ENZYMOLOGY OF THE REDUCTION OF THE POTENT BENZOTRIAZINE-DI-N-OXIDE HYPOXIC CELL CYTOTOXIN SR-4233 (WIN-59075) BY NAD(P)H - (QUINONE ACCEPTOR) OXIDOREDUCTASE (EC 1.6.99.2) PURIFIED FROM WALKER 256 RAT-TUMOR CELLS

3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233; WIN 59075) is a highly selective hypoxic cell cytotoxin soon to enter phase 1 clinical trial. The compound is thought to exert its action through a toxic one-electron reduced free radical intermediate. Preliminary data have suggested that SR 4233 may be metabolized by DT-diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2)] to both two- and four-electron reduced products and that this route of biotransformation may represent a bioprotection pathway. In this study, a highly purified enzyme preparation was employed in order to investigate further the metabolism of SR 4233 by DT-diaphorase and to examine the mechanism of reduction in more detail. Spectrophotometric analysis showed that SR 4233 underwent reduction by DT-diaphorase with an apparent K(m) of 1.23 +/- mM and V(max) of 8.55 +/- 1.67 nmol/min/mu-g protein. This reaction was inhibited completely by dicoumarol (100-mu-M) and partially by an antiserum raised against the purified enzyme. Characterization of the products of SR 4233 reduction by reverse-phase HPLC confirmed that both two- (SR 4317) and four- (SR 4330) electron reduction products were generated, the latter being the predominant metabolite, particularly in prolonged incubation. Further experiments showed that the four-electron reduction product, but not the two-electron reduction product, was also a substrate for DT-diaphorase with an apparent K(m) of 1.14 mM and a V(max) of 57.12 nmol/min/mu-g protein. The results presented confirm that SR 4233 is indeed a substrate for DT-diaphorase and that a mixture of two-, four- and six-electron reduced products may be formed. The possible toxicological and pharmacodynamic significance of this metabolism is discussed.