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

Toxicology and Applied Pharmacology 202 (2005) 160 – 171 www.elsevier.com/locate/ytaap

Contribution of acetaminophen-cysteine to acetaminophen nephrotoxicity II. Possible involvement of the g-glutamyl cycle Stephan T. Sterna,1, Mary K. Brunoa, Robert A. Hortonb, Dennis W. Hillc, Jeanette C. Robertsb,2, Steven D. Cohena,d,* a

Toxicology Program, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06268, USA b Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA c Toxicology Program, Department of Pathobiology, University of Connecticut, Storrs, CT 06268, USA d Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, School of Pharmacy-Worcester, Worcester, MA 01608, USA Received 12 March 2004; accepted 7 June 2004 Available online 25 September 2004

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 g-glutamyl acceptor substrates for g-glutamyl transpeptidase (g-GT). The present study sought to determine if APAP-CYS-induced renal glutathione depletion is the result of disruption of the g-glutamyl cycle through interaction with g-GT. The results confirmed that APAP-CYS-induced renal GSH depletion was antagonized by the g-glutamyl transpeptidase (g-GT) inhibitor acivicin. In vitro analysis demonstrated that APAP-CYS is a g-glutamyl acceptor for both murine and bovine renal g-GT. Analysis of urine from mice pretreated with acivicin and then treated with APAP, APAP-CYS, or acetaminophen-glutathione identified a g-glutamyl-cysteinylacetaminophen 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 g-glutamyl cycle. D 2004 Elsevier Inc. All rights reserved. Keywords: Acetaminophen nephrotoxicity; Glutathione-conjugate-mediated toxicity; Acetaminophen-cysteine; g-Glutamyl transpeptidase; g-Glutamyl cycle; Acivicin

Introduction * Corresponding author. Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences, School of Pharmacy-Worcester, 19 Foster Street, Worcester, MA 01608, USA. Fax: +1 508 890 5618. E-mail address: [email protected] (S.D. Cohen). 1 Present address: Department of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA. 2 Present address: School of Pharmacy, University of Wisconsin, Madison, WI 53705-2222, USA. 0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2004.06.029

Acetaminophen (APAP) is a commonly used over-thecounter 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

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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 g-glutamyl transpeptidase (gglutamyl transpeptidase; g-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 acetaminophencysteine (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 kidneyselective 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 g-glutamyl acceptor substrate resulting in enhancement of GSH catabolism by g-GT and perturbation of the g-glutamyl cycle (Scheme 1). The gglutamyl cycle is responsible for maintenance of GSH

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homeostasis and may play a role in amino acid transport (Meister et al., 1979; Smith et al., 1991). Administration of g-glutamyl acceptor substrates (e.g., glycylglycine) to mice and rats increases transpeptidation by brush border g-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 g-GT levels, were considerably less susceptible to such GSH depletion after these substrates. Because APAPCYS is similar to benzylamine and amino acid g-glutamyl acceptors, it may also selectively deplete renal GSH by acting as a g-glutamyl acceptor (Scheme 1). If APAP-CYS were to function as a g-glutamyl acceptor and decrease renal GSH stores, this could explain how APAP-GSHderived metabolites contribute to APAP nephrotoxicity. The present study explores the hypothesized role of APAP-CYS as a g-glutamyl acceptor substrate with consequent implications for the mode of action in APAP-induced kidney toxicity.

Materials and methods Materials. Acivicin, 2-amino-methyl-1,3-propanediol, acetaminophen (APAP), bovine renal g-glutamyl transpeptidase (g-GT), bovine serum albumin (BSA), l-cysteine, 5,5V-dithiobis(2-nitrobenzoic acid) (DTNB), l-glutamic acid g ( p-nitroanilide), glutathione (reduced), glutathione reductase, glycylglycine, l-lysine, reduced nicotinamide adenine dinucleotide phosphate (NADPH), disodium ethyl-

Scheme 1. The g-glutamyl cycle is important in maintaining glutathione (GSH) homeostasis. The key enzyme in the cycle g-glutamyl transpeptidase (g-GT) is most abundant in the renal proximal tubule, the site of APAP’s acute renal injury. In mice and rats, administration of amino acid g-glutamyl acceptor substrates (e.g., glycylglycine (Gly-Gly)) results in renal-specific GSH depletion. In this scheme, APAP-CYS is shown as a hypothetical g-glutamyl acceptor substrate in place of Gly-Gly.

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inediaminetetraacetic acid (Na2EDTA), potassium carbonate, propylene glycol, o-phthaldehyde, sodium phosphate (monobasic and dibasic), and 2-vinylpyridine were purchased from Sigma (St. Louis, MO). Lowry-based detergent-compatible (DC) protein assay from BIO-RAD Laboratories (Hercules, CA). Meta-phosphoric acid and perchloric acid were obtained from Baker Chemical Co. (Phillipsburg, NJ). Acetic acid (AcOH), butanol (BuOH), hydrochloric acid (HCl), and methanol (MeOH) were obtained from Fischer Chemical Co. (Fair Lawn, NJ). Physiological saline was purchased from Butler (Columbus, OH). All other routine reagents were purchased from one of the above suppliers and all chemicals used were of reagent grade or better. Animals and treatments. Three- to five-month-old male Crl:CD-1(1CR)BR mice were purchased from Charles River Laboratories (Wilmington, MA). They were housed in a temperature and humidity controlled facility with 12-h light–dark cycles. Animals were held in steel wire-bottomed cages hanging over hardwood chips (Sani-Chips, P.J. Murphey Forest Products) and were fed Purina Rodent chow #5001 (Ralston Purina Co., St Louis, MO) ad libitum. Animals were allowed to acclimate to housing conditions for at least 1 week before initiation of experiments. Mice were fasted for 18 h before treatment between 8 and 11 am. All chemicals were administered in 50% propylene glycol/50% deionized distilled water vehicle at an injection volume of 5 ml kg
1, unless otherwise noted. For g-GT inhibition studies, the g-GT inhibitor acivicin (50 mg kg
1 in physiological saline) was administered intraperitoneally 30 min before intraperitoneal administration of APAP-CYS (50 mg kg
1) (Reed et al., 1980). For the urinary metabolite study, APAP (600 mg kg
1), APAP-CYS (50 mg kg
1), and acetaminophen-glutathione (APAP-GSH) (100 mg kg
1) were dosed intraperitoneally with or without 30 min of acivicin (50 mg kg
1, ip) pretreatment. All experiments included appropriate, vehicle-treated controls. Synthesis of APAP-CYS and APAP-GSH. APAP-CYS was synthesized and purified as described in the previous paper (Stern et al., 2004) based on the methods of Hammer and Kleinberg (1953), Focella et al. (1972), and Prasad et al. (1990). To prepare APAP-GSH, an identical procedure was employed substituting GSH (10.2 g, 33 mmol) for lcysteine. After reverse-phase purification of crude APAPGSH, the fractions containing the conjugate were lyophilized for 24–36 h to give white fluffy solid. Yield: 475 mg ˆ 8C (d). R f = 0.22. Nuclear magnetic (16%). mp = 164 A resonance (NMR, 200 MHz, D2O) d 1.95–2.01 (m, SH, glutamyl b + CH3), 2.31 (t, 2H, glutamyl g), 3.04–3.29 (m, 2H, cysteinyl b), 3.61–3.68 (m, 3H, glycyl a + glutamyl a), 4.30–4.37 (m, 1H, cysteinyl a), 6.80 (d, 1H), 7.08 (dd, 1H) ppm. MS (FAB+) m/e 456.1 (theoretical: 456.5). Anal. Calcd. for C18H24N4O8S Iˆz 1.5 H2O: C, 44.73; H, 5.63; N, 11.60. Found: C, 44.54; H, 5.69; N, 11.32.

Nuclear magnetic resonance (NMR) spectra were collected on an IBM Instruments 200-MHz FT-NMR spectrometer. Fast atom bombardment mass spectrometry (FAB MS) was conducted at the University of Utah Department of Chemistry using a Finnegan MAT 95. Elemental analysis was performed by Galbraith Laboratories (Knoxville, TN). Utilization of APAP-CYS as a c-glutamyl acceptor in vitro. The buffer and incubation conditions for this in vitro reaction were based on the g-GT assay method of Tate and Meister (1985). Incubations contained 1 unit ml
1 bovine renal g-GT, 1 mM APAP-CYS, 2 mM GSH in a total volume of 200 Al of 0.1 M Tris–HCl (pH 8.0, 378C). The reactions were terminated by addition of 200 Al of ice-cold serine-borate (10 mM each in 0.1 M Tris–HCl (pH 8.0)), followed by 400 Al ice-cold methanol. l-serine in the presence of borate has been shown to function as a transition state inhibitor of g-GT (Tate and Meister, 1978). The incubations were centrifuged at 1400  g for 10 min (48C) and filtered through a 0.2-Am filter. A Beckman System Gold HPLC with 166 NM detector, 128 NM solvent module, and Rheodyne rotary injector (model 7725i) were used for metabolite quantitation [20 Al injection, flow rate 1 ml min
1, A 254, aqueous mobile phase 12.5% MeOH/1% acetic acid, 3.9  250 mm Zorbax C-18 column]. Metabolites were identified by comparison of retention times to synthetic standards or by HPLC/MS/MS analysis. Metabolites were quantified by use of an APAP standard curve (Howie et al., 1977). Purification of murine renal brush border membrane. Murine (CD-1) renal brush border was prepared by the method of Malathi et al. (1979). Briefly, mouse renal cortical tissue was removed following cervical dislocation, and then washed with isotonic saline and homogenized (10% w/v) in ice-cold 50 mM mannitol/2 mM Tris–HCl buffer (pH 7.0). Following homogenization, 1 M CaCl2 was added to arrive at a final concentration of 10 mM CaCl2 in the homogenate. The homogenate was allowed to stir in an ice bath for 10 min before centrifugation at 3000  g for 15 min. The resulting supernatant was further centrifuged at 43 000  g for 20 min. The pellet fraction, containing the renal brush border, was then assayed for g-GT activity as described in the preceding paragraph. Protein determinations were performed by the method of Lowry et al. (1951) using the BIO-RAD DC Protein Assay (BIO-RAD Laboratories) and BSA as a surrogate protein standard. In vitro assessment of c-GT acceptors and renal c-GT activity. This in vitro reaction was based on the methods of Thompson and Meister (1975). The incubation mixture contained either 0.016 units of Sigma crude bovine renal gGT or 0.0025 units of purified CD-1 renal g-GT from renal brush borders of male CD-1 mice, 0.5 mM l-glutamic acid and p-nitroanilide (donor), and 0.13–40 mM of acceptor

