Free 3-nitrotyrosine Research Articles

Analytical methods for 3-nitrotyrosine quantification in biological samples: the unique role of tandem mass spectrometry

Reactive-nitrogen species, such as peroxynitrite (ONOO(-)) and nitryl chloride (NO(2)Cl), react with the aromatic ring of tyrosine in soluble amino acids and in proteins to form 3-nitrotyrosine. The extent of nitration can be quantified by measuring 3-nitrotyrosine in biological matrices, such as blood, urine, and tissue. This article reviews and discusses current analytical methodologies for the quantitative determination of 3-nitrotyrosine in their soluble and protein-associated forms, with the special focus being on free 3-nitrotyrosine. Special emphasis is given to analytical approaches based on the tandem mass spectrometry methodology. Pitfalls and solutions to overcome current methodological problems are emphasized and requirements for quantitative analytical approaches are recommended. The reliability of current analytical methods and the suitability of 3-nitrotyrosine as a biomarker of nitrative stress are thoroughly examined.

Circulating free nitrotyrosine and cognitive decline

To determine if the circulating nitrotyrosine level significantly correlates with parameters measuring cognitive abilities. One-hundred and twelve community-living subjects (ranging in age from 27 to 98 years) were evaluated for cognitive abilities [Mini Mental State Examination (MMSE) score] and circulating free nitrotyrosine plasma level, as well as for several variables that might influence cognitive abilities (age, education) and nitrotyrosine level (body mass index, haematological parameters, cardiovascular and inflammatory indices). In the sub-group of cognitively impaired subjects (score at MMSE < 23.9), but not in that of cognitively not impaired subjects, a significant inverse correlation exists between nitrotyrosine level and MMSE score (r = -0.378; P < 0.02). The finding, if confirmed by longitudinal studies, could play a role in the management of the subjects with Mild Cognitive Impairment, the clinical condition considered as a transitional state between the changes of cognitive ability in normal aging and dementia.

Reliable Quantification of Endogenous Free 3-Nitrotyrosine in Human Plasma by GC–ECD or GC–MS in Health and Disease?

Reliable Quantification of Endogenous Free 3-Nitrotyrosine in Human Plasma by GC–ECD or GC–MS in Health and Disease?

Rapeseed Protein in a High-Fat Mixed Meal Alleviates Postprandial Systemic and Vascular Oxidative Stress and Prevents Vascular Endothelial Dysfunction in Healthy Rats

High-saturated fat and high-sucrose meals induce vascular endothelial dysfunction, the early hallmark of atherogenesis. The impact of dietary protein on vascular homeostasis remains misunderstood. In this study, we investigated whether rapeseed protein, an emergent arginine- and cysteine-rich protein, can acutely modulate the onset of adverse effects induced by a high-saturated fat meal (HFM). In a series of crossover experiments, healthy rats received 3 HFM (saturated fat: 60%; sucrose: 20%; protein: 20% energy) with the protein source being either total milk protein (MP; control), rapeseed protein (RP), or MP supplemented with cysteine and arginine to the same level as in RP (MP+AA). Endothelium-related vascular reactivity, measured as an acetylcholine-induced transient decrease in blood pressure, and plasma triglycerides, hydroperoxides, cyclic GMP (cGMP), and free 3-nitrotyrosine were measured before and 2, 4, and 6 h after meals. Superoxide anion production, expressed as ethidine fluorescence, was measured in the aorta 6 h after meals. Whereas plasma triglycerides rose similarly in all meals, the decrease in vascular reactivity after MP was attenuated after MP+AA and entirely prevented after RP. The type of meal had no consistent effect on plasma cGMP and free 3-nitrotyrosine over the postprandial period. The postprandial increase in plasma hydroperoxides differed according to the meal, and concentrations were 43% lower 6 h after MP+AA and RP than after MP. Aortic superoxide anion production was 36% lower 6 h after RP than MP. These results show that substituting rapeseed protein for milk protein markedly reduces vascular and oxidative disturbances induced by an HFM and this may be mediated in part by cysteine and arginine.

