METABOLISM OF DIAZIQUONE BY NAD(P)H-(QUINONE ACCEPTOR) OXIDOREDUCTASE (DT-DIAPHORASE) - ROLE IN DIAZIQUONE-INDUCED DNA DAMAGE AND CYTOTOXICITY IN HUMAN COLON-CARCINOMA CELLS

  • 15 November 1990
    • journal article
    • research article
    • Vol. 50  (22) , 7293-7300
Abstract
Reduction of 2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzo-quinone (diaziquone; AZQ) by purified rat hepatic DT-diaphorase was 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 AZQH2 reoxidized and oxygen consumed. The steady state kinetics of AZQ reduction were consistent with a ping-pong mechanism and a high Km for AZQ. There was no evidence for saturation in the range of 25-200 .mu.M AZQ at 200 .mu.M NADH. AZQ (0-20 .mu.M) 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 .mu.M) in HT-29 cytosol occurred, which was either NADH or NADPH dependent and could be inhibited by dicumarol. Little metabolism 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.