Peroxisome Proliferator-Activated Receptor Alpha-Null Mice Lack Resistance to Acetaminophen Hepatotoxicity following Clofibrate Exposure

Chuan Chen*, Gayle E. Hennig{dagger}, Herbert E. Whiteley{dagger}, J. Christopher Corton{ddagger} and José E. Manautou*,1

* Toxicology Program, Department of Pharmaceutical Sciences and {dagger} Department of Pathobiology, University of Connecticut, Storrs, Connecticut 06269–2092; and {ddagger} Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709–2137

Received April 21, 2000; accepted June 8, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of this study was to investigate whether activation of the nuclear receptor PPAR{alpha} is needed for protection from acetaminophen (APAP) hepatotoxicity produced by repeated administration of the peroxisome proliferator clofibrate (CFB). Female wild-type and PPAR{alpha}-null mice received corn oil vehicle or 500 mg CFB/kg, ip, daily for 10 days. They were then fasted overnight (18 h) and either killed at 4 or 24 h after challenge with 400 mg APAP/kg. Controls received 50% propylene glycol vehicle only. In this model of CFB hepatoprotection, liver injury was assessed by measuring plasma sorbitol dehydrogenase activity and by histopathology at 24 h after APAP challenge. Significant hepatocellular necrosis was evident in both corn oil-pretreated PPAR{alpha}-null and wild-type mice at 24 h after APAP challenge. In agreement with previous studies, CFB-pretreated wild-type mice showed marked protection against APAP toxicity. In contrast, CFB did not provide protection against APAP hepatotoxicity in the PPAR{alpha}-null mice. Similarly, at 4 h after APAP challenge, hepatic glutathione depletion and selective arylation of cytosolic proteins were reduced significantly in CFB-pretreated wild-type mice, but not in PPAR{alpha}-null mice. The lack of changes in APAP binding and NPSH depletion in CFB-pretreated, PPAR{alpha}-null mice is consistent with the presence of significant liver injury at 24 h in this treatment group. These findings demonstrate that the protection against APAP hepatotoxicity by peroxisome proliferator treatment is mediated by the activation of PPAR{alpha}.

Key Words: acetaminophen (APAP); hepatoprotection; hepatotoxicity; PPAR{alpha}; clofibrate.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to peroxisome proliferation, the hypolipidemic drug clofibrate (CFB) has also been shown to protect mice against acetaminophen (APAP) hepatotoxicity (Manautou et al., 1994Go; Nicholls-Grzemski et al., 1992Go). Hepatoprotection is not limited to APAP, since the toxicity of other model hepatotoxicants such as carbon tetrachloride, bromobenzene, and chloroform can also be reduced by CFB treatment (Manautou et al., 1998Go). Many of the effects of peroxisome proliferators in rodent liver have been shown to be receptor-mediated. Activation of the peroxisome proliferator-activated receptor-alpha (PPAR{alpha}), a member of the nuclear receptor superfamily, has been strongly correlated with peroxisome proliferation and liver cancer (Corton et al., 2000Go; Gonzalez et al., 1998Go; Holden and Tugwood, 1999Go; Vanden Heuvel, 1999Go). Null mice lacking this PPAR isoform do not show the morphological and biochemical changes that are typically observed in rodents following chronic administration of peroxisome proliferators (Lee et al., 1995Go).

Although several mechanistic aspects of the hepatoprotection afforded by CFB have been investigated (e.g., alterations in bioactivating and detoxifying reactions for APAP and changes in glutathione homeostasis), the precise mechanism of this protection is not fully understood (Manautou et al., 1994Go, 1996aGo, bGo). Although an association between hepatoprotection and peroxisome proliferation has not been clearly established, peroxisome proliferators of different chemical composition (fibrate-type drugs, phthalate plasticizers, and herbicides) have been shown to be equally effective in blocking APAP toxicity (Nicholls-Grzemski et al., 1992Go). This suggests that the hepatoprotection might somehow be linked to the pleiotropic response produced by these chemicals. Since PPAR{alpha} is known to regulate the transcription of a wide variety of peroxisomal, microsomal, mitochondrial and cytosolic proteins in liver such as acyl coenzyme A oxidase, peroxisomal bifunctional enzyme, cytochrome P450 CYP4A, and liver fatty acid-binding protein, it is possible that genes involved in this cytoprotective effect might also be under transcriptional regulation by PPAR{alpha}. The availability of a PPAR{alpha}-deficient animal model serves as a useful tool to examine the involvement of this nuclear receptor in the hepatoprotection afforded by peroxisome proliferators.

