Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity II. Possible involvement of the γ-glutamyl cycle

Abstract

Acetaminophen (APAP) nephrotoxicity has been observed both in humans and research animals. Our recent investigations have focused on the possible involvement of glutathione-derived APAP metabolites in APAP nephrotoxicity and have demonstrated that administration of acetaminophen-cysteine (APAP-CYS) potentiated APAP-induced renal injury with no effects on APAP-induced liver injury. Additionally, APAP-CYS treatment alone resulted in a dose-responsive renal GSH depletion. This APAP-CYS-induced renal GSH depletion could interfere with intrarenal detoxification of APAP or its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI) and may be the mechanism responsible for the potentiation of APAP nephrotoxicity. Renal-specific GSH depletion has been demonstrated in mice and rats following administration of amino acid γ-glutamyl acceptor substrates for γ-glutamyl transpeptidase (γ-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the γ-glutamyl cycle through interaction with γ-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the γ-glutamyl transpeptidase (γ-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a γ-glutamyl acceptor for both murine and bovine renal γ-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a γ-glutamyl-cysteinyl-acetaminophen metabolite. 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.

Introduction

Acetaminophen (APAP) is a commonly used over-the-counter analgesic and antipyretic. Though acetaminophen is recognized as a safe and efficacious drug, overdose can result in both nephrotoxicity and hepatotoxicity (Curry et al., 1982, Proudfoot and Wright, 1970). In a recent review of pediatric overdose cases, 9% of the patients had renal injury (Boutis and Shannon, 2001). Though acetaminophen-induced nephrotoxicity is less common than hepatotoxicity, the fact that nephrotoxicity can occur in the absence of hepatotoxicity requires that kidney function always be monitored following overdose (Davenport and Finn, 1988, Kher and Maker, 1987). The apparent independence of acetaminophen-induced nephrotoxicity and hepatotoxicity suggests that different mechanisms may be involved. Our earlier investigations demonstrated the possible role of glutathione-conjugate-derived APAP metabolites in APAP nephrotoxicity. Thus, pretreatment with acivicin, an inhibitor of γ-glutamyl transpeptidase (γ-glutamyl transpeptidase; γ-GT), or probenecid, an organic anion transport inhibitor, protected against APAP nephrotoxicity but not hepatotoxicity in male CD-1 mice (Emeigh et al., 1996). Our accompanying report (Stern et al., 2004) demonstrated the ability of acetaminophen-cysteine (APAP-CYS) to potentiate APAP-induced renal injury. The present report provides a possible mechanism for the observed potentiation.

In the accompanying manuscript, APAP-CYS administration to male CD-1 mice resulted in a dose-dependent decrease in renal GSH levels, while sparing hepatic thiols (Stern et al., 2004). Established mechanisms for other nephrotoxic glutathione conjugates have involved a kidney-selective uptake or bioactivation (Dekant, 2001) and cannot adequately explain the APAP-CYS phenomenon. Recently, Mutlib et al. (2001) proposed a novel mechanism for renal GSH depletion by benzylamine. In this mechanism, benzylamine functions as a γ-glutamyl acceptor substrate resulting in enhancement of GSH catabolism by γ-GT and perturbation of the γ-glutamyl cycle (Scheme 1). The γ-glutamyl cycle is responsible for maintenance of GSH homeostasis and may play a role in amino acid transport (Meister et al., 1979, Smith et al., 1991). Administration of γ-glutamyl acceptor substrates (e.g., glycylglycine) to mice and rats increases transpeptidation by brush border γ-GT and results in catabolism of intracellular GSH, presumably following luminal translocation (Griffith et al., 1978, Palekar et al., 1975). Organs such as the liver, with much lower γ-GT levels, were considerably less susceptible to such GSH depletion after these substrates. Because APAP-CYS is similar to benzylamine and amino acid γ-glutamyl acceptors, it may also selectively deplete renal GSH by acting as a γ-glutamyl acceptor (Scheme 1). If APAP-CYS were to function as a γ-glutamyl acceptor and decrease renal GSH stores, this could explain how APAP-GSH-derived metabolites contribute to APAP nephrotoxicity. The present study explores the hypothesized role of APAP-CYS as a γ-glutamyl acceptor substrate with consequent implications for the mode of action in APAP-induced kidney toxicity.