8-nitroguanine and peroxynitric-acid

Nitric oxide (NO) and superoxide rapidly react to yield peroxynitrite. Peroxynitrite is a potent oxidant which reacts with proteins, lipids, and DNA. The present paper overviews the various DNA modifications induced by exposure to peroxynitrite or NO and superoxide concurrently, with special reference to the formation of 8-nitroguanine and 8-oxoguanine as well as the induction of DNA single strand breakage. In addition, we review the secondary processes that may follow the process of DNA damage, such as activation of the nuclear enzyme, poly(ADP-ribose) synthetase, apoptosis, and carcinogenesis.

Topics: Animals; Apoptosis; Cell Transformation, Neoplastic; DNA; DNA Damage; Guanine; Humans; Inflammation; Nitrates; Nitric Oxide; Oxidants; Poly(ADP-ribose) Polymerases; Superoxides

Peroxynitrite reacts with 2'-deoxyguanosine to yield several major products, including 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-nitroguanine (8-nitroGua). While the terminal products formed during the reaction of 8-oxodG with peroxynitrite have been previously characterized, those formed from 8-nitroGua have not. To identify these products, 9-ethyl-8-nitroxanthine was used as a model for 8-nitroGua, since the former could be easily synthesized in high yield, and facilitated reversed-phase HPLC separation of the resulting products. Using this model substrate, the products formed during the peroxynitrite reaction were identified as the ethyl derivatives of oxaluric acid, 5-iminoimidazolidin-2,4-dione, III, [N-nitro-N'-[2,4-dioxo-imidazolidine-5-ylidene]-urea, V, dehydroallantoin, parabanic acid, cyanuric acid, and uric acid. Upon the basis of the previous studies with 8-oxodG, these products were recognized as those expected to arise from peroxynitrite-mediated uric acid oxidation. Furthermore, the presence of uric acid in the reaction mixture led us to propose a model in which the 8-nitropurine is first converted to the 8-oxopurine which is further oxidized by peroxynitrite to give the observed final products. We have also provided evidence suggesting that the peroxynitrite anion, acting as a nucleophile, might be responsible for the initial conversion of the 8-nitropurine to the 8-oxopurine and that a hydroxyl radical or oxidative process is less likely to explain this conversion.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA Adducts; Guanine; Nitrates; Xanthine; Xanthines

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; DNA; DNA Damage; Electrochemistry; Fruit; Gas Chromatography-Mass Spectrometry; Guanine; Nitrates; Nitrites; Phenols; Reference Standards; Spectrophotometry, Ultraviolet; Vegetables

Tissue inflammation and chronic infection lead to the overproduction of nitric oxide and superoxide. These two species rapidly combine to yield peroxynitrite (ONOO(-)), a powerful oxidizing and nitrating agent that is thought be involved in both cell death and an increased cancer risk observed for inflamed tissues. ONOO(-) has been shown to induce single-strand breaks and base damage in DNA and is mutagenic in the supF gene, inducing primarily G to T transversions clustered at the 5' end of the gene. The mutagenicity of ONOO(-) is believed to result from chemical modifications at guanine nucleobases leading to miscoding DNA lesions. In the present work, we applied a combination of molecular and analytical techniques in an attempt to identify biologically important DNA modifications induced by ONOO(-). pUC19 plasmid treated with ONOO(-) contained single-strand breaks resulting from direct sugar damage at the DNA backbone, as well as abasic sites and nucleobase modifications repaired by Fpg glycosylase. The presence of carbon dioxide in the reaction mixture shifted the ONOO(-) reactivity towards reactions at nucleobases, while suppressing the oxidation of deoxyribose. To further study the chemistry of the ONOO(-) interactions with DNA, synthetic oligonucleotides representing the mutation-prone region of the supF gene were treated with ONOO(-), and the products were analyzed by liquid chromatography-negative ion electrospray ionization mass spectrometry (LC-ESI(-) MS) and tandem mass spectrometry. 8-Nitroguanine (8-nitro-G) was formed in ONOO(-)-treated oligonucleotides in a dose-dependent manner with a maximum at a ratio of [ONOO(-)]: [DNA]=10 and a decline at higher ONOO(-) concentrations, suggesting further reactions of 8-nitro-G with ONOO(-). 8-Nitro-G was spontaneously released from oligonucleotides (t(1/2)=1 h at 37 degrees C) and, when present in DNA, was not recognized by Fpg glycosylase. To obtain more detailed information on ONOO(-)-induced DNA damage, a restriction fragment from the pSP189 plasmid containing the supF gene (135 base pairs) was [32P]-end-labeled and treated with ONOO(-). PAGE analysis of the products revealed sequence-specific lesions at guanine nucleobases, including the sites of mutational "hotspots." These lesions were repaired by Fpg glycosylase and cleaved by hot piperidine treatment, but they were resistant to depurination at 90 degrees C. Since 8-nitro-G is subject to spontaneous depurination, and 8-oxo-guanine is not efficie

