Enzymatic single-electron reduction and aerobic cytotoxicity of tirapazamine and its 1-oxide and nor-oxide metabolites

Aerobic cytotoxicity of 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ), a bioreductively activated hypoxia-specific anticancer agent, is responsible for TPZ side effects in chemotherapy. In order to clarify its mechanisms, we examined the aerobic cytotoxicity of TPZ and its main metabolites, 3-amino-1,2,4-benzotriazine-1-oxide and 3-amino-1,2,4-benzotriazine in murine hepatoma MH22a cells, and their reduction by NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP+ reductase (FNR). Analogous studies of several quinones and nitroaromatic compounds with similar values of single-electron reduction midpoint potentials (E17) were carried out. In single-electron reduction by P-450R and FNR, the reactivity of TPZ and its monoxide was similar to that of quinones and nitroaromatics, and increased with an increase in their E17. The cytotoxicity of TPZ and its metabolites possessed a prooxidant character, because it was partly prevented by an antioxidant N,N’-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. Importantly, the cytotoxicity of TPZ and, possibly, its 1-N-oxide, was much higher than that of quinones and nitroaromatics with similar values of E17 and redox cycling activities. A possible additional factor in the aerobic cytotoxicity of TPZ is its reductive activation in oxygen-poor cell nuclei, leading to the formation of DNA-damaging species similar to those forming under hypoxia.