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(glycylglycine, l-lysine, l-cysteine or APAP-CYS) in a total reaction volume of 200 Al 2-amino-methyl-1,3propanediol buffer (200 mM, pH 8.0, 378C). The formation of p-nitroanilide (e 405 nm = 9900 M
1 cm
1 ) was determined spectrophotometrically with measurements at 0–10 min. The assays included a buffer blank, g-GT negative control, and acceptor-free blank (to account for hydrolysis). Samples were run in triplicate. K m and V max values were estimated from Lineweaver–Burke plots. To determine renal g-GT activity, pre-purification homogenate and post-purification brush border test samples were diluted with reagent buffer to within the assay’s linear range. Incubations containing 40 mM glycylglycine acceptor and 10 Al of diluted test sample were assayed in triplicate under identical conditions as described for the acceptor studies above. Renal and urine thiol determination. Renal GSH was determined by the spectrophotometric method of Hissin and Hilf (1976) as described in Emeigh et al. (1996). This fluorometric assay is minimally affected by g-glutamyl cycle thiol products, such as cysteine and cysteinylglycine, which have approximately 0.25% the response of GSH (Cohn and Lyle, 1966). Total renal and urine glutathione (reduced and oxidized) were determined by the method of Griffith (1980) adapted for the microtiter plate, as described (Stern et al., 2004). Urine samples were diluted 1:2 with 2 N perchloric acid. Urine creatinine assay. Urine samples were stored at 48C and analyzed for creatinine within 1 week of collection. Creatinine levels were determined using a microtiter plate version of Sigma Diagnostics Creatinine kit (#555-A). Though creatinine clearance is commonly used as an indicator of renal injury, the purpose of creatinine determination in the metabolism study was to normalize urinary metabolite quantities. There was no significant variation in urinary creatinine excretion between any of the treatment groups used in the metabolism studies. Urine samples were diluted appropriately with distilled deionized water and used in the assay. Urine sample (18 Al), 3.0 mg dl
1 standard, or water blank was added to sets of duplicate wells in 96-well microtiter plates. To all wells, 176 Al of alkalinized picric acid solution was added, followed by 6 Al of acid reagent to one set of wells and 6 Al of water to the other set. The microtiter plate was then gently shaken and allowed to incubate at room temperature for 5 min. The plate was then read at 490 nm on a BIOTEK Ceres 900 microtiter plate reader (Winkooski, VT). Urine metabolites. Six-hour urine samples were diluted 1:2 with MeOH, centrifuged at 1200  g for 10 min at 48C (Sorvall RMC 14 refrigerated microcentrifuge, Dupont Co., Wilmington, DE, USA), and filtered through a 0.20-Am nylon filter. The samples were then further diluted 1:5 with distilled deionized water. A Beckman System Gold HPLC

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(Beckman Instruments, Inc., Fullerton, CA, USA) with a 166 NM detector, 128 NM solvent module, and Rheodyne rotary injector (model 7725i) were used for metabolite quantitation [20 Al injection, flow rate 1.7 ml min
1, A 254, 4.6  250 mm Zorbax SB 5 Am C18 reverse-phase column (MAC-MOD Analytical, Inc., Chadds Ford, PA, USA)]. A gradient mobile phase system was utilized (Moldeus, 1978). The gradient system consisted of mobile phase A, 1% acetic acid, and mobile phase B, 1% acetic acid-methanolethyl acetate (84.9–15–0.1). An initial isocratic system consisting of 75% A and 25% B was run from 0 to 7 min, followed by a gradient system from 7 to 27 min, ending in 1% A and 99% B run. To regenerate the initial isocratic system, a second gradient was run from 27 to 35 min ending in 75% A and 25% B. Metabolites were quantified by use of an APAP standard curve, as the molar extinction coefficient of APAP and its conjugated metabolites at 254 nm are essentially the same (Howie et al., 1977). The identification of metabolites was achieved by comparison of retention times to synthetic standards, except for the gglutamylcysteine-acetaminophen metabolite, which was characterized by HPLC/MS analysis. HPLC/MS and MS/MS. Atmospheric pressure mass spectra were determined on a Fison’s Quattro (Micromass Inc., Beverly, MA) tandem mass spectrometer. HPLC analyses were performed on a Hewlett Packard 1090 (Hewlett Packard, Palo Alto, CA) HPLC system using a Zorbax stable bond octyldecyl reverse phase column (2.1  150 mm, 3.5-Am particle size) (Mac Mod, Chatam Ford, PA). The column temperature was maintained at 308C. The mobile phase consisted of 0.05% (v/v) trifluoroacetic acid (TFA) in water as solvent A and 0.05% (v/v) TFA in acetonitrile as solvent B. Analyses were performed using a solvent gradient of 100% A to 100% B in 15 min with a flow rate of 0.417 ml min
1. Approximately one-tenth of the column effluent was diverted to the source of the mass spectrometer while the remainder was passed to the diode array detector. The diode array detector collected spectral data from A 198 to A 402 at a rate of 320 ms scan
1. The mass spectrometer source was operated in the positive ion electrospray (ESP) mode. The cone potential was set at 30 V and the source temperature was maintained at 1208C. Mass spectral data were collected from 100 to 600 Da at a rate of 600 ms scan
1. Five microliters of the in vitro isolate dissolved in water was analyzed by HPLC/MS to determine the molecular weight of the major constituent in the isolate. One major compound with a protonated molecular ion of m/z 400 was detected in the sample. One hundred microliters of the isolate dissolved in 0.05% TFA in water/acetonitrile (1:1) was further subjected to MS/MS analysis to determine the product ion spectrum of the m/z 400 ESP ion. The sample was introduced into the ESP source at a flow rate of 10 Al min
1. The ESP source was operated under the same conditions described for the

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HPLC/MS analysis. The protonated molecular ion of the compound of interest (m/z 400) was subjected to collisioninduced dissociation (CID) in argon (3.0  10
3 mBar) at a collision energy of 30 eV. HPLC/MS analysis of urinary g-GLU-CYS-APAP was analyzed by direct injection of urine samples, without prior isolation. Urine was diluted 1:10 with 0.05% HFBA. MS/ MS analysis of the urine metabolite samples was not possible do to concentration limitations. Statistical analyses. Statistical differences (n z 3, P V 0.05) were determined by Student’s t test or ANOVA, with post hoc comparisons made by Neuman–Keuls test, where appropriate.

