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Expression Profile of Early Estradiol-responsive Genes in Cynomolgus Macaque Liver: Implications for Drug-metabolizing Enzymes
Drug Metab. Pharmacokinet. 27 (4): 451455 (2012).
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
Note Expression Profile of Early Estradiol-responsive Genes in Cynomolgus Macaque Liver: Implications for Drug-metabolizing Enzymes Ryota I SE 1 , Go K ITO 2 and Yasuhiro U NO 2,3, * 1
Drug Safety Research Center, Shin Nippon Biomedical Laboratories, Ltd., Kagoshima, Japan 2 Laboratory of Translational Research, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 3 Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., Kainan, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Estrogen plays important roles in estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, the major drug metabolizing organ, estrogen is known to regulate expression of some drug-metabolizing enzymes. Due to the lack of information on the role of estrogen in hepatic gene expression in primate species, we previously investigated the late response of hepatic gene expression to estradiol in cynomolgus macaques. To understand the early response of hepatic gene expression to estradiol, in this study, microarray analysis was conducted using cynomolgus macaque liver samples collected at 1 h and 5 h after estradiol injection. Comparison of expression profiles in estradiol and solvent (control)-treated ovariectomized cynomolgus macaques revealed 27 differentially expressed genes (>2.0-fold), including 18 at 1 h and 9 at 5 h after estradiol injection. As indicated by Gene Ontology analysis, these genes were related to oxidoreductase activity and transferase activity, partly representing important aspects of drug-metabolizing enzymes. Further analysis by quantitative polymerase chain reaction revealed that estradiol down-regulated CYP2A24, CYP2C76, and CYP2E1 (>2.0-fold) at 1 h and up-regulated GSTM5 (>2.0-fold) at 5 h after estradiol injection. These results suggest that the short-term estradiol treatment influenced expression of hepatic genes, including drug-metabolizing enzyme genes, in cynomolgus macaque liver. Keywords: cynomolgus macaque; cytochrome P450; liver; gene expression; metabolic activity
In the liver, estrogen plays roles in the regulation of lipid metabolism, bile acid excretion, and blood coagulation.6®8¥ Estrogen also regulates drug metabolism by affecting expression of drug-metabolizing enzyme genes in rodents9¥. Similarly, in the cynomolgus macaque, a primate species widely used in drug metabolism studies due to its evolutionary closeness to humans, we previously identified several estradiol-responsive genes for drug-metabolizing enzymes in cynomolgus macaque liver after a 4-day estradiol treatment, including CYP3A4, CYP4F12, and GSTM5.10¥ However, the early response of estradiol to hepatic genes remained to be investigated in cynomolgus macaques. In this study, therefore, expression profiles of hepatic genes were analyzed in ovariectomized cynomolgus macaques by microarray analysis at 1 h and 5 h after estradiol
Introduction Estrogen, including 17Ç-estradiol, estriol, and estrone, plays an important role in the biological events in many organs such as mammary glands, ovaries, and the uterus. Most estrogenic chemicals are biological response modulators mediated by direct interaction with estrogen receptors.1¥ Estrogen receptors form a transcriptional complex by recruiting coactivators such as CREB-binding protein, RNA polymerase II, steroid receptor coactivator 1, and TATA element binding protein, which in turn bind to estrogen responsive elements in the upstream region of the target genes ¤late estrogen-responsive genes¥.2®4¥ In addition, rapid non-genomic responses can be mediated by membrane estrogen receptors, stimulating signal transduction.5¥
Received December 5, 2011; Accepted February 3, 2012 J-STAGE Advance Published Date: February 21, 2012, doi:10.2133/dmpk.DMPK-11-NT-147 *To whom correspondence should be addressed: Yasuhiro UNO, D.V.M., Ph.D., Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., 16-1 Minami Akasaka, Kainan 642-0017, Japan. Tel. +81-73-483-8881, Fax. +81-73-483-7377, E-mail: uno-yasuhiro@snbl. co.jp 451
