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Communications Nitrotyrosine-Protein Adducts in Hepatic Centrilobular Areas following Toxic Doses of Acetaminophen in Mice Jack A. Hinson,* Sherryll L. Pike, Neil R. Pumford, and Philip R. Mayeux Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Received February 26, 1998
Treatment of mice with a toxic dose of acetaminophen (300 mg/kg, ip) significantly increased hepatotoxicity at 4 h, as evidenced by histological necrosis in the centrilobular areas of the liver, and increased serum levels of alanine aminotransferase (ALT) (from 8 ( 1 IU/L in salinetreated mice to 3226 ( 892 IU/L in the acetaminophen-treated mice). Serum levels of nitrate plus nitrite (a marker of nitric oxide synthesis) were also increased from 62 ( 8 µM in salinetreated mice to 110 ( 14 µM in acetaminophen-treated mice (P < 0.05). Regression analysis of serum ALT levels to serum nitrate plus nitrite levels in individual mice revealed a positive, linear relationship between serum ALT levels and serum nitrate plus nitrite levels with a correlation coefficient of 0.9 (P < 0.05). The y intercept value (nitrate plus nitrite level) was 63 ( 15 µM. Immunohistochemical analysis of liver sections from acetaminophen-intoxicated mice using an anti-3-nitrotyrosine antibody indicated tyrosine nitration in the proteins of the centrilobular cells. Tyrosine nitration has been shown to occur by peroxynitrite, a reactive intermediate formed by an extremely rapid reaction of nitric oxide and superoxide and a species which also has hydroxyl radical-like activity. Analysis of liver sections using an antiacetaminophen antiserum indicated the centrilobular cells also contained acetaminophenprotein adducts, a reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine residues on proteins. These data are consistent with acetaminophen metabolic activation leading to increased synthesis of nitric oxide and superoxide and to peroxynitrite as an important intermediate in the toxicity.
Introduction Large doses of the commonly used analgesic acetaminophen (paracetamol) are known to produce necrosis in the centrilobular cells in the liver of humans as well as experimental animals (1). A significant amount of evidence has been presented that the toxicity is mediated by cytochrome P450 metabolism of the drug to N-acetylp-benzoquinone imine (2, 3). Following a low dose of acetaminophen, the metabolite is preferentially detoxified by glutathione. However, following a toxic dose, glutathione is depleted and the metabolite covalently binds to cysteine residues on proteins as 3-(cystein-S-yl)acetaminophen adducts (4, 5). Both the histological site of binding as well as the relative amount of covalent binding has been shown to correlate with the development of the toxicity (6). Even though the prevailing theory of toxicity is that the metabolite covalently binds to critical protein, a growing body of literature suggests that the oxidant stress is involved. Nakae et al. reported that administration of superoxide dismutase to rats decreased their susceptibility to the toxic effects of * Corresponding author address: Division of Toxicology, Slot 638, University of Arkansas for Medical Sciences, Little Rock, AR 72205. E-mail:
[email protected]. Tel: 501-686-7036. Fax: 501-686-8970.
acetaminophen (7). Also, it has been shown that when mice are pretreated with ferrous sulfate their susceptibility to the toxic effects of acetaminophen is increased (8), and the iron chelator deferoxamine has been reported to decrease the toxicity (9). In addition, compounds which decrease macrophage/Kupffer cell functioning, dramatically decrease acetaminophen toxicity (10-12). In this communication we have examined for evidence of peroxynitrite formation in the toxicity. Peroxynitrite is an intermediate formed by reaction of nitric oxide with superoxide (13-15).
