Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Received February 14, 2001; accepted May 7, 2001
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ABSTRACT |
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Key Words: peroxynitrite; nitrotyrosine; acetaminophen; allopurinol; liver failure; mitochondria; oxidant stress.
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INTRODUCTION |
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More recently, the concept emerged that nitric oxide and peroxynitrite may be critical mediators for acetaminophen hepatotoxicity (Gardner et al., 1998, 1999
; Hinson et al., 1998
; Michael et al., 1999
). Peroxynitrite is a strong oxidant generated by the spontaneous reaction of nitric oxide and superoxide (Koppenol, 1998
). Peroxynitrite or secondary metabolites can cause tyrosine nitration as well as induce oxidative damage to proteins, DNA and lipids (Squadrito and Pryor, 1998
). During acetaminophen toxicity, the inducible nitric oxide synthase (iNOS) is upregulated in the liver (Gardner et al., 1998
) and there is evidence for increased formation of NO (Gardner et al., 1998
, Hinson et al., 1998
). Reduced liver injury after acetaminophen was observed in animals treated with the iNOS inhibitor aminoguanidine (Gardner et al., 1998
) and in iNOS knock-out mice (Gardner et al., 1999
). 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., 1995
), attenuated hepatocellular nitrotyrosine staining (Michael et al., 1999
) and acetaminophen-induced liver injury (Goldin et al., 1996
; Laskin et al., 1995
; Michael et al., 1999
). 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, 1990
). 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.
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MATERIALS AND METHODS |
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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, 1990). 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., 1999) using an anti-nitrotyrosine antibody (Hinson et al., 2000
) or an anti-acetaminophen antiserum (Matthews et al., 1996
). 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., 2000; Lawson et al., 1999
). 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 420% SDSpolyacrylamide 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., 2000). 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.
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RESULTS |
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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 1). This significant intracellular oxidant stress induced by acetaminophen was prevented by the high dose of allopurinol (Table 1
). Since previous data suggest that most of the GSSG is located in mitochondria at 24 h (Jaeschke, 1990
), 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 1
). However, GSSG levels were increased 6-fold and the GSSG-to-GSH ratio was increased 8-fold above baseline values after acetaminophen (Table 1
), 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 1
). 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, 1990
), the mitochondrial glutathione content decreased rapidly to levels 2025% of controls at 0.5 to 2 h after acetaminophen (Fig. 7
). After 2 h, mitochondrial glutathione levels began to recover. The mitochondrial GSSG content decreased to 49% at 15 min but then increased to 7080% of baseline between 0.5 and 2 h (Fig. 7
). 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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DISCUSSION |
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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 12 h after acetaminophen administration (Donnelly et al., 1994; Esterline et al., 1989
; Meyers et al., 1988
; Ramsay et al., 1989
). 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, 1990
; Tirmenstein and Nelson, 1990
). The enhanced state-4 (resting) respiration, with succinate as a substrate (Donnelly et al., 1994
; Meyers et al., 1988
), 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, 1990
). The baseline GSSG levels measured in mitochondria were higher than the level in the intact liver (Table 1
). 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 12 h after acetaminophen treatment (Esterline et al., 1989
; Donnelly et al., 1994
; Meyers et al., 1988
; Ramsay et al., 1989
).
