* Toxicology Program, Department of Pharmaceutical Sciences and
Department of Pathobiology, University of Connecticut, Storrs, Connecticut 062692092; and
Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 277092137
Received April 21, 2000; accepted June 8, 2000
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ABSTRACT |
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Key Words: acetaminophen (APAP); hepatoprotection; hepatotoxicity; PPAR; clofibrate.
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INTRODUCTION |
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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., 1994, 1996a
, b
). 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., 1992
). This suggests that the hepatoprotection might somehow be linked to the pleiotropic response produced by these chemicals. Since PPAR
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
. The availability of a PPAR
-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-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
-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.
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MATERIALS AND METHODS |
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Animals.
Three to 4-month-old female homozygous PPAR-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., 1995
). 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-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
-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, 1025% necrotic cells or mild diffuse degenerative changes, 2; moderate, 2540% necrotic or degenerative cells, 3; marked, 4050% 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., 1989; Beierschmitt et al., 1989
; Hart et al., 1994
; Manautou et al., 1994
, 1996a
, 1998
).
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-null mice killed at 4 h following APAP or vehicle challenge, as previously described (Manautou et al., 1994
, 1996a
). Briefly, livers were homogenized in STM buffer (0.25 M sucrose, 10 mM TrisHCl, 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% SDSpolyacrylamide electrophoresis slab gels using a 4% stacking gel (Laemmli, 1970
), 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., 1988
), 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.
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RESULTS |
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NPSH concentration was measured at 4 h after APAP challenge in corn oil or CFB-pretreated wild-type and PPAR-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. 1
). The level of NPSH depletion was virtually identical in corn oil-pretreated wild-type and PPAR
-null mice (approximately 80%). The degree of NPSH depletion in response to 400 mg APAP/kg in corn oil-pretreated wild-type and PPAR
-null female mice is similar to values previously reported for female CD-1 mice receiving a toxic dose of APAP (Hoivik et al., 1995
). 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
-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
expression.
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DISCUSSION |
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Since a large number of the hepatic effects produced by peroxisome proliferators in liver have been attributed to the transcriptional regulation of PPAR-responsive genes, it was of interest to investigate if the hepatoprotective effects of these chemicals might also be under the control of PPAR
. The results of these studies support the involvement of PPAR
activation in this response. Mice lacking PPAR
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
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., 1994, 1996b
; Nicholls-Grzemski et al., 1996
; Nguyen et al., 1999
). 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
-null mice when compared to corn oil-pretreated wild-type mice, CFB-pretreated PPAR
-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., 1998
; Nguyen et al., 1999
).
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 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. 1996a
). 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., 1994
). 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
-responsive genes in hepatoprotection.
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ACKNOWLEDGMENTS |
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NOTES |
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Presented in part at the 39th Annual Meeting of the Society of Toxicology, Philadelphia, PA, 2000.
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