Sex and Circadian Modulatory Effects on Rat Liver As Assessed by Transcriptome Analyses
The Journal of Toxicological Sciences (J. Toxicol. Sci.) 9 Vol.36, No.1, 9-22, 2011
Original Article Sex and circadian modulatory effects on rat liver as assessed by transcriptome analyses
Jun Hirao1, Masatoshi Nishimura2, Shingo Arakawa1, Noriyo Niino1, Kazuhiko Mori1, Tadashi Furukawa1, Atsushi Sanbuissho1, Sunao Manabe3, Masugi Nishihara4 and Yuji Mori5
1Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 717 Horikoshi, Fukuroi, Shizuoka 437-0065, Japan 2Exploratory Research Laboratories I, Daiichi Sankyo Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan 3Global Project Management Department, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan 4Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 5Laboratory of Veterinary Ethology, Animal Resource Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
(Received September 15, 2010; Accepted October 15, 2010)
ABSTRACT — The present study was designed to fully uncover sex and circadian modulatory effects on rat liver. Hepatic transcriptome analyses were performed at 4 hr intervals of a day-night cycle using \RXQJDGXOWPDOHDQGIHPDOHUDWV6H[XDOO\GLPRUSKLFJHQHVZKLFKZHUHLGHQWL¿HGE\DFURVVVH[FRP- parison of time series data, included representative sex-predominant genes such as male- or female-pre- dominant cytochrome P450 subfamilies (Cyp2c11, Cyp2c12, Cyp2c13, and Cyp3a2), sulfotransferases, DQGJOXWDWKLRQH6WUDQVIHUDVH
Key words: Sex, Circadian, Transcriptome, Liver, Rats
INTRODUCTION toxicity. Factors that affect the liver metabolism include genetic polymorphisms, age, biological sex, dietary fac- The liver is the major organ of drug metabolism, which tors, endogenous hormonal factors, pregnancy, dis- can alter the balance between therapeutic response and eased states, epigenetic factors, and environmental fac- Correspondence: Jun Hirao (E-mail: [email protected])
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J. Hirao et al. tors (Claudel et al., 2007; Kaiser, 2005; Waxman and ulate circadian functions (Karatsoreos et al., 2007; Vida Holloway, 2009). Among them, genetic and environmen- et al., 2008). Moreover, we have previously reported that tal factors such as biological sex and light-dark cycles can the circadian rhythms of hepatic P450 activities in rats LQÀXHQFHDGYHUVHGUXJUHVSRQVHVLQH[SHULPHQWDODQLPDOV are obvious in males but not in females (Furukawa et al., (Kato et al., 1962; Matsunaga, 2009). Therefore, atten- 1999c). In mice, the circadian clock components, Crypto- tion should be focused on these inter- and intra-individual chrome 1 and 2 (Cry1 and Cry2), are suggested as a pre- confounders to reinforce the proper use of pharmaceutical requisite for sustaining sex dimorphism in the liver metab- products aimed at tailor-made treatment, including gen- olism (Bur et al., 2009). Furthermore, sex dependency of GHUVSHFL¿FPHGLFDWLRQDQGFKURQRWKHUDS\ circadian pharmacology has been observed in a Phase III Biological sex is characterized by differences in phys- clinical trial for chronomodulated therapy of metastat- iological, anatomical, and behavioral traits resulted from ic colorectal cancer (Giacchetti et al., 2006). Therefore, natural or sexual selection. The characterization of sex- sexual dimorphism could participate in circadian rhyth- ually dimorphic genes would be a key towards a better micity; however, the interaction between sex and circadi- XQGHUVWDQGLQJRIVH[VSHFL¿FSK\VLRORJLFDOWUDLWV6H[ an modulatory effects on rat liver has not been fully focused VSHFL¿FJHQHVWUDQVFULSWLRQLQWKHURGHQWOLYHULVUHJXODWHG on. by secretory patterns of growth hormone (GH), which is To fully uncover sex and circadian modulatory effects episodic in males and continuous in females (Ahluwalia on rat liver, genome-wide analyses of liver genes expres- et al., 2004; Hirao et al., 2010; Rinn and Snyder, 2005; sion were performed at 4 hr intervals of a day-night cycle Waxman and Holloway, 2009). Notably, several cyto- using young adult male and female rats at 10 weeks of chrome P450 enzymes are differentially expressed age. Thereafter, a cross-sex comparison of time series between the sexes. Moreover, the sexually dimorphic GDWDLGHQWL¿HGJHQHVZLWKVH[XDOGLPRUSKLVPWKDWZHUH genes have been demonstrated to be regulated by several sustained at every measured time point. In addition, uni- transcription factors including signal transducer and acti- versal circadian-regulated genes in both sexes were iden- vator of transcription 5b (STAT5b) and hepatocyte nucle- WL¿HGE\DFURVVVH[FRPSDULVRQRIFLUFDGLDQUHJXODWHG ar factor 4-alpha (HNF4-alpha) (Ahluwalia et al., 2004; genes, which were analyzed by Fourier transform. Since Isensee and Ruiz Noppinger, 2007). Sexually dimorohic WKHLQWHJUDWLRQRIWKHLGHQWL¿HGJHQHVLQWRDELRORJLFDOQHW- genes