Acetaminophen (APAP) is the most widely used. Selective Mitochondrial Glutathione Depletion by Ethanol Enhances Acetaminophen Toxicity in Rat Liver

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1 Selective Mitochondrial Glutathione Depletion by Ethanol Enhances Acetaminophen Toxicity in Rat Liver Ping Zhao, Thomas F. Kalhorn, and John T. Slattery Chronic alcohol consumption may potentiate acetaminophen (APAP) hepatotoxicity through enhanced formation of N-acetyl-p-benzoquinone imine (NAPQI) via induction of cytochrome P450 2E1 (CYP2E1). However, CYP2E1 induction appears to be insufficient to explain the claimed magnitude of the interaction. We assessed the role of selective depletion of liver mitochondrial glutathione (GSH) by chronic ethanol. Rats were fed the Lieber- DeCarli diet for 10 days or 6 weeks. APAP toxicity in liver slices (% glutathione-s-transferase released to the medium, GST release) and NAPQI toxicity in isolated liver mitochondria (succinate dehydrogenase inactivation, SDH) from these rats were compared with pair-fed controls. Ethanol induced CYP2E1 in both the 10-day and 6-week groups by 2-fold. APAP toxicity in liver slices was higher in the 6-week ethanol group than the 10-day ethanol group. Partial inhibition of NAPQI formation by CYP2E1 inhibitor diethyldithiocarbamate to that of pair-fed controls abolished APAP toxicity in the 10-day ethanol group only. Ethanol selectively depleted liver mitochondrial GSH only in the 6-week group (by 52%) without altering cytosolic GSH. Significantly greater GSH loss and APAP covalent binding were observed in liver slice mitochondria of the 6-week ethanol group. Isolated mitochondria of the 6-week ethanol group were 50% more susceptible to NAPQI ( mol/l) induced SDH inactivation. This increased susceptibility was reproduced in pairfed control mitochondria pretreated with diethylmaleate. In conclusion, 10-day ethanol feeding enhances APAP toxicity through CYP2E1 induction, whereas 6-week ethanol feeding potentiates APAP hepatotoxicity by inducing CYP2E1 and selectively depleting mitochondrial GSH. (HEPATOLOGY 2002;36: ) Acetaminophen (APAP) is the most widely used analgesic in the United States, being consumed by 23% of Americans each week. 1 Over the last several decades, more than 100 case reports have described a therapeutic misadventure in which alcoholics were claimed to be unusually susceptible to APAP hepatotoxicity. 2,3 These reports asserted that alcohol abusers displayed very high serum aspartate aminotransferase values after taking APAP at doses considered nontoxic. 3 When Abbreviations: APAP, acetaminophen; NAPQI, N-acetyl-p-benzoquinone imine; GSH, glutathione; CYP2E1, cytochrome P450 2E1; DEM, diethylmaleate; DDC, diethyldithiocarbamate; GS-APAP, GSH conjugate of APAP; HPLC, highperformance liquid chromatography; 6-OH CLZ, 6-hydroxy chlorzoxazone; ATP, adenosine-5-triphosphate; GST, glutathione-s-transferase ; SDH, succinate dehydrogenase; ELISA, enzyme-linked immunosorbent assay. From the Department of Pharmaceutics, University of Washington, Seattle, WA. Received January 28, 2002; accepted May 23, Supported by NIH GM Address reprint requests to: John T. Slattery, Ph.D., University of Washington, Department of Pharmaceutics, Box , Seattle, WA jts@u.washington.edu; fax: Copyright 2002 by the American Association for the Study of Liver Diseases /02/ $35.00/0 doi: /jhep liver biopsies were performed, most showed the central lobular necrosis characteristic of APAP-induced acute liver injury. 3 However, the existence of the syndrome is controversial and difficult to prove unequivocally in humans. 4-6 APAP hepatotoxicity is mediated by its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which is generated by liver cytochrome P450s and is detoxified by conjugation with hepatic glutathione (GSH). 7,8 The major P450 isoform that is responsible for NAPQI formation, cytochrome P450 2E1 (CYP2E1), 9 is induced by ethanol. This induction effect has been considered to be the mechanism for the enhanced APAP toxicity after chronic ethanol ingestion both in humans and experimental animals. 3,10 Ethanol induces CYP2E1 in part by a mechanism through which binding of ethanol to the active site protects the enzyme from degradation, 11,12 although increased de novo synthesis of CYP2E1 is also observed at high plasma ethanol concentrations. 13,14 The binding of ethanol to the active site of CYP2E1 competitively inhibits the oxidation of other substrates, including the for- 326

