1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. BD is metabolically activated by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA to form a range of nucleobase adducts. Among these, the most abundant are the hydrolytically labile N7-guanine adducts such as N7-hydroxybuten-1-yl-dG (N7-EB-dG). We now report that N7-EB-dG can be converted to the corresponding ring open N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-(2-hydroxybut-3-en-1-yl)-formamidopyrimidine (EB-Fapy-dG) adducts. We for the first time report the synthesis of EB-Fapy-dG and the EB-Fapy-dG lesions were detected in EB-treated calf thymus DNA and in EB-treated mammalian cells using quantitative isotope dilution nano HPLC-NSI-MS/MS. EB-Fapy-dG adduct formation in EB-treated calf thymus DNA was concentration dependent and was greatly accelerated at increased pH. EB-FAPy-dG adduct amounts were 2-fold higher in BER deficient NEIL1-/- mouse embryonic fibroblasts (MEF) as compared to isogenic controls (NEIL1+/+), suggesting that this lesion may be a substrate for NEIL1. However, NEIL1-/- cells were only slightly sensitized to EB as compared to NEIL1+/+ fibroblasts, suggesting that EB-FAPy-dG lesions are not the main cytotoxic species. To elucidate the biological effects of EB-FAPy lesions, they were site-specifically incorporated into DNA oligomers. Overall, our results indicate that ring open EB-FAPy-dG adducts do form under physiological conditions, prompting future studies to determine their contributions to genotoxicity and mutagenicity of 1,3-butadiene.

EB-Fapy-dG adducts of 1,3-butadiene: Synthesis, structural identification, and detection in human cells

256th National Meeting (2018)

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