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Dual Action of Tirapazamine in the Induction of DNA Strand Breaks¹

Department of Radiobiology, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada

Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide, SR 4233) is the lead compound of a new class of hypoxic cell cytotoxins showing considerable antitumor activity. Hypoxic cytotoxicity of tirapazamine is believed to be mediated by free radical attack of its one-electron reduced metabolite on DNA, but little is known about the DNA lesions induced by the drug. Using the anoxic xanthine/xanthine oxidase system to effect one-electron reduction of tirapazamine under controlled conditions, we studied the action of the drug toward pUC18 and calf thymus DNA. Agarose gel electrophoresis indicated that tirapazamine causes substantially higher levels of single-strand breakage than double-strand breakage. The 5' DNA termini at the single-strand breaks were shown to be phosphorylated. Little, if any, base damage was observed when the damaged DNA was analyzed by a ³²P-postlabeling assay. The major detectable lesion (comprising 32% of the 3' ends of tirapazamine-induced single-strand breaks) was the phosphoglycolate moiety, which is caused by deoxyribose fragmentation. Since phosphoglycolate formation requires the addition of oxygen, we conclude that tirapazamine acts in a dual fashion to produce phosphoglycolates: (a) to generate a free radical in the deoxyribose ring (i.e., °C-4') and (b) then to donate an oxygen atom. The oxygen donation by tirapazamine was confirmed by anoxic irradiation of DNA in the presence of the unmetabolized drug. Increasing the concentration of the drug (up to 50 µM) led to a dramatic increase in the yield of phosphoglycolate.

¹ This work was supported by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (M. W.) and by a McCartney Research Fellowship from the Alberta Cancer Foundation (G. D. D. J.).

² Present address: Centre for Mechanisms of Human Toxicity, University of Leicester, Hodgkin Building, P. O. Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom. Phone: 0116-252-5527; Fax: 0116-252-5616; E-mail: gdj2@le.ac.uk.

³ To whom requests for reprints should be addressed. Phone: (403) 492-8066; Fax: (403) 439-5744; E-mail: mweinfel@gpu.srv.ualberta.ca.

Received 10/31/95. Accepted 1/22/96.

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