2 HEPATOLOGY, Vol. 36, No. 2, 2002 ZHAO, KALHORN, AND SLATTERY 327 mation of NAPQI from APAP, 15,16 regardless of the mechanism by which the abundance of CYP2E1 is actually increased. Enhanced CYP2E1 activity is observed only between the time ethanol is eliminated from the active site and the time CYP2E1 returns to the basal level dictated by the normal CYP2E1 turnover. The magnitude of CYP2E1 induction by ethanol is moderate, and the window of the increased activity appears to be narrow. 11,16,17 CYP2E1 induction by ethanol does not seem to be sufficient in itself to explain the magnitude of enhanced APAP toxicity that has been claimed in ethanol abusers, 3 although the apparent window of vulnerability to enhanced CYP2E1 activity may partially explain the relative rareness of the clinical observation given the ubiquity of ethanol abuse ( 18 million Americans according to National Institute of Alcoholics and Alcohol Abuse) and APAP use (see above). In addition to inducing CYP2E1, ethanol feeding for more than 3 weeks to rats depleted mitochondrial GSH without altering the GSH content in cytosol. 18 Collel et al. have found that chronic ethanol consumption alters mitochondrial membrane fluidity thereby interfering with GSH transport machinery located at the inner mitochondrial membrane. The subsequent loss of mitochondrial GSH can be corrected by coadministration of compounds that normalize the increased mitochondrial membrane tension, such as S-adenosine methionine or tauroursodeoxycholic acid. 19,20 Mitochondria are the critical target of NAPQI and become more susceptible to toxicity when GSH is depleted. 8,21-23 Chronic ethanol feeding selectively depletes mitochondrial GSH without altering cytosolic GSH. 18 Since NAPQI is formed by CYP2E1 at the endoplasmic reticulum, readily reacts with GSH in aqueous solution, 24 and the reaction is accelerated by cytosolic glutathione S-transferases, it is possible that normal cytosolic levels of GSH will prevent NAPQI from reaching GSH deprived mitochondria. In this study, we took advantage of the rat as a model of both human alcohol-induced liver disease and APAP hepatotoxicity to further evaluate the mechanisms by which chronic ethanol potentiates APAP-induced liver toxicity, 7,25 focusing on the separate roles played by induction of CYP2E1 (which occurs within 10 days of ethanol administration) and selective depletion of liver mitochondrial GSH (which requires 3-6 weeks of ethanol administration). Materials and Methods Chemicals Chloroform, 2,6-dichloroindophenol, diethylmaleate (DEM), silver oxide, and succinic acid were purchased from Aldrich Chemical Company (Milwaukee, WI). Monobromobiamine (Thiolate reagent) was bought from Calbiochem (La Jolla, CA). Acetonitrile, ethyl ether, and methanol were obtained from Fisher Scientific (Pittsburgh, PA). Ketamine and xylazine were obtained from Phoenix Pharmaceutical, Inc. (St. Joseph, MO). Ethanol was purchased from Pharmaco Products, Inc. (Brookfield, CT). All other reagents were from Sigma Chemical Co. (St. Louis, MO). NAPQI was synthesized by oxidation of APAP using silver oxide in chloroform and purified as described previously. 24 Identity was confirmed by 500 MHz 1-H NMR in chloroform: 2.34 (s, 3H), 6.65 (d, 2H), 7.00 (d, 2H). Animals Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were fed the Lieber-DeCarli liquid diet, in which 36% of total energy was contributed by ethanol. Ethanol-free animals were pair-fed an iso-caloric liquid diet (Bioserv Inc., Frenchtown, NJ). Rats were fed the liquid diet for either 10 days (starting at 250 g) or 6 weeks (starting at 180 g). The rats were maintained according to the Guidelines of Animal Care described by the National Academy of Sciences and published by the National Institutes of Health. At the end of the pretreatment period (at which point rat body weight was 300 to 350 g), rats were euthanized with ketamine/xylazine, and livers were removed and flushed with ice-cold Krebs-Henseleit buffer (Sigma Chemical Co.) supplemented with 5 mmol/l CaCl 2 and 40 mmol/l HEPES, ph 7.4. Livers to be used for mitochondria incubations were frozen in liquid nitrogen immediately upon removal and stored at 80 C. Livers used for liver slice experiments were kept on ice and processed for incubations within 30 minutes of harvesting the liver. Liver Slice Incubation Liver slices were prepared using a Krumdieck tissue slicer (Alabama R&D, Munford, AL) in ice-cold Krebs- Henseleit buffer by standard methods. 26 Slices ( 25 mg, 8 mm diameter) were incubated in oxygenated Krebs- Henseleit buffer containing 50 g/ml gentamicin (Life Technologies, Grand Island, NY) in 12-well tissue culture plates, two slices per milliliter of medium per well. The plates were placed into a 37 C shaking water bath under 95% O 2 /5% CO 2. After a 35-minute preincubation, slices were washed and incubated in the fresh buffer containing varying concentrations of APAP or 1 mmol/l chlorzoxazone. Preincubation of the liver slices from ethanol pretreated rats with diethyldithiocarbamate (DDC), a CYP2E1 inhibitor, 27 was accomplished by adding the