Experimental Procedures Caution: Formaldehyde was used to preserve the tissues. Formaldehyde has been reported to be a carcinogen. Hydrogen peroxide is caustic. All operations were performed with caution, and with volatile chemicals the operations were performed in an efficient fume hood. Chemicals. Anti-nitrotyrosine antibody and peroxynitrite were purchased from Upstate Biotechnology (Lake Placid, NY). Assay kit for serum levels of nitrate plus nitrite was purchased from Oxford Biomedical Research (Oxford, MI). Acetaminophen, nitrotyrosine, and serum ALT assay kit were purchased from Sigma Chemical Co. (St. Louis, MO). Peroxidase-labeled goat anti-rabbit IgG (H+L) was procured from Gibco BRL (Gaithersburg, MD). Universal DAKO LSAB+ (Labeled Streptavidin-
S0893-228x(98)00034-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/23/1998
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Figure 1. Comparison of acetaminophen-induced hepatotoxicity to nitric oxide formation in mice treated with a toxic dose of acetaminophen. Mice (n ) 6) were treated with acetaminophen (300 mg/kg) and sacrificed at 4 h. Serum samples were analyzed for ALT and nitrate plus nitrite levels. The data points are serum levels of ALT and nitrate plus nitrite in the individual mice. Biotin) Peroxidase Kit was purchased from DAKO Corp. (Carpinteria, CA). Mice. Male B6C3F1 mice (23 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Mice were acclimatized for 1 week before use. The day before acetaminophen administration the food was removed from the mice at 5:00 p.m., and at 9:00 a.m. the next morning the mice were injected with acetaminophen (300 mg/kg, ip in saline) (n ) 6). Control mice received saline only (n ) 5). At the designated time mice were anesthetized in a carbon dioxide chamber, and blood was removed from the retro-orbital sinus. Mice were subsequently anesthetized and sacrificed by decapitation. Livers were surgically removed. Analyses. For immunohistochemical analyses fresh liver tissues, previously trimmed to approximately 2-mm thickness, were placed in plastic cassettes and immersed in neutral buffered formalin for 24 h. Paraffin-embedded tissue sections were deparaffinized with xylene (2 × 5 min, 25 °C) then rehydrated in a series of graded ethanol washes and deionized water. The sections were then placed in Immunopure peroxidase suppressor for 30 min to quench endogenous peroxidase activity. Next, Dako Protein Block (serum-free) was added to each tissue section for 30 min to block nonspecific binding. After being washed in PBS, the sections were incubated with an antiacetaminophen antiserum (16) (1:1000) or a polyclonal anti-3nitrotyrosine antibody (17) (1:100) for 60 min at room temperature. For color development the protocol described in the DAKO LSAB+ kit was used. The slides were counterstained with Gills Hematoxylin II for 2 min and following rinsing in deionized water were immersed in ammonia blue for 2 min. The slides were dehydrated and mounted with Permount. For the blocked antibody experiment an identical procedure was followed except the anti-3-nitrotyrosine antibody was preincubated with authentic 3-nitrotyrosine (10 mM). Serum alanine aminotransferase (ALT) was determined spectrophotometrically as described in the Sigma Chemical Co. kit. Nitrate plus nitrite levels in serum were determined by precipitation of protein, reduction of the nitrate to nitrite by cadmium, and quantitation of nitrite using Griess reagent utilizing Oxford Biomedical kit NB 88. Statistical Analysis. Data are reported as mean ( SE. Comparisons among treatment groups were by one-way analysis of variance followed by the Student-Newman-Keuls post hoc test. Data in Figure 1 were analyzed by linear regression analysis. P < 0.05 was considered statistically significant.
Results and Discussion To determine if nitric oxide synthesis was altered following toxic doses of acetaminophen, mice (n ) 6) were
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treated with a moderately hepatotoxic dose of acetaminophen (300 mg/kg). Control mice received only saline (n ) 5). After 4 h mice were anesthetized under a carbon dioxide atmosphere and blood samples taken from the retro-orbital sinus. Serum ALT levels (a marker of hepatic cell lysis) were increased from 8 ( 1 IU/L in saline-treated mice to 3226 ( 891 IU/L in acetaminophen-treated mice (P < 0.05). Serum levels of nitrate plus nitrite (a marker of NO synthesis) were also increased from 62 ( 8 µM in saline-treated mice to 110 ( 14 µM in acetaminophen-treated mice (P < 0.05). Regression analysis of serum ALT levels to serum nitrate plus nitrite levels in individual mice was performed (Figure 1). This analysis revealed a positive, linear relationship between serum ALT levels and serum nitrate plus nitrite levels with a corrrelation coefficient of 0.9 (P < 0.05). The y intercept value (nitrate plus nitrite level) was 63 ( 15 µM. This value agrees well with