Metabolism of Bioreductive Antitumor Compounds by Purified Rat and Human DT-Diaphorases

Abstract

The metabolisms of two standard electron acceptors and a series of bioreductive antitumor compounds by purified rat and human DT-diaphorases (DTD) were compared. DTD was purified from rat liver cytosol and from Escherichia coli in which rat liver or human lung tumor DTD complementary DNA was expressed. K_m and k_cat values for menadione and 2,6-dichlorophenolindophenol reduction were similar for the three enzyme preparations except that rat E. coli DTD had 2–3-fold higher k_cat values for both menadione and 2,6-dichlorophenolindophenol and a 2–3-fold higher K_m for menadione than either rat liver or human E. coli DTD. Reduction of the antitumor compounds was 1.9–4.9 times faster with rat E. coli DTD than with human E. coli DTD. The antitumor compounds were reduced in the following order by rat E. coli DTD: 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone > streptonigrin > mitomycin A > diaziquone > mitomycin C (MC) > 5-(aziridin-1-yl)-2,4-dinitrobenzamide. The order was the same for human E. coli DTD with one exception; diaziquone was reduced slightly faster than mitomycin A. Metabolism of doxorubicin could not be detected using rat or human E. coli DTD. MC-induced DNA cross-linking was also more efficient using rat E. coli DTD relative to human E. coli DTD. Metabolism of MC by rat and human E. coli DTD was also compared under aerobic and hypoxic conditions. Rates of reduction of MC and metabolite formation were similar under aerobic and hypoxic conditions, and the toxicity of MC to DTD-rich HT-29 cells was also similar in aerobic and hypoxic conditions. In contrast, the toxicity of MC to DTD-deficient BE cells was potentiated markedly under hypoxia. These data show that although small catalytic differences between rat and human E. coli DTD can be observed, compounds such as 2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone and streptonigrin are still excellent substrates for the human enzyme and may be useful in the therapy of tumors high in DTD activity. In addition, metabolism of MC by rat and human E. coli DTD was similar in aerobic and hypoxic conditions; in agreement with these data, cytotoxicity of MC to a DTD-rich cell line was oxygen independent. Increased MC cytotoxicity under hypoxia appears to be mediated by enzymes other than DTD.