RELATIONSHIP BETWEEN HEPATIC DNA-DAMAGE AND METHYLENE CHLORIDE-INDUCED HEPATOCARCINOGENICITY IN B6C3F1 MICE

Methylene chloride (MC) induced DNA damage in freshly isolated hepatoc ytes from mice and rats, which was detectable as single-strand (ss) br eaks by alkaline elution. The lowest in vitro concentration of MC need ed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower t han for rat hepatocytes (30 mM), and is close to the calculated steady -state concentration of MC in the mouse liver (1.6 mM) at a carcinogen ic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectabl e in hepatocyte DNA from mice which had inhaled 4000 p.p.m. MC for 6 h , but not in hepatocyte DNA from rats similarly exposed. In studies wi th hepatocytes cultured overnight in the presence of buthionine sulfox imine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjuga tion of MC with GSH is important in its activation to DNA-damaging spe cies in the liver. The GSH pathway of MC metabolism produces two poten tial DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH(2)Cl). Formaldehyde is known to cause DNA ss breaks in cells. Ho wever, the lowest concentration of formaldehyde required to induce a s ignificant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is u nlikely to be formed following,ving in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That f ormaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activati on system from mouse liver (S9 fraction). Formaldehyde was responsible for the DNA-protein cross-linking effect of MC, but did not cause the DNA damage leading to ss breaks. These DNA ss breaks are likely to be caused by GSCH(2)Cl. The results suggest a genotoxic mechanism for MC carcinogenicity in the mouse liver, and support the proposal that the observed species differences in liver carcinogenicity result from dif ferences in the amount of MC metabolism via the GSH pathway in the tar get organ.