Metabolism of tirapazamine by multiple reductases in the nucleus.

Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide, SR4233, Tirazone), a bioreductive drug currently in clinical trials, is selectively toxic to hypoxic cells commonly found in solid tumors. The toxicity results from the intracellular metabolism of TPZ to a highly toxic radical. When oxygen levels are low, the TPZ radical reacts with cellular molecules, producing DNA damage and cell death. The much lower toxicity towards aerobic cells results from the back-oxidation of the TPZ radical by oxygen. A major unresolved aspect of the mechanism of TPZ is the identity of the reductase(s) in the cell responsible for activating the drug to its toxic form. We have studied both the metabolism of the drug using HPLC and the formation of the TPZ radical with a fluorescence assay using dihydrorhodamine 123. We also measured DNA double- and single-strand breaks produced by TPZ, using the comet assay. We demonstrated that multiple reductases in the nucleus metabolize TPZ under hypoxia. Using the cofactor dependence of the reductases for metabolizing TPZ and of the DNA damage caused by TPZ, we show that DNA single-strand breaks after TPZ metabolism are probably caused by the most abundant source of reductase in the nucleus. DNA double-strand breaks, on the other hand, are formed by TPZ metabolism by an unknown nuclear reductase that requires only NADPH for its activity. This study is the first to characterize multiple nuclear reductases capable of activating TPZ.

Mitochondrial dysfunction after aerobic exposure to the hypoxic cytotoxin tirapazamine.

Tirapazamine (TPZ) is a bioreductive drug that exhibits a high degree of selective toxicity toward hypoxic cells, and at doses that are used clinically, little or no cell killing is observed in aerobic cells. Nonetheless, the effects of TPZ on aerobic tissues are still responsible for the dose limitations on the clinical administration of this drug. Clinical side effects include fatigue, muscle cramping, and reversible ototoxicity. We have investigated TPZ-induced changes in the mitochondria in aerobically exposed cells as a potential mediator of these side effects. Our data show that aerobic administration of TPZ at clinically relevant doses results in a profound loss in the mitochondrial membrane potential (MMP). We show that loss in the MMP occurs in a variety of cell lines in vitro and also occurs in muscle tissues in vivo. The loss in MMP is temporary because recovery occurs within 2 h. TPZ is directly metabolized within mitochondria to a DNA-damaging form, and this metabolism leads to both the cell-killing effects of TPZ on aerobic cells at high doses and to the loss in MMP at clinically relevant doses. Using cell lines derived from genetically modified mice with a targeted deletion in manganese superoxide dismutase, we have further distinguished the phenotypic effects of TPZ in mitochondria at high toxic doses versus those at clinically relevant doses. We have investigated several potential mechanisms for this TPZ-induced loss in MMP. Our results indicate no change in the rate of cellular respiration in TPZ-treated cells. This implies that the loss in MMP results from an inability of the inner mitochondrial membrane to sustain a potential across the membrane after TPZ treatment. Incubation of cells with an inhibitor of the mitochondrial permeability transition suggests that the loss of MMP may result from the regulated opening of a large mitochondria channel.

Tirapazamine is metabolized to its DNA-damaging radical by intranuclear enzymes.

Tirapazamine (TPZ), a new anticancer drug that is currently in Phase II and III clinical trials, has a unique mechanism of action. Its cytotoxicity is selective for hypoxic cells in solid tumors and results from DNA damage produced by a free radical, which is generated by enzymatic reduction of the parent molecule. However, there is no agreement as to which enzyme(s) are involved. Here, we have measured both DNA damage and TPZ metabolism in A549 human lung cancer cells and in isolated nuclei derived from the cells. We show that, although the nuclei metabolize TPZ at a rate that is only 20% of that of whole cells, they have levels of DNA damage that are similar to those of the cells. We also show that TPZ radicals that are formed outside nuclei do not contribute to intranuclear DNA damage. Thus, essentially all of the DNA damage from TPZ results from radicals generated within the nucleus, and the 80% of the drug metabolism that occurs in the cytoplasm is probably irrelevant for the activity of this drug in killing hypoxic cells.