Results Role of c-GT in APAP-CYS-induced reduction in renal GSH 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). To verify that the effect of APAP-CYS on renal GSH is mediated by gGT, groups of mice were pretreated with the g-GT inhibitor acivicin (50 mg kg
1, ip), 30 min before administration of APAP-CYS (Reed et al., 1980). If the mechanism behind APAP-CYS-induced renal GSH depletion involved enhancement of g-GT-dependent GSH catabolism, then acivicin pretreatment should block such depletion. Indeed, pretreatment of mice with acivicin resulted in approximately 20% less APAP-CYS-induced renal GSH depletion at 30 min than APAP-CYS alone

Fig. 2. Utilization of APAP-CYS as a g-glutamyl acceptor in vitro. Incubations contained 1 unit ml
1 bovine renal g-GT, 1 mM APAP-CYS, 2 mM GSH in a total volume of 200 Al of 0.1 M Tris–HCL (pH 8.0, 378C). The reactions were terminated by addition of 200 Al ice-cold serine-borate (10 mM–10 mM) in 0.1 M Tris–HCl (pH 8.0), followed by 400 Al ice-cold methanol. Metabolites in the incubation mixture were quantified by HPLC and identified by either mass spectrometry or by comparison of retention times to synthetic standards. Loss of APAP-CYS (solid bar) from the incubation is balanced by formation of g-glutamyl-cysteinyl-APAP, gGLU-CYS-APAP (striped bar). Data are presented as the average of replicate samples with variability b5%.

(Fig. 1). Interestingly, acivicin treatment alone also resulted in minor reductions in renal GSH. This phenomenon has been observed previously in studies utilizing perfused rat kidneys (Heuner et al., 1989). Given the short half-life of GSH in the kidney, approximately 30 min, it is likely that the acivicin-induced renal GSH depletion reflects decreased GSH precursor availability (Griffith and Meister, 1979a,b). Purification of the brush border fraction from renal homogenates resulted in an approximately 100-fold increase in g-GT specific activity (0.09 F 0.02 vs. 9.00 F 1.70 units mg
1 protein (mean F SE) for pre- and post-purification samples, respectively). Comparison of brush border g-GT activity from acivicinor vehicle-treated mice indicated that the acivicin treatment produced 99% inhibition of enzyme activity 30 min post-treatment. Assessment of APAP-CYS as a c-glutamyl acceptor substrate

Fig. 1. Effect of acivicin (AT-125) pretreatment on APAP-CYS-induced depletion of renal glutathione (GSH). Mice were pretreated intraperitoneally with 50 mg acivicin kg
1 or phosphate-buffered saline vehicle (V) 30 min before intraperitoneal treatment with 50 mg APAP-CYS kg
1. The mice were killed 30 min later and renal cortical tissue was analyzed for GSH. Data are presented as the mean F SE for groups of at least three animals. Bars with different letters are significantly different from one another ( P V 0.05), as determined by ANOVA.

An in vitro reaction was used to demonstrate that APAP-CYS is a suitable g-glutamyl acceptor substrate for the renal brush border enzyme g-GT. APAP-CYS was incubated with bovine g-GT and GSH for up to 10 min (Fig. 2). APAP metabolites in the reaction mixture were quantified by HPLC and identified either by comparison of retention time to synthetic standards or by mass spectrometry. The observed decrease of incubate concentration of APAP-CYS was balanced by the appearance of g-glutamylcysteinyl-APAP (g-GLU-CYS-APAP). Equilibrium between the substrate, APAP-CYS, and the product gGLU-CYS-APAP occurred within 2–5 min. At 10 min APAP-CYS was apparently regenerated via g-GT mediated cleavage of g-GLU-CYS-APAP. This apparent reversal of

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Table 1 Comparison of APAP-CYS with known g-glutamyl acceptors Acceptor

V max (Amol min
1 mg
1) Mean F SE, bovine/murine

K m (mM) Mean F SE, bovine/murine

(V max/K m) (ml min
1 mg
1) Mean F SE, bovine/murine

Glycylglycine APAP-CYS Cysteinea Lysine

2.2 1.0 1.0 0.9

6.6 F 2.0/6.2 F 2.7 8.3 F 1.1/9.0 F 1.8 6.9 F 0.5/5.6 F 2.4 25 F 7.2/51 F 5.1

0.36 0.12 0.14 0.04

F F F F

0.1/0.9 0.3/0.4 0.2/0.3 0.2/0.7

F F F F

0.2 0.1 0.1 0.2

F F F F

0.11/0.17 F 0.02/0.04 F 0.04/0.06 F 0.02/0.01 F

0.05 0.01 0.01 0.01

Note. The incubation mixtures contained either commercial bovine renal g-GT or purified murine CD-1 renal g-GT, l-glutamic acid g ( p-nitroanilide) (donor), and acceptor (glycylglycine, l-lysine, l-cysteine, or APAP-CYS) in a total reaction volume of 200 Al 2-amino-methyl-1,3-propanediol buffer (pH 8.0, 378C). The formation of p-nitroanilide (e 405 nm = 9900 M
1 cm
1) was determined spectrophotometrically at 405 nm with measurements at 0 and 5 min. Apparent Michaelis–Menten constants were determined using Lineweaver–Burke plots. a Cysteine was analyzed with an equal quantity of dithiothreitol to prevent cystine formation.

