Analysis of peroxynitrite reactions with guanine, xanthine, and adenine nucleosides by high-pressure liquid chromatography with electrochemical detection: C8-nitration and -oxidation.

Peroxynitrite, the reaction product of nitric oxide and superoxide anion, and a powerful oxidant, was found to nitrate as well as oxidize adenine, guanine, and xanthine nucleosides. A highly sensitive reverse-phase HPLC method with a dual-mode electrochemical detector, which reduces the nitro product at the first electrode and detects the reduced product by oxidation at the second electrode, was applied to detect femtomole levels of 8-nitroguanine and 8-nitroxanthine. This method was used to separate and identify the products of nitration and oxidation from the reactions of nucleosides with peroxynitrite. Peroxynitrite nitrates deoxyguanosine at neutral pH to give the very unstable 8-nitrodeoxyguanosine, in addition to 8-nitroguanine. 8-Nitrodeoxyguanosine, with a half-life of approximately 10 min at room temperature and

Determination of 3-nitrotyrosine by high-pressure liquid chromatography with a dual-mode electrochemical detector.

3-Nitrotyrosine, a product of tyrosine nitration, is useful as a marker for the generation of reactive nitrogen oxide species with short half-lives such as peroxynitrite. A reverse-phase high-pressure liquid chromatographic method using a dual-mode electrochemical detector in series with a photodiode array detector has been developed to determine the levels of 3-nitrotyrosine in biological samples. The principle of this method involves reduction of 3-nitrotyrosine at an upstream gold amalgam electrode and oxidation of the resulting product(s) at a downstream glassy carbon electrode. 3-Nitrotyrosine is quantified by the amount of the current generated at the downstream electrode, and a femtomole detection level can be achieved. The disappearance of the corresponding peak when the electrochemical detector is used only in the single oxidative mode provides additional evidence for the identity of 3-nitrotyrosine in the sample. Tyrosine from the same sample is determined by its UV absorption at 280 nm, thus eliminating the need for an internal standard. With this method a dose-dependent increase of 3- to 10-fold in the levels of protein 3-nitrotyrosine was observed in the blood plasma, and a 2- to 4-fold increase in the lung cytosols, of rats treated with the lung carcinogen and nitrating agent tetranitromethane.

N2-amination of guanine to 2-hydrazinohypoxanthine, a novel in vivo nucleic acid modification produced by the hepatocarcinogen 2-nitropropane.

2-Nitropropane, an industrial chemical and a hepatocarcinogen in rats, induces aryl sulfotransferase-mediated liver DNA and RNA base modifications [Sodum, R. S., Sohn, O. S., Nie, G., and Fiala, E. S. (1994) Chem. Res. Toxicol. 7, 344-351]. Two of these modifications were previously identified as 8-aminoguanine and 8-oxoguanine. We now report that the base moiety of the so far unidentified third nucleic acid modification, namely RX1 in RNA and DX1 in DNA, is 2-hydrazinohypoxanthine (N2-aminoguanine). 2-Hydrazinoinosine and 2-hydrazinodeoxyinosine, synthesized by adapting published procedures, cochromatographed with RX1 and DX1 of liver RNA and DNA, respectively, from 2-nitropropane-treated rats. 2-Hydrazinoinosine and 2-hydrazinodeoxyinosine are unstable in solution like the in vivo products RX1 and DX1. At neutral pH, hypoxanthine nucleoside is the major product of decomposition, while at pH 10 or above, xanthine nucleoside is also formed. RX1 and DX1 could be generated in the anaerobic reactions of hydroxylamine-O-sulfonic acid, an intermediate in the proposed activation pathway of 2-nitropropane, with guanine nucleosides. These results provide further evidence for the activation of 2-nitropropane and other carcinogenic secondary nitroalkanes to a reactive species capable of aminating nucleic acids and proteins.

Inhibition of 2-nitropropane-induced rat liver DNA and RNA damage by benzyl selenocyanate.

