite (97). Sev- agents are likely to prevent or reduce the peroxyni-trite-induced DNA injury, and also reduce other cy-eral recent studies have shown the importance of

catechol–estrogens in DNA damage. Because NO is totoxic processes triggered by peroxynitrite. OnceDNA injury has occurred, inhibitors of PARS mayalso produced by constitutive and inducible types of

NO synthases in human breast tissues (98, 99), it reduce acute cellular injury, without preventingapoptotic cell death.may react with superoxide generated from catechol–

estrogens to produce peroxynitrite, which can inducethe G:C r T:A transversions observed frequently in

ACKNOWLEDGMENTbreast tumors.Other sites, where inflammation, peroxynitrite This work was supported, in part, by a grant from the National

production, and DNA injury have been linked to car- Institutes of Health (R29GM54773) to C.S.

cinogenesis, include Helicobacter pylori infection,gastritis, and gastric cancer (100–103), as well as

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