Topics: Base Sequence; DNA; DNA Damage; Dose-Response Relationship, Drug; Genes, Suppressor; Guanine; Mass Spectrometry; Mutation; Nitrates; Oligonucleotides; Oxidants; Plasmids; RNA, Transfer

Guanine (Gua) modification by nitrating and hydroxylating systems was investigated in DNA. In isolated calf thymus DNA, 8-NO(2)-Gua and 8-oxo-Gua were dose-dependently formed with peroxynitrite, and 8-NO(2)-Gua was released in substantial amounts. Myeloperoxidase (MPO) with H(2)O(2) and NO(2)(-) reacted with calf thymus DNA to form 8-NO(2)-Gua dose dependently without release of 8-NO(2)-Gua. The frequency of strand breaks was higher than the sum of 8-NO(2)-Gua and 8-oxo-Gua, particularly in the MPO-treated DNA, indicating the importance of other types of damage. The activation of human neutrophils and lymphocytes with phorbol ester did not induce 8-NO(2)-Gua and 8-oxo-Gua in their nuclear DNA. However, 8-NO(2)-Gua was found in calf thymus DNA co-incubated with activated neutrophils in the presence of NO(2)(-). No significant formation of 8-NO(2)-Gua was found in liver DNA from mice treated with Escherichia coli lipopolysaccharide. The incubation of peroxynitrite or MPO-H(2)O(2)-NO(2)(-)-treated DNA with formamidopyrimidine glycosylase (Fpg) released 8-oxo-Gua, but not 8-NO(2)-Gua, indicating that 8-NO(2)-Gua is not a substrate for Fpg. Although 8-NO(2)-Gua was generated in isolated DNA by different nitrating systems, other types of damage were formed in abundance, and the lesion could not be found reliably in nuclear DNA, suggesting that the biological importance is limited.

Topics: Animals; Cattle; DNA; Escherichia coli; Guanine; Humans; Hydrogen Peroxide; In Vitro Techniques; Lipopolysaccharides; Liver; Lymphocytes; Male; Mice; Mice, Inbred Strains; Neutrophils; Nitrates; Nitrosation; Oxidants; Peroxidase; Tetradecanoylphorbol Acetate

Peroxynitrite (ONOO-) is a powerful oxidizing agent that forms in a reaction of nitric oxide (NO*) and superoxide (O2-*). We have investigated ONOO--induced DNA damage using deoxynucleosides and oligonucleotides as model substrates, with particular attention paid to the oxidation of 8-oxodG by ONOO-. With regard to deoxynucleosides, ONOO- was found to have significant reactivity only with dG; dA, dC, and dT showed minimal reactivity. However, two of the major products of ONOO--induced oxidation of dG (8-oxodG and 8-nitroG) were both found to be significantly more reactive with ONOO- than with dG. In the context of an oligonucleotide, we observed a concentration-dependent oxidation of 8-oxodG to at least two types of products, one appearing at ONOO- concentrations of /=500 microM. We also examined the susceptibility of these oxidation products to repair by FaPy glycosylase, endonuclease III, uracil glycosylase, and MutY. FaPy glycosylase, which recognizes 8-oxoG as its primary substrate, was the only enzyme that exhibited an efficient reaction with 8-oxodG oxidation products at low ONOO- concentrations (/=500 microM either was not recognized or was poorly repaired by the enzymes. While processing of the lesions was inefficient with endonuclease III and not apparent with uracil glycosylase, the excision of A opposite an 8-oxoG lesion by the enzyme MutY was not affected by the reaction of 8-oxoG with ONOO-. In addition to demonstrating the complexity of ONOO- DNA damage chemistry, these results suggest that 8-oxodG may be a primary target of ONOO- in DNA.

Topics: Deoxyribonuclease (Pyrimidine Dimer); Deoxyribonucleosides; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Endodeoxyribonucleases; Guanine; N-Glycosyl Hydrolases; Nitrates; Nucleic Acid Conformation; Oligonucleotides; Oxidants; Substrate Specificity; Uracil-DNA Glycosidase

We investigated the reaction of 2'-deoxyguanosine (dGuo) with NO/O2 gas mixture under physiological condition and detected 8-nitroguanine, which is known as a novel DNA lesion caused by peroxynitrite (ONOO-). The yield increased with increase in the ratio of O2 and pH. The reaction mechanism is discussed.

Topics: Deoxyguanosine; Guanine; Models, Chemical; Nitrates; Nitric Oxide; Oxidation-Reduction; Oxygen

Carbon dioxide has been reported to react with peroxynitrite (ONO2CO2-), altering the reactivity characteristic of peroxynitrite. We found that carbon dioxide caused a dose-dependent increase in 8-nitroguanine formation in calf thymus DNA incubated with peroxynitrite, whereas there was not apparent effect on 8-oxoguanine formation. In contrast, carbon dioxide inhibited peroxynitrite-induced strand breakage in plasmid pBR322 DNA and thymine-propenal formation from thymidine.

