This proposal is designed to test the hypothesis that the nephrotoxicity of para-aminophenol (PAP) is mediated by formation of cytotoxic metabolites in the liver. These metabolites are proposed to be generated by sequential cytochrome P450-dependent oxidation and glutathione (GSH) conjugation. GSH-PAP conjugates are secreted into bile, reabsorbed in the gastrointestinal tract, and delivered to the kidney, where cytotoxicity occurs. While this mechanism is proposed to account for cytotoxicity of PAP, the scheme of sequential oxidation and glutathione conjugation may be a general pathway for metabolism of para-substituted organic compounds. The long-term goals of the present project are to determine if PAP-GSH conjugates can account for the cytotoxicity of PAP and to define the mechanisms involved in the formation of PAP-GSH conjugates. To accomplish these goals, the following specific aims will be addressed: (1) identification of early renal functional changes in vivo following PAP administration; (2) evaluation of PAP-induced cytotoxicity in renal tubules in vitro in the presence and absence of hepatic S9 fraction and the presence and absence of NADPH; (3) determination of the source and enzymatic processes involved in the formation of reactive PAP intermediates; (4) evaluation of the cytotoxicity of PAP-GSH metabolites in renal tubules in vitro; and (5) evaluation of the rates of formation of PAP-GSH conjugates in liver and kidney in vitro. Renal functional changes induced by PAP will be evaluated early following PAP administration and in freshly isolated renal tubules incubated with PAP in the presence and absence of hepatic S9 fraction and the presence and absence of NADPH. The site of formation of cytotoxic PAP metabolites will be determined by selectively inhibiting oxidation or GSH conjugation in either hepatic S9 fraction or renal tubules and assessing PAP cytotoxicity in renal tubules. PAP-GSH conjugates will be synthesized, isolated, and purified by semi- preparative HPLC. Identification will be confirmed by NMR and mass spectral analyses. PAP-GSH conjugates will be tested for cytotoxicity using renal tubules in suspension. Cytotoxicity of PAP-GSH conjugates will be compared to cytotoxicity of PAP to determine relative potencies. Rates of formation of PAP-GSH conjugates in liver and kidney homogenates will be measured to determine if PAP-GSH conjugates are formed in quantities sufficient to account for nephrotoxicity. The information obtained in this project will help establish a general model of bioactivation of chemicals that selectively injure the kidney. Further, establishing the role of glutathione conjugates in PAP-induced nephrotoxicity will strengthen the hypothesis that glutathione conjugates serve as transport forms of nephrotoxic agents.
