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 high-performance 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 (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, 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 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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.006593.
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ABBREVIATIONS: NDPS, N-(3,5-dichlorophenyl)succinimide; AOAA, (aminooxy)acetic acid hemihydrochloride; CID, collision-induced dissociation; 2-NDASA, N-(3,5-dichlorophenyl)-2-aminosuccinamic acid; N-acetyl-2-NDASA, N-(3,5-dichlorophenyl)-2-N-acetylaminosuccinamic acid; 2- and 3-NDHSA, N-(3,5-dichlorophenyl)-2- and -3-hydroxysuccinamic acid; NDPSA, N-(3,5-dichlorophenyl)succinamic acid; Z-Asp-OMe, O-methyl-N-carbobenzoxyaspartic acid; Ac-Asp-OMe, O-methyl-N-acetylaspartic acid; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; MS, mass spectrometry.
- Received July 14, 2005.
- Accepted September 14, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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