© 1993 Oxford University Press
research-article |
Effects of 1,1-Dichloroethene and of Some of Its Metabolites on the Functional Viability of Mouse Hepatocytes
*Mechanisms of Drug Toxicity Research Group, Pharmaceutical Sciences Institute, Aston University Aston Triangle, Birmingham B4 7ET, United Kingdom
School of Biochemistry, University of Birmingham Birmingham B15 2TT, United Kingdom
Received January 6, 1993; accepted May 6, 1993
1,1-Dichloroethene (DCE) is hepatotoxic in rodents, and the expression of its toxicity involves probably its metabolism. In this study the role of DCE metabolites in the generation of the hepatotoxic lesion was investigated. Hepatocytes from male BALB/c mice in suspension were used as the experimental model. Cells were incubated with DCE for up to 5 hr and cellular viability was assessed by measurement of the release of lactate dehydrogenase into the medium and by alterations in the reduction of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. After incubation for 3 hr DCE at 0.5 mM caused maximal toxicity, whereas at 0.1 mM DCE was only marginally toxic. Cytotoxicity was exacerbated by pretreatment of mice with buthionine sulfoximine (1.6 g/kg), an inhibitor of glutathione biosynthesis, given 4 hr prior to hepatocyte isolation. Inclusion of N-acetylcysteine (10 mM) into the incubate protected cells against DCE-induced cytotoxicity. Coincubation with octylamine (0.5 mM), an inhibitor of cytochrome P450, abolished the cytotoxic potential of 0.5 mM DCE during incubation for 3 hr. DCE toxicity was increased in hepatocytes from mice which had received ethanol or acetone in their drinking water, both of which induce levels of the hepatic cytochrome P450 isozyme P450 2E1. Incubation of cells with the P450 2E1 inhibitors N,N-dimethylformamide (10 mM) or diethyldithiocarbamate (100 µM) protected liver cells against the detrimental effect of DCE. Pretreatment of animals with phenobarbital, which induces the P450 2B subfamily, or 3-methylcholan-threne, which induces P450 1A1, did not affect the degree of hepatocytotoxicity elicited by DCE. The DCE metabolites chloroacetic acid and dichloroacetaldehyde at 0.75 mM were toxic toward the cells; however, their toxic potency was inferior to that of DCE. Dichloroacetic acid, another product of metabolic DCE oxidation, and S-(chloroacetyl)glutathione and glutathionylacetylglutathione, both of which are generated by conjugation of DCE metabolites with glutathione, at concentrations of up to 5 mM did not interfere with hepatocyte viability. The results suggest that (i) DCE undergoes metabolic toxification in mouse hepatocytes, (ii) P450 2E1 is responsible for the metabolic activation of DCE, and (iii) conjugation with glutathione is a detoxification step.