have also been demonstrated to cause the sex differ- work is essential to gain an in-depth understanding of the ence in both pharmacological and toxicological effects of underlying physiological mechanism (Curtis et al., 2005; several drugs such as pentobarbital, carisoprodol, strych- Jimenez-Marin et al., 2009; Werner, 2008), the biologi- nine, and octamethylpyrophosphoramide (Franconi et al., cal network analyses were conducted using the sexually 2007; Kato et al., 1962). dimorphic or circadian-regulated gene lists. The present Circadian rhythms control myriads of physiological UHVXOWVFODUL¿HGVH[DQGFLUFDGLDQPRGXODWRU\HIIHFWVRQ processes, including liver metabolism and behavior, in rat liver, and would provide deeper insights into the eval- synchrony with an environmental light-dark cycle (Froy, uations of rat toxicological studies and its clinical impli- 2010; Green et al., 2008; Takahashi et al., 2008). The cations. central pacemaker in mammals is located in the supra- chiasmatic nucleus (SCN) of the hypothalamus (Ralph et MATERIALS AND METHODS al., 1990; Rusak and Zucker, 1979). Circadian oscillators exist not only in the SCN, but also in peripheral organs Animal maintenance and sample preparation such as the liver (Balsalobre et al., 1998; Yamazaki et al., Male and female F344/DuCrlCrlj rats (n = 30/sex) 2000). The hepatic transcriptome and liver metabolism aged 7 weeks, weighing 125 to 155 g in males and DUHLQÀXHQFHGE\FLUFDGLDQFRPSRQHQWVLQUDWVDQGWKH 100 to 130 g in females, were purchased from Charles circadian-regulated genes have been well characterized River Laboratories Japan, Inc. (Kanagawa, Japan). Ani- in males (Boorman et al., 2005; Desai et al., 2004; Hirao mals were acclimatized to an animal room controlled at et al., 2006). Period homolog 2 (Per2), which is a key a room temperature of 23 ± 3°C, relative humidity of 55 component of the core clock oscillator, is reported to be ± 15%, and ventilated 10 to 15 times/hr with 12 hr light- involved in both incidence and severity of hepatotoxici- dark cycles (ZT, zeitgeber time; ZT0, light on; ZT12, ty due to acetaminophen or carbon tetrachloride (Chen et light off). All animals were given tap water and solid feed al., 2009; Kakan et al., 2010). &HUWL¿HG5RGHQW'LHW 30,1XWULWLRQ,QWHUQDWLRQDO The presence of androgen and estrogen receptors with- Inc., Tokyo, Japan) sterilized by radiation (irradiated with in the SCN indicates that sexual hormones might mod- a 60Co-J ray of 30 kGy) ad libitum. After acclimatizing
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Sex and circadian modulatory effects on rat liver
DOODQLPDOVIRUDWOHDVWZHHNV¿YHDQLPDOVSHUVH[ZHUH link the most important transcription factors in the select- sacrificed at six time points (ZT2, ZT6, ZT10, ZT14, ed gene sets with the closest receptors using a shortest ZT18, and ZT22). The left lateral lobes of the liver were path algorithm (Ekins et al., 2006). dissected from each animal and immediately frozen in liq- uid nitrogen. The liver samples were stored at -80°C until Fourier transform analyses use. All animal procedures were performed as per the rec- Cosinor methodology was applied to analyze the bio- ommendations of the Animal Care and Use Committee of logical time series rhythmicity (Nelson et al., 1979). To Daiichi Sankyo Co., Ltd., Tokyo, Japan. UHSUHVHQWWKHGDLO\ÀXFWXDWLRQDVWKHVXPRIWKUHHVLQXVRL- dal waves, the Z-score normalized expression data were Microarray analyses further processed by Fourier transform using MATLAB Total RNA was isolated from the liver sample, labeled, (The MathWorks, Inc., Natick, MA, USA). Mathemati- and hybridized to the microarray as follows. The liver cally, each converted expression data y(t) was expressed sample was homogenized with an RLT buffer supplied as the following function of time t (hr). in the RNeasy mini kit (QIAGEN, Valencia, CA, USA), and the total RNA was isolated according to the manu-
IDFWXUHU¶VLQVWUXFWLRQV7KH51$VDPSOHVIURP¿YHDQL- in which mn and tdn represent the amplitude and the mals at each time point were pooled using equal amounts phase shift (hr) of the sinusoidal wave signal whose fre- IURPHDFKDQLPDOWRPDNHDWRWDORIȝJRI51$IRU quency is n times per one day, respectively. The basal males and females separately. The pooled samples were period T was set at a daily cycle of 24 (hr). These calcula- used as starting material. The cDNA was synthesized tions were performed for males and females separately. using a T7-(dT)24 primer (Amersham Biosciences Corp., Using the Fourier transform analyses, the following Piscataway, NJ, USA). The biotin-labeled cRNA criterion was adopted to identify circadian-regulated tran- was transcribed using a BioArray High Yield RNA scripts from microarray data in males and females sepa- Transcription Labeling Kit (Enzo Diagnostics, rately.
