Diaziquone-induced cytotoxicity in isolated rat hepatocytes

2,5-Diaziridinyl-3,6-bis(carboethoxyamine)-1,4-benzoquinone (AZQ) is a lipid-soluble antitumor agent. The following evidence using isolated rat hepatocytes as a model for cytotoxicity studies suggests that, under aerobic conditions, AZQ participates in futile oxidation-reduction cycling and oxygen activation. The H2O2 formed can mediate oxidative stress cytotoxicity in compromised cells. (a) Addition of AZQ to hepatocytes causes the stoichiometric oxidation of glutathione (GSH) to oxidized glutathione. No subsequent reduction back to GSH occurred. This was found to be the result of reversible inactivation of glutathione reductase by AZQ. The extent of GSH oxidation increased with AZQ concentration. (b) Cytotoxicity occurred when AZQ concentrations were sufficient to completely deplete GSH. (c) Addition of AZQ to hepatocytes enhanced cyanide-resistant respiration. (d) If the hepatocytes were compromised with azide or cyanamide to inhibit catalase, cytotoxicity was increased 10-fold or 100-fold if ascorbate was also present. (e) AZQ readily induced Ca2+ release by energized mitochondria. Ascorbate markedly enhanced the effectiveness of AZQ, and catalase delayed Ca2+ release. H2O2 formed by aerobic oxidation-reduction cycling of AZQ may therefore cause mitochondrial Ca2+ release.