the mean serum level observed in saline-treated mice (62 ( 8 µM). These data suggest that nitric oxide synthesis is increased in acetaminophen-induced hepatotoxicity and its formation correlates with the relative amount of injury in individual mice. The increased nitric oxide level synthesis in acetaminophen-treated mice may be important in the previously reported decrease in blood pressure (18) and decrease in temperature (19) with acetaminophen (20, 21). Nitric oxide reacts very rapidly with superoxide to produce peroxynitrite. Peroxynitrite may oxidize lipids, DNA, and protein. Also, it may nitrate tyrosine residues on protein (13-15). Livers from acetaminophen-treated mice were examined by immunohistochemical analyses for 3-nitrotyrosine-protein adducts. All acetaminophentreated mouse livers stained positive for nitrotyrosine adducts in all the observed centrilobular areas. In the individual mice the relative amount of staining appeared to correlate with the relative severity of the toxicity. Figure 2A is a representative positive staining section for nitrotyrosine-protein adducts from the liver of an acetaminophen-treated mouse. The brown-staining areas represent nitrotyrosine-protein adducts in the centrilobular areas of the liver. Addition of authentic nitrotyrosine (10 mM) to the staining system eliminated the brown stain which indicated that the antibody was recognizing the nitrotyrosine epitope (Figure 2B). Note that the staining intensity of the background blue stain is decreased in the centrilobular areas compared to the periportal areas, a finding consistent with toxic damage to the centrilobular cells. Removal of the primary antibody from the staining system eliminated the stain (data not shown). Figure 2C is a representative liver section from a saline-treated mouse and shows that these adducts are not present in the control liver. A liver section from an adjacent cut to that in Figure 2A,B was stained for the presence of acetaminophenprotein adducts. We have previously shown that these adducts occur in the centrilobular areas of the liver and that peak levels are observed at 1-2 h. The relative intensity of the stain decreases by 4 h as a result of cellular lysis with release of cytosolic adducts into the serum (22). Figure 2D is a representative positive staining section for acetaminophen-protein adducts from the liver of an acetaminophen-treated mouse. These data are consistent with our previous time and dose-response study for acetaminophen-protein adducts (22). Comparison of Figure 2A,D indicates that nitrotyrosine
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Figure 2. Immunochemical analysis of liver sections for nitrotyrosine adducts or acetaminophen adducts. (A, top left) Liver section from a mouse treated with acetaminophen (300 mg/kg) at 4 h and stained with an antibody specific for nitrotyrosine adducts. The brown stain in the centrilobular regions indicates nitrotyrosine adducts. The unstained areas are the periportal areas. (B, top right) Adjacent liver section stained with the anti-nitrotyrosine antibody as in panel A, but the anti-nitrotyrosine antibody was previously incubated with nitrotyrosine (10 mM) to block the antibody specificity. The lack of brown stain indicates the specificity for the stain in panel A was for nitrotyrosine adducts. (C, bottom left) Liver section from a control mouse treated with saline. Liver sections were stained using anti-nitrotyrosine. The lack of brown stain indicates the absence of nitrotyrosine adducts in control animals. (D, bottom right) Adjacent liver section to the sections in panels A and B stained with an antibody specific for acetaminophen adducts. The brown stain in the centrilobular regions indicates acetaminophen-protein adducts. The unstained areas are the periportal areas.
adducts occur in the same regions as the acetaminophen-protein adducts, the centrilobular areas, the site of the toxicity. Neither nitrotyrosine-protein adducts nor acetaminophen-protein adducts were observed in the periportal areas. Consistent with our previous observations (22) acetaminophen-protein adducts were not observed in saline-treated control animals (data not shown). Peroxynitrite is a reactive oxidant which has hydroxyl radical-like activity and also leads to tyrosine nitration. It is formed by a very rapid reaction between nitric oxide and superoxide anion. It has been postulated to be important in a variety of pathological states including human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis (13-15); however, its role of chemical-induced tissue necrosis is not clear. The data presented in this communication indicate that peroxynitrite is formed in the centrilobular areas of the liver following toxic doses of acetaminophen. Since peroxynitrite is formed by reaction of superoxide and nitric oxide (13-15, 17), the data are consistent with increased synthesis of these substances at the site of the toxicity.
