The Journal of Toxicological Sciences (J. Toxicol. Sci.) 613 Vol.36, No.5, 613-623, 2011

Original Article Altered expression of during the development of chemical-mediated hypertrophy and liver tumor promotion in rats

Shogo Ozawa1,*, Toshie Gamou1,*, Wataru Habano1, Kaoru Inoue2, Midori Yoshida2, Akiyoshi Nishikawa2, Kiyomitsu Nemoto3 and Masakuni Degawa3

1Department of Pharmacodynamics and Molecular Genetics, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan 2Division of Pathology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan 3Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka 422-8526, Japan

(Received June 30, 2011; Accepted August 5, 2011)

ABSTRACT — The purpose of our study was to examine the altered expression associated with nongenotoxic chemical-mediated liver hypertrophy and successive liver tumor promotion. Five-week-old male rats were fed a basal diet or a diet containing phenobarbital (PB) or clofibrate (CF) for 3 days, 4 weeks, and 13 weeks. Hepatic expression profiling of cell growth- and stress-related genes, as well as those involved in xenobiotic metabolism, was performed by DNA microarray and/or real time quantita- tive reverse transcription-polymerase chain reaction. The induction of liver hypertrophy and hepatic cyto- chrome P450 (CYP) isoforms (CYP2B1/2B2 for PB and CYP4A1 for CF) by PB and CF were clearly observed at all the treatment periods examined. Genes encoding DNA damage-inducible 45 (GADD45) family , in particular GADD45g (GADD45 gamma) were down-regulated by treatment with either PB or CF for 4 and 13 weeks. The chemical-mediated development of liver hypertrophy, induction of hepatic CYPs, and suppression of hepatic GADD45g gene at week 13 disappeared at 4 weeks following cessation of the chemical treatment. Additionally, DNA microarray data indicated that -relat- ed genes such as cyclins CCNB1 and CCNA2 and cyclin-dependent inhibitor CDKN3 were also down-regulated by treatment with either PB or CF at 13 weeks. Since GADD45 functions as a chemical and radiation stress sensor by interacting with cyclins and cyclin-dependent kinase inhibitors, the decrease in the of GADD45g mRNA observed in this study, may be associated with nongenotoxic chemical-induced tumor promotion of hepatocarcinogenesis rather than liver hypertrophy.

Key words: Liver tumor promotion, Liver hypertrophy, Growth arrest and DNA damage-inducible gene 45, Constitutive androstane receptor, Phenobarbital, Clofibrate

INTRODUCTION nogen, which, through its agonistic effect on the consti- tutive androstane receptor (CAR), causes liver hypertrophy A number of xenobiotics that cause hypertrophy and and successive promotion of liver tumors (Feldman et al., altered foci in the liver are often reported to induce cyto- 1981; Yamamoto et al., 2004). Clofibrate (CF), a typical chrome P450s (CYPs) in rodents (Chen and Eaton, 1993; peroxisome proliferator-activated receptor α (PPARα) Huang et al., 2005; Deguchi et al., 2009). Phenobarbi- agonist, is also a nongenotoxic hepatocarcinogen and tal (PB), an inducer of the CYP2B subfamily enzymes, shows an ability to induce hypertrophy, hyperplasia, has been characterized as a typical nongenotoxic carci- and tumor formation in the rodent liver (Holden and Correspondence: Shogo Ozawa (E-mail: [email protected]) *These authors equally contributed to this work.

