Formation and mass spectrometric analysis of DNA and nucleoside adducts by S-(1-acetoxymethyl)glutathione and by glutathione S-transferase-mediated activation of dihalomethanes

The dihalomethane CH(2)Cl(2) is an industrial solvent of potential concern to humans because of its potential genotoxicity and carcinogenicity. To characterize DNA damage by dihalomethanes, a rapid DNA digestion under acidic conditions was developed to identify alkali labile DNA-dihalomethane nucleoside adducts using HPLC-electrospray mass spectrometry. DNA digestion worked best using pH 5.0 sodium acetate buffer, a 30 min incubation with DNase II and phosphodiesterase II, and a 2 h acid phosphatase digest. DNA was modified with S-(1-acetoxymethyl)glutathione (GSCH(2)OAc), a reagent modeling activated dihalomethanes. Adducts to G, A, and T were detected at high ratios of GSCH(2)OAc/DNA following digestion of the DNA with the procedure used here. The relative efficacy of adduct formation was G > T > A > C. The four DNA nucleosides were also reacted with the dihalomethanes CH(2)Cl(2) and CH(2)Br(2) in the presence of glutathione (GSH) and GSH S-transferases from bacteria (DM11), rat (GST 5-5), and human (GST T1-1) under conditions that produce mutations in bacteria. All enzymes formed adducts to all four nucleosides, with dGuo being the most readily modified nucleoside. Thus, the pattern paralleled the results obtained with the model compounds GSCH(2)OAc and DNA. CH(2)Cl(2) and CH(2)Br(2) yielded similar amounts of adducts under these conditions. The relative efficiency of adduct formation by GSH transferases was rat 5-5 > human T1-1 > bacterial DM11, showing that human GSH transferase T1-1 can form dihalomethane adducts under the conditions used. Although the lability of DNA adducts has precluded more sophisticated experiments and in vivo studies have not yet been possible, the work collectively demonstrates the ability of several GSH transferases to generate DNA adducts from dihalomethanes, with G being the preferred site of adduction in both this and the GSCH(2)OAc model system.     

Isolation and characterization of N7-guanyl adducts derived from 1,2-dibromo-3-chloropropane

1,2-Dibromo-3-chloropropane is a potent renal and testicular toxicant and has been shown to induce tumor formation in laboratory animals. The toxic effects of the compound are thought to be a result of a bioactivation step in which a glutathione conjugate is formed and subsequently reacts with cellular DNA. The L-glutathione conjugate of 1,2-dibromo-3-chloropropane was chemically synthesized and used to alkylate DNA: following incubations of the conjugate with calf thymus DNA and neutral thermal hydrolysis (to release N7-guanyl adducts) four major fluorescent products were observed. Three of these were isolated and characterized, the structures being determined as S-[bis(N7-guanylmethyl)methyl]glutathione and the two diastereomers of S-[1-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione. The fourth fluorescent product was unstable and formed in low yield and thus could not be characterized. The formation of these N7-guanyl adducts can be explained by a mechanism that includes the formation of two consecutive episulfonium ion intermediates followed by nucleophilic attack at the unsubstituted methylene carbon. These adducts bear structural and mechanistic similarities to the major adduct derived from 1,2-dibromoethane, S-[2-(N7-guanyl)ethyl]glutathione. The same adducts were also formed when DBCP was incubated with rat liver cytosol, GSH, and DNA. In vivo experiments with DBCP yielded very low levels of the N7-guanyl adducts formed in rat liver compared to the levels seen after treatments with 1,2-dibromoethane. The bis-guanyl adduct represents a cross-linked structure that may be important in the toxicity of this compound. The conjugate was not found to be mutagenic to Salmonella typhimurium TA100 but rather showed a toxic effect toward the bacteria.     

Characterization of S-[2-(N1-adenyl)ethyl]glutathione as an adduct formed in RNA and DNA from 1,2-dibromoethane.