We observed that pretreatment of male F344 rats with benzyl selenocyanate, a versatile organoselenium chemopreventive agent in several animal model systems, decreases the levels of DNA and RNA modifications produced in the liver by the hepatocarcinogen 2-nitropropane. To clarify the mechanisms involved, we pretreated male F344 rats with either benzyl selenocyanate, its sulfur analog benzyl thiocyanate, phenobarbital or cobalt protoporphyrin IX; the latter is a depletor of P450. We then determined (1) the ability of liver microsomes to denitrify 2-nitropropane, (2) effects on 2-nitropropane-induced liver DNA and RNA modifications and (3) amount of nitrate excreted in rat urine following administration of the carcinogen. Pretreatment with benzyl selenocyanate or phenobarbital increased the denitrification activity of liver microsomes by 217 and 765%, respectively, increased liver P4502B1 by 31- and 435-fold, respectively, decreased the levels of 2-nitropropane-induced modifications in liver DNA (29-70% and 17-30%, respectively) and RNA (67-85% and 30-50%, respectively), and increased the 24-h urinary excretion of nitrate by 157 and 209%, respectively. Pretreatment with benzyl thiocyanate had no significant effect on any of these parameters. Pretreatment with cobalt protoporphyrin IX decreased liver P4502B 1 by 87%, decreased the denitrification activity of liver microsomes by 76%, decreased the 24 h urinary excretion of nitrate by 88.5%, but increased the extent of 2-nitropropane-induced liver nucleic acid modifications by 17-67%. These results indicate that the metabolic sequence from 2-nitropropane to the reactive species causing DNA and RNA modifications does not involve the removal of the nitro group. Moreover, they suggest that benzyl selenocyanate inhibits 2-NP-induced liver nucleic acid modifications in part by increasing its detoxication through induction of denitrification, although it is evident that other mechanisms must also be involved.

Amination of tyrosine in liver cytosol protein of male F344 rats treated with 2-nitropropane, 2-nitrobutane, 3-nitropentane, or acetoxime.

Previously, the secondary nitroalkane 2-nitropropane, a strong hepatocarcinogen in rats, had been shown to induce the formation of 8-aminoguanine in both DNA and RNA of rat liver through a sulfotransferase-mediated pathway. This pathway was postulated to convert the carcinogen into an aminating species [Sodum, R. S., et al. (1994) Chem. Res. Toxicol. 7, 344-351]. To submit this postulate to further test, we examined liver proteins of rats treated with 2-nitropropane, other carcinogenic secondary nitroalkanes, or the related rat liver tumorigen acetoxime for the presence of 3-aminotyrosine, the expected product of tyrosine amination. Using ion-pair and/or cation-exchange high-performance liquid chromatography with electrochemical detection, we found that the liver cytosolic proteins of these animals contained 0.1-1.5 mol of 3-aminotyrosine/10(3) mol of tyrosine. Treatment with the noncarcinogenic primary nitroalkane 1-nitropropane or with other primary nitroalkanes did not produce an analogous increase in the aminated amino acid (level of detection estimated at approximately 0.01 mol/10(3) mol of tyrosine). To our knowledge, this is the first report of the modification of protein tyrosine in vivo by a carcinogen. In vitro studies with acetoxime-O-sulfonate and hydroxylamine-O-sulfonate showed that these proposed intermediates in the activation pathway of 2-nitropropane react with guanosine to give 8-aminoguanosine, N1-aminoguanosine, and 8-oxoguanosine and also react with tyrosine to give 3-aminotyrosine and 3-hydroxytyrosine. The in vitro amination and oxidation of guanosine at C8 were also produced by acetophenoxime-O-sulfonate and 2-heptanoxime-O-sulfonate. These results provide additional evidence for the production of a reactive species capable of aminating nucleic acids and proteins from 2-nitropropane and other carcinogenic secondary nitroalkanes by a pathway involving oxime- and hydroxylamine-O-sulfonates as intermediates.

(-)-Epigallocatechin gallate, a polyphenolic tea antioxidant, inhibits peroxynitrite-mediated formation of 8-oxodeoxyguanosine and 3-nitrotyrosine.

