Persistence of N7-(2,3,4-trihydroxybutyl)guanine adducts in the livers of mice and rats exposed to 1,3-butadiene

Liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) and stable isotope methodology was employed for the analysis of the N7-guanine (Gua) adducts derived from 1,2:3, 4-diepoxybutane (BDO2) a reactive metabolite of 1,3-butadiene (BD). Two diastereomeric forms of N7-(2,3,4-trihydroxybutyl)guanine (THBG) were identified in the livers of both mice and rats. One of the diastereomers [(+/-)-THBG] was formed by reaction of DNA with (+/-)-BDO2, and the other diastereomer (meso-THBG) was formed by reaction of DNA with meso-BDO2. There was significantly more (+/-)-THBG and meso-THBG in the liver DNA of the mice when compared with those of the rats during the 10 days of exposure to BD and the 6 days of postexposure that were monitored. There was a 2-fold excess of (+/-)-THBG over meso-THBG in the rat liver at all the time points. In the mouse liver after 10 days of exposure to BD, the (+/-)-THBG (3.9 adducts/10(6) normal bases) was also present in an almost 2-fold excess over meso-THBG (2.2 adducts/10(6) normal bases). However, 6-days after exposure to BD, (+/-)-THBG (1.2 adducts/10(6) normal bases) and meso-THBG (1.0 adduct/10(6) normal bases) were present in almost equal amounts in the mouse liver. Furthermore, there was an almost 5-fold excess of the two THBG diastereomers in the mouse liver DNA 6 days after exposure to BD when compared with rat liver DNA. The half-lives of (+/-)-THBG and meso-THBG appeared to be slightly longer in mouse liver (4.1 and 5.5 days, respectively) than in rat liver (3.6 and 4.0 days, respectively). The apparent persistence of these adducts in the mouse may contribute to the increased susceptibility of this species to BD-induced carcinogenesis. It is possible that (+/-)-THBG and meso-THBG could have also been derived from the reaction of DNA with the hydrolysis product of BDO2, 1,2-dihydroxy-3,4-epoxybutane (DHEB). Surprisingly, a vast majority of the studies in which the mutagenic and carcinogenic potential of BDO2 have been examined have only employed the commercially available (+/-)-BDO2. In light of the present findings, additional studies will be required to determine the potency of meso-BDO2 and the DHEB that is the precursor to meso-THBG as mutagens and carcinogens.     

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 the reactivity, regioselectivity, and stereoselectivity of the reactions of butadiene monoxide with valinamide and the N-terminal valine of mouse and rat hemoglobin.

Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of hemoglobin N-terminal valine adduct formation with the primary reactive metabolite, butadiene monoxide (BMO). However, mechanistic details concerning the relative reactivity, regioselectivity, and stereospecificity of BMO with the N-terminal valine of hemoglobin are lacking. In the studies presented here, L-valinamide was used as a model for the N-terminal valine of hemoglobin to compare the nucleophilic reactivity, regioselectivity, and stereoselectivity of the reaction both in aqueous solution and within a protein microenvironment. Four products produced by the reaction of L-valinamide with racemic BMO (two pairs of diastereomers produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and characterized by (1)H NMR and GC/MS. These four reaction products were used as analytical standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4) and temperature (37 degrees C). The results show that the adducts formed by reaction at C-2 were formed at a ratio of approximately 2:1 compared to the adducts formed by reaction at C-1. The stereoisomers of each respective regioisomer were produced with similar rates of formation. The reaction of BMO with the N-terminal valine of hemoglobin was also studied in vitro using intact erythrocytes from Sprague-Dawley rats and B6C3F1 mice. After cleavage of the N-modified valine by the N-alkyl Edman degradation procedure using pentafluorophenylisothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and quantitation of the four expected products by silylation of the PFPTH-valine-BMO derivatives. The hemoglobin results contrast with the valinamide results in that the reaction of BMO with the N-terminal valine residue in both rat and mouse hemoglobin produced mostly C-1 adducts. The rates obtained with rat hemoglobin were much slower than the rates obtained with mouse hemoglobin or with valinamide. These results, and the finding that the reaction with rat hemoglobin produced a higher ratio of C1:C2 adducts in comparison with the reaction with mouse hemoglobin, indicate the importance of measuring all four adducts when comparing the relative rates of adduct formation both with model compounds and among different species.     