In the present study, we investigated the susceptibility of PPAR{alpha}-null mice to toxic doses of APAP following 10 days of CFB pretreatment. The response of the null mice to APAP was compared to that of wild-type mice. The results clearly show that PPAR{alpha}-null mice pretreated with CFB are not resistant to APAP-induced liver injury, suggesting that activation of this nuclear receptor by CFB is a required, initiating event leading to biochemical changes responsible for blocking the toxic response to APAP and perhaps to other model hepatotoxic agents.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
4-Acetamidophenol (APAP), propylene glycol, glutathione, trichloroacetic acid, EDTA, 5,5'-dithio-bis(2-nitrobenzoic acid), trizma hydrochloride, trizma base, sucrose, magnesium chloride, ß-nicotinamide adenine dinucleotide (reduced form), D-L-dithiothreitol, sodium dodecyl sulfate (SDS), teleostean (fish) gelatin, 2-mercaptoethanol, glycine, ammonium persulfate, anti-rabbit IgG peroxidase conjugate, TEMED, bromophenol blue, acrylamide (electrophoretic grade), and N,N'-methylene-bis-acrylamide were purchased from Sigma Chemical Co. (St. Louis, MO); Coomassie brilliant blue was from Bio-Rad Laboratories (Richmond, CA); and reagent grade methanol from Fisher Scientific (Springfield, NJ). All other chemicals used were obtained from the above suppliers and were of reagent grade or better.

Animals.
Three to 4-month-old female homozygous PPAR{alpha}-null (-/-) or wild-type (+/+) mice on an Sv/129 background were obtained from a colony maintained at the Chemical Industry Institute of Toxicology (Research Triangle Park, NC). Original breeding pairs were obtained from Dr. F. J. Gonzalez (Metabolism Branch, National Institutes of Health, Bethesda, MD). Production and characterization of this null mouse have been previously described (Lee et al., 1995Go). Upon arrival, mice were allowed to acclimate for 10 days prior to the beginning of experiments. They were housed in community stainless steel cages on hardwood shavings (Sani-Chip, P.J. Murphey Forest Products, Montville, NJ) with free access to Purina Rodent Chow 5002 (PMI Nutrition International, St. Louis, MO) and water. Lights were maintained on a 12:12-h light:dark cycle, and the room temperature was maintained at 22°C.

Treatment regimen.
Pilot studies were initially conducted to determine the dose of APAP to be used in toxicity studies. Only wild-type mice were used in pilot studies, due to the limited supply of PPAR{alpha}-null mice. Mice received corn oil vehicle (2.5 ml/kg), ip, daily for 10 days, and then were challenged with doses of APAP ranging from 250 to 700 mg/kg in 50% propylene glycol vehicle. A dose of 400 mg APAP/kg was selected for hepatoprotection and covalent binding studies in both wild-type and PPAR{alpha}-null mice. This dose of APAP produced significant, but not excessive, hepatocellular necrosis. No mortalities were observed at this dose. For covalent binding and toxicity studies, mice were given 500 mg CFB/kg, ip, daily for 10 days. Controls received corn oil vehicle (2.5 ml/kg). Mice were fasted overnight (18 h) prior to challenge with 400 mg APAP/kg in 50% propylene glycol/water vehicle by gavage. Controls were challenged with propylene glycol vehicle only (5 ml/kg). Animals received their daily dose of CFB or corn oil vehicle between the hours of 9:00 and 11:00 A.M.. The same time frame was used for APAP challenge. Mice were killed by decapitation at 4 and 24 h after challenge. Mice in the 24-h hepatoprotection studies were re-fed at 10 h after APAP administration. The University of Connecticut Institutional Animal Care and Use Committee (IACUC) have approved all animal protocols.

