• Induction of cytochrome P-4501A and toxicity of carbon tetrachloride and chloroform in primary cultures of rainbow trout hepatocytes

      Rabergh, Christina Maria I.; Lipsky, Michael M. (1995)
      In vitro mammalian system for the study of mechanisms of toxicity have been widely used for several years. It is, however fairly recently that in vitro models of aquatic animals have been developed and the mechanisms of toxicity of xenobiotics on the cellular level are still poorly understood. In this study a cell system based on primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes was established. The hepatocytes were cultured on polylysine coated dishes in serum-free medium. The maintenance of differentiation was studied over time in culture, by measuring the inducibility of tyrosine aminotransferase (TAT), by dexamethasone (DEX). Cells maintained TAT inducibility up to 19 days in culture. In order to further evaluate the model system, the induction of cytochrome P4501A mRNA by various xenobiotics, {dollar}\beta{dollar}-naphthoflavone (BNF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), isosafrole (ISF) and carbon tetrachloride {dollar}\rm (CCl\sb4){dollar} was studied using a CYP1A probe and Northern blotting. TCDD was shown to be the most potent inducer of CYP1A, followed by 3-MC and BNF. ISF was the weakest inducer of CYP1A. Inducibility of CYP1A was observed up to 14 days in culture. Furthermore, this study reports the first results of differential induction of two CYP1A genes in trout by using the RNase protection assay and riboprobes specific for the two genes, CYP1A1 and CYP1A2. Both genes were induced with the model substances; BNF and ISF preferentially induced CYP1A1, while TCDD showed a higher induction of CYP1A2. 3-MC induced both genes, with a preference for CYP1A1 that was less pronounced than that seen with BNF and ISF, while {dollar}\rm CCl\sb4{dollar} induced CYP1A1 exclusively. The toxicity of two chlorinated hydrocarbons, carbon tetrachloride {dollar}\rm (CCl\sb4){dollar} and chloroform {dollar}\rm (CHCl\sb3){dollar} was studied. As observed in mammals, {dollar}\rm CCl\sb4{dollar} was five times more toxic than {dollar}\rm CHCl\sb3.{dollar} In the presence of antioxidants, cells were protected from chemical induced injury at low level toxicity. Glutathione (GSH) was depleted in the presence of both chemicals but was protected by antioxidants in cells treated with {dollar}\rm CHCl\sb3.{dollar} These results indicate that fish hepatocytes are as sensitive as mammalian hepatocytes in their response to {dollar}\rm CCl\sb4{dollar} and {dollar}\rm CHCl\sb3.{dollar}