CCl4-induced toxicity in isolated hepatocytes: the importance of direct solvent injury.
CCl4 is proposed to induce cellular injury through its metabolites that are generated by a cytochrome P-450 dependent step. These free radical products can interact with membrane structures, thereby generating lipid peroxides. The latter process has been implicated as a major mechanism of CCl4 hepatoxicity, although this relationship has been difficult to demonstrate when using isolated hepatocyte preparations. This report demonstrates that there are at least two mechanisms by which CCl4 induces injury in isolated hepatocytes. One occurs within minutes of exposure to CCl4 and is characterized by modest malondialdehyde formation and no decline in cellular-reduced glutathione. SKF 525A, metyrapone and promethazine did not protect against this early damage. A second phase of damage, evident particularly after 3 hr, is characterized by a marked increase in malondialdehyde formation, a fall in cellular glutathione and substantial further cellular damage. These changes could be moderated by the cytochrome P-450 inhibitors and promethazine, and antioxidant. Further examination of the initial phase of damage reveals an immediate dose-related inhibition of O2 consumption. This could not be prevented by SKF 525A or metyrapone and was associated with loss of ability to stimulate mitochondrial respiration with dinitrophenol. Rapid recovery to initial O2 consumption rates occurred with time as CCl4 evaporated from the incubation system. This was associated with a partial return of dinitrophenol stimulation of mitochondrial O2 consumption despite significant glutamate dehydrogenase release. A portion of this recovery could be inhibited by SKF 525A, suggesting that some O2 consumption was due to CCl4 metabolism and ensuing lipid peroxidation. These data suggest that early CCl4 toxicity is a direct consequence of its solvent properties and is partially reversible; subsequent damage may be mediated by lipid peroxidation. This solvent injury has not been previously recognized and may have relevance not only to a reversible toxicity as demonstrated with isolated hepatocytes but also in vivo as well.