Reduction of sulfamethoxazole and dapsone hydroxylamines by a microsomal enzyme system purified from pig liver and pig and human liver microsomes

Abstract

Biotransformation involving nitrogen are of pharmacological andn toxicological relevance. In principle, nitrogen containing functional groups can undergo all the known biotransformation processes such as oxidation, reduction, hydrolysis and formation of conjugates. For the N-reduction of benzamidoxime an oxygen-insensitive liver microsomal enzyme system that required cytochrome b 5, NADH-cytochrome b 5 reductase and a cytochrome P450 isoenzyme of the subfamily 2D has been described. In previous studies it was demonstrated that N-hydroxylated derivates of strongly basic functional groups are easily reduced by this enzyme system. The N-hydroxylation of sulfonamides such sulfamethoxazole (SMX) and dapsone (DDS) to sulfamethoxazole-hydroxylamine (SMX-HA) and dapsone-hydroxylamine (DDS- N-OH), respectively is the first step in the formation of reactive metabolites. Therefore it seemed reasonable to study the potential of cytochrome b 5, NADH-cytochrome b 5 reductase and CYP2D to detoxify these N-hydroxylated metabolites by N-reduction. Metabolites were analysed by HPLC analysis. SMX-HA and DDS- N-OH are reduced by cytochrome b 5, NADH-cytochrome b 5 reductase and CYP2D but also only by cytochrome b 5 and NADH-cytochrome b 5 reductase without addition of CYP2D. The reduction rate for SMX-HA by cytochrome b 5, NADH-cytochrome b 5 reductase and CYP2D was 0,65 ± 0,1 nmol SMX/min/mg protein. The reduction rate by b 5 and b 5 reductase was 0,37 ± 0,15 nmol SMX/min/mg protein. For DDS- N-OH the reduction rate by cytochrome b 5, NADH-cytochrome b 5 reductase and CYP2D was 1.79 ± 0.85 nmol DDS/min/mg protein and by cytochrome b 5 and NADH-cytochrome b 5 reductase 1.25 ± 0.15 nmol DDS/min/mg protein. Cytochrome b 5, NADH-cytochrome b 5 reductase are therefore involved in the detoxification of these reactive hydroxylamines and CYP2D increased the N-reduction.