Western Blot Analysis for Nitrotyrosine Protein Adducts in Livers of Saline-Treated and Acetaminophen-Treated Mice

Jack A. Hinson1, Sherryll L. Michael, Subrena G. Ault and Neil R. Pumford

Division of Toxicology, Department of Pharmacology and Toxicology, Slot 638, University of Arkansas for Medical Sciences, Little Rock, AR 72205

Received July 1, 1999; accepted September 21, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.

Key Words: nitrotyrosine; Western blot; peroxynitrite; acetaminophen; hepatotoxicity; necrosis..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In overdose, the analgesic/antipyretic acetaminophen produces centrilobular hepatic necrosis in man and experimental animals. The metabolism of acetaminophen by cytochrome P-450 to the reactive species N-acetyl-p-benzoquinone imine is an important step in the development of the hepatotoxicity. At low doses, this reactive metabolite is efficiently detoxified by glutathione conjugation, forming the 3-(glutathion-S-yl)acetaminophen conjugate. In contrast, large doses result in hepatic glutathione depletion, and the metabolite covalently binds to proteins as 3-(cystein-S-yl)acetaminophen-protein adducts. Treatment of overdose victims with N-acetylcysteine to increase hepatic glutathione has proven to be an excellent antidote (reviewed in Hinson et al., 1995).

It is well established that metabolism of acetaminophen to a reactive metabolite is a necessary step in hepatotoxicity. However, recent work conducted in our laboratory indicates that a second step may also be important in this toxicity. It was shown that shortly after the treatment (4 h) of mice with a toxic dose of acetaminophen (300 mg/kg), nitrotyrosine protein adducts and acetaminophen protein adducts were produced within the same hepatic centrilobular cells (Hinson et al., 1998Go). These cells represent the site of necrosis. Nitration of tyrosine is mediated by peroxynitrite, a reactive species formed by the rapid reaction of nitric oxide with superoxide (Beckman, 1996Go; Pryor and Squadrito,1995Go). In mice the relative amount of nitric oxide synthesis (serum nitrate plus nitrite) was directly proportional to hepatotoxicity (serum ALT) (correlation coefficient = 0.9) (Hinson et al., 1998Go). Because it has been reported that pretreatment of rats or mice with macrophage (Kupffer cell) inactivators decreases acetaminophen-induced hepatotoxicity (Blazka et al., 1996; Goldin et al.,1996Go; Laskin et al., 1995Go) and the activation of these cells may lead to the formation of excess levels of nitric oxide and superoxide (Laskin and Perdino, 1995Go; Winwood and Arthur, 1993Go), the importance of peroxynitrite within this animal mode was investigated. Pretreatment of mice with the macrophage inactivators gadolinium chloride and dextran sulfate dramatically decreased acetaminophen-induced hepatotoxicity by 99% and 85%, respectively. This pretreatment eliminated nitrotyrosine protein adducts; however, a decrease in acetaminophen covalent binding to proteins did not occur. Thus, it was postulated that acetaminophen metabolic activation results in macrophage/Kupffer cell activation in the liver, leading to formation of excess levels of nitric oxide and superoxide. These species react rapidly to form peroxynitrite, and nitration of proteins and/or peroxynitrite-mediated oxidant mechanisms is a critical step in the toxicity (Michael et al., 1999Go). This postulation is presented in Figure 1Go.



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FIG. 1. Postulated mechanism of acetaminophen-induced hepatotoxicity. Metabolic activation is metabolism of acetaminophen to N-acetyl-p-benzoquinone imine, leading to GSH depletion and covalent binding to protein, and superoxide/hydrogen peroxide formation by P-450.

