REDUCTIVE METABOLISM OF DIAZIQUONE (AZQ) IN THE S9 FRACTION OF MCF-7 CELLS .2. ENHANCEMENT OF THE ALKYLATING ACTIVITY OF AZQ BY NAD(P)H - QUINONE-ACCEPTOR OXIDOREDUCTASE (DT-DIAPHORASE)

The alkylating activity of reduced diaziquone was studied by the nitrobenzylpyridine (NBP) assay and was compared to those of the parent compound and aziridine-containing N,N;,N"-triethylenethiophosphoramide (Thio-TEPA). Diaziquone (AZQ) was reduced enzymatically by 2e- using S9 cell fraction from MCF-7 cells which is rich in NAD(P)H:quinone-acceptor oxidoreductase (DT-diaphorase) (QAO) activity. One electron enzymatic reduction was performed with NADPH-cytochrome c reductase. The alkylating activity of AZQ increased 3-fold when reduced by 2e-. This increase was inhibited by dicumarol, an inhibitor of QAO. In contrast, the alkylating activity of AZQ did not increase beyond that of the parent compound when reduced by 1e- using purified NADPH-cytochrome c reductase. Similar results were obtained when AZQ was reduced chemically with borohydride (2e-) and with NADPH (1e-). Anaerobic incubations of AZQ with the S9 fraction of MCF-7 cells (2e- reduction) resulted in an increase in NBP alkylation over its aerobic counterpart (1.8-fold) while maintaining the near 3-fold increase in alkylation over untreated AZQ. In contrast, AZO incubations with NADPH-cytochrome c reductase (1e- reduction) under the same conditions did not result in an NBP alkylation increase over untreated AZQ. These results indicate that AZO hydroquinone is most likely the responsible species for the observed alkylation of this antitumor agent to DNA and other nucleophiles. The results also suggest that NAD(P)H:quinone-acceptor oxidoreductase is a very important enzyme in the bioactivation of AZQ.