Nitrotyrosine promotes human aortic smooth muscle cell migration through oxidative stress and ERK1/2 activation

Nitrotyrosine is a new biomarker of atherosclerosis and inflammation. The objective of this study was to determine the direct effects of free nitrotyrosine on human aortic smooth muscle cell (AoSMC) migration and molecular mechanisms. By a modified Boyden chamber assay, nitrotyrosine significantly increased AoSMC migration in a concentration-dependent manner. For example, nitrotyrosine at 300 nM increased AoSMC migration up to 152% compared with l-tyrosine-treated control cells ( P < 0.01). Cell wound healing assay confirmed this effect. Nitrotyrosine significantly increased the expression of some key cell migration-related molecules including PDGF receptor-B, matrix metalloproteinase 2 (MMP2) and integrins αV and β3 at both mRNA and protein levels in AoSMC ( P < 0.01). In addition, nitrotyrosine increased reactive oxygen species (ROS) production in AoSMC by staining with fluorescent dye DCFHDA. Furthermore, nitrotyrosine induced transient phosphorylation of ERK2 by Bio-Plex luminex immunoassay and western blot analysis. AoSMC were able to uptake nitrotyrosine. Antioxidants including seleno- l-methionine and superoxide dismutase mimetic (MnTBAP) as well as ERK1/2 inhibitor PD98059 effectively blocked the promoting effect of nitrotyrosine on AoSMC migration and the mRNA expression of above cell migration-related molecules. Thus, nitrotyrosine directly increases AoSMC migration in vitro and the expression of migration-related molecules through overproduction of ROS and activation of ERK1/2 pathway. Nitrotyrosine may contribute to cardiovascular pathogenesis.

Bioenergetic and oxidative effects of free 3‐nitrotyrosine in culture: selective vulnerability of dopaminergic neurons and increased sensitivity of non‐dopaminergic neurons to dopamine oxidation

Protein bound and free 3-nitrotyrosine (3NT) levels are elevated in neurodegenerative diseases and have been used as evidence for peroxynitrite generation. Intrastriatal injection of free 3NT causes dopaminergic neuron injury and represents a new mouse model of Parkinson's disease (PD). We are investigating the nature of free 3NT neurotoxicity. In primary ventral midbrain cultures, free 3NT damaged dopaminergic neurons, while adjacent non-dopaminergic neurons were unaffected. Combined treatment with free 3NT and subtoxic amounts of dopamine caused extensive death of non-dopaminergic forebrain neurons in culture. Free 3NT alone directly inhibited mitochondrial complex I, decreased ATP, sensitized neurons to mitochondrial depolarization, and increased superoxide production. Subtoxic concentrations of rotenone (instead of free 3NT) caused similar results. Additionally, free 3NT and dopamine combined increased extraneuronal hydrogen peroxide and decreased intraneuronal glutathione levels more than dopamine alone. Oxidative and bioenergetic processes have been proposed to contribute to neurodegeneration in PD. As free 3NT is a compound that is increased in PD, damages dopamine neurons in vivo and in vitro and has detrimental effects on neuronal bioenergetics, it is possible that free 3NT is an endogenous contributing factor to neuronal loss, in addition to being a marker of oxidative and nitrative processes.

Generation and release of nitrotyrosine O-sulfate by HepG2 human hepatoma cells upon SIN-1 stimulation: identification of SULT1A3 as the enzyme responsible

In addition to serving as a biomarker of oxidative/nitrative stress, elevated levels of nitrotyrosine have been shown to cause DNA damage or trigger apoptosis. Whether the body is equipped with mechanisms for protecting against the potentially harmful nitrotyrosine remains unknown. The present study was designed to investigate the possibility that sulfation serves as a pathway for the metabolism/regulation of nitrotyrosine. Using metabolic labelling, nitrotyrosine O-[35S]sulfate was found to be produced and released into the medium of HepG2 human hepatoma cells labelled with [35S]sulfate in the presence of nitrotyrosine. To identify the enzyme(s) responsible for nitrotyrosine sulfation, a systematic study of all eleven known human cytosolic SULTs (sulfotransferases) was performed. Of the 11 enzymes tested, only SULT1A3 displayed sulfating activity toward nitrotyrosine. The pH-dependence and kinetic constants of SULT1A3 with nitrotyrosine or dopamine as substrate were determined. To examine whether the sulfation of nitrotyrosine occurs in the context of cellular physiology, HepG2 cells labelled with [35S]sulfate were treated with SIN-1 (morpholinosydnonimine), a peroxynitrite generator. Increments of nitrotyrosine O-[35S]sulfate were detected in the medium of HepG2 cells treated with higher concentrations of SIN-1. To gain insight into the physiological relevance of nitrotyrosine sulfation, a time-course study was performed using [3H]tyrosine-labelled HepG2 cells treated with SIN-1. The findings confirm that the bulk of free [3H]nitrotyrosine inside the cells was present in the unconjugated form. The proportion of sulfated [3H]nitrotyrosine increased dramatically in the medium over time, implying that sulfation may play a significant role in the metabolism of free nitrotyrosine.

