Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice

Abstract

Methylene chloride (MC) induced DNA damage in freshly isolated hepatocytes from mice and rats, which was detectable as single-strand (ss) breaks by alkaline elution. The lowest in vitro concentration of MC needed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower than 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 carcinogenic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectable 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 with hepatocytes cultured overnight in the presence of buthionine sulfoximine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjugation of MC with GSH is important in its activation of DNA-damaging species in the liver. The GSH pathway of MC metabolism produces two potential DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH2Cl). Formaldehyde is known to cause DNA ss breaks in cells. However, the lowest concentration of formaldehyde required to induce a significant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikely to be formed following in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That formaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activation 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 GSCH2Cl. 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 differences in the amount of MC metabolism via the GSH pathway in the target organ.

Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse: Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human

“…The Ni-NTA resin was treated with lysis buffer, pH 6.0 (5 bed vol.) before being washed extensively with lysis buffer containing stepwise increases of imidazole (20,30,40 and 60 mM) at pH 8.0. Finally, when no protein was detected in the eluate by absorbance at 280 nm, the recombinant protein was eluted with 8 bed vol.…”

“…Certain of the chemicals that are activated by GST are widely distributed in the environment ; there is therefore justified concern about the risk they pose to human health. For example, DCM, which is a potent hepatic and pulmonary carcinogen in mouse [20,21], is found in paint strippers, aerosol preparations and is used extensively as an industrial solvent [22].…”

The distribution of theta-class glutathione S-transferases in the liver and lung of mouse, rat and human

“…Some of these activation reactions have resulted in the development of tumours in laboratory animals. For example, methylene chloride is known to cause cancer in the lungs and livers of mice by a mechanism that involves a glutathione metabolite formed by the mouse Theta enzyme GSTT1-1 [7,9,10]. Although enzymes equivalent to this mouse enzyme are known to exist in the rat (rGSTT1-1) and in humans (hGSTT1-1), increases in lung and liver cancer are not seen in rats (or hamsters) exposed to methylene chloride [11], nor is there any evidence for an increase in cancer in exposed human populations, suggesting that either the activity or distribution of these enzymes differs markedly between species.…”

Isolation of a mouse theta glutathione S-transferase active with methylene chloride

“…of methylene chloride for 2 years an increased incidence of liver and lung tumours was seen, which was not seen in rats or hamsters exposed under the same or similar conditions [1,2]. The tumours seen in mice have been attributed to metabolites of methylene chloride produced by glutathione conjugation in a reaction catalysed by a glutathione S-transferase (GST) enzyme [3,4]. The specificity is believed to arise from the markedly higher enzyme activity found in mice than in other species including humans [5,6].…”