Regio- and stereospecific DNA adduct formation in mouse lung at N

Pertti Koivisto, Ilse-Dore Adler, Francesca Pacchierotti, Kimmo Peltonen

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Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N6adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified32P-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 N6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7-dGMP and N6-dAMP adducts were formed 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 exposed 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 μg DNA, respectively. The level of N6- 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 μg DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N6-dAMP adducts was not affected. N7-dGMP adducts were 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 N6position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69±3% of the total N6adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation - Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.
Original languageEnglish
Pages (from-to)385 - 397
Number of pages13
Issue number6
Publication statusPublished - Nov 1998
Externally publishedYes


All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Clinical Biochemistry
  • Health, Toxicology and Mutagenesis

Cite this

Koivisto, P., Adler, I-D., Pacchierotti, F., & Peltonen, K. (1998). Regio- and stereospecific DNA adduct formation in mouse lung at N. Biomarkers, 3(6), 385 - 397.