Reaction with peroxynitrite at pH 7.4 and 37 degrees C was found to increase the 8-oxodeoxyguanosine levels in calf thymus DNA 35- 38-fold. This oxidation of deoxyguanosine, as well as the peroxynitrite-mediated nitration of tyrosine to 3-nitrotyrosine, was significantly inhibited by ascorbic acid, glutathione and (-)-epigallocatechin gallate, a polyphenolic antioxidant present in tea. For 50% inhibition of the oxidation of deoxyguanosine to 8-oxodeoxyguanosine, 1.1, 7.6 or 0.25 mM ascorbate, glutathione or (-)-epigallocatechin gallate, respectively, was required. For 50% inhibition of tyrosine nitration, the respective concentrations were 1.4, 4.6 or 0.11 mM. Thus, (-)-epigallocatechin gallate is a significantly better inhibitor of both reactions than either ascorbate or glutathione. Reaction of (-)-epigallocatechin gallate with peroxynitrite alone resulted in the formation of a number of products. Ultraviolet spectra of two of these suggest that the tea polyphenol and/or its oxidation products are nitrated by peroxynitrite.

Secondary nitroalkanes: induction of DNA repair in rat hepatocytes, activation by aryl sulfotransferase and hepatocarcinogenicity of 2-nitrobutane and 3-nitropentane in male F344 rats.

The secondary nitroalkanes, 2-nitropropane, 2-nitrobutane, 3-nitropentane, 2-nitroheptane, nitrocyclopentane and nitrocyclohexane, as well as the primary nitroalkanes, 1-nitropropane, 1-nitrobutane, 1-nitropentane and 1-nitroheptane, were examined for their ability to induce DNA repair in rat hepatocytes and to serve as substrates for activation by partially purified rat liver aryl sulfotransferase in vitro. All of the secondary, but none of the primary nitroalkanes examined, induced significant DNA repair in rat hepatocytes. Also, the nitronates of all of the secondary nitroalkanes, but none of the primary nitroalkanes, served as substrates for the aryl sulfotransferase-catalysed production of 8-aminoguanosine and 8-oxoguanosine from guanosine in vitro. In a carcinogenicity assay using male F344 rats, the secondary nitroalkanes, 2-nitrobutane and 3-nitropentane, produced a highly significant incidence of hepatocarcinoma with metastases to the lungs, whereas the primary nitroalkane, 1-nitrobutane, was not carcinogenic. While a low incidence of hepatocarcinoma was also produced by cyclopentanone oxime, the results were not statistically significant. Since the secondary nitroalkane, 2-nitropropane, in contrast to the primary nitroalkane, 1-nitropropane, was also previously shown to be hepatocarcinogenic in rats, it is probable that secondary nitroalkanes constitute a hitherto unrecognized class of chemical carcinogens.

Activation of the liver carcinogen 2-nitropropane by aryl sulfotransferase.

8-Aminoguanine had previously been identified as one of the nucleic acid base modifications produced in livers of rats by treatment with the hepatocarcinogen 2-nitropropane (2-NP), and a hypothetical mechanism of activation of 2-NP to hydroxylamine-O-sulfonate or acetate that would lead to NH2+, an aminating species, was proposed [Sodum et al. (1993) Chem. Res. Toxicol. 6, 269-276]. We now present in vivo and in vitro experimental evidence for the activation of 2-NP to an aminating species by rat liver aryl sulfotransferase. Pretreatment of rats with the aryl sulfotransferase inhibitors pentachlorophenol or 2,6-dichloro-4-nitrophenol significantly decreased the levels of liver nucleic acid modifications produced by 2-NP treatment. Furthermore, partially purified rat liver aryl sulfotransferase was shown to activate 2-NP and 2-NP nitronate in vitro at neutral pH and 37 degrees C, to a reactive species that aminated guanosine at the C8 position. This activation was dependent on the presence of the enzyme, its specific cofactor adenosine 3'-phosphate 5'-phosphosulfate, and mercaptoethanol. As in the case of the in vitro studies, pentachlorophenol and 2,6-dichloro-4-nitrophenol inhibited the in vitro formation of 8-aminoguanosine and 8-oxoguanosine. The corresponding primary nitroalkane, 1-nitropropane, which is not mutagenic and does not appear to be carcinogenic, was not a substrate for aryl sulfotransferase in the in vitro amination of guanosine.

