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Drug Metabolism and Disposition Fast Forward
First published on September 20, 2005; DOI: 10.1124/dmd.105.006593

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Received for publication July 15, 2005.
Revised September 13, 2005.
Accepted for publication September 14, 2005.

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

Donghui (Dan) Cui 1, Gary O. Rankin 2, Peter J Harvison 3*

1 Bristol Myers Squibb 2 Marshall University School of Medicine 3 Univ. of the Sciences in Philadelphia

* Address correspondence to: E-mail: p.harvis{at}usip.edu

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 14C-NDPS in rats was previously evaluated using HPLC-electrospray ionization (ESI)-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 LC-MS/MS and synthetic standards, the two metabolites were identified as N-(3,5-dichlorophenyl)-2-aminosuccinamic acid (2-NDASA) and its N-acetylated derivative (N-acetyl-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 (2-NDHSA), 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 (AOAA), an inhibitor of pyridoxal 5'-phosphate (PLP)-containing enzymes such as aminotransferases, almost completely abolished the formation of 2-NDASA. Using LC-MS, the transamination mechanism was further supported by the incorporation of an 15N-amino group in 2-NDASA when 15N-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.


Key words: HPLC, mass spectrometry, metabolite identification, renal toxicity, stable isotopes




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