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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

NADH Cytochrome b₅ Reductase and Cytochrome b₅ Catalyze the Microsomal Reduction of Xenobiotic Hydroxylamines and Amidoximes in Humans

Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin

Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine drugs, and amidoximes, used as pro-drugs, are metabolized by an as yet incompletely characterized NADH-dependent microsomal reductase system. We hypothesized that NADH cytochrome b₅ reductase and cytochrome b₅ were responsible for this enzymatic activity in humans. Purified human soluble NADH cytochrome b₅ reductase and cytochrome b₅, expressed in Escherichia coli, efficiently catalyzed the reduction of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and benzamidoxime, with apparent K_m values similar to those found in human liver microsomes and specific activities (V_max) 74 to 235 times higher than in microsomes. Minimal activity was seen with either protein alone, and microsomal protein did not enhance activity other than additively. All three reduction activities were significantly correlated with immunoreactivity for cytochrome b₅ in individual human liver microsomes. In addition, polyclonal antibodies to both NADH cytochrome b₅ reductase and cytochrome b₅ significantly inhibited reduction activity for sulfamethoxazole hydroxylamine. Finally, fibroblasts from a patient with type II hereditary methemoglobinemia (deficient in NADH cytochrome b₅ reductase) showed virtually no activity for hydroxylamine reduction, compared with normal fibroblasts. These results indicate a novel direct role for NADH cytochrome b₅ reductase and cytochrome b₅ in xenobiotic metabolism and suggest that pharmacogenetic variability in either of these proteins may effect drug reduction capacity.

Address correspondence to: Dr. Lauren Trepanier, Department of Medical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706. E-mail: latrepanier{at}svm.vetmed.wisc.edu

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