Conjugation of butadiene diepoxide with glutathione yields DNA adducts in vitro and in vivo

1,2,3,4-Diepoxybutane (DEB) is reported to be the most potent mutagenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant. DEB is capable of inducing the formation of monoalkylated DNA adducts and DNA-DNA and DNA-protein cross-links. We previously reported that DEB forms a conjugate with glutathione (GSH) and that the conjugate is considerably more mutagenic than several other butadiene-derived epoxides, including DEB, in the base pair tester strain Salmonella typhimurium TA1535 [Cho et al. (2010) Chem. Res. Toxicol. 23, 1544-1546]. In the present study, we determined steady-state kinetic parameters of the conjugation of the three DEB stereoisomers-R,R, S,S, and meso (all formed by butadiene oxidation)-with GSH by six GSH transferases. Only small differences (<3-fold) were found in the catalytic efficiency of conjugate formation (k(cat)/K(m)) with all three DEB stereoisomers and the six GSH transferases. The three stereochemical DEB-GSH conjugates had similar mutagenicity. Six DNA adducts (N(3)-adenyl, N(6)-adenyl, N(7)-guanyl, N(1)-guanyl, N(4)-cytidyl, and N(3)-thymidyl) were identified in the reactions of DEB-GSH conjugate with nucleosides and calf thymus DNA using LC-MS and UV and NMR spectroscopy. N(6)-Adenyl and N(7)-guanyl GSH adducts were identified and quantitated in vivo in the livers of mice and rats treated with DEB ip. These results indicate that such DNA adducts are formed from the DEB-GSH conjugate, are mutagenic regardless of sterochemistry, and are therefore expected to contribute to the carcinogenicity of DEB.     

Identification of paraldol-deoxyguanosine adducts in DNA reacted with crotonaldehyde

Crotonaldehyde (1) is a mutagen and carcinogen, but its reactions with DNA have been only partially characterized. In a previous study, we found that substantial amounts of 2-(2-hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 7), the dimer of 3-hydroxybutanal (8), were released upon enzymatic or neutral thermal hydrolysis of DNA that had been allowed to react with crotonaldehyde. We have now characterized two paraldol-deoxyguanosine adducts in this DNA: N(2)-[2-(2-hydroxypropyl)-6-methyl-1,3-dioxan-4-yl]deoxyguanosine (N(2)-paraldol-dG, 13) and N(2)-[2-(2-hydroxypropyl)-6-methyl-1, 3-dioxan-4-yl]deoxyguanylyl-(5'-3')-thymidine [N(2)-paraldol-dG-(5'-3')-thymidine, 14]. Four diastereomers of N(2)-paraldol-dG (13) were observed. Their overall structures were determined by (1)H NMR, by MS, and by reaction of paraldol with deoxyguanosine and DNA. (1)H NMR data showed that two diastereomers had all equatorial substituents in the dioxane ring, while two others had an axial 6-methyl group. Preparation of paraldol with the (R)- or (S)-configuration at the 6-position of the dioxane ring and the carbinol carbon of the 2-(2-hydroxypropyl) group allowed partial assignment of the absolute configurations of N(2)-paraldol-dG (13). Four diastereomers of N(2)-paraldol-dG-(5'-3')-thymidine (14) were observed. Their overall structure was determined by (1)H NMR, MS, and hydrolysis with snake venom or spleen phosphodiesterase. Reactions of nucleosides and nucleotides with paraldol demonstrated that adducts were formed only from deoxyguanosine and its monophosphates. Experiments with DNA that had been reacted with crotonaldehyde indicated that N(2)-paraldol-dG-containing adducts in DNA are relatively resistant to enzymatic hydrolysis. The results of this study demonstrate that the reaction of crotonaldehyde with DNA is more complex than previously recognized and that stable N(2)-paraldol-dG adducts are among those that should be considered in assessing mechanisms of crotonaldehyde mutagenicity and carcinogenicity.     

