Modulation of Trenimon-induced Cytotoxicity by DT-Diaphorase in Isolated Rat Hepatocytes under Aerobic versus Hypoxie Conditions

Trenimon belongs to a class of aziridinylbenzoquinone anticancer drugs that cross the blood-brain barrier. In this study we have investigated the molecular mechanisms for trenimon-induced toxicity in aerobic versus hypoxic conditions with the use of freshly isolated rat hepatocytes. The following evidence suggests the mechanisms for trenimon detoxification involves reduction by DT-diaphorase, while the cytotoxic mechanism involves macromolecule alkylation under hypoxic conditions as well as oxidative stress under aerobic conditions, (a) Hepatocyte cytotoxicity induced by trenimon (250 ¿IM) under aerobic conditions ensued following an initial induction of cyanide-resistant respiration and partial oxidation of glutathione to oxidized glutathione. Trenimon reduction to the hydroquinone by the hepatocytes was rapid. Inhibition of hepatocyte DTdiaphorase by dicumarol increased trenimon-induced cytotoxicity by ap proximately 10-fold, and markedly inhibited hydroquinone formation. Furthermore, both cyanide-resistant respiration and oxidized glutathione formation were markedly increased, resulting in depletion of oxygen in the media. Trenimon reduction to the hydroquinone then occurred. This suggests that DT-diaphorase in normal hepatocytes prevents the forma tion of the semiquinone that causes cytotoxic protein alkylation and oxidative stress, (b) Hepatocyte cytotoxicity induced by trenimon (350 UM) under hypoxic conditions ensued following glutathione depletion without oxidized glutathione formation. Inactivation of hepatocyte DTdiaphorase by dicumarol under hypoxic conditions increased trenimoninduced cytotoxicity by approximately 3.5-fold and increased semiquinone radical levels 2-fold without affecting its reduction rate. This suggests that the cytotoxic mechanism involves protein alkylation by semiquinone radicals formed by reductases catalyzing a one-electron reduction of