The in vitro effects and metabolism-mediated cytotoxicity of phorone, a glutathione-depleting agent

The use of S-9 liver fractions to examine metabolism-mediated cytotoxicity in vitro has the inherent problem that not only are activating enzyme systems added, but also deactivation pathways, such as that involving glutathione. The in vivo manipulation of these activating and deactivating systems prior to S-9 preparation is possible with animals, but not with humans. Hence, the possibility of depleting glutathione in target cells and S-9 fractions was evaluated using phorone, a known glutathione-depleting agent. 3T3-L1 mouse fibroblast-like cells and V79 Chinese hamster lung fibroblasts were used as the target cells, and cytotoxicity was assessed by the FRAME (Fund for the Replacement of Animals in Medical Experiments) kenacid blue method. Phorone was found to have a moderate intrinsic cytotoxicity and to effectively deplete cellular glutathione. When phorone was used in the two-component S-9 fraction/target cell system, its toxicity to 3T3-L1 cells was markedly increased, which suggests transformation to a toxic metabolite. The use of S-9 fractions from animals pretreated with phenobarbitone and beta-naphthoflavone resulted in greatly increased phorone toxicity, which indicates the involvement of cytochrome P-450 enzymes in its metabolism. The metabolism-mediated toxicity of phorone was reduced by the addition of exogenous glutathione.