Conjugation of Butadiene Diepoxide with Glutathione Yields DNA Adducts in Vitro and in Vivo

Abstract

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 crosslinks. 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., Chem. Res. Toxicol. 23, 1544–1546 (2010)). 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 (kcat/Km) with all three DEB stereoisomers and the six GSH transferases. The three stereochemical DEB-GSH conjugates had similar mutagenicity. Six DNA adducts (N3-adenyl, N6-adenyl, N7-guanyl, N1-guanyl, N4-cytidyl, and N3-thymidyl) were identified in the reactions of DEB-GSH conjugate with nucleosides and calf thymus DNA using LC-MS and UV and NMR spectroscopy. N6-Adenyl and N7-guanyl GSH adducts were identified and quantitated in vivo in the livers of mice and rats treated with DEB i.p.. These results indicate that such DNA adducts are formed from the DEB-GSH conjugate, are mutagenic irregardless of sterochemistry, and therefore expected to contribute to the carcinogenicity of DEB.

Replication past the Butadiene Diepoxide-Derived DNA Adduct S-[4-(N6-Deoxyadenosinyl)-2,3-dihydroxybutyl]glutathione by DNA Polymerases

“…27 Further, the different isomers of DEB had very similar mutagenicities when their GSH conjugates were prepared and used with S. typhimurium . 27 The N 6 dA-butanetriol adduct has been reported in vivo in humans exposed to BD 50 but the stereochemistry is unknown. Individual stereoisomers of the N 6 dA-butanetriol adduct have been examined and concluded to be only very weakly mutagenic, 51 consistent with our findings with these polymerases.…”

“…25,26 Six adducts containing GSH have been identified, but only two—the N 6 dA-(OH) 2 butyl-GSH and the N 7 G-(OH) 2 butyl-GSH adducts—were detected in mice and rats. 27 Detailed studies with the N 6 -dA adducts were possible, as presented here, but studies with the N 7 -dG adducts are not currently possible because of the instability of the glycosidic bond. The labile N 7 dG adducts, susceptible to spontaneous depurination, would be expected to produce G:C to T:A transversions.…”

“…26 Six DNA adducts were identified from the reaction of DEB-GSH conjugate with free deoxyribonucleosides or calf thymus DNA and among these six adducts, N 6 dA-(OH) 2 butyl-GSH and N 7 G-(OH) 2 butyl-GSH adducts were identified and quantitated in vivo in livers of mice and rats treated with DEB. 27 …”

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

“…In fact, four histidine and two tyrosine residues present in human CYP2E1 react with EB to form adducts (Boysen et al, 2007), although their impact on activity was not investigated. As a bifunctional electrophile, DEB can modify individual residues and form intra- and inter-protein crosslinks (Cho and Guengerich, 2012), as well as protein-DNA crosslinks (Loecken et al, 2009). In this study, we did not specifically investigate the covalent modification of rodent CYP2E1 enzymes by BD metabolites; however, we did not observe any irreversible inhibition/inactivation of CYP2E1 by EB and DEB that would suggest covalent binding.…”

1,3-Butadiene-induced mitochondrial dysfunction is correlated with mitochondrial CYP2E1 activity in Collaborative Cross mice

“…The toxicity of butadiene arises from the well-characterized metabolism of butadiene by CYP2E1 to reactive epoxide metabolites (3,4-epoxy-1-butene, 1,2:3,4-diepoxybutane, 1,2-butenediol, and 1,2-epoxybutane-3,4-diol), shown in Figure 1. These epoxide metabolites have been demonstrated to form covalent adducts with glutathione, DNA, and proteins in vitro and in vivo (Boysen et al 2007a; Cho and Guengerich 2012; Georgieva et al 2010; Koivisto et al 1999; Koivisto et al 1997). While the importance of activation/metabolism by microsomal CYP2E1 has been established, less is known about the contribution of mitochondrial CPY2E1.…”