the reaction direction most likely occurs following degradation of GSH, the glutamyl donor substrate, to its constituent amino acids by g-GT-mediated transpeptidation and hydrolysis. Because GSH would no longer be available to compete with g-GLU-CYS-APAP for the donor site of g-GT, the g-GLU-CYS-APAP could then be cleaved, by gGT-mediated transpeptidation or hydrolysis, and thus regenerate APAP-CYS. These results document that APAP-CYS can function as a g-glutamyl acceptor and also suggest that g-GLU-CYS-APAP may function as a gglutamyl donor for bovine renal g-GT. The mass spectra and fragmentation analysis for the g-GLU-CYS-APAP isolate are described below. Spectrophotometric analysis demonstrated that apparent V max and K m values for bovine and murine g-GT utilizing APAP-CYS were within the range of endogenous gglutamyl acceptor substrates (Table 1). Bovine g-GT catalytic efficiencies (V max/K m) were 0.12 ml min
1 mg
1 utilizing APAP-CYS and 0.14 ml min
1 mg
1 utilizing lcysteine, and for murine g-GT the corresponding values were 0.04 and 0.06 ml min
1 mg
1, respectively. It appears that APAP arylation of l-cysteine has very little effect on the apparent Michaelis–Menten constants under these conditions. The K m values and order of the catalytic efficiencies for the acceptors in this study were similar to those determined for rat renal g-GT (Allison, 1985; Griffith et al., 1981).

Analysis of urinary metabolites Identification of the g-glutamyl cycle metabolite gGLU-CYS-APAP in vivo following APAP, APAP-CYS, and APAP-GSH treatment would be highly consistent with APAP-CYS functioning as a g-glutamyl acceptor in vivo. Previous studies have shown that partial inhibition of gGT increases urinary excretion of g-glutamyl derivatives of amino acid acceptors in mice (Anderson and Meister, 1986; Griffith et al., 1981). It was hypothesized that this phenomenon results from competition between GSH, which is elevated upon g-GT inhibition, and the gglutamyl derivatives for the g-GT g-glutamyl donor site or transport (Griffith et al., 1981). To determine if partial inhibition of g-GT with acivicin results in excretion of the g-GLU-CYS-APAP metabolite, mice were treated with either APAP (600 mg kg
1, 10 ml kg
1 in 50% PG, ip), APAP-CYS (50 mg kg
1, 5 ml kg
1 in 50% PG, ip), or APAP-GSH (100 mg kg
1, 5 ml kg
1 in 50% PG, ip) with or without 30-min pretreatment with acivicin (50 mg kg
1, 5 ml kg
1 in 50% PG). Mice were held in individual metabolism cages and urine was collected on ice for the ensuing 6 h. APAP metabolites were quantified by HPLC analysis and identified by comparison of their retention times to those of synthetic standards or by LC/MS analysis (see below). The 6-h urine metabolite profile for the specified treatment regimens is displayed in Table 2. The

Table 2 Urine metabolite study Treatment

APAP

Metabolite (lmol mg
1 creatinine) APAP-GLUC 380 F 31 APAP-SULF 20 F 1.8 APAP-CYS 73 F 5.8 APAP 62 F 4.0 g-GLU-CYS-APAP ND APAP-GSH ND APAP-NAC 2.8 F 1.6

Acivicin/APAP

APAP-CYS

Acivicin/APAP-CYS

APAP-GSH

Acivicin/APAP-GSH

360 29 93 81 4.7 1.6 2.9

ND ND 27 F 1.7 Trace ND ND 1.9 F 0.2

ND ND 28 F 3.4 Trace 4.5 F 0.4 ND 1.4 F 0.4

ND ND 41 F 4.7 Trace ND ND ND

ND ND 38 F 3.9 Trace 9.6 F 0.7 1.0F 0.2 ND

F F F F F F F

12 1.2 1.5 4.0 0.1 0.3 0.9

Note. Mice were treated with APAP (600 mg kg
1), APAP-CYS (50 mg kg
1), or APAP-GSH (100 mg kg
1) with or without 30 min of acivicin (50 mg kg
1) pretreatment. Animals were held in individual metabolism cages and urine was collected over ice for 6 h. Metabolites were quantified by HPLC analysis and identified by comparison to synthetic standards, except for g-GLU-CYS-APAP which was characterized by HPLC/MS. Abbreviations: acetaminophencysteine, APAP-CYS; acetaminophen-glucuronide, APAP-GLUC; acetaminophen-glutathione, APAP-GSH; acetaminophen-mercapturate, APAP-NAC; acetaminophen-sulfate, APAP-SUL; g-glutamylcysteinylacetaminophen, g-GLU-CYS-APAP. Data are expressed as the mean F SE for groups of at least three mice. ND = not detected.

