nucleotides stored in the cells not only serve as substrates for nucleic acid biosynthesis but also participate in the energy metabolism and signal transduction . nitric oxide ( no ) is a gaseous free radical that is synthesized by nitric oxide synthases ( noss ) . no plays important roles in the regulation of diverse physiological phenomena such as vascular and neuronal signal transduction , host defense , and cell death regulation . signal transduction by no primarily involves a nucleotide signal molecule , guanosine 3',5'-cyclic monophosphate ( cgmp ) , generated by soluble guanylate cyclase ( sgc ) from guanosine triphosphate ( gtp ) . cgmp thus formed binds to allosteric regulatory domains of target proteins , including protein kinases , ion channels , and phosphodiesterases , with various downstream biological consequences that allow cells to adapt to changes and stresses occurring under different environmental conditions and metabolic demands . excess production of no has been suggested to be a cause of diverse pathophysiological conditions , such as inflammation , neurodegenerative and cardiovascular diseases , and cancer . these detrimental effects of no are attributed to reactive nitrogen oxide species ( rnos ) , including nitrogen dioxide ( no2 ) and peroxynitrite ( onoo ) , which are formed by the reaction of no with molecular oxygen and reactive oxygen species ( ros ) such as the superoxide anion radical o2 . peroxynitrite is thought to act as a strong oxidizing and nitrating agent in different pathophysiological situations . such rnos include nitryl chloride ( no2cl ) , which is formed from nitrite ( no2 ) and hypochlorous acid ( hocl ) , and no2 , which is generated by oxidation of no with molecular oxygen or by catalysis of peroxidases such as myeloperoxidase ( mpo ) and eosinophil peroxidase using hydrogen peroxide ( h2o2 ) and nitrite as substrates . when production of rnos exceeds the cellular antioxidant capacity , these molecules can cause nitrative and oxidative damage of nucleic acids , proteins , lipids , and carbohydrates by nitrosation , nitration , and oxidation reactions . rnos are known to have a strong potential to oxidize and nitrate nucleic acids at the level of their base structures , e.g. , guanine and adenine . during the past several years , oxidized and nitrated guanine derivatives , including 8-oxoguanine and 8-nitroguanine , were identified in diverse cultured cells , in tissues and organs from humans with cancer or degenerative diseases , and in different organisms with viral pneumonia , cancer , and other inflammatory conditions . not only the mutagenic potential but also the redox - active property of 8-nitroguanine derivatives suggested that guanine nitration may have significant biological effects yet to be identified . in fact , we recently discovered a nitrated cyclic nucleotide , 8-nitroguanosine 3',5'-cyclic monophosphate ( 8-nitro - cgmp ) , that was produced in cells expressing inducible nos ( inos ) . 8-nitro - cgmp is an extremely potent signaling molecule in biological systems because of its dual nature in signal transduction , i.e. , in the canonical no / cgmp pathway and in noncanonical electrophilic signaling . among the nitrated guanine derivatives studied , 8-nitro - cgmp possessed the strongest redox - active and electrophilic properties . because of its electrophilic behavior , 8-nitro - cgmp reacts effectively with highly nucleophilic sulfhydryl groups of certain cysteine ( cys ) residues and formed a protein - s - cgmp adduct via a unique posttranslational 
 modification named s - guanylation . in addition , some particular biological effects , e.g. , cell death induction , rather than mutagenic potential are now well recognized to be caused by 8-oxoguanine accumulated in the nucleotide pool in the cells . here , we will review current knowledge about endogenous nucleotides chemically modified via oxidative / nitrative stress with respect to their formation and biological significance . there is now ample evidence from a number of data indicating relatively frequent formation of 8-oxoguanine in various cells and tissues under oxidative stress . ros derived from both endogenous origins such as mitochondria , leukocytes ( oxidative burst ) , peroxisomes ( degradation of fatty acids ) and cytochrome p450 system ( mixed function oxidative system ) , as well as exogenous origins such as cigarette smoking , uv radiation , and ionizing radiation can contribute to the formation of 8-oxoguanine . epidemiological studies showed the increased formation of 8-oxoguanine as a risk factor for cancer , atherosclerosis , diabetes and neurodegenerative disorders . there are two pathways for the accumulation of 8-oxoguanine in dna or rna : one is a result of the incorporation of oxidized ( deoxy)guanosine triphosphate ( 8-oxo - dgtp ) generated in nucleotide pools while the other is a result of the direct oxidation of guanine in dna or rna . recent progress in studies of the sanitization of nucleotide pools , as well as dna repair , has revealed that the impact of oxidation of free nucleotides such as dgtp is unexpectedly large , in comparison with the direct oxidation of dna . similarly , in vitro and in vivo experiments have shown possible nitration of nucleic acids , more specifically guanine derivatives , that have been associated with various inflammatory conditions . yermilov et al . found that peroxynitrite reacted with the guanine base of nucleic acids in vitro to form 8-nitroguanine . our group was the first to report in vivo evidence of guanine nitration : we found marked guanine nitration in the lungs of influenza virus - infected mice and in the lungs of patients with idiopathic pulmonary fibrosis and lung cancer , with the nitration depending on production of no by inos . we also observed formation of 8-nitroguanosine in mice infected with bacteria such as salmonella typhimurium . in addition , hoki et al . detected 8-nitroguanine formation in malignant fibrous histiocytoma specimens from patients . it is also interesting that formation of 8-nitroguanine was recently suggested to be linked to diabetic retinopathy , which is a major cause of blindness . our chemical analyses using high - performance liquid chromatography - based electrochemical detection and tandem mass spectrometry ( lc - ms / ms ) revealed that , from among a series of 8-nitroguanine derivatives and related compounds , only a nitrated derivative of cgmp , 8-nitro - cgmp , was generated in significant amounts in cell culture models with different types of cells . for example , by using lc - ms / ms , we identified 8-nitro - cgmp in murine macrophage raw 264.7 cells that had been stimulated with interferon- and lipopolysaccharide to produce no via inos . as just mentioned , infection of murine macrophages with the gram - negative bacterium salmonella also facilitated formation of 8-nitro - cgmp , which was reported to be involved in host defense against infection . formation of 8-nitro - cgmp and 8-nitroguanine derivatives can be easily detected by means of conventional immunocytochemistry with the use of anti-8-nitro - cgmp monoclonal antibodies . it was intriguing that intracellular 8-nitro - cgmp formation and 8-nitroguanine formation had similar immunostaining profiles for time and location . this may suggest that a major nitrated guanine derivatives formed in the cells is likely to be 8-nitro - cgmp rather than other nitrated nucleotides and dna / rna . we recently precisely quantified the no - dependent formation of 8-nitro - cgmp in c6 glioma cells via lc - ms / ms . treatment of cultured rat c6 glial cells with the no donor s - nitroso - n - acetylpenicillamine ( snap ) led to a rapid and transient increase in cgmp , but the level of 8-nitro - cgmp gradually increased linearly up to a peak value comparable to that of cgmp at 24  h and declined thereafter . markedly high levels ( reaching up to 100  m ) of 8-nitro - cgmp were also evident in c6 cells that had been stimulated to express inos and produce excessive no . the amount of 8-nitro - cgmp generated was much higher than that of cgmp , whose production profile slightly preceded 8-nitro - cgmp formation in the activated inos - expressing cells . because of these unexpectedly large amounts of 8-nitro - cgmp , we suspected that gtp ( a substrate of cgmp biosynthesis ) , rather than cgmp per se , may undergo guanine nitration . this idea was indeed supported by the fact that 8-nitroguanosine 5'-triphosphate ( 8-nitro - gtp ) , produced in a cell - free chemical reaction of gtp with peroxynitrite , served as an effective substrate for sgc . among the pathological effects associated with oxidative and nitrative stress , the mutagenic potential of ros and rnos is of great interest . rnos such as peroxynitrite that commonly generated during infection and inflammation nonselectively affect a host s cells and tissues . obviously , such host defense molecules are produced to kill invading pathogens , which then suffer oxidative stress because of the host s antimicrobial attack . it may therefore be logical to expect that mutagenesis of various microbial pathogens occurs during infections in biological systems as a result of host defense . evidence of this mutagenesis includes the finding that human leukocytes producing o2 , but not leukocytes from patients with chronic granulomatous disease , were shown to be mutagenic for s. typhimurium ta100 . our earlier study also confirmed that oxidative and nitrative stress induced by a high output of no and ros accelerated mutation of the rna virus . related to this in vivo rna virus mutation , our investigations also found that 8-nitroguanine formed by rnos in the viral genome led to an increased frequency of mutations in an rna virus ( fig .  