Also, the increased serum nitrate plus nitrite levels are indicative of increased nitric oxide synthesis. What role acetaminophen, a phenol, may have in inhibiting nitration of tyrosine is unclear. In an in vitro system acetaminophen has been reported to inhibit tyrosine nitration by peroxynitrite (23). Synthesis of peroxynitrite is consistent with activation of Kupffer cells (hepatic macrophages). It has been recently shown that inactivators of Kupffer cells (and other macrophages) decrease the toxicity of acetaminophen (10-12). It is known that activation of Kupffer cells may lead to increased synthesis of nitric oxide and superoxide (24, 25). The fact that peroxynitrite is highly reactive and has hydroxyl radical-like oxidation activity (15) makes it an excellent candidate as a toxic intermediate leading to cell lysis. Hydroxyl radical activity has been characterized as an important oxidant in many toxicities. The previous report that encapsulated superoxide dismutase inhibited the toxicity of acetaminophen in the rat is consistent with peroxynitrite as an ultimate toxic species (7). The data suggest a multistep mechanism leading to the toxicity. It has been clearly shown that there is an
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excellent correlation between covalent binding of Nacetyl-p-benzoquinone imine (NAPQI) to protein in hepatocytes and development of the toxicity (22). However, the important step leading to peroxynitrite formation may be either covalent binding and/or oxidative stress. P4502E1, a major enzyme in NAPQI formation, may also form superoxide and peroxide (26). Since GSH depletion occurs as a result of NAPQI formation, GSH peroxidase activity may be decreased. Thus, covalent binding and oxidative stress occur simultaneously in the centrilobular cells, and either may be important in induction of increased nitric oxide synthesis (27).
Acknowledgment. Financial support from the National Institutes of Health (GM-48749 to J.A.H. and DK44716 to P.R.M.) is gratefully acknowledged.
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(11) Blazka, M. E., Germolec, D. R., Simeonova, P., Bruccoleri, A., Pennypacker, K. R., and Luster, M. I. (1996) Acetaminopheninduced hepatotoxicity is associated with early changes in NFkB and NF-IL6 DNA binding activity. J. Inflamm. 47, 138-150. (12) 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, 432-435. (13) Beckman, J. S., Ye, Y. Z., Anderson, P. G., Chen, J., Accavitti, M. A., Tarpey, M. M., and White, C. R. (1994) Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol. Chem. Hoppe-Seyler 375, 81-88. (14) Beckman, J. S., and Koppenol, W. H. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am. J. Physiol. 271, C1424-1437. (15) Pryor, W. A., and Squadrito, G. L. (1995) The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268, L699-L722. (16) 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, 1192-1196. (17) Crow, J. P., and Ischiropoulos, H. (1996) Detection and quantitation of nitrotyrosine residues in protein: in vivo marker of peroxynitrite. Methods Enzymol. 269, 185-194. (18) Loscalzo, J., and Welch, G. (1995) Nitric oxide and its role in the cardiovascular system. Prog. Cardiovas. Dis. 38, 87-104. (19) Branco, L. G., Carnio, E. C., and Barros, R. C. (1997) Role of the nitric oxide pathway in hypoxia-induced hypothermia of rats. Am. J. Physiol. 273, R967-971. (20) Brown, G. (1996) Acetaminophen-induced hypotension. Heart Lung. 25, 137-140. (21) Walker, R. M., Massey, R. E., McElligott, T. F., and Racz, W. J. (1981) Acetaminophen-induced hypothermia, hepatic congestion, and modification by N-acetylcysteine in mice. Toxicol. Appl. Pharmacol. 59, 500-507. (22) Roberts, D. W., Bucci, T. J., Benson, R. W., Warbritton, A. R., McRae, T. A., Pumford, N. R., and Hinson, J. A. (1991) Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adduct in acetaminophen hepatotoxicity. Am. J. Pathol. 138, 359371. (23) Whiteman, M., Kaur, H., and Halliwell, B. (1996) Protection against peroxynitrite dependent tyrosine nitration and -antiproteinase inactivation by some antiinflammatory drugs and by the antibiotic tetracycline. Ann. Rheum. Dis. 55, 383-387. (24) Xia, Y., and Sweier, J. L. (1997) Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc. Natl. Acad. Sci. U.S.A. 94, 6954-6958. (25) Baggiolini, M., and Wymann, M. P. (1990) Turning on the respiratory burst. Trends Biol. Sci. 15, 69-72. (26) Dai, Y., and Cederbaum, A. I. (1995) Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells. J. Pharmacol. Exp. Ther. 273, 1497-1505. (27) Nakamura, H., Nakamura, K., and Yodoi, J. (1997) Redox regulation of cellular activation. Annu. Rev. Immunol. 15, 351-369.
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