This proposal is designed to test the hypothesis that the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (3,5-NDPS) is metabolically activated to a reactive intermediate. Although this compound is active against a wide variety of pathogenic plant fungi, it is also capable of producing nephrotoxicity in rats. The mechanism by which 3,5-NDPS produces renal damage is unknown, although it is extensively metabolized in both rats and dogs. An unusual aspect of this compound is that glutathione potentiates the nephrotoxicity. Interestingly, the close structural analogue N-(2,6-dichlorophenyl)succinimide (2,6-NDPS) is inactive as a fungicide and not nephrotoxic. The long term goals of this proposal are to fully elucidate the metabolic pathway of 3,5-NDPS in rats and to determine if a reactive intermediate is responsible for the observed renal damage. Studies will be conducted in parallel with 2,6-NDPS to determine if metabolic differences can account for the lack of toxicity of this analogue. To accomplish these goals the following specific aims have been established: synthesize the known and potential metabolites of 3,5-NDPS and 2,6-NDPS; develop an HPLC assay system to separate the parent compounds from their respective metabolites; study the metabolism of the two compounds in vitro and in vivo; and measure covalent binding to proteins as an index of formation of a reactive intermediate. Radiolabelled substrates will be used to quantitate the metabolites that are produced and to assess covalent binding. In vitro metabolism studies will be conducted with subcellular fractions and isolated cells prepared from rat liver and kidney. In the in vivo experiments, animals will be dosed with 3,5-NDPS or 2,6-NDPS and the metabolites present in the urine, feces, bile and tissues will be determined. Nephrotoxicity will be simultaneously assessed by urinalysis and histological examination. The metabolites produced in the in vitro and in vivo experiments will be tentatively identified by co- chromatography with synthetic standards. For definitive identification the metabolites will be isolated by semi-preparative HPLC and characterized by NMR and mass spectrometry. Since 3,5-NDPS has been suggested as a good model of human interstitial nephritis, it is important to fully characterize the metabolic profile of this compound. The information obtained in this project may be of value in determining what factors contribute to the expression of chemically-induced nephrotoxicity in humans and animals.