Free 3-nitrotyrosine in exhaled breath condensates of children fails as a marker for oxidative stress in stable cystic fibrosis and asthma

3-Nitrotyrosine (3-NT) is considered as a marker of oxidative stress, which occurs during inflammation. Since 3-NT levels in exhaled breath condensate (EBC) are very low, we applied a specific and sensitive gas chromatography–negative ion chemical ionization–mass spectrometry (GC–NICI–MS) method and high performance liquid chromatography (HPLC) with electrochemical detection for the analysis of free 3-NT in EBC. A total of 42 children (aged 5–17 years) were enrolled in this study, including children with asthma ( n = 12), cystic fibrosis ( n = 12), and healthy controls ( n = 18). Additionally, 14 healthy non-smoking adults (aged 18–59 years) were included. An EcoScreen system was used for the collection of EBC samples. Free 3-NT levels in EBC ranged from 0.54–6.8 nM. Median (interquartile range) concentrations (nM) were similar in all groups: 1.46 (0.97–2.49) in healthy adults, 2.51 (1.22–3.51) in healthy children, 1.46 (0.88–2.02) in children with asthma, and 1.97 (1.37–2.35) in CF children, respectively ( p = 0.24, Kruskall–Walis test). No difference was found between the children with airway disease and age-matched healthy controls. In healthy subjects, there was no effect of age on 3-NT concentrations. HPLC analyses provided similar concentration ranges for EBC 3-NT when compared with GC–NICI–MS. Our study has clearly demonstrated that free 3-NT in EBC fails as a marker for oxidative stress in children with stable CF and asthma.

Interaction of peroxynitrite with myoglobin and hemoglobin

Peroxynitrite, a biological toxin produced in vivo by the nearly diffusion-controlled reaction of nitrogen monoxide with superoxide, can nitrate and oxidize various biomolecules. Modifications caused by peroxynitrite have been linked to many human diseases, in particular, increased levels of free or protein-bound 3-nitrotyrosine, a biomarker for peroxynitrite in vivo, have been detected in a variety of pulmonary and cardiovascular diseases as well as in neurodegenerative and chronic inflammatory disorders. These observations have led to the search for a drug that can scavenge this powerful nitrating and oxidizing agent. Heme proteins, in particular myoglobin and hemoglobin, present in large amounts in muscles and red blood cells, respectively, have been proposed to serve as sinks for peroxynitrite in these cells. This report reviews the current knowledge of the reactions of different forms of myoglobin and hemoglobin with peroxynitrite and discusses their physiological role on the basis of measured rate constants.Key words: myoglobin, hemoglobin, peroxynitrite, tyrosine nitration, iron(III) peroxynitrite complex.

Electrochemical detection of free 3-nitrotyrosine: Application to microdialysis studies

3-Nitrotyrosine (3-NT) is formed by the reaction of peroxynitrite with either free or protein-bound tyrosine residues and has been proposed as a biomarker of oxidative stress caused by reactive nitrogen species. This study describes the development of an HPLC electrochemical detection assay for free 3-NT capable of measuring this metabolite at the very low (nanomolar) levels encountered physiologically. We employed a dual-cell coulometric approach in which 3-NT is first reduced at an upstream cell to 3-aminotyrosine, which itself is then oxidized at the downstream cell. The method was shown to be linear over the range of 1–500 nM ( r = 0.999), with a detection limit (signal/noise ratio of 3) of 0.5 nM (25 fmol on column). Ten consecutive injections of 2 and 20 nM 3-NT standards produced coefficients of variation of 5.88 and 1.87%, respectively. Validation of the identity of the 3-NT peak was confirmed by coelution with authentic standards and by the in vitro production of 3-NT by incubation of 3-morpholinylsydnoneimine (SIN-1, 100 μM), a molecule releasing nitric oxide and superoxide in solution at a pH of 7.0 or higher with tyrosine (10 μM). Using this method, 3-NT was detected in human liver microdialysate (levels up to 2.6 nM), although levels in rat spinal cord dialysate were below the limit of detection.