 
In previous work, a commercial antinitrotyrosine antiserum was used for immunohistochemical analyses. However, this antiserum was not useful in Western blot analysis to determine the specific protein that becomes nitrated in acetaminophen hepatotoxicity. The commercial antinitrotyrosine was produced by immunizing rabbits with nitrated KLH. Therefore, in the present work a new approach was used to raise a polyclonal antiserum in rabbits. The resulting antiserum has an extremely high specificity for nitrotyrosine residues and has been used to analyze for specific nitrated protein in liver homogenates from saline-treated and acetaminophen-treated mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Acetaminophen (APAP, paracetamol) and bovine serum albumin (Fraction V, lot 12H0183) were obtained from Sigma Chemical Company (St. Louis, MO). 3-Nitro-4-hydroxybenzoic acid was purchased from Aldrich Chemical Company (Milwaukee, WI). Peroxidase-labeled goat anti-rabbit IgG (H+L) was procured from Gibco BRL (Gaithersburg, MD). Universal DAKO LSAB + (Labeled Streptavidin-Biotin) Peroxidase Kit and DAKO protein block (serum free) were acquired from DAKO Corporation (Carpinteria, CA). Precast 4–20% polyacrylamide mini gels were purchased from Owl Scientific (Portsmouth, NH). Enhanced chemiluminescence (ECL) kit and ECL Hyperfilm were obtained from Amersham Life Science, Inc. (Arlington Heights, IL). Imject Keyhole Limpet Hemocyanin (KLH), 1-ethyl-3-(3-dimethlyaminopropyl) carbodiimide-HCL (EDC), Immunopure Peroxidase Suppresser, Coomassie Plus Protein Assay Reagent, and Superblock were purchased from Pierce Chemical Company (Rockford, IL). Complete and Incomplete Freunds Adjuvent were acquired from Life Technologies (Grand Island, NY). Peroxynitrite and rabbit polyclonal antinitrotyrosine (lot # 16402) were obtained from Upstate Biotechnology (Lake Placid, NY).

Animals.
All animal experimentation followed humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Eight-week-old male C57Bl/6 mice having an average weight of 23 g were obtained from Harlan Sprague Dawley. Animals were housed in clear plastic cages with five animals per cage. Mice were fed ad libitum and maintained on a 12-h light/dark cycle. The mice were acclimatized for 1 week before use. The day before experimentation all food was removed at 5:00 p.m. The next morning at 9:00 a.m., the mice were treated with either 0.4 ml of saline (ip) or acetaminophen (300 mg/kg, ip). At the indicated time, the mice were anesthetized with CO2 and blood was taken from the retro-orbital sinus. The collected blood was allowed to coagulate at room temperature. After coagulation occurred, the samples were centrifuged. The serum was then removed and stored at 4°C prior to analysis. Alanine aminotransferase levels (ALT) in serum were determined using a diagnostic kit (505-OP) obtained from Sigma Chemical Co. (St. Louis, MO). Immediately after bleeding, the mice were euthanized in a CO2 atmosphere and subsequently their livers were removed. A small section was removed from each liver and placed in formalin to be used in immunohistochemical analysis. The remaining portion of each liver was weighed and homogenized in a 3:1 v/w of 0.25 M sucrose, 10 mM HEPES, 1 mM EDTA (pH 7.5) buffer. The protein concentration in each sample was determined using Pierce Coomassie Plus Protein Assay Reagent. Other aliquots of the homogenate were stored at –80°C.

Synthesis of immunogen and solid-phase antigen.
The immunogen, 3-nitro-4-hydroxybenzoic acid-KLH, was synthesized using a two-step method previously described by Davis and Preston (1981) and modified by Matthews et al., (1997). Briefly, 3-nitro-4-hydroxybenzoic acid (27.47 mg) was dissolved in 2.5 ml of methanol and combined with EDC (144 mg) in 2.5 ml of 20 mM potassium phosphate buffer (pH 5.0) at room temperature for 2 min. This reaction mixture was then added to a suspension of KLH (20 mg) in 8 ml of 200 mM phosphate buffer (pH 8.0) and allowed to incubate overnight at room temperature. EDC and unreacted 3-nitro-4-hydroxybenzoic acid were removed from the mixture by dialysis against 10 mM phosphate buffered saline (PBS) (pH 7.2) at 4°C for time intervals of 2 h, 4 h, and finally overnight. One of the solid-phase antigens used in the ELISA was made in the same manner as the immunogen with the substitution of bovine serum albumin for KLH. The other solid-phase antigen was prepared by nitrating bovine serum albumin (2 mg/ml) in 60 mM carbonate buffer (pH 9.6) with peroxynitrite.