The major DNA adduct derived from 1,2-dibromoethane is known to be S-[2-(N7-guanyl)-ethyl]glutathione; minor nucleic acid DNA adducts were characterized in view of the possibility that some might be unusually persistent or biologically active. RNA was modified in vitro by treatment with 1,2-dibromoethane and glutathione in the presence of rat liver cytosol, and bases were released by mild acid hydrolysis, which liberated greater than 99% of the bound radioactivity. One of the minor adducts was identified as S-[2-(N1-adenyl)ethyl]glutathione on the basis of its UV, mass, and NMR spectra. This adduct could be synthesized by reaction of S-(2-chloroethyl)-glutathione with adenosine. The material was desulfurized by treatment with Raney Ni to give N1-ethyladenine in low yield. The Raney Ni reaction was accompanied by considerable formation of the corresponding N6-adenine derivative via Dimroth rearrangement. Another adduct was identified as S-[2-(N7-guanyl)ethyl]cysteinylglycine by its UV, mass, and NMR spectra, but the material was demonstrated to be formed from the major DNA adduct, S-[2-(N7-guanyl)-ethyl]glutathione under conditions of mild acid hydrolysis. The imidazole ring opened derivative of S-[2-(N7-guanyl)ethyl]glutathione was synthesized and found not to be formed in DNA in vitro or in vivo. The two remaining minor adducts account for 1-2% of the total binding, but insufficient quantities were recovered to allow for structure determination; however, neither of these (uncharacterized) minor products are seen after the reaction of S-(2-chloroethyl)glutathione with guanosine or adenosine. S-[2-(N1-Adenyl)ethyl]glutathione was formed in rat liver RNA and DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation spectra of S-(2-hydroxy-3,4-epoxybutyl)glutathione: comparison with 1,3-butadiene and its metabolites in the Escherichia coli rpoB gene

S-(2-Hydroxy-3,4-epoxybutyl)glutathione (DEB-GSH conjugate) is formed from the reaction of 1,2:3,4-diepoxybutane (DEB) with glutathione (GSH), and the conjugate is considerably more mutagenic than several other butadiene-derived epoxides-including DEB-in Salmonella typhimurium TA1535 [Cho, S.-H., (2010) Chem. Res. Toxicol. 23, 1544-1546]. We previously identified six DNA adducts in the reaction of the DEB-GSH conjugate with nucleosides and calf thymus DNA and two DNA adducts in livers of mice and rats treated with DEB [Cho, S.-H. and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 706-712]. To define the role of GSH conjugation in 1,3-butadiene (BD) metabolism and characterize the mechanism of GSH transferase (GST)-enhanced mutagenicity of DEB, mutation spectra of BD and its metabolites in the absence and presence of GST/GSH and mouse liver microsomes were compared in the rpoB gene of Escherichia coli TRG8. The presence of GST considerably enhanced mutations. The mutation spectra derived from the DEB-GSH conjugate, the DEB/GST/GSH system, and the BD/mouse liver microsomes/GST/GSH system matched each other and were different from those derived from the other systems devoid of GSH. The major adducts in E. coli TRG8 cells treated with the DEB/GST/GSH system, the BD/mouse liver microsomes/GST/GSH system, or the DEB-GSH conjugate were S-[4-(N(7)-guanyl)-2,3-dihydroxybutyl]GSH, S-[4-(N(3)-adenyl)-2,3-dihydroxybutyl]GSH, and S-[4-(N(6)-deoxyadenosinyl)-2,3-dihydroxybutyl]GSH, indicating the presence of the GSH-containing DNA adducts in the systems. These results, along with the strong enhancement of mutagenicity by GST in this system, indicate the relevance of these GSH-containing DNA adducts.     

Identification of O2-substituted pyrimidine adducts formed in reactions of 4-(acetoxymethylnitrosamino)- 1-(3-pyridyl)-1-butanone and 4-(acetoxymethylnitros- amino)-1-(3-pyridyl)-1-butanol with DNA

Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2'-position of the tobacco specific carcinogen N'-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALCH(2)OAc) with DNA, dCyd, and dThd. NNKCH(2)OAc and NNALCH(2)OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH(2)OAc and NNALCH(2)OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 degrees C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH(2)OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH(2)OAc were similarly characterized. The dThd adduct of NNKCH(2)OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH(4) gave material, which was identical to that produced upon reaction of NNALCH(2)OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH(2)OAc with DNA are O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH(2)OAc are O(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) andO(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH(2)OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH(2)OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH(2)OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.     

Identification of adducts formed in the reactions of 5'-acetoxy-N'-nitrosonornicotine with deoxyadenosine, thymidine, and DNA

N'-Nitrosonornicotine (NNN) is the most prevalent of the carcinogenic tobacco-specific nitrosamines found in all tobacco products. Previous studies have demonstrated that cytochrome P450-mediated 5'-hydroxylation of NNN is a major metabolic pathway leading to mutagenic products, but to date, DNA adducts formed by this pathway have been only partially characterized, and there have been no studies reported on adducts formed with bases other than dGuo. Because adducts with dAdo and dThd have been identified in the DNA of the livers of rats treated with the structurally related carcinogen N-nitrosopyrrolidine, we investigated dAdo and dThd adduct formation from 5'-acetoxyNNN (3), a stable precursor to 5'-hydroxyNNN (2). Reaction of 3 with dAdo gave diastereomeric products, which were identified by their spectral properties and LC-ESI-MS/MS-SRM analysis as N(6)-[5-(3-pyridyl)tetrahydrofuran-2-yl]dAdo (9). This adduct was further characterized by NaBH(3)CN reduction to N(6)-[4-hydroxy-4-(3-pyridyl)but-1-yl]dAdo (17). A second dAdo adduct was identified, after NaBH(3)CN treatment, as 6-[2-(3-pyridyl)pyrrolidin-1-yl]purine-2'-deoxyriboside (18). Reaction of 3 with dThd, followed by NaBH(3)CN reduction, gave O(2)-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (11). Adducts 9, 11, 17, and 18 were all identified by LC-ESI-MS/MS-SRM comparison to synthetic standards. The reaction of 3 with calf thymus DNA was then investigated. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the resulting mixture was treated with NaBH(3)CN and analyzed by LC-ESI-MS/MS-SRM. Adducts 11, 17, and 18, as well as the previously identified dGuo adducts, were identified. The results of this study provide a more comprehensive picture of DNA adduct formation by the quantitatively important 5'-hydroxylation pathway of NNN and will facilitate investigation of the presence of these adducts in laboratory animals treated with NNN or in people who use tobacco products.     

Characterization of covalently modified deoxyribonucleosides formed from dibenz[a,j]anthracene in primary cultures of mouse keratinocytes

Identification of various deoxyribonucleoside adducts formed in primary cultures of mouse keratinocytes exposed to dibenz[a,j]anthracene (DB[a,j]A) is presented. A preliminary analysis of the DNA adducts formed from 7-methyldibenz[a,j]anthracene (7MeDB[a,j]A) also is presented. Cultures of keratinocytes obtained from dorsal skins of female SENCAR mice were exposed to 0.5 microgram of tritium-labeled hydrocarbons/mL of medium for 24 h. The total DNA binding was 2.23 +/- 0.54 and 5.28 +/- 0.97 pmol of hydrocarbon/mg of DNA for DB[a,j]A and 7MeDB[a,j]A, respectively. These binding values represented the radioactivity associated with the modified deoxyribonucleosides separated from the normal deoxyribonucleosides on Sephadex LH-20 columns following enzymatic digestion of isolated DNA. Treatment of keratinocytes with DB[a,j]A produced adduct peaks corresponding to marker adducts derived from trans addition of both deoxyguanosine as well as deoxyadenosine residues to the (+) enantiomer of the anti-diol epoxide where the deoxyadenosine adducts were predominant. In addition, DNA adduct peaks corresponding to markers of trans and cis addition, respectively, of deoxyguanosine and deoxyadenosine to the (+)-syn-diol epoxide were also noted in these chromatograms. A major DNA adduct in cells exposed to DB[a,j]A was tentatively identified as resulting from the addition of deoxyadenosine to DB[a,j]A-5,6-oxide. Several other later eluting DNA adduct peaks, not corresponding to any of the marker adducts, were also present in these chromatograms. In comparison, when cells were exposed to the more biologically potent 7-methyl analogue, at least 12 DNA adduct peaks were consistently observed in HPLC chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the adducts formed in the reactions of glycidamide with thymidine and cytidine

