Nitroimidazole bioreductive metabolism. Quantitation and characterisation of mouse tissue benznidazole nitroreductases in vivo and in vitro.
We have investigated the nitroreduction of the 2-nitroimidazole benznidazole (BENZO) to its corresponding amine by murine normal tissues and tumours. In vivo concentrations of BENZO and its amine metabolite were measured by HPLC 3 hr after BENZO, 2.5 mmoles kg-1 i.p. This gave plasma and tissue BENZO concentrations of 96-160 micrograms ml-1 or g-1. Mouse plasma, KHT and RIF-1 tumour BENZO amine concentrations were very low (0.3-1.4 micrograms g-1); kidney and EMT6 tumours had intermediate levels; and liver contained very high amine levels (approximately 50 micrograms g-1). Three per cent of the BENZO dose was recovered as amine in the 24 hr urine, compared to 5% for the parent compound. Nitroreduction to the amine was demonstrated with liver and tumour preparations under N2 in vitro. The reaction was highly dependent on NADPH, and inhibited extensively in air. With liver microsomes and whole homogenates 2 and 3 moles respectively of BENZO were consumed per mole of amine formed. Inhibitor studies showed that NADPH: cytochrome P-450 (cytochrome c) reductase and cytochrome P-450 were both involved in BENZO reduction, predominantly at early and late reduction steps respectively. Aldehyde oxidase contributed to the cytosolic nitroreduction. Purified buttermilk xanthine oxidase also reduced BENZO to its amine under anaerobic conditions in vitro, but very inefficiently. The apparent Km and Vmax for BENZO amine production by whole liver homogenates were 0.148 mM and 1.45 nmole min-1 mg-1 protein respectively. Tumour homogenates were less active than liver; e.g. Vmax for the KHT tumour was 6-10-fold lower.
D042931:Aldehyde Oxidase; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000201:enzymology*; D009593:Nitroimidazoles / Q000378:metabolism*; D009601:Nitroreductases / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*
D009593:Nitroimidazoles; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C029492:aromatic NADH-dependent nitroreductase; D000445:Aldehyde Oxidoreductases; D042931:Aldehyde Oxidase; D009251:NADPH-Ferrihemoprotein Reductase; D009601:Nitroreductases; C009999:benzonidazole