3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6-5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6-2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity.
Keywords: cell resistance; cytotoxicity; oxidative stress; reductive activation; tirapazamine.