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GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE
Citation:
Ross, M. K. AND R A. Pegram. GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE. CHEMICAL RESEARCH IN TOXICOLOGY 16:216-226, (2002).
Description:
ABSTRACT
Bromodichloromethane (BDCM), a prevalent drinking water disinfection by-product, was previously shown to be mutagenic in Salmonella expressing glutathione S-transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the reaction kinetics and isoform specificity of the GST-pathway. BDCM-GSH conjugation activity in mouse liver cytosol was time- and protein-dependent; was not significantly inhibited by S-hexyl-GSH; and correlated with GST T1-1 activity toward the substrate 1,2-epoxy-3-(4?-nitrophenoxy)propane. GST T1-1 activity (but not GST alpha, mu, and pi activity) was competitively inhibited by BDCM (Kic = 4.2 mM). Compared with CH2Cl2, the catalytic efficiency (kcat/Km) of BDCM-GSH conjugation by pure rat GST T1-1 was ~3?6-fold lower. Taken together, this suggests that GST T1-1 is the primary catalyst for BDCM-GSH conjugation and that flux through this pathway is less than for CH2Cl2. The initial BDCM-GSH conjugate formed was unstable and degrades to several metabolites, including GSCH2OH, S-formyl-GSH, and HCOOH. Addition of NAD+ to cytosol did not alter the rate of BDCM-GSH conjugation; however, it did increase the amount of 14C-formate produced (~10-fold). A similar result was seen in a reaction containing pure GST T1-1 and GSH-dependent formaldehyde dehydrogenase. These findings suggest that a fraction (~10?14%) of the initial conjugate was non-enzymatically reduced to CH2Cl2. Subsequent conjugation of CH2Cl2 by GSH likely yielded formate (via S-formyl-GSH). The half-life of synthetic S-formyl-GSH in pH 7.4 buffer was ~1 h at room temperature and decreased to ~7 min in pH 9.0 buffer, and it did not react with deoxyguanosine. In conclusion, GST T1-1 conjugation of BDCM has been definitively demonstrated and the kinetics of BDCM-GSH conjugation characterized. The significance of this GST pathway is that toxic GSH conjugates can form resulting in possible formation of DNA adducts. Comparisons with CH2Cl2 suggest that the reactive intermediates specific to BDCM-GSH conjugation are more mutagenic/genotoxic than those derived from CH2Cl2.