Title

APAP nephrotoxicity: Contribution of APAP-cysteine and possible involvement of the gamma-glutamyl cycle

Date of Completion

January 2001

Keywords

Health Sciences, Toxicology|Health Sciences, Pharmacology

Degree

Ph.D.

Abstract

Acetaminophen (APAP) nephrotoxicity has been observed both in man and research animals. Inhibitors of either γ-glutamyl transpeptidase γ-GT or the probenecid-sensitive organic anion transporter protect against APAP-induced nephrotoxicity but not hepatotoxicity in mice and rats, and suggest that transport of a glutathione (GSH)-derived APAP conjugate may contribute to the toxicity. Acetaminophen-cysteine (APAP-CYS) is an ideal candidate for involvement in APAP nephrotoxicity since it is both a product of the γ-GT pathway and a likely substrate for the organic anion transporter. The objective of this dissertation was to determine if APAP-CYS can contribute to APAP nephrotoxicity and elucidate its underlying mechanism of action. ^ Pretreatment of mice with APAP-CYS potentiated the nephrotoxicity of APAP at 24 hr, as determined by renal histopathology and blood urea nitrogen (BUN) analysis. Hepatotoxicity, as measured by plasma sorbitol dehydrogenase (SDH) activity and hepatic histopathology, was unaffected by pretreatment with APAP-CYS. APAP-CYS treatment alone depleted renal GSH and total glutathione (GSH+ glutathione disulfide (GSSG)) in a dose-responsive manner, and this depletion was antagonized by the γ-GT inhibitor acivicin. Hepatic non-protein sulfhydryl levels were unaffected by APAP-CYS treatment. ^ Renal specific, γ-GT-dependent GSH depletion has been demonstrated in mice and rats following administration of amino acids that serve as γ-glutamyl acceptor substrates for γ-GT. Given that APAP-CYS is structurally similar to amino acid γ-glutamyl acceptor substrates, it was hypothesized that APAP-CYS functioned as a γ-glutamyl acceptor to increase catabolism of GSH by γ-GT. In vitro analysis demonstrated that APAP-CYS is a suitable γ-glutamyl acceptor substrate for both murine and bovine renal γ-GT. Urine metabolite studies identified γ-glutamylcysteinylacetaminophen as a urinary metabolite of acetaminophen-glutathione, APAP-CYS and APAP, suggesting that APAP-CYS functions as a γ-glutamyl acceptor substrate in vivo as well. These findings are consistent with the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by depletion of renal GSH stores through interaction with the γ-glutamyl cycle. ^