Inhalation exposure of rats and mice to 1,3-butadiene induces N-6-adenine adducts of epoxybutene detected by P-32-postlabeling and HPLC

In this paper we report DNA binding of butadiene monoepoxide, a first metabolite of 1,3-butadiene catalyzed by monooxygenases. We prepared alkylated purines as marker compounds for P-32-postlabeling and electrochemical analysis and developed methods to measure the corresponding products. The traditional postlabeling assay was modified by incorporating a solid phase extraction column and high-performance liquid chromatography (HPLC) enrichment steps to the assay prior to labeling. The final analysis of adducted N-6 adenines is based on two dimensional thin-layer chromatography (TLC) and an on-line HPLC/radioactivity analysis. The qualitative and quantitative results are based on positively identified marker compounds. Alkylated N-7 guanines were released from DNA by neutral thermal hydrolysis, prepurified by HPLC, and analyzed by HPLC with a sensitive electrochemical detection procedure. By using these methods, we found alkylation of calf thymus DNA exposed to butadiene monoepoxide in vitro at adenine N-6 and guanine N-7 sites. Analysis of lung DNA samples from mice and rats exposed to butadiene through inhalation showed that adenine N-6 adducts were formed in vivo in a dose responsive manner.