Detection of sulfur mustard-induced DNA modifications

Abstract

Sulfur mustard is acutely toxic to the skin, eyes, and respiratory tract, and is considered carcinogenic to humans by the IARC. Since all of these toxicities are thought to be initiated by DNA alkylation, the level of DNA damage should serve as a biomarker for exposure. To develop methods of detecting this damage, DNA was modified by [ 14C]-labeled sulfur mustard and DNA adducts were released by mild acid hydrolysis. Radioactivity co-eluted on HPLC analysis with marker 7-(2-hydroxyethylthioethyl) guanine and 3-(2-hydroxyethylthioethyl) adenine synthesized from 2-chloroethyl 2-hydroxy-ethyl sulfide. Unambiguous identification of the major adduct, 7-(2-hydroxy-ethylthioethyl) guanine, was provided by gas chromatography combined with mass spectrometric detection. The most abundant adduct, 7-(2-hydroxyethyl-thioethyl) guanine, accounted for 61% of the total alkylation and could be detected as a fluorescent HPLC peak with a detection limit of 10 pmol. To demonstrate the applicability of this method to biological samples, DNA was extracted from the white blood cells of human blood exposed to 131 μM sulfur mustard in vitro and shown to contain 470 pmol of 7-(2-hydroxyethylthio-ethyl) guanine per mg of DNA.