2-Nitropropane-induced liver DNA and RNA base modifications: differences between Sprague-Dawley rats and New Zealand white rabbits.

2-Nitropropane (2-NP), a hepatocarcinogen in male Sprague-Dawley rats but not, under the same conditions, in male New Zealand White rabbits, induces characteristic base modifications in rat liver DNA and RNA including increases in 8-oxoguanine and the formation of 8-aminoguanine. We compared the levels of these modifications in the two animal species at 6, 18 and 42 h after a single i.p. treatment with 1.12 mmol/kg 2-NP. Significantly less nucleic acid base modifications were found to be produced in rabbit liver than in rat liver. Thus, the relative resistance of the rabbit to the hepatocarcinogenicity of 2-NP correlates with decreased levels of 2-NP-induced liver DNA and RNA base damage.

8-Aminoguanine: a base modification produced in rat liver nucleic acids by the hepatocarcinogen 2-nitropropane.

2-Nitropropane (2-NP), an important industrial chemical and a hepatocarcinogen in rats, had previously been found to produce several modifications of nucleosides in rat liver RNA and DNA that are discernible using HPLC with electrochemical detection. While one of these modifications has been identified as an increase in the levels of 8-oxoguanosine and 8-oxo-2'-deoxyguanosine in RNA and DNA, respectively, the others had not been identified. We now present evidence that a major modification in rat liver nucleic acids due to the administration of 2-NP is the amination of guanine at C8, apparently a completely novel in vivo reaction. 8-Aminoguanosine, isolated from hydrolysates of liver RNA from 2-NP-treated rats, cochromatographed with synthetic or commercially-obtained standard on reverse-phase as well as cation-exchange HPLC, and its UV spectral characteristics at acidic, neutral, and basic pH were identical to those of the standard. Acid hydrolysis produced 8-aminoguanine, which had a retention time and fragmentation pattern identical to that of the standard on gas chromatography-mass spectrometry of the trimethylsilyl derivatives. Evidence for the presence of 8-aminodeoxyguanosine in liver DNA of rats treated with 2-NP was also obtained by cochromatography with synthetic standard on HPLC. Hydroxylamine-O-sulfonic acid was found to react with RNA and DNA to give 8-oxo- and 8-amino-substituted guanines. We propose, as a working hypothesis, that 2-NP may be metabolized to hydroxylamine-O-sulfonate or acetate, which yield the reactive nitrenium ion, NH2+, capable of aminating cellular macromolecules in vivo.

Acute toxicity of trans-4-hydroxy-2-nonenal in Fisher 344 rats [corrected].

The potential toxicity of trans-4-hydroxy-2-nonenal (HNE), a product formed in vivo during lipid peroxidation, which is also present in foods, was investigated in Fisher 344 rats. Five groups of five male rats each were given by gavage 1000, 300, 100, 30 or 10 mg/kg body weight HNE dissolved in 0.5 mL corn oil. The sixth group, the control, received corn oil alone. Two rats died 6 and 8 hr after being treated with 1000 mg/kg HNE. These two rats showed extensive acute tubular necrosis of the kidney, but had very little liver damage. Diffuse liver cell necrosis was observed in a dose dependent manner in all the rats killed 14 days after treatment, whereas renal change was mild. Interestingly, body weight of the lowest dosage group was significantly higher than that of the control group at termination of the experiment. The results of this study show that HNE has almost the same toxicity as other enals, such as trans-2-heptenal, and that kidney and liver are the main organs affected by toxicity of HNE. Although animals may have efficient defense systems, such as glutathione, to detoxify low to moderate dosages of HNE, at high doses of HNE this defense system is overwhelmed, resulting in serious renal and hepatic damage.

Stereoselective formation of in vitro nucleic acid adducts by 2,3-epoxy-4-hydroxynonanal.