1 ) . in addition , authentic 8-nitroguanosine added exogenously to an rna virus - infected cells caused a dose - dependent increase in the frequency of viral mutations , especially c to u transitions . an earlier study by wogan s group documented that a high no output induced mutations in an endogenous hypoxanthine - guanine phosphoribosyltransferase gene ( hprt ) in murine macrophages expressing inos . genetic analysis of the no - induced mutated gene indicated that the no - associated mutational spectrum was similar to that arising spontaneously , but small deletions and insertions were found in the no - induced mutants . the same group showed that mutagenicity was enhanced by no overproduction in vivo , as assessed by mutation of an exogenously expressed lacz gene in lacz - containing pur288 plasmid - transgenic mice . excess production of no by inos induced by inammatory cytokines , possibly through rnos ( particularly peroxynitrite ) , caused dna damage and impaired dna repair in human cholangiocarcinoma cells , as assessed by the comet assay , which suggests an no - dependent development and progression of cholangiocarcinoma . it is important to note that guanine nitration appears to cause dna mutagenesis as well , with a mutation spectrum similar to that induced by peroxynitrite . thus , 8-nitroguanine in dna may be rapidly depurinated from dna in vitro , within 14  h under physiological conditions , the result being the formation of mutagenic abasic sites and release of free 8-nitroguanine . formation of 8-nitroguanine in dna may therefore facilitate g to t transversion via abasic site formation . in addition , suzuki et al . used photochemical synthesis to obtain an oligodeoxynucleotide containing a single 8-nitrodeoxyguanosine at a specific position and utilized the oligodeoxynucleotide as a template in primer extension reactions catalyzed by mammalian dna polymerases . this finding suggests that 8-nitrodeoxyguanosine in dna can mispair with adenine , thereby directly inducing a g to t transversion in mammalian cells . treatment of chinese hamster ovary as52 cells with 8-nitroguanosine significantly increased the mutation frequency of the xanthine - guanine phosphoribosyltranserase gene , with g to t transversion at the gene locus . concomitant increase of abasic sites in dna further supports the notion that depurination - dependent mutagenesis may be operative for 8-nitroguanosine - induced mutation . a similar mutation spectrum is observed for oxidative damaged nucleic acids caused by peroxynitrite and ros . specifically , 8-oxoguanine can pair with both cytosine and adenine during dna synthesis , and this base mismatching could contribute significantly to spontaneous mutations in genomic dna . direct oxidation in g : c pair can result in g to t transversion during replication , whereas 8-oxo - dgtp can be incorporated in dna opposite to the adenine , leading to the a to c transversion . for example , 8-oxoguanine dna glycosylase1 ( ogg1 ) excises 8-oxoguanine from 8-oxo - g : c pairs in dna , thus initiating base excision repair . mutt homolog-1 ( mth1 ) is an enzyme that hydrolyzes 8-oxo - dgtp to the monophosphate form , thus preventing incorporation of 8-oxo - dgtp into dna during replication . immunohistochemical studies demonstrated that 8-oxoguanine and 8-nitroguanine are also present in the cytosol of various cells and tissues suffering from oxidative / nitrative stress , which suggests that not only dna but also the nucleotide pool , rna , and other cytosolic compartments are modified chemically via oxidative / nitrative stress . for example , because certain enzymes such as mpo are unlikely to nitrate dna directly , nitrated nucleic acids may be formed in the nucleotide pool or in rna by reactive nitrogen species that are generated by such enzyme systems . as shown for certain oxidized and halogenated nucleosides , 8-nitroguanine and related nucleosides and nucleotides may be misincorporated into dna , which can result in mutations . similarly , 8-nitroguanine and related nucleosides and