Spectrophotometric analyses.
The concentration of 3-nitro-4-hydroxybenzoic acid covalently linked to KLH and BSA was determined spectrophometrically as the intensely yellow phenolate ion at pH 8.0. Briefly, the 3-nitro-4-hydroxybenzoic acid-protein adducts and authentic 3-nitro-4-hydroxybenzoic acid were scanned from 190 nm to 820 nm using a HP8452A diode array spectrophotometer. The absorbance maximum at 408 nm, which was observed only at pH 8.0, was attributed to the phenolate ion. One µmole of the authentic compound was determined to have an absorbance of 4.36 and this value was used to calculate the approximate amount of 3-nitro-4-hydroxybenzoic acid adducted to a known amount of KLH or BSA. The amount of nitrotyrosine in the peroxynitrite-treated BSA was calculated by a similar method. The absorbance maximum of the phenolate of nitrotyrosine was at 438 nm and 1 µmole gave an absorbance of 4.4.

Immunization procedure.
The rabbit was immunized with 380 µl of 3-nitro-4-hydroxybenzoic acid-KLH (500 µg/animal) emulsified in 3 volumes of Freund's complete adjuvent. The mixture was administered by subcutaneous injection at multiple sites along the back and one intramuscular injection in each hind quarter. The rabbit was given five booster injections at 4-week intervals following the primary immunization. The rabbit was boosted with 500 µg of 3-nitro-4-hydroxybenzoic acid-KLH immunogen in Freund's incomplete adjuvant, using the original injection scheme. Seven to 10 days after each injection, approximately 15 ml of arterial blood was collected from the rabbit ear and allowed to clot at room temperature. Serum was separated by centrifugation at 1000 x g for 20 min at 4°C. Additional rabbits were not immunized, as the single rabbit yielded an excellent antiserum.

ELISA.
All ELISAs were performed in Immulon II 96-well plates (Dynatech, Burlington, MA). Both solid-phase antigens, at several different concentrations in 60 mM carbonate buffer (pH 9.6), were adsorbed to the plates. The plates were then incubated overnight in a humidified chamber at 4°C. The plates were then washed four times with 0.05% Tween 20/PBS. To block nonspecific binding sites, washed plates were incubated in a humidified chamber with 0.25% BSA in 0.05% Tween 20/PBS (100 µl) for 1 h at room temperature. The blocked plates were then incubated with serial dilutions of rabbit serum in 0.25% BSA/PBS (100 µl/well) for 90 min in a humidified chamber at room temperature. The plates were washed as described above, then incubated with secondary antibody (Goat Anti-Rabbit AP Conjugated, Gibco BRL) at a dilution of 1:2000 in 0.25% BSA/PBS (100 µl/well) for 1 h at room temperature. The plates were once again washed, then developed using a colometric assay kit (Bio-Rad, Hercules, CA). Upon optimal color development, the reaction was stopped by adding 0.4N NaOH (100 µl/well) to the plates. Absorbance was determined using a microplate reader at 410 nm.

Immunohistochemistry.
Paraffin-embedded tissue sections were deparaffinized with Xylene (2 x 5 min, 25°C) then rehydrated in a series of graded ethanol washes and deionized H2O. The sections were then placed in Pierce Immunopure Peroxidase suppresser for 60 min at room temperature to quench endogenous peroxidase activity. Next, DAKO protein block was added to each tissue section for 30 min to block nonspecific binding. After washing in PBS, the sections were incubated with the primary antiserum, anti-3-nitro-4-hydroxybenzoic acid (1:250), for 60 min at room temperature. From this point until color development, the DAKO LSAB+ kit was used as well as the suggested protocol. The slides were counterstained with Gills Hematoxylin II for 2 min and following rinsing in deionized H2O were immersed in ammonia blue for 2 min. The slides were dehydrated and mounted with Permount.