Mutagenic spectrum of butadiene-derived N1-deoxyinosine adducts and N6,N6-deoxyadenosine intrastrand cross-links in mammalian cells

Reactive metabolites of 1,3-butadiene, including 1,2-epoxy-3-butene (BDO), 1,2:3,4-diepoxybutane (BDO(2)), and 3,4-epoxy-1,2-butanediol (BDE), form both stable and unstable base adducts in DNA and have been implicated in producing genotoxic effects in rodents and human cells. N1 deoxyadenosine adducts are unstable and can undergo either hydrolytic deamination to yield N1 deoxyinosine adducts or Dimroth rearrangement to yield N(6) adducts. The dominant point mutation observed at AT sites in both in vivo and in vitro mutagenesis studies using BD and its epoxides has been A --> T transversions followed by A --> G transitions. To understand which of the butadiene adducts are responsible for mutations at AT sites, the present study focuses on the N1 deoxyinosine adduct at C2 of BDO and N(6),N(6)-deoxyadenosine intrastrand cross-links derived from BDO(2). These lesions were incorporated site-specifically and stereospecifically into oligodeoxynucleotides which were engineered into mammalian shuttle vectors for replication bypass and mutational analyses in COS-7 cells. Replication of DNAs containing the R,R-BDO(2) intrastrand cross-link between N(6) positions of deoxyadenosine yielded a high frequency (59%) of single base substitutions at the 3' adducted base, while 19% mutagenesis was detected using the S,S-diastereomer. Comparable studies using the R- and S-diastereomers of the N1 deoxyinosine adduct gave rise to approximately 50 and 80% A --> G transitions with overall mutagenic frequencies of 59 and 90%, respectively. Collectively, these data establish a molecular basis for A --> G transitions that are observed following in vivo and in vitro exposures to BD and its epoxides, but fail to reveal the source of the A --> T transversions that are the dominant point mutation.     

Quantitative analysis of trihydroxybutyl mercapturic acid, a urinary metabolite of 1,3-butadiene, in humans

1,3-Butadiene (BD) is a known human carcinogen present in cigarette smoke and in automobile exhaust, leading to widespread exposure of human populations. BD requires cytochrome P450-mediated metabolic activation to electrophilic species, e.g. 3,4-epoxy-1-butene (EB), hydroxymethyl vinyl ketone (HMVK), and 3,4-epoxy-1,2-diol (EBD), which form covalent adducts with DNA. EB, HMVK, and EBD can be conjugated with glutathione and ultimately excreted in urine as monohydroxybutenyl mercapturic acid (MHBMA), dihydroxybutyl mercapturic acid (DHBMA), and trihydroxybutyl mercapturic acid (THBMA), respectively, which can serve as biomarkers of BD exposure and metabolic processing. While MHBMA and DHBMA have been found in smokers and nonsmokers, THBMA has not been previously detected in humans. In the present work, an isotope dilution HPLC-ESI(-)-MS/MS methodology was developed and employed to quantify THBMA in urine of known smokers and nonsmokers (19-27 per group). The new method has excellent sensitivity (LOQ, 1 ng/mL urine) and achieves accurate quantitation using a small sample volume (100 μL). Mean urinary THBMA concentrations in smokers and nonsmokers were found to be 21.6 and 13.7 ng/mg creatinine, respectively, suggesting that there are sources of THBMA other than exposure to tobacco smoke in humans, as is also the case for DHBMA. However, THBMA concentrations are significantly greater in urine of smokers than that of nonsmokers (p < 0.01). Furthermore, THBMA amounts in human urine declined 25-50% following smoking cessation, suggesting that smoking is an important source of this metabolite in humans. The HPLC-ESI(-)-MS/MS methodology developed in the present work will be useful for future epidemiological studies of BD exposure and metabolism.     

