Characterization and Quantification of Cysteinyl Adducts of Benzene Diol Epoxide

Abstract

The production of macromolecular adducts of benzene diol epoxide (BDE), a toxic metabolite of benzene, has received little attention despite the demonstrated mutagenicity and carcinogenicity of BDE in rodents. Syn and anti enantiomers of BDE were relatively stable in 0.1 M ammonium acetate buffer, pH 7.6 (half times were greater than 5 h), and showed evidence of pseudo-first-order reactions with albumin (half times were about 4 h) and glutathione (GSH) (half times were about 0.3-0.4 h). Reaction products of BDE isomers with l-cysteine, N-acetyl-l-cysteine, N-acetyl-l-cysteine methyl ester, and GSH were characterized by a combination of electrospray ionization mass spectrometry and/or gas chromatography-mass spectrometry with electron impact ionization of trimethylsilyl derivatives of the adducts. Products corresponded to 1:1 addition of BDE isomers with each nucleophilic species, suggesting that adduction occurred primarily at the free sulfhydryl group. To investigate the disposition of the BDEs in vivo, we developed an assay for cysteinyl BDE-protein adducts. The assay involves enzymatic hydrolysis of the protein followed by derivatization of the released adducts and gas chromatography-negative ion chemical ionization-mass spectrometry. Preliminary applications of the assay showed linear increases in the formation of BDE-GSH adducts in samples of GSH incubated with increasing concentrations of BDE (10-300 microM) and showed the presence of BDE-albumin following incubation of albumin with 10 microM BDE.