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Primary cultures of rat hepatocytes were used to explore the mechanisms of the toxicity of aryl halides. The sensitivity of the hepatocytes to chloro-, bromo-, and iodobenzene was enhanced by inhibition of glutathione reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In each case, the increased cell killing depended on the metabolism of the toxicant, a result shown by the protective effect of SKF-525A, an inhibitor of mixed function oxidation. BCNU decreased the metabolism of [14C]bromobenzene and the covalent binding of its metabolites by 20%. Chelation by deferoxamine of a cellular source of ferric iron prevented the cell killing in the presence or absence of BCNU. Deferoxamine had no effect on the metabolism or the covalent binding of [14C]bromobenzene. Similarly, the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) reduced the cell killing and had no effect on the metabolism of [14C]bromobenzene. Thus, the toxicity of the three aryl halides was manipulated in ways that modify the sensitivity of hepatocytes to an oxidative stress, and the changes in cell killing occurred without parallel changes in the metabolism of [14C]bromobenzene or the covalent binding of its metabolites.

Rats treated with a single 0.5 ml/kg dose (ip) of CCl4 exhibited a threefold increase in liver microsomal phospholipase C (PLC) activity that was enhanced by phenobarbital and diminished by metyrapone pretreatment, respectively. Hepatocytes and hepatocellular fractions exposed to 0.5 mM CCl4 in vitro also exhibited a rapid rise in PLC activity that was reduced by metyrapone. Metyrapone also reduced the CCl4-related increase in the PLC-mediated reductions in cellular phosphatidylcholine content. The influence of CCl4 biotransformation on the activation of liver cell PLC was assessed in vitro. Covalent binding of 14CCl4 metabolites to isolated hepatocyte proteins and lipids was linear through 20 min of incubation and then quickly plateaued. The association of CCl4 metabolites with cellular phospholipids was inhibited by metyrapone and preceded the CCl4-dependent rise in PLC activity. CCl4-mediated increases in PLC activity were rapid and preceded reductions in cell viability. The translocation of cytosolic PLC to membranes such as the endoplasmic reticulum may explain the rapid, metabolite-dependent activation of PLC.PLC activation by haloalkanes was proportional to dose and incubation time in the order of CBrCl3 greater than CCl4 greater than CHCl3 greater than CFCl3 which corresponds to the observed hepatotoxic potential of these agents in vivo and in vitro. Haloalkane-dependent increases in PLC activity were inhibited by metyrapone. These results suggest that chemical metabolites activate PLC in vitro and in vivo. Therefore, the activation of a PLC that degrades membrane phospholipids may represent an important step in the pathogenic scheme of chemical-mediated liver cell necrosis.

Carbon tetrachloride (CCl4) biotransformation and covalent binding was measured in 1000g liver fractions by determining the amount of 14CCl4 metabolites covalently bound to proteins and lipids at various (5-60 min) incubation times. Reactive intermediate binding to proteins and phospholipids peaked at 20 min, whereas CCl4 metabolites associated with neutral lipids (primarily diacylglycerol) were initially low (0-15 min) and then gradually increased from 20 to 60 min. The rise in labeled diacylglycerol was associated with a decrease in phospholipids containing covalently bound CCl4 metabolites, since CCl4 bioactivation increased phospholipase C (PLC) activity three- to fourfold. The major rise in PLC activity occurred after the plateau of CCl4 metabolite binding to cellular phospholipids. In contrast, when CCl4 bioactivation is absent, 0.5 mM CCl4 has little effect on PLC activity. At CCl4 concentrations of 1 mM and greater, the NADPH-dependent activation of PLC by CCl4 is reduced because CCl4 biotransformation is inhibited. Nevertheless, PLC is still activated by CCl4; however, PLC activation by high doses of CCl4 occurs by bioactivation-independent mechanisms. Therefore, there are two components of CCl4-induced PLC activation: one which is dependent on CCl4 biotransformation and one which is not. Under both conditions (+/- biotransformation), the activation of PLC may be a key event in CCl4 hepatotoxicity since PLC disrupts the functional and structural integrity of membranes by degrading membrane phospholipids.