Western blot and densitometric analysis.
Liver homogenate (100 µg) was separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes (Matthews et al., 1997). Membranes were blocked with Superblock overnight at room temperature. Membranes were then incubated with either antinitrotyrosine (1:250) (Upstate Biotechnology) or anti-3-nitro-4-hydroxybenzoic acid antiserum (1:500) for 120 min. Membranes were next incubated with peroxidase-labeled goat anti-rabbit IgG (1:4000) for 90 min. All membranes were visualized using ECL and exposure to ECL Hyperfilm. The immunoblots of the homogenates that were incubated with antinitrotyrosine showed no bands; however, the immunoblots that were incubated with anti-3-nitro-4-hydroxybenzoic acid gave blots with multiple bands. Densitometric analysis of the film was performed using a Model GS-710 imaging densitometer (Bio-Rad Laboratories, Hercules, CA) in transmittance mode and analyzed using Bio-Rad Discovery software.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recently, we had excellent success in developing a polyclonal antiserum that recognized 3-(cystein-S-yl)acetaminophen protein adducts by synthesizing 4-acetamidobenzoic-keyhole limpet hemocyanin and using it as an immunogen in rabbits. We found that the benzoic acid derivative gave a high derivatization level with keyhole limpet hemocyanin, and we thought that this derivitization was a significant factor in developing an excellent antiserum (Matthews et al., 1997). Thus, to develop a new antiserum that would recognize 3-nitro-4-hydroxyphenyl adducts, as occur with nitrated tyrosine, we purchased 3-nitro-4-hydroxybenzoic acid and coupled it to KLH with a water-soluble carbodiimide. The procedure for synthesis of this immunogen is described in Materials and Methods, and it is shown schematically in Figure 2Go. The level of derivatization of this immunogen (3-nitro-4-hydroxybenzoic acid-KLH) was approximately 171 µmoles/mg protein. This number was calculated by determination of its absorbance at 408 nm as a nitrophenolate ion in pH 8.0 buffer as described in the Materials and Methods section. The rabbit immunized with this KLH adduct developed a high serum antibody titer (Figure 3Go). For evaluation of the antibody titer, a solid-phase antigen was synthesized by adducting 3-nitro-4-hydroxybenzoic acid to bovine serum albumin using a modification of the procedure described for synthesis of the immunogen. The relative level of derivatization was the same as for the KLH adduct, approximately 171 µmoles/mg protein. As shown in the ELISA (Figure 3AGo), the immune antiserum recognized 3-nitro-4-hydroxybenzoic acid adducted to bovine serum albumin. A significant absorbance above background was observed at serum dilutions of 1:16,000 (log 4.2) and greater (Figure 3AGo). In addition to evaluation of the 3-nitro-4-hydroxybenzoic acid-BSA adduct (Figure 3AGo), BSA was treated with peroxynitrite to nitrate tyrosine residues. The relative amount of nitrotyrosine in the BSA was determined from its absorbance at 438 nm as the nitrophenolate ion at pH 8.0. This procedure is described in the Materials and Methods section. The level of nitrotyrosine present in the BSA was approximately 55 µmoles/mg protein. As shown in the ELISA in Figure 3BGo, the antiserum had high recognition of nitrated BSA. A significant absorbance above background was observed at serum dilutions of 1:16,000 and greater (Figure 3BGo). The antiserum did not recognize untreated bovine serum albumin (Figures 3A and 3BGo). Preimmune serum did not recognize either the 3-nitro-4-hydroxybenzoic acid-BSA adduct or the nitrated BSA.



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FIG. 2. Procedure for synthesis of immunogen and antigens. The details of the synthetic procedures are presented in Materials and Methods

 


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FIG. 3. ELISA for anti-3-nitro-4-hydroxybenzoic acid. Serum from rabbit treated with 3-nitro-4-hydroxybenzoic acid-KLH was assayed for ability to recognize either (A) 3-nitro-4-hydroxybenzoic-BSA or (B) nitrated (peroxynitrite-treated)-BSA. The assay was performed as described in Materials and Methods. The x-axis indicates antibody dilution.