Farmingdale, NY, USA) according to the manufactur- Criterion for circadian-regulated transcripts: rmax = max er’s instructions. Labeled-cRNA was hybridized to the (r21, r31) < 0.5, under at least 2 Presence calls of 6 time
Rat Genome U34A Array (Affymetrix, Inc., Santa Clara, points. In this equation, r21 and r31 represent m2/m1 and
CA, USA) at 45°C for 16 hr. GeneChip was washed and m3/m1, respectively. stained using Fluidics Station (Affymetrix, Inc.) and 7KHLGHQWL¿HGFLUFDGLDQUHJXODWHGJHQHVLQFOXGHGFORFN scanned with a GeneArray Scanner (Hewlett-Packard, or clock-controlled “guide” genes such as aryl hydrocar- Palo Alto, CA, USA). The microarray imaging data was bon receptor nuclear translocator-like (Arntl), Per2, and analyzed with Microarray Suite (Affymetrix, Inc.). All D site albumin promoter binding protein (Dbp) (Green et the expression data were normalized by a trimmed mean al., 2008), which support the validity of criterion. (trim value: 4%) and then converted into Z-scores using Spotfire (Spotfire Inc., Somerville, MA, USA) (Nadon Measurements of hepatic phase I drug-metabolizing and Shoemaker, 2002). enzyme activities To verify the sex difference in the circadian rhythms of Biological network analyses hepatic P450 activities in the present study, P450 activi- To identify and rank relevant biological path- ties and P450 content were measured using microsomal ways enriched among selected gene sets, the differen- fractions which were prepared from the liver samples col- tially expressed genes were analyzed using the Data- lected at ZT6 (nadir time of the rhythmic P450 activities) base for Annotation, Visualization and Integrated and ZT18 (peak time of the rhythmic P450 activities) in Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/) males and females (Furukawa et al., 1999c). The thawed (Dennis et al., 2003; Huang da et al., 2009; Huang da et liver samples were mixed with 1.15% potassium chloride al., 2007; Sherman et al., 2007). The following settings of 3-fold volume of the sample and homogenized on ice. were applied: Functional Annotation Chart; thresholds: The homogenate was centrifuged at 9,000 g for 20 min at &RXQW ($6( 8QOHVVRWKHUZLVHVWDWHGRI¿FLDO 4°C, and the supernatant fraction was further centrifuged gene symbols were used to represent molecules. at 105,000 g for 1 hr at 4°C. The obtained precipitate was Furthermore, transcription factor targets modeling suspended with 1.15% potassium chloride of equal volume within the GeneGO MetaCore software (http://www.gene- of each supernatant and centrifuged at 105,000 g for 1 hr at go.com/) was used to calculate a list of networks which 4°C again. The resultant precipitate was resuspended with
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1.15% potassium chloride containing 20% glycerol of equal Statistical analysis volume of each supernatant and used as the microsomal For ACD (MCD, ECD, and PCD) activities and P450 fraction. The 7-methoxycoumarin, 7-ethoxycoumarin, and content, the homogeneity of variance between the val- 7-hydroxycoumarin were purchased from Aldrich. The 7- ues in the middle of the light and dark periods (ZT6 and propoxycoumarin was synthesized as previously described ZT18) was estimated by the FWHVW DQGVLJQL¿FDQWGLIIHU- (Matsubara et al., 1983). Using these substrates, the 7- ence of the mean value was evaluated by the Student’s alkoxycoumarin O-dealkylase (ACD) activities [7-meth- t-test (for homogeneous data) or the Aspin-Welch’s t-test oxycoumarin O-dealkylase (MCD), 7-ethoxycoumarin O- (for heterogeneous data) (Yoshimura, 1987). Statistical dealkylase (ECD), and 7-propoxycoumarin O-dealkylase analysis was performed with SAS System Version 6.1.2 (PCD) activities] were measured in the microsomal frac- (SAS Institute Inc., Cary, NC, USA). A 5% level of prob- tions by the method of Matsubara et al. (1983). The P450 DELOLW\ZDVFRQVLGHUHGWREHVLJQL¿FDQW content and protein concentrations in the microsomal frac- tions were determined by the methods of Omura and Sato (1964) and Lowry et al. (1951), respectively.
Table 1. Male-predominant genes in rat liver Accession Number Gene Symbol Gene Name NM_198784 - alpha-2u-globulin NM_172320 Afm Afamin NM_138510 Akr1c18 Aldo-keto reductase family 1, member C18 NM_019292 Ca3 Carbonic anhydrase 3 NM_053977 Cdh17 Cadherin 17 NM_133295 Ces3 Carboxylesterase 3 NM_013086 Crem cAMP responsive element modulator NM_021750 Csad &\VWHLQHVXO¿QLFDFLGGHFDUER[\ODVH NM_017148 Csrp1 Cysteine and glycine-rich protein 1 NM_012693 Cyp2a2 Cytochrome P450, 2a2 NM_019184 Cyp2c11 Cytochrome P450, 2c11 NM_138514 Cyp2c13 Cytochrome P450, 2c13 NM_153312 Cyp3a2 Cytochrome P450, 3a2 NM_145782 Cyp3a18 Cytochrome P450, 3a18 NM_021653 Dio1 Deiodinase, type I NM_012552 Ela1 Elastase 1 NM_057104 Enpp2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 NM_012883 Estsul Estrogen sulfotransferase NM_012959 Gfra1 Glial cell line-derived neurotrophic factor family receptor alpha 1 NM_017014 Gstm1 Glutathione S-transferase, mu 1 NM_032082 Hao2 Hydroxyacid oxidase 2 (long chain) NM_053301 Hfe Hemochromatosis NM_017080 Hsd11b1 Hydroxysteroid 11-beta dehydrogenase 1 NM_012584 Hsd3b Steroid delta-isomerase, 3 beta NM_031817 Omd Osteomodulin NM_019125 Pbsn Probasin NM_012621 Pfkfb1 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 NM_199208 Rdh2 Retinol dehydrogenase 2 NM_033231 Sah SA rat hypertension-associated gene XM_216782 Serpina3m Serine (or cysteine) proteinase inhibitor, clade A, member 3M NM_152936 Spink3 Serine protease inhibitor, Kazal type 3 NM_031732 Sult1c1 Sulfotransferase family, cytosolic, 1C, member 1
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RESULTS sion under Presence call at any time point in males were regarded as male-predominant genes (Table 1). On the ,GHQWL¿FDWLRQRIVH[XDOO\GLPRUSKLFJHQHV other hand, genes with 1.5-fold higher expression under To identify genes with sexual dimorphism that were Presence call at any time point in females were regarded sustained at every measured time point, gene expression as female-predominant genes (Table 2). levels normalized by a trimmed mean were compared between males and females at ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22. Genes with 1.5-fold higher expres-
Table 2. Female-predominant genes in rat liver Accession Number Gene Symbol Gene Name NM_053607 Acsl5 Acyl-CoA synthetase long-chain family member 5 NM_019286 Adh1 Alcohol dehydrogenase 1 NM_134329 Adh7 Alcohol dehydrogenase 7 NM_030985 Agtr1 Angiotensin II receptor, type 1 NM_022407 Aldh1a1 Aldehyde dehydrogenase family 1, member A1 NM_053019 Avpr1a Arginine vasopressin receptor 1A NM_031561 Cd36 Fatty acid translocase/CD36 (predicted) NM_016998 Cpa1 Carboxypeptidase A1 NM_001013137 Cxcl14 Chemokine (C-X-C motif) ligand 14 NM_012541 Cyp1a2 Cytochrome P450, 1a2 NM_012692 Cyp2a1 Cytochrome P450, 2a1 NM_031572 Cyp2c12 Cytochrome P450, 2c12 NM_147206 Cyp3a9 Cytochrome P450, 3a9 NM_153307 Cyp4a10 Cytochrome P450, 4a10 NM_017274 Gpam Glycerol-3-phosphate acyltransferase, mitochondrial NM_031509 Gsta3 Glutathione S-transferase A3 NM_001009920 Gsta5 Glutathione S-transferase Yc2 subunit NM_017060 Hrasls3 HRAS-like suppressor NM_001136124 ,¿WP Interferon induced transmembrane protein 3 NM_013144 Igfbp1 Insulin-like growth factor binding protein 1 NM_013122 Igfbp2 Insulin-like growth factor binding protein 2 NM_012696 Kng1 Kininogen 1 NM_144748 Ks .LGQH\VSHFL¿FSURWHLQ NM_012732 Lal Lysosomal acid lipase 1 NM_019196 Mpdz Multiple PDZ domain protein XM_240417 Mtmr7 Myotubularin-related protein 7 (predicted) NM_017000 Nqo1 NAD(P)H dehydrogenase, quinone 1 NM_053551 Pdk4 Pyruvate dehydrogenase kinase, isoenzyme 4 NM_134449 Prkcdbp Protein kinase C, delta binding protein NM_012630 Prlr Prolactin receptor NM_031841 Scd2 Stearoyl-Coenzyme A desaturase 2 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, NM_001009663 Serpina6 antitrypsin), member 6 (mapped) NM_053424 Slc4a4 Solute carrier family 4, member 4 NM_012695 Smp2a Rat senescence marker protein 2A gene NM_017070 Srd5a1 Steroid 5 alpha-reductase 1 Sulfotransferase family 2A, dehydroepiandrosterone (DHEA)-preferring, NM_001025131 Sult2a2 member 2 (predicted) NM_001039549 Ugt1a5 UDP glucuronosyltransferase 1 family, polypeptide A5
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Table 3. Biological pathways relevant to sexually dimorphic genes in rat liver Pathways P-Value Molecules Retinol metabolism 2.89E-16 CYP3A18, CYP3A2, ADH7, CYP1A2, ALDH1A1, CYP4A10, ADH1, UGT1A5, CYP2C12, CYP2C13, CYP2A1, CYP3A9, CYP2A2, CYP2C11 Drug metabolism - cytochrome P450 2.83E-14 CYP3A18, GSTA3, GSTA5, CYP3A2, ADH7, CYP1A2, ADH1, UGT1A5, CYP2C12, CYP2C13, CYP2A1, CYP3A9, CYP2A2, CYP2C11 Metabolism of xenobiotics by cytochrome 3.69E-12 CYP3A18, GSTA3, GSTA5, ADH1, CYP3A2, UGT1A5, ADH7, P450 CYP2C12, CYP1A2, CYP2C13, CYP3A9, CYP2C11 Linoleic acid metabolism 5.83E-08 CYP3A18, CYP3A2, CYP2C12, CYP1A2, CYP2C13, CYP3A9, CYP2C11 Drug metabolism - other enzymes 9.79E-07 CES3, CYP3A18, CYP3A2, UGT1A5, CYP2A1, CYP3A9, CYP2A2 Androgen and estrogen metabolism 4.75E-06 SMP2A, HSD3B, SULT2A2, UGT1A5, HSD11B1, SRD5A1, ESTSUL Bile acid biosynthesis 1.07E-03 ADH1, LAL, ADH7, SRD5A1 Caffeine metabolism 2.15E-03 CYP1A2, CYP2A1, CYP2A2 C21-Steroid hormone metabolism 4.16E-03 HSD3B, AKR1C18, HSD11B1 Fatty acid metabolism 5.36E-03 CYP4A10, ADH1, ADH7, ACSL5 gamma-Hexachlorocyclohexane 6.76E-03 CYP3A18, CYP3A2, CYP3A9 degradation Arachidonic acid metabolism 1.30E-02 CYP4A10, CYP2C12, CYP2C13, CYP2C11 PPAR signaling pathway 2.37E-02 CYP4A10, SCD2, CD36, ACSL5 1- and 2-Methylnaphthalene degradation 4.44E-02 ADH1, ADH7 2I¿FLDOJHQHV\PEROVZHUHXVHGWRUHSUHVHQWPROHFXOHV
