Metabolism of the Nephrotoxicant N-(3,5-Dichlorophenyl)succinimide in Rats: Evidence for Bioactivation through Alcohol-O-glucuronidation and O-Sulfation

Abstract

The agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS), was shown to be nephrotoxic in rats. Previous studies have indicated that the metabolism of NDPS contributes to its mechanism of toxicity and both phase I and phase II pathways may be involved. In the current report, we investigated the detailed biotransformation of [(14)C]NDPS in rats using HPLC-ESI-MS. The chemical reactivity of the phase II NDPS metabolites was also evaluated. In vivo studies were conducted by administering [(14)C]NDPS to male Fischer 344 rats. Urine, tissue (liver and kidney), and plasma samples were analyzed. The mechanism of formation and chemical reactivity of the glucuronide and sulfate metabolites of NDPS were investigated in vitro using liver subcellular preparations. Major in vivo metabolites of NDPS were identified as the oxidative [N-(3,5-dichlorophenyl)-2- and 3-hydroxysuccinamic acid, 2-/3-NDHSA] and hydrolytic products [N-(3,5-dichlorophenyl)succinamic acid]. N-Acetylcysteine and cysteine (with intramolecular aminolysis) conjugates were also detected in rat urine and fecal extracts, respectively, suggesting the formation of reactive intermediate(s) in the metabolism of NDPS. Small amounts of the alcohol-O-glucuronide and O-sulfate of 2-/3-NDHSA were detected in rat urine, plasma, and tissue homogenates. The formation of these phase II metabolites was found to be mediated through the initial conjugation of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) followed by hydrolysis. As compared to NDHS, NDHS-O-sulfate is approximately 500-fold more reactive toward GSH conjugation. In rat liver S9, fortifying phase II cofactors (UDPGA or PAPS) in incubation mixtures with NDHS also significantly increased the amount of GSH adducts produced. Results of this research demonstrate that phase II metabolites of NDPS were produced in rats. The formation of the alkyl alcohol-O-glucuronide and O-sulfate conjugates represents bioactivation pathways in the metabolism of NDPS that could potentially contribute to its mechanism of nephrotoxicity.