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retention times were APAP-glucuronide approximately 6 min, APAP-sulfate approximately 9 min, APAP-CYS approximately 10 min, APAP approximately 11 min, gGLU-CYS-APAP approximately 16 min, APAP-GSH approximately 17 min, and APAP-N-acetylcysteine approximately 27 min. The g-GLU-CYS-APAP metabolite was identified in urine from mice pretreated with acivicin followed by APAP, APAP-CYS, or APAP-GSH (Table 2), suggesting that APAP-CYS can function as a g-glutamyl acceptor in vivo. Interestingly, this metabolite was not detected in the absence of the acivicin pretreatment. Also, it does not appear the g-GLU-CYS-APAP metabolite was excreted at the expense of other metabolites, because the amount of the individual metabolites was generally similar for both vehicle and acivicin pretreatment groups (Table 2). However, detection of g-GLU-CYS-APAP in the urine of mice given acivicin followed by APAP-CYS suggests that the gGLU-metabolite detected after APAP may be formed from APAP-CYS. As expected, acivicin pretreatment also increased urinary excretion of APAP-GSH because catabolism of this metabolite by g-GT would also have been diminished (Table 2). These data clearly support the hypothesis that APAP-CYS can act as a g-glutamyl acceptor for g-GT in vivo. The profile of the 6-h urinary APAP metabolites from the vehicle-pretreated APAP-dosed group agrees with a previous study by Fischer et al. (1981). In both studies, the order of APAP urinary metabolites, in decreasing quantity, was APAP-glucuronide N APAP N APAP-CYS N APAPsulfate N APAP-mercapturate (Table 2). Likewise, the profile of 6-h urinary APAP metabolites from the vehiclepretreated, APAP-CYS-, and APAP-GSH-treated groups were similar to a previous 2-h urine metabolite study by Fischer et al. (1985). In the present study, as in the previous Fischer et al. (1985) study, the order of APAP-CYS and APAP-GSH urinary metabolites, in decreasing quantity, was APAP-CYS N APAP-GSH N APAP-mercapturate (Table 2).

Identification of c-GLU-CYS-APAP In vitro isolate A major peak with a retention time of 4.77 min was observed in the HPLC analysis of the suspected APAP adduct isolate (data not shown). The UV spectrum of this compound had wavelength maxima at 244 and 298 nm (data not shown). The ESP mass spectrum of the compound had a base ion at m/z 400 that appeared to be the protonated molecular ion (data not shown). This is consistent with the molecular weight of 399 Da for g-GLU-CYS-APAP. Fig. 3 shows the CID spectrum of the m/z 400 ion obtained from the analysis of this isolate. Fig. 4 illustrates possible fragmentation mechanisms that would account for the major ions in the CID spectrum. The CID spectrum in Fig. 3 contains many ions in common with the spectrum generated by Mutlib et al. (2000) for synthetic g-GLU-CYS-APAP, including m/z 182, 208, 225, 254, 271, 337, and 382. These data suggest that the compound isolated from the in vitro reaction was g-GLU-CYS-APAP. Urine samples Though LC/MS/MS analysis of the urine samples was not possible, LC/MS data for the LC peak tentatively identified as g-GLU-CYS-APAP in the urine were identical to that of the in vitro isolate concerning retention time and fragmentation pattern (data not shown). Thus, these data suggest that the tentatively identified urinary g-GLU-CYSAPAP peak was in fact g-GLU-CYS-APAP.

Discussion We have documented that 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). The present study demonstrates that this APAPCYS-induced renal GSH depletion is antagonized by the

Fig. 3. CID spectrum of the m/z 400 ion isolated from the in vitro reaction mixture. The parent ion (m/z 400) was generated in the ESP mode at a cone potential of 30 V. The CID spectrum was generated in argon gas (3.0  10
4 mBar) at a collision energy of 30 eV.

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Fig. 4. Possible fragmentation mechanism of protonated g-glutamylcysteinylacetaminophen that could account for the major ions in the CID spectrum of the m/z 400 ion of the suspected acetaminophen adduct isolated from the in vitro reaction mixture. The symbols b1, y1Q, z1 represent the respective backbone fragmentation mechanism of a peptide as described by Biemann (1988) and Snyder (2000).

g-GT inhibitor acivicin (Fig. 1). There are many established mechanisms by which GSH-conjugate-derived APAP metabolites may alter renal homeostasis. In searching for potential mechanisms that could explain APAPCYS-induced renal GSH depletion, it is reasonable to first examine mechanisms attributed to other aromatic thioethers. Cysteine conjugate h-lyase has been shown to utilize aromatic substrates and some nephrotoxic phenolic GSH conjugates are thought to be bioactivated via the hlyase pathway (Cooper, 1998; Hong et al., 1997). The reactive thiols formed via the h-lyase pathway can covalently bind macromolecules, deplete nonprotein sulfhydryls, and cause lipid peroxidation (Chen et al., 1990). However, because pretreatment with aminooxyacetic acid, AOAA, an inhibitor of renal cysteine conjugate h-lyase, does not alter APAP nephrotoxicity, it is unlikely that the h-lyase pathway is involved (Emeigh et al., 1996). Alternatively, APAP thioethers may act like quinone thioether conjugates, which are structurally similar. Quinone thioethers can be targeted to the kidney by mercapturic acid pathway coupled transport processes (Lau et al., 1988). The mercapturic acid pathway has also been shown to regulate the redox potential of quinone thioethers (Monks and Lau, 1990). Once distributed to the kidney, the toxicity of quinone thioethers results from their

electrophilic properties and their ability to redox cycle, resulting in protein or DNA alkylation and lipid peroxidation (Bolton et al., 2000). These mechanisms, however, would not explain the g-GT dependency of APAP-CYSinduced renal GSH depletion. The mechanism of APAP-CYS-induced renal GSH depletion likely involves disruption of GSH homeostasis. GSH homeostasis could be altered by changes in GSH synthesis or degradation. The rate-limiting enzyme in GSH synthesis is g-glutamylcysteine synthase (g-GCS) (Meister et al., 1979). Inhibition of g-GCS by buthionine sulphoximine (BSO) results in a rapid depletion of rodent renal and hepatic GSH (Standeven and Wetterhahn, 1991). While it is conceivable that APAP-CYS could occupy the cysteine site of g-GCS and thereby inhibit GSH synthesis, this would not explain the g-GT-dependency or renal-selectivity of APAPCYS-induced GSH depletion (Fig. 1; Stern et al., 2004). A more likely g-glutamyl cycle target for APAP-CYS is g-GT, the enzyme responsible for GSH catabolism (Meister et al., 1979). Administration of g-glutamyl acceptor substrates to mice and rats results in a g-GT-dependent, renal-selective GSH depletion (Griffith et al., 1978; Palekar et al., 1975) similar to that observed following APAP-CYS treatment in the present study. This effect of g-glutamyl acceptor substrates on renal GSH levels has also been observed in

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freshly isolated rat kidney cells and perfused kidneys (Ormstad et al., 1980a, 1980b). Because APAP-CYS is structurally similar to amino acid g-glutamyl acceptor substrates, we theorized that APAP-CYS might also serve as a g-glutamyl acceptor and, thereby, decrease renal GSH. Consistent with this theory is the observation that some quinone thioethers with structural similarity to native substrates also interact with enzymes that process GSH and its derivatives (van Ommen et al., 1988; van Ommen et al., 1991). APAP-CYS functioned in vitro as a g-glutamyl acceptor substrate for both murine and bovine g-GT (Table 1 and Fig. 2). Given that the murine K m for APAP-CYS is 9.0 F 1.8 mM (mean F SE) and urinary APAP-CYS values were in the millimolar range following 600 mg APAP kg
1 ip, it is probable that APAP-CYS can function as an acceptor in vivo as well. The detection of g-GLU-CYS-APAP in the urine of acivicin-pretreated mice given APAP, APAP-CYS, or APAP-GSH suggests that APAP-CYS does indeed function as a g-glutamyl acceptor in vivo (Table 2). A similar conversion of cysteinyl leukotrienes to g-glutamyl derivatives has been observed previously in rat liver perfusion studies (Wettstein et al., 1989). The identification of the g-GLU-CYS-APAP metabolite is consistent with the findings of Mutlib et al. (2000), who have also identified g-GLU-CYS-APAP, as well as other gglutamyl acetaminophen derivatives, in the bile of rats given APAP. Mutlib et al. (2000) proposed that g-GT transpeptidation was involved in formation of the g-glutamyl APAP derivatives. In a separate study by Fischer et al. (1985), intravenous treatment of mice with APAP-CYS resulted in urinary excretion of a metabolite tentatively identified as APAP-GSH. In that study, the metabolite was identified from its reverse-phase HPLC retention time and the ability of g-GT to convert the metabolite to the APAPCYS conjugate. Both APAP-GSH and g-GLU-CYS-APAP have similar retention times, and g-GLU-CYS-APAP would be converted to APAP-CYS by such g-GT treatment. Therefore, it is possible that the metabolite reported by Fischer et al. (1985) was in fact g-GLU-CYS-APAP rather than APAP-GSH. In the present study, acivicin pretreatment was required in order for g-GLU-CYS-APAP to be detected in urine from mice given APAP or its conjugates (Table 2). It is possible that low concentrations of g-GLU-CYS-APAP were in fact present in urine samples from vehicle-pretreated mice, but were below the limits of detection. Fischer et al. (1985), using radiolabeled APAP-CYS, would have had greater sensitivity for detection of low quantities of g-GLU-CYS-APAP. The in vitro g-GT assay (Fig. 2) and APAP-CYS urine metabolite study (Table 2) suggest that g-GLU-CYS-APAP can also function as a g-glutamyl donor. The regeneration of APAP-CYS at the 10-min time point in Fig. 2 was most likely the result of g-GT-mediated cleavage of g-GLU-CYSAPAP. The APAP-CYS urine metabolite study (Table 2) suggests that under normal physiological conditions g-