Topics: Animals; Carbon Dioxide; Cattle; DNA; Dose-Response Relationship, Drug; Guanine; Nitrates; Thymus Gland

Antioxidant and pro-oxidant activities of flavonoids have been reported. We have studied the effects of 18 flavonoids and related phenolic compounds on DNA damage induced by nitric oxide (NO), peroxynitrite, and nitroxyl anion (NO-). Similarly to our previous findings with catecholamines and catechol-estrogens, DNA single-strand breakage was induced synergistically when pBR322 plasmid was incubated in the presence of an NO-releasing compound (diethylamine NONOate) and a flavonoid having an ortho-trihydroxyl group in either the B ring (e.g., epigallocatechin gallate) or the A ring (e.g., quercetagetin). Either NO or any of the above flavonoids alone did not induce strand breakage significantly. However, most of the tested flavonoids inhibited the peroxynitrite-mediated formation of 8-nitroguanine in calf-thymus DNA, measured by a new HPLC-electrochemical detection method, as well as the peroxynitrite-induced strand breakage. NO- generated from Angeli's salt caused DNA strand breakage, which was also inhibited by flavonoids but at only high concentrations. On the basis of these findings, we propose that NO- and/or peroxynitrite could be responsible for DNA strand breakage induced by NO and a flavonoid having an ortho-trihydroxyl group. Our results indicate that flavonoids have antioxidant properties, but some act as pro-oxidants in the presence of NO.

Topics: Antioxidants; Chromatography, High Pressure Liquid; DNA Damage; DNA, Single-Stranded; Flavins; Free Radical Scavengers; Free Radicals; Guanine; Molecular Structure; Nitrates; Nitric Oxide; Nitrites; Oxidants; Phenols; Plasmids

Carbon dioxide has been reported to react with peroxynitrite (ONOO-), a strong oxidant and nitrating agent, to form an ONO2CO2- adduct, altering the reactivity characteristic of peroxynitrite. We found that bicarbonate (0-10 mM) caused a dose-dependent increase of up to 6-fold in the formation of 8-nitroguanine in calf-thymus DNA incubated with 0.1 mM peroxynitrite, whereas it produced no apparent effect on 8-oxoguanine formation. In contrast, bicarbonate inhibited peroxynitrite-induced strand breakage in plasmid pBR322 DNA and thymine-propenal formation from thymidine. We conclude that C02/HCO3- reacts with peroxynitrite to form a potent nitrating agent, but also to inactivate hydroxyl-radical-like activity of peroxynitrous acid.

Topics: Animals; Bicarbonates; Carbon Dioxide; Cattle; DNA Damage; DNA, Single-Stranded; Guanine; Nitrates; Thymine

Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide in inflamed tissues. We have demonstrated that 8-nitroguanine is formed dose-dependently in calf thymus DNA incubated with low concentrations of peroxynitrite in vitro. 8-Nitroguanine in acid-hydrolyzed DNA was chemically reduced into 8-aminoguanine, which was analyzed using high performance liquid chromatography with electrochemical detection. Only peroxynitrite, but not nitrite, tetranitromethane nor NO-releasing compounds, formed 8-nitroguanine. Antioxidants and desferrioxamine inhibited the reaction. 8-Nitroguanine was depurinated from DNA incubated at pH 7.4, 37 degrees C (t1/2 = approximately 4 h). Peroxynitrite did not increase 8-oxoguanine levels in DNA.

Topics: Animals; Antioxidants; Apurinic Acid; Cattle; Deferoxamine; DNA; Free Radicals; Guanine; Nitrates

Nitric oxide and superoxide anion, both formed in inflamed tissues, react rapidly to form the peroxynitrite anion (ONOO-), a strong oxidant which can initiate reactions characteristic of hydroxyl radical (HO.), nitronium ion (NO2+) and nitrogen dioxide radical (NO2.). Peroxynitrite, therefore, may cause DNA or tissue damage, contributing to the multistage carcinogenesis process. We have studied reactions of various bases, nucleosides or deoxynucleosides with peroxynitrite in vitro. Guanine reacted rapidly with peroxynitrite under physiological conditions and formed several substances, two of which were yellow, a characteristic of nitro and nitroso compounds. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for approximately 80% of all compounds formed) as 8-nitroguanine. Its formation was maximal at approximately pH 8 and increased dose-dependently with peroxynitrite concentration, but was not dependent on guanine concentration. The presence of ferric ions, which has been shown to catalyse nitration of tyrosine, did not affect nitration of guanine. 8-Nitroguanine could act as a specific marker for DNA damage induced by peroxynitrite in inflamed tissues.

Topics: Carcinogens; DNA; DNA Damage; Guanine; Hydrogen-Ion Concentration; Kinetics; Nitrates; Nucleosides