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Tugwood, 1999; Reddy et al., 1979). Peroxisome pro- tion-polymerase chain reaction (qRT-PCR) analysis indi- liferator-mediated activation of PPARα results in induc- cated that liver hypertrophy and induction of CYP(s) tion of various enzymes, including CYP4A1, fatty acid (CYP2B1/2B2 for PB and CYP4A1 for CF) resulted β-oxidation enzyme, acyl-CoA oxidase, apolipoproteins, from treatment with either PB or CF for all periods exam- fatty acid transporters, lipoprotein lipase, and thioesterases ined and, notably, suppression of the GADD45g gene was (Peters et al., 2005). observed from 4 to 13 weeks after dietary treatment with Toxicogenomic studies conducted to identify molec- either chemical. ular markers of hepatotoxicity and hepatocarcinogenici- ty in rodents reveal that hepatotoxicants may be classified MATERIALS AND METHODS into 3 types of chemicals: CYP inducers, hepatocellular necrosis inducers, and hepatocellular cholestasis induc- Chemicals ers (Hamadeh et al., 2002a, 2002b; de Longueville et al., PB and CF were purchased from Wako Pure Chemicals 2003; Jessen et al., 2003; Kramer et al., 2004; Elrick et Co. Ltd. (Tokyo, Japan). All other chemicals used were of al., 2005; Stierum et al., 2005). PB and CF are classi- the highest grade available. fied as CYP inducer-type hepatotoxicants (de Longueville et al., 2003); however, little is known regarding the key Animals and treatments molecular events involved in the process of chemical- Sixty male F344 rats (4-week-old), weighing 80 ± 5 g, mediated development of liver hypertrophy and hepato- were purchased from Charles River Co. Ltd. (Tokyo, carcinogenesis. Japan) and were housed 5 rats per cage with access to tap Elrick et al. (2005) reported that PB activates sever- water and a commercial basal diet (MF; Oriental Yeast al stress response genes, including growth arrest and Co. Ltd., Tokyo, Japan) during the acclimatization peri- DNA damage-inducible 45β gene (GADD45b) in rats. od (one week). During the 1-week acclimatization peri- GADD45 was originally identified to be associated with od, daily clinical signs and body weights were checked. p53 in a radiation-induced DNA damage response (Zhan At 5 weeks of age, healthy animals were randomly et al., 1993); however, it has since been confirmed that assigned into 3 groups (20 rats per group) to avoid sig- GADD45 family members GADD45α, GADD45β, and nificant differences of group mean body weights among GADD45γ interact with cyclins and cyclin-dependent control (untreated), PB-treated, and CF-treated groups at kinase inhibitors, and are implicated in cell cycle arrest as the beginning of experiment. The rats were fed a basal a cellular stress response. Therefore, GADD45α, β, and diet (CRF-1; Oriental Yeast Co. Ltd.) or CRF-1 containing γ play an important role in the stress signaling pathways 500 ppm of PB or 2,500 ppm of CF for up to 13 weeks, controlling cell cycle arrest, DNA repair, cell survival, as illustrated in Figure 1. Both doses are reported to and/or (Liebermann and Hoffman, 2008). cause liver neoplasms after long-term treatment (Feldman GADD45α is involved in the DNA repair and surviv- et al., 1981; Reddy et al., 1979), and therefore were al of damaged cells (Hollander et al., 2001; Gupta et al., selected as liver hypertrophy and tumor-inducible doses 2006). GADD45β is an antiapoptotic factor, inhibiting for this experiment. After 3 days, 4 weeks, and 13 weeks c-Jun N-terminal kinase (JNK) signaling in cooperation of administration of PB or CF, the of 5 rats from with NF-κB (Papa et al., 2004, 2007), and GADD45γ each treatment group were excised. At 13 weeks, 5 rats inhibits the cell cycle progression from S to G1 phase from each treatment group entered a 4-week recovery through interactions with proliferating cell nuclear anti- period, during which they were fed a basal diet (CRF-1), gen and (Zhao et al., 2000; Azam et al., 2001). There- and subsequently, at 17 weeks after the start of the treat- fore, the next logical step is to examine the expression of ment, the livers of the 5 rats were excised. After sacrifice, GADD45 family members during the process of chemi- absolute and relative weights of the liver were measured; cal-mediated development of liver hypertrophy and hepa- the livers were cut into small pieces and stored at -80°C tocarcinogenesis. until use. All experiments were performed in accordance In the present study, we administered the CYP inducer- with the guidelines for animal experiments set forth by type liver hypertrophic chemicals PB and CF to Fischer the National Institute of Health Sciences. 344 (F344) rats for 3 days, 4 weeks, and 13 weeks. Sub- sequent hepatic gene expression profiling of rats treat- Histopathology and morphology ed with PB or CF for 13 weeks revealed down-regula- The rat livers from the control and chemical-treated tion of some cyclins, cyclin-dependent kinase inhibitors, groups were examined for histopathology at day 3, and and GADD45g. Real-time quantitative reverse transcrip- weeks 4, 13, and 17. After fixation in 10% neutral buff-

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Fig. 1. Experimental study design. Schedules for the administration of phenobarbital and clofibrate are illustrated. A total of 20 ani- mals were used for each chemical, 5 animals were used for each treatment group and period, and were sacrificed at 3 days, 4 weeks, 13 weeks, and 17 weeks (13 weeks plus an additional 4-week chemical-free period).

