Abstract In Vitro Cytotoxicity of Mono-, Di-, and Trichloroacetate and Modulation by Hepatic Peroxisome Proliferation. Bruschi, S. A., and Bull, R. J. (1993). Fundam. Appl. Toxicol. 21, 366-375. Dichloroacetate (DCA) and trichloroacetate (TCA) are major by-products of drinking water chlorination. Recent experiments have shown that both of these compounds produce hepatic tumors in B6C3F1 mice. There was evidence that these effects may be associated with cytotoxic effects and/or peroxisomal proliferation. Therefore, in the present study the in vitro cytotoxicity of monochloroacetate (MCA), DCA, TCA and a metabolite, glycolate (GLY), was determined in hepatocyte suspensions prepared from naive and clofibric acid-pretreated male Sprague-Dawley rats and B6C3F1 mice. Cytotoxic responses, measured by release of lactic dehydrogenase and/or trypan blue exclusion, were only observed with high concentrations (5.0 mM) of MCA and GLY in hepatocytes from naive animals ( p = 0.025 and 0.008, respectively, Sprague-Dawley rat; p = 0.033 and 0.001, respectively, B6C3F1 mouse). The cytotoxic responses to both compounds were observed much earlier and at much lower concentrations in hepatocytes taken from mice and rats that had been pretreated with clofibric acid ( p ⩽ 0.001, Sprague-Dawley rat and B6C3F1 mouse). DCA and TCA produced no evidence of cytotoxicity in hepatocytes from naive or clofibric acid-pretreated animals of either species at concentrations up to 5.0 mM. Increasing concentrations of MCA and GLY resulted in dose-related depletion of intracellular reduced glutathione (GSH) that closely paralleled the cytotoxic responses. Only GLY (0.25-5.0 mM) produced increased intracellular oxidized glutathione. Neither DCA nor TCA was found to alter cellular GSH status in hepatocytes isolated from either Sprague-Dawley rats or B6C3F1 mice. It was concluded from these in vitro observations that DCA and TCA are not highly cytotoxic to hepatocytes. Moreover, the rates of their conversion to MCA or GLY may be insufficient to induce cytotoxic effects in hepatocytes in vivo.