Mutagenicity of 5-hydroxymethylfurfural in V79 cells expressing human SULT1A1: identification and mass spectrometric quantification of DNA adducts formed

5-Hydroxymethylfurfural (HMF), a heterocyclic product of the Maillard reaction, is a ubiquitous food contaminant. It has demonstrated hepatocarcinogenic activity in female mice. This effect may originate from sulfo conjugation of the benzylic alcohol yielding 5-sulfooxymethylfurfural (SMF), which is prone to react with DNA via nucleophilic substitution. Indeed, we showed that HMF induces gene mutations in Chinese hamster V79 cells engineered for the expression of human (h) sulfotransferase (SULT)1A1 but not in parental V79 cells. In order to identify potential DNA adducts, we incubated DNA samples with SMF or HMF in aqueous solution. Modified DNA was digested and surveyed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for adducts that may be formed by nucleosides either via nucleophilic substitution at the electrophilic carbon atom of SMF or via imine formation with the aldehyde group present in HMF and SMF. The most abundant adducts formed from SMF, N(6)-((2-formylfuran-5-yl)methyl)-2'-deoxyadenosine (N(6)-FFM-dAdo) and N(2)-((2-formylfuran-5-yl)methyl)-2'-deoxyguanosine (N(2)-FFM-dGuo), were synthesized, purified, and characterized by (1)H NMR. Imine adducts were only detected when DNA was incubated with very high levels of HMF following reduction of the imines to corresponding secondary amines by NaBH(3)CN. Sensitive techniques based on LC-MS/MS multiple reaction monitoring for the quantification of the adducts in DNA samples were devised using isotope-labeled [(15)N(5)]N(6)-FFM-dAdo and [(13)C(10),(15)N(5)]N(2)-FFM-dGuo as internal standards. Both 5-methylfurfuryl adducts were detected in DNA from V79-hSULT1A1 treated with HMF but not in DNA from V79 control cells. Considering the lack of other known mutagenic metabolites, we hypothesize that the hepatocarcinogenic potential of HMF originates from the formation of mutagenic SMF.     

Hydroxymethylvinyl ketone: a reactive Michael acceptor formed by the oxidation of 3-butene-1,2-diol by cDNA-expressed human cytochrome P450s and mouse, rat, and human liver microsomes.

The metabolic fate of 3-butene-1,2-diol (BDD), a secondary metabolite of the industrial carcinogen, 1,3-butadiene, is unclear. The current study characterizes BDD oxidation to hydroxymethylvinyl ketone (HMVK), a reactive Michael acceptor. Because of its instability in aqueous medium, HMVK was trapped by conjugation with GSH, a reaction that occurred readily at physiological conditions (pH 7.4, 37 degrees C) to yield 1-hydroxy-2-keto-4-(S-glutathionyl)butane. The results show that BDD was oxidized to HMVK by mouse, rat, and human liver microsomes and by cDNA-expressed human cytochrome P450s. Eadie-Hofstee plots demonstrated biphasic kinetics of BDD oxidation with mouse and rat liver microsomes and one of three individual human liver microsomes; BDD oxidation by the other two human liver microsomal samples was best described by monophasic kinetics. Of the human P450 enzymes examined, only P450 2E1 exhibited activity at 1 mM BDD. P450 3A4 was capable of catalyzing the reaction at a high BDD (10 mM) concentration; P450 1A1, 1A2, 1B1, 2D6-Met, and 2D6-Val produced only trace amounts of HMVK-GSH whereas P450 2A6, 2C8, 2C9, and 4A11 had no detectable activity. Detection of HMVK or the HMVK-GSH conjugate was dependent on reaction time, protein, and BDD concentrations, and the presence of NADPH. Collectively, the results provide clear evidence for BDD bioactivation to yield HMVK. Because mouse, rat, and human liver microsomes exhibited K(m) values of 50-80 microM, the results also suggest that HMVK could be formed after rodent or human exposure to BDD or its parent compound, BD.     

Identification of DNA adducts of acetaldehyde

Acetaldehyde is a mutagen and carcinogen which occurs widely in the human environment, sometimes in considerable amounts, but little is known about its reactions with DNA. In this study, we identified three new types of stable acetaldehyde DNA adducts, including an interstrand cross-link. These were formed in addition to the previously characterized N(2)-ethylidenedeoxyguanosine. Acetaldehyde was allowed to react with calf thymus DNA or deoxyguanosine. The DNA was isolated and hydrolyzed enzymatically; in some cases, the DNA was first treated with NaBH(3)CN. Reaction mixtures were analyzed by HPLC, and adducts were isolated and characterized by UV, (1)H NMR, and MS. The major adduct was N(2)-ethylidenedeoxyguanosine (1), which was identified as N(2)-ethyldeoxyguanosine (7) after treatment of the DNA with NaBH(3)CN. The new acetaldehyde adducts were 3-(2-deoxyribos-1-yl)-5,6,7, 8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (9), 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N(2)-deoxyguanosyl+ ++)- 6-methylpyrimido[1,2-a]purine-10(3H)one (12), and N(2)-(2, 6-dimethyl-1,3-dioxan-4-yl)deoxyguanosine (11). Adduct 9 has been previously identified in reactions of crotonaldehyde with DNA. However, the distribution of diastereomers was different in the acetaldehyde and crotonaldehyde reactions, indicating that the formation of 9 from acetaldehyde does not proceed through crotonaldehyde. Adduct 12 is an interstrand cross-link. Although previous evidence indicates the formation of cross-links in DNA reacted with acetaldehyde, this is the first reported structural characterization of such an adduct. This adduct is also found in crotonaldehyde-deoxyguanosine reactions, but in a diastereomeric ratio different than that observed here. A common intermediate, N(2)-(4-oxobut-2-yl)deoxyguanosine (6), is proposed to be involved in formation of adducts 9 and 12. Adduct 11 is produced ultimately from 3-hydroxybutanal, the major aldol condensation product of acetaldehyde. Levels of adducts 9, 11, and 12 were less than 10% of those of N(2)-ethylidenedeoxyguanosine (1) in reactions of acetaldehyde with DNA. As nucleosides, adducts 9, 11, and 12 were stable, whereas N(2)-ethylidenedeoxyguanosine (1) had a half-life of 5 min. These new stable adducts of acetaldehyde may be involved in determination of its mutagenic and carcinogenic properties.     

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.     

Identification and characterization of 2'-deoxyadenosine adducts formed by isoprene monoepoxides in vitro

Isoprene, the 2-methyl analogue of 1,3-butadiene, is ubiquitous in the environment, with major contributions to total isoprene emissions stemming from natural processes despite the compound being a bulk industrial chemical. Additionally, isoprene is a combustion product and a major component in cigarette smoke. Isoprene has been classified as possibly carcinogenic to humans (group 2B) by IARC and as reasonably anticipated to be a human carcinogen by the National Toxicology Program. Isoprene, like butadiene, requires metabolic activation to reactive epoxides to exhibit its carcinogenic properties. The mode of action has been postulated to be that of a genotoxic carcinogen, with the formation of promutagenic DNA adducts being essential for mutagenesis and carcinogenesis. In rodents, isoprene-induced tumors show unique point mutations (A→T transversions) in the K-ras protooncogene at codon 61. Therefore, we investigated adducts formed after the reaction of 2'-deoxyadenosine (dAdo ) with the two monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (IP-1,2-O) and propen-2-yloxirane (IP-3,4-O), under physiological conditions. The formation of N1-2'-deoxyinosine (N1-dIno) due to the deamination of N1-dAdo adducts was of particular interest, since N1-dIno adducts are suspected to have high mutagenic potential based on in vitro experiments. Major stable adducts were identified by HPLC, UV-spectroscopy, and LC-MS/MS and characterized by (1)H NMR and (1)H,(13)C HSQC and HMBC NMR experiments. Adducts of IP-1,2-O that were fully identified are R,S-C1-N(6)-dAdo, R-C2-N(6)-dAdo, and S-C2-N(6)-dAdo; adducts of IP-3,4-O are S-C3-N(6)-dAdo, R-C3-N(6)-dAdo, R,S-C4-N(6)-dAdo, S-C4-N1-dIno, R-C4-N1-dIno, R-C3-N1-dIno, S-C3-N1-dIno, and C3-N7-Ade. Both monoepoxides formed adducts on the terminal and internal oxirane carbons. This is the first study to describe adducts of isoprene monoepoxides with dAdo. Characterization of adducts formed by isoprene monoepoxides with deoxynucleosides and subsequently with DNA represent the first step toward evaluating their potential for being converted into a mutation or as biomarkers of isoprene metabolism and exposure.     