Glycidamide (GA) is a mutagenic epoxide metabolite of acrylamide (AM), a high production chemical with many industrial uses. Moreover, recent findings have shown that AM is formed in starchy foods cooked at high temperatures. This has refocused the attention on this chemical and its metabolite and on their possible mutagenicity and carcinogenicity. In this study, we have reacted GA with cytidine and thymidine in aqueous-buffered solutions. The adducts from the nucleosides have been isolated by reversed phase HPLC and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with thymidine yielded one adduct, N3-(2-carbamoyl-2-hydroxyethyl)thymidine (N3-GA-dThd), while the reaction with cytidine yielded three adducts. Two adducts were identified as a diastereomeric pair of N3-(2-carboxy-2-hydroxyethyl)cytidine (N3-GA-Cyd-1 and N3-GA-Cyd-2). The third adduct from the cytidine reaction was identified as N3-(2-carboxy-2-hydroxyethyl)uridine (N3-GA-Urd).     

Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline at the C-8 and N2 atoms of guanine.

The covalent binding of the carcinogenic N-hydroxy metabolites of 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to deoxynucleosides and DNA was investigated in vitro. Two major adducts were formed by the reaction of the N-acetoxy derivatives of IQ and MeIQx with deoxyguanosine (dG); however, no adducts were formed with deoxycytidine, deoxyadenosine, or thymidine. From proton NMR and mass spectroscopic characterization the adducts were identified as 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ),N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]q uinoline (dG-C8-IQ), 5-(deoxyguanosin-N2-yl)-2-amino-3,8-dimethylimidazo[4,5-f]qu inoxaline (dG-N2-MeIQx), and N-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]qui noxaline (dG-C8-MeIQx). The level of dG-C8 adducts was approximately 8-10 times greater than the amount of dG-N2 adducts formed from the reaction of dG with the N-acetoxy derivatives of IQ and MeIQx. The C-8-substituted dG adduct was also the major adduct formed from reactions of DNA with N-acetoxy-IQ and N-acetoxy-MeIQx. Approximately 60-80% of the bound carcinogens were recovered from DNA as dG-C8 adducts upon enzymatic digestion. The dG-N2 adducts also were detected and accounted for approximately 4% of the bound IQ and 10% of the bound MeIQx. These results suggest that the relative contributions of the nitrenium and carbenium ion resonance forms as well as DNA macromolecular structure are major determinants for DNA adduct substitution sites. Investigations on adduct conformation of 1H NMR spectroscopy revealed that the anti form is preferred for the dG-N2 adducts of IQ and MeIQx, while the syn form is preferred for the dG-C8 adducts. The possible role of these adducts in the initiation of carcinogenesis is discussed.     

Identification of adducts derived from reactions of (1-chloroethenyl)oxirane with nucleosides and calf thymus DNA

(1-Chloroethenyl)oxirane is a major mutagenic metabolite of chloroprene, an important large-scale petrochemical used in the manufacture of synthetic rubbers. The reactions of (1-chloroethenyl)oxirane with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The adducts from the nucleosides were isolated by reversed-phase HPLC, and characterized by their UV absorbance and (1)H and (13)C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one major adduct, N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGI), and eight minor adducts which were identified as diastereoisomeric pairs of N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyguanosine (dGII, dGIII), N3,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGIV, dGV), N7,N9-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVI, dGVII), and N1,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVIII, dGIX). The reaction of 2'-deoxyadenosine with (1-chloroethenyl)oxirane gave two adducts: N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAI) and N(6)-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAII). The adduct dAII was shown to arise via a Dimroth rearrangement of adduct dAI. The HPLC analyses of the reaction mixtures of (1-chloroethenyl)oxirane with 2'-deoxycytidine and thymidine showed the formation of one major product in each reaction. The adduct from 2'-deoxycytidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (dCI) derived by alkylation at N-3 followed by deamination. The adduct from thymidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-thymidine (TI). Reaction of (1-chloroethenyl)oxirane with calf thymus DNA gave all of the adducts observed from the individual nucleosides except dGII and dGIII. However, there was selectivity for the formation of dGI and dCI. The adduct levels in DNA were 9,630 (dGI), 240 (dCI), 83 (dAI), 6 (dAII), and 28 (TI) pmol/mg DNA, respectively. The preferred formation of dCI may be relevant to chloroprene mutagenesis.     