3 328 ZHAO, KALHORN, AND SLATTERY HEPATOLOGY, August 2002 reagent to the preincubation medium. The final concentration of DDC was 25 mol/l. Drug Metabolism. Metabolites of APAP and chlorzoxazone in the liver slice incubation were assayed after 90 minutes of incubation. Slices were homogenized in their incubation media. NAPQI formation in the liver slice incubation was quantified by APAP 3-GSH conjugate (GS-APAP) formation using the high performance liquid chromatography (HPLC) method of Chen et al. 28 CYP2E1 activity in the liver slice incubation was quantified by 6-hydroxychlorzoxazone formation (6-OH CLZ, a CYP2E1 activity marker) using HPLC (pentoxifylline as internal standard). 29,30 Hepatocellular Toxicity. After the 35-minute preincubation, slices were incubated with or without APAP for an additional 6 hours. Slices were then removed from the incubation medium and homogenized in the fresh Krebs- Henseleit buffer (2 slices in 500 L). One part of the homogenate was immediately extracted with trichloroacetic acid for the analysis of adenosine-5-triphosphate (ATP) using a Roche Diagnostics commercial kit (Mannheim, Germany). 31 APAP-induced ATP depletion was expressed as the ratio of ATP content with APAP to the ATP content without APAP. The remaining homogenate was stored at 80 C for the analysis of enzyme activities and protein concentration. APAP-induced hepatic necrosis in liver slices was measured by glutathione-s-transferase (GST) leakage. GST release has been found to be a good marker for liver cell injury due to its abundance in cytosol (accounting for up to 5% of total protein) and uniform distribution throughout the liver. 20,32 GST activity was measured using 1-chloro 2,4-dinitrobenzene as substrate. 33 GST released into the medium was expressed as a percentage of total enzyme activity (sum of medium slice activity). There was no decline in the total GST activity over the 6 hours of slice incubation; after 6 hours, the GST activities and ATP contents were not different between ethanol and pair-fed control groups in the absence of APAP (data not shown). Protein concentration was determined using Bio-Rad DC protein assay kit (Richmond, CA). Mitochondria Isolation and Incubation With NAPQI Liver mitochondria were isolated by homogenization and differential centrifugation in a buffer containing 200 mmol/l mannitol, 50 mmol/l sucrose, 10 mmol/l KCl, 1 mmol/l EDTA, 10 mmol/l HEPES-KOH (ph 7.4). 34 Liver was homogenized in 5 volume of mannitol-sucrose buffer and centrifuged at 1,000g for 5 minutes. The supernatant was then centrifuged at 8,000g for 10 minutes, and the resulting mitochondrial pellet was washed three times and reconstituted in the same buffer. The final protein concentration of the mitochondrial suspension was 4 mg/ml. All operations were performed at 0 to 4 C. Integrity of mitochondria was checked by the release of citrate synthase (mitochondrial matrix enzyme), which had to be less than 15% for the preparation to be accepted. The absence of cytosolic contamination of mitochondria was determined by the ratio of lactate dehydrogenase activities (LDH assay kit; Sigma Chemical Co.) in the mitochondria and homogenates ( 0.5%). In the case of DEM preincubation, homogenizing buffer was supplemented with 75 mol/l DEM, and mitochondria were washed and reconstituted in DEM-free buffer. Mitochondria were incubated with NAPQI according to the method of Burcham and Harman. 21 Briefly, mitochondria were preincubated at 37 C for 30 seconds before NAPQI was added. Mitochondria were incubated with NAPQI at 37 C for 90 seconds and were immediately tested for enzyme activity or derivatized for GSH measurement or GS-APAP and APAP measurement. Acetonitrile was used as solvent for NAPQI. The final volume of acetonitrile was 4% (vol/vol). Incubations without NAPQI also contained 4% acetonitrile. All incubation experiments were finished within 3 hours after mitochondria were isolated. Assays. Mitochondrial citrate synthase activity was measured according to Srere. 35 NAPQI-induced mitochondrial toxicity was measured by the inactivation of succinate dehydrogenase (SDH). SDH is a key mitochondrial enzyme involved in both the citrate cycle and oxidative phosphorylation. It has been found to be inactivated by NAPQI. 21 SDH was measured using succinic acid as substrate and 2, 6-dichloroindophenol to indicate reducing equivalents formed. 36 The SDH activities were not different between ethanol-treated and pair-fed control rats at the time mitochondria were isolated (data not shown). SDH inactivation was presented as the percentage of the NAPQI-free mitochondrial sample. Reduced GSH in mitochondria and homogenate were derivatized with monobromobimane at ph After acid precipitation (5% trichloroacetic acid) to remove protein, samples were subjected to HPLC analysis (Rainin microsorb column, C18, 3 ; 8% acetonitrile in 0.5% acetic acid, ph 2.6, 0.9 ml/min) with fluorometric detection ( Ex: 394 nm; Em: 480 nm). APAP and GS-APAP were measured as described for drug metabolism studies in the liver slice experiment. APAP-Protein Adduct Formation in Liver Slice Mitochondria Two hours after incubation with APAP, liver slices (6 slices) were homogenized with mannitol buffer and mito-

4 HEPATOLOGY, Vol. 36, No. 2, 2002 ZHAO, KALHORN, AND SLATTERY 329 chondria were isolated as described above. APAP-protein adduct formation in the mitochondria from liver slices was assayed by a competitive enzyme-linked immunosorbent assay (ELISA) according to Matthews et al. 38 The anti-apap antiserum and solid phase antigen, 4-acetamidobenzoic acid coupled to bovine serum albumin were generous gifts from Dr. Stephen M. Roberts of the University of Florida. The antiserum was raised in rabbits against 4-acetamidobenzoic acid coupled to keyhole limpet hemocyanin. 38 Solid-phase antigen was used as a standard. APAP adduct formation was also assessed in isolated liver mitochondria from 6-week fed rats treated with NAPQI. Statistical Analysis All data are reported as mean SD of varying numbers of animals. A two-tailed student s t test was used to examine the significance of differences between groups. P.05 is used as the criterion for significance. Results Six Weeks of Ethanol Caused Greater APAP Toxicity in Liver Slices Than 10 Days of Ethanol The effect of APAP on the leakage of GST from liver slices to the incubation medium over the 6-hour period of incubation is shown in Fig. 1. Minimal GST release was observed in the liver slices prepared from rats fed the ethanol-free diet at APAP concentrations up to 2 mmol/l (roughly the peak concentration after a human takes 20 g APAP). GST release in the 6-week ethanol group increased as APAP concentration increased. At 1 mmol/l APAP, GST release was 36% 16% for the liver slices prepared from rats fed ethanol for 10 days, and 69% Fig. 1. Percentage of GST released from liver slices incubated with varying concentrations of APAP for 6 hours. ( ) 10-day pair-fed control; ( ) 10-day ethanol pretreatment; ({) 6-week pair-fed control; (}) 6-week ethanol. *P.05 vs. 10-day ethanol group; **P.01 vs. 10-day ethanol group. N 4 to 7 rats. Fig. 2. Liver slice GS-APAP formation after 90 minutes of incubation with APAP. ( ) 10-day pair-fed control; ( ) 10-day ethanol; ({) 6-week pair-fed control; (}) 6-week ethanol. N 4 to 8 rats. 11% for rats fed ethanol for 6 weeks (P.01). At 2 mmol/l APAP, GST release was 41% 23% for the 10-day ethanol group and 92% 3% for the 6-week ethanol group (P.05). Ten-Day and 6-Week Ethanol Induced CYP2E1 Activity Similarly The effect of ethanol feeding on NAPQI formation in liver slices was assessed by analyzing GS-APAP formation in liver slices over the 90-minute incubation period. GS- APAP formation in liver slices prepared from both 10-day and 6-week ethanol groups was about 2-fold higher than their respective pair-fed controls (Fig. 2). Figure 3 shows the effect of the CYP2E1 inhibitor DDC on GS-APAP formation in liver slices prepared from ethanol-pretreated groups. At 1 mmol/l APAP, DDC decreased the formation rate of GS-APAP in liver slices from ethanol-fed groups to the level of pair-fed control groups (10-day group: vs nmol/mg protein, not significant; 6-week group: vs nmol/mg protein, not significant). CYP2E1 activity in liver slice incubations was assessed by the formation of 6-OH CLZ at a chlorzoxazone concentration of 1 mmol/l (Fig. 4). Ethanol feeding increased 6-OH CLZ formation (10-day group: 1.6-fold; 6-week group: 1.5-fold; P.05 vs. respective pair-fed controls). DDC decreased liver slice 6-OH CLZ formation rate in the ethanol-fed rats to the level of the pair-fed control groups (10-day group: vs nmol/mg protein, not significant; 6-week group: vs nmol/mg protein, not significant).

