NADH-dependent reduction of sulphamethoxazole hydroxylamine in dog and human liver microsomes

Abstract

1. Reduction of hydroxylamine drug metabolites by NADH-dependent hydroxylamine reductase (NDHR) has been suggested to be involved in the pathogenesis of idiosyncratic sulphonamide toxicity in humans. The dog represents a naturally occurring clinical model for sulphonamide toxicity in humans. The purpose of these studies, therefore, was to characterize the presence of hepatic NADH-dependent hydroxylamine reductase activity in the dog and to compare this activity with that found in humans. 2. NDHR activity was characterized by the presence of two enzymes in both dog and human liver microsomes, with comparable estimates of K_m (K_m1 = 75 μM, K_m = 404 μM in dog; K_m1 = 69 μM, K_m = 503 μM in human). Estimates of maximal velocity were significantly, but not dramatically, higher for dog NDHR (V_max = 2.09 nmole mg⁻¹ min⁻¹, V^max2 = 4.58 nmole mg⁻¹ min⁻¹) compared with human NDHR (V_max1 = 0.42 nmole mg⁻¹ min⁻¹, V_max2 = 1.56 nmole mg⁻¹ min⁻¹). NDHR in dog, as in humans, preferred NADH to NADPH, was more active at pH 6.3 than at 7.4 and was not inhibited by carbon monoxide, azide, anaerobic conditions, the CYP substrate inhibitors tolbutamide, dextromethorphan, or erythromycin, or antibodies directed against CYP2C, CYP2D or CYP3A. 3. It is concluded that two forms of NDHR are present in dog and humans with similar biochemical characteristics. Although NDHR activity has been attributed to a CYP2D isoform in pig, there is no evidence for involvement of CYP450 in the reduction of sulphamethoxazole hydroxylamine in either dogs or humans.