Reaction of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA: identification of DNA adducts

Glyoxal (ethanedial) is an increasingly used industrial chemical that has been found to be mutagenic in bacteria and mammalian cells. In this study, the reactions of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The nucleoside adducts were isolated by reversed-phase liquid chromatography and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one adduct, the previously known 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one adduct. The reaction of 2'-deoxyadenosine with glyoxal resulted in the formation of a previously not reported N6-(hydroxyacetyl)-2'-deoxyadenosine adduct. In the reaction of glyoxal with 2'-deoxycytidine and cytidine at neutral conditions and 37 degrees C, 5-hydroxyacetyl pyrimidine derivatives were obtained. When the cytidine reaction was performed at pH 4.5 and 50 degrees C, the 5-hydroxyacetyl derivative of uridine was formed through deamination of cytidine-glyoxal. Adducts in the thymidine reaction could not be detected. In the reaction of glyoxal with calf thymus DNA, the 2'-deoxyguanosine-glyoxal and 2'-deoxyadenosine-glyoxal adducts were obtained, the former being the major adduct.     

Analysis of DNA adducts formed in vivo in rats and mice from 1,2-dibromoethane, 1,2-dichloroethane, dibromomethane, and dichloromethane using HPLC/accelerator mass spectrometry and relevance to risk estimates

Dihaloalkanes are of toxicological interest because of their high-volume use in industry and their abilities to cause tumors in rodents, particularly dichloromethane and 1,2-dichloroethane. The brominated analogues are not used as extensively but are known to produce more toxicity in some systems. Rats and mice were treated i.p. with (14)C-dichloromethane, -dibromomethane, -1,2-dichloroethane, or -1,2-dibromoethane [5 mg (kg body weight)(-1)], and livers and kidneys were collected to rapidly isolate DNA. The DNA was digested using a procedure designed to minimize processing time, because some of the potential dihalomethane-derived DNA-glutathione (GSH) adducts are known to be unstable, and the HPLC fractions corresponding to major adduct standards were separated and analyzed for (14)C using accelerator mass spectrometry. The level of liver or kidney S-[2-(N(7)-guanyl)ethyl]GSH in rats treated with 1,2-dibromoethane was approximately 1 adduct/10(5) DNA bases; in male or female mice, the level was approximately one-half of this. The levels of 1,2-dichloroethane adducts were 10-50-fold lower. None of four known (in vitro) GSH-DNA adducts was detected at a level of >2/10(8) DNA bases from dibromomethane or dichloromethane. These results provide parameters for risk assessment of these compounds: DNA binding occurs with 1,2-dichloroethane but is considerably less than from 1,2-dibromoethane in vivo, and low exposure to dihalomethanes does not produce appreciable DNA adduct levels in rat or mouse liver and kidney of the doses used. The results may be used to address issues in human risk assessment.     

Synthesis, characterization, and 32p-postlabeling analysis of DNA adducts derived from the environmental contaminant 3-nitrobenzanthrone

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. 3-NBA forms DNA adducts in rodent tissues that arise principally through reduction to N-hydroxy-3-aminobenzanthrone (N-OH-ABA), esterification to its acetate or sulfate ester, and reaction of this activated ester with DNA. We detected 3-NBA-derived DNA adducts in rodent tissues by (32)P-postlabeling and generated them chemically by acid-catalyzed reaction of N-OH-ABA with DNA, but their structural identification has not yet been reported. We have now prepared 3-NBA-derived adducts by reaction of a possible reactive metabolite, N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA), with purine nucleosides and nucleotides, characterized them, and have shown that they are present in DNA treated with this 3-NBA derivative. Three of these adducts have been characterized as the C-C adduct N-acetyl-3-amino-2-(2'-deoxyguanosin-8-yl)benzanthrone, the C-N adduct N-acetyl-N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone, and an unusual 3-acetylaminobenzanthrone adduct of deoxyadenosine, which involves a double linkage between adenine and benzanthrone (N1 to C1, N(6) to C11b), creating a five-membered imidazo type ring system. According to IUPAC fused ring conventions, we propose the following systematic name for this adduct: (9'-(2' '-deoxyribofuranosyl))purino[6',1':2,3]imidazo[5,4-p](1,11b-dihydro-(N-acetyl-3-amino))benzanthrone. The 3'-phosphates of these novel adducts could be 5'-postlabeled using [gamma-(32)P]ATP, although the efficiency of labeling was found to be low (less than 20%). However, none of these adducts could be detected in DNA from 3-NBA-treated rats by (32)P-postlabeling. Two of these synthetic adducts were treated with alkali to generate nonacetylated adducts, and these were also shown by HPLC to differ from those adducts found in rat DNA. Therefore, a different approach to the synthesis of authentic standards is needed for the structural characterization of 3-NBA-derived DNA adducts formed in vivo.     

