Toxic interactions between carbon tetrachloride and chloroform in cultured rat hepatocytes

Abstract

Primary cultures of adult rat hepatocytes were incubated (1.5–16 hr) with various concentrations of CCl 4 (≤0.5 m m) and/or CHCl 3 (≤2.5 m m). Agent-dependent alterations in hepatocyte functions were assessed by measuring (1) [ 3H]choline incorporation into phosphatidylcholine (endoplasmic reticulum), (2) MTT (tetrazolium salt) reduction (mitochondria), and (3) AST release into medium (plasma membrane). Cultured hepatocytes incubated with 0.5 m m CCl 4 displayed a significant ( p ≤ 0.001) and rapid (1.5 hr) reduction (40%) in endoplasmic reticulum function that preceded significant ( p ≤ 0.001) alterations in mitochondria (6–16 hr) and plasma membrane (6–16 hr) functions. CCl 4-dependent alterations in liver cell functions are a result of CCl 4 bioactivation since metyrapone inhibits the CCl 4-mediated changes in cell functions. Response surface methods (RSM) were used to determine the influence of combinations of CCl 4 and CHCl 3 on liver cell MTT reduction and [ 3H]choline incorporation. Regression coefficients were determined for CCl 4, CHCl 3, and CCl 4-CHCl 3. All results were significant ( p < 0.0001) and implied that CCl 4 was a more potent hepatotoxin in vitro than CHCl 3. The RSM analysis also suggested that combinations of CHCl 3 and CCl 4 have greater than additive effects on MTT reduction and [ 3H]choline incorporation. These effects of CCl 4 and/or CHCl 3 on liver cell functions in vitro are consistent with liver alterations observed in vivo. Therefore, primary cultures of adult rat hepatocytes may be an appropriate model in vitro to assess the hepatotoxic potential of agents alone or in combination.