The role of specific reductases in the intracellular activation and binding of 2-nitroimidazoles.

Abstract

To determine the relative effectiveness of specific cellular reductases for the activation and binding of 2-nitroimidazoles in vivo. Monkey kidney cells were transfected with recombinant plasmids to effect intracellular overexpression of P450 reductase and DT-diaphorase. The covalent binding of 2-nitroimidazoles to cellular macromolecules was measured as a function of time of cell incubation at various oxygen concentrations. The effect of allopurinol on cellular binding of radiolabeled 2-nitroimidazoles was also measured. A 1,000-fold overexpression of DT-diaphorase resulted in a small but significant increase in 2-nitroimidazole binding rate. An 80-fold overexpression of cytochrome P450 reductase resulted in a 5-7-fold increase in the binding rate of 2-nitroimidazole. The inhibition of xanthine oxidase by allopurinol had no effect on 2-nitroimidazole binding rates. The amplification of P450 reductase activity within cells was always much larger than the resultant increase in 2-nitroimidazole binding rate, suggesting an enzyme kinetic process less than first order and possibly of 1/2-order. These data suggest that cytochrome P450 reductase is the most important enzyme in these cells for reducing 2-nitroimidazoles to intermediates which can covalently bind to cellular macromolecules. Furthermore, since this cellular process demonstrates approximately 1/2-order kinetics, a tissue's capacity for binding 2-nitroimidazole drug in hypoxia should be proportional to the square root of its intracellular P450 reductase level.