Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial andmammalian dichloromethane-active glutathione S-transferases

The kinetic properties of bacterial and rat liver glutathione S-transferase s (GST) active with dichloromethane (DCM) were compared. The theta class gl utathione S-transferase (rGSTT1-1) from rat liver had an affinity for dihal omethanes lower by three orders of magnitude (K-app > 50 mM) than the bacte rial DCM dehalogenase;GST from Methylophilus sp. DM11. Unlike the bacterial DCM dehalogenase, the rat enzyme was unable to support growth of the dehal ogenase minus Methylobacterium sp. DM4-2cr mutant with DCM. Moreover, the p resence of DCM inhibited growth with methanol of the DM4-2cr transconjugant expressing the rat liver GSTT1-1. In Salmonella typhimurium TA1535, expres sion of rat and bacterial DCM-active GST from a plasmid in the presence of DCM yielded up to 5.3 times more reversions to histidine prototrophy in the transconjugant expressing the rat enzyme. Under the same conditions, howev er, GST-mediated conversion of DCM to formaldehyde was lower in cell-free e xtracts of the transconjugant expressing the rat GSTT1 than in the correspo nding strain expressing the bacterial DCM dehalogenase. This provided new e vidence that formaldehyde was not the main toxicant associated with GST-med iated DCM conversion, and indicated that an intermediate in the transformat ion of DCM by GST, presumably S-chloromethylglutathione, was responsible fo r the observed effects. The marked differences in substrate affinity of rat and bacterial DCM-active GST, as well as in the toxicity and genotoxicity associated with expression of these enzymes in bacteria, suggest that bacte rial DCM dehalogenases/GST have evolved to minimise the toxic effects assoc iated with glutathione-mediated catalysis of DCM conversion.