5 330 ZHAO, KALHORN, AND SLATTERY HEPATOLOGY, August 2002 Fig. 3. Effect of DDC on GS-APAP formation in liver slices prepared from rats fed ethanol diet for 10 days and 6 weeks. Liver slices from ethanol pretreated rats were incubated with DDC 25 mol/l for 35 minutes. DDC was washed out and slices were incubated with 1 mmol/l APAP for 90 minutes. ( ) Ethanol pretreatment; ( ) DDC ethanol pretreated group; ( ) pair-fed control group. *P.05 vs. pair-fed control. N 5 to 8 rats. Partial CYP2E1 Inhibition Did Not Completely Block APAP Toxicity in the 6-Week Ethanol Group The effect of DDC on APAP-induced ATP depletion in liver slices is shown in the upper panels of Fig. 5. When APAP was added to liver slice incubations prepared from ethanol-fed rats, ATP content was significantly diminished (Fig. 5, 10-day group: 60% and 69% ATP depletion at 0.5 and 1 mmol/l APAP, P.05 vs. pair-fed control; 6-week group: 61% at 0.5 mmol/l APAP, P.05, and 85% at 1 mmol/l APAP, P.01, vs. pair-fed Fig. 4. Effect of DDC on 6-OH CLZ formation in liver slices prepared from rats fed ethanol diet for 10 days and 6 weeks. Liver slices from ethanol pretreated rats were incubated with DDC 25 mol/l for 35 minutes. DDC was washed out and slices were incubated with 1 mmol/l chlorzoxazone for 90 minutes. ( ) Ethanol pretreatment; ( ) DDC ethanol pretreated group; ( ) pair-fed control group. *P.05 vs. pair-fed control. N 4 to 6 rats. Fig. 5. Effect of DDC on APAP induced liver slice ATP depletion (upper panels) and GST release (lower panels) from rats fed ethanol for 10 days and 6 weeks. Liver slices from ethanol pretreated rats were incubated with DDC 25 mol/l for 35 minutes. DDC was washed out and slices were incubated with 0.5 and 1 mmol/l APAP for 6 hours. ATP depletion was presented as the ratio of ATP content with APAP to ATP content without APAP. GST released into the medium was expressed as a percentage of enzyme activity in both medium and slices. ( ) Ethanol pretreated group; ( ) DDC ethanol pretreated group; ( ) ethanol-free group. *P.05; **P.01 vs. pair-fed control. N 4 to 6 rats. control). The addition of DDC to incubations almost completely prevented ATP depletion in the 10-day ethanol group (not significant vs. pair-fed control group). However, APAP-induced ATP depletion was only partially prevented by DDC in the 6-week ethanol group (26% at 0.5 mmol/l APAP; 46% at 1 mmol/l APAP, P.05 vs. pair-fed control group). The lower panels of Fig. 5 show the effect of DDC on APAP-induced GST release from liver slices. Ethanol feeding for 6 weeks caused a significant increase in GST release at both 0.5- and 1-mmol/L APAP concentrations (33% 13% and 69% 11%, P.05 and P.01 vs. the pair-fed control group). DDC pretreatment dampened GST release in rats fed ethanol for 6 weeks, but GST release was still significantly elevated above the pair-fed control group levels (25% 9% and 37% 19% at 0.5 and 1 mmol/l APAP, P.05). APAP-induced GST release was significantly elevated in liver slices from rats fed the ethanol diet for 10 days (23% 2% and 36% 16% at 0.5 and 1 mmol/l APAP, P.05). However, DDC pretreatment completely blocked GST release in the 10-day ethanol group (not significant vs. the pair-fed control group).

