DNA Damage and Cytotoxicity in Human Colon Carcinoma Oxidoreductase (DT-Diaphorase): Role in Diaziquone-induced Metabolism of Diaziquone by NAD(P)H:(Quinone Acceptor)

Reduction of 2,5-diaziridinyl-3,6-bis(carboethoxyamino)-l,4-benzoquinone (diaziquone; AZQ) by purified rat hepatic DT-diaphorase »as NADH and enzyme dependent and was inhibited by prior boiling of the enzyme or by dicumarol. Under aerobic conditions some of the hydroquinone (AZQH2) formed by reduction oxidized to regenerate AZQ and an approximate 1:1 stoichiometry was observed between V/(.)l I reoxidized and oxygen consumed. The steady state kinetics of AZQ reduction were consistent with a ping-pong mechanism and a high A',,,for AZQ. There was no evidence for saturation in the range of 25-200 MMAZQ at 200 MMNADH. AZQ (0-20 MM)induced dicumarol-inhibitable DNA interstrand cross-linking and cytotoxicity in HT-29 human colon carcinoma cells which have high DT-diaphorase activity but not in BE cells which have low DT-diaphorase activity. Extensive metabolism (>90%) of AZQ (100 MM) in HT-29 cytosol occurred, which was either NADH or NADPH dependent and could be inhibited by dicumarol. Little metabo lism of AZQ could be detected in BE cell cytosols. DT-diaphorase was purified from HT-29 cells and metabolism of AZQ by this enzyme was confirmed. These data show that AZQ can be metabolized by purified rat hepatic and human HT-29 DT-diaphorase and suggest that in HT-29 cells, DT-diaphorase catalyzed reduction of AZQ represents a bioactivation process leading to the production of genotoxic and cytotoxic metabolites.