Heat-stimulated nitroreductive bioactivation of the 2-nitroimidazole benznidazole in vitro.

Hyperthermia enhances nitroimidazole cytotoxicity, possibly through increased nitroreductive bioactivation. Using C3H/He mouse liver microsomes and KHT tumour homogenates, we have investigated the effects of temperature (33-44 degrees) on the anaerobic nitroreduction of benznidazole (BENZO) to its amine metabolite in vitro. Microsomal nitroreductase activity was unaltered after 2 hr anaerobic incubation at 37 and 41 degrees. However at 44 degrees and 47 degrees, inactivation occurred with half-lives of 68 and 17 min respectively. At 33 degrees microsomal reduction rates were 45% lower than at 37 degrees. Reduction rates were increased by 22% at 41 compared to 37 degrees, and by 0-54% depending on substrate concentration at 44 degrees. Microsomal amine formation followed Michaelis-Menten kinetics up to 41 degrees. The 4 degrees rise from 33 to 37 degrees increased the apparent Vmax by 45% (from 0.54 to 0.98 nmol min-1 mg-1 protein) with a further increase of 32% occurring at 41 degrees. Apparent Km values were unaltered. Deviation from Michaelis-Menten kinetics was seen for amine formation at 44 degrees. The kinetics of parent drug disappearance exhibited deviation from the Michaelis-Menten relationship at all temperatures studied. KHT tumour BENZO amine formation rates were also markedly increased at elevated temperatures, e.g. by 26% at 37 degrees compared to 33 degrees and by a further 35% from 42.5 to 57.4 pmol min-1 mg-1 protein over the range 37-41 degrees. In contrast to the microsomal results, tumour reduction rates were enhanced by an average of 54% (range, 26-79%) at 44 degrees compared to 37 degrees at low as well as high substrate concentrations. These results support the hypothesis that hyperthermia-enhanced nitroimidazole cytotoxicity may be a result of increased nitroreductive bioactivation.