Identification of adducts formed in the reaction of alpha-acetoxy-N-nitrosopyrrolidine with deoxyribonucleosides and DNA

N-Nitrosopyrrolidine (NPYR) is a well-established hepatocarcinogen in the rat. NPYR requires metabolic activation by cytochrome P450-catalyzed alpha-hydroxylation to express its carcinogenic activity. This produces alpha-hydroxyNPYR (2), which spontaneously ring opens to 4-oxobutanediazohydroxide (4), a highly reactive intermediate, which may itself modify DNA or yield a cascade of electrophiles that react with DNA to produce adducts. Multiple dGuo adducts formed in this reaction have been previously characterized, but there are no examples of adducts formed with other DNA nucleobases. In this study, we used alpha-acetoxyNPYR (3) as a stable precursor to 2 and 4. Compound 3 was allowed to react with DNA. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the products were analyzed by LC-ESI-MS and LC-ESI-MS/MS. Reactions of 3 with individual deoxyribonucleosides were also carried out. The products were identified by their MS, UV, and NMR spectra as N6-(tetrahydrofuran-2-yl)dAdo (16) and N4-(tetrahydrofuran-2-yl)dCyd (17) in addition to the previously characterized N2-(tetrahydrofuran-2-yl)dGuo (13). Unstable dThd adducts were also formed. Further characterization of the adducts was achieved by NaBH3CN reduction of the reaction mixtures of 3 with deoxyribonucleosides or DNA. This produced N6-(4-hydroxybut-1-yl)dAdo (21), N4-(4-hydroxybut-1-yl)dCyd (22), O2-(4-hydroxybut-1-yl)dThd (23), O4-(4-hydroxybut-1-yl)dThd (24), and 3-(4-hydroxybut-1-yl)dThd (25). Adducts 21 and 22 were characterized by their spectral properties, while the dThd adducts 23-25 were identified by comparison to synthetic standards. The results of this study demonstrate that 3 forms adducts with dAdo, dCyd, and dThd in DNA, in addition to the previously characterized dGuo adducts. These newly characterized standards can be used to investigate DNA adduct formation in rats treated with NPYR.     

Analysis of crotonaldehyde- and acetaldehyde-derived 1,n(2)-propanodeoxyguanosine adducts in DNA from human tissues using liquid chromatography electrospray ionization tandem mass spectrometry

Crotonaldehyde, a mutagen and carcinogen, reacts with deoxyguanosine (dGuo) in DNA to generate a pair of diastereomeric 1,N(2)()-propanodeoxyguanosine adducts (Cro-dGuo, 2), which occur in (6S,8S) and (6R,8R) configurations. They can also be formed through the consecutive reaction of two acetaldehyde molecules with dGuo. Cro-dGuo adducts inhibit DNA synthesis and induce miscoding in human cells. Considering their potential role in carcinogenesis, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to explore the presence of Cro-dGuo adducts in DNA from various human tissues, such as liver, lung, and blood. DNA was isolated from human tissues and enzymatically hydrolyzed to deoxyribonucleosides. [(15)N(5)]Cro-dGuo was synthesized and used as an internal standard. The Cro-dGuo adducts were enriched from the hydrolysate by solid-phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring (SRM). This method allows the quantitation of the Cro-dGuo adducts at a concentration of 4 fmol/micromol dGuo, corresponding to about 1 adduct per 10(9) normal nucleosides starting with 1 mg of DNA, with high accuracy and precision. DNA from human liver, lung, and blood was analyzed. The Cro-dGuo adducts were detected more frequently in human lung DNA than in liver DNA but were not detected in DNA from blood. The results of this study provide quantified data on Cro-dGuo adducts in human tissues. The higher frequency of Cro-dGuo in lung DNA than in the other tissues investigated is potentially important and deserves further study.     

