Glutathione transferase theta 1-1-dependent metabolism of the water disinfection byproduct bromodichloromethane.

Abstract

Bromodichloromethane (CHBrCl(2)), a prevalent drinking water disinfection byproduct, was previously shown to be mutagenic in Salmonella that express rat GSH transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the kinetics of CHBrCl(2) reactions mediated by GST in different species as well as the isoform specificity and reaction products of the GST pathway. Conjugation activity of CHBrCl(2) with GSH in mouse liver cytosol was time- and protein-dependent, was not inhibited by the GST alpha, mu and pi inhibitor S-hexyl-GSH, and correlated with GST T1-1 activity toward the substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. Conjugation activities in hepatic cytosols of different species toward CHBrCl(2) followed the order mouse > rat > human. As compared with CH(2)Cl(2), the catalytic efficiency (k(cat)/K(m)) of conjugation of CHBrCl(2) with GSH by pure recombinant rat GST T1-1 was approximately 3-6-fold less. Taken together, this suggests that GST T1-1 is the primary catalyst for conjugation of CHBrCl(2) with GSH and that flux through this pathway is less than for CH(2)Cl(2). The initial GSCHCl(2) conjugate formed was unstable and degraded to several metabolites, including GSCH(2)OH, S-formyl-GSH, and HCOOH. Addition of NAD(+) to cytosol did not alter the rate of conjugation of CHBrCl(2) with GSH; however, it did increase the amount of [(14)C]HCOOH produced ( approximately 10-fold). A similar result was seen in a reaction containing pure rat GST T1-1 and GSH-dependent formaldehyde dehydrogenase, indicating that GSCH(2)OH was formed as a precursor to S-formyl-GSH. The half-life of synthetic S-formyl-GSH in pH 7.4 buffer was approximately 1 h at ambient temperature and decreased to approximately 7 min in pH 9.0 buffer, and it does not react with deoxyguanosine. In conclusion, GST T1-1 conjugation of CHBrCl(2) has been definitively demonstrated and the kinetics of conjugation of CHBrCl(2) with GSH characterized in mouse, rat, and human hepatic cytosols. The significance of this GST pathway is that reactive GSH conjugates are produced resulting in possible formation of DNA adducts. Comparisons with CH(2)Cl(2) suggest that the reactive intermediates specific to GSH conjugation of CHBrCl(2) are more mutagenic/genotoxic than those derived from CH(2)Cl(2).