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Hepatocarcinogenic Susceptibility of Fenofibrate and Its Possible Mechanism of Carcinogenicity in a Two-Stage Hepatocarcinogenesis Model of rasH2 Mice

¹ Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
² Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, Gifu, Gifu 501-1193, Japan
³ Department of Applied Biological Science, United Graduate School of Agricultural Sciences, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan

Correspondence: Address correspondence to: Masaomi Kawai, Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan; e-mail:m_kawai{at}cc.tuat.ac.jp.

	Abstract

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Fenofibrate (FF) has previously been shown to induce hepatocellular neoplasia in a conventional mouse bioassay (NDA 1993), but there has been no report to examine the carcinogenic susceptibility of rasH2 mice to this chemical. In the present study, male rasH2 mice were subjected to a two-thirds partial hepatectomy (PH), followed by an N-diethylnitrosamine (DEN) initiation twenty-four hours after PH, and given a diet containing 0, 1200, or 2400 ppm FF for seven weeks. The incidences of preneoplastic foci were significantly increased in mice from the FF-treated groups. Immunohistochemistry revealed that significant increases in proliferating cell nuclear antigen (PCNA)-positive cells and cytokeratin 8/18 positive foci were observed in FF-treated groups. In addition, the transgene and several downstream molecules such as c-myc, c-jun, activating transcription factor 3 (ATF3), and cyclin D1 were overexpressed in these groups. These results suggest that the hepatocarcinogenic activity of rasH2 mice to FF can be detected in this hepatocarcinogenesis model and that up-regulation of genes for the ras/MAPK pathway and cell cycle was probably involved in the hepatocarcinogenic mechanism of rasH2 mice.

Key Words: rasH2 mouse • fenofibrate • cytokeratin 8/18 • liver

Abbreviations: ATF3, activating transcription factor 3 • DEHP, diethyl hexylphalate • DEN, N-diethylnitrosamine • ENU, N-ethyl-N-nitrosourea • FDA, Food and Drug Administration • FF, fenofibrate • KO, knockout • MAPK, mitogen activated protein kinase • PBS, phosphate-buffered saline • PCNA, proliferating cell nuclear antigen • PH, partial hepatectomy • PPAR, peroxisome proliferator-activated receptor • real-time RT-PCR, quantitative real-time reverse transcription-polymerase chain reaction • TG, transgenic

	Introduction

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In 1997, the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH) proposed new guidelines on carcinogenicity testing, since it was concluded that the carcinogenicity of drugs can be evaluated based on data from six-month carcinogenicity studies performed using transgenic (TG) or knockout (KO) mice or using data from a 2-year conventional carcinogenicity study of one rodent species (D’Arcy et al. 1998). So far, many six-month studies using p53 KO mice have been conducted to analyze the carcinogenicity of newly developed drugs in accordance with the recommendation of the Food and Drug Administration (FDA) in the United States. It was confirmed by the FDA that p53 KO mice are not susceptible to non-genotoxic carcinogens but are susceptible to genotoxic carcinogens, although positive results were obtained in two-year carcinogenicity studies of chemicals that were regarded as non-genotoxic carcinogens in rodents (Storer et al. 2001). Accumulated data from two-year carcinogenicity studies in rodents reviewed by the FDA showed carcinogenicity findings that indicated that continued clinical development of some peroxisome proliferator-activated receptor (PPAR) agonists (, / agonists) could not be supported because adequate margins of safety were not demonstrated (Elangbam et al. 2002). Based on this information, the new guidelines provided by the FDA for compounds in this class indicate that the carcinogenic potential of these PPAR agonists cannot be evaluated in TG or KO mice, and clinical studies longer than six months in duration cannot be initiated until two-year rodent carcinogenicity studies are completed and submitted for the agency to review (El-Hage 2004). On the other hand, it is well recognized that rasH2 mice, hemizygous transgenic mice carrying the human prototype c-Ha-ras gene with its own promoter and enhancer, are susceptible not only to genotoxic carcinogens but also to some non-genotoxic carcinogens classified as PPAR agonists, such as clofibrate, diethyl hexylphalate (DEHP), and Wy-14643 (Nesfield et al. 2005; Toyosawa et al. 2001; Yamamoto et al. 1996). This means that in the absence of a database on rasH2 mice, the FDA recognizes the insusceptibility of these TG and KO mice to PPAR agonists.

In general, chronic administration of many PPAR ligands induces hepatocellular tumors in rats and mice (Kluwe et al. 1982; Reddy et al. 1976). For example, PPAR agonists such as fenofibrate (FF) and clofibrate induce liver tumors in mice (Nesfield et al. 2005). In many countries FF, a member of the fibrate class of hypolipidemic drugs, has been extensively used for a long time to treat hypertriglyceridemia and mixed hyperlipidemia (Staels et al. 1998). FF has also been reported to potentially exert hepatocarcinogenic and hepatocellular tumor-promoting effects in rats (Nishimura et al. 2007), and the possible mechanism of hepatocarcinogenicity of FF has gradually been elucidated. In their study, FF treatment increased 8-OHdG level in liver DNA, lipofuscin deposition in hepatocytes, and in vitro production of reactive oxygen species in microsomes. These results suggest that oxidative stress is involved in the development of FF-induced hepatocellular preneoplastic foci in rats (Nishimura et al. 2008). Hepatocellular neoplasms were induced in male rasH2 mice given clofibrate for six months (Nesfield et al. 2005), but there is no report dealing with the molecular mechanism of hepatocarcinogenesis of PPAR agonists in rasH2 mice.

In the present study, we performed a short-term carcinogenicity study of FF using an eight-week, two-stage hepatocarcinogenesis model to examine the carcinogenic susceptibility of rasH2 mice to this chemical and to gain insight into the possible mechanism of hepatocarcinogenesis.

	Materials and Methods

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Chemicals
FF (CAS no.49562-28-9, purity > 99%) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and N-diethylnitrosamine (DEN) was purchased from Nacarai-tesque Co. (Kyoto, Japan). All other chemicals were of analytical grade and obtained commercially.

Animals
Male rasH2 mice, six weeks of age, were obtained from CLEA Japan, Inc. (Tokyo, Japan). They were housed in plastic cages (each cage contained five animals) with absorbent paper chip bedding in an animal room maintained under standard conditions (room temperature, 22 ± 2°C; relative humidity, 55% ± 5%; and light/dark cycle, 12 hours) and given free access to a powdered diet (Oriental MF; Oriental Yeast, Tokyo, Japan) and tap water. The animals were acclimatized for one week prior to the beginning of the experiment. The experiment was performed in accordance with the guidelines for animal experimentation of the Faculty of Agriculture, Tokyo University of Agriculture and Technology.

Experimental Design
In this experiment, we used a short-term, two-stage liver carcinogenesis model (Moto et al. 2006) in rasH2 mice. To enhance hepatocellular proliferation, mice were subjected to two-thirds partial hepatectomy. Twenty-four hours after the hepatectomy, mice were given a single intraperitoneal (ip) injection of DEN (30 mg/kg body weight) dissolved in saline to initiate hepatocarcinogenesis. One week after the injection, they were subdivided into 3 groups and administered a powdered diet containing FF at a concentration of 0, 1200, or 2400 ppm for seven weeks. The dosage in our study was selected based on the information on a carcinogenicity study of FF published by the U.S. FDA, in which liver carcinomas were induced by the oral administration of 200 mg/kg/day of FF in both sexes of mice (http://www.fda.gov/medwatch/; search word is Triglide tablets). On completion of the treatment period, the mice were killed by exsanguination from the posterior vena cava under ether anesthesia, and livers were immersed in 4% paraformaldehyde solution for microscopic examination. Some of the livers were cut into small pieces and frozen in RNAlater (Qiagen, Hilden, Germany) and stored at 80°C until analysis.

Histopathology, Immunohistochemistry, and Quantitative Analysis for Positive Cells of the Liver
After mice were sacrificed, five pieces of each liver fixed in 4% paraformaldehyde were embedded in paraffin, sectioned at a thickness of 3 µm, and all samples were stained with hematoxylin and eosin (HE) for histopathological examination. For each mouse, the numbers of hepatocellular-altered foci in all of the HE-stained sections were counted under a light microscope. The area of these altered foci was not measured. The amount of tissue examined was the same for all groups, and the evaluation was done in a blinded fashion. For immunohistochemical analyses, liver sections were randomly selected from five animals from each group. Serial liver sections were made for immunohistochemical stainings of cytokeratin 8/18 and PCNA.

Cytokeratin 8/18
Anti-cytokeratin 8/18 polyclonal antibody was purchased from PROGEN Biotechnik GmbH (Heidelberg, Germany). The liver sections were deparaffinized in xylene and rehydrated in ethanol. For antigen retrieval, liver sections were incubated with citrate buffer (0.1 mol/L citrate [pH 6.0], 0.1% NP-40) in an autoclave at 120°C for ten minutes and then heated in a microwave oven at low power for five minutes. The sections were then allowed to cool at room temperature for forty minutes. Endogenous peroxidase activity was blocked by incubation for ten minutes in 3% hydrogen peroxide. Nonspecific binding of Ig was prevented by incubating the sections in phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA), 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.5% casein. The sections were washed and then incubated with a guinea pig peroxidase-conjugated secondary antibody (Fitzgerald Industries International, Inc., Concord, MA, USA) diluted in PBS supplemented with 0.5% casein.

Subsequently, 3, 3'-diaminobenzidine (DAB, Dojindo Laboratories, Kumamoto, Japan) was applied as a chromogen. Finally, the sections were counterstained with hematoxylin. Five sections from each liver of five animals were examined. The positive foci (over three cells) counted were divided according to various size ranges, and the results were expressed as the area and number of foci per mm². The number and areas of cytokeratin 8/18-positive foci and the total areas of the liver sections were quantified using a computer-assisted image analyzer (WinRoof version 5.7.2, 2006, Mitani Corporation, Tokyo, Japan).

PCNA
PCNA (Dako, Carpinteria, CA, USA) immunohistochemistry was performed to determine cell proliferation. The sections were deparaffinized in xylene and rehydrated in ethanol. To enhance immunostaining, the sections were placed in citrate buffer (pH 6.0) and heated in a microwave oven for thirty minutes (low power) for antigen unmasking and then allowed to cool at room temperature for twenty minutes. Endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide in PBS for twenty minutes. After gradual quenching at room temperature, nonspecific binding sites were blocked with blocking solution A (Histofine, Nichirei, Tokyo, Japan), and the specimens were incubated overnight with the primary antibody at a dilution of 1:300 in 0.5% casein-PBS at 4°C. Each section was then incubated with dextran polymers conjugated with the secondary antibodies and peroxidase molecules (Envision, Nichirei, Tokyo, Japan). Subsequently, DAB was applied as a chromogen. The sections were then counterstained with hematoxylin. For quantification of cell proliferation, sections from five mice in each group were analyzed. Multiple different parts of the liver were examined. The number of cells that reacted positively with PCNA antibodies was determined in a blinded fashion by counting at least 3000 hepatocytes in each liver section, and the PCNA-positive index was calculated as the percentage of positive cells.

Real-Time RT-PCR Analysis
For real-time RT-PCR, total RNA was isolated from five animals of each group using Trizol (Invitrogen Corp., Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. cDNA was synthesized from 2 µg total RNA by using SuperScript III reverse transcriptase and a random primer (Invitrogen Corp., Carlsbad, CA, USA) in 20 µL reaction mixture. Quantitative real-time RT-PCR with SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The oligonucleotide primers for PCR were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). The amount of target genes normalized to an endogenous reference (β-actin) and relative to a control was obtained using the 2Ct method (Livak and Schmittgen 2001). The sequences of PCR primers for mouse ATF3 were as follows: forward, 5'-CAGCATTTGATATACATGCTCAACCT-3' and reverse 5'-TCCGGTGTCCGTCCATTCT-3'. The other primers (human Ha-ras, mouse Ha-ras, c-fos, raf, c-myc, and cyclinD1) were described in our previous study (Okamura et al. 2006; Okamura et al. 2007).

Statistical Analysis
Statistical analyses were performed using a statistical software package (StatLight version 2.0, 2003, Yukms Co. Ltd., Tokyo, Japan), and all results are presented as mean ± SD. Multigroups were used to test the homogeneity of variance between the groups using Bartlett’s test. Next, Dunnett’s multiple comparison test in the same package was applied. When the data were homogeneous, Dunnett’s test was used, and when heterogeneous, Dunnett’s rank sum test was used. A p value of less than .05 was considered statistically significant.

	Results

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Body and Liver Weights and Macroscopic Examinations
The final body weights and absolute and relative liver weights are shown in Table 1. During the experimental period, neither death nor clinical symptoms relating to the treatment with FF were observed in any of the groups, but a significant decrease in body weight gain was found in the FF-treated groups (data not shown). Macroscopically, all rasH2 mice in the FF-treated groups had liver enlargement. Measurement of organ weights revealed that the absolute and relative liver weights of the FF-treated groups were significantly increased, as compared to the DEN-alone group.

View larger version (131K): [in this window] [in a new window]   Figure 1 Microscopic findings of HE stained-section and cytokeratin 8/18 immunohistochemistry. HE staining of the liver sections from rasH2 mice given 0 ppm (a) or 2400 ppm FF (b). Hypertrophy and vacuolar degeneration of centrilobular hepatocytes are evident in the 2400 ppm FF group. Bar = 200 µm. Immunohistochemical staining of cytokeratin 8/18 antibody in the liver from rasH2 mice given 0 ppm (PH control: c and DEN alone: d), 1200 ppm (e), or 2400 ppm FF (f). Altered foci are strongly stained with cytokeratin 8/18 in the 1200 and 2400 ppm FF groups (e and f), as compared with the PH-control (c) and DEN-alone (d) groups. Bar = 100 µm.

	Discussion

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FF is one of the representative peroxisome proliferators (PPs) in rodents. It has been reported that PPs such as clofibrate, one of the fibrate drugs, and Wy-14,643, a widely used PP-representative compound, increase the number of peroxisomes, up-regulate peroxisomal beta-oxidation, and cause hepatocellular hypertrophy and hyperplasia when administered to rats and mice (Takashima et al. 2008). In addition, Takashima et al. (2008) added that PPAR-dependent alterations in cell cycle regulatory proteins are likely to contribute to the hepatocarcinogenicity of PPs. In the present study, all of the FF-treated mice exhibited centrilobular hypertrophy and vacuolar degeneration in hepatocytes, and some animals of this group had altered foci. The centrilobular hypertrophy of hepatocytes observed has been recognized to be attributable to the increases in the number of peroxisomes (Pruimboom-Brees et al. 2005). In our previous study, FF treatment for thirteen weeks induced DNA damage owing to oxidative stresses resulting from reactive oxygen species (ROS) generation, cell proliferation, apoptosis suppression, and increased number of altered foci in a DEN-initiated hepatocarcinogenesis model in partially hepatectomized rats (Nishimura et al. 2008). Oxidative stress has been recognized as an important factor causing lipid peroxidation (Wiseman and Halliwell 1996) and affecting degenerative and inflammatory diseases, aging, and cancer (Trush et al. 1991).

Therefore, the vacuolar degeneration in hepatocytes observed in FF-treated mice is considered to be owing to lipid peroxidation resulting from ROS production, although we did not measure the generation of ROS in our study. In addition, the number of PCNA-positive hepatocytes slightly increased in mice of the DEN + FF group. Such a cell proliferation is likely a result of the mitogenic effect of this drug and/or owing to increased cell turnover from vacuolar degeneration resulting in cell death. ROS is also believed to play a pivotal role in the etiology of liver cancer, and ROS overproduction and subsequent oxidative DNA damage have been implicated to enhance the development of hepatocellular carcinomas that are caused by carcinogenic agents including FF (Dewa et al. 2008; Nishimura et al. 2008). Marsman et al. (1988) demonstrated that the hepatocarcinogenic potential of the PPAR agonists di (2-ethylhexyl) phthalate and Wy-14643 correlated with the ability to cause sustained cell proliferation. Moreover, with regard to the tumor-promoting mechanism of PPAR agonists, it has been reported that increased replicative DNA synthesis, cell proliferation, and apoptosis suppression were involved in the development of hepatocellular tumors (Klaunig et al. 2003). Based on the above references and the results of our study, it can be considered that vacuolar degeneration of hepatocytes observed in the liver causes cell injury owing to oxidative stresses generated, and such increased cell turnover from cell injury and/or sustained generation of ROS probably results in the formation of altered foci.

It is generally recognized that point mutations and overexpression of the transgene are the most probable mechanisms of enhanced hepatocarcinogenesis in rasH2 mice, based on the molecular analyses of several tumors induced by genotoxic carcinogens (Maruyama et al. 2001; Tamaoki 2001). Okamura et al. (2004) reported that activation of the ras/MAPK cascade following both overexpression of the transgene and up-regulation of endogenous mouse ras genes appears to be involved in the enhanced tumorigenesis of N-ethyl-N-nitrosourea (ENU)-induced forestomach squamous cell carcinomas in rasH2 mice. In addition, they reported that overexpression of the transgene plays an important role in carcinogenesis in rasH2 mice, and the genes that show a similar expression pattern in both ENU-and urethane-induced tumors are probably the candidate genes responsible for the enhanced carcinogenesis in these mice (Okamura et al. 2006). Therefore we analyzed mRNA expression of the transgene and some molecules involved in the ras pathway in the liver of rasH2 mice given FF.

In the present study, we confirmed the overexpression of the transgene and certain downstream molecules of the ras pathway, such as c-myc, c-jun, and cyclin D1, in the liver of FF-treated rasH2 mice. Since overexpression of transgenes and molecules of the ras pathway is common in tumors of rasH2 mice induced by various carcinogens, it is suggested that the overexpression of these genes plays an important role in the increased preneoplastic foci in rasH2 mice given FF. Expression levels of c-fos (one of the downstream molecules of the ras pathway) varied among the samples, but the reason why only the c-fos gene showed such a discrepancy in its expression level is unknown. ATF3 is a member of the ATF/CREB family of transcription factors, and it is induced by stimuli such as carbon tetrachloride, ischemia/reper-fusion, radiation, and PPAR activators (Nawa et al. 2000). ATF3 is rapidly induced in the regenerating liver (Hsu et al. 1991; Mohn et al. 1991), in which the c-myc transcript is also induced (Makino et al. 1984). Thus, c-myc and ATF3 together may regulate liver regeneration. Moreover, ATF3 is overexpressed in murine melanoma cells with high metastatic potential (Ishiguro et al. 1996), and its gene is amplified in esophageal cancer cells (Prmkhaokham et al. 2000). Therefore, it may be possible that ATF3 is also involved in the FF-induced hepatocarcinogenesis in rasH2 mice. The other genes, including c-myc, c-jun, and cyclin D1, are thought to play a role in the process of tumorigenesis and cellular transformation with activation of the ras/MAPK cascade (Cook et al. 1999; Yu et al. 2005). Consequently, activation of these genes, together with overexpression of the transgene and mouse endogenous ras genes in rasH2 mice, may contribute to the formation of preneoplastic foci. Further studies are now in progress to examine the roles of ATF3 and other factors in hepatocellular tumors that will be induced by FF treatment for twenty-six weeks after DEN initiation.

Cytokeratin 8/18 is one of the intermediate filaments of the epithelium (Gonsebatt et al. 2007). It has been reported that cytokeratin 8/18 expression is maintained in hepatocellular carcinomas (Athanassiadou et al. 2007), and cytokeratin 8/18 were diffusely positive in 70% of hepatocellular carcinomas of human liver tumors removed surgically (Stroescu et al. 2006). In mice, the overexpression of cytokeratin 18 was elevated in hepatocellular carcinomas that were developed in mice exposed transplacentally to arsenic during gestation (Liu et al. 2004). Moreover, cytokeratin 18 synthesis in liver cells is tightly correlated with the differentiation program and with several cellular processes such as apoptosis and cell proliferation (Gonsebatt et al. 2007). Gonsebatt et al. (2007) reported that the altered cytokeratin 18 expression could modify the differentiation pattern in the liver during chronic inorganic arsenic exposure, and expression of cytokeratin 18 was modulated by the oxidative stress in hepatocytes of mice. As described above, FF had the potential to generate oxidative stress, and its effect was involved in the development of hepatocarcinogenesis in rats (Nishimura et al. 2007). Based on these reasons, it is likely that cytokeratin is associated with increases in preneoplastic foci that may be enhanced by the generation of oxidative stress. In the present study, the number and areas of cytokeratin 8/18 positive foci/cells were significantly increased in the FF-treated groups. Therefore, the findings obtained in our study and from the published literature suggest that cytokeratin 8/18 may become a specific marker of altered foci of hepatocytes of mice induced by FF, although further investigation is necessary.

In conclusion, our data suggest that the hepatocarcinogenic activity of FF in rasH2 mice can be detected in the eight-week, two-stage hepatocarcinogenesis model. The overexpression of the transgene and several downstream molecules—such as c-myc, c-jun, ATF3, and cyclin D1, which are categorized as genes related to the ras/MAPK pathway and cell cycle—plays an important role in the enhanced hepatocarcinogenesis induced by FF in rasH2 mice.

	Acknowledgments

 
This research was supported in part by grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan.

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This version was published on December 1, 2008

Toxicologic Pathology, Vol. 36, No. 7, 950-957 (2008)
DOI: 10.1177/0192623308327118


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