6 HEPATOLOGY, Vol. 36, No. 2, 2002 ZHAO, KALHORN, AND SLATTERY 331 Table 1. Total Hepatic GSH (nmol/mg protein) and Liver Mitochondrial GSH From Rats Receiving Ethanol for Different Time Periods Duration of Liquid Diet 6 Weeks 10 Days Control-Fed Ethanol-Fed Control-Fed Ethanol-Fed Total hepatic GSH (nmol/mg protein) Mitochondrial GSH (nmol/mg protein) * *P.05 compared with ethanol-free control (n 4-7 rats). Six Weeks of Ethanol Selectively Depleted Mitochondrial GSH Hepatic and mitochondrial GSH in rats treated with the liquid diets for 10 days and 6 weeks are summarized in Table 1. Total hepatic GSH levels were slightly lower in the ethanol-treated rats in both subchronic (10-day) and chronic (6-week) ethanol-fed conditions, but the values were not significantly different from the respective pairfed control groups. Liver mitochondrial GSH from the 6-week ethanol group was 52% lower (P.05) than the pair-fed control group. Ten days of ethanol feeding did not decrease liver mitochondrial GSH. These results agreed with those of Hirano et al. that selective depletion of liver mitochondrial GSH is caused by chronic ethanol ingestion. 18 APAP Covalent Binding Was Increased in Liver Slice Mitochondria of the 6-Week Ethanol Group Table 2 shows the results of APAP protein adduct formation and GSH levels in mitochondria of liver slices incubated with 1 mmol/l APAP for 2 hours. Mitochondrial GSH in the liver slices from both the 10-day and 6-week ethanol groups were significantly lower than respective control groups (0.9 and 0.4 nmol/mg protein, P.05 vs. pair-fed controls). GSH in liver slices of the 6-week ethanol group was also significantly lower than that in the 10-day ethanol group (P.05), which was 10% of the pair-fed control group in the absence of APAP. Accordingly, APAP adduct formation in mitochondria was significantly increased in liver slices from 6-week ethanol fed rats as compared with pair-fed controls and 10-day ethanol groups ( pmol adduct/mg protein, P.05 vs. controls and 10-day ethanol groups). GSH-Depleted Liver Mitochondria From 6-Week Ethanol-Fed Rats Were More Susceptible to NAPQI Toxicity NAPQI caused a concentration-dependent decrease of GSH in liver mitochondria isolated from rats given either ethanol or ethanol-free diet (Fig. 6, top panels). At each NAPQI concentration (25 to 165 mol/l), the 6-week ethanol treatment group had significantly lower GSH levels than the pair-fed control (P.05), whereas no difference was found between the 10-day ethanol group and its respective control group. Mitochondrial susceptibility to NAPQI was assessed by SDH inactivation (Fig. 6, bottom panels). Mitochondrial SDH was not affected by NAPQI concentrations below 50 mol/l in either the ethanol-fed group or the pair-fed control group. In the 6-week ethanol group, SDH inactivation was significantly greater than its pair-fed control group at concentrations of 75 mol/l or greater NAPQI (P.05). At 165 mol/l NAPQI, the inactivation was about 90% in the 6-week ethanol group as compared with 70% in the 6-week pair-fed control group. Ten days of ethanol did not alter mitochondrial susceptibility to NAPQI. Mitochondrial GSH in the 6-week ethanol-free group was decreased to about 50% by the GSH-depleting agent DEM, which was similar to the initial GSH level of the 6-week ethanol group. NAPQI-induced GSH depletion and Table 2. Mitochondria APAP Protein Adduct Formation and Mitochondrial GSH in Liver Slices Incubated With 1 mmol/l APAP for 2 Hours Duration of Liquid Diet 6 Weeks 10 Days Control-Fed Ethanol-Fed Control-Fed Ethanol-Fed Adduct formation pmol/mg mitochondrial protein * Mitochondrial GSH nmol/mg protein * * % of pair-fed control without APAP (66%) (12%) (63%) (22%) NOTE. N 5-7 rats for GSH; n 5-7 measurements for adduct formation in mitochondria from pooled slices from each group. *P.05 compared with ethanol-free control. P.05 vs. 10-day ethanol group.