Persistence of benzo[a]pyrene--DNA adducts in hematopoietic tissues and blood of the mummichog, Fundulus heteroclitus

The formation and persistence of benzo[a]pyrene (B[a]P)-DNA adducts were investigated in blood, liver and two hematopoietic tissues (anterior kidney and spleen) of the mummichog (Fundulus heteroclitus). Fish were injected with a single, sublethal dose of B[a]P (12 mg/kg body weight) and sampled from 8 to 96 days post-injection. 32P-Postlabeling analysis and storage phosphor imaging were used to resolve and quantify hydrophobic DNA adducts. One major DNA adduct was present in each of the examined tissues at all sampling times. This adduct had similar chromatographic characteristics to those of the adduct standard, 7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydro-benzo[a]pyrene (B[a]PDE-dG). Minor DNA adduct spots, representing less than 2% of the total DNA adducts, were observed in some liver, anterior kidney and spleen samples for up to 32 days post-injection. The B[a]P-DNA adducts reached maximal levels at 32 days post-injection and persisted for at least 96 days in all examined tissues. B[a]P-DNA adduct levels were significantly higher in the liver and anterior kidney than in the spleen from 16 to 96 days (P<0.001), although liver and anterior kidney DNA adduct levels were not significantly different at any time. This is the first controlled study to demonstrate the formation and persistence of B[a]P-DNA adducts in hematopoietic tissues and blood of fishes exposed to the prototypical polycyclic aromatic hydrocarbon, B[a]P. Although persistent DNA adducts are generally recognized as potential initiators of carcinogenic processes, adducts in these vital tissues may also lead to disruption of physiological functions such defense mechanisms and hematopoiesis.     

Synthesis and characterization of deoxyguanosine-benzoquinone adducts

Benzene expresses its carcinogenic potential in humans largely in the form of acute leukemia. Because an understanding of the formation of DNA adducts by benzene metabolites may help to explain the etiological role they play in benzene-induced bone marrow disease, we have synthesized, isolated and characterized adducts formed by the reaction of deoxyguanosine with hydroquinone and p-benzoquinone, two toxic metabolites of benzene. [3H]Deoxyguanosine and [14C]hydroquinone reacted in neutral aqueous buffer containing iron to form two dual-labeled products, which were separated using HPLC. When p-benzoquinone was substituted for hydroquinone, the same adducts were formed in the absence of added iron. The ultraviolet and fluorescence spectra of the less polar adduct, called Adduct 2, were distinctly different from the spectra of the starting materials. NMR and mass spectrometry suggested a compound with a mass of 357 with the p-benzoquinone moiety bound to the N-1 and N2 positions of deoxyguanosine. Based on these data it is proposed that Adduct 2 is (3'OH)benzetheno(1,N2)deoxyguanosine. The more polar product, Adduct 1, was found to have a unique ultraviolet spectrum but did not appear to be fluorescent. Both adducts were observed after calf thymus DNA was incubated with hydroquinone and digested to its constituent nucleosides.     

Synthesis and characterization of nucleosides and oligonucleotides with a benzo[a]pyren-6-ylmethyl adduct at adenine N6 or guanine N2

Benzo[a]pyrene (1) can be converted to reactive electrophilic species by a number of metabolic pathways, of which the route to the mutagenic and carcinogenic diol epoxide(s) is the best studied. An alternative and interesting pathway to a highly genotoxic electrophile is through alkylation at the 6 position to 6-methylbenzo[a]pyrene (2) followed by oxidation of the methyl group to give 6-hydroxymethylbenzo[a]pyrene (3). Esterification of 3, especially to sulfate ester 4, gives compounds which are both mutagenic and carcinogenic. The major DNA adduct identified from exposure of rats and mice to 4 is the guanine N(2) adduct [2'-deoxy-N(2)-(benzo[a]pyren-6-ylmethyl)guanosine, 5] which is also formed via activation of 2 to a radical cation species by horseradish peroxidase/H(2)O(2) or iodine. To study the biological and structural properties of this adduct and the analogous adenine N(6) adduct (6), a nonbiomimetic synthesis of the adducted nucleosides 5 and 6 has been developed and has been extended to preparation of oligonucleotides containing 5 or 6 at a single site.     

