ADENINE ADDUCTS WITH DIEPOXYBUTANE - ISOLATION AND ANALYSIS IN EXPOSED CALF THYMUS DNA

1,3-Butadiene (ED) is a high-volume industrial chemical and a common e nvironmental pollutant. Although ED is classified as a ''probable huma n carcinogen'', only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of E D compared to rats (approximate to 10(3)-fold), making interspecies ex trapolations difficult. Identification and quantitation of specific ED -induced DNA adducts are important for improving our understanding of the mechanisms of ED biological effects and for explaining the observe d species differences. Covalent binding of ED to DNA is probably due t o its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepo xybutane (DEB). Both EE and DEB are direct mutagens producing frameshi ft and point mutations at both A:T and G:C base pairs. DEB is 100 time s more mutagenic than EB and is found in quantity only in tissues of t he most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to ED could be due to greater exposure to DEB. The present work was initiated in order to isolate and struct urally characterize DEB-induced adenine adducts. The adducts were form ed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo ), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectr ometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hyd roxy-3',4'-epoxybut-1'-yl)ade These adducts underwent acid-catalyzed h ydrolysis to their corresponding (2',3', 4'-trihydroxybut-1'-yl)adenin es upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N-6-(2',3',4'-trihydroxybut-1 '-yl)adenine (NG-DEB-Ade). N-3-DEE-Ade and N-6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3 -DEB-Ade (13/10(3) normal Ade) and N-6-DEB-Ade (5/10(3) normal Ade) we re slightly lower than those of the corresponding EB-induced adducts i n similar experiments, suggesting comparable reactivity of the two epo xy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of ED-induced adenyl DNA adducts in vitro and in vivo.