7 332 ZHAO, KALHORN, AND SLATTERY HEPATOLOGY, August 2002 Fig. 6. Effect of ethanol-feeding on NAPQI induced GSH depletion (upper panels) and SDH inactivation (lower panels) in isolated liver mitochondria. Left: mitochondria from rats pretreated liquid diet for 10 days; right, 6 weeks. NAPQI was incubated with mitochondria for 90 seconds after a 30-second preincubation at 37 C. ( ) Pair-fed control; ( ) ethanol; (}) 6-week pair-fed control preincubated with 75 mol/l DEM during the mitochondrial preparation. *P.05 vs. 6-week ethanol group and DEM pretreated 6-week control group. N 5 to 7 rats. SDH inactivation in the DEM-treated mitochondria was similar to that observed in rats fed ethanol for 6 weeks. At the end of the mitochondrial incubations with NAPQI, APAP and GS-APAP were quantified (Fig. 7). The amount of NAPQI added was 18.8, 25.0, and 41.3 nmol/mg mitochondrial protein for 75, 100, and 165 mol/l NAPQI, respectively. The formation rates of APAP by reduction of NAPQI did not vary by ethanol pretreatment (Fig. 7, bottom panel). Within 90 seconds, more than 50% of NAPQI was reduced to APAP; most of the remaining NAPQI was recovered in the form of GS- APAP in the 10-day ethanol group and pair-fed control groups. However, GS-APAP formation in the 6-week ethanol group was 34%, 35%, and 40% lower than its pair-fed control at 75, 100, and 165 mol/l NAPQI (P.05), indicating that a significant fraction of NAPQI dose was lost to a process other than reduction or GSH conjugation. APAP covalent binding in mitochondria treated with NAPQI were tested by competitive ELISA. APAP adduct formation in the 6-week ethanol group was higher than the respective ethanol-free control at 165 mol/l NAPQI ( vs pmol/mg protein, P.01). These results also show that NAPQI was completely lost over the 90-second period of mitochondrial incubation. Discussion The major findings of this study include (1) 6-week but not 10-day ethanol feeding selectively depleted rat liver mitochondrial GSH, which enhanced NAPQI-induced mitochondrial toxicity; (2) ethanol feeding enhanced APAP-induced hepatotoxicity in liver slices, but the degree of toxicity was less severe following 10 days of ethanol than 6 weeks; (3) both 10-day and 6-week ethanol feeding increased liver slice NAPQI formation by about 2-fold; (4) DDC pretreatment decreased liver slice NAPQI formation in both ethanol treatment groups to that of ethanol-free groups, completely blocking APAP toxicity in the 10-day ethanol group, but only partially preventing APAP toxicity in liver slices prepared from 6-week ethanol treated rats; (5) APAP protein adduct formation was greater in mitochondria of liver slices prepared from 6-week ethanol-fed rats than from liver slices prepared from rats fed ethanol for 10 days. The induction of CYP2E1 by ethanol has been suggested to be the major mechanism contributing to the enhanced APAP toxicity in both chronic ethanol-fed animals and alcoholics. 3,10 In this study, APAP hepatotoxicity was much greater after 6 weeks of ethanol administration than after 10 days of ethanol, despite a similar magnitude of increased NAPQI formation by CYP2E1. Our observations of a 2-fold CYP2E1 induction are consistent with previous reports. 17 DDC, a CYP2E1 inhibitor, 27 blocked the enhanced APAP toxic- Fig. 7. APAP (upper panels) and GS-APAP (lower panels) formations in mitochondria incubated with NAPQI. Left: mitochondria from rats pretreated liquid diet for 10 days; right, 6 weeks. NAPQI was incubated with mitochondria for 90 seconds after a 30-second preincubation at 37 C. NAPQI doses were 18.8, 25.0, and 41.3 nmol/mg mitochondrial protein for 75, 100, and 165 mol/l, respectively. ( ) Pair-fed control; ( ) ethanol. *P.05. N 4 to 5 rats.