This paper describes the reactions of purine nucleosides and nucleic acids with 2,3-epoxy-4-hydroxynonanal. 2,3-Epoxy-4-hydroxynonanal was produced with tert-butyl hydroperoxide by epoxidation of trans-4-hydroxy-2-nonenal, a lipid peroxidation product. The epoxy aldehyde exists as a pair of diastereomers, I and II. Because these isomers could not be completely separated under the chromatographic conditions used, reactions were carried out with a mixture of known proportions of isomers I and II. Reaction of adenine nucleosides with the epoxy aldehyde yielded diastereomers A1 and A2, which structures were assigned on the basis of their spectroscopic data and by chemical synthesis as 1,N6-etheno adducts possessing a heptyl group at C8. These adducts were formed from isomers I and II in a stereoselective manner. Isomer I appeared to be responsible for the formation of A2, whereas isomer II favored the production of A1. Stereoselectivity of isomers I and II was also observed upon reaction with guanine nucleosides in the formation of adducts G1, G2, G3, G4, G5, and G6, G2, G3, G5, and G6 were unstable in base and could be converted quantitatively to G1. The structures of these adducts were reported (Sodum, R. S., and Chung, F-L. Chem. Res. Toxicol., 2: 23-28, 1989). G5 and G6 were the products formed predominantly from reactions in which isomer I was in excess, whereas G1 and G4 were the major products in reactions enriched with isomer II. Incubation of DNA with the epoxy aldehyde at 37 degrees and pH 7.0 yielded a modified DNA containing 1,N2-ethenodeoxyguanosine (G1) at levels of 10 pmol/mg DNA. Although G2, G3, G5, and G6 were not readily detected in this DNA hydrolysate, base conversion of fractions corresponding to these adducts to G1 indicated that the total yield of these adducts was equivalent to approximately 20% of that of G1. A1 and A2 were not found in this DNA. Contrary to the reactions with native DNA, reactions of single-stranded DNA resulted in the formation of primarily A1 and A2, with a total adduct level of 30 nmol/mg DNA. In this DNA, the yield of guanine adducts was relatively small, estimated at 0.73 nmol/mg DNA based on conversion to G1. RNA was extensively modified by the epoxy aldehyde, yielding both adenine and guanine nucleosides. The levels of adenine nucleoside adducts formed in RNA were greater than 50 nmol/mg RNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural characterization of adducts formed in the reaction of 2,3-epoxy-4-hydroxynonanal with deoxyguanosine.

Six adducts were isolated by reverse-phase high-performance liquid chromatography from the reaction of deoxyguanosine at 50 degrees C in pH 7.0 buffer with the epoxide of trans-4-hydroxy-2-nonenal, a major alpha,beta-unsaturated aldehyde from lipid peroxidation. These adducts were designated as adducts 1-6. Structures of these adducts were fully characterized by spectroscopic methods such as UV, proton NMR, MS, and CD and by chemical reactions. Adduct 1 was previously identified as 1,N2-ethenodeoxyguanosine. Adducts 2, 3, 5, and 6 are four diastereomeric 1,N2-ethanodeoxyguanosine derivatives possessing two five-membered fused rings at the 1- and N2-positions of guanine. The systematic name of these adducts is 3-(2-deoxy-beta-D-erythropentofuranosyl)-3,5,5a,7,8,8a-hexahydro-8 -hydroxy-7- pentyl-10H-furo[2',3':4,5]imidazo[1,2-a]-purin-10-one. Acid hydrolysis of adducts 5 and 6 yielded the corresponding guanine products which were identical in all respects except having opposite CD. Similar results were obtained with adducts 2 and 3, suggesting they are two pairs of enantiomers. The stereochemical characteristics of these adducts were elucidated. Adduct 4 was characterized by spectroscopic methods and chemical reactions as 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,9-dihydro-7-(1,2- dihydroxyheptyl)-9H-imidazo[1,2-a]purin-9-one, a 1,N2-ethenodeoxyguanosine derivative. Upon mild base treatment, adducts 2, 3, 5, and 6 were readily converted to adduct 1. The mechanisms for the formation of these adducts and the conversion of adducts 2, 3, 5, and 6 to adduct 1 are discussed.