nucleotides , like oxidized and halogenated nucleosides , may be incorporated into rna and interfere with rna function and metabolism . the studies summarized here therefore clearly show that oxidative and nitrative dna damage , as evidenced by increased formation of 8-oxoguanine and 8-nitroguanine , can be induced under various inflammatory conditions . in fact , chronic inflammation induced by various biological , chemical , and physical factors has also been associated with an increased risk of human cancer at many locations . however , to establish a causal relationship between this type of dna damage and human cancer , additional studies that utilize a molecular epidemiological approach in a large human population are required . 8-nitro - cgmp has the potential to activate cgmp - dependent protein kinase ( pkg ) in vascular smooth muscle cells ; in addition , 8-nitroguanosine and its derivatives possess a significant redox activity . 8-nitro - cgmp had the highest redox activity among the 8-nitroguanosine derivatives we tested , with redox activity decreasing in the following order : 8-nitro - cgmp > 8-nitroguanosine > 8-nitroguanosine 5'-monophosphate ( 8-nitro - gmp )  8-nitro - gtp . 8-nitroguanine had only negligible redox activity . in the presence of certain oxidoreductases and electron donors such as nadph , 8-nitroguanosine derivatives were readily reduced to form their anion radicals , after which a single electron was transferred to molecular oxygen to form superoxide anion radical . because of their electrophilicity , nitro - nucleotides readily react with nucleophilic thiol compounds of low and high molecular weight to form 8-thioalkoxy - guanosine ( 8-rs - cgmp ) adducts ( fig .  2 ) . this reaction seems to occur via a nucleophilic attack by the thiol group of a protein cys or gsh on c8 of 8-nitro - cgmp , the results being release of the nitro moiety and formation of the 8-rs - cgmp adduct ( fig .  2 ) . the second - order rate constant for the reaction of 8-nitro - cgmp with the sulfhydryl of gsh was determined to be 0.03  m s at ph  7.4 and 37c . this value is much smaller than values for other electrophiles such as 4-hydroxynonenal , 15-deoxy--prostaglandin j2 , and nitrolinoleic and nitrooleic acids . those compounds have reaction rate constants with gsh of 1.3 , 0.7 , 355 , and 183  m s , respectively , at ph  7.4 and 37c . this comparatively lower second - order rate constant may account for the stable nature of this novel compound in the cellular compartment where gsh is abundant ( at ~ mm levels ) and may be responsible for the fact that 8-nitro - cgmp causes very selective s - guanylation with sulfhydryls possessing high nucleophilicity , as determined , at least in part , by low pka values of sulfhydryls of the cys moiety . because of its electrophilicity , 8-nitro - cgmp may mediate electrophilic signaling by means of induction of s - guanylation of redox sensor proteins . among this class of proteins , kelch - like ech - associated protein 1 ( keap1 ) was identified as a highly sensitive s - guanylation target . keap1 is a negative regulator of nf - e2-related factor 2 ( nrf2 ) , which is a transcription factor that regulates phase-2 detoxifying and antioxidant enzymes for electrophiles and ros . our chemical analyses revealed that keap1 expressed by various cultured cells was highly susceptible to s - guanylation induced by no - dependent 8-nitro - cgmp . in fact , we found that no and rnos could activate the keap1-nrf2 pathway in macrophages during bacterial infections and in rat c6 glial cells in culture after treatment with proinflammatory stimuli . that 8-nitro - cgmp may act as an endogenous electrophilic ligand and affect keap1 sulfhydryls via s - guanylation , which would lead to antioxidant signaling , is therefore highly plausible . cytoprotection and host defense conferred by 8-nitro - cgmp were clearly associated with increased expression of heme oxygenase 1 ( ho-1 ) in cultured macrophages and in vivo during salmonella infection . ho-1 is an enzyme with various physiological roles including vasoregulation , cytoprotection , and anti - inflammatory effects . we also reported earlier that ho-1 expression induced by no contributed to cell survival in certain solid tumor models . we recently found , in rat c6 glial cells , that keap1 is the major target that is s - guanylated by no exposure and that its s - guanylated structure derives primarily from 8-rs - cgmp adducts . s - guanylated keap1 led to nrf2 activation and subsequent induction of antioxidant enzymes including ho-1 , so 8-nitro - cgmp protected cells against the cytotoxic effects of hydrogen peroxide . proteomic analysis for endogenously modified keap1 with matrix - assisted laser desorption / ionization time - of - flight - ms / ms revealed that 8-nitro - cgmp s - guanylated the cys434 of keap1 ( fig .  