MOUSE-LIVER GLUTATHIONE-S-TRANSFERASE MEDIATED METABOLISM OF METHYLENE-CHLORIDE TO A MUTAGEN IN THE CHO HPRT ASSAY/

Although methylene chloride (MC) is readily detectable as a bacterial mutagen, published studies in mammalian cells have been inconclusive. We have previously shown (Graves et al., 1995) that glutathione S-tran sferase (GST)-mediated metabolism of MC by mouse liver cytosol (S100 f raction) causes DNA single-strand (ss) breaks in CHO cells. In this st udy, MC GST metabolites were shown to cause mutations at the HPRT locu s of CHO cells. The mutagenicity of MC was enhanced by exposing the ce lls in suspension rather than as attached cultures. The MC GST metabol ite formaldehyde was mutagenic in independent experiments, although th e number of mutants induced was lower than with the MC. CHO HPRT mutat ions were also induced by the reference genotoxin 1,2-dibromoethane (1 ,2-DBE), which is activated to a mutagen by GST-mediated metabolism. A ssay of DNA ss breaks and DNA-protein cross-links at mutagenic concent rations of MC, formaldehyde or 1,2-DBE, showed that all three compound s induced DNA ss breaks, but only formaldehyde induced significant DNA -protein cross-linking. These results suggest that whilst formaldehyde may play a role in MC mutagenesis, its weak mutagenicity and the abse nce of significant DNA-protein cross-linking after MC exposure, leads to the conclusion that the MC DNA damage and resulting mutations are i nduced by the glutathione conjugate of MC, S-chloromethylglutathione.