Sensitive and accurate analyses of free 3-nitrotyrosine in exhaled breath condensate by LC–MS/MS

The quantitative determination of 3-nitro-l-tyrosine, a biological marker for inflammatory processes, in exhaled breath condensate (EBC) is described. The clean-up and preconcentration was performed by solid phase extraction (SPE). After liquid chromatography the specific detection was performed by tandem mass spectrometry using electron spray ionisation and selected reaction monitoring (SRM). 13C9-3-nitrotyrosine was used as an internal standard. For reliability, tests for the precision of the method, the losses during preparation, a test for nitrating artifacts and the comparibility of calibrants in EBC and buffer solution were performed. The calibration of the method was linear over a range of 10-500 pg/mL. The within-run coefficients of variation (CV) of the samples were found to be 8.4% at 25 pg/mL and 8.3% at 250 pg/mL. The day-to-day CV was found to be 11.2%. The limit of quantification was 3.9 pg/mL. The losses during preparation were 15%. The discrepancy between the calibration with EBC and buffer solution was below 10%. No artificial production of 3-nitrotyrosine was observed during the procedure. The application of the method on the EBC samples of healthy smokers (N=10) and non-smokers (N=10) showed no difference between the two groups. The concentration of 3-nitrotyrosine ranged between the limit of quantification and 184 pg/mL and was distinctly lower than data detected by an immunoassay procedure. The procedure was proven to be accurate, sensitive and in contrast to GC methods less elaborate and is recommended for the determination of 3-nitrotyrosine in exhaled breath condensate.

Selective quantification of free 3-nitrotyrosine in exhaled breath condensate in asthma using gas chromatography/tandem mass spectrometry

Reactive nitrogen species can cause oxidative modifications of certain amino acid residues in proteins, notably the modification of tyrosine to 3-nitrotyrosine (3-NT), which is a potentially useful marker of oxidative stress. Since lung diseases are associated with airway inflammation and oxidative stress, quantification of 3-NT in exhaled breath condensate (EBC) may provide a non-invasive means for monitoring ongoing inflammatory processes. 3-NT-like immunoreactivity has previously been detected in EBC, but no definitive evidence for the presence of 3-NT in EBC is available. Here, a method based on gas chromatography/negative ion chemical ionization/tandem mass spectrometry was established for the quantification of free 3-NT in EBC. The detection limit was 0.56 pM (corresponding to 3.0 amol microl(-1) sample injected) and the method was found to give linear results (r2 > 0.999) in the concentration range of 0-5.0 nM. The coefficient of variation (CV) for within-day and between-day precision were 11 and 12%, respectively. No artifactual nitration was observed during sample processing. The method was applied to study subjects with asthma (n = 8), and healthy subjects (n = 10), but only a slight non-significant increase in 3-NT levels was found in the former group (median [interquartile ranges]; 99 [50-547] amol s(-1) vs. 75 [35-147] amol s(-1)). No correlation with exhaled nitric oxide (NO), pulmonary function or EBC levels of total protein was observed. The 3-NT levels were much lower compared to previously reported levels, based on immunochemical measurements. The method does not allow the simultaneous quantification of tyrosine in samples.

Protein Nitration in a Mouse Model of Familial Amyotrophic Lateral Sclerosis: POSSIBLE MULTIFUNCTIONAL ROLE IN THE PATHOGENESIS

Multiple mechanisms have been proposed to contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence of a role for oxidative damage was based on the finding, in patients and murine models, of high levels of markers, such as free nitrotyrosine (NT). However, no comprehensive study on the protein targets of nitration in ALS has been reported. We found an increased level of NT immunoreactivity in spinal cord protein extracts of a transgenic mouse model of familial ALS (FALS) at a presymptomatic stage of the disease compared with age-matched controls. NT immunoreactivity is increased in the soluble fraction of spinal cord homogenates and is found as a punctate staining in motor neuron perikarya of presymptomatic FALS mice. Using a proteome-based strategy, we identified proteins nitrated in vivo, under physiological or pathological conditions, and compared their level of specific nitration. alpha- and gamma-enolase, ATP synthase beta chain, and heat shock cognate 71-kDa protein and actin were overnitrated in presymptomatic FALS mice. We identified by matrix-assisted laser desorption/ionization mass spectrometry 16 sites of nitration in proteins oxidized in vivo. In particular, alpha-enolase nitration at Tyr(43), target also of phosphorylation, brings additional evidence on the possible interference of nitration with phosphorylation. In conclusion, we propose that protein nitration may have a role in ALS pathogenesis, acting directly by inhibiting the function of specific proteins and indirectly interfering with protein degradation pathways and phosphorylation cascades.