Treatment with a high dose of allopurinol attenuated the acetaminophen-induced mitochondrial oxidant stress (Table 1), prevented the decline of hepatic ATP levels (Jaeschke, 1990
), and abolished cell injury (Fig. 5
). Furthermore, allopurinol prevented NT staining in hepatocytes but had only a moderate effect on vascular NT adduct formation (Fig. 6
). 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., 1998
) and an induction of iNOS in the liver (Gardner et al., 1998
) 68 h after acetaminophen. In addition, ex vivo stimulation with endotoxin and interferon-
of hepatic macrophages and hepatocytes from acetaminophen-treated rats showed enhanced formation of nitric oxide compared to control cells (Gardner et al., 1998
, 1999
). 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, 1990), which can be a significant source of superoxide under certain pathophysiological conditions (Jaeschke and Mitchell, 1989
). Allopurinol is a potent inhibitor of xanthine oxidase; doses as low as 510 mg/kg completely inhibit this enzyme activity in the liver in vivo (Jaeschke, 1990
). 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, 1989
). 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, 1990). 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, 1990
). 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, 1990
). 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., 1990
). Since then, a number of mitochondrial proteins have been identified as targets for covalent binding (Cohen and Khairallah, 1997
; Pumford et al., 1997
; Qiu et al., 1998
). The time course of acetaminophen protein adduct formation (Pumford et al., 1990
) correlated well with the onset of mitochondrial dysfunction (Donnelly et al., 1994
; Meyers et al., 1988
). The fact that allopurinol strongly reduced covalent binding (Fig. 6
) and eliminated the decline in hepatic ATP levels (Jaeschke, 1990
; Tirmenstein and Nelson, 1990
) as well as the mitochondrial oxidant stress (Table 1
), 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. 9). 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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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Cohen, S. D., and Khairallah, E. A. (1997). Selective protein arylation and acetaminophen-induced hepatotoxicity. Drug Metab. Rev. 29, 5977.[ISI][Medline]
Dahlin, D. C., Miwa, G. T., Lu, A. Y. H., and Nelson, S. D. (1984). N-acetyl-p-benzoquinone imine: A cytochrome P-450-dependent oxidation product of acetaminophen. Proc. Natl. Acad. Sci. U.S.A. 81, 13271331.[Abstract]
Donnelly, P. J., Walker, R. M., and Racz, W. J. (1994). Inhibition of mitochondrial respiration in vivo is an early event in acetaminophen-induced hepatotoxicity. Arch. Toxicol. 68, 110118.[ISI][Medline]
Esterline, R. L., Ray, S. D., and Ji, S. (1989). Reversible and irreversible inhibition of hepatic mitochondrial respiration by acetaminophen and its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Biochem. Pharmacol. 38, 23872390.[ISI][Medline]
Gardner, C. R., Heck, D. E., Chiu, H., Laskin, J. D., Durham, S. K., and Laskin, D. L. (1999). Decreased hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase. In Cells of the Hepatic Sinusoid (E. Wisse, D.L. Knook, R. de Zanger, and R. Fraser, Eds.), Vol. 7, pp. 104105. Kupffer Cell Foundation, Leiden.
Gardner, C. R., Heck, D. E., Yang, C. S., Thomas, P. E., Zhang, X. J., DeGeorge, G. L., Laskin, J. D., and Laskin, D. L. (1998). Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat. Hepatology 26, 748754.[ISI]
Goldin, R. D., Ratnayaka, I. D., Breach, C. S., Brown, I. N., and Wickramasinghe, S. N. (1996). Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity. J. Pathol. 179, 432435.[ISI][Medline]
Hinson, J. A., Michael, S. L., Ault, S. G., and Pumford, N. R. (2000). Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice. Toxicol. Sci. 53, 467473.
Hinson, J. A., Pike, S. L., Pumford, N. R., and Mayeux, P. R. (1998). Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic doses of acetaminophen in mice. Chem. Res. Toxicol. 11, 604607.[ISI][Medline]
Jaeschke, H. (1990). Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxicity in mice in vivo: The protective effect of allopurinol. J. Pharmacol. Exp. Ther. 255, 935941.[Abstract]
Jaeschke, H., Kleinwaechter, C., and Wendel, A. (1987). The role of acrolein in allyl alcohol-induced lipid peroxidation and liver cell damage in mice. Biochem. Pharmacol. 36, 5157.[ISI][Medline]
Jaeschke, H., and Mitchell, J. R. (1989). Mitochondria and xanthine oxidase both generate reactive oxygen species after hypoxic damage in isolated perfused rat liver. Biochem. Biophys. Res. Commun. 160, 140147.[ISI][Medline]
Jaeschke, H., and Mitchell, J. R. (1990). Use of isolated perfused organs in hypoxia and ischemia/reflow oxidant stress. Methods Enzymol. 186, 752759.[Medline]
Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis: II. Role of covalent binding in vivo. J. Pharmacol. Exp. Ther. 187, 195202.[ISI][Medline]
Koppenol, W. H. (1998). The basic chemistry of nitrogen monoxide and peroxynitrite. Free Radic. Biol. Med. 25, 385391.[ISI][Medline]
Laskin, D. L., Gardner, C. R., Price, V. F., and Jollow, D. J. (1995). Modulation of macrophage functioning abrogates the acute hepatotoxicity of acetaminophen. Hepatology 21, 10451050.[ISI][Medline]
Laskin, D. L., and Pilaro, M. (1986). Potential role of activated macrophages in acetaminophen hepatotoxicity: I. Isolation and characterization of activated macrophages from rat liver. Toxicol. Appl. Pharmacol. 86, 204215.[ISI][Medline]
Lawson, J. A., Farhood, A., Hopper, R. D., Bajt, M. L., and Jaeschke, H. (2000). The hepatic inflammatory response after acetaminophen overdose: Role of neutrophils. Toxicol. Sci. 54, 509516.
Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A., and Jaeschke, H. (1999). Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice. Toxicol. Appl. Pharmacol. 156, 179186.[ISI][Medline]
Liu, P., McGuire, G. M., Fisher, M. A., Farhood, A., Smith, C. W., and Jaeschke, H. (1995). Activation of Kupffer cells and neutrophils for reactive oxygen formation is responsible for endotoxin-enhanced liver injury after hepatic ischemia. Shock 3, 5662.[ISI][Medline]
Matthews, A. M., Roberts, D. W., Hinson, J. A., and Pumford, N. R. (1996). Acetaminophen-induced hepatotoxicity. Analysis of total covalent binding vs. specific binding to cysteine. Drug Metab. Dispos. 24, 11921196.[Abstract]
Meyers, L. L., Beierschmitt, W. P., Khairallah, E. A., and Cohen, S. D. (1988). Acetaminophen-induced inhibition of mitochondrial respiration in mice. Toxicol. Appl. Pharmacol. 93, 378387.[ISI][Medline]
Michael, S. L., Pumford, N. R., Mayeux, P. R., Niesman, M. R., and Hinson, J. A. (1999). Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species. Hepatology 30, 186195.[ISI][Medline]
Mirochnitchenko, O., Weisbrot-Lefkowitz, M., Reuhl, K., Chen, L., Yang, C., and Inouye, M. (1999). Acetaminophen toxicity: Opposite effects of two forms of glutathione peroxidase. J. Biol. Chem. 274, 1034910355.
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973). Acetaminophen-induced hepatic necrosis: IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211217.[ISI][Medline]
Pumford, N. R., Halmes, N. C., and Hinson, J. A. (1997). Covalent binding of xenobiotics to specific proteins in the liver. Drug Metab. Rev. 29, 3957.[ISI][Medline]
Pumford, N. R., Roberts, D. W., Benson, R. W., and Hinson, J. A. (1990). Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen. Biochem. Pharmacol. 40, 573579.[ISI][Medline]
Qiu, Y., Benet, L. Z., and Burlingame, A. L. (1998). Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry. J. Biol. Chem. 273, 1794017953.
Ramsay, R. R., Rashed, M. S., and Nelson, S. D. (1989). In vitro effects of acetaminophen metabolites and analogs on the respiration of mouse liver mitochondria. Arch. Biochem. Biophys. 273, 449457.[ISI][Medline]
Sies, H., Sharov, V. S., Klotz, L. O., and Briviba, K. (1997). Glutathione peroxidase protects against peroxynitrite-mediated oxidations. J. Biol. Chem. 272, 2781227817.
Smith, C. V., and Jaeschke, H. (1989). Effect of acetaminophen on the hepatic content and biliary efflux of glutathione disulfide in mice. Chem. Biol. Interactions 70, 241248.[ISI][Medline]
Squadrito, G. L., and Pryor, W. A. (1998). Oxidative chemistry of nitric oxide: The role of superoxide, peroxynitrite and carbon dioxide. Free Radic. Biol. Med. 25, 392403.[ISI][Medline]
Tirmenstein, M. A., and Nelson, S. D. (1990). Acetaminophen-induced oxidation of protein thiols. Contribution of impaired thiol-metabolizing enzymes and the breakdown of adenine nucleotides. J. Biol. Chem. 265, 30593065.