Identification of adducts formed by pyridyloxobutylation of deoxyguanosine and DNA by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone,a chemically activated form of tobacco specific carcinogens

The tobacco specific carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are metabolically activated to 4-oxo-4-(3-pyridyl)-1-butanediazohydroxide (7), which is known to pyridyloxobutylate DNA. A substantial proportion of the adducts in this DNA releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 11) under various hydrolysis conditions, including neutral thermal hydrolysis. These HPB-releasing DNA adducts have been detected in target tissues of animals treated with NNK and NNN as well as in lung tissue from smokers. Although their presence in pyridyloxobutylated DNA was conclusively demonstrated 15 years ago, their structures have not been previously determined. We investigated this question in the present study by determining the structures of products formed in reactions with dGuo and DNA of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc, 3), a stable precursor to 7. Reaction mixtures from NNKCH(2)OAc and dGuo were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) with selected ion monitoring at m/z 415. A major peak was detected and identified as 7-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (37) by its ESI-MS fragmentation pattern and by neutral thermal hydrolysis, which converted it to 11 and 7-[4-oxo-4-(3-pyridyl)but-1-yl]Gua (26). The latter was identified by comparison to synthetic 26 using LC-ESI-MS with selected ion monitoring at m/z 299, M + 1 of 26. Further evidence was obtained by NaBH(4) reduction of 26 to 7-[4-hydroxy-4-(3-pyridyl)but-1-yl]Gua, which was also matched with a standard. Adduct 37 was similarly identified in enzyme hydrolysates of DNA reacted with NNKCH(2)OAc, accounting for 30-35% of the HPB-releasing adducts in this DNA. Several other adducts resulting from pyridyloxobutylation of the N(2)- and O(6)-positions of Gua were also identified as products in the dGuo or DNA reactions by comparison to standards; their concentrations were considerably less than that of 37. These adducts were N(2)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (23), N(2)-[4-oxo-4-(3-pyridyl)but-2-yl]dGuo (25), N(2)-[2-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (31a) (or its open chain tautomer 31b), and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]dGuo (10). Adducts 23, 25, and 10 did not release HPB upon neutral thermal hydrolysis. The results of this study provide the first structural identification of an HPB-releasing DNA adduct of the tobacco specific nitrosamines NNK and NNN.     

Supercoiled DNA promotes formation of intercalated cis-N2-deoxyguanine adducts and base-stacked trans-N2-deoxyguanine adducts by (+)-7R,8S-dihydrodiol-9S,10R-epoxy-7,8,9,10-tetra- hydrobenzo[a]pyrene

The highly reactive and mutagenic benzo[a]pyrene metabolite, (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), forms predominantly N2-deoxyguanine DNA adducts in two stereoisomeric configurations (cis and trans). In previous in vitro assays using oligonucleotide substrates site specifically modified with cis- and trans-BPDE adducts, the nucleotide excision repair (NER) systems of eukaryotes and prokaryotes incise cis-BPDE adducts more efficiently than trans-BPDE adducts [Hess, et al. (1997) Mol. Cell Biol 17, 7069; Zou, et al. (2001) Biochemistry 40, 2923). We investigated the influence of DNA secondary structure on stereospecificity of BPDE adduct formation, and incision of BPDE adducts by the prokaryotic UvrABC NER endonuclease was examined. BPDE adducts formed at low density on supercoiled plasmids were incised 6-7-fold better by the thermoresistant Bacillus caldotenaxUvrABC than were BPDE adducts formed on linear DNA. Linearizing supercoiled plasmid DNAs after BPDE adduct formation did not diminish incision efficiency. These results suggested that configuration and/or conformation of adducts formed on linear and supercoiled DNAs differed. This hypothesis was confirmed by low temperature fluorescence spectroscopy of adducted supercoiled and linear DNAs. Spectroscopic results indicated that intercalated cis-BPDE adducts as well as base-stacked trans-BPDE adducts formed more abundantly in supercoiled DNA than in linear DNA. A higher cis to trans adduct ratio in supercoiled DNA was confirmed by high resolution [32P]postlabeling analyses. These results demonstrate that DNA secondary structure influences both configuration and conformation of BPDE adducts formed at low density (approximately 1 adduct/kbp) and suggests that the ratio of cis- to trans-BPDE adducts and amount of base-stacked trans adducts formed under physiological exposure conditions may be higher than inferred from high dose experiments.