IDENTIFICATION AND QUANTITATION OF DNA-ADDUCTS FROM CALF THYMUS DNA EXPOSED TO 3,4-EPOXY-1-BUTENE

3,4-Epoxy-1-butene (EB) is the major mutagenic metabolite of butadiene (BD), an important industrial chemical classified as a probable human carcinogen. Although the mechanism of carcinogenicity of EB is not kn own, its reactions with nucleophilic sites of DNA giving pro-mutagenic lesions are likely to constitute the early crucial step in multistage carcinogenesis. This study was conducted to characterize the adducts formed from reactions of EB with the most nucleophilic DNA nucleobases , adenine (Ade) and guanine (Gua), as free nucleobases, 2'-deoxyribonu cleosides and constituents of calf thymus DNA (CT DNA) in order to pro vide insight into the nature of DNA modification by EB. The adducts we re isolated using HPLC separation coupled with diode array detection ( DAD) and structurally characterized from their electronic, mass- and n uclear magnetic resonance spectra. Four EB-adenine products were ident ified as N-1-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade I), N-1-(1-hydro xy-3-buten-2-yl) adenine (EB-Ade II, N-3-(2-hydroxs-3-buten-1-yl) aden ine (EB-Ade III) and N-3-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade IV). Two previously reported guanine adducts: N-7-(2-hydroxy-3-buten-1-yl) guanine (EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-Gua I I) were also collected. The purified adducts were used as reference co mpounds to detect and quantitate the corresponding adduct species form ed in calf thymus DNA incubated with EB. All six adducts mere detected in treated DNA, The N-7 position of guanine was the most reactive in DNA followed by N-3 of adenine and N-1 of adenine. The formation of N- 1 and N-3-adenine adducts (EE-Ade I, 1.2 +/- 0.36; EB-Ade II, 0.8 +/- 0.27; EB-Ade III, 2.7 +/- 0.38; EB-Ade IV, 5.9 +/- 0.68 nmol/mu mol Ad e) in CT DNA was approximately one-tenth that of EB-guanine adducts (5 0.7 +/- 2.37 and 47.9 +/- 3.6 nmol/mu mol Gua, respectively). The N-1- EB-Ade adducts detected in this study are likely to be the precursors of previously reported N-6 - EB-adenine adducts (Koivisto et al., 1995 ) through Dimroth rearrangement. Since BD and EB induce significant nu mbers of point mutations at A:T base pairs, the EB-adenine adducts may represent important lesions involved in BD-induced mutagenesis and ca rcinogenesis.