Contribution of metabolites to carbon tetrachloride-induced hepatotoxicity in rat liver microsomes in vitro.

Abstract

Maximal incorporation of labeled carbon from 14C-CCl4 into isolated rat hepatocytes occurred 5 min after exposure to 14C-CCl4. The radioactivity (expressed as unit per milligram of protein) in the microsomal fraction was found to be higher than that in the nuclear, mitochondrial or soluble fraction of the cell. Exposure of liver microsomes of rats pretreated with phenobarbital (PB), 3-methylcholanthrene (3 MC) or polychlorinated biphenyls (PCBs) to CCl4 inactivated cytochromes P-450 and b5, and aminopyrine N-demethylase, aniline hydroxylase and glucose-6-phosphatase. The contents of the two cytochromes and the activities of the three microsomal enzyme declined to levels similar to those in CCl4-exposed microsomes from rat liver untreated with the inducers of microsomal enzymes. The contents of lipid peroxide in microsomes of rats pretreated with PB and PCBs were increased by exposure to CCl4 more than those in untreated and 3 MC-pretreated rat liver microsomes. Piperonyl butoxide, which inactivates cytochrome P-450, suppressed the destruction of cytochrome and the production of lipid peroxidation induced by CCl4, while cysteine had no effect on the reduction of the two cytochrome contents, the inactivation of the three microsomal enzymes or the production of lipid peroxide. These results suggest (1) that the species of cytochrome P-450 induced by PB and PCBs mainly participate in the biotransformation of CCl4 to trichloromethyl radical, and are involved in the inactivation of P-450 and the initiation of lipid peroxidation by CCl4, (2) that a CCl4 metabolite, probably trichloromethyl radical, causes the destruction of cytochrome P-450 and lipid peroxidation, and (3) that the formation of phosgene does not play a significant role in the inactivation of cytochrome P-450 and microsomal enzymes by CCl4.