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Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes
Citation:
MAZUR, C. S., J. F. KENNEKE, M. GOLDSMITH, AND C. BROWN. Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes. DRUG METABOLISM AND DISPOSITION. American Society for Pharmacology and Experimental Therapeutics, Bethesda, MD, 37(9):1801-1805, (2009).
Impact/Purpose:
Our research objective is to develop and apply innovative techniques for elucidating the kinetics and mechanisms of xenobiotic metabolism, and apply these techniques to the understanding and modeling of chemical exposure for informing human health and ecological risk assessment.
Description:
Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative P450 transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose 6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the P450-mediated transformation of propiconazole and 11 beta-hydroxysteroid dehydrogenase type 1 (HSD1) carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate that the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas P450-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and P450 processes may not be properly assessed with direct addition of NADPH. In addition, we used in silico predictions as follows: 1) to show that 11 beta-HSD1 carbonyl reduction was energetically more favorable than oxidative P450 transformation; and 2) to calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11 beta-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.
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Contrasting Influence of NADPH and a NADPH-regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes