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A primary route of metabolism of dihalomethanes occurs via glutathione (GSH) transferase-catalyzed conjugation. Mammalian theta class GSH transferases and a group of bacterial dichloromethane dehalogenases are able to catalyze the hydrolytic dehalogenation of dihalomethanes via GSH conjugation and subsequent formation of HCHO. Dihalomethanes have been shown to induce revertants in Salmonella typhimurium TA 1535 expressing theta class GSH transferases. Two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and the bacterial dehalogenase DM11 were compared in the in vitro conjugation of CH(3)Cl and using in vitro assays (HCHO formation) and the S. typhimurium mutagenesis assay with the dihalomethanes CH(2)Cl(2), CH(2)Br(2), CH(2)BrCl, CH(2)ICl, CH(2)I(2), and CH(2)ClF. GSTs 5-5 and T1 had similar characteristics and exhibited first-order rather than Michaelis-Menten kinetics for HCHO formation over the range of dihalomethane concentrations tested. In contrast, the DM11 enzyme displayed typical hyperbolic Michaelis-Menten kinetics for all of the compounds tested. A similar pattern was observed for the conjugation of CH(3)Cl. The reversion tests with S. typhimurium expressing DM11 or GST 5-5 showed a concentration-dependent increase in revertants for most of the dihalomethanes, and DM11 produced revertants at dihalomethane concentrations lower than GST 5-5. Collectively, the results indicate that rates of conversion of dihalomethanes to HCHO are not correlated with mutagenicity and that GSH conjugates are genotoxic. The results are compared with the conjugation and genotoxicity of haloethanes in the preceding paper in this issue [Wheeler, J. B., Stourman, N. V., Armstrong, R. N., and Guengerich, F. P. (2001) Chem. Res. Toxicol. 14, 1107-1117]. The halide order appears most important in the dihalomethane conjugation reactions catalyzed by GST 5-5 and less so in GST T1 and DM11, probably due to changes in the rate-limiting steps.

Glutathione (GSH) transferases are generally involved in the detoxication of xenobiotic chemicals. However, conjugation can also activate compounds and result in DNA modification. Activation of 1,2-dihaloethanes (BrCH(2)CH(2)Br, BrCH(2)CH(2)Cl, and ClCH(2)CH(2)Cl) was investigated using two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and a bacterial dichloromethane dehalogenase (DM11). Although the literature suggests that the bacterial dehalogenase does not catalyze reactions with CH(3)Cl, ClCH(2)CH(2)Cl, or CH(3)CHCl(2), we found a higher enzyme efficiency for DM11 than for the mammalian GSH transferases in conjugating CH(3)Cl, CH(3)CH(2)Cl, and CH(3)CH(2)Br. Enzymatic rates of activation of 1,2-dihaloethanes were determined in vitro by measuring S,S-ethylene-bis-GSH, the major product trapped by nonenzymatic reaction with the substrate GSH. Salmonella typhimurium TA 1535 systems expressing each of these GSH transferases were used to determine mutagenicity. Rates of formation of S,S-ethylene-bis-GSH by the GSH transferases correlated with the mutagenicity determined in the reversion assays for the three 1,2-dihaloethanes, consistent with the view that half-mustards are the mutagenic products of the GSH transferase reactions. Half-mustards [S-(2-haloethyl)GSH] containing either F, Cl, or Br (as the leaving group) were tested for their abilities to induce revertants in S. typhimurium, and rates of hydrolysis were also determined. GSH transferases do not appear to be involved in the breakdown of the half-mustard intermediates. A halide order (Br > Cl) was observed for both GSH transferase-catalyzed mutagenicity and S,S-ethylene-bis-GSH formation from 1,2-dihaloethanes, with the single exception (both assays) of BrCH(2)CH(2)Cl reaction with DM11, which was unexpectedly high. The lack of substrate saturation seen for conjugation of dihalomethanes with GSTs 5-5 and T1 was also observed with the mono- and 1,2-dihaloethanes [Wheeler, J. B., Stourman, N. V., Thier, R., Dommermuth, A., Vuilleumier, S., Rose, J. A., Armstrong, R. N., and Guengerich, F. P. (2001) Chem. Res. Toxicol. 14, 1118-1127], indicative of an inherent difference in the catalytic mechanisms of the bacterial and mammalian GSH transferases.

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