Plasma and urinary free 3-nitrotyrosine following cardiac angiography procedures with non-ionic radiocontrast media

Plasma and urinary free 3-nitrotyrosine following cardiac angiography procedures with non-ionic radiocontrast media

Effects of diabetes on plasma nitrotyrosine levels.

Oxidative stress plays a major role in disease processes such as atherosclerosis and diabetes. Peroxynitrite is a reaction product of nitric oxide (NO) and superoxide and a potent oxidant. The peroxynitrite-mediated tyrosine nitration, which forms nitrotyrosine (NT), is associated with several pathological conditions. We measured plasma NT levels using the HPLC method in 40 Mexican Americans with diabetes, but not taking medications, and 40 age- and sex-matched euglycaemic controls. Plasma-free NT levels were not different between subjects with diabetes (11.0 +/- 1.7 nmol/l, n = 40) and with non-diabetes (10.4 +/- 1.5 nmol/l, n = 40). There was also no association with levels of fasting glucose (r = -0.049, P = 0.663) or 2-h glucose (r = -0.099, P = 0.390). However, females had significantly lower free NT level (7.6 +/- 1.4 nmol/l, n = 40) than males (13.8 +/- 1.7 nmol/l, n = 40, P = 0.005), which were not affected by age, smoking status, BMI and glucose levels. In contrast to some earlier reports, our study shows that diabetes has no effect on plasma NT levels in Mexican Americans. We have also demonstrated lower free NT levels in females than males, which may partly explain the lower risk profile to vascular disease in women.

Circulating free nitrotyrosine in obstructive sleep apnea.

Obstructive sleep apnea (OSA) has been increasingly linked to cardiovascular disease, endothelial dysfunction, and oxidative stress, generated by repetitive nocturnal hypoxemia and reperfusion. Circulating free nitrotyrosine has been reported as a novel biomarker of nitric oxide (NO)-induced oxidative/nitrosative stress. Nitrosative stress has been implicated as a possible mechanism for development of cardiovascular diseases. We tested the hypothesis that repetitive severe hypoxemia resulting from OSA would increase NO-mediated oxidative stress. We studied 10 men with newly diagnosed moderate to severe OSA who were free of other diseases, had never been treated for OSA, and were taking no medications. Nitrotyrosine measurements, performed by liquid chromatography-tandem mass spectrometry, were made before and after untreated apneic sleep. We compared free nitrotyrosine levels in these patients with those obtained at similar times in 10 healthy male control subjects without OSA, with similar age and body mass index. Evening baseline nitrotyrosine levels were similar before sleep in the control and OSA groups [0.16 +/- 0.01 and 0.15 +/- 0.01 ng/ml, respectively, P = not significant (NS)]. Neither normal nor disturbed apneic sleep led to significant changes of plasma nitrotyrosine (morning levels: control group 0.14 +/- 0.01 ng/ml; OSA group 0.15 +/- 0.01 ng/ml, P = NS). OSA was not accompanied by increased circulating free nitrotyrosine either at baseline or after sleep. This observation suggests that repetitive hypoxemia during OSA does not result in increased NO-mediated oxidative/nitrosative stress in otherwise healthy subjects with OSA.

Induction of motor neuron apoptosis by free 3-nitro-L-tyrosine.