Analysis of plasma sorbitol dehydrogenase (SDH) activity as an indicator of APAP-induced liver injury.
Trunk blood was collected from mice killed at 24 h after APAP administration into heparinized tubes. Plasma was separated by centrifugation at 8,800 x g for 5 min using a Sorvall RMC-14 refrigerated microcentrifuge (DuPont Company, Wilmington, DE). Plasma sorbitol dehydrogenase activity (SDH) was measured as a biochemical indicator of hepatocellular necrosis according to the procedure of Gerlach and Hiby (1974).

Histopathology.
The results of plasma SDH analysis were supported by histopathological analysis of liver sections. The left lateral liver lobes from mice killed at 24 h after APAP were fixed and stored in 10% phosphate-buffered formalin. These samples were then embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. Sections were examined by light microscopy and graded for presence and intensity of lesions, using a severity scale from 0 to 5 (none, 0; minimal, involving single to few necrotic cells, 1; mild, 10–25% necrotic cells or mild diffuse degenerative changes, 2; moderate, 25–40% necrotic or degenerative cells, 3; marked, 40–50% necrotic or degenerative cells, 4; severe, more than 50% necrotic or degenerative cells, 5). Historically, sections with scores higher than 2 are considered as having significant liver injury (Bartolone et al., 1989Go; Beierschmitt et al., 1989Go; Hart et al., 1994Go; Manautou et al., 1994Go, 1996aGo, 1998Go).

Analysis of hepatic non-protein sulfhydryls (NPSH).
Liver samples obtained from mice killed at 4 h after APAP challenge were homogenized (20% w/v) in 5% trichloroacetic acid/ethylenediamine tetraacetic acid (TCA/EDTA). Homogenates were centrifuged at 1,500 x g for 15 min. NPSH concentration in supernatants was determined as an indicator of reduced glutathione (GSH) following the colorimetric procedure of Ellman (1959). NPSH concentration was quantified by comparison with a GSH standard curve.

Immunochemical analysis of APAP selective arylation of cytosolic proteins.
Liver cytosol was prepared from wild-type and PPAR{alpha}-null mice killed at 4 h following APAP or vehicle challenge, as previously described (Manautou et al., 1994Go, 1996aGo). Briefly, livers were homogenized in STM buffer (0.25 M sucrose, 10 mM Tris–HCl, 1 mM MgCl2, pH 7.4) and centrifuged at 9000 x g for 20 min at 40C. Supernatants were centrifuged at 105,000 x g for 60 min at 40C, and the resulting cytosolic fractions were assayed immunochemically for APAP-selective protein arylation. Protein concentration was determined by the method of Bradford (1976) using the Coomassie protein reagent (Pierce Chemical Co., Rockford, IL). Proteins (30 µg/lane) were resolved on 10% SDS–polyacrylamide electrophoresis slab gels using a 4% stacking gel (Laemmli, 1970Go), followed by electrotransfer to PVDF-Plus membrane (Micron Separations, Westboro, MA). Immunochemical detection of APAP-bound proteins was carried out with affinity purified anti-APAP antibody (Bartolone et al., 1988Go), followed with peroxidase-conjugated anti-rabbit IgG. Immunoreactive bands were detected using the ECL chemiluminescent kit (Amersham Life Science, Arlington Heights, IL). APAP-adducted proteins were visualized by exposure to Fuji Medical X-Ray film. Immunoreactive intensity of APAP-bound proteins in the Western blot was quantified using a PDI Image Analyzer (Protein and DNA ImageWare System, PDI, Inc., Huntington Station, NY).

Statistical analysis.
Results are expressed as means ± SE of 3 or more mice per treatment group. NPSH content and quantitative data from Western blots were analyzed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. SDH data were log-transformed first, and then subjected to the same analysis. Histopathology scores were ranked and then subjected to ANOVA, followed by Duncan's test. Differences were considered significant at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Consistent with previous observations documenting the lack of hepatomegaly in PPAR{alpha}-null mice treated with peroxisome proliferators (Lee et al., 1995Go), no changes in liver mass were detected in female PPAR{alpha}-null mice after the administration of 500 mg CFB/kg for 10 days, in the present study, while CFB pretreatment resulted in a 25% increase in liver mass in wild-type mice. Mice in these groups did not receive any challenge (APAP or propylene glycol vehicle) after CFB treatment. This observation illustrates the resistance of PPAR{alpha}-null mice to the effects of CFB in liver.

NPSH concentration was measured at 4 h after APAP challenge in corn oil or CFB-pretreated wild-type and PPAR{alpha}-null mice as an indicator of reduced GSH content in the liver. As expected, administration of 400 mg APAP/kg resulted in significant depletion of NPSH in wild-type mice pretreated with corn oil in comparison to propylene glycol-challenged controls (Fig. 1Go). The level of NPSH depletion was virtually identical in corn oil-pretreated wild-type and PPAR{alpha}-null mice (approximately 80%). The degree of NPSH depletion in response to 400 mg APAP/kg in corn oil-pretreated wild-type and PPAR{alpha}-null female mice is similar to values previously reported for female CD-1 mice receiving a toxic dose of APAP (Hoivik et al., 1995Go). These observations serve as an indirect indication that the lack of this nuclear receptor does not affect the capacity of the liver to generate APAP's toxic intermediate. In agreement with our previous observations with male CD-1 mice, wild-type female mice pretreated with CFB showed significantly less NPSH depletion than corn oil-pretreated controls. Interestingly, NPSH in CFB-pretreated PPAR{alpha}-null mice was depleted to levels comparable to those observed in both groups of corn oil-pretreated control mice (wild-type and null mice). These results suggest that the capacity of CFB treatment to reduce GSH depletion in response to APAP-reactive intermediate generation is dependent on PPAR{alpha} expression.



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FIG. 1. Hepatic non-protein sulfhydryl (NPSH) content at 4 h after APAP challenge. Female PPAR{alpha}-null and wild-type mice were pretreated with corn oil or CFB (500 mg/kg), ip, daily for 10 days. Mice were then challenged with 400 mg APAP/kg, po, after overnight fasting. Controls received 50% propylene glycol (PG) vehicle only. Mice were killed at 4 h after challenge for determination of hepatic NPSH content. Results are the means ± SE for 3 to 5 mice per treatment group. Values with different letters are significantly different from each other.

 
Results of the immunochemical analysis of APAP-selective protein arylation parallel those of the NPSH analysis (Fig. 2Go). In groups of wild-type and PPAR{alpha}-null female mice pretreated with corn oil vehicle, proteins of approximately 58, 56, and 44 kDa were the most prominent APAP-bound targets detected in cytosolic samples from livers obtained at 4 h after challenge with 400 mg APAP/kg (Fig. 2AGo, lanes 3–8). This binding pattern is comparable to that reported in other studies (Bartolone et al., 1989Go; Pumford et al., 1992Go). We previously showed that pretreatment with CFB for 10 days produces a marked reduction in APAP arylation (covalent binding) of cytosolic and microsomal target proteins in male CD-1 mice (Manautou et al., 1994Go). Consistent with this observation, CFB-pretreated wild-type female mice in the present study showed a significant decrease in selective covalent binding (Fig. 2AGo, lanes 9–11). However, this diminution in APAP binding was not seen in CFB-pretreated PPAR{alpha}-null mice (Fig. 2AGo, lanes 12–14). The quantitative image analysis of immunoreactive bands for the principal APAP adducts (58, 56, and 44 kDa) in liver cytosol confirmed these findings (Fig. 2BGo).



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FIG. 2. Immunochemical analysis of APAP covalent binding to cytosolic proteins at 4 h after challenge. Female PPAR{alpha}-null and wild-type mice were pretreated with corn oil or CFB (500 mg/kg), ip, daily for 10 days. Mice were then challenged with 400 mg APAP/kg, po, after overnight fasting. Controls received 50% propylene glycol (PG) vehicle only. Mice were killed at 4 h after APAP challenge, and selective arylation of cytosolic proteins was determined by Western blotting, using an affinity purified polyclonal anti-APAP antibody as described in Materials and Methods. (A) Immunoblot: Lanes 1 and 2 represent individual corn oil-pretreated wild-type, and PPAR{alpha}-null mice challenged with 50% PG vehicle, respectively; lanes 3–5 represent individual corn oil-pretreated wild-type mice challenged with APAP; lanes 6–8 represent individual corn oil-pretreated, PPAR{alpha}-null mice challenged with APAP; lanes 9–11 represent individual CFB-pretreated wild-type mice challenged with APAP; and lanes 12–14 represent individual CFB-pretreated, PPAR{alpha}-null mice challenged with APAP. Arrows on the left side of the immunoblot indicate the approximate molecular weight of the most prominent APAP-arylated protein targets. (B) Analysis of optical density of target proteins: The intensity of immunoreactive bands with molecular weights of 44, 56, and 58 KDa was quantified using a PDI image analyzer and expressed as OD x mm2. Results are the means ± SE for 3 to 5 mice per treatment group. Values with different letters are significantly different from each other.

 
Our previous studies documenting the hepatoprotective effect of CFB on APAP-induced liver injury by CFB were conducted using male CD-1 mice (Manautou et al., 1994Go, 1996aGo). Although female mice have been shown to be equally sensitive to the hepatotoxic effects of APAP as male mice (Hoivik et al., 1995Go), the gender specificity of the hepatoprotective response induced by CFB was never investigated. Protection against APAP hepatotoxicity was also observed in the present study in wild-type female mice using the same CFB pretreatment regimen (Fig. 3Go). At 24 h after challenge with 400 mg APAP/kg, sorbitol dehydrogenase activity was increased to approximately 2717 ± 802 (U/ml) in wild-type female mice pretreated with corn oil vehicle. In contrast, CFB-pretreated wild-type mice receiving the same APAP challenge showed negligible levels of plasma SDH activity, as previously reported in CD-1 male mice (Manautou et al., 1994Go), thus indicating that the protection afforded by CFB is not gender-specific. Plasma SDH activity in PPAR{alpha}-null mice pretreated with corn oil vehicle and then challenged with APAP, was similar to that measured in their respective corn oil-pretreated wild-type controls (2,373 ± 746 U/ml). This indicates that both wild-type and null mice respond to the same toxic dose of APAP in a similar manner. Interestingly, CFB did not provide protection against APAP-induced liver injury in the PPAR{alpha}-null mice. Plasma SDH activity in this group of mice (1823 ± 476 U/ml) was similar to that measured in either group of corn oil-pretreated mice. As expected, plasma SDH activity in groups of propylene glycol vehicle-challenged mice was negligible (ranging from 6 to 19 U/ml).



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FIG. 3. Plasma sorbitol dehydrogenase (SDH) activity determined at 24 h after APAP challenge. Female PPAR{alpha}-null and wild-type mice were pretreated with corn oil or CFB (500 mg/kg), ip, daily for 10 days. Mice were then challenged with 400 mg APAP/kg, po, after overnight fasting. Controls received 50% propylene glycol (PG) vehicle only. Mice were killed at 24 h after challenge and blood was collected for measurement of plasma SDH activity. Results are the means ± SE for 3 to 5 mice per treatment group. Values with different letters are significantly different from each other.

 
Histopathological analysis of liver samples served to validate the plasma SDH data. Figure 4Go shows representative photomicrographs of liver sections from all groups of wild-type and null mice challenged with APAP. These photomicrographs are oriented such that the centrilobular regions are located on the right side of the photographs and the periportal regions on the left side. Severe centrilobular degeneration and necrosis were observed in corn oil-pretreated wild-type mice at 24 h after APAP dosing (A). As expected, wild-type mice receiving CFB for 10 days prior to APAP challenge were protected against hepatic centrilobular injury (B). However, both corn oil- and CFB-pretreated PPAR{alpha}-null mice (C and D, respectively) showed severe liver damage in response to APAP. The severity of APAP-induced liver damage in both groups of PPAR{alpha}-null mice was comparable to that observed in corn oil-pretreated wild-type mice. No hepatic lesions were present in any of the mice receiving 50% propylene glycol vehicle (not shown). Lee et al. (1995) has reported that peroxisome proliferator treatment results in accumulation of fatty droplets in PPAR{alpha}-null mice hepatocytes. In the present studies, evident hepatocyte vacuolation was observed in both corn oil and CFB-pretreated PPAR{alpha}-null mice, possibly indicating the accumulation of fatty droplets (not shown).



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FIG. 4. Histopathological changes in the liver at 24 h following challenge with APAP. Female PPAR{alpha}-null and wild-type mice were pretreated with corn oil and CFB (500 mg/kg), ip, daily for 10 days. Mice were then challenged with 400 mg APAP/kg, po, after overnight fasting. Controls received 50% propylene glycol (PG) vehicle only. Mice were killed at 24 h after challenge, and liver samples were collected and processed for histopathological analysis. (A) Liver section from corn oil-pretreated wild-type mice challenged with APAP. Note severe centrilobular hepatocellular necrosis (arrow). (B) Liver section from CFB-pretreated wild-type mice challenged with APAP. Note the absence of APAP-induced hepatocellular necrosis, which demonstrates the hepatoprotective effect of CFB in wild-type mice. (C) Liver section from corn oil-pretreated PPAR{alpha}-null mice challenged with APAP. Note presence of centrilobular hepatocellular necrosis (arrow). (D) Liver section from CFB-pretreated PPAR{alpha}-null mice challenged with APAP. Note presence of centrilobular hepatocellular necrosis (arrow) as seen in A and C, which demonstrates that CFB-pretreated PPAR{alpha}-null mice lack resistance to APAP-induced liver injury. (Bar = 70 µm; hematoxylin and eosin stain).

 
Sections of liver from all treatment groups were given a score from 0 to 5, depending on the degree of the liver injury detected. Cumulative data from our laboratory show that the histopathological scoring analysis performed in sections of liver obtained at either 12 or 24 h after APAP challenge is consistently in agreement with the biochemical analysis of hepatotoxicity (Manautou et al., 1994Go, 1996aGo, 1998Go; Nguyen et al., 1999Go; Silva et al., 2000Go). In this analysis, sections of liver with scores above 2 are considered to have significant hepatocellular necrosis (Bartolone et al., 1989Go; Beierschmitt et al., 1989Go; Manautou et al., 1994Go). In the present studies, all corn oil-pretreated wild-type mice challenged with 400 mg APAP/kg had histology scores higher than 2 (Table 1Go). Scores in this group of mice ranged from 3 to 5. Similarly, 4 out of 5 corn oil-pretreated PPAR{alpha}-null mice showed scores higher than 2, thus confirming the presence of significant hepatocellular necrosis in both groups of vehicle-pretreated mice. Consistent with the SDH data, all CFB-pretreated wild-type mice challenged with APAP received a score of 0. On the other hand, CFB-pretreated PPAR{alpha}-null mice had scores ranging from 2 to 5, with the percentage of animals with scores higher than 2 being equal to that in corn oil-pretreated PPAR{alpha}-null mice (80%). Statistical analysis of ranked scores showed no difference in severity of liver injury between corn oil-pretreated wild-type, corn oil-pretreated PPAR{alpha}-null, and CFB-pretreated PPAR{alpha}-null mice. This confirms that CFB pretreatment does not protect PPAR{alpha}-null mice against the toxicity produced by 400 mg APAP/kg.


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TABLE 1 Effect of Clofibrate (CFB) Pretreatment on Liver Histopathology following Acetaminophen (APAP) Challenge in Wild-Type and PPAR{alpha}-Null Mice
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A mouse model deficient in the ligand-activated transcription factor PPAR{alpha} was recently developed to investigate, in depth, its role in the multiple responses produced by peroxisome proliferators in rodent livers (Lee et al., 1995Go). This mutant mouse was shown to be resistant to peroxisome proliferation, hepatomegaly, induction of peroxisome proliferator-responsive enzymes, and hepatocellular adenomas and carcinomas induced by these compounds (Corton et al., 1998Go; Lee et al., 1995Go; Peters et al., 1997Go). Another less-known effect of peroxisome proliferators is their capacity to confer protection against liver injury produced by the administration of high doses of model hepatotoxic agents such as APAP and carbon tetrachloride (Manautou et al., 1994Go, 1998Go; Nicholls-Grzemski et al., 1992Go). The mechanism of this hepatoprotection is not fully understood. Furthermore, an association between peroxisome proliferation and hepatoprotection has not been established.

Since a large number of the hepatic effects produced by peroxisome proliferators in liver have been attributed to the transcriptional regulation of PPAR{alpha}-responsive genes, it was of interest to investigate if the hepatoprotective effects of these chemicals might also be under the control of PPAR{alpha}. The results of these studies support the involvement of PPAR{alpha} activation in this response. Mice lacking PPAR{alpha} that were pretreated with CFB for 10 days showed marked liver injury at 24 h following administration of 400 mg APAP/kg, while biochemical and histological evidence of liver necrosis was virtually absent in wild-type mice receiving the same CFB pretreatment and APAP challenge. This marked contrast in response to APAP between wild-type and null mice pretreated with CFB serves to document a dependency on PPAR{alpha} for this response.

Previous studies using male CD-1 mice and isolated hepatocytes in culture have shown that alterations in APAP bioactivation and detoxification are not responsible for the CFB-mediated hepatoprotection (Manautou et al., 1994Go, 1996bGo; Nicholls-Grzemski et al., 1996Go; Nguyen et al., 1999Go). Despite the lack of changes in APAP biotransformation, male CD-1 mice receiving CFB for 10 days and then challenged with a toxic dose of APAP, had much higher residual NPSH levels and less covalent binding at 4 h after APAP administration than vehicle-pretreated controls. This suggests that the time frame for this hepatoprotective response lies between the generation of the reactive metabolite and its subsequent binding to proteins and other cellular constituents. Although higher APAP binding appear to be present in both groups of PPAR{alpha}-null mice when compared to corn oil-pretreated wild-type mice, CFB-pretreated PPAR{alpha}-null mice showed greater protein binding and lower residual NPSH content by 4 h after APAP administration than CFB-pretreated wild-type mice receiving APAP. This pronounced difference in binding and NPSH depletion between CFB-pretreated wild-type and null mice correlates well with the amount of liver damage observed at 24 h seen in these 2 groups of mice. In theory, assessment of liver injury at a single time point does not rule out the possibility of a shift or delay in the time course of APAP-induced liver injury in CFB-pretreated animals. However, previous studies from our laboratory indicate that the hepatoprotection seen under the experimental conditions used is not the result of a shift in the time course of toxicity. This has been properly addressed in previous contributions (Manautou et al., 1998Go; Nguyen et al., 1999Go).

Although the present studies do not elucidate the precise mechanism by which CFB and other peroxisome proliferators protect the liver against APAP toxicity, they do document the mechanistic involvement of PPAR{alpha} in this response. It is very difficult to single out one cytoprotective cellular constituent which may account for this effect of CFB, since peroxisome proliferators are known to regulate the expression of a wide variety of hepatic enzymes and proteins. The complexity of this hepatoprotection is further illustrated by previous studies from our laboratory, which suggest the existence of multiple mechanisms of protection. A single dose of CFB administered 24 h before APAP challenge also prevents APAP-induced liver injury (Manautou et al. 1996aGo). In contrast to the 10 day CFB dosing regimen, no differences in NPSH depletion or APAP binding to cytosolic protein targets were seen in male CD-1 mice pretreated with a single dose of CFB. Mechanistically, this implies that a single dose of CFB protects against liver injury through intervention during the progression of toxicity, beyond the point of protein arylation and GSH depletion. This is in contrast with the findings of the 10-day pretreatment regimen, in which binding and GSH depletion were both greatly reduced in CFB animals (Manautou et al., 1994Go). Again, these divergent observations illustrate the mechanistic complexity of the hepatoprotective effects of CFB. Additional studies are needed to further explore the role of PPAR{alpha}-responsive genes in hepatoprotection.


    ACKNOWLEDGMENTS
 
This study was supported in part by a grant from the Pharmaceutical Research and Manufactures of America (PhRMA) Foundation and a Boehringer Ingelheim Pharmaceutical Predoctoral Fellowship in Toxicology to Mr. Chuan Chen. The authors would like to thank Mr. Paul W. Ross (CIIT) for his assistance with the care and shipment of PPAR{alpha}-null and wild-type mice.


    NOTES
 
1 To whom correspondence should be addressed at the University of Connecticut Toxicology Program, Department of Pharmaceutical Sciences, School of Pharmacy, 372 Fairfield Road, Box U-92, Storrs, CT 06269–2092. Fax: (860) 486-4998. E-mail: manautou{at}uconnvm.uconn.edu. Back

Presented in part at the 39th Annual Meeting of the Society of Toxicology, Philadelphia, PA, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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