8 HEPATOLOGY, Vol. 36, No. 2, 2002 ZHAO, KALHORN, AND SLATTERY 333 *The NAPQI concentration in the mitochondrial incubations ranged from 25 to 165 M. It is difficult to estimate the concentration of NAPQI present in the hepatocyte following a toxic dose of APAP. However, Figure 7 shows that more than half of the NAPQI added was reduced to APAP and the rest was conjugated with GSH over the 90-second incubation period. Thus, while M concentrations of NAPQI are likely to exceed in vivo levels initially, it is lost very rapidly from the incubation. In any event, the results clearly show that mitochondria with diminished GSH stores are at greater risk of NAPQI toxicity. ity in liver slices prepared from rats fed ethanol for 10 days, but not in liver slices prepared from 6-week ethanoltreated rats. Mechanism(s) other than CYP2E1 induction must contribute to the enhanced APAP toxicity in rats fed ethanol for 6 weeks. GSH is a major defense mechanism against oxidative stress within mitochondria. 39 GSH also serves as scavenger to toxic electrophiles such as NAPQI. 8,40 Mitochondrial GSH depletion by NAPQI is followed by increased covalent binding of NAPQI in this organelle, disruption of mitochondrial structure and function, and ATP depletion. These events immediately precede hepatocellular necrosis Chronic ethanol has been found to profoundly alter mitochondria, 41 including the selective depletion of mitochondrial GSH 18 as observed in this study. Cytosolic GSH was not influenced by ethanol, which is also consistent with previous findings. 18 The importance of mitochondrial GSH was confirmed in isolated mitochondria incubated with NAPQI. NAPQI more readily depleted GSH to critical levels in liver mitochondria isolated from rats fed ethanol for 6 weeks than in rats fed an ethanol-free diet due to the significantly lower initial GSH levels. Consequently, NAPQI-induced mitochondrial damage, as measured by SDH inactivation, was more pronounced. In isolated liver mitochondria incubated with NAPQI,* GS-APAP formation was significantly lower in the 6-week ethanol group, and competitive ELISA detected more extensive covalent binding in the 6-week ethanol group. To evaluate the potential role of mechanisms other than GSH depletion as contributors to the enhanced susceptibility of mitochondria to NAPQI, we depleted mitochondiral GSH with DEM from the pair-fed control group, which is otherwise nontoxic. DEM depleted mitochondrial GSH to the level observed in mitochondria isolated from livers of rats that received ethanol for 6 weeks, simultaneously rendering them equal susceptibility to NAPQI toxicity. This result shows that the enhanced susceptibility of hepatocellular mitochondria to NAPQI following 6 weeks of ethanol is largely, if not completely, due to mitochondrial GSH depletion (Fig. 6). We have not directly addressed the other effects associated with long-term ethanol administration that may participate in enhancing APAP hepatotoxicity. The activation of Kupffer cells by endotoxin and the consequent up-regulation of proinflammatory cytokines, down-regulation of anti-inflammatory cytokines, and increased reactive oxygen species associated with chronic alcohol consumption also may contribute to the enhanced APAP toxicity However, the first insult leading to APAPinduced hepatotoxicity is to mitochondria. Thus, if mechanisms related to Kupffer cell activation and regulation of cytokines contribute to the enhanced hepatotoxicity, their roles would seem to be to amplify the damage beyond that through increased NAPQI formation and decreased NAPQI detoxification (CYP2E1 induction and selective mitochondrial GSH depletion, respectively). We conclude that 10-day ethanol feeding enhances APAP toxicity by inducing CYP2E1, whereas 6 weeks of ethanol potentiates APAP hepatotoxicity via both selective depletion of mitochondrial GSH and CYP2E1 induction, despite unchanged cytosolic GSH stores. The two mechanisms appear to contribute equally and largely explain the enhanced APAP toxicity in liver slices of rats fed ethanol for 6 weeks. If this mechanism holds in humans, depletion of hepatic mitochondrial GSH by longterm ethanol could account for potentiation of APAP toxicity even when ethanol is taken simultaneously with APAP, thereby inhibiting NAPQI formation. The combined mechanisms of CYP2E1 induction and mitochondrial GSH depletion may explain the unusually high serum aspartate transaminase described in human abusers of ethanol who also consumed APAP. 2,3 While the mechanisms by which ethanol enhances the toxicity of APAP are becoming clearer, the interaction does not appear to render therapeutic doses of APAP dangerous to the alcoholic patient. We observed enhanced APAP toxicity in 6-week ethanol-fed rats at APAP concentrations of 0.5 mmol/l and 1 mmol/l. These concentrations translate into approximately 5- and 10-g single doses in humans, which are considerably higher than the recommended dose for adults (1 g every 4-6 hours with a maximum daily dose of 4 g according to the label). Kuffner et al. have recently shown that 1 g APAP given 4 times daily for 2 days to 102 alcoholic patients entering detoxification had no effect on the coagulation indices, serum aspartate aminotransferase, or alanine aminotransferase, which were essentially identical to a group of 99 alcoholics who received placebo. 6 It is likely that chronic ethanol potentiates APAP hepatotoxicity in humans by the mechanisms observed in rats (a very good model species for liver changes induced by both APAP and ethanol). However, the lower limit of APAP dose that will produce toxicity in the human abuser of alcohol is not known and will be difficult to establish.

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