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
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ABSTRACT |
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Key Words: nitrotyrosine; Western blot; peroxynitrite; acetaminophen; hepatotoxicity; necrosis..
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INTRODUCTION |
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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., 1998). 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, 1996
; Pryor and Squadrito,1995
). 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., 1998
). 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.,1996
; Laskin et al., 1995
) and the activation of these cells may lead to the formation of excess levels of nitric oxide and superoxide (Laskin and Perdino, 1995
; Winwood and Arthur, 1993
), 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., 1999
). This postulation is presented in Figure 1
.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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DISCUSSION |
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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., 1998). 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., 1991
). These combined data suggest that peroxynitrite may play a role in toxicity (Hinson et al., 1998
). Very recently, we showed that macrophage inactivators decrease toxicity and nitrotyrosine adducts without affecting acetaminophen-protein adducts (Michael et al., 1999
). 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 6
).
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., 1998), 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, 1999
). Additional work is necessary to determine if this is one of the nitrated proteins in acetaminophen-induced hepatotoxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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