Conjugation of Haloalkanes by Bacterial and Mammalian Glutathione Transferases:  Mono- and Vicinal Dihaloethanes

Abstract

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.

Metabolism and Mutagenicity of Source Water Contaminants 1,3-Dichloropropane and 2,2-Dichloropropane

“…Interestingly, neither S,SЈ-propyl-bis(GSH) nor S-(3-hydroxypropyl)GSH was detected in reaction mixtures containing 1,3-DBP or 1,3-DCP following a 1-h reaction. This was in marked contrast to GSH reactions with 1,2-dihaloethanes, which produced abundant amounts of both S,SЈ-ethylene-bis(GSH) and S-(2-hydroxyethyl)GSH (Cmarik et al, 1990;Wheeler et al, 2001b). The half-mustards (GSCH 2 CH 2 X) were not detectable because they rearrange to a highly reactive episulfonium ion that is rapidly hydrolyzed or, alternatively, can react with cellular nucleophiles such as GSH or DNA (Peterson et al, 1988).…”

“…These secondary reactions are not catalyzed by GST enzyme (Wheeler et al, 2001b). The half-lives of S-(2-chloroethyl)GSH and S-(2-bromoethyl)GSH in aqueous buffer were previously estimated to be 5.3 and 0.44 min, respectively (Wheeler et al, 2001b), and this gave direct evidence of the instabilities of these half-mustards. Furthermore, formation of a sulfonium ion intermediate derived from GSH conjugation of 1,3-dihalopropanes is unlikely to be an energetically favored pathway (Smit et al, 1979).…”

“…The half-mustards (GSCH 2 CH 2 X) were not detectable because they rearrange to a highly reactive episulfonium ion that is rapidly hydrolyzed or, alternatively, can react with cellular nucleophiles such as GSH or DNA (Peterson et al, 1988). These secondary reactions are not catalyzed by GST enzyme (Wheeler et al, 2001b). The half-lives of S-(2-chloroethyl)GSH and S-(2-bromoethyl)GSH in aqueous buffer were previously estimated to be 5.3 and 0.44 min, respectively (Wheeler et al, 2001b), and this gave direct evidence of the instabilities of these half-mustards.…”

“…Thus, this GST pathway may be considered a detoxication mechanism for 1,3-DCP. This reduced reactivity may account for the much lower mutagenic activity of 1,3-dihalopropane, compared with 1,2-dihaloethanes, in S. typhimurium that express rat or human glutathione transferase T1-1 (Thier et al, 1996;Wheeler et al, 2001b).…”

“…GSTs catalyze the conjugation of glutathione (GSH) with numerous haloalkanes to form potentially genotoxic compounds (Thier et al, 1993;Hallier et al, 1994;Wheeler et al, 2001a,b). With respect to 1,2-dihaloethanes, the GST pathway is regarded as the principal mechanism of activation and mutagenesis (Wheeler et al, 2001b). The mutagenicity of 1,2-dichoroethane has been demonstrated in Salmonella TA1535 supplemented with rat liver cytosol and GSH, but not with rat liver microsomes and NADPH (Guengerich et al, 1980).…”