2 ) . this finding is therefore the first compelling corroboration of the potential roles of 8-nitro - cgmp in the nrf2-dependent antioxidant response . recent studies have suggested that 8-oxoguanine formation not only can contribute to mutagenesis , but also may play an important role in the regulation of cell death . excessive formation of 8-oxoguanine was found to induce cell death via nuclear dna - dependent and mitochondrial dna - dependent mechanisms . in addition to ogg1 , muty homolog ( mutyh ) excises adenine opposite 8-oxoguanine in template dna , with concomitant formation of single strand breaks ( ssbs ) during the base excision repair . the accumulation of ssbs in nuclear dna by the action of mutyh leads to poly - adp - ribose polymerase - dependent nuclear translocation of apoptosis - inducing factor , and triggers cell death . on the other hand , the accumulation of ssbs in mitochondrial dna , which is also dependent on mutyh , caused mitochondrial dysfunction and calcium ion release , thereby activating calpain , and finally triggers cell death . recent study also demonstrated that excessive production of 8-oxo - dgtp in the nucleotide pool is a major source of oxidized bases , which may in turn cause accumulation of 8-oxoguanine in dna , leading to activate the cell death pathway as mentioned above . taken together , 8-oxoguanine may function as a signaling molecule triggering the cell death , which appears to cause a completely opposite effect compared with the downstream cytoprotective effect of 8-nitro - cgmp signaling as described above ( fig .  2 ) . in this article , we overviewed available data on the physiology and pathophysiology of oxidized and nitrated guanine nucleotides , with emphasis on information on mutagenesis and cell signaling related to these nucleotides reported in the last decades . different rnos produced under various pathophysiological conditions may oxidize and nitrate guanine and its related nucleosides and nucleotides , which exist as part of dna or rna or in free form as an abundant component of the intracellular nucleotide pool . not only do 8-oxoguaine and 8-nitroguanine function biologically as an endogenous mutagen but it may also serve as a biomarker for ros- and rnos - induced nucleic acid damage . more important , the major nitrated guanine nucleotide product , 8-nitro - cgmp , may play a critical role in signal transduction during cellular responses to oxidative stress that are primarily mediated by no and ros . this concept was unambiguously confirmed by our recent observation that no - dependent formation of 8-nitro - cgmp is a potent contributor to activation of the antioxidant signaling pathway controlled by the keap1-nrf2 system via unique site - specific s - guanylation of keap1 in cells . also , structural evidence of keap1 s - guanylation at cys434 in vivo indicated that 8-nitro - cgmp , formed from the major nucleotide sensor gtp that exists in an intracellular pool , appears to act as a critical signaling molecule in the initial defense against nitrative and oxidative stress . of importance is that , while guanine oxidation to form 8-oxoguanine may activate the cell death pathway , either dependent or independent of the p53 regulation , guanine nitration involving 8-nitro - cgmp - mediated antioxidant responses exhibits a completely opposite cellular protective effect ( fig therefore , the nucleotides in the intracellular pool seem to be sensing the oxidative and nitrative stress occurring in cells , as being differentially recognized based on the altered chemical structures of nucleotides ( e.g. , oxo- and nitro - moieties ) modified by ros and rnos . in other words , oxidative and nitrative nucleotide modifications may not be simple chemical damages , which mostly lose their biological functions because of the altered nucleotide structures , but may be physiologically relevant phenomena , which allow the cells to evoke the versatile cell signaling for adaptive responses to the various chemical stress . further clarification of the signaling functions via oxidized and nitrated nucleotides may shed light on the chemical biology and mutation research and may support an emerging paradigm for oxidative stress - related chemical sensing and signaling of no and ros via cellular nucleotides functioning potentially as nitrative / oxidative stress sensors .