Role of peroxisome proliferator-activated receptor-
(PPAR
) in bezafibrate-induced hepatocarcinogenesis and cholestasis
Thomas Hays1,
Ivan Rusyn2,
Amanda M. Burns1,
Mary J. Kennett1,
Jerrold M. Ward3,
Frank J. Gonzalez4 and
Jeffrey M. Peters1,5
1 Department of Veterinary Science and Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, PA 16802, USA, 2 Department of Environmental Sciences and Engineering, The University of North Carolina, Chapel Hill, NC 27599, USA, 3 Infectious Disease Pathogenesis Section, Comparative Medicine Branch and SoBran, Inc., National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, USA and 4 Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
5 To whom correspondence should be addressed Email: jmp21{at}psu.edu
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Abstract
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Prolonged administration of peroxisome proliferators to rodents typically leads to hepatocarcinogenesis. Peroxisome proliferator-activated receptor-
(PPAR
) is required to mediate alterations in PPAR
target gene expression, repress apoptosis, enhance replicative DNA synthesis, oxidative stress to DNA and hepatocarcinogenesis induced by the relatively specific PPAR
agonist, Wy-14,643. Interestingly, administration of the less specific PPAR
agonist, bezafibrate, leads to a modest induction of PPAR
target genes in the absence of PPAR
expression. In these studies, the role of PPAR
in modulating hepatocarcinogenesis induced by long-term feeding of 0.5% bezafibrate was examined in wild-type (+/+) and PPAR
-null (/) mice. The average liver weight was significantly higher in (+/+) and (/) mice fed bezafibrate than controls, but this effect was considerably less in (/) mice as compared with similarly treated (+/+) mice. Increased levels of mRNA encoding cell cycle regulatory proteins and DNA repair enzymes were found in (+/+) mice fed bezafibrate, and this effect was not found in (/) mice. In mice fed bezafibrate for 1 year, preneoplastic foci, adenomas and a hepatocellular carcinoma were found in (+/+) mice, while only a single microscopic adenoma was found in one (/) mouse. This effect was observed in both Sv/129 and C57BL/6N strains of mice, although only preneoplastic foci were observed in the latter strain. Interestingly, hepatic cholestasis was observed in 100% of the bezafibrate-fed (/) mice, and this was accompanied by significantly elevated hepatic expression of mRNA encoding bile salt export pump and lower expression of mRNA encoding cytochrome P450 7A1, consistent with enhanced activation of the bile acid receptor, farnesoid X receptor. Results from these studies demonstrate that the PPAR
is required to mediate hepatocarcinogenesis induced by bezafibrate, and that PPAR
protects against potential cholestasis.
Abbreviations: ACO, acyl CoA oxidase; BSEP, bile salt export pump; CDK4, cyclin-dependent kinase-4; CYP4A, cytochrome P450 4A1; CYP7A1, cytochrome P450 7A1; FXR, farnesoid X receptor; GADPH, glyceraldehyde-3-phosphate dehydrogenase; PCNA, proliferating cellular nuclear antigen; PPRE, peroxisome proliferator responsive elements
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Introduction
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Since its initial discovery in 1990 (1), the diverse roles for the peroxisome proliferator-activated receptor-
(PPAR
) have been elucidated. In response to ligand activation, PPAR
heterodimerizes with retinoid X receptor
(RXR
), and after recruitment of co-activators, modulates transcription of genes containing peroxisome proliferator responsive elements (PPREs) in their promoter sequence. PPAR
can be activated by a broad class of chemicals, collectively known as peroxisome proliferators, that include phthalates, fatty acids, dehydroepiandrostenedione sulfate, trichloroethylene and the hypolipidemic fibrate drugs (e.g. bezafibrate, clofibrate, ciprofibrate, etc.). In recent years, it was determined that PPAR
has a critical regulatory role in lipid homeostasis as its target genes include many enzymes and proteins involved in the transport and catabolism of fatty acids (2). However, while PPAR
is essential for regulating lipid homeostasis, it is also known to mediate hepatocarcinogenesis typically observed in rodents fed peroxisome proliferators. This conclusion is based on the fact that PPAR
-null mice do not exhibit hepatocarcinogenesis after long-term feeding of Wy-14,643 (3). Given the fact that the fibrate class of hypolipidemic drugs is still in use today, and that other chemicals known to act as PPAR
agonists are present in the environment, there is concern about the relative risk for humans exposed to these agents.
The mode of action underlying PPAR
agonist-induced hepatocarcinogenesis has not yet been fully elucidated. In response to ligand activation, PPAR
mediates alterations in gene expression that lead to increased hepatic cell proliferation, decreased apoptosis and increased signaling for replicative DNA synthesis, that ultimately causes mutant cell populations to proliferate and become neoplastic (4). While PPAR
is required to mediate Wy-14,643-induced hepatocarcinogenesis, the specific target genes modulated by this nuclear receptor that lead to discordant cell cycle regulation have not been identified. It is known that a number of proteins required for transition into the S-phase of the cell cycle are increased by peroxisome proliferators, and this event requires PPAR
(5), although functional PPREs have not been characterized in any of these critical regulatory proteins. Additionally, oxidative stress induced by activated Kupffer cells in response to peroxisome proliferators may contribute to increased signaling for hepatocyte proliferation (6). Whether or not increased intracellular H2O2 resulting from elevated acyl CoA oxidase (ACO) expression in liver parenchymal cells contributes to oxidative stress-mediated signaling for increased cell proliferation is uncertain, but increased expression of ACO and resulting generation of H2O2 may lead to oxidative DNA damage (7). However, the evidence linking peroxisome proliferators to secondary oxidative DNA damage is conflicting, and therefore the mechanisms underlying the initiation of DNA damage in response to this class of chemicals have not been elucidated (8).
The observation that PPAR
-null mice are refractory to Wy-14,643-induced hepatocarcinogenesis and alterations in cell proliferation associated with the early events that probably contribute to the mechanisms of peroxisome proliferator-induced liver cancer (3,5), provides convincing evidence that PPAR
is required to mediate this effect. However, the role of this receptor in peroxisome proliferator-induced hepatocarcinogenesis has only been examined using one relatively specific PPAR
agonist, Wy-14,643. Interestingly, a recent report demonstrated that bezafibrate could induce the expression of a number of different PPAR
target genes in PPAR
-null mice. While bezafibrate markedly increased the hepatic expression of mRNAs encoding peroxisomal and mitochondrial fatty acid oxidizing enzymes in wild-type mice, a marginal increase in the same mRNAs was observed in the absence of PPAR
expression (9). In contrast, these changes in mRNA markers are completely absent in PPAR
-null mice fed Wy-14,643 (10). It is of interest to note that the EC50 for receptor activation is at least two orders of magnitude different for Wy-14,643 and bezafibrate with values of 0.63 and 90 µM for mouse PPAR
being reported, respectively (11). Because the effect of feeding only one prototype peroxisome proliferator (Wy-14,643) to PPAR
-null mice has been examined to date, the goal of the present studies was to examine the hypothesis that PPAR
is required for bezafibrate-induced hepatocarcinogenesis using a peroxisome proliferator with inherently different PPAR
activity. Additionally, a secondary goal of these studies was to examine the effect of marginally induced liver ACO on peroxisome proliferator-induced hepatocarcinogenesis, as the less specific PPAR
agonist bezafibrate (as compared with Wy-14,643), is capable of inducing a PPAR
target protein (ACO) in the absence of PPAR
expression, and has been postulated to contribute to oxidative DNA damage during peroxisome proliferator-induced hepatocarcinogenesis.
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Materials and methods
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Diet
2-{4-[2-(4-Chlorobenzamido)ethyl]phenoxy}-2-methylpropanoic acid (bezafibrate) was purchased from Sigma Chemical (St Louis, MO). The pelleted mouse diet was prepared by Bioserv (Frenchtown, NJ) containing 0 (control) and 0.5% bezafibrate. Diets were available to mice ad libitum.
Short-term study
PPAR
(+/+) and (/) male mice with an Sv/129 genetic background (10,12) were housed in a temperature (25°C) and light (12-h light/12-h dark) controlled environment. Mice were fed either the control or 0.5% bezafibrate diet for 1 or 5 weeks. Following this treatment, the mice were killed by overexposure of carbon dioxide after a 2-h fast. Body and liver weights were recorded, and the liver was snap-frozen in liquid nitrogen for subsequent analysis of mRNAs. A separate cohort of PPAR
(+/+), PPAR
(/), PPAR
(/)/PPARß (/) or PPAR
(/)/PPAR
(/) [liver-specific deletion using albumin-Cre/floxed PPAR
alleles (13)] mice were fed 0.5% bezafibrate for 2 weeks, killed after a 2-h fast, and their liver collected and analyzed for mRNA as described above.
