TOXICITY OF CHLOROFORM AND CARBON-TETRACHLORIDE IN PRIMARY CULTURES OF RAINBOW-TROUT HEPATOCYTES

The toxicity of two chlorinated hydrocarbons, carbon tetrachloride (CC l4) and chloroform (CHCl3) was studied in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes. The aim was to study the cyto toxicity of these two chemicals in vitro in fish hepatocytes and to ev aluate the sensitivity of the model system in comparison to mammalian systems. Both chemicals showed a steep dose response in this system wi th CCl4 being five times more toxic than CHCl3 measured as LDH release . Glutathione levels were rapidly depleted in the presence of CCl4 and CHCl3. There was a difference, however, in the pattern of GSH depleti on. CCl4-induced GSH depletion followed the release of LDH, while CHCl 3 caused a significant decrease in the GSH content prior to LDH releas e. The cytochrome P450 inhibitor, SKF-525A (12 mu M) decreased the tox icity of 5 mM CCl4 and of doses ranging from 10 to 25 mM CHCl3. The ad dition of the antioxidant DPPD (20 mu M) decreased the toxicity of bot h CCl4 and CHCl3. The protection was more pronounced with CHCl3 than w ith CCl4. When the time course of GSH depletion with EC50 concentratio ns of each chemical was studied, DPPD effectively protected the cells from CHCl3-induced GSH depletion, while no significant maintenance of GSH was seen in cells treated with DPPD and CCl4. The relationship bet ween DNA single strandbreaks (SSB) and cytotoxicity may provide some i nsight into potential carcinogenicity of non-genotoxic chemicals. In c ells treated with CHCl3, DNA SSB were seen only concommitant with high toxicity. With CCl4, however, DNA SSB were seen in the absence of LDH release at 2.5 mM. This study indicates that the toxicity of CCl4 and CHCl3 in trout hepatocytes is due to metabolism and the formation of free radicals at doses causing low toxicity and to a combination of me tabolism dependency and solvent effect at doses causing high toxicity. This study also indicates that there are differences in the mechanism s of toxicity of CHCl3 and CCl4 in rainbow trout hepatocytes. Furtherm ore, the model system based on primary cultures of rainbow trout hepat ocytes is a sensitive tool in toxicological studies and is comparable to model systems based on mammalian hepatocytes.