Replication past the Butadiene Diepoxide-Derived DNA Adduct <i>S</i>‑[4‑(<i>N</i>⁶‑Deoxyadenosinyl)-2,3-dihydroxybutyl]glutathione by DNA Polymerases

1,2,3,4-Diepoxybutane (DEB), a metabolite of the carcinogen butadiene, has been shown to cause glutathione (GSH)-dependent base substitution mutations, especially A:T to G:C mutations in <i>Salmonella typhimurium</i> TA1535 [Cho, S. H., et al. (2010) <i>Chem. Res. Toxicol. 23</i>, 1544] and <i>Escherichia coli</i> TRG8 cells [Cho, S. H., and Guengerich, F. P. (2012) <i>Chem. Res. Toxicol. 25</i>, 1522]. We previously identified <i>S</i>-[4-(<i>N</i>⁶-deoxyadenosinyl)-2,3-dihydroxybutyl]­GSH [<i>N</i>⁶dA-(OH)₂butyl-GSH] as a major adduct in the reaction of <i>S</i>-(2-hydroxy-3,4-epoxybutyl)­glutathione (DEB-GSH conjugate) with nucleosides and calf thymus DNA and <i>in vivo</i> in livers of mice and rats treated with DEB [Cho, S. H., and Guengerich, F. P. (2012) <i>Chem. Res. Toxicol. 25</i>, 706]. For investigation of the miscoding potential of the major DEB-GSH conjugate-derived DNA adduct [<i>N</i>⁶dA-(OH)₂butyl-GSH] and the effect of GSH conjugation on replication of DEB, extension studies were performed in duplex DNA substrates containing the site-specifically incorporated <i>N</i>⁶dA-(OH)₂butyl-GSH adduct, <i>N</i>⁶-(2,3,4-trihydroxybutyl)­deoxyadenosine adduct (<i>N</i>⁶dA-butanetriol), or unmodified deoxyadenosine (dA) by human DNA polymerases (Pol) η, ι, and κ, bacteriophage polymerase T7, and <i>Sulfolobus solfataricus</i> polymerase Dpo4. Although dTTP incorporation was the most preferred addition opposite the <i>N</i>⁶dA-(OH)₂butyl-GSH adduct, <i>N</i>⁶dA-butanetriol adduct, or unmodified dA for all polymerases, the dCTP misincorporation frequency opposite <i>N</i>⁶dA-(OH)₂butyl-GSH was significantly higher than that opposite the <i>N</i>⁶dA-butanetriol adduct or unmodified dA with Pol κ or Pol T7. LC–MS/MS analysis of full-length primer extension products confirmed that Pol κ or Pol T7 incorporated the incorrect base C opposite the <i>N</i>⁶dA-(OH)₂butyl-GSH lesion. These results indicate the relevance of GSH-containing adducts for the A:T to G:C mutations produced by DEB

Conjugation of Butadiene Diepoxide with Glutathione Yields DNA Adducts in Vitro and in Vivo

1,2,3,4-Diepoxybutane (DEB) is reported to be the most potent mutagenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant. DEB is capable of inducing the formation of monoalkylated DNA adducts and DNA–DNA and DNA–protein cross-links. We previously reported that DEB forms a conjugate with glutathione (GSH) and that the conjugate is considerably more mutagenic than several other butadiene-derived epoxides, including DEB, in the base pair tester strain <i>Salmonella typhimurium</i> TA1535 [Cho (2010) Chem. Res. Toxicol. 23, 1544−1546]. In the present study, we determined steady-state kinetic parameters of the conjugation of the three DEB stereoisomers<i>R</i>,<i>R</i>, <i>S</i>,<i>S</i>, and <i>meso</i> (all formed by butadiene oxidation)with GSH by six GSH transferases. Only small differences (<3-fold) were found in the catalytic efficiency of conjugate formation (<i>k</i>_cat/<i>K</i>_m) with all three DEB stereoisomers and the six GSH transferases. The three stereochemical DEB–GSH conjugates had similar mutagenicity. Six DNA adducts (<i>N</i>³-adenyl, <i>N</i>⁶-adenyl, <i>N</i>⁷-guanyl, <i>N</i>¹-guanyl, <i>N</i>⁴-cytidyl, and <i>N</i>³-thymidyl) were identified in the reactions of DEB–GSH conjugate with nucleosides and calf thymus DNA using LC-MS and UV and NMR spectroscopy. <i>N</i>⁶-Adenyl and <i>N</i>⁷-guanyl GSH adducts were identified and quantitated in vivo in the livers of mice and rats treated with DEB ip. These results indicate that such DNA adducts are formed from the DEB–GSH conjugate, are mutagenic regardless of sterochemistry, and are therefore expected to contribute to the carcinogenicity of DEB