1,N2-ethenodeoxyguanosine as a potential marker for DNA adduct formation by trans-4-hydroxy-2-nonenal.

The reaction of trans-4-hydroxy-2-nonenal, a major alpha, beta-unsaturated aldehyde released during lipid peroxidation, with deoxyguanosine under physiological conditions was investigated in order to assess its DNA damaging potential. This aldehyde was dissolved in tetrahydrofuran (THF) prior to addition to the reaction mixture. The results showed that structurally different adducts were formed in these reactions depending on the THF used. Using THF unprotected from light, reactions yielded adducts 1 to 6. Adduct 1 was characterized as 1,N2-ethenodeoxyguanosine (5,9-dihydro-9-oxo-3-beta-D-deoxyribofuranosylimidazo[1,2-alpha]pu rine) by its UV, proton nuclear magnetic resonance, and mass spectrum and by comparison to the corresponding guanosine and guanine adducts reported in the literature. The UV spectrum of adduct 4 was indicative of a substituted 1,N2-etheno derivative. Adducts 2,3,5, and 6 were essentially identical in UV spectra and appeared to be N2-substituted deoxyguanosine diastereomers. At room temperature adducts 2,3,5, and 6 were converted quantitatively to a single product at pH 10.5. This product was shown to be identical to 1,N2-ethenodeoxyguanosine (adduct 1). Analogous conversions to 1,N2-ethenoguanine were also observed for the corresponding guanine adducts. Using THF that had been protected from the light, however, the reactions of trans-4-hydroxy-2-nonenal with deoxyguanosine gave three major adducts, 7,8, and 9. These adducts possessed UV spectra similar to that of 1,N2-propanodeoxyguanosine and were not converted to 1,N2-ethenodeoxyguanosine upon base treatment. Evidence obtained suggests that adducts 1 to 6 were formed from the reaction of deoxyguanosine with the epoxide of trans-4-hydroxy-2-nonenal generated in the presence of hydroperoxide in the light unprotected THF, whereas adducts 7 to 9 were formed by direct Michael addition. Adducts 1 to 6 were formed presumably as a result of nucleophilic addition of the exo-amino of deoxyguanosine to the aldehydic group of the epoxide of trans-4-hydroxy-2-nonenal. Base treatment of these adducts facilitated subsequent cyclization and eliminations and finally gave 1,N2-ethenodeoxyguanosine. These results demonstrated that trans-4-hydroxy-2-nonenal readily forms adducts with deoxyguanosine either by direct Michael addition or via its epoxide formation. The facile conversion of some of these adducts to a single adduct suggests that 1,N2-ethenodeoxyguanosine may provide a simple and useful marker for assessing potential DNA damage by trans-4-hydroxy-2-nonenal and related alkenals associated with lipid peroxidation.

Reactions of nucleosides with glyoxal and acrolein.

The structural determination of the products formed by the reaction of derivatives of glyoxal with guanine are reviewed, as are the applications of these reactions. Conditions have now been defined for the use of glyoxal as a probe for microdetermination of the structure of unidentified nucleic acid components. Unexpectedly, 3-methylguanine shows a positive reaction with glyoxal. The adduct produced has been isolated and characterized. A new ring is formed by substitution at the 1- and N2-positions of 3-methylguanine in this product. Acrolein reacts smoothly with 1-methylcytosine, cytidine and deoxycytidine at pH 4. The structures of the adducts have been determined. The amino group of cytosine adds to the double bond of acrolein, while the aldehyde binds to the 3-position, forming a new ring. Adenosine, deoxyadenosine and 9-methyladenine react in an analogous manner with acrolein, with ring formation involving the amino group and the N-1 of adenine. The orientation of addition is similar to that in the cytosine series. Guanosine reacts with excess acrolein at pH 4 to form a bis-adduct. Two new rings are fused to the guanine structure, one at N-1 and the amino group, and the other involving N-7 and C-8. A number of derivatives of this product have been obtained by glycosyl cleavage and imidazole-ring-opening reactions. However, the assignment of the orientation of addition for both acroleins remains tentative in this series.