Differences in butadiene adduct formation between rats and mice not due to selective inhibition of CYP2E1 by butadiene metabolites

CYP2E1 metabolizes 1,3-butadiene (BD) into genotoxic and possibly carcinogenic 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EB-diol). The dose response of DNA and protein adducts derived from BD metabolites increases linearly at low BD exposures and then saturates at higher exposures in rats, but not mice. It was hypothesized that differences in adduct formation between rodents reflect more efficient BD oxidation in mice than rats. Herein, we assessed whether BD-derived metabolites selectively inhibit rat but not mouse CYP2E1 activity using B6C3F1 mouse and Fisher 344 rat liver microsomes. Basal CYP2E1 activities toward 4-nitrophenol were similar between rodents. Through IC₅₀ studies, EB was the strongest inhibitor (IC₅₀ 54 μM, mouse; 98 μM, rat), BD-diol considerably weaker (IC₅₀ 1200 μM, mouse; 1000 μM, rat), and DEB inhibition nonexistent (IC₅₀ > 25 mM). Kinetic studies showed that in both species EB and BD-diol inhibited 4-nitrophenol oxidation through two-site mechanisms in which inhibition constants reflected trends observed in IC₅₀ studies. None of the reactive epoxide metabolites inactivated CYP2E1 irreversibly. Thus, there was no selective inhibition or inactivation of rat CYP2E1 by BD metabolites relative to mouse Cyp2e1, and it can be inferred that CYP2E1 activity toward BD between rodent species would similarly not be impacted by the presence of BD metabolites. Inhibition of CYP2E1 by BD metabolites is then not responsible for the reported species difference in BD metabolism, formation of BD-derived DNA and protein adducts, mutagenicity and tumorigenesis.     

Roles of etheno-DNA adducts in tumorigenicity of olefins

Cyclic DNA adducts bearing an "etheno" structure have been described to occur after interaction with metabolites of halogenated olefins. Extensive work has been published on adducts of vinyl chloride, both in vitro and in vivo. The major DNA adduct of vinyl chloride is 7-(2-oxoethyl)guanine, but an important minor adduct appears to be N2,3-ethenoguanine. Other etheno adducts, i.e., 1, N6-ethenoadenine and 3, N4-ethenocytosine, are readily formed with DNA, vinyl chloride, and a metabolizing system in vitro and with RNA in vivo, but usually are not detected as DNA adducts in vivo. Other compounds that have been studied with respect to possible formation of etheno DNA adducts are vinyl bromide (which is more or less completely analogous to vinyl chloride), acrylonitrile, vinyl acetate and vinyl carbamate. Proposals of possible structures of DNA adducts with an etheno structure have been promutagenic potential of these lesions which may lead to misincorporation of wrong DNA bases in newly synthesized DNA.     

Reactions of formaldehyde plus acetaldehyde with deoxyguanosine and DNA: formation of cyclic deoxyguanosine adducts and formaldehyde cross-links

We investigated the reactions of formaldehyde plus acetaldehyde with dGuo and DNA in order to determine whether certain 1,N(2)-propano-dGuo adducts could be formed. These adducts-3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-(3H)-one (1) and 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-(3H)-one (3a,b)-have been previously characterized as products of the reaction of acrolein with dGuo and DNA. Adduct 1 predominates in certain model lipid peroxidation systems [Pan, J., and Chung, F. L. (2002) Chem. Res. Toxicol. 15, 367-372]. We hypothesized that this could be due to stepwise reactions of formaldehyde and acetaldehyde with dGuo, rather than by reaction of acrolein with dGuo. The results demonstrated that adducts 1 and 3a,b were relatively minor products of the reaction of formaldehyde and acetaldehyde with dGuo and that there was no selectivity in their formation. These findings did not support our hypothesis. However, substantial amounts of previously unknown cyclic dGuo adducts were identified in this reaction. The new adducts were characterized by their MS, UV, and NMR spectra as diastereomers of 3-(2'-deoxyribosyl)-6-methyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (10a,b). Adducts 10a,b were apparently formed by addition of formaldehyde to N1 of N(2)-ethylidene-dGuo, followed by cyclization. An analogous set of four diastereomers of 3-(2'-deoxyribosyl)-6,8-dimethyl-1,3,5-diazinan[4,5-a]purin-10(3H)-one (12a-d) were formed in the reactions of acetaldehyde with dGuo. These products are the first examples of exocyclic dGuo adducts of the pyrimido[1,2-a]purine type in which an oxygen atom is incorporated into the exocyclic ring. Formaldehyde-derived adducts were the other major products of the reactions of formaldehyde plus acetaldehyde with dGuo. Prominent among these were N(2)-hydroxymethyl-dGuo (9) and the cross-link di-(N(2)-deoxyguaonosyl)methane (13). We did not detect adducts 1, 3a,b, or 10a,b in enzymatic hydrolysates of DNA that had been allowed to react with formaldehyde plus acetaldehyde. However, we did detect substantial amounts of the formaldehyde cross-links di-(N(6)-deoxyadenosyl)methane (17), with lesser quantities of (N(6)-deoxyadenosyl-N(2)-deoxyguanosyl)methane (18), di-(N(2)-deoxyguanosyl)methane (13), and N(6)-hydroxymethyl-dAdo (19). Schiff base adducts of formaldehyde and acetaldehyde were also detected in these reactions. These results demonstrate that the reactions of formaldehyde plus acetaldehyde with dGuo are dominated by newly identified cyclic adducts and formaldehyde-derived products whereas the reactions with DNA result in the formation of formaldehyde cross-link adducts. The carcinogens formaldehdye and acetaldehyde occur in considerable quantities in the human body and in the environment. Therefore, further research is required to determine whether the adducts described here are formed in animals or humans exposed to these agents.     