 
Western blot assays were performed to determine the utility of the antiserum to recognize specific proteins that contained nitrated tyrosine residues. In these assays, the 3-nitro-4-hydroxybenzoic-BSA adduct was compared to nitrated BSA (Figure 4Go). The amount of nitrophenol in each adduct was quantified as described in Materials and Methods, and the same amount was loaded onto each side of the gel. This allowed comparison of the relative sensitivity to each antigen. The left side of this figure shows the 3-nitro-4-hydroxybenzoic acid-BSA adduct, and the right side shows the nitrated BSA adduct. Lanes 1, 2, 3, and 4 contained 170 pmoles, 17 pmoles, 1.7 pmoles, and 0.17 pmoles, respectively, of 3-nitro-4-hydroxybenzoic acid residues attached to BSA. Lanes 6, 7, 8, and 9 contained 170 pmoles, 17 pmoles, 1.7 pmoles, and 0.17 pmoles, respectively, of nitrotyrosine residues in BSA. The blots were overstained to determine the lower limit of sensitivity of the assay. As shown in Figure 4Go, the assay easily detected 17 pmoles of nitrophenol adducts in both protein samples.



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FIG. 4. Western Blot analysis for 3-nitro-4-hydroxybenzoic acid-BSA and nitrated BSA. The sensitivity of the antiserum for detection of 3-nitro-4-hydroxybenzoic acid-BSA and nitrated BSA were compared in a Western blot analysis as described in Materials and Methods. The presence of multiple bands is augmented as a result of overstaining to determine the lowest level of sensitivity. Under these conditions, linearity of the stain is not expected.

 
Our laboratory has been interested in mechanisms of hepatotoxicity of acetaminophen. In recent work, we utilized a commercial antiserum in immunohistochemical analyses to show that nitrotyrosine adducts in proteins correlate with acetaminophen protein adducts and toxicity (Hinson et al., 1998Go; Michael et al., 1999Go). To determine if our new antiserum would recognize nitrated tyrosine residues in acetaminophen, we treated four mice with a toxic dose of acetaminophen (300 mg/kg) and four mice with saline. Livers from the mice were removed at 8 h and stained for the presence of nitrotyrosine protein adducts as described in Materials and Methods. Figure 5AGo is a histology slide that is representative of a liver from a mouse treated with saline. The histology slide in Figure 5BGo is representative of a liver from a mouse treated with acetaminophen (300 mg/kg, 8 h). The brown stain indicates the presence of nitrotyrosine protein adducts. In the saline-treated control animals, a low level of nitrotyrosine adducts is present in both the centrilobular and periportal regions of the liver. In contrast, the liver from an acetaminophen-treated mouse yielded an intense brown stain present in the centrilobular cells, the site of toxicity.



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FIG. 5. Immunohistochemical analysis from (A) saline-treated mouse liver and (B) acetaminophen-treated mouse liver for nitrotyrosine protein adducts. (A) Liver from a saline-treated mouse. (B) Liver from a mouse treated with a toxic dose of acetaminophen (300 mg/kg) at 8 h. Livers were stained for the presence of nitrotyrosine protein adducts as described in Materials and Methods.

 
Individual livers from this experiment were homogenized and analyzed for the presence of nitrotyrosine protein adducts using Western blot analysis. The ALT levels in the individual mice (5–8) were 10,053 IU/L, 7,320 IU/L, 5,640 IU/L, and 6,405 IU/L, respectively. These high values indicated significant hepatotoxicity in all mice treated with acetaminophen. In contrast, ALT levels from saline-treated mice (1–4) were less than 10 IU/L, a value that correlates with no hepatotoxicity. We were unsuccessful in obtaining a Western blot that showed protein bands when we utilized the commercial antinitrotyrosine (data not shown); however, successful Western blots were obtained using the newly developed anti-3-nitro-4-hydroxybenzoic acid. As shown in Figure 6Go, the liver homogenates from all of the mice had nitrotyrosine protein adducts. The predominant nitrated proteins had molecular weights of 36 kDa, 44 kDa, and 85 kDa. A protein with a molecular weight of 85 kDa appeared to have the greatest concentration of nitrotyrosine. Comparison of the relative densities of the nitrotyrosine protein adducts from both groups indicated that the livers from the acetaminophen-treated mice had greater amounts of nitrotyrosine protein adducts.



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FIG. 6. Western blot analysis of liver homogenates from saline-treated and acetaminophen-treated mice for nitrotyrosine protein adducts. Mice were treated with either saline or acetaminophen (300 mg/kg, ip) and sacrificed at 8 h. Serum ALT levels (hepatotoxicity) indicated no hepatotoxicity in all saline-treated mice and significant hepatotoxicity in all acetaminophen-treated mice. Western blot analysis was as described in Materials and Methods. No protein bands were observed at molecular weights above 200 kDa and this portion of the blot is not shown. The densitometric analyses are for the total lanes.