ered formalin, the livers were embedded in paraffin wax, Biosystems) following the manufacturer’s protocol sup- cut into 3 μm thick sections, routinely processed, and plied with the SYBR Green PCR Master Mix. stained with hematoxylin and eosin (H & E). The mRNA levels of GADD45a, GADD45b, GADD45g, CYP2B1, CYP2B2, and CYP4A1 were determined, DNA microarray and RT-PCR analyses using GAPDH as an internal control. PCR primers were Hepatic gene expression was analyzed by DNA micro- designed using the Primer3Plus software and the prim- array (Agilent Technologies) and qRT-PCR. Total RNA er sequences are listed in Table 1; the PCR efficiency for was extracted from a frozen rat liver (approximately all target genes was almost equal. Target mRNA levels

100 mg), using TriPure reagent (Roche) according to the were determined according to the 2ЧЧCt method (Livak manufacturer’s instructions; total concentration and quali- and Schmittgen, 2001). Ct values for the specific target ty of the purified RNA were measured by spectrophotom- gene (Ct target) and GAPDH (Ct GAPDH) were obtained for the etry. RNA quality was evaluated using an Agilent 2100 chemical-treated and control (chemical-untreated) rats.

Bioanalyzer (Agilent Technologies, Tokyo, Japan) as well Assuming these values as Ct GAPDH, as ratio between Absorbance unit at 260 nm and that at Using A for chemical-treated group = (Ct target г Ct GAPDH) treatment.

280 nm, which should be higher than 1.9. Using B for control group = (Ct target г Ct GAPDH) control.

For DNA microarray analysis, total RNA (500 ng), We took the average (Bmean) of the control group (n = 5) which comprised total hepatic RNA (100 ng per rat) for each target gene and mRNA levels of the target gene from 5 individual rats in each experimental group, was relative to the control group were calculated as 2(AгBmean). labeled with cyanine-3 using a Quick-Amp Labeling Kit The mRNA levels of each chemical-treated group (n = 5) (Agilent Technologies). The labeled targets were hybrid- were expressed as mean (S.D.). ized to Whole Rat Genome 4x44K oligo DNA microarrays In the present qRT-PCR analysis, we examined differ- (Agilent Technologies) and hybridized microarrays were ences in Ct values for GAPDH among the untreated, PB- scanned using an Agilent Microarray Scanner (Agilent and CF-administered study groups. As a result, we did not Technologies). Image analysis and raw array data observe any significant difference in Ct values for GAP- generation were processed using Feature Extraction soft- DH. Therefore, GAPDH was considered as an appropri- ware (Agilent Technologies). Normalization of the raw ate internal control for the present 2ЧЧCt analysis to eval- array data was performed using Genespring software uate effects of PB and CF on various gene expressions. Ver. 11. For qRT-PCR analysis, cDNA was prepared from 2 μg Statistical analysis of total RNA by reverse transcription using an RT-PCR The data on the body weights, organ weights, and kit (Invitrogen) according to the manufacturer’s instruc- hepatic mRNA levels were expressed as mean (S.D.) tions. (n = 5). Statistically significant difference between the qRT-PCR with SYBR Green PCR Master Mix (Applied control and chemical-treated groups was evaluated by Biosystems, Foster City, CA, USA) was performed using one-way ANOVA with a post hoc test (Dunnett’s test). the ABI PRISM 7500 Real-time PCR System (Applied

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RESULTS Treatment with PB for 4 weeks resulted in development of centrilobular hypertrophy of hepatocytes (Fig. 3B), Increases in the absolute and relative liver which occurred in a time-dependent manner from day 3 weights up to week 13. The hepatocytes displayed a ground-glass The chemical treatments did not result in a remarkable appearance in their cytoplasm; these histological chang- decrease in the body weights of rats using the experimen- es in the liver resulting from PB treatment for 13 weeks tal conditions illustrated in Fig. 1. Changes in the abso- disappeared 4 weeks after the cessation of the chemi- lute and relative liver weights (g liver weight/100 g body cal treatment. The CF-dependent hepatocellular hyper- weight) are illustrated in Figs. 2A and B for each treat- trophy characterized by eosinophilic cytoplasm was also ment period. The absolute liver weights of rats fed 500 observed at week 4 (Fig. 3C) and week 13, and the hepa- ppm PB and 2,500 ppm CF signifi cantly increased at day tocellular hypertrophy displayed a diffuse distribution 3, week 4, and week 13 as compared with controls. Per- throughout the lobules of the liver. cent increases in PB-fed rats were 25%, 24%, and 35% at day 3, week 4, and week 13, respectively; those in CF- Effects of PB and CF on hepatic gene expression fed rats were 24%, 36%, and 31%, respectively. Statisti- Altered gene expression in the liver of the rats treated cally signifi cant increases in relative liver weights were with PB or CF for 13 weeks were initially examined by observed for up to 13 weeks after the PB and CF treat- DNA microarray analyses and revealed changes in gene ments. Percent increases at day 3, week 4, and week 13 expression levels, including the GADD45 family genes were 24%, 24%, and 33% in PB-treated rats, and 26%, and the CYP2B/4A subfamily genes, which were further 49%, and 35% in CF-treated rats, respectively. After the examined by qRT-PCR. 4-week recovery period, the absolute and relative liver weights had returned to those of the control rats. Altered expression of genes related to cell cycle arrest and DNA damage Histopathology Changes in the hepatic gene expression profile of Representative H & E-stained liver sections of the genes related to cell cycle arrest and DNA damage were control and chemical-treated rats are shown in Fig. 3. first examined in rats treated with PB and CF for 13

Fig. 2. Liver weights of rats administered with phenobarbital (PB) or clofi brate (CF). Absolute liver weights (A) and relative liver weights (g/100 g body weight, B) are shown as a mean (S.D.) for 5 rats from each experimental group. The difference in the values from those of the corresponding controls was evaluated by Dunnett’s test. *, **Statistically signifi cant differences from the corresponding controls: *P < 0.05, **P < 0.01.

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Fig. 3. Representative liver hypertrophy observed microscopically in control rats and rats treated with phenobarbital (PB) and clofi brate (CF) for 4 weeks. Representative photographs (H & E staining) of livers of control rats (A) and those administered with phenobarbital (B) or clofi brate (C) for 4 weeks are shown. Doses of chemicals are 500 ppm for phenobarbital and 2,500 ppm for clofi brate. PB induced marked hepatocellular hypertrophy with ground-glass appearance in centrilobular area (B). Diffuse hepatocellular hypertrophy with eosinophilic cytoplasm was a feature of CF treatment(C). *, Central vein.

Table 1. Nucleotide sequence of oligonucleotide primers used for determining mRNA levels in quantitative real-time PCR Accession number Gene symbol Forward primer (5′ to 3′) Reverse primer (3′ to 5′) NM_024127 GADD45a TCTGTTGCGAGAACGACATC TGTGATGAATGTGGGTTCGT NM_001008321 GADD45b GCTGGCCATAGACGAAGAAG TGACAGTTCGTGACCAGGAG AB020978 GADD45g TCGCACAATGACTCTGGAAG AGGGTCCACATTCAGGACT NM_001134844 CYP2B1 CTTGTCCCTATTGGAGTACC AGACAAATGCGCTTTCCTGTG XM_341808 CYP2B2 CATCCCTTGATGATCGTACC CAGACACCTTCAATCCTGAG NM_175837 CYP4A1 TTGCTCTTGCTGAATGGACA CCAGCTGTTCCCATTTGTCT NM_017008 GAPDH AGACAGCCGCATCTTCTTGT CTTGCCGTGGGTAGAGTCAT

weeks. The representative results (change in > 2.0-fold or treatments (data not shown). < 0.5-fold by the chemical treatments) are summarized in Since cyclins and cyclin-dependent kinase are known Table 2. The genes of cyclins (CCNB1andCCNA2), cyc- to function as chemical- and radiation-induced stress sen- lin-dependent kinase inhibitor CDKN3, and GADD45g sors by interacting with GADD45 proteins, the altered were down-regulated by both PB and CF treatments. As gene expression of hepatic GADD45 family members, previously reported (Motojima et al., 1998; Tamura et al., GADD45α, GADD45β, and GADD45γ, by PB and CF 2006), the genes LPL, CD36, and LEPR, associated with treatments were further examined by qRT-PCR. fatty acid metabolism and energy generation (Holden and Tugwood, 1999), were up-regulated by both chemical

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Altered gene expression of GADD45α (2.1-fold, P < 0.05). Dietary treatment with PB for 4 and 13 weeks result- ed in decreases in levels of hepatic GADD45a mRNA Altered gene expression of GADD45γ by 43% and 26%, respectively (Fig. 4). Treatment with Statistically significant and persistent down-regula- CF for 4 and 13 weeks led to signifi cant decreases in the tion of GADD45g gene expression by PB and CF was GADD45a mRNA level (by 81% and 59%, respectively). observed at weeks 4 and 13 (Fig. 6). Treatment with PB The CF-dependent decrease in GADD45a mRNA level for 4 and 13 weeks significantly decreased the level of was restored after the 4-week recovery (basal diet) treat- GADD45g mRNA by 82% and 64%, respectively. Sim- ment. ilarly, CF treatment for 4 and 13 weeks decreased the mRNA level by 80% and 85%, respectively. The CF- Altered gene expression of GADD45β dependent decreases in the level of GADD45g mRNA Expression levels of GADD45b, a CAR-regulated gene were restored after the 4-week recovery (basal diet) peri- (Yamamoto and Negishi, 2008), at 3 days and 4 weeks od. In the case of PB, signifi cant, but rather deviated, ele- after PB treatment were 2.8-fold (P < 0.01) and 4.1-fold vation in GADD45g mRNA level was detected after the (P < 0.001) higher than corresponding controls, respec- 4-week recovery (basal diet) period. tively (Fig. 5). In contrast, in CF-treated rats, no such sig- nificant increases at day 3 and week 4 were observed, Altered gene expression of CYP2B1 and CYP2B2 while signifi cant down-regulation of GADD45b occurred Signifi cant increases in the level of hepatic CYP2B1 at week 13. By DNA microarray analysis, GADD45b was mRNA were observed in the rats treated with PB by down-regulated at week 13 (0.46-fold of control). After 82-fold at day 3, 65-fold at week 4, and 26-fold at week the 4-week recovery (basal diet) period, slight, but signif- 13 (Fig. 7). The increased level at week 13 returned to icant elevation of GADD45b mRNA level was observed the level of the corresponding controls after the 4-week

Fig. 4. GADD45a mRNA expression in livers of rats admin- Fig. 5. GADD45b mRNA expression in livers of rats admin- istered with phenobarbital (PB) or clofibrate (CF). istered with phenobarbital (PB) or clofibrate (CF). Hepatic GADD45a mRNA levels were measured in Hepatic GADD45b mRNA levels were measured in rats dosed with 500 ppm phenobarbital or 2,500 ppm rats dosed with 500 ppm phenobarbital or 2,500 ppm clofibrate. GADD45a mRNA levels are shown as a clofibrate. GADD45b mRNA levels are shown as a mean (S.D.) for 5 rats from each experimental group. mean (S.D.) for 5 rats from each experimental group. The differences in the GADD45a mRNA levels were The differences in the GADD45b mRNA levels were compared between the control and the chemical-ad- compared between the control and the chemical-ad- ministered groups for each time point. **, ***Statis- ministered groups for each time point. *, **, ***Statis- tically signifi cant differences from the corresponding tically signifi cant differences from the corresponding controls: ** P < 0.01, *** P < 0.001. controls: * P < 0.05, ** P < 0.01, *** P < 0.001.

Vol. 36 No. 5 Table 2. Expression changes of cyclins and related genes (> 2.0- or < 0.5-fold) in livers of rats after administration of PB or CF for 13 weeks together with known up-regulation of metabolism genes Fold Gene name Accession number change Description (biological process) (Probe ID) Systematic name

PB CF GADD45g down-regulationbynongenotoxiclivercarcinogens

GO:0000074 (regulation of progression through CCNA2 (A_44_P527480) NM_053702 Cyclin A2 (Ccna2), mRNA 0.36 0.26 cell cycle)

GO:0000086 (G2/M transition of mitotic cell CCNB1 (A_44_P534089) NM_171991 Cyclin B1 (Ccnb1), mRNA 0.29 0.26 cycle)

BC167026 GO:0000079 (regulation of cyclin-dependent CDKN3 (A_44_P513360) Cyclin-dependent kinase inhibitor 3, mRNA 0.25 0.13 NM_001106028 kinase activity) GO:0000074 (regulation of progression through Growth arrest and DNA- damage- inducible GADD45a (A_44_P216395) NM_024127 cell cycle) ;GO:0000079 (regulation of cyclin- 0.95 0.64 45 alpha (Gadd45a), mRNA dependent protein kinase activity) GO:0000074 (regulation of progression Growth arrest and DNA- damage- inducible GADD45b (A_43_P16529) NM_001008321 through cell cycle) ; GO:0006915 (apoptosis) 1.67 0.46 45 beta (Gadd45b), mRNA ;GO:0006950 (response to stress) Growth arrest and DNA- damage- inducible GO:0006469 (negative regulation of protein GADD45g (A_44_P365379) NM_001077640 0.49 0.27 45 gamma (Gadd45g), mRNA kinase activity)

CYP2B1 (A_44_P316194) ENSRNOT00000047540 Cytochrome P450 2B1 GO:0006118 (electron transport) 20 1.1

CYP4A1 (A_44_P1059556) NM_175837 Cytochrome P450 4A1, mRNA GO:0006118 (electron transport) 1.0 3.1

Fold change represents ratio of hepatic mRNA levels of chemical treated rats to those of control diet-fed rats. Vol. 36No.5 619 620

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Fig. 6. GADD45g mRNA expression in livers of rats admin- Fig. 7. CYP2B1 mRNA expression in livers of rats admin- istered with phenobarbital (PB) or clofibrate (CF). istered with phenobarbital (PB) or clofibrate (CF). Hepatic GADD45g mRNA levels were measured in Hepatic CYP2B1 mRNA levels were measured in rats dosed with 500 ppm phenobarbital or 2,500 ppm rats dosed with 500 ppm phenobarbital or 2,500 ppm clofi brate. GADD45g mRNA levels are shown as mean clofi brate. CYP2B1 mRNA levels are shown as mean (S.D.) for 5 rats from each experimental group. The (S.D.) for 5 rats from each experimental group. differences in the GADD45g mRNA levels were com- CYP2B1 mRNA levels were compared between the pared between the control and chemical-administered control and the chemical-administered groups for each groups for each time point. *, **, ***Statistically sig- time point. ***Statistically signifi cant differences from nifi cant differences from the corresponding controls: the corresponding controls: *** P < 0.001. * P < 0.05, ** P < 0.01, *** P < 0.001.

recovery (basal diet) treatment. In addition, treatment cals. Liver hypertrophy developed in rats treated with with PB resulted in an increase in the level of CYP2B2 either PB or CF for 3 days, 4 weeks, and 13 weeks. Sim- mRNA: 10.3-fold at day 3, 6.1-fold at week 4, and 8.3- ilarly, the induction of hepatic CYP2B1/2B2 and CYP4A1 fold at week 13 (data not shown). No signifi cant increases was observed in PB- and CF-treated rats, respective- were observed with CF treatment. ly, for all the treatment periods examined. These chem- ical-induced increases in liver weight and liver hyper- Altered gene expression of CYP4A1 trophy and levels of hepatic CYPs (CYP2B1/2B2 or Up-regulation of the CYP4A1 gene is reported in CYP4A1) were returned to those of the control rats at male rat livers following exposure to CF (Baker et al., 4 weeks after the cessation of the chemical treatment. 2004). Therefore, we examined the effect of dietary CF Thus, the nongenotoxic chemicals displayed reversi- on expression of CYP4A1, a PPAR-α target gene, and ble effects on induction of liver hypertrophy and hepatic confirmed a significant increase in the level of hepatic CYPs, which is consistent with a known good correlation CYP4A1 mRNA by CF (Fig. 8). The CYP4A1 mRNA lev- between the development of liver hypertrophy and the els at day 3, week 4, and week 13 were 2.1-, 2.5-, and induction of hepatic CYPs, such as PB (CAR activation)- 2.8-fold higher than the corresponding controls, respec- dependent CYP2B induction and CF (PPARα activation)- tively. No signifi cant increase in the CYP4A1 mRNA lev- dependent CYP4A1 induction (Chen and Eaton, 1993; el was observed in any PB-treated rats. Honkakoski et al., 1998a, 1998b; Wei et al., 2000; Sueyoshi and Negishi, 2001; Yoshinari et al., 2001; DISCUSSION Yamamoto et al., 2004; Huang et al., 2005). Gene expression profi ling in the liver of the rats treated The ultimate goal of our study was to determine signa- with either PB or CF for 13 weeks revealed the down-reg- ture gene expression for liver hypertrophy and successive ulation of genes associated with cell cycle regulation and liver tumor promotion induced by nongenotoxic chemi- DNA damage repair: cyclins CCNB1and CCNA2, cyc-

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treatment, the elevation of hepatic GADD45g mRNA level was detected in the rats of PB-administered group. Those rats would probably be destined to liver malig- nancy after longer-term exposure. We currently speculate that repair process from any possible PB-caused toxicity might have started in those rats during the recovery treat- ment to result in the elevation of GADD45g mRNA level. The elevation varied depending on stochastic PB-damage repair process occurred in the individual animals. Further studies are needed to give an answer to this speculation. Furthermore, in our preliminary experiments, admin- istration of the nongenotoxic hepatocarcinogen piperonyl butoxide (PBO) to rats resulted in a marked decrease in gene expression of GADD45g together with an increase in CYP2B1 expression (Gamou et al., unpublished). Stud- ies have shown that the hepatocarcinogenicity of PB, Fig. 8. CYP4A1 mRNA expression in livers of rats admin- CF, and PBO may involve oxidative stress (Elrick et istered with phenobarbital (PB) or clofibrate (CF). al., 2005; Muguruma et al., 2007; de Longueville et al., Hepatic CYP4A1 mRNA levels were measured in 2003). This reported oxidative stress by PB, CF and PBO, rats dosed with 500 ppm phenobarbital or 2,500 ppm and our present results on the sustained down-regulation clofi brate. CYP4A1 mRNA levels are shown as mean (S.D.) for 5 rats from each experimental group. of GADD45g may constitute a common cellular stress CYP4A1 mRNA levels were compared between the response pathway. We further consider that the down- control and the chemical-administered groups for each regulation of Gadd45γ may be associated with the process time point. ***Statistically signifi cant differences from involved in triggering liver tumor-associated cell growth. the corresponding controls: *** P < 0.001. Further investigation is needed to prove these hypotheses by investigating influence of oxidative stress and GADD45g expression on liver tumor promotion. Thus we raise hypothesis that cell growth proliferation lin-dependent kinase inhibitorCDKN3, and GADD45g. toward malignancy might be triggered by or associated In particular, signifi cant decreases in the expression lev- with the down-regulation of Gadd45γ. Besides Gadd45γ, els of GADD45g were persistently observed by treatment down-regulation of Gadd45α and Gadd45β should also with either PB or CF for 4 and 13 weeks. It is notewor- be considered with respect to tumor promotion. Down- thy that these chemicals showed different characteristics regulation of Gadd45α, which is involved in cell surviv- in terms of the activation of CYP genes (CYP2B1/2B2 al in response to DNA damage (Hollander et al., 2001; and CYP4A1) and their transcription factors (CAR and Liebermann and Hoffman, 2008), was observed in rats PPARα). GADD45g has an inhibitory effect on cell cycle treated with CF for 4 weeks and 13 weeks. GADD45a progression to adapt to cellular stress (Zhao et al., 2000; and GADD45b have also been shown to interact with Azam et al., 2001; Vairapandi et al., 2002; Togo et al., cdc2-cyclin B1 complex to exert an inhibitory effect on 2004), and also functions as a tumor suppressor (Ying et cell cycle progression to adapt cellular stress (Vairapandi al., 2005). Genotoxic or cytokine-causing stress was load- et al., 2002). These findings also raise possibility that ed in many studies as variety of “cellular stress” where down-regulation of Gadd45α has some role in direct- GADD45g was involved. In contrast, PB and CF are non- ing hepatocytes to malignancy during the liver tumor genotoxic carcinogens. Thus PB- and CF-mediated sup- promotion process in CF-treated rats. Up-regulation of pression of GADD45g gene may be key events that occur Gadd45β, which is activated along with CYP2B subfami- during the promotion of hepatocarcinogenesis. No induc- ly genes through activation of CAR by PB (Yamamoto tion of liver cell growth-associated marker (i.e., Ki-67) and Negishi, 2008), occurred in rats treated with was observed in rats treated with either chemical for up to PB for 3 days and 4 weeks, but not for 13 weeks. 13 weeks (Inoue, Yoshida, Nishikawa, unpublished), sug- Signifi cant increases in the expression levels of hepatic gesting that the liver tumor-associated cell proliferation CYP2B1/2B2 were observed in the rats treated with PB may occur after longer-term treatment with nongenotoxic for 13 weeks; however, no significant up-regulation of carcinogens. After the 4-week recovery (normal diet) GADD45b was observed, indicating that up-regulation of

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GADD45b might not occur solely through activation of gene expression changes in rat liver: A cross-laboratory analy- CAR. GADD45b might have been down-regulated through sis using membrane cDNA arrays. Environ. Health Persp., 112, 428-438. currently unknown mechanisms other than CAR around Chen, Z.Y. and Eaton, D.L. (1993): Association between respon- the period of 13 week-administration of PB to return siveness to phenobarbital induction of CYP2B1/2 and 3A1 in its expressed level to that of untreated rats. We hereby rat hepatic hyperplastic nodules and their zonal origin. Environ. point out a possible role of the Gadd45β down-regulation Health Persp., 101, 185-190. in liver tumor promotion in rats treated either with PB Deguchi, Y., Yamada, T., Hirose, Y., Nagahori, H., Kushida, M., Sumida, K., Sukata, T., Tomigahara, Y., Nishioka, K., Uwagawa, (cancellation of the observed up-regulation at 4week) S., Kawamura, S. and Okuno, Y. (2009): Mode of action anal- or CF (down-regulation) for 13 week, as Vairapandi et ysis for the synthetic pyrethroid metofluthrin-induced rat liver al. showed interaction of Gadd45β with cdc2-cyclin B1 tumors: evidence for hepatic CYP2B induction and hepatocyte complex to exert an inhibitory effect on cell cycle proliferation. Toxicol. Sci., 108, 69-80. progression (Vairapandi et al., 2002). Accordingly, the de Longueville, F., Atienzar, F.A., Marcq, L., Dufranc, S., Evrard, S., Wouters, L., Leroux, F., Bertholet, V., Gerin, B., Whomsley, altered gene expression of GADD45a and GADD45b R., Arnould, T., Remacle, J. and Canning, M. (2003): Use of a by PB and CF might not necessarily be related to the low-density microarray for studying gene expression patterns development of liver hypertrophy. Nor that of GADD45g induced by hepatotoxicants on primary cultures of rat hepato- might be, as GADD45g down-regulation did not occur by cytes. Toxicol. Sci., 75, 378-392. treatment with either PB or CF for 3 days. Elrick, M.M., Kramer, J.A., Alden, C.L., Blomme, E.A.G., Bunch, R.T., Cabonce, M.A., Curtiss, S.W., Kier, L.D., Kolaja, K.L., In conclusion, the present study showed changes in the Rodi, C.P. and Morris, D.L. (2005): Differential display in rat mRNA levels of the GADD45 gene family members in livers treated for 13 weeks with phenobarbital implicates a role the livers of rats treated with nongenotoxic chemicals, PB for metabolic and oxidative stress in nongenotoxic carcinogenic- and CF. Decreases in the expression of GADD45g were ity. Toxicol. Pathol., 33, 118-126. Feldman, D., Swarm, R.L. and Becker, J. (1981): Ultrastructural commonly observed by the treatment with PB and CF for study of rat liver and liver neoplasms after long-term treatment 4 and 13 weeks, and GADD45a and GADD45b were also with phenobarbital. Res., 41, 2151-2162 down-regulated especially after the treatment with CF for Gupta, M., Gupta, S.K., Hoffman, B. and Liebermann, D.A. (2006): 13 weeks, despite that PB and CF show different charac- Gadd45a and Gadd45b protect hematopoietic cells from UV- teristics in terms of induction of hepatic CYP isoforms. induced apoptosis via distinct signaling pathways, including p38 activation and JNK inhibition. J. Biol. Chem., 281, 17552- We herein propose that down-regulation of GADD45g 17558. and, possibly, GADD45a and GADD45b might be one Hamadeh, H.K., Bushel, P.R., Jayadev, S., DiSorbo, O., Bennett, of the causes of development of liver tumor promotion L., Li, L., Tennant, R., Stoll, R., Barrett, J.C., Paules, R.S., mediated by some kind of nongenotoxic chemicals Blanchard, K. and Afshari, C.A. (2002a): Prediction of com- rather than liver hypertrophy, which should be elucidated pound signature using high density gene expression profiling. Toxicol. Sci., 67, 232-240. through further study. It is very important to determine Hamadeh, H.K., Bushel, P.R., Jayadev, S., Martin, K., DiSorbo, what kind of nongenotoxic chemical carcinogens O., Sieber, S., Bennett, L., Tennant, R., Stoll, R., Barrett, J.C., show similar changes in expression of genes including Blanchard, K., Paules, R.S. and Afshari, C.A. (2002b): Gene GADD45a, GADD45b, and GADD45g. expression analysis reveals chemical-specific profiles. Toxicol. Sci., 67, 219-231. Holden, P.R. and Tugwood, J.D. (1999): Peroxisome proliferator- ACKNOWLEDGMENTS activated receptor alpha: role in rodent and species differences. J. Mol. Endocrinol., 22, 1-8. 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