Biochemical Pharmacology, Vol. 37. No. 8. pp. 1487-1495, 1988. tX06-295&k? $3.00 + 0.00 Printed in Great Britain. @I 1988. Pergamon Press plc MOLECULAR MECHANISMS FOR THE HYPOXIA- DEPENDENT ACTIVATION OF 3-AMINO-1,2,4- BENZOTRIAZINE-1,6DIOXIDE (SR 4233) KEITH LADEROUTE*, PETER WARDMAN? and A. MICHAEL RAUTH*$ *Ontario Cancer Institute, Physics Division, Toronto, Ontario, Canada M4X lK9; and tGray Laboratory of the Cancer Research Campaign, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, U.K. (Received 6 April 1987; accepted 1 September 1987) Abstract-The reduction of the hypoxic cell toxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) was investigated using pulse radiolysis, radiation chemical reduction, and xanthine oxidase. Evidence was found that the one-electron reduction product of the parent compound is an oxidizing radical that caused single- and double-strand breaks in plasmid DNA and that produced a malondialdehyde-like thiobarbituric acid adduct from 2-deoxy-D-ribose. Possible forms of the reactive radical, either carbon- or nitrogen-centered, are suggested. The “natural” lifetime of the radical was sufficiently long that it could diffuse over significant distances within hypoxic cells and thus inflict oxidative damage on cellular targets. The radical reacted with O2 at a rate comparable to those of the nitroimidazoles misonidazole and metronidazole. Thus, the selectivity for hypoxic cells is probably due to the elimination of “futile” reduction when the cellular oxygen concentration is sufficiently low. The compound 3-amino-1,2,4-benzotriazine-1,4- dioxide (SR 4233; NSC 130181) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1 has been shown to have significant selective toxicity for hypoxic mam- malian cells in uitro and in uiuo [l]. Recently the reduction chemistry of 1 was investigated using radi- ation chemical, electrochemical and enzymatic methods [2]. Two major reduction products were found, 3-amino-1,2,4-benzotriazine-l-oxide 2 (SR 4317) and 3-amino-1,2,4_benzotriazine 3 (SR 4330). The reduction stoichiometry was determined using the one-electron reductants, CO; and e,, at pH 7, and an apparent stoichiometry of approximately 0.5 one-electron donors per molecule of 1 was found. Two-electron stoichiometry is the expected result since 2 is a two-electron reduction product of 1. Since 2 has been reported to be not measurably toxic to hypoxic cells in vitro at levels significantly greater than those which are toxic for 1 [l], it is likely that the damaging species exists at the one-electron reduction level. This result raised the possibility that the peculiar reduction stoichiometry observed earlier is relevant to the hypoxic cell toxicity. In the present work an attempt has been made to characterize the properties of the one-electron intermediate. $ Address correspondence to: Dr. A. Michael Rauth, Ontario Cancer Institute, Physics Division, 500 Sherbourne St., Toronto, Ontario, Canada M4X lK9. MATERIALSAND METHODS The compounds sodium cyanamide and malon- dialdehyde bis (dimethylacetal) were obtained from the Aldrich Chemical Co., Milwaukee, WI. Xan- thine oxidase (grade III), xanthine, 2-deoxy-D-ribose and thiobarbituric acid (TBA) were purchased from the Sigma Chemical Co., St. Louis, MO. Barium [14C]cyanamide (5 mg; 1 mCi/mg) was from Amer- sham, Oakville, Ontario, and desferrioxamine mesy- late (Desferal) was from CIBA, Dorval, P.Q. Other materials, including Sepharose 4B (Pharmacia, Mon- treal, P.Q.) and pre-purified argon (Matheson Gas Products Canada, Toronto, Ontario), were com- mercial samples of high purity. Plasmid pDHd33 (3.3 kilobases) was provided by Dr. D. E. Pulleyblank of the University of Toronto, and plasmid pPM17 (5.5 kilobases) was obtained from Dr. G. F. Whitmore of the Ontario Cancer Institute. The latter plasmid was grown and purified by conventional micro- biological techniques [3]. Both plasmids were >80% supercoiled. Compound 1 was synthesized according to the procedure of Mason and Tennant [4]. Samples of 2 were obtained from this synthetic sequence. The labelled compound 3-14C]-3-amino-1 ,2,4_benzotria- zine-1,4-dioxide r (l- 4C) was also produced in this way, but the required precursor, [14C]cyanamide, was synthesized from barium [14C]cyanamide by a modification of the procedure of Zbarsky and Fischer [5] in which 5 mg of labelled material was diluted with 2 g of sodium cyanamide. The yield of crude [14C]cyanamide was 43%, and this was used directly in the synthesis of 1-14C. Purification of 1-14C was accomplished using a semi-preparative, Waters @ondapak C1s HPLC column and a mobile phase of 10% CH3CN/20% CH30H/H20. The specific activity of the purified l-14C was 2 pCi/mg. 1487