Molecular basis of sex differences in hepatic Molecular basis of circadian-regulated genes in transcriptome the liver To understand the molecular basis of the sex differenc- 7KHLGHQWL¿HGFLUFDGLDQUHJXODWHGJHQHVLQFOXGHGFORFN es in hepatic transcriptome, the above sexually dimorphic or clock-controlled genes such as Arntl, Per2, and Dbp. genes were allocated on relevant biological pathways, The biological pathway analyses demonstrated that the which were notably enriched in the metabolism of retin- circadian-regulated gene sets were enriched in the urea ols, xenobiotics, linoleic acids, or androgen and estrogen, cycle and the metabolism of amino acids, fatty acids, or or bile acid biosynthesis (Table 3). Furthermore, tran- glucose (Table 5). In addition, transcription factor targets scription factor targets modeling was performed to cal- modeling suggested that transcription factors SP1, HNF4- culate a list of networks which link the most important alpha, and c-Myc proto-oncogene protein (c-MYC) were transcription factors in the sexually dimorphic gene sets involved in the circadian-regulatory networks (Figs. 3A with the closest receptors. In particular, transcription fac- and B). tors SP1, HNF4-alpha, and STAT5b were suggested to be core nodes in the regulatory networks among the sexually Sex and circadian effects on hepatic phase I dimorphic genes (Fig. 1). drug-metabolizing enzyme activities Hepatic ACD activities and P450 content were meas- ,GHQWL¿FDWLRQRIFLUFDGLDQUHJXODWHGJHQHV ured at ZT6 and ZT18 in males and females, and the Periodically expressed transcripts were obviously results are depicted in Fig. 4. The ACD activities at ZT18 extracted using the Fourier transform analyses based on were significantly higher than those at ZT6 in males. the criteria for circadian-regulated transcripts, as shown The percent differences at ZT18 compared to ZT6 were in Fig. 2. Consequently, genes which met the criteria in 23.8%, 22.0%, and 19.0% in the activities of MCD, ECD, ERWKVH[HVZHUHLGHQWL¿HGDVXQLYHUVDOFLUFDGLDQUHJXODW- DQG3&'UHVSHFWLYHO\,QFRQWUDVWQRVLJQL¿FDQWGLIIHU- ed genes (Table 4). ences were detected in the ACD activities between the light and dark periods in females. The P450 content at
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DISCUSSION
Genetic and environmental factors such as biological VH[DQGOLJKWGDUNF\FOHVFDQLQÀXHQFHOLYHUPHWDEROLVP and adverse drug responses in experimental animals (Kato et al., 1962; Matsunaga, 2009). Indeed, the sex difference in both pharmacological and toxicological effects of sev- eral drugs such as pentobarbital, carisoprodol, strych- nine, and octamethylpyrophosphoramide has been dem- onstrated (Kato et al., 1962). In addition, a key circadian clock component Per2 is reported to be involved in both incidence and severity of hepatotoxicity due to acetami- nophen or carbon tetrachloride (Chen et al., 2009; Kakan et al., 2010). Comprehension of sex and circadian modu- latory effects on rat liver is inevitable to assess the poten- tial toxicity of the drugs. Cross-sex comparison of time series data identified genes with sexual dimorphism that were sustained at eve- ry measured time point. The sexually dimorphic genes included representative male- or female-predominant genes such as cytochrome P450 subfamilies (Cyp2a1, Cyp2a2, Cyp2c11, Cyp2c12, Cyp2c13, Cyp3a2, and Cyp3a18), alcohol dehydrogenase, hydroxysteroid 11- beta dehydrogenase 1, carbonic anhydrase 3, sulfotrans- ferases (Estsul and Sult1c1), glutathione S-transferase Yc2, and prolactin receptor (Ahluwalia et al., 2004; Mugford and Kedderis, 1998; Waxman and Holloway, 7KHLGHQWL¿HGVH[XDOO\GLPRUSKLFJHQHVZHUHDOOR- Fig. 1. Representative regulatory networks for sexually di- cated on relevant biological pathways, which were over- morphic genes. represented in the metabolism of retinols, xenobiotics, Male-predominant genes are marked with red circles; linoleic acids, or androgen and estrogen, or bile acid bio- female-predominant genes with blue circles. Arrow- shaped lines between genes indicate relationships synthesis. Furthermore, transcription factor targets mode- between the two genes. Official gene symbols were ling suggested that transcription factors SP1, HNF4-alpha, used to represent molecules. The differently shaped and STAT5b might serve as core nodes in the regulato- nodes represent transcription factors (HIF1A, HNF4- ry networks among the sexually dimorphic genes. HNF4- alpha, NUR77, RXRA, SP1, STAT5A, and STAT5B), alpha is a highly conserved member of the nuclear recep- enzymes (ADH1, AKR1C18, ALDH1A1, CES3, CYP1A2, CYP2C12, CYP3A2, CYP3A9, CYP3A18, tor superfamily (NR2A1) and it regulates multiple liver DIO1, GSTA3, HSD11B1, LAL, NQO1, PFKFB1, and functions, including lipid homeostasis, lipoprotein produc- UGT1A5), a kinase (PDK4), generic receptors (CD36 tion, and bile acid biosynthesis (Waxman and Holloway, and PRLR), a G-protein-coupled receptor (AGTR1), a 2009). STAT5b mediates the signal transduction triggered transporter (SLC4A4), a protein (IFITM3), or generic by GH (Clodfelter et al., 2006). Indeed, deletion of Hnf4- binding proteins (HFE and SERPINA6). alpha or Stat5b in the mouse liver has been shown to lead WRDPDUNHGORVVRIVH[VSHFL¿FLW\ &ORGIHOWHU et al., 2006; ZT18 was slightly lower in females by 14.4% percent dif- Holloway et al., 2008; Waxman and Holloway, 2009). ference as compared to that at ZT6; however, the P450 Using Fourier transform analyses, periodically content in males was not different between the light and expressed transcripts were obviously extracted for circa- dark periods. GLDQUHJXODWHGWUDQVFULSWV7KHLGHQWL¿HGFLUFDGLDQUHJ- ulated genes included clock or clock-controlled genes such as Arntl, Per2, and Dbp (Green et al., 2008). The circadian-regulated gene sets were over-represented in the urea cycle and the metabolism of amino acids, fatty
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acids, or glucose, indicating that the major liver functions glycine). Xenobiotic metabolism is sexually dimorphic are under circadian control. In addition, transcription fac- (Ahluwalia et al., 2004; Rinn and Snyder, 2005; Waxman tor targets modeling suggested that transcription factors and Holloway, 2009) and is also subjected to circadi- SP1, HNF4-alpha, and c-MYC serve as major hubs in an regulation (Froy, 2010; Green et al., 2008; Takahashi the circadian-regulatory gene networks. Carbamoyl-phos- et al., 2008). Consistent with our previous reports phate synthetase 1, argininosuccinate lyase, and argin- (Furukawa et al., 1999a, 1999b and 1999c; Hirao et al., ase 1 are involved in the main pathways for urea cycle 2006), the hepatic phase I drug-metabolizing enzyme (Desvergne et al., 2006), and those genes were circadi- activities in the present study showed obvious differenc- an-regulated. HNF4-alpha has a crucial role for the reg- es between day and night cycles with high values dur- ulation of ureagenesis, and liver-specific deletion of ing the dark period and low values during the light peri- Hnf4-alpha results in hyperammonemia and hypouremia od in males. On the other hand, females did not show (Desvergne et al., 2006; Inoue et al., 2002). A proto-onco- any obvious fluctuations. We have shown that sexual- gene protein c-MYC regulates the G0 to G1 transition of ly dimorphic GH pulsatility is responsible for the circa- the cell cycle, and c-Myc has shown to be under circadian dian rhythms of hepatic ACD activities (Furukawa et al., regulation in mice (Lee, 2006). Accumulating evidence in 1999c). In addition, male-predominant P450 subfamilies mammals reveals that some essential checkpoint elements such as Cyp2c11 and Cyp3a2 are known to be under the in the cell cycle, including c-Myc (G0/G1 transition), Cyc- control of GH secretion (Ahluwalia et al., 2004; Hirao et lin D1 (G1/S transition), and WEE1 (G2/M transition) are al., 2010). Our transcriptome analysis, however, indicated under circadian control (Chen-Goodspeed and Lee, 2007; that male-predominant P450 genes were not expressed in Hunt and Sassone-Corsi, 2007; Sahar and Sassone-Corsi, a circadian fashion. Rather, P450 oxidoreductase, i.e. the 2009). Overall, peripheral circadian clock-mediated tran- redox partner of P450 monooxigenases (Lu et al., 1969), VFULSWLRQDOUHJXODWLRQVDUHOLNHO\WRLQÀXHQFHNH\FHOOX- and Cyp2e1 were circadianly expressed in the present lar proliferation and major tissue activities for optimizing study. Therefore, these genes might be important caus- local rhythms. es for the circadian rhythms of hepatic ACD activities. The liver is the major organ of xenobiotic metabolism, Regarding a phase II xenobiotic metabolism, glutamate which is broadly divided into phase I (oxidation, reduc- cysteine ligase (GCL) is the major determinant of glutath- tion, and hydrolysis) and phase II (conjugation reactions ione synthesis. GCL is composed of a catalytic (GCLC) with sulfate, glucuronic acid, glutathione, acetate, and DQGPRGL¿HU *&/0 VXEXQLW /X DQGWKHJHQH
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Sex and circadian modulatory effects on rat liver
Table 4. Circadian-regulated genes in rat liver Accession Number Gene Symbol Gene Name NM_019286 Adh1 Alcohol dehydrogenase 1 NM_001007144 Adrp Adipose differentiation-related protein NM_031835 Agt2 Alanine-glyoxylate aminotransferase 2 NM_030985 Agtr1 Angiotensin II receptor, type 1 NM_017201 Ahcy S-adenosylhomocysteine hydrolase NM_138510 Akr1c18 Aldo-keto reductase family 1, member C18 NM_031731 Aldh3a2 Aldehyde dehydrogenase family 3, subfamily A2 NM_012496 Aldob Aldolase B M31322 Aplp2 Amyloid beta (A4) precursor-like protein 2 NM_019158 Aqp8 Aquaporin 8 NM_022960 Aqp9 Aquaporin 9 NM_024151 Arf4 ADP-ribosylation factor 4 NM_017134 Arg1 Arginase 1 NM_024362 Arntl Aryl hydrocarbon receptor nuclear translocator-like NM_021577 Asl Argininosuccinate lyase NM_013113 Atp1b1 ATPase, Na+/K+ transporting, beta 1 polypeptide NM_017290 Atp2a2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 NM_030850 Bhmt Betaine-homocysteine methyltransferase NM_013087 Cd81 CD 81 antigen NM_017127 Chka Choline kinase alpha XM_235878 Chordc1 Cysteine and histidine-rich domain (CHORD)-containing, zinc-binding protein 1 (predicted) NM_053698 Cited2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 NM_001007802 Cklfsf6 Chemokine-like factor super family 6 NM_133558 Cml1 Camello-like 1 NM_134345 Cox8a Cytochrome c oxidase, subunit VIIIa NM_017072 Cps1 Carbamoyl-phosphate synthetase 1 NM_031559 Cpt1a Carnitine palmitoyltransferase 1 NM_012930 Cpt2 Carnitine palmitoyltransferase 2 NM_031987 Crot Carnitine O-octanoyltransferase NM_017074 Cth CTL target antigen NM_031543 Cyp2e1 Cytochrome P450, family 2, subfamily e, polypeptide 1 NM_012543 Dbp D site albumin promoter binding protein NM_012788 Dlg1 Discs, large homolog 1 (Drosophila) NM_001025411 Dnaja4 DnaJ (Hsp40) homolog, subfamily A, member 4 XM_341663 Dnajb1 DnaJ (Hsp40) homolog, subfamily B, member 1 (predicted) NM_134382 Elovl5 Elongation of long chain fatty acids (yeast) NM_198742 Etfdh (OHFWURQWUDQVIHUULQJÀDYRSURWHLQGHK\GURJHQDVH XM_341119 Etnk2 Ethanolamine kinase 2 (predicted) NM_030832 Fabp7 Fatty acid binding protein 7 NM_019238 Fdft1 Farnesyl diphosphate farnesyl transferase 1 NM_017126 Fdx1 Ferredoxin 1 NM_001008724 Fga Fibrinogen, alpha polypeptide NM_017005 Fh1 Fumarate hydratase 1 NM_001009632 G0s2 G0/G1 switch gene 2 NM_031589 G6pt1 Glycerol-6-phosphate transporter NM_001013089 Galt Galactose-1-phosphate uridyl transferase NM_024356 Gch GTP cyclohydrolase 1
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Table 4. (Continued.)Circadian-regulated genes in rat liver Accession Number Gene Symbol Gene Name NM_012815 Gclc Glutamate-cysteine ligase, catalytic subunit NM_017305 Gclm *OXWDPDWHF\VWHLQHOLJDVHPRGL¿HUVXEXQLW NM_031776 Gda Guanine deaminase NM_013222 Gfer Growth factor, erv1 homolog (S. cerevisiae) NM_013120 Glre Glucokinase regulatory protein NM_012565 Gluka Glucokinase NM_053660 Gng10 Guanine nucleotide binding protein (G protein), gamma 10 NM_012571 Got1 Glutamate oxaloacetate transaminase 1 NM_017274 Gpam Glycerol-3-phosphate acyltransferase, mitochondrial NM_012578 H1f0 H1 histone family, member 0 NM_032082 Hao2 Hydroxyacid oxidase 2 (long chain) NM_013134 Hmgcr 3, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase NM_053493 Hpcl2 2-hydroxyphytanoyl-Coenzyme A lyase NM_024391 Hsd17b2 Hydroxysteroid (17-beta) dehydrogenase 2 NM_001011901 Hsp105 Heat shock protein 105 NM_022229 Hsp60 Heat shock protein 1 (chaperonin) NM_175761 Hsp90 Heat shock 90kDa protein 1, alpha-like 3 (predicted) NM_013058 Id3 Inhibitor of DNA binding 3 XM_220451 Igtp Interferon gamma induced GTPase NM_022392 Insig1 Insulin-induced gene 1 NM_012591 Irf1 Interferon regulatory factor 1 NM_053902 Kynu Kynureninase (L-kynurenine hydrolase) U53184 Litaf LPS-induced TN factor NM_017061 Lox Lysyl oxidase NM_053541 Lrp3 Low density lipoprotein receptor-related protein 3 NM_012860 Mat1a Methionine adenosyltransferase I, alpha NM_134410 Mg87 Mg87 protein NM_001007714 Morf412 MORF-related gene X NM_138826 Mt1a Metallothionein 1a NM_053986 Myo1b Myosin Ib AY325167 Ndufa8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8 NM_012988 1¿D Nuclear factor I/A NM_031073 Ntf3 Neurotrophin 3 NM_053598 Nudt4 Nudix (nucleoside diphosphate linked moiety X)-type motif 4 NM_022585 Oazi Ornithine decarboxylase antizyme inhibitor NM_012615 Odc Ornithine decarboxylase 1 NM_139081 Oaz1 Ornithine decarboxylase antizyme 1 NM_030996 Oprs1 Opioid receptor, sigma 1 NM_172062 P4ha1 Proline 4-hydroxylase, alpha 1 polypeptide NM_017225 Pctp Phosphatidylcholine transfer protein NM_031678 Per2 Per2, period homolog 2 (Drosophila) NM_053487 Pex11a Peroxisomal biogenesis factor 11A NM_012621 Pfkfb1 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 NM_053291 Pgk1 Phosphoglycerate kinase 1 NM_031576 Por P450 (cytochrome) oxidoreductase NM_013196 Ppara Peroxisome proliferator activated receptor alpha NM_033096 Ppm1b Protein phosphatase 1B, magnesium dependent, beta isoform NM_053576 Prdx6 Peroxiredoxin 6 NM_134449 Prkcdbp Protein kinase C, delta binding protein
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Table 4. (Continued.) Circadian-regulated genes in rat liver Accession Number Gene Symbol Gene Name NM_173322 Prol2 Proline rich 2 NM_022390 Qdpr Quinoid dihydropteridine reductase NM_032617 Rab11b RAB11B, member RAS oncogene family NM_001024331 Rab43 Ras-related protein RAB43 NM_053664 Sardh Sarcosine dehydrogenase NM_001012213 Sfxn1 6LGHURÀH[LQ NM_012716 Slc16a1 Solute carrier family 16 (monocarboxylic acid transporters), member 1 NM_133418 Slc25a10 Solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 NM_133315 Slc39a1 Solute carrier family 39 (iron-regulated transporter), member 1 NM_017206 Slc6a6 Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 NM_022667 Slco2a1 Solute carrier organic anion transporter family, member 2a1 NM_031683 Smc1l1 Structural maintenance of chromosomes 1-like 1 (S. cerevisiae) NM_012695 Smp2a Rat senescence marker protein 2A gene NM_031122 St13 Suppression of tumorigenicity 13 XM_341540 Svil Supervillin (predicted) NM_013026 Synd1 Syndecan 1 NM_019194 Tef Thyrotroph embryonic factor XM_342851 Tle1 Transducin-like enhancer of split 1, homolog of Drosophila E(spl) (predicted) NM_053331 Txn2 Thioredoxin 2 NM_080887 Txnl Thioredoxin-like (32kD) NM_031614 Txnrd1 Thioredoxin reductase 1 NM_053768 Uox Urate oxidase NM_057097 Vamp3 Vesicle-associated membrane protein 3 NM_031836 Vegfa Vascular endothelial growth factor A NM_001009686 Wdr23 WD repeat domain 23
Table 5. Biological pathways relevant to circadian-regulated genes in rat liver Pathways P-Value Molecules Urea cycle and metabolism of amino groups 2.49E-04 ARG1, ASL, CPS1, ODC, ALDH3A2 Arginine and proline metabolism 6.05E-04 ARG1, GOT1, P4HA1, ASL, CPS1 Glycine, serine and threonine metabolism 2.19E-03 AGT2, CHKA, CTH, BHMT, SARDH Methionine metabolism 2.90E-03 CTH, AHCY, MAT1A, BHMT Fatty acid metabolism 1.57E-02 CPT2, ADH1, ALDH3A2, CPT1A Selenoamino acid metabolism 2.09E-02 CTH, AHCY, MAT1A Glycolysis/Gluconeogenesis 2.22E-02 ADH1, ALDOB, PGK1, GLUKA, ALDH3A2 2I¿FLDOJHQHV\PEROVZHUHXVHGWRUHSUHVHQWPROHFXOHV
expressions of Gclc and Gclm were circadian-regulated cancer (Giacchetti et al., 7KHVH¿QGLQJVLPSO\WKDW in both sexes. In mice, the circadian clock components, sexual dimorphism and the circadian timing system are Cry1 and Cry2, are suggested as a prerequisite for sus- generally interconnected in both humans and rodents. taining sex dimorphism in the liver metabolism (Bur et In conclusion, our transcriptome analyses identified al., 2009). In human beings, sex dependency of circadi- sexually dimorphic and circadian-regulated genes, as well an pharmacology has been observed in a Phase III clinical as the relevant biological pathways in rat liver. Moreover, trial for chronomodulated therapy of metastatic colorectal sexual dimorphism was considered likely to interact with
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A B
Fig. 3. Representative regulatory networks for circadian-regulated genes. Circadian-regulated genes are marked with a “checkerboard” color. Arrow-shaped lines between genes indicate relationships EHWZHHQWKHWZRJHQHV2I¿FLDOJHQHV\PEROVZHUHXVHGWRUHSUHVHQWPROHFXOHV $ 7KHGLIIHUHQWO\VKDSHGQRGHVUHSUHVHQW transcription factors (ARNTL, CITED2, DBP, HNF4-alpha, IRF1, p53, and SP1), enzymes (ADH1, AGT2, AKR1C18, AL- DOB, BHMT, CPT-1A, CPT II, CROT, CTH, CYP2E1, FDFT1, FH1, GALT, GCH, GCLC, HPCL2, HSD17B2, MAT1A, ODC, P4HA1, PFKFB1, POR, and TXNRD1), kinases (ETNK2 and PGK1), a GTPase (ARF4), generic receptors (CD81 and SYND1), a G-protein-coupled receptor (AGTR1), transporters (PCTP and SLC16A1), a protein (G0S2), a generic channel (AQP9), a ligand-gated ion channel (ATP2A2), or generic binding proteins (APLP2, DLG1, FDX1, GFER, HSP60, HSP90 alpha, HSP105, ID3, MT1A, OAZ1, SMC1, and WDR23). (B) The differently shaped nodes represent transcrip- tion factors (c-Myc, DBP, E4BP4, IRF1, and NFIA), enzymes (ARG1, GALT, GCLC, HMGCR, NDUFA8, ODC, P4HA1, QDPR, and TXNRD1), a kinase (PGK1), a GTPase (ARF4), a ligand-gated ion channel (ATP2A2), a protein (VAMP3), a G protein adaptor (GNG10), or generic binding proteins (DLG1, HSP60, HSP90 alpha, HSP105, ID3, MYO1B, OAZ1, and OAZI).
MCD activity ECD activity ) 1 ) 1 ZT6 ZT6 0.8 ZT18 0.8 ZT18 0.6 0.6 0.4 0.4 0.2 0.2 (nmol/min/mg protein (nmol/min/mg 0 protein (nmol/min/mg 0 Male Female Male Female
PCD activity P450 content ) 1 1 ZT6 ZT6 0.8 ZT18 0.8 ZT18 0.6 0.6 0.4 0.4 0.2 0.2 (nmol/mg protein) (nmol/mg
(nmol/min/mg protein (nmol/min/mg 0 0 Male Female Male Female
Fig. 4. Sex and circadian effects on hepatic ACD activities and P450 content. MCD, ECD, and PCD activities, and P450 content were measured in the liver of rats (n = 5/sex/ZT). Columns and bars rep- resent the means ± S.D. of each value. Light bar indicates the value at ZT6, whereas black bar indicates the value at ZT18. 6LJQL¿FDQWO\GLIIHUHQWIURP=7JURXS P P < 0.01 (Student’s t-test); #P < 0.05 (Aspin-Welch’s t-test).
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Sex and circadian modulatory effects on rat liver circadian rhythmicity via overlapping gene regulatory net- Clodfelter, K.H., Holloway, M.G., Hodor, P., Park, S.H., Ray, W.J. works. Interestingly, transcription factors SP1 and HNF4- and Waxman, D.J. (2006): Sex-dependent liver gene expression is extensive and largely dependent upon signal transducer and alpha are likely to orchestrate not only sexually dimor- activator of transcription 5b (STAT5b): STAT5b-dependent acti- phic, but also circadian-regulated genes even though each vation of male genes and repression of female genes revealed by criterion was rather mutually exclusive. This suggests microarray analysis. Mol. Endocrinol., 20, 1333-1351. WKHFURVVWDONEHWZHHQWKRVHUHJXODWLRQVDQGWKHVH¿QG- Curtis, R.K., Oresic, M. and Vidal-Puig, A. (2005): Pathways to the ings would provide deeper insights into the evaluations of analysis of microarray data. Trends Biotechnol., 23, 429-435. Dennis, G.Jr., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., rat toxicological studies and its clinical implications. Sex Lane, H.C. and Lempicki, R.A. (2003): DAVID: Database for differences can manifest differently at different stages in Annotation, Visualization and Integrated Discovery. Genome the animal life cycle (Gochfeld, 2007). In particular, the Biol., 4, R60. VH[XDOO\GLPRUSKLF*+SUR¿OHLQUDWVVKRZVDJHGHSHQG- Desai, V.G., Moland, C.L., Branham, W.S., Delongchamp, R.R., ent changes; and the low GH nadir is eliminated in senes- Fang, H., Duffy, P.H., Peterson, C.A., Beggs, M.L. and Fuscoe, J.C. (2004): Changes in expression level of genes as a function cent male rats, followed by feminization and demasculi- of time of day in the liver of rats. Mutat. Res., 549, 115-129. nization of the sex-dependent isoforms of P450 (Dhir and Desvergne, B., Michalik, L. and Wahli, W. (2006): Transcriptional Shapiro, 2003; Lee et al., 2008; Mori et al., 2007). Addi- regulation of metabolism. 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