Transamination in the Metabolism of the Nephrotoxicant N-(3,5-Dichlorophenyl)succinimide in Rats

Abstract

ABSTRACT:The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) is nephrotoxic in rats. Due to the involvement of NDPS metabolism in its mechanism of toxicity, the detailed biotransformation of 14 C-NDPS in rats was previously evaluated using highperformance liquid chromatography-electrospray ionization-mass spectrometry. In the present report, we describe the identification of two novel amino metabolites of NDPS, which were present in significant amounts in rat kidney tissues. Using liquid chromatography-tandem mass spectrometry and synthetic standards, the two metabolites were identified as N-(3,5-dichlorophenyl)-2-aminosuccinamic acid (2-NDASA) and its N-acetylated derivative (Nacetyl-2-NDASA). The mechanism of formation of 2-NDASA was studied in vitro. Incubations were carried out in rat liver and kidney cytosols using the major oxidative metabolite of NDPS, N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid, as the substrate. Formation of 2-NDASA in vitro was confirmed using mass spectrometry. Inhibitors of alcohol dehydrogenase (4-methylpyrazole) and aldehyde dehydrogenase (disulfiram) reduced 2-NDASA formation by 40 to 50%. Menadione (an inhibitor of aldehyde oxidase) and quercetin (an inhibitor of carbonyl reductase) did not show any effects. (Aminooxy)acetic acid, an inhibitor of pyridoxal 5-phosphate-containing enzymes such as aminotransferases, almost completely abolished the formation of 2-NDASA. Using liquid chromatography-mass spectrometry, the transamination mechanism was further supported by the incorporation of a 15 N-amino group in 2-NDASA when 15 N-glutamic acid was included in the incubation mixture. Results from these studies show that transamination is a metabolic pathway in the clearance of NDPS in rats, and that cytosolic dehydrogenases and aminotransferases may be involved in this process. succinimide (NDPS; Fig. 1) was originally synthesized as an agricultural antifungal agent (Fujinami et al., 1972). In subsequent testing, NDPS was shown to be nephrotoxic in male rats at doses Ն0.4 mmol/kg following acute administration (Rankin, 1982;Rankin et al., 1985). Although not widely used, NDPS remains on the market as an agricultural fungicide, and it has been studied extensively as a model compound for chemically induced kidney damage (Rankin, 2004).Studies in rats have shown that NDPS is well absorbed, widely distributed (highest in kidney), and rapidly eliminated, mainly as metabolites in urine (Ohkawa et al., 1974;Griffin and Harvison, 1998). The major in vivo metabolites of NDPS are N- (3,5-dichlorophenyl)succinamic acid (NDPSA), N-(3,5-dichlorophenyl)-2-and 3-hydroxysuccinamic acids (2-and 3-NDHSA), and N-(3,5-dichlorophenyl)malonamic acid (Ohkawa et al., 1974;Griffin and Harvison, 1998). Glucuronide/sulfate and glutathione-derived conjugates of 2-/3-NDHSA and NDPS/NDPSA, respectively, have also been reported (Cui et al., 2005). Both phase I and II metabolic pathways may be involved in the formation of a nephrotoxic species from NDPS (Rankin et al., 1987;Nyarko et al., 1997; Ho...

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