Long-term study
PPAR
(+/+) and (/) mice with either Sv/129 or C57BL/6N genetic backgrounds were housed as described above. Mice from both genotypes were fed either the control or 0.5% bezafibrate diet for up to 12 months. One of the (/) mice, and three (+/+) mice fed bezafibrate died unexpectedly after 10 months of feeding, and another two (/) mice fed bezafibrate for 11 months were killed because of signs of morbidity. The livers from the four former mice were examined for grossly visible signs of liver tumors, but were not examined histologically due to autolysis. Tissues from the latter two (/) mice that were killed early were examined for grossly visible signs of liver tumors and included for additional histological analysis as described below. At the end of the 12-month feeding period, mice were killed after a 2-h fast; livers were removed and rinsed in phosphate-buffered saline, and weighed. Gross lesions were counted, the location and diameter were recorded, and representative samples were prepared for histological examination (see below).
Pathology
Each liver was examined for the presence of grossly visible lesions. For each mass, the diameter was measured and the location noted. Representative liver samples were removed and fixed in 10% phosphate-buffered formalin (Fisher Scientific, Fair Lawn, NJ) and embedded in paraffin. Paraffin sections were prepared from these samples, sections were stained with hematoxylin and eosin and examined morphologically for the presence of carcinomas, adenomas or preneoplastic lesions using established criteria (14). Control sections were also examined from mice lacking grossly visible lesions.
Northern blot analysis
Total RNA was isolated from liver sections lacking gross lesions by the Trizol method using the manufacturer's procedure (Invitrogen Life Technologies). Five to ten micrograms of RNA was separated by gel electrophoresis on 1% agarose gels containing 0.22 M formaldehyde, and transferred to nylon membrane in 10x SSC (1x SSC is 0.15 M NaCl and 0.18 M sodium citrate) overnight. Membranes were dried, UV cross-linked, washed twice in 2x SSC (1x SSC is 0.15 M NaCl and 0.18 M sodium citrate) and 0.5% sodium dodecyl sulfate for 15 min at 65°C. The following previously described cDNA probes were used for northern blotting: ACO, cytochrome P450 4A1 (CYP4A), cyclin-dependent kinase-4 (CDK4), proliferating cellular nuclear antigen (PCNA), cyclin B1, catalase, farnesoid X receptor (FXR), bile salt export pump (BSEP), cytochrome P450 7A1 (CYP7A1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control (5,10,15,16). Probes were labeled with [32P]dCTP using a random priming method and hybridized in ULTRAhybTM buffer (Ambion, Austin, TX) at 42°C overnight, and washed. Quantification of hybridization signals was performed using phosphor imaging and expression data were normalized relative to the GAPDH signal. Representative liver RNA samples from between 3 and 4 mice were used for analysis per treatment group.
Hepatic bile acids
Frozen liver samples were homogenized in 75% ethanol, and incubated at 50°C for 2 h. After centrifugation, the supernatant was obtained and used for enzymatic determination of bile acids using the Sigma Diagnostics Bile Acid colorimetric assay (St Louis, MO).
Ribonuclease protection assays
Total RNA from frozen liver samples was isolated using RNeasy total RNA extraction kit (Qiagen, Valencia, CA) and dissolved in RNase-free water. Samples were stored at 80°C until assayed for no longer than 2 months to minimize degradation. The quality of preparations was determined using an Agilent Bio-Analyzer®. Expression of base excision DNA repair genes was determined using an RNase protection assay using mouse multi-probe RNA probe template set (mBER-1, BD PharMingen, San Diego, CA) as described previously (17). Riboprobes were synthesized in the presence of [32P]dUTP to yield labeled antisense RNA probes. The RNase protection assays were performed using 30 µg of individual total RNA samples using a RiboQuantTM multi-probe RNase protection assay kit. Protected fragments were separated on 5% polyacrylamide nucleic acid separation gels, dried and exposed to phosphor-imaging screen. The intensity of protected bands was quantified using a phosphor image analyzer and normalized to the intensity of housekeeping gene L32.
Statistical analysis
Differences between treatments were determined using ANOVA followed by Dunnett's post-hoc test (Prism 4.0, GraphPad Software, San Diego, CA). Significant differences were determined with P
0.05.
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Results
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Consistent with previous work, administration of bezafibrate caused a large fold increase in the expression of mRNAs encoding ACO and CYP4A in the liver of (+/+) mice after 1 or 5 weeks as compared with the control. In contrast, induction of ACO and CYP4A was still observed in the liver of (/) mice fed bezafibrate as compared with similarly treated (+/+) mice but the fold induction was considerably lower (Figure 1A). Mice nullizygous for both PPAR
and PPARß, or PPAR
and PPAR
were fed bezafibrate to determine if this marginal increase in ACO and CYP4A mRNA was mediated by either PPARß and/or PPAR
. Consistent with previous studies demonstrating that PPARß does not mediate bezafibrate-induced increases in liver ACO and CYP4A (9), mice nullizygous for both PPAR
and PPARß exhibited a marginal induction of mRNA encoding ACO and CYP4A (Figure 1B). Additionally, mice nullizygous for both PPAR
and PPAR
exhibited a similar, marginal increase in both ACO and CYP4A mRNAs (Figure 1B). Bezafibrate feeding resulted in a similar increase in the mRNA encoding catalase in both (+/+) and (/) mice (Figure 1A). In contrast to the results observed with ACO and CYP4A expression, the level of mRNA encoding CDK4 and PCNA were significantly higher in (+/+) mice fed bezafibrate for 1 or 5 weeks, and the level of mRNA encoding cyclin B1 was significantly higher in this group as compared with (+/+) controls, but this effect was not found in similarly treated (/) mice (Figure 1A). This is consistent with the
2-fold increase in average liver weight observed in (+/+) mice fed bezafibrate for 1 or 5 weeks (data not shown). Despite no changes in mRNAs encoding markers of cell cycle progression in the (/) mice fed bezafibrate for either 1 or 5 weeks, the average liver weight was marginally increased as compared with the control by
1.5-fold (data not shown).

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Fig. 1. Representative northern blot of hepatic mRNAs encoding ACO, CYP4A, catalase and cell cycle regulatory proteins, including, CDK4, PCNA and cyclin B1. (A) Total RNA was isolated from wild-type (+/+) and PPAR -null (/) mice with an Sv/129 genetic background, fed either a control diet or one containing 0.5% bezafibrate, for 1 (1 wk) or 5 weeks (5 wk). Total RNA was separated by gel electrophoresis, transferred to nylon membranes and probed with the indicated cDNAs. Representative samples from two independent samples per treatment are shown; a total of four independent samples were analyzed. Hybridization signals were normalized to GAPDH and are presented as the fold-increase as compared with respective control. *Significantly different than controls, P 0.05. (B) Representative northern blot of hepatic mRNAs encoding ACO, CYP4A or peroxisome proliferator-activated receptors (PPARs) from control (+/+) or bezafibrate-fed (+/+), PPAR -null (/), PPAR /PPARß-null [ /ß (/)], or PPAR /PPAR -null [ / (/)]. Note the absence of endogenous mRNA encoding PPAR , PPARß or PPAR as shown previously (10,13,35), in the (/), /ß (/) and / (/) mice. Representative samples from two independent samples per treatment are shown; a total of at least three samples were analyzed. Hybridization signals were normalized to GAPDH and are presented as the fold-increase as compared with respective control. #Significantly different than wild-type control, P 0.05. *Significantly different than wild-type control and bezafibrate-treated wild-type, P 0.05.
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After 1 year of treatment with 0.5% bezafibrate in the diet, the average liver weight was
4-fold higher in (+/+) mice than in the control (+/+) mice, while the average liver weight was only marginally increased (
2-fold) in (/) mice fed bezafibrate as compared with the control (/) mice (Table I). Grossly visible lesions consistent with the presence of adenomas or carcinomas were found in 100% of the (+/+) mice fed bezafibrate for 1 year whereas grossly visible lesions were not found in the (/) mice fed bezafibrate for the same timeframe (Table I). Average tumor multiplicity was 5 ± 2 lesions per liver in bezafibrate-fed (+/+) mice. Microscopic examination of liver sections was highly consistent with the gross examinations, as histological evidence of hepatic neoplasias was observed in 100% of the (+/+) mice fed bezafibrate for 1 year (Table II). The histological examination revealed that the majority of liver lesions were either adenomas or preneoplastic foci, but one well-differentiated carcinoma was observed in the (+/+) mice fed bezafibrate for 1 year (Figure 2). Interestingly, one adenoma was found in a (/) mouse fed bezafibrate (Figure 2), however, this was also accompanied by a unique phenotype not observed previously in response to long-term Wy-14,643 feeding. In addition to significant lipid accumulation (steatosis), liver pathology consistent with cholestasis was found in 100% of the (/) mice fed bezafibrate for 1 year that was not found in similarly treated (+/+) mice. Pigmented hepatocytes, consistent with bile acid accumulation, were noted in 100% of the (/) mice fed bezafibrate (Figure 2).
