Toxic interactions between carbon tetrachloride and chloroform in cultured rat hepatocytes

Primary cultures of adult rat hepatocytes were incubated (1.5-16 hr) with various concentrations of CCl4 (less than or equal to 0.5 mM) and/or CHCl3 (less than or equal to 2.5 mM). 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 mM CCl4 displayed a significant (p less than or equal to 0.001) and rapid (1.5 hr) reduction (40%) in endoplasmic reticulum function that preceded significant (p less than or equal to 0.001) alterations in mitochondria (6-16 hr) and plasma membrane (6-16 hr) functions. CCl4-dependent alterations in liver cell functions are a result of CCl4 bioactivation since metyrapone inhibits the CCl4-mediated changes in cell functions. Response surface methods (RSM) were used to determine the influence of combinations of CCl4 and CHCl3 on liver cell MTT reduction and [3H]choline incorporation. Regression coefficients were determined for CCl4, CHCl3, and CCl4-CHCl3. All results were significant (p less than 0.0001) and implied that CCl4 was a more potent hepatotoxin in vitro than CHCl3. The RSM analysis also suggested that combinations of CHCl3 and CCl4 have greater than additive effects on MTT reduction and [3H]choline incorporation. These effects of CCl4 and/or CHCl3 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.