GLU-CYS-APAP is generated and catabolized by g-GT in a cyclical process, in which APAP-CYS acts as a g-glutamyl acceptor and g-GLU-CYS-APAP acts as a g-glutamyl donor. In the in vivo study with acivicin one would expect tubular GSH to be elevated and thus to compete with gGLU-CYS-APAP for the g-GT donor site allowing g-GLUCYS-APAP to pass into the urine in detectable levels. Presumably, residual g-GT activity accounts for the synthesis of g-GLU-CYS-APAP, as treatment of mice with acivicin does not inhibit g-GT activity completely (Griffith and Meister, 1979). The g-glutamyl acceptor function of APAP-CYS may be responsible for the g-GT-dependent renal GSH depletion observed following APAP-CYS treatment (Fig. 1; Stern et al., 2004), because similar g-GT-dependent renal GSH depletion profiles have been observed following treatment of mice and rats with known g-glutamyl acceptor substrates (e.g., glycylglycine (GLY-GLY)) (Griffith et al., 1978; Palekar et al., 1975). In these previous studies, treatment of rats with 450 mg GLY-GLY kg
1 resulted in a rapid depletion of renal GSH to around 50% of control by 1 h, with only a minor 10% decline in hepatic GSH. This depletion was antagonized by pretreatment with the g-GT inhibitor serine-borate. Recently, Mutlib et al. (2001) have proposed that renal GSH depletion by 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2V-(methylsulfonyl)-[1-1V-biphenyl]-4-YL]-1-(trifuoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) is also the result of enhanced g-GT-dependent catabolism. Administration of DPC 423 to mice results in 50% depletion of total renal glutathione (reduced and oxidized) and excretion of a urinary g-glutamyl derivative of the benzylamine moiety. Further in vitro studies demonstrated that the g-glutamyl derivative of DPC 423 could be generated using renal S9 fractions and radiolabeled GSH. Acivicin treatment suppressed generation of the gglutamyl derivative both in vivo and in vitro, suggesting gGT involvement. Based on these data, Mutlib et al. (2001) have hypothesized that the benzylamine moiety of DPC 423 serves as a surrogate for a g-glutamyl acceptor amino acid by g-GT, resulting in formation of the g-glutamyl derivative and diminution of the endogenous renal GSH pool. Taken together with the accompanying study (Stern et al., 2004), the present results provide support for the hypothesis that APAP-CYS contributes to APAP nephrotoxicity by functioning as a g-glutamyl acceptor substrate with subsequent diminution of renal GSH. This would make less GSH available for renal detoxification of N-acetyl-p-benzoquinoneimine (NAPQI). This is depicted in Scheme 2. NAPQI is conjugated with cellular GSH in renal proximal tubule cells. This conjugate then enters the tubular lumen and is broken down to the cysteinyl derivative APAP-CYS by the sequential actions of g-GT and dipeptidase. The resulting elevation in the g-glutamyl acceptor APAP-CYS then increases g-GT catabolism of GSH, thereby interfering with further GSH-dependent detoxification of NAPQI. The g-GLU-CYS-APAP intermediate is free to cycle back to APAP-CYS either by the

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Scheme 2. Hypothesized role of g-glutamyl cycle involvement in APAP nephrotoxicity. APAP-CYS may potentiate APAP nephrotoxicity by functioning as a g-glutamyl acceptor and increasing renal GSH catabolism, leading to reduced detoxification of N-acetyl-pbenzoquinonimine (NAPQI) by glutathione (GSH) conjugation (CYP450 = cytochrome, DP = dipeptidase, GS = glutathione synthetase, g GC = g-glutamylcyclotransferase, g-GLUCYS-APAP = gglutamylcysteinylacetaminophen, g-GCS = g-glutamylcysteine synthetase, g-GT = g-glutamyltranspeptidase, NAT = n-acetyl transferase, 5-OX = oxoprolinase).

action of intracellular g-glutamylcyclotransferase or extracellular g-GT. Transport of g-GLU-CYS-APAP across the brush border into the proximal tubule could be mediated by amino acid carriers, as has been suggested for other cysteine conjugates (Wright et al., 1998). Because N-acetylation of APAP-CYS would prevent further transpeptidation, mercapturate formation can be thought of as a termination step in this renal GSH depletion cycle. Indeed, one of the physiological roles of mercapturate formation may be to prevent enhancement of g-GT-mediated catabolism by xenobiotic and endogenous cysteine conjugates. There is significant evidence in the clinical literature linking APAP use with perturbation of the g-glutamyl cycle, for example, oxoproline generated metabolic acidosis of unknown etiology (Creer et al., 1989; Pitt, 1990; Pitt and Hauser, 1998). It is important to note that in almost all the clinical cases identified the doses of APAP were in the therapeutic range. Obviously, the idiosyncratic nature of this metabolic acidosis would suggest the presence of sensitive subpopulations. However, a random sampling of pediatric urine specimens submitted for routine metabolic screening demonstrated a significant elevation in oxoproline excretion in specimens that were also identified as positive for APAP (Pitt, 1990). The mechanism underlying the elevation in oxoproline may therefore be universal. This association is further supported by a study in rats, in which chronic APAP exposure resulted in oxoprolinuria (Ghauri et al., 1993). Evidence has surfaced that early metabolic acidosis is also a common feature of APAP overdose and this condition often precedes detectable liver injury (Roth et al., 1999). To date,

no reasonable theory has been presented to account for APAP-induced oxoproline elevation. Because treatment of mice with g-glutamyl acceptor substrates results in elevation of renal oxoproline, it is intriguing to speculate that this phenomenon is due to the g-glutamyl acceptor function of APAP metabolites such as APAP-CYS (van der Werf et al., 1974). While other, as yet undiscovered, mechanisms may exist, the proposed g-glutamyl cycle pathway for the contribution of APAP-CYS to APAP-induced kidney injury represents a novel mechanism with potentially far reaching toxicological implications. Because Mutlib et al. (2000) claim to have identified g-glutamyl metabolites of several other undisclosed compounds, as well as APAP, this renal GSH depletion pathway could conceivably extend to other xenobiotics as well. Acknowledgment Supported in part by NIH ES07163 and the University of Utah College of Pharmacy. Underlying support for facilities from NCI grant 5 P30 CA42014 and NIH grant 1 S10 RR06262 is greatly appreciated. References Allison, D., 1985. g-Glutamyl transpeptidase: kinetics and mechanism. Methods Enzymol. 113, 419 – 437. Anderson, M.E., Meister, A., 1986. Inhibition of g-glutamyl transpeptidase and induction of glutathionuria by g-glutamyl amino acids. Proc. Natl. Acad. Sci. U.S.A. 83, 5029 – 5032.

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