Peroxynitrite-dependent tyrosine nitration has been postulated to be involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Evidence supporting this supposition includes the appearance of both free and protein-linked 3-nitro-l-tyrosine (nitrotyrosine) in both sporadic and familial ALS, as well as of increased free nitrotyrosine levels in the spinal cord of transgenic mice expressing ALS-linked superoxide dismutase mutants at symptom onset. Here we demonstrate that incubation with clinically relevant concentrations of nitrotyrosine induced apoptosis in motor neurons cultured with trophic factors. Nitrotyrosine was bound to proteins, but it was not incorporated into alpha-tubulin, as previously demonstrated for other cell types. Neither inhibition of nitric oxide production nor scavenging of superoxide and peroxynitrite prevented increases in cell nitrotyrosine immunoreactivity or motor neuron death, suggesting that these effects are not due to the endogenous formation of reactive nitrogen species. In contrast, some populations of astrocytes incorporated nitrotyrosine into alpha-tubulin, but free nitrotyrosine had no effect on the viability and phenotype of astrocytes in culture, as evaluated by glial fibrillary acidic protein immunoreactivity, cell growth and morphology. Co-culture of motor neurons on astrocyte monolayers delayed, but did not prevent, nitrotyrosine-induced motor neuron death. These results suggest that free nitrotyrosine may play a role in the induction of motor neuron apoptosis in ALS.

Plasma and urinary free 3-nitrotyrosine following cardiac angiography procedures with non-ionic radiocontrast media.

Radiocontrast media (RCM) administration is a common cause of hospital-acquired acute renal failure, especially in high-risk patients, but mechanisms of nephrotoxicity have not been fully elucidated. Reactive oxidant species recently have been shown to play a role in experimental RCM nephropathy, while there is clinical evidence that acetylcysteine, an antioxidant drug, has a protective effect against RCM nephropathy in humans. However, no study has been published showing that RCM administration elicits oxidative stress in humans. In an unselected series of patients undergoing elective cardiac catheterization for coronary artery angiography and/or angioplasty, we monitored the time course of plasma and urinary levels of free 3-nitrotyrosine (3-NT), a stable marker of peroxynitrite generation resulting from the in vivo reaction of superoxide and nitric oxide. Urinary 3-NT levels were measured as the ratio of urinary 3-NT to urinary creatinine. Measurements were taken at baseline, immediately after the procedure and at 24, 48 and 72 h. Twenty-six patients were studied (median age 67.5 years, range 42-86; baseline serum creatinine 1.0 mg/dl, 0.6-1.5; RCM dose 215 ml, 100-580). Plasma 3-NT levels slightly increased over the 72 h following the procedure (P<0.001), while urinary 3-NT levels peaked at the end of the procedure (P<0.001). Urinary 3-NT levels reached at the end of the procedure were proportional to the RCM dose administered (P = 0.017). The present study provides indirect evidence that RCM administration in humans is associated with an increased production of 3-NT. Further studies are needed to ascertain whether oxygen- and nitrogen-derived radical species play a major role in the pathogenesis of RCM-associated nephrotoxicity in the clinical setting.

Cerebrospinal fluid levels of free 3-nitrotyrosine are not elevated in the majority of patients with amyotrophic lateral sclerosis or Alzheimer’s disease

The mechanisms behind the degeneration of neurons in diseases such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) are not fully understood. However, oxidation of certain amino acid residues in proteins may contribute to cell injury and some of these oxidized amino acids may also be suitable as biomarkers for oxidative injury. Therefore, it is suggested that the reaction between peroxynitrite (ONOO −) and tyrosine in vivo can be monitored by monitoring the formation of 3-nitrotyrosine (3-NT). In this work, a newly developed gas chromatographic–mass spectrometric method was applied to human cerebrospinal fluid (CSF). The free 3-NT levels were determined in the CSF from 19 controls, 17 patients with AD and 14 patients with ALS. The levels of free 3-NT in the CSF were considerably lower than those previously reported. The majority of the patients with AD or ALS had free 3-NT levels in the same range as seen in the control individuals and only a few patients showed increased levels of free 3-NT.

Oxidative DNA damage induced by nitrotyrosine, a biomarker of inflammation

Inflammation has been postulated as a risk factor for several cancers. 3-Nitrotyrosine is a biochemical marker for inflammation. We investigated the ability of nitrotyrosine and nitrotyrosine-containing peptides (nitroY-peptide) to induce DNA damage by the experiments using 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and an HPLC-electrochemical detector. Nitrotyrosine and nitroY-peptide caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Catalase inhibited DNA damage, suggesting the involvement of H 2O 2. Nitrotyrosine and nitroY-peptide increased 8-oxo-7,8-dihydro-2 ′-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage. Nitrotyrosine-containing peptides of histone induced 8-oxodG formation more efficiently than free nitrotyrosine. We propose the possibility that nitrotyrosine-induced H 2O 2 formation and DNA damage contribute to inflammation-associated carcinogenesis.