Human in vitro glutathionyl and protein adducts of carbamazepine-10,11-epoxide, a stable and pharmacologically active metabolite of carbamazepine.

The clinical use of carbamazepine (CBZ), an anticonvulsant, is associated with a variety of idiosyncratic adverse reactions that are likely related to the formation of chemically reactive metabolites. CBZ-10,11-epoxide (CBZE), a pharmacologically active metabolite of CBZ, is so stable in vitro and in vivo that the potential for the epoxide to covalently interact with macromolecules has not been fully explored. In this study, two glutathione (GSH) adducts were observed when CBZE was incubated with GSH in the absence of biological matrices and cofactors (e.g., liver microsomes and NADPH). The chemical reactivity of CBZE was further confirmed by the in vitro finding that [14C]CBZE formed covalent protein adducts in human plasma as well as in human liver microsomes (HLMs) without NADPH. The two GSH adducts formed in the chemical reaction of CBZE were identical to the two major GSH adducts observed in the HLM incubation of CBZ, indicating that the 10,11-epoxidation represents a bioactivation pathway of CBZ. The two GSH adducts were isolated and identified as two diastereomers of 10-hydroxy-11-glutathionyl-CBZ by NMR. In addition, the covalent binding of [14C]CBZE was significantly increased in the HLM incubation upon addition of NADPH, indicating that CBZE can be further bioactivated by HLMs. To our knowledge, this is the first time the metabolite CBZE has been confirmed for its ability to form covalent protein adducts and the identity of the two CBZE-glutathionyl adducts has been confirmed by NMR. These represent important findings in the bioactivation mechanism of CBZ.     

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 and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro metabolism and DNA adduct formation from the mutagenic environmental contaminant 2-nitrofluoranthene.

The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) were studied. Incubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded trans-7,8-dihydroxy-7,8-dihydro-2-nitrofluoranthene, trans-9,10-dihydroxy-9,10-dihydro-2-nitrofluoranthene, and 7-, 8-, and 9-phenolic metabolites. When the epoxide hydrolase inhibitor 3,3,3-trichloropropylene was present in the incubation, only phenolic metabolites were detected. Under hypoxic conditions, 2-aminofluoranthene was obtained, together with a trace of the ring-oxidized metabolites. The activated metabolite, N-hydroxy-2-aminofluoranthene, was prepared in situ and reacted with calf thymus DNA. Upon enzymatic hydrolysis of the DNA and purification by HPLC, a C8-substituted deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-2-aminofluoranthene, was identified by mass and proton NMR spectral analysis. This adduct was also formed at a level of 10 pmol/mg of DNA when 2-NFA was metabolized by xanthine oxidase, 6 pmol/mg of DNA from incubation with liver microsomes of rats pretreated with 3-methylcholanthrene, and 3-pmol/mg of DNA from metabolism by liver microsomes of rats pretreated with phenobarbital.