Toxicity of chloroform and carbon tetrachloride in primary cultures of rainbow trout hepatocytes

Abstract

The toxicity of two chlorinated hydrocarbons, carbon tetrachloride (CCl 4) and chloroform (CHCl 3) was studied in primary cultures of rainbow trout ( Oncorhynchus mykiss) hepatocytes. The aim was to study the cytotoxicity of these two chemicals in vitro in fish hepatocytes and to evaluate the sensitivity of the model system in comparison to mammalian systems. Both chemicals showed a steep dose response in this system with CCl 4 being five times more toxic than CHCl 3 measured as LDH release. Glutathione levels were rapidly depleted in the presence of CCl 4 and CHCl 3. There was a difference, however, in the pattern of GSH depletion. CCl 4-induced GSH depletion followed the release of LDH, while CHCl 3 caused a significant decrease in the GSH content prior to LDH release. The cytochrome P450 inhibitor, SKF-525A (12 μM) decreased the toxicity of 5 mM CCl 4 and of doses ranging from 10 to 25 mM CHCl 3. The addition of the antioxidant DPPD (20 μM) decreased the toxicity of both CCl 4 and CHCl 3. The protection was more pronounced with CHCl 3 than with CCl 4. When the time course of GSH depletion with EC 50 concentrations of each chemical was studied, DPPD effectively protected the cells from CHCl 3-induced GSH depletion, while no significant maintenance of GSH was seen in cells treated with DPPD and CCl 4. The relationship between DNA single strandbreaks (SSB) and cytotoxicity may provide some insight into potential carcinogenicity of non-genotoxic chemicals. In cells treated with CHCl 3, DNA SSB were seen only concommitant with high toxicity. With CCl 4, however, DNA SSB were seen in the absence of LDH release at 2.5 mM. This study indicates that the toxicity of CCl 4 and CHCl 3 in trout hepatocytes is due to metabolism and the formation of free radicals at doses causing low toxicity and to a combination of metabolism dependency and solvent effect at doses causing high toxicity. This study also indicates that there are differences in the mechanisms of toxicity of CHCl 3 and CCl 4 in rainbow trout hepatocytes. Furthermore, the model system based on primary cultures of rainbow trout hepatocytes is a sensitive tool in toxicological studies and is comparable to model systems based on mammalian hepatocytes.