 
The nature of the hepatic adducts was investigated in a Western blot experiment. Liver homogenates (100 µg) from acetaminophen-treated mice were combined and treated as described above except the antiserum was preincubated with either 20 mM nitrotyrosine, o-nitrophenol, tyrosine, phosphotyrosine, or nitrobenzene. The only compounds to effectively block the staining of the adducts in the saline- or the acetaminophen-treated liver homogenates were nitrotyrosine and o-nitrophenol (data not shown). These data are consistent with the conclusion that the liver adducts are nitrotyrosine.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies aimed at determining the role of peroxynitrite in cellular toxicity comprise an area of active research. This species is a potent oxidant in vitro and nitrates tyrosine residues both in vitro and in vivo. Whereas a low level of this species has been implicated in apoptosis, high levels are believed to cause necrotic cell injury (Spear et al., 1997Go). Immunohistochemical studies for nitrotyrosine protein adducts have revealed that nitration of tyrosine occurs during the pathogenesis of a large number of different disease states. These pathologies include Alzheimer's disease (Smith et al., 1997Go), heart disease (Beckman et al., 1994Go; Kooy et al., 1997Go), acute lung disease (Kooy et al., 1995Go), and renal ischaemia reperfusion (Walker et al., 1998Go).

We have been investigating the role of peroxynitrite in the necrosis produced by large doses of the commonly used drug acetaminophen. Recently, we observed immunohistochemical staining for nitrotyrosine adducts, as well as acetaminophen-protein adducts, in the hepatic centrilobular cells of mice treated with a hepatotoxic dose of acetaminophen (300 mg/kg, 4 h) (Hinson et al., 1998Go). Analysis of sequential liver sections indicated that the cells that contained acetaminophen-protein adducts also contained nitrotyrosine protein adducts. In earlier work, we showed that there is an excellent correlation between formation of acetaminophen-protein adducts and cellular necrosis (Roberts et al., 1991Go). These combined data suggest that peroxynitrite may play a role in toxicity (Hinson et al., 1998Go). Very recently, we showed that macrophage inactivators decrease toxicity and nitrotyrosine adducts without affecting acetaminophen-protein adducts (Michael et al., 1999Go). Even though our data suggested that peroxynitrite may be important in acetaminophen-induced liver necrosis, our studies were limited by the fact that we could not study specific nitrated proteins. Thus, in this manuscript we report the development of a new antiserum, which we have used in a Western blot to assay for nitrotyrosine adducts in livers (Figure 4 and Figure 6GoGo).

At present, we have not identified any of the nitrated proteins; however, the major nitrotyrosine-containing protein with a molecular weight of 85 kDa is most interesting. In a very recent report (Hellberg et al., 1998Go), peroxynitrite (1 mM) was added to the mouse macrophage cell line RAW 264.7 and subsequently the proteins were immunoprecipitated with antinitrotyrosine. One of the precipitated proteins was immunologically identified to be the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), a protein important in a cell-signaling pathway (Rameh and Cantley, 1999Go). Additional work is necessary to determine if this is one of the nitrated proteins in acetaminophen-induced hepatotoxicity.


    ACKNOWLEDGMENTS
 
This work was supported by a grant from the National Institute of General Medical Science (GM-58884).


    NOTES
 
1 To whom correspondence should be addressed. Fax: (501) 686–8970. E-mail: HinsonJackA{at}exchange.UAMS.edu. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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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 formation of reactive oxygen and nitrogen species. Hepatology 30, 186–195.[ISI][Medline]

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Roberts, D. W., Bucci, T. J., Benson, R. W., Warbritton, A. R., McRae, T. A., Pumford, N. R., and Hinson, J. A.(1991). Immunohistochemical localization and quantitation of the 3-(cystein-S-yl)acetaminophen protein adduct in acetaminophen hepatotoxicity. Am. J. Path. 138, 359–371.[Abstract]

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