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Table I. Effect of bezafibrate on average relative liver weight and grossly visible hepatic lesions in wild-type (+/+) and PPAR -null (/) mice with an Sv/129 genetic background
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Table II. Effect of bezafibrate on the incidence of hepatocellular neoplasms in the wild-type (+/+) and PPAR -null (/) mice with an Sv/129 genetic background
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Fig. 2. Representative histopathology of the liver in response to bezafibrate treatment. Wild-type (+/+) or PPAR -null (/) mice were fed either a control or 0.5% bezafibrate diet for 1 year, and livers examined. (A) Representative photomicrograph of peroxisome proliferation in (+/+) mouse liver. This phenotype was observed in both (+/+) Sv/129 and (+/+) C57BL/6N mouse liver, but not in the (/) liver from either strain. Note the dense presence of peroxisomes (eosinophilic cytoplasmic granularity) in the hepatocyte cytosol (arrows). Magnification = 400x. (B) Representative photomicrograph of preneoplastic irregular clear cell focus in the (+/+) mouse liver. The focus is indicated by f and the normal tissue surrounding the focus is indicated by n. These lesions were observed in both (+/+) Sv/129 and (+/+) C57BL/6N mouse liver, but not in the (/) liver from either strain. Magnification = 200x. (C) Representative photomicrograph of hepatocellular adenoma from the (+/+) mouse. The adenoma and adjacent liver are both eosinophilic. The adenoma is indicated by a and the surrounding normal tissue is indicated by n. Magnification = 200x. (D) Representative photomicrograph of well-differentiated hepatocellular carcinoma from the (+/+) mouse. Magnification = 400x. (E) Representative photomicrograph of lipid accumulation observed in the (/) mice fed bezafibrate with an Sv/129 genetic background. Magnification = 400x. (F) Representative photomicrograph of cholestasis present in 100% of the (/) mice, both strains. Arrows indicate the presence of pale, brown finely granular pigment in the cytoplasm of hepatocytes. Magnification = 400x. (G) Photomicrograph of the hepatocellular adenoma from the one (/) mouse. The adenoma is basophilic and the adjacent liver is much less eosinphilic than the (+/+) mice fed bezafibrate. The adenoma is indicated by a and the surrounding normal tissue is indicated by n. Magnification = 200x.
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The presence of cholestasis in the (/) mice fed bezafibrate suggests an imbalance in bile acids, which was further evaluated by examining the concentration of hepatic bile acids and mRNAs encoding key regulators of bile acid homeostasis. Bezafibrate feeding resulted in a significant decrease in the concentration of hepatic bile acids in (+/+) mice (Figure 3A). A significant decrease in hepatic bile acid concentration was also observed in bezafibrate-fed (/) mice, but the percentage decrease was lower and the average concentration was significantly greater than that found in bezafibrate (+/+) mice (Figure 3A). To determine the biological significance of this difference in bile acid concentration, mRNA markers of bile acid homeostasis were examined. No difference in the expression pattern of mRNA encoding FXR was found between genotypes or treatment (Figure 3B). However, the expression of mRNA encoding BSEP that is known to be increased as a result of bile acid activation of FXR was significantly increased in the (/) mice after 1 week, 5 weeks and 1 year of feeding the bezafibrate diet (Figure 3B). In contrast, expression of mRNA encoding BSEP was reduced in (+/+) mice fed bezafibrate at similar time points (Figure 3B). Further, the expression of CYP7A1, which is known to be down-regulated by FXR was significantly lower at earlier time points in the bezafibrate-fed (/) mice as compared with similarly treated (+/+) mice (Figure 3B). These results are consistent with enhanced bile acid/FXR-mediated signaling in the (/) mice fed bezafibrate.
As hepatic ACO mRNA expression was marginally increased in the (/) fed bezafibrate for 5 weeks, an RNase protection assay was performed to examine the relative expression of mRNA markers for oxidative stress to DNA. In (+/+) mice, bezafibrate treatment led to a significant time-dependent increase in expression of mRNAs encoding 8-oxoguanine DNA glycosylase/lyase 1 (Ogg1), apurinic/apyrimidinic endonuclease 1 (Ape), O6-methyl guanine methyltransferase (Mgmt), N-methylpurine DNA glycosylase (Mpg), thymine DNA glycosylase (Tdg) and endonuclease III homolog 1 (Nth1) (Figure 4). However, expression of mRNAs encoding DNA repair proteins was not affected in the bezafibrate-treated (/) mice (Figure 4).
All of the previous work examining the role of PPAR
in mediating peroxisome proliferator-induced hepatocarcinogenesis, including these studies and prior results (3), have utilized mice with an Sv/129 genetic background. In these studies, a small cohort of mice with the C57BL/6N genetic background were also examined after 1 year of feeding 0.5% bezafibrate. Hepatomegaly was found in (+/+) mice with a C57BL/6N genetic background fed bezafibrate for 1 year, and similar to results observed in the Sv/129 mice, a small increase in average liver weight was also found in (/) mice with a C57BL/6N genetic background fed bezafibrate for 1 year (Table III). In contrast to the high incidence of grossly visible lesions found in the Sv/129 (+/+) mice fed bezafibrate for 1 year, no grossly visible lesions were observed in the bezafibrate-fed (+/+) mice with a C57BL/6N genetic background, nor in the (/) mice on the same genetic background (Table III). While no adenomas or carcinomas were found in the C57BL/6N (+/+) mice fed bezafibrate, microscopic examination revealed preneoplastic foci in 75% of the livers examined (Table IV). No microscopic evidence of neoplasias was found in any of the (/) mice with the C57BL/6N genetic background fed bezafibrate for 1 year (Table IV), although cholestasis was noted in all of these samples in the absence of significant lipid accumulation (Figure 2).
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Table III. The effect of bezafibrate on average relative liver weight and grossly visible hepatic lesions in wild-type (+/+) and PPAR -null (/) mice with a C57BL/6N genetic background
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Table IV. Effect of bezafibrate on the incidence of hepatocellular neoplasms in wild-type (+/+) and PPAR -null (/) mice with a C57BL/6N genetic background
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Discussion
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Results from these studies show that the wild-type mice fed bezafibrate exhibit many of the hallmark changes associated with activation of PPAR
including induction of ACO and CYP4A, induction of mRNAs encoding cell cycle regulatory proteins and DNA repair enzymes, hepatomegaly, and the resulting high incidence of hepatocellular neoplasms. While the hepatocarcinogenic effects of long-term bezafibrate feeding have not been reported in mice, it is well established that long-term feeding of other fibrates and PPAR
agonists to rodents results in liver hepatocarcinogenesis (reviewed in ref. 4). Previous studies have demonstrated that the hepatocarcinogenic effect of a prototypical PPAR
agonist, Wy-14,643, is mediated by PPAR
, as PPAR
-null mice are refractory to PPAR
-induced changes including induction of cell cycle regulatory proteins (5), oxidative stress to DNA (18), and more importantly, hepatocellular carcinogenesis (3). Results from present studies that used the less specific PPAR
agonist, bezafibrate, are relatively consistent with results from the former study. For example, no changes in hepatic levels of mRNAs encoding CDK4, PCNA and cyclin B1 or DNA repair enzymes were observed in bezafibrate-fed PPAR
-null mice, and the incidence of hepatocellular neoplasms was significantly lower in the bezafibrate-fed PPAR
-null mice as compared with similarly treated wild-type mice. However, in contrast to the previous studies demonstrating the lack of liver carcinogenesis in Wy-14,643-fed PPAR
-null mice, one adenoma was found in a PPAR
-null fed bezafibrate for 1 year. While this result suggests that PPAR
may not be essential for mediating the hepatocarcinogenic effect of bezafibrate, the collective evidence from the present studies are inconsistent with this hypothesis, as no changes in markers of hepatocyte cell proliferation (e.g. CDK4, cyclin B1 and PCNA mRNAs) and oxidative DNA damage were found in bezafibrate-fed PPAR
-null mice. As these changes are causally linked to the mode of action for peroxisome proliferator-induced hepatocarcinogenesis (4), the absence of these changes suggests that other mechanisms probably underlie the presence of the single adenoma in the PPAR
-null mouse fed bezafibrate for 1 year. There are a number of possible variables that could account for this observation, including the presence of a rare, spontaneous neoplasm in an older mouse, differences in receptor/ligand affinity and the presence of altered bile acid homeostasis observed in the PPAR
-null mice fed bezafibrate.
It is well documented that reporter constructs containing ACO PPREs (a PPRE from an endogenous PPAR
-regulated target gene) can be activated by all three PPARs when treated with specific ligands (19). There is also some promiscuity associated with ligand and receptor specificity as PPAR
ligands can also activate ACO PPREs when co-transfected with either PPARß or PPAR
expression vectors (2022). This ligand/receptor promiscuity also occurs with PPARß and PPAR
ligands. Therefore, it is possible that the biological effects induced by specific PPAR ligands could be mediated by more than one PPAR. The EC50 for mouse PPAR activation by Wy-14,643 based on cell-based transactivation assays is 0.63, >100 and 32 µM, while the EC50 for mouse PPAR activation by bezafibrate is 90, 110 and 55 µM, for mouse PPAR
, PPARß(
) and PPAR
, respectively (11). Given the known ability for PPAR
ligands to activate more than one PPAR, the fact that all three PPARs can transactivate similar response elements, and that the relative affinity for bezafibrate for PPAR
is less specific as compared with Wy-14,643, suggests that bezafibrate-induced changes in gene expression could be mediated by either PPARß or PPAR
. This is of particular interest because in the absence of PPAR
expression, known PPAR
target genes are still marginally induced by bezafibrate (9). The present studies indicate that this could be due to either PPARß or PPAR
as a marginal induction of ACO and CYP4A is observed in the absence of both PPAR
and PPAR
, or PPAR
and PPARß expression in the liver. This suggests that PPARß or PPAR
could mediate this effect, but this hypothesis will require examination of mice nullizygous for all three PPARs. These combined observations suggest that the presence of an adenoma in the PPAR
-null mouse fed bezafibrate could be the result of a marginal induction of PPAR
target genes; however, results from the present studies are inconsistent with this hypothesis as no changes in mRNAs encoding cell cycle regulatory proteins or DNA repair enzymes were found in the PPAR
-null mice fed bezafibrate. While the marginal induction of ACO and CYP4A observed in the PPAR
-null mice fed bezafibrate could be due to activation of PPARß or PPAR
[and increased expression of PPAR
is reported to occur in PPAR
-null mice (23)], it is unlikely that this is causally related to the one adenoma observed in the PPAR
-null mouse, as similar marginal induction of ACO that occurs with dietary modulation has never been shown to lead to hepatocarcinogenesis. Further, the lack of increased markers of oxidative DNA damage suggests that the marginal induction of ACO by bezafibrate in PPAR
-null mice probably does not lead to DNA damage. As ligand activation of PPAR
is reported to cause hepatotoxicity (24), it is possible that the adenoma found in the bezafibrate-fed PPAR
-null mouse resulted from activation of this PPAR isoform. The possibility that bezafibrate activates PPAR
in the PPAR
-null mice is also supported by the observed steatosis in PPAR
-null mice, since it is known that PPAR
is required for lipid accumulation in liver (25).
The presence of cholestasis in 100% of the PPAR
-null mice fed bezafibrate indicates that the concentration of bile acids is considerably higher in these mice than similarly treated wild-type mice. While bezafibrate feeding resulted in a significant reduction in hepatic bile acid concentration in wild-type mice, the percentage decrease and the average concentration of hepatic bile acids were significantly greater in PPAR
-null mice. These observations are consistent with the detected changes in gene expression of BSEP and CYP7A1 in the PPAR
-null mice fed bezafibrate. Interestingly, these genes are known to be regulated by FXR, the nuclear receptor that controls hepatic bile acid homeostasis (16). Others have demonstrated previously that bile acids can function as tumor promoters (26,27), suggesting that a bezafibrate-induced increase in bile acids in the PPAR
-null mice could promote tumor formation. Others have also shown that PPAR
agonists, including Wy-14,643 and ciprofibrate, can inhibit bile acid synthesis (28), which is consistent with the observed increase in cholestasis found in PPAR
-null mice fed bezafibrate. There is also evidence that conjugation of bile acids can be modulated by PPAR
(29), thus, the absence of this regulation could lead to accumulation of conjugated bile acids that may contribute to the observed phenotype. However, it is important to point out that cholestasis is not observed in Wy-14,643-fed PPAR
-null mice after long-term feeding (3), which suggests that this putative mechanism is probably not functional for all PPAR
agonists and could be related to the differences in receptor affinity described above. It is also important to note that these alterations in bile acid homeostasis are not found in bezafibrate-fed wild-type mice that express a functional PPAR
, suggesting that with this low affinity ligand, PPAR
actually protects against cholestasis. The possibility remains that bezafibrate or one of its metabolites may actually be an FXR agonist. It is also of interest to note that bile acids (natural FXR ligands) antagonize PPAR
agonist-induced modulation of target gene expression in liver indicating the possibility of cross-talk between PPAR
and FXR (30). This observation suggests that in the absence of PPAR
expression, bile acids could possibly activate FXR with greater efficiency. It will be interesting to determine whether other lower affinity fibrate drugs produce cholestasis in the absence of PPAR
expression. Combined, these observations suggest that there is enhanced accumulation of bile acids and activation of FXR that would not normally occur in the presence of a functional PPAR
, indicating a protective role for this nuclear receptor in preventing cholestasis in response to bezafibrate. This is of great interest because cholestasis is uncommon in mice, but is often observed in humans in response to therapeutic drugs (31).
An interesting strain difference in response to bezafibrate was also determined from these studies. Preneoplastic lesions were found in a high percentage (75%) of bezafibrate-fed wild-type mice with the C57BL/6N genetic background, but no adenomas or carcinomas were observed in these mice suggesting that in this genetic background, hepatocellular tumors develop more slowly than in the Sv/129 background. No evidence of neoplasia was found in bezafibrate-fed PPAR
-null mice in the C57BL/6N genetic background, despite the presence of cholestasis (but no steatosis) in all of these mice. C57BL/6N mice fed the potent PPAR
agonist ciprofibrate for 21 months exhibited an approximate incidence of 57% of hepatic neoplasia, while similar treatment with ciprofibrate for 18 months resulted in an approximate incidence of 62% (32). In contrast, all other published reports examining peroxisome proliferator-induced hepatocarcinogenesis have used mouse strains other than C57BL/6N, and a very high incidence of neoplasia (>90%) is typically observed in these lines, dependent on the duration of exposure, dose and potency of PPAR
agonist (reviewed in ref. 4). As the C57BL/6 strain of mouse is more resistant to hepatocarcinogenesis-induced peroxisome proliferators (32), and other chemicals (33,34), results from the present studies examining bezafibrate-induced hepatocarcinogenesis in C57BL/6N mice provide additional evidence that illustrates that the PPAR
-null in an Sv/129 genetic background are more appropriate for examination of PPAR
agonist-induced hepatocarcinogenesis. Additionally, the lack of preneoplastic foci in the PPAR
-null mice with the C57BL/6N genetic background suggests that PPAR
is required for bezafibrate-induced hepatocarcinogenesis in this mouse strain.
In summary, the extent of bezafibrate-induced changes in hepatic expression of mRNAs encoding ACO, cell cycle regulatory proteins and DNA repair enzymes, hepatomegaly, and the incidence of hepatocellular neoplasms is either completely lacking or significantly lower in PPAR
-null mice as compared with similarly treated wild-type mice. These results are consistent with a previous study demonstrating the lack of these similar changes in PPAR
-null mice fed Wy-14,643 for 11 months. This suggests that PPAR
is essential for bezafibrate-induced hepatocarcinogenesis. The distinctly unique presence of altered bile acid homeostasis in PPAR
-null mice treated with this relatively less specific PPAR
agonist, coupled with the known tumor-promoting capabilities of bile acids, suggests that these alterations probably underlie the mechanisms leading to a minor incidence of neoplasia in the absence of PPAR
expression. As the role of PPAR
in peroxisome proliferator-induced hepatocarcinogenesis had previously only been examined using the prototypical peroxisome proliferator Wy-14,643, results from the present studies provide a new data set that strengthens the argument that PPAR
is essential to mediate peroxisome proliferator-induced hepatocarcinogenesis.
 |
Acknowledgments
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The authors gratefully acknowledge Debra Wolgemuth, Christopher Sinal and Grace Guo for providing the cDNA probes. This work supported in part by CA89607 and CA97999 (J.M.P.).
 |
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Received July 5, 2004;
revised September 9, 2004;
accepted September 10, 2004.