ToxSci Advance Access originally published online on January 29, 2007
Toxicological Sciences 2007 97(1):44-54; doi:10.1093/toxsci/kfm011
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Effect of Fenofibrate on Oxidative DNA Damage and on Gene Expression Related to Cell Proliferation and Apoptosis in Rats
,1,
* Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan
Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan
Division of Pathology, National Institute of Health Sciences, 18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
Biochemistry and Biotechnology, United Graduate School of Agricultural Sciences, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan
1 To whom correspondence should be addressed. Fax: +81 42 367 5771. E-mail: j_nisimr{at}cc.tuat.ac.jp.
Received October 11, 2006; accepted January 18, 2007
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ABSTRACT |
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To investigate the relationship between fenofibrate (FF) and oxidative stress, enzymatic, histopathological, and molecular biological analyses were performed in the liver of male F344 rats fed 2 doses of FF (Experiment 1; 0 and 6000 ppm) for 3 weeks and 3 doses (Experiment 2; 0, 3000, and 6000 ppm) for 9 weeks. FF treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl–CoA oxidizing system, and catalase in the liver. However, it decreased those of superoxide dismutase in the liver in both experiments. Increased 8-hydroxy-2'-deoxyguanosine levels in liver DNA and lipofuscin accumulation were observed in the treated rats of Experiment 2. In vitro measurement of reactive oxygen species (ROS) in rat liver microsomes revealed a dose-dependent increase due to FF treatment. Microarray (only Experiment 1) or real-time reverse transcription–polymerase chain reaction analyses revealed that the expression levels of metabolism and DNA repair–related genes such as Aco, Cyp4a1, Cat, Yc2, Gpx2, Apex1, Xrcc5, Mgmt, Mlh1, Gadd45a, and Nbn were increased in FF-treated rats. These results provide evidence of a direct or indirect relationship between oxidative stress and FF treatment. In addition, increases in the expression levels of cell cycle–related genes such as Chek1, Cdc25a, and Ccdn1; increases in the expression levels of cell proliferation–related genes such as Hdgfrp3 and Vegfb; and fluctuations in the expression levels of apoptosis-related genes such as Casp11 and Trp53inp1 were observed in these rats. This suggests that cell proliferation induction, apoptosis suppression, and DNA damage due to oxidative stresses are probably involved in the mechanism of hepatocarcinogenesis due to FF in rats.
Key Words: peroxisome proliferators-activated receptor alpha agonist; fenofibrate; rat; liver; oxidative stress; DNA damage; hepatocarcinogenesis; ROS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Peroxisome proliferators are a structurally diverse group of pharmaceutical and industrial chemicals that include hypolipidemic drugs, herbicides, plasticizers, steroids, and solvents. They activate specific transcription factors belonging to the nuclear hormone receptor superfamily, that is, the peroxisome proliferator-activated receptors (PPARs; Schoonjans et al., 1996
). Thus far, three distinct PPARs—
, 
/ß, and 
—have been identified, and activation of the PPAR isoform leads to persistent peroxisome proliferation in hepatocytes and the induction of hepatocellular tumors in rodents (Klaunig et al., 2003). Aside from being potent hepatotoxicants, PPAR agonisits and their metabolites are neither direct genotoxic substances (Ashby et al., 1994; Butterworth et al., 1984) nor tumor initiators (Ward et al., 1986), although they possess tumor-promoting activity. It has been reported that oxidative stress due to excessive hydrogen peroxide (H2O2) generation due to the administration of PPAR agonists leads to lipid peroxidation and oxidative DNA damage (Seo et al., 2004); hence, oxidative stress is involved in the mechanism of PPAR agonist–mediated carcinogenesis in rodents (O'Brien et al., 2005). However, DNA damage or lipid peroxidation by other PPAR agonist remains to be identified (Elliott and Elcombe, 1987). The evidence linking PPAR agonist to secondary oxidative DNA damage is conflicting, and therefore, the mechanisms underlying the initiation of DNA damage in response to this class of chemicals remain to be elucidated.
Fenofibrate (FF), a member of the fibrate class of hypolipidemic drugs, has been extensively used in many countries to treat hypertriglyceridemia and mixed hyperlipidemia for a long time (Staels et al., 1998). FF has also been identified as a PPAR agonist and liver tumor promoter in rodents; however, there are no detailed reports of its hepatocarcinogenicity. In addition, despite numerous reports on PPAR agonist, the molecular mechanism of hepatocarcinogenesis by PPAR agonist is not completely understood. In particular, molecular investigations on the involvement of oxidative stress in PP-induced liver tumors remain to be performed.
In the present study, in order to investigate the relationship between FF and oxidative stress and to clarify the hypothesis that oxidative stress is involved in FF-induced hepatocarcinogenesis, we performed 3- and 9-week repeated dose toxicity studies of FF in rats as a preliminary study to clarify the mode of action of its early stage of the hepatocarcinogenesis. The liver of FF-treated rats was subjected to histopathological examinations, enzyme activity measurement, gene expression profile analyses using a large-scale oligonucleotide microarray, mRNA expression analyses of the metabolism, DNA repair–, cell proliferation–, cell cycle–, and apoptosis-related genes using real-time reverse transcription–polymerase chain reaction (RT-PCR), and measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the liver, a marker of oxidative stress. In addition, microsomal reactive oxygen species (ROS) products were measured in vitro to measure ROS production during FF metabolism.
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MATERIALS AND METHODS |
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Chemicals.
The test compound FF (CAS no. 49562-28-9, purity > 99%) was purchased from Sigma-Aldrich Chemical Co. (St Louis, MO). All other chemicals were of analytical grade and obtained commercially.
Animals.
Male F344/N Slc rats aged 5 weeks were purchased from Japan SLC Inc. (Shizuoka, Japan). The rats were housed in stainless steel cages with three or four animals per cage and allowed ad libitum access to tap water and a commercial powdered basal diet (MF, Oriental Yeast Industries Co., Ltd., Tokyo, Japan). All the animals were handled under standard conditions (room temperature, 23 ± 3°C; relative humidity, 55 ± 15%; 12-h light and dark cycle). The rats were acclimatized for 1 week before dosing and randomly allocated to two test groups (five or six rats per group) for a 3-week study (Experiment 1; Exp. 1) using stratification methods based on their body weight just before dosing. Animal care and experiments were carried out in accordance with the Guide for Animal Experimentation of Tokyo University of Agriculture and Technology. Based on Exp. 1, a 9-week study (Experiment 2; Exp. 2) to investigate the effect of further long-term administration of FF was performed. New rats were obtained and acclimatized by the same methods and randomly divided into three groups (six or seven rats per group).
Experimental design (in vivo)
In Exp. 1, rats were fed the powdered basal diet containing FF at 0 and 6000 ppm for 3 weeks. This dosage was selected based on the information on carcinogenicity studies of FF announced by the U.S. FDA, where liver carcinomas were induced by the oral administration of 200 mg/kg/day of FF in both sexes of rats (http://www.fda.gov/medwatch/; search word is Triglide tablets). In Exp. 2, a dose of 3000 ppm was administered in addition to that in Exp. 1 because a significant inhibition in body weight gain was observed in the 6000-ppm group in Exp. 1, as described later.
Both body weight and food intake were measured once a week. Necropsy was performed under anesthesia with ether at the end of the experiment after starvation for 16 h. Blood samples were collected from the abdominal aorta, and the liver of each rat was removed and weighed. The liver samples were sectioned, and one section was used for histopathological examinations, while the other sections were frozen in liquid nitrogen and stored at –80°C for future analyses.
Biochemical examination.
In Exps. 1 and 2, the blood samples collected at necropsy were centrifuged and sera obtained. Biological examinations were performed in five animals of each group at SRL Co., Ltd. (Tokyo, Japan), and the following parameters were measured: aspirate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP).
Histopathology.
In Exps. 1 and 2, one section comprising the left lateral and quadrate lobes of the liver obtained from each liver sample was fixed using 10% natural-buffered formalin and embedded in paraffin. Paraffin sections were prepared from these samples, stained with hematoxylin and eosin (H&E), and examined morphologically. Serial sections of the H&E preparations were used for other stainings by the Schmorl reaction, immunohistochemistry for Ki-67, and terminal deoxynucleotidyl–mediated nick-end labeling (TUNEL) method.
Schmorl reaction.
In Exps. 1 and 2, lipofuscin was stained by the Schmorl reaction. Tissue sections were deparaffinized in xylene, rehydrated through a graded series of ethanol solutions, rinsed in distilled water, and incubated for 5 min at room temperature in a solution containing 0.75% ferric chloride and 0.25% potassium ferricyanide.
Immunohistochemistry for Ki-67.
In Exp. 2, the proliferative activities of hepatocytes were measured by immunohistochemical staining using an anti-Ki-67 mouse monoclonal antibodies (DAKO Japan, Kyoto, Japan) and Histofine SAB-PO kit (Nichirei, Tokyo, Japan). Tissue sections were first immersed in 0.3% H2O2 in methanol for 20 min to block endogenous peroxidase and heated by autoclave in citrate buffer for antigen retrieval and then incubated at 4°C overnight with primary antibody at dilution of 1:30, followed by incubation with biotinylated secondary antibody for 10 min and the avidin-biotin-peroxidase complexes for 10 min. Then, 3,3-diaminobenzidine was applied as a chromogen. The sections were finally counterstained with hematoxylin. The labeling index of Ki-67 was expressed as the percentage of positive cells after counting 
1000 hepatocytes in five to six fields (x200) selected randomly in each specimen.
TUNEL methods.
In Exps. 1 and 2, apoptosis was detected by TUNEL assay using an ApopTag Kit (CHEMICON International, Inc., CA) according to the manufacturer's instructions. The labeling index of TUNEL was expressed as the percentage of positive cells after counting 3000 hepatocytes in 15 fields (x200) selected randomly in each specimen.
Determination of enzyme activities.
In Exps. 1 and 2, the rat liver tissue used for the enzyme assay was homogenized in ice-cold 10mM Tris-HCl (pH 7.4) containing 0.25M sucrose and 1mM ethylene diamine tetraacetic acid (EDTA) using an all-glass Potter Elvehjem homogenizer. Each homogenate was centrifuged for 20 min at 800 x g. The resulting supernatant fraction was used to determine enzyme activities. Protein concentrations (mg/ml) were determined using the BCA Protein Assay Kit (Pierce Biotechnology, IL).
Carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (CPT) that are involved in mitochondrial ß-oxidation were measured spectrophotometrically by the methods of Markwell et al. (1973) following the release of a CoA-SH from acetyl-CoA and palmitoyl-CoA each, using the general thiol reagent 5,5'-dithio-bis-(2-nitrobenzoic acid). The fatty acyl–CoA oxidizing system (FAOS) involved in peroxisomal ß-oxidation was measured spectrophotometrically by the methods of Markwell et al. (1973). Activity was defined as micromole per minute per milligram of protein. Superoxide dismutase (SOD) and catalase levels were determined using the SOD assay kit-WST (Dojindo Molecular Technologies, Inc., Gaithersburg, MD) and Amplex Red catalase assay kit (Molecular Probes, Inc., Eugene, OR), respectively, according to the manufacturer's protocol. Activity was defined as units per milligram of protein.
RNA isolation and oligonucleotide microarray analysis.
In Exp. 1, total RNA was isolated from three animals of each group using the TRIzol reagent (Invitrogen, Corp., CA) according to the manufacturer's protocol. Analysis of gene expression using CodeLink Bioarray Rat Whole Genome (GE Healthcare Bio-Sciences, NJ) was performed to identify 35,129 genes in one animal of each group. The production of a labeled oligonucleotide probe, hybridization, and imaging data analysis for the comparison between individuals of the 0- and 6000-ppm groups were performed by KURABO Industries Ltd. by using the CodeLink Expression Bioarray System. To minimize the effects of the measurement variation introduced by artificial sources in analyzed data of the microarrays in Exp. 1, genes that were up- or downregulated by at least more than twofold or less than 0.5-fold, respectively, were included. Genes were grouped into functional classes using BioCompass (NEC Engineering, Ltd., Tokyo, Japan) and selected using DAVID Bioinformatic Resources (available on the World Wide Web at http://niaid.abcc.ncifcrf.gov/) and Gene Ontology (available on the World Wide Web at http://www.geneontology.org/).
Real-time RT-PCR.
First-strand cDNA was synthesized from 2 µg total RNA with random primers and ThermoScript reverse transcriptase (Invitrogen, Corp.). In Exp. 1, cDNA synthesis was performed using the RNA samples obtained from three animals of each group including the animals subjected to the microarray in 3-week study. Quantitative real-time RT-PCR with SYBR Green was performed using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Ltd., CA) to validate the microarray results. PCR was performed according to the SYBR Green PCR master mix protocol. To obtain the relative quantitative values for gene expression, ß-actin was used as an endogenous control, and its expression levels were calculated according to the 2–
Ct method.
The PCR primers listed in Table 1 were designed by using the Primer Express software (Applied Biosystems, Ltd.). Besides, although microarray analysis revealed that the expression levels of tumor protein p53 (Tp53), 8-oxoguanine-DNA-glycosylase (Ogg1), growth arrest and DNA damage–inducible 45 alpha (Gadd45a), proliferating cell nuclear antigen (Pcna), and tumor necrosis factor-alpha (Tnfa) were not changed, real-time RT-PCR analysis was performed. In Exp. 2, six animals of each group were tested in the same manner as Exp. 1 to assess the effects of the administration period and dose response.
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Determination of 8-OHdG in liver DNA.
In Exp. 2, the measurement of 8-OHdG levels in liver DNA was performed according to the method of Nakae et al. (1995)
. Nuclear DNA was extracted using a DNA Extractor WB kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). During the extraction, an iron chelator was used to prevent DNA oxidation. The DNA was digested to deoxynucleotides using nuclease P1 and ALP, and the level of 8-OHdG (8-OHdG/105 deoxyguanosine) was assessed by high-performance liquid chromatography (HPLC) using an electrochemical detection system (Coulochem II; ESA Biosciences, Inc., MA).
Determination of microsomal ROS production of the liver (in vitro)
The nonfluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate, (DCFH-DA; Molecular Probes, Inc.) has been used as a sensitive intracellular probe for detecting ROS formation during cellular metabolism, and in the presence of ROS, it is oxidized to highly fluorescent DCF (Serron et al., 2000). In this study, assays were performed using this probe. The liver tissue from 10-week-old rats fed basal diet was homogenized in ice-cold 1.15% KCl buffer (pH 7.4) containing 0.2mM EDTA, 0.1mM dithiothreitol, 0.1mM phenylmethylsulfonyl fluoride, and 20% glycerine. Hepatic microsomes obtained from the homogenates were routinely purified by differential centrifugation (Sequeira et al., 1992), suspended in the abovementioned buffer, and stored at –80°C until evaluation. ROS was measured by partially modifying the method of Serron et al. (2000). Microsomes (final concentration 0.5 mg/ml) were incubated in the dark at 37°C in 40mM Tris buffer (pH 7.4) and DCFH-DA (5µM). At the end of the incubation period, FF (0.001–1mM), H2O2 (1mM; positive control) or the vehicle (methanol), and 0.6mM NADPH were added, and the mixture was incubated at 37°C for 30 min under dark. The rate at which ROS formed the fluorescent product was measured using a microplate reader (excitation 485 nm; emission 528 nm). The data were normalized to control values, and the control was expressed as a value of 100%.
Statistical evaluation.
Statistical analyses were performed using statistical software (StatLight; Yukms Co., Ltd., Japan), and all results are presented as mean ± SD. The two corresponding groups were compared by analyzing the data using the F-test for homogeneity of variance between the control and FF-treated groups. If the variance was homogeneous, the Student's t-test was applied for comparisons, and if it was heterogeneous, Aspin-Welch t-test was used. Multigroups were compared by using Dunnett's test to isolate the groups that were significantly different from the control group. A p value of less than 0.05 was considered statistically significant.
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RESULTS |
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Biochemistry and Body and Liver Weights
During the experimental period, neither death nor FF-related clinical symptoms were observed in any of the groups (data not shown). However, a significant decrease in body weight gain was found in the FF groups in Exps. 1 and 2 (Table 2). Macroscopically, all the rats of these treated groups sacrificed at weeks 3 and 9 showed liver enlargement and discoloration (data not shown).
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With regard to biochemistry, rats administered 6000 ppm FF in Exp. 1 did not show changes in any parameters compared to the control value. On the other hand, rats administered 3000 and 6000 ppm FF in Exp. 2 showed significant increases in the serum activities of AST (128–153%), ALT (164–170%), and ALP (233–288%) as compared to the corresponding control values (Table 2).
Measurement of organ weights revealed that the absolute and relative liver weights of all the FF-treated groups in Exps. 1 and 2 had increased by 300–330% (Table 2).
Histopathological Examinations and Schmorl Reaction
Histopathological examinations revealed that in both experiments, all the FF-treated rats exhibited severe hypertrophy and eosinophilic cytoplasmic changes in hepatocytes (Figs. 1A-2, A-4, and A-5). Brown pigments were also observed in the 3000-ppm group or more in Exp. 2. These pigments exhibited a positive Schmorl reaction and were believed to be lipofuscin (Figs. 1B-4 and B-5).
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Immunohistochemistry for Ki-67
The Ki-67–positive indices in the liver of rats administered 3000 and 6000 ppm FF in Exp. 2 showed a significant increase as compared to the control group (Fig. 2A).
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TUNEL Method
The TUNEL-positive indices in the liver of rats administered 6000 ppm FF in Exp. 1 and 3000 ppm FF in Exp. 2 were not significantly different from those of the corresponding controls; however, that of rats administered 6000 ppm FF in Exp. 2 showed a significant decrease (Fig. 2B).
Enzyme Activity Measurements
In the 6000-ppm group in Exp. 1, the activities of CAT (37.8-fold), CPT (15.4-fold), and FAOS (27.2-fold) were markedly higher, while that of catalase was slightly higher (1.7-fold) and that of SOD was slightly lower (0.75-fold) compared to the corresponding values of the control group. In Exp. 2, the fluctuations in the enzymatic activities observed in the FF-treated groups were identical to those observed in the FF-treated group in Exp. 1. However, the degree of enzymatic activities in Exp. 2 was higher: 97.8-/83.0-fold increase in CAT, 24.9-/28.2-fold in CPT, 78.6-/79.9-fold in FAOS, and 2.4-/2.8-fold in catalase in the 3000-/6000-ppm group, respectively (Table 3). The SOD activity in the treated groups in Exp. 2 was almost equal to that in Exp. 1.
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Analysis of FF-Induced Gene Expression by Microarray and Real-Time RT-PCR
In the microarray analysis on the liver of rats administered FF in Exp. 1, a total of 1637 genes were either up- or downregulated. Of these, 1166 genes were upregulated by twofold or more than that of the control group, and the expression of genes related to the metabolism, transport, stress response, apoptosis, regulation of cell proliferation, regulation of cell cycle, regulation of cell growth, cell growth and/or maintenance, and regulation of transcription-related genes fluctuated predominantly (supplemental data). In this study, to clarify the relationship between FF and oxidative stress, metabolism-related genes involved in oxidative stress and DNA repair–related genes, some apoptosis-related genes, and cell cycle– and cell proliferation–related genes were investigated by real-time RT-PCR using the primer sets summarized in Table 1. Acyl-Coenzyme A oxidase 1 (Aco) and cytochrome P450, 4A1 (Cyp4a1), genes were investigated as positive PPAR
agonists. Although some genes such as Ogg1, Gadd45a, Tp53, Pcna, and Tnfa showed no significant fluctuations in expression levels in the microarray analysis, real-time RT-PCR analyses were performed for these genes. Three animals of each group in Exp. 1 and six animals of each group in Exp. 2 were examined. These results are summarized in Table 4.
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The gene expression levels by real-time RT-PCR were approximately similar to those in the microarray, except for some genes such as superoxide dismutase 2, mitochondrial (Sod2), DNA polymerase delta (Pold1), proliferating cell nuclear antigen (Pcna), and tumor necrosis factor-alpha (Tnfa). In Exps. 1 and 2, compared to the control group, significant upregulation of metabolism and DNA repair–related genes such as catalase (Cat), glutathione peroxidase 2 (Gpx2), glutathione S-transferase (GST), Yc2 (Yc2), 1,25-dihydroxyvitamin D-3 (Txnip), apurinic/apyrimidinic endonuclease 1 (Apex1), nibrin (Nbn), and X-ray repair complementing defective repair in Chinese hamster cells 5 (Xrcc5) was predominantly observed in all FF-treated groups. Upregulation of cell cycle–, apoptosis-, and cell proliferation–related genes such as hepatoma-derived growth factor–related protein 3 (Hdgfrp3), cell division cycle 25A (Cdc25a), vascular endothelial growth factor B mRNA (Vegfb), and checkpoint kinase 1 homolog (Chek1) was also observed in all FF-treated groups compared to the control group. In addition, significant increases in the expression levels of apoptosis-related genes such as tumor protein p53 (Tp53), transformation-related protein 53 inducible nuclear protein 1 (Trp53inp1), caspase11 (Casp11), and BTB (POZ) domain containing 14B (Btbd 14b, other name: Nac-1)were observed in the 6000-ppm group of Exp. 1. However, except for the change in the expression level of Trp53inp1, prolonging the administration period in Exp. 2 caused statistically significant increases in the expression levels of the abovementioned genes of the 3000-ppm group, but no alteration in the 6000-ppm group. The expression level of Trp53inp1 showed a significant decrease as compared with the control group. In addition, significant increases in O-6-methylguanine-DNA methyltransferase (Mgmt), mutL homolog 1 (Mlh1), and cyclin D1 (Ccnd1) were observed in the 6000-ppm group of Exp. 2, and a significant increase in (Gadd45a) was observed in the 3000-ppm group of Exp. 2. There were no significant changes in the expression levels of Ogg1, Pcna, and Tnfa in both experiments compared with the corresponding controls.
Formation of 8-OHdG in Liver DNA
HPLC analysis revealed that the level of 8-OHdG in the 6000-ppm group in Exp. 2 was significantly higher than that of the control group, and the degree of change was approximately three times that of the control group. The levels of 8-OHdG in the control and 6000-ppm groups in Exp. 2 were 0.21 ± 0.03 and 0.59 ± 0.09 (unit: 8-OHdG/105 deoxyguanosine), respectively.
Formation of Microsomal ROS in the Liver
As shown in Figure 3, in the presence of 1mM H2O2 (positive control), significant increases were observed, and the percentage of increase (120%) was calculated. In the case of FF administration, a concentration-dependent increase in ROS production was observed; a significant increase being evident when the concentration of FF was 0.1mM or more.
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DISCUSSION |
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In the present enzyme assay, there were significant increases in the activities of CAT, CPT, and FAOS, which are distributed in peroxisomes and/or mitochondria and are associated with fatty acid ß-oxidation, a slight increase in antioxidant enzymatic activity of catalase, and a slight decrease in SOD activity in the FF-treated groups of Exps. 1 and 2. It has been reported that treating rodents with PPAR
agonists causes significant activation of genes encoding several enzymes involved in peroxisomal ß-oxidation, including fatty acyl–Coenzyme A oxidase and the microsomal cytochrome p450 (CYP) 4A isoform, which produces ROS comprising mainly H2O2 as a by-product (Yeldandi et al., 2000). In contrast, it has been reported that induction of the H2O2-degrading enzyme catalase increased slightly (approximately twofold of the control value) in a study in which male rats were fed a basal diet containing nafenopin (0.03 mmol/kg basal diet) for 24 weeks (Furukawa et al., 1985). Additionally, in our study, a significant production of ROS in microsomes was observed in an in vitro study when FF was present at a concentration of 0.1mM or more. These data support the findings obtained in the study by Jiao and Zhao (2002) in which FF at a concentration of 0.15mM or more caused an elevation in ROS in the human hepatoma cell line HepG2. However, there were differences in test systems and the concentration of FF in which ROS were produced between our studies and that of Jiao and Zhao (2002). Our data indicate that various cell organelles in hepatocytes of FF-treated rats generate excess ROS that leads to a shift in the balance toward oxidative stress. In addition, gene expression analyses revealed significant increases in the expression levels of oxidative stress–related genes such as Txnip, which binds to and inhibits thioredoxin (Junn et al., 2000), Gpx2, which is a potent detoxifier of ROS (Chu et al., 2004), Cat, and Yc2, which has a high conjugating activity of GST toward AFB1-8,9-epoxide (Zangar et al., 1992) in the FF-treated groups of Exps. 1 and 2. On the other hand, although Anderson et al. (2004) reported that there is little overlap between genes regulated by Wy-14,643 and PPAR and Nrf2-regulating genes, we obtained upregulation of some Nrf2-regulating genes, Cat, Gpx2, and Gst-Yc subunits (Cho et al., 2002). As a future study, we need to examine whether the treatment of FF activates the transactivational response of Nrf2 in rats.
ROS induces oxidative damage to various cell constituents such as DNA, proteins, and lipids through oxidative stress. In particular, oxidative DNA damage causes mutations and abnormal gene expressions that are involved in carcinogenesis. It has been reported that 8-OHdG, produced by the oxidation of deoxyguanosine, is believed to be the most sensitive marker of oxidative DNA adducts, and an elevation in 8-OHdG levels is believed to play an important role in chemically induced carcinogenesis (Kinoshita et al., 2002). In the present study, the HPLC analysis showed a significant elevation in 8-OHdG levels in the liver DNA of the 6000-ppm group compared to the control group in Exp. 2. With regard to 8-OHdG, there have been some reports that its DNA adducts are repaired by OGG1 (Dybdahl et al., 2003). Rusyn et al. (2004) reported that the WY-14,643 treatment resulted in the increased expression of some base excision repair (BER) genes (Ogg1; uracil-DNA glycosylase; N-methylpurine-DNA glycosylase; thymine-DNA glycosylase; Apex1, polymerase, ß; flap structure–specific endonuclease 1; Pcna; ligase I, DNA, ATP dependent) in the liver of mice. Thus, we anticipated an elevation of the expression of BER genes, including Ogg1. However, except for Apex1, which is induced by oxidative stress (Grosch et al., 1998), no change in the expression levels of these genes was observed in the FF-treated groups of either experiment. This contradicted a previous report (Rusyn et al., 2004). As the other DNA repair genes which were not observed in a previous report (Rusyn et al., 2004), upregulations of Xrcc5 which is related to DNA double-strand break repairs and genetic stability (Thacker and Zdzienicka, 2004), Nbn which is a member of MRN complex (Mre11/Rad50/Nbn) and has a well-documented DNA repair and S-phase checkpoint function in both yeast and mammalian cells (D'Amours and Jackson, 2002), Mgmt which removes alkyl groups from DNA and has no role in removal oxidative DNA damage (Rafferty et al., 1996), and Mlh1 which is related to the DNA mismatch repair (Hegan et al., 2006), and Gadd45a which is involved in the maintenance of genomic stability and positive regulation of apoptosis (Smith et al., 2000) were observed in FF-treated rats. Our data may suggest that FF induces more severe oxidative stress than WY-14,643 (Rusyn et al., 2004) or FF causes no DNA damage other than oxidative stress. With regard to DNA damage induced by the treatments of PPAR agonists, there have been contradictory observations. However, judging from our data in this study, it is difficult to deny possible involvements of DNA oxidative stress due to ROS by the treatments of FF in rats. On the other hand, since it is unclear whether the gene mutations and modulations are caused by DNA damages originating from the FF-induced oxidative stress, further studies to clarify these points are necessary.
Increase in lipid peroxidation is one of the damages due to oxidative stress. Further, lipofuscin is known as an age-associated pigment that is regarded as cellular debris derived from lipid peroxides formed by oxidative stress (Tsuchida et al., 1987) and has been reported to be increased markedly by the treatment of PPAR agonists in the livers of rats (Yeldandi et al., 2000). In our study, abundant lipofuscin accumulation was observed in the hepatocytic cytoplasm of the treated groups in Exp. 2 but rarely in the 6000-ppm group of Exp. 1. These results may suggest that treatment with FF induces oxidative stress, but it takes longer to be recognized as cellular debris derived from lipid peroxides.
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). Along with these findings, an increase in liver weight and the upregulation of cell cycle–related genes such as Ccnd1, Cdc25a, and Chek1 and cell proliferation–related genes such as Hdgfrp3 and Vegfb was observed in the FF-treated groups in both experiments of our study. It has been reported that genotoxic stress activates the cell cycle checkpoints (Hartwell and Weinert, 1989) leading to diverse cellular responses such as cell cycle arrest, DNA repair, and cell death. Chek1 is a serine/threonine kinase that plays the role of a DNA damage–induced checkpoint regulator by enforcing cell cycle arrest (Zhang et al., 2006). It is also responsible for the instability of Cdc25a during an unperturbed cell cycle. In addition, Ccdn1 is known as an oncogene and a key regulator of cell cycle progression; translocation or amplification of the gene and its subsequent overexpression have been described in various human cancers (Hunter and Pines, 1991). Hdgfrp3 is one of the hepatoma-derived growth factor 1–related protein families, and the expression of mRNA is prominent in the nervous system (Abouzied et al., 2004). Vegfb is a mitogen for the endothelial cells and its mRNA or protein is observed in the cytoplasm of the tumor cells (Salven et al., 1998). The facts that Ki-67 index is significantly increased after the treatment of FF will support that the induction of cell proliferation was expressed. TUNEL methods in our study showed a suppression of apoptosis in the 6000-ppm group of Exp. 2. On the other hand, the gene expression analyses in apoptosis-related genes showed different fluctuations between Exp. 1 and Exp. 2; significant increases in Tp53, Trp53inp1, Casp11, and Nac-1 mRNA observed in the 6000-ppm group of Exp. 1 disappeared with time, while these genes except for Trp53inp1 conversely increased with time in the 3000-ppm group of Exp. 2. Casp11 plays an important regulatory role as activation of caspase1 which is related to cytokine maturation and caspase3 which is related with apoptosis (Kang et al., 2000), and Trp53inp1 is a Tp53-inducible gene that regulates p53-dependent apoptosis (Okamura et al., 2001). In addition, it has been reported that the overexpression of Nac-1 was followed by downregulation of the antiapoptotic proteins such as Bcl-2 and Bcl-2-x1 and upregulation of proapoptotic proteins such as Bax and p53 in PC-12 cells (Korutla et al., 2003). Youssef et al. (2003) reported that PPAR agonists alter age dependently the balance between pro- and antiapoptotic genes in the livers. Our data support the findings obtained in the study of Youssef et al. (2003) and suggest that the suppression of apoptosis in the 6000-ppm group is probably involved in the liver tumor promotion effect of FF, although there are slight differences in the fluctuated genes. On the other hand, Klaunig et al. (2003) suggest that the suppressions of apoptosis by PPAR agonists are likely to return to the background except for Wy-14,643 when once a steady state of the liver enlargement is reached. As a future study, we need to clarify whether the suppression of apoptosis is involved in the hepatocarcinogenesis of FF in rats.
In conclusion, our data from the present study suggest that FF has the potential to generate ROS and induces oxidative stress including the formation of 8-OHdG and fluctuations of DNA repair–related genes. These findings may have a great contribution to the clarification of mechanism of the PPAR agonist–induced hepatocarcinogenesis in rodents. In addition, the results of our study suggest that cell proliferation induction and apoptosis suppression, which were observed in the early stage of repeated dose toxicity of FF, is probably involved in the hepatocarcinogenesis mechanism of FF in rats. Further investigations are now in progress to clarify whether oxidative stress is involved in the development of hepatocellular preneoplastic foci induced by FF in a two-stage hepatocarcinogenesis rat model.
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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