Evaluation of the effect of oxygen exposure on human liver microsomal metabolism of mitomycin C in the presence of glutathione using liquid chromatography-quadrupole time of flight mass spectrometry.
This article describes a metabolite profiling method for evaluating the effect of oxygen exposure on human liver microsomal metabolism of mitomycin C (MC) in the presence of glutathione (GSH) and NADPH under hypoxic (100% helium), limitedly and fully aerobic, and hyperoxic (100% oxygen) conditions. MC and its metabolite(s) were characterized and the relative percentages of these components were estimated at different incubation times using liquid chromatography and quadrupole time-of-flight mass spectrometry. The MC metabolite profiles were confirmed using purified human cytochrome P450 reductase, acidic activation, and UV-Vis detection at 550 nm. In hypoxia, MC was exclusively metabolized into 2,7-diaminomitosene-10-glutathione-S-conjugate (2,7-DAM-10-SG) within 30 min, whereas approximately 5% of this conjugate, 16% of 2,7-diaminomitosene (2,7-DAM), and 77% of MC were observed under a fully aerobic condition at 90 min. Under limitedly aerobic conditions, the relative percentages of the two metabolites in incubations varied greatly depending on the volume ratio of air to liquid. In hyperoxia, 2% of 2,7-DAM-10-SG, 9% of 2,7-DAM, and 86% of MC were obtained at 90 min. The results indicate that oxygen strongly inhibits the in vitro metabolism of MC. These data suggest that GSH may serve a dual function in facilitating the formation of a leucoaziridinomitosene followed by electron rearrangement giving intermediate metabolite 2,7-DAM, and then trapping this intermediate giving rise to 2,7-DAM-10-SG. These findings provide direct evidence for understanding the fate of oxygen-sensitive metabolic deactivation of MC by GSH.
Lang
Wensheng
W
Johnson & Johnson Pharmaceutical Research and Development, Welsh and McKean Roads, Spring House, PA 19477, USA. wlang@prdus.jnj.com
Caldwell
Gary W
GW
Masucci
John A
JA
D002853:Chromatography, Liquid; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism*; D016685:Mitomycin / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors
D016685:Mitomycin; D009249:NADP; D005978:Glutathione; D010100:Oxygen