Regio- and stereospecific DNA adduct formation in mouse lung at N-6 and N7position of adenine and guanine after 1,3-butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N-6 adducts we re prepared and enriched by solid phase extraction and HPLC. The purified a dducts were analysed by a modified P-32-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N-6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7-dGMP and N-6-dAMP adducts were f ormed in a ratio of 200:1. In dGMP and in dAMP 48% and 86% of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation expose d to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8+/-0.9 fmol, 11 +/-2.0 fmol and 30+/-6.7 fmol in 10 mu g DNA, respectively. The level of N- 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/-0.06 fmol and 0.11+/-0.05 fmol in 10 mu g DNA. At 200 ppm the adduct lev el was below the detection limit. A sub-group of animals exposed to 1300 pp m was killed 3 weeks after the last exposure. N7-dGNIP adducts were not det ected but the level of N-6-dAMP adducts was not affected. N7-dGMP adducts w ere formed in a clear stereospecific manner in vivo. S-BMO adducts were the main product and represented 77% (n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N-6 position of dAMP, because of the poor separation of the enantiomers. H owever, we could separate regioisomeric adducts which indicated that C-2 ad ducts represented 69+/-3 % of the total N-6 adducts formed in mice lung DNA . This observation is supported by the data derived from in vitro DNA exper iments but is different to our previously published data, which indicates t he 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosid es. We suggest that the data presented in this communication indicate a dif ferent mechanism between nucleotides and DNA in BMO-derived adduct formatio n - Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation .