Vascular and Hepatocellular Peroxynitrite Formation during Acetaminophen Toxicity: Role of Mitochondrial Oxidant Stress

Tamara R. Knight, Angela Kurtz, Mary Lynn Bajt, Jack A. Hinson and Hartmut Jaeschke,1

Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Received February 14, 2001; accepted May 7, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peroxynitrite may be involved in acetaminophen-induced liver damage. However, it is unclear if peroxynitrite is generated in hepatocytes or in the vasculature. To address this question, we treated C3Heb/FeJ mice with 300 mg/kg acetaminophen and assessed nitrotyrosine protein adducts as indicator for peroxynitrite formation. Vascular nitrotyrosine staining was evident before liver injury between 0.5 and 2 h after acetaminophen treatment. However, liver injury developed parallel to hepatocellular nitrotyrosine staining between 2 and 6 h after acetaminophen. The mitochondrial content of glutathione disulfide, as indicator of reactive oxygen formation determined 6 h after acetaminophen, increased from 2.8 ± 0.6% in controls to 23.5 ± 5.1%. A high dose of allopurinol (100 mg/kg) strongly attenuated acetaminophen protein-adduct formation and prevented the mitochondrial oxidant stress and liver injury after acetaminophen. Lower doses of allopurinol, which are equally effective in inhibiting xanthine oxidase, were not protective and had no effect on nitrotyrosine staining and acetaminophen protein adduct formation. In vitro experiments showed that allopurinol is not a direct scavenger of peroxynitrite. We conclude that there is vascular peroxynitrite formation during the first 2 h after acetaminophen treatment. On the other hand, reactive metabolites of acetaminophen bind to intracellular proteins and cause mitochondrial dysfunction and superoxide formation. Mitochondrial superoxide reacts with nitric oxide to form peroxynitrite, which is responsible for intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite for hepatocellular peroxynitrite formation and liver injury remains to be established.

Key Words: peroxynitrite; nitrotyrosine; acetaminophen; allopurinol; liver failure; mitochondria; oxidant stress.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acetaminophen is a commonly used analgesic and antipyretic, which is considered very safe in the therapeutic range. In overdose, it produces centrilobular hepatic necrosis in humans and animals. It is well established that the formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) during the metabolism of acetaminophen by cytochrome P-450 is an important step in the development of the hepatotoxicity (Dahlin et al., 1984Go). NAPQI is detoxified by glutathione, resulting in the depletion of this sulfhydryl reagent (Mitchell et al., 1973Go). If NAPQI formation exceeds the capacity of cellular glutathione, NAPQI covalently binds to intracellular proteins (Jollow et al., 1973Go). In recent years, a substantial number of proteins have been identified as targets for NAPQI (Pumford et al., 1997Go; Cohen and Khairallah, 1997Go; Qui et al., 1998). However, none of these proteins appeared to be inactivated to the extent that it could explain massive cell necrosis. Therefore, the molecular mechanism of acetaminophen-induced hepatocellular necrosis is not completely understood.

More recently, the concept emerged that nitric oxide and peroxynitrite may be critical mediators for acetaminophen hepatotoxicity (Gardner et al., 1998Go, 1999Go; Hinson et al., 1998Go; Michael et al., 1999Go). Peroxynitrite is a strong oxidant generated by the spontaneous reaction of nitric oxide and superoxide (Koppenol, 1998Go). Peroxynitrite or secondary metabolites can cause tyrosine nitration as well as induce oxidative damage to proteins, DNA and lipids (Squadrito and Pryor, 1998Go). During acetaminophen toxicity, the inducible nitric oxide synthase (iNOS) is upregulated in the liver (Gardner et al., 1998Go) and there is evidence for increased formation of NO (Gardner et al., 1998Go, Hinson et al., 1998Go). Reduced liver injury after acetaminophen was observed in animals treated with the iNOS inhibitor aminoguanidine (Gardner et al., 1998Go) and in iNOS knock-out mice (Gardner et al., 1999Go). However, the location of peroxynitrite formation remains unclear. Inhibitors of Kupffer cell activity, which reduce the capacity of Kupffer cells to generate reactive oxygen (Liu et al., 1995Go), attenuated hepatocellular nitrotyrosine staining (Michael et al., 1999Go) and acetaminophen-induced liver injury (Goldin et al., 1996Go; Laskin et al., 1995Go; Michael et al., 1999Go). This suggests that the resident macrophages of the liver could be a source of superoxide and/or nitric oxide after acetaminophen treatment. However, intracellular sources of reactive oxygen such as mitochondria cannot be excluded (Jaeschke, 1990Go). Thus, the objective of this investigation was to differentiate formation of peroxynitrite in the vascular space and in hepatocytes during the development of acetaminophen-induced liver injury and to address whether mitochondria-derived superoxide could be responsible for hepatocellular peroxynitrite generation and injury.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male C3Heb/FeJ mice (Jackson Laboratory, Bar Harbor, Maine), with an average weight of 18 to 20 g, were housed in an environmentally controlled room with 12-h light/dark cycle and allowed free access to food and water. The animals were fasted overnight before the experiments. Animals received an intraperitoneal injection of 300 mg/kg or 150 mg/kg acetaminophen (Sigma Chemical Co., St. Louis, MO) dissolved in warm saline solution (15 mg/ml). Some of the animals were treated twice with allopurinol (10 or 100 mg/kg) (Sigma) suspended in water. The first dose of allopurinol was administered by gavage 18 h before, and the second dose 1 h before acetaminophen. Control animals received only water (16 ml/kg). Some animals were treated ip with 250 mg/kg phorone (Aldrich Chemical Co., Inc. Milwaukee, WI) dissolved in corn oil to deplete hepatic glutathione levels (Jaeschke et al., 1987Go).

Experimental protocols.
At selected times after acetaminophen treatment, the animals were killed by cervical dislocation. Blood was drawn from the vena cava into heparinized syringes and centrifuged. The plasma was used for determination of alanine aminotransferase (ALT) activities. Immediately after collecting the blood, the livers were excised and rinsed in saline. A small section from each liver was placed in 10% phosphate-buffered formalin to be used in immunohistochemical analysis. A portion of the remaining liver was homogenized for isolation of mitochondria or frozen in liquid nitrogen and stored at –80°C for later analysis of glutathione.

Isolation of mitochondria.
A portion of the liver was homogenized in ice-cold isolation buffer (pH 7.4) containing 220 mM mannitol, 70 mM sucrose, 2.5 mM Hepes, 10 mM EDTA, 1 mM EGTA, and 0.1% bovine serum albumin.

The liver homogenate was centrifuged at 600 x g for 8 min at 4°C to remove nuclei and cellular debris. The supernatant was removed and centrifuged at 10,000 x g for 10 min at 4°C to pellet the mitochondria; the pellet was washed once with 2 ml of isolation buffer. The mitochondrial pellet was resuspended in 3% sulfosalicylic acid containing 0.1 mM EDTA, vigorously vortexed, and centrifuged to sediment the precipitated protein. A part of the supernatant was diluted in 100 mM potassium phosphate buffer (pH 6.5) for the determination of total glutathione (GSH + GSSG) and another part was added to 10 mM N-ethylmaleimide (NEM) in potassium phosphate buffer for the determination of glutathione disulfide (GSSG).

Methods.
Plasma ALT activities were determined with the test kit DG 159- UV (Sigma Chem. Co., St. Louis, MO) and expressed as IU/liter. Protein concentrations were assayed using the bicinchoninic acid kit (Pierce, Rockford, IL). Plasma levels of nitrite/nitrate were determined with the Colorimetric Non-Enzymatic Nitric Oxide Assay Kit (Oxford Biochemical Research, Inc., Oxford, MI). Total soluble GSH and GSSG were measured in the liver homogenate and mitochondrial homogenate with a modified method of Tietze, as described in detail (Jaeschke and Mitchell, 1990Go). Briefly, the frozen tissue or isolated mitochondria were homogenized at 0°C in 3% sulfosalicylic acid containing 0.1 mM EDTA. An aliquot of the homogenate was added to 10 mM NEM in phosphate buffer (KPP) and another aliquot was added to 0.01 N HCl. The NEM-KPP sample was centrifuged and the supernatant was passed through a C18 cartridge to remove free NEM and NEM-GSH adducts (Sep-pak; Waters Associates, Waltham, MA). The HCl sample was centrifuged and the supernatant was diluted with KPP. All samples were assayed using dithionitrobenzoic acid (DTNB). All data are expressed in GSH-equivalents.

Immunohistochemistry.
Protein adducts of acetaminophen and nitrotyrosine were assessed by immunohistochemistry as described in detail previously (Michael et al., 1999Go) using an anti-nitrotyrosine antibody (Hinson et al., 2000Go) or an anti-acetaminophen antiserum (Matthews et al., 1996Go). Some of the stained sections were evaluated independently by two of the authors. The extent of the staining and the intensity of the staining was graded separately for vascular staining and parenchymal cell staining. The maximal staining in each compartment was set as 100% (2-h time point for the vasculature; 6 h for parenchymal cells) and the percentage estimated for earlier time points.

Western blotting.
The expression of iNOS protein in the liver was determined by Western blotting as previously described for other proteins (Bajt et al., 2000Go; Lawson et al., 1999Go). Briefly, liver tissue was homogenized in 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.5) containing 5 mM EDTA, 2 mM DTT (dithiothreitol), 0.1% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 1 µg/ml pepstatin, leupeptin, and aprotinin. Homogenates were centrifuged at 14,000 x g at 4°C for 20 min. Cytosolic extracts (10 µg per lane) were resolved by 4–20% SDS–polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to polyvinylidine difluoride membranes (PVDF, Immobilin-P, Millipore, Bedford, MA). The membranes were first blocked with 5% milk in TBS (20 mM Tris, 15 M NaCl, 0.1% Tween 20, and 0.1% BSA) overnight at 4°C, followed by incubation with primary antibody for 2 h at room temperature. A rabbit polyclonal iNOS antibody (Upstate Biotechnology, Lake Placid, NY) was used as a primary antibody. The membranes were washed and then incubated with the secondary antibody anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology). Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech. Inc., Piscataway, NJ), according to the manufacturer's instructions. Densitometric analysis of the gels was performed with a GS170 Calibrated Imaging Densitometer (Biorad, Hercules, CA) using Quantity One 4.0.3 software (Biorad).

Nitration of BSA in vitro.
The nitration of proteins by peroxynitrite (Upstate Biotechnology, Lake Placid, NY) was determined spectrophotometrically at 438 nm as the intensely yellow phenolate of nitrotyrosine (Hinson et al., 2000Go). Briefly, BSA (Sigma Chem. Co., St. Louis, MO) was added to 60 mM carbonate buffer (pH 9.6) with a final concentration of 2 mg/ml. Varying concentrations (50 µM, 200 µM, 500 µM, 1 mM) of allopurinol or N-acetylcysteine were added to the BSA-carbonate buffer. Peroxynitrite was then added to each solution (final concentration of 1.40 mM) to nitrate BSA. A spectrum was recorded and the amount of nitrotyrosine in the peroxynitrite-treated BSA was determined at the absorbance maximum of the phenolate ion at 438 nm.

Statistics.
All results were expressed as mean ± SE. Comparisons between multiple groups were performed with one-way ANOVA followed by Bonferroni t-test. If the data were not normally distributed, we used the Kruskal-Wallis Test (nonparametric ANOVA) followed by Dunn's Multiple Comparisons Test; p < 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Administration of a moderately toxic dose of 300 mg/kg acetaminophen caused liver injury in C3Heb/FeJ mice. Based on the release of ALT, cell damage was first evident at 2 h (Fig. 1Go). The injury progressed to severe damage at 6 h. Immunohistochemical staining for nitrotyrosine (NT) protein adducts in liver sections of controls indicated no adduct formation (Fig. 2AGo). However, at 1 h after acetaminophen administration, an exclusive staining in the vasculature around central veins was detectable (Fig. 2BGo). There was no staining of hepatocytes at that time. In contrast, at 4 h after acetaminophen treatment, individual hepatocytes in the pericentral area were intensely stained for NT protein adducts (Fig. 2CGo). However, a significant number of hepatocytes were unstained. At 6 h, a confluent staining of all hepatocytes in the central area was observed (Fig. 2DGo). The staining in the vascular compartment and in hepatocytes was then evaluated in all samples of each time point. The extent and intensity of NT staining in each compartment was visually assessed by 2 investigators. For each time point, the percent of the maximal staining in the vasculature (2 h) and in hepatocytes (6 h) was estimated. Vascular staining for NT was negligible at 30 min after acetaminophen injection but then increased steeply up to 2 h (Fig. 3Go). In contrast, hepatocellular staining was not detectable up to 1 h (Figs. 2B and 3GoGo). After that time, the number of NT-positive hepatocytes increased progressively up to 6 h with the confluent staining of all hepatocytes in the centrilobular area (Fig. 3Go). A comparison of the liver injury data in Figure 1Go with the staining pattern showed that the vascular staining preceded liver injury. However, hepatocellular staining increased parallel to the ALT release. To evaluate nitric oxide formation, plasma levels of nitrite/nitrate were measured. No significant change of basal nitrite/nitrate concentrations (43 ± 2 µM, n = 5) was observed up to 6 h after acetaminophen treatment (data not shown). Consistent with these results, the baseline expression of iNOS protein was not increased in livers of acetaminophen-treated animals (Fig. 4Go). In contrast, a decrease of hepatic iNOS protein levels was evident at 6 h after acetaminophen.



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FIG. 1. Time course of plasma ALT activities in C3Heb/FeJ mice after administration of acetaminophen (300 mg/kg, ip). Data represent means ± SE of n = 5 animals per group; *p < 0.05 (compared to t = 0).

 


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FIG. 2. Immunohistochemical staining of liver sections for nitrotyrosine (NT) protein adducts in controls and 1, 4, and 6 h after 300 mg/kg of acetaminophen. Control (A): The liver was histologically normal with no evidence of NT staining or hepatocyte injury (400 x). One-hour acetaminophen (B): NT staining present in vascular lining cells but not in hepatocytes of centrilobular areas (400 x). Four-hour acetaminophen (C): Intensely NT stained hepatocytes were scattered among unstained cells in centrilobular areas (400 x). Six-hour acetaminophen (D): Confluent hepatocellular staining for NT in centrilobular areas (400 x); CV, central vein.

 


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FIG. 3. Time course of nitrotyrosine (NT) staining in vascular lining cells and hepatocytes after treatment with acetaminophen (300 mg/kg, ip). Staining intensity and extent of staining in the vasculature and hepatocytes was estimated as percent of maximal staining in the respective compartment, i.e., at 2 h in the vasculature and at 6 h in hepatocytes. Five high-power fields (400 x) in each of 3–4 slides per time point were evaluated.

 


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FIG. 4. Western-blot analysis of the inducible nitric oxide synthase (iNOS; molecular weight 125 kDa) in livers from controls and acetaminophen (AAP)-treated animals. Liver samples were obtained at different time points after 300 mg/kg of AAP. Each lane represents a sample from a single animal. The bar graph shows the densitometric analysis of each lane. The dashed line indicates the control level.

 
The xanthine oxidase inhibitor allopurinol has been shown to prevent a mitochondrial oxidant stress and protect against acetaminophen-induced liver injury (Jaeschke, 1990Go). Therefore, animals were pretreated with low doses (2x 10 mg/kg) or high doses (2x 100 mg/kg) of allopurinol. In previous studies it was shown that both doses completely inhibited xanthine oxidase activities in the liver. However, only the high dose of allopurinol protected against acetaminophen-induced liver injury (Jaeschke, 1990Go). Based on plasma ALT values, these findings were confirmed (Fig. 5Go). A dose of 100 mg/kg allopurinol completely prevented acetaminophen-induced liver injury at 6 h. The lower dose of allopurinol was ineffective (Fig. 5Go). NT staining showed again the confluent staining of hepatocytes around the centrilobular area in livers of animals treated with acetaminophen alone (Fig. 6BGo) or with acetaminophen and the low dose of allopurinol (Fig. 6CGo). In contrast, treatment with the high dose of allopurinol prevented hepatocellular staining (Fig. 6DGo). However, the vascular staining was only attenuated (Fig. 6DGo). Evaluation of NT staining at 1 h after acetaminophen confirmed that the high dose of allopurinol did not eliminate vascular staining (data not shown).



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FIG. 5. Effect of allopurinol (AP) on acetaminophen-induced liver injury. Plasma ALT activities were measured 6 h after a single dose of 300 mg/kg acetaminophen (AAP). Vehicle-treated controls are compared with animals that received 2x 10 mg/kg AP or 2x100 mg/kg AP 18 h and 1 h before AAP injection. Data represent means ± SE of n = 5 animals per group; *p < 0.05 (compared to controls); #p < 0.05 (compared to AAP/H2O).

 


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FIG. 6. Immunohistochemical analyses of liver sections for nitrotyrosine (NT) protein adducts (A–D) and acetaminophen protein adducts (E, F) 6 h after acetaminophen administration. Control (A): The liver was histologically normal with no evidence of NT staining or hepatocyte injury (400 x). Six-hour acetaminophen (B): Confluent hepatocellular staining for NT in centrilobular areas (200 x). Six-hour acetaminophen/10 mg/kg allopurinol (C): Similar confluent hepatocellular staining in centrilobular areas as after acetaminophen alone (200 x). Six-hour acetaminophen/100 mg/kg allopurinol (D): NT staining present in vascular lining cells but not in hepatocytes of centrilobular areas (400 x). Six-hour acetaminophen (E): Staining for acetaminophen protein adducts showed a confluent hepatocellular staining in centrilobular areas (200 x). Six-hour acetaminophen/100 mg/kg allopurinol (F): Reduced acetaminophen protein-adduct formation in livers of animals treated with the high dose of allopurinol (200 x); CV, central vein.

 
To address the mechanism of the protective effect of allopurinol against acetaminophen hepatotoxicity, acetaminophen protein adducts were visualized by immunohistochemistry. Consistent with previous findings (Matthews et al., 1996Go; Michael et al., 1999Go), extensive centrilobular staining for acetaminophen protein adducts was observed 6 h after acetaminophen treatment (Fig. 6EGo). The low dose of allopurinol did not affect this staining pattern (data not shown); however, the high dose of allopurinol substantially reduced (Fig. 6FGo) or in some cases completely eliminated staining for acetaminophen protein adducts.

Since increased hepatocellular superoxide formation is a prerequisite for intracellular peroxynitrite generation, liver GSSG levels were measured as indicator for an increased oxidant stress 6 h after acetaminophen treatment. Compared to control animals, the liver content of total glutathione (GSH + GSSG) was reduced by 37% in acetaminophen-treated animals, but the levels of GSSG and the GSSG-to-GSH ratio were significantly increased (Table 1Go). This significant intracellular oxidant stress induced by acetaminophen was prevented by the high dose of allopurinol (Table 1Go). Since previous data suggest that most of the GSSG is located in mitochondria at 24 h (Jaeschke, 1990Go), mitochondria were isolated for determination of GSH and GSSG at 6 h. The total glutathione content of hepatic mitochondria was similar in control and acetaminophen-treated animals (Table 1Go). However, GSSG levels were increased 6-fold and the GSSG-to-GSH ratio was increased 8-fold above baseline values after acetaminophen (Table 1Go), indicating a mitochondrial oxidant stress. Treatment with the high dose of allopurinol had no significant effect on the total glutathione content but prevented the increase of mitochondrial GSSG levels (Table 1Go). Thus, allopurinol eliminated the mitochondrial oxidant stress induced by acetaminophen. To determine the time when the oxidant stress was first evident, mitochondria were isolated at different time points after acetaminophen treatment. Similar to the total glutathione content in the liver (Jaeschke, 1990Go), the mitochondrial glutathione content decreased rapidly to levels 20–25% of controls at 0.5 to 2 h after acetaminophen (Fig. 7Go). After 2 h, mitochondrial glutathione levels began to recover. The mitochondrial GSSG content decreased to 49% at 15 min but then increased to 70–80% of baseline between 0.5 and 2 h (Fig. 7Go). At 4 h, mitochondrial GSSG levels were significantly higher than control values. Therefore, the GSSG-to-GSH ratio of 0.050 ± 0.009 in controls did not change significantly at 15 min but increased to 0.156 ± 0.0124 (p < 0.05) at 30 min. This increased ratio was maintained for the duration of the experiment. To address the concern that the increased GSSG-to-GSH ratio may not reflect an enhanced oxidant stress but merely be due to the decline in GSH, animals were treated with 250 mg/kg phorone, a known GSH-depleting agent. Phorone treatment reduced hepatic glutathione levels by 70% at 90 min. The mitochondrial GSH content deceased by 85% to 0.41 ± 0.09 nmol/mg protein and the GSSG levels dropped below the detection limit of the assay (0.01 nmol/mg protein). Thus, the GSSG-to-GSH ratio observed in controls was not increased after phorone treatment. Similar to treatment with phorone, a nontoxic dose of acetaminophen (150 mg/kg) did not increase the mitochondrial GSSG-to-GSH ratio (data not shown).


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TABLE 1 Effect of Allopurinol on Hepatic and Mitochondrial Glutathione Levels after Acetaminophen Overdose
 


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FIG. 7. Time-dependent changes of the mitochondrial content of glutathione (GSH + GSSG) and glutathione disulfide (GSSG) after a single dose of acetaminophen (300 mg/kg). All results are given in GSH equivalents. Data represent means ± SE of n = 4 animals per group; *p < 0.05 (compared to t = 0).

 
To determine if allopurinol was protective by acting as a direct scavenger of peroxynitrite, we performed an in vitro experiment using a BSA-carbonate buffer containing various concentrations of allopurinol or N-acetyl cysteine. Peroxynitrite was then added to each solution and the nitration of BSA was determined by reading the absorbance maximum of phenolate at a wavelength of 438 nm. As shown in Figure 8Go, 1 mM allopurinol had no effect on the nitration of BSA by peroxynitrite. Similar results were obtained with lower concentrations of allopurinol (data not shown). In contrast, 1 mM N-acetyl cysteine completely prevented BSA nitration (Fig. 8Go). The effect of N-acetyl cysteine was dose-dependent (data not shown).



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FIG. 8. Effect of allopurinol (AP) on protein nitration by peroxynitrite (PN). Nitration of bovine serum albumin (BSA) was determined spectrophotometrically as the intensely yellow phenolate of nitrotyrosine at 438 nm. PN was added to BSA-carbonate buffer (final concentration of 2 mg/ml; pH 9.6) containing 1 mM of AP or N-acetyl cysteine (NAC). A spectrum was recorded and the amount of nitrotyrosine in the peroxynitrite-treated BSA was calculated by reading the absorbance maximum of the phenolate ion at 438 nm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major objective of this investigation was to localize peroxynitrite formation during acetaminophen-induced liver injury. Our data demonstrated a clear initial nitrotyrosine staining of vascular lining cells of the centrilobular areas. This suggests that peroxynitrite was formed in sinusoids during the early phase after acetaminophen overdose. Peroxynitrite could be formed in the vascular lumen by superoxide derived from Kupffer cells and nitric oxide from Kupffer and endothelial cells. Since basal levels of NO are present in the vasculature, increased peroxynitrite formation may depend to a large degree on superoxide formation by Kupffer cells. In fact, use of gadolinium chloride, which inhibits Kupffer cell-derived superoxide formation (Liu et al., 1995Go), attenuated acetaminophen-induced liver injury (Laskin et al., 1995Go; Michael et al., 1999Go). Isolation of Kupffer cells from acetaminophen-treated animals demonstrated the activation and priming of these cells for superoxide formation (Laskin et al., 1986Go). Gadolinium chloride treatment also prevented the later hepatocellular NT staining (Michael et al., 1999Go), which suggests an important role of Kupffer cell-derived vascular oxidant stress in acetaminophen hepatotoxicity. Other vascular sources of reactive oxygen formation, e.g., neutrophils, can be excluded, because these cells accumulate at later time points (Lawson et al., 2000Go). Further support for the relevance of sinusoidal peroxynitrite generation was also provided by the observation that the overexpression of vascular glutathione peroxidase reduced liver injury and prolonged survival after acetaminophen overdose (Mirochnitchenko et al., 1999Go). Glutathione peroxidase can not only metabolize peroxides but is also an effective reductase for peroxynitrite (Sies et al., 1997Go). Thus, vascular peroxynitrite formation occurs early in the pathophysiology of acetaminophen-induced liver injury and may be relevant for later hepatocellular events.

In addition to the initial vascular NT staining, our data showed a progressive intracellular staining in hepatocytes. Based on the magnitude of NT staining in hepatocytes, it appears unlikely that vascular peroxynitrite could be responsible for the intracellular NT adduct formation in hepatocytes. Thus, peroxynitrite must have been generated in hepatocytes. Previous data indicated the development of an intracellular oxidant stress, located mainly in mitochondria, during acetaminophen hepatotoxicity (Jaeschke et al., 1990). Mitochondrial dysfunction occurs rapidly, i.e., within 1–2 h after acetaminophen administration (Donnelly et al., 1994Go; Esterline et al., 1989Go; Meyers et al., 1988Go; Ramsay et al., 1989Go). All studies showed an inhibition of state-3 (ADP-stimulated) respiration of mitochondria. Consistent with these findings, hepatic ATP levels declined rapidly after acetaminophen treatment in vivo (Jaeschke, 1990Go; Tirmenstein and Nelson, 1990Go). The enhanced state-4 (resting) respiration, with succinate as a substrate (Donnelly et al., 1994Go; Meyers et al., 1988Go), suggests uncoupling of the mitochondrial respiratory chain with electron leakage, which may result in superoxide formation. These findings are consistent with the dramatic increase in mitochondrial GSSG content (Jaeschke, 1990Go). The baseline GSSG levels measured in mitochondria were higher than the level in the intact liver (Table 1Go). This may reflect some potential thiol oxidation during the isolation process. However, the significantly higher GSSG levels at 4 and 6 h after acetaminophen, and the higher GSSG-to-GSH ratio compared to identically isolated control mitochondria, suggests a higher oxidant stress at 0.5 to 6 h after drug treatment. In addition, glutathione depletion with phorone did not increase the GSSG-to-GSH ratio in mitochondria. These data further support the conclusion that the increase of the GSSG-to-GSH ratio at 0.5 to 2 h reflects an oxidant stress. These findings are consistent with earlier observations showing a rapid mitochondrial dysfunction 1–2 h after acetaminophen treatment (Esterline et al., 1989Go; Donnelly et al., 1994Go; Meyers et al., 1988Go; Ramsay et al., 1989Go).

Treatment with a high dose of allopurinol attenuated the acetaminophen-induced mitochondrial oxidant stress (Table 1Go), prevented the decline of hepatic ATP levels (Jaeschke, 1990Go), and abolished cell injury (Fig. 5Go). Furthermore, allopurinol prevented NT staining in hepatocytes but had only a moderate effect on vascular NT adduct formation (Fig. 6Go). These results suggest that the mitochondrial superoxide formation may promote peroxynitrite generation and protein nitration. Interestingly, no significant increase in iNOS protein levels or plasma nitrite/nitrate concentrations were observed, suggesting no relevant increase over baseline in NO formation during the first 6 h after acetaminophen administration. Thus, the enhanced formation of superoxide may have primarily determined the increase in peroxynitrite generation. There are reports showing a moderate increase in plasma nitrite/nitrate levels (Hinson et al., 1998Go) and an induction of iNOS in the liver (Gardner et al., 1998Go) 6–8 h after acetaminophen. In addition, ex vivo stimulation with endotoxin and interferon-{gamma} of hepatic macrophages and hepatocytes from acetaminophen-treated rats showed enhanced formation of nitric oxide compared to control cells (Gardner et al., 1998Go, 1999Go). Our data do not contradict these findings. We can not rule out a moderate increase in nitric oxide formation, which may not result in higher plasma nitrite/nitrate levels. It appears that under our experimental conditions, basal or modestly increased levels of nitric oxide are sufficient to react with superoxide and generate peroxynitrite.

Acetaminophen overdose induces conversion of xanthine dehydrogenase to the oxidase (Jaeschke, 1990Go), which can be a significant source of superoxide under certain pathophysiological conditions (Jaeschke and Mitchell, 1989Go). Allopurinol is a potent inhibitor of xanthine oxidase; doses as low as 5–10 mg/kg completely inhibit this enzyme activity in the liver in vivo (Jaeschke, 1990Go). However, only the high dose (100 mg/kg) of allopurinol prevented the intracellular oxidant stress and NT staining in hepatocytes. This indicates that xanthine oxidase is not a relevant contributor to the intracellular reactive oxygen formation after acetaminophen treatment and, therefore, is unlikely to be involved in peroxynitrite formation. Other intracellular sources such as the microsomal P450 system can be excluded because no oxidant stress could be detected during the initial metabolism of acetaminophen in mouse livers (Smith and Jaeschke, 1989Go). Thus, based on the cellular localization of GSSG, it can be concluded that mitochondria are the main source of superoxide.

Although it was previously shown that allopurinol can prevent acetaminophen-induced liver injury, the molecular mechanism of this hepatoprotection remained unclear (Jaeschke, 1990Go). Protein nitration by peroxynitrite in vitro was not inhibited by allopurinol. These results suggest that allopurinol did not directly react with peroxynitrite. Furthermore, allopurinol treatment had no effect on hepatic glutathione depletion or biliary acetaminophen-glutathione conjugate excretion (Jaeschke, 1990Go). This suggests that allopurinol does not affect the kinetics or extent of NAPQI formation. Consistent with these findings, allopurinol did not enhance the pentobarbital sleeping time in mice (Jaeschke, 1990Go). This indicated that allopurinol does not inhibit the isoenzymes of P450 responsible for pentobarbital and, at least in part, acetaminophen metabolism. Thus, there is no evidence to suggest that allopurinol inhibited the metabolic activation of acetaminophen. However, our current data indicate that allopurinol reduced or eliminated acetaminophen protein adduct formation. The molecular mechanism of how allopurinol interferes with the binding of NAPQI to cellular proteins is unclear at present and requires further investigation. Covalent binding of acetaminophen to mitochondrial proteins was first reported by Jollow et al. (1973). Subsequent studies showed the highest cellular levels of 3-(cystein-S-yl) acetaminophen protein adducts in the plasma membrane and mitochondrial fractions (Pumford et al., 1990Go). Since then, a number of mitochondrial proteins have been identified as targets for covalent binding (Cohen and Khairallah, 1997Go; Pumford et al., 1997Go; Qiu et al., 1998Go). The time course of acetaminophen protein adduct formation (Pumford et al., 1990Go) correlated well with the onset of mitochondrial dysfunction (Donnelly et al., 1994Go; Meyers et al., 1988Go). The fact that allopurinol strongly reduced covalent binding (Fig. 6Go) and eliminated the decline in hepatic ATP levels (Jaeschke, 1990Go; Tirmenstein and Nelson, 1990Go) as well as the mitochondrial oxidant stress (Table 1Go), suggests that covalent binding of NAPQI to these proteins is responsible for mitochondrial dysfunction. Thus, the reduced NT staining and hepatoprotective effect of allopurinol after acetaminophen overdose is likely related to the reduced mitochondrial superoxide formation due to the lack of NAPQI protein binding. However, our data do not allow us to distinguish whether mitochondrial dysfunction per se is sufficient to cause cell death or if the subsequent peroxynitrite formation is a critical event in the pathophysiology. More specific intervention studies are necessary to clarify this issue.

In summary, our investigation demonstrated an initial vascular nitrotyrosine staining after acetaminophen treatment, which indicates that peroxynitrite formation took place in the sinusoids. The subsequent intracellular peroxynitrite generation in hepatocytes increased in parallel with parenchymal cell injury. Mitochondria are the most likely source of superoxide after acetaminophen overdose. Allopurinol, which attenuated acetaminophen protein-adduct formation and mitochondrial dysfunction and oxidant stress, eliminated hepatocellular nitrotyrosine staining and injury. We conclude that after acetaminophen treatment, the reactive metabolite NAPQI binds to intracellular proteins and causes mitochondrial dysfunction and superoxide formation (Fig. 9Go). Mitochondrial superoxide reacts with nitric oxide to form peroxynitrite, which is responsible for intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite for hepatocellular peroxynitrite formation and liver injury remains to be established.



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FIG. 9. Current hypothesis of the mechanism of acetaminophen hepatotoxicity; EC, endothelial cells; NAPQI, N-acetyl-p-benzoquinone imine.

 


    ACKNOWLEDGMENTS
 
This work was supported in part by NIH grants ES-06091 and GM-58884.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Mailslot 638, Little Rock, AR 72205–7199. Fax: (501) 686-8970. E-mail: jaeschkehartmutw{at}uams.edu. Back


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 DISCUSSION
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