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injection. The estradiol-responsive genes identified were classified by Gene Ontology ¤GO¥ analysis. Further analysis by quantitative polymerase chain reaction ¤qPCR¥ was conducted for drug-metabolizing enzyme genes identified to be estradiol-responsive in addition to 11 cytochrome P450 ¤P450¥ genes relevant to drug metabolism, including CYP2A23, CYP2A24, CYP2B6, CYP2C20, CYP2C43, CYP2C75, CYP2C76, CYP2D17, CYP2E1, CYP3A4, and CYP3A5. Materials and Methods Animals, treatments, tissues, and RNA extraction: Twelve female cynomolgus macaques ¤from Indochina, 14®16 years of age, 3®6 kg¥ were ovariectomized according to the standard procedure established at Shin Nippon Biomedical Laboratories, Ltd. ¤Kagoshima, Japan¥. Treatment was carried out at least 30 days after the ovariectomy, using subcutaneous injections of estradiol benzoate ¤50 µg/ kg body weight, 8 animals¥ or sesame oil ¤4 animals as control¥, accompanied by injection of 17Ç-estradiol ¤5 µg/kg body weight, i.v.¥ for a rapid increase in blood estrogen levels, as described previously.10¥ Liver samples were collected from 4 treated and 2 control animals at 1 h or 5 h, and flash-frozen in liquid nitrogen. The study was reviewed and approved by the institutional ethics committee. Total RNA was extracted from these 12 liver samples and treated with DNase I as described previously.11¥ The extracted RNAs were subsequently purified using RNeasy Mini Kit ¤Qiagen, Valencia, CA¥, according to the manufacturerös protocols. Microarray analysis: Total RNA from 8 liver samples ¤2 treated and 2 control animals at 1 h and 5 h¥ were used for microarray analysis, which was performed using the Human Genome U130 GeneChip set ¤Affymetrix, Santa Clara, CA¥ by Bio Matrix Research, Inc. ¤Nagareyama, Japan¥ in accordance with the procedures established by Affymetrix. These include verification of RNA quality, labeling, hybridization with biotin-labeled cRNAs ¤15 µg¥, scanning of the arrays, and data analysis. GeneChip Operating Software ¤GCOS; Affymetrix¥ was used for image processing and data acquisition. Differentially expressed genes were selected at a threshold of 1.5-fold difference between estradiol-treated and control animals ¤p g 0.05¥. Bioinfomatics: Estradiol-responsive genes were subjected to GO classification, to describe the roles of the genes identified using precisely defined GO terms.12¥ Overrepresented biological processes were determined using DAVID 2.0 ¤http://david.abcc.ncifcrf.gov/¥ according to the protocol. The analysis included 250 up- and 111 downregulated genes ¤h1.5-fold, p g 0.05¥ at 1 h and 5 h after estradiol injection, respectively. Significantly overrepresented Biological process and Molecular function categories were selected ¤p g 0.05¥. Quantitative PCR: Real-time reverse transcriptionPCR was performed using liver total RNAs as described previously for GSTM5, CYP2A23, CYP2A24, CYP2B6,
CYP2C20, CYP2C43, CYP2C75, CYP2C76, CYP2D17, CYP2E1, CYP3A4, CYP3A5, and PXR.10¥ Relative expression levels were determined by normalizing the raw data to the 18S ribosomal RNA level based on three independent amplifications. Statistical significance for differences in gene expression was determined using a two-tailed unpaired Studentös t test. For the statistical analysis, mean values of all 4 control animals ¤1 h and 5 h¥ were compared with those of 4 estradiol-treated animals ¤1 h or 5 h¥. Values were presented as mean + S.D. from the 4 animals and were considered significant if p was g0.05. Results and Discussion To identify early estradiol-responsive genes in cynomolgus macaque liver, gene expression was measured using DNA microarrays at 1 h and 5 h after estradiol injection. Comparison of gene expression between estradiol-treated and control liver showed that 250 genes were up- and 111 genes were down-regulated by estradiol treatment ¤h1.5-fold¥ at 1 h and 5 h after estradiol injection. The genes expressed differently ¤h2.0-fold¥ between estradiol-treated and control liver included 10 and 1 up-regulated genes; and 8 and 8 down-regulated genes at 1 h and 5 h after estradiol injection, respectively ¤Table 1¥. GO analysis of the estradiol-responsive genes ¤h1.5-fold¥ with level 2 GO annotation using DAVID 2.0 revealed that 17 and 6 functional categories were enriched for Biological process and Molecular function ¤p g 0.05¥, respectively, at 1 h after injection ¤Table 2¥. At 5 h after injection, 5 and 4 functional categories were enriched for Biological process and Molecular function ¤p g 0.05¥, respectively ¤Table 2¥. The Biological process categories included various metabolic processes and oxidation reduction, while the Molecular function categories included oxidoreductase activity and transferase activity, which included drug-metabolizing enzyme genes. Microarray data showed that expression of drugmetabolizing enzyme genes was also changed ¤h1.5-fold¥ by estradiol treatment, including GSTM5, CYP2B6, and CYP3A4. qPCR analysis showed that GSTM5 was up-regulated at 1 h ¤2.2-fold¥ and 5 h ¤3.9-fold, p g 0.05¥ ¤Fig. 1¥, but differential expression at 1 h did not reach statistical significance. qPCR analysis also showed up-regulation of CYP2B6 at 1 h ¤1.5-fold¥ and 5 h ¤2.9-fold¥ and downregulation of CYP3A4 at 5 h ¤2.5-fold¥, but none of these differential expressions reached statistical significance. Further qPCR analysis of P450 genes relevant to drug metabolism ¤CYP2A23, CYP2A24, CYP2C20, CYP2C43, CYP2C75, CYP2C76, CYP2D17, CYP2E1, and CYP3A5¥ and PXR ¤an important transcriptional regulator of P450 genes¥ revealed that CYP2A24 ¤5 h¥, CYP2C20 ¤5 h¥, CYP2C75 ¤1 h, 5 h¥, CYP2E1 ¤5 h¥, CYP4A11 ¤1 h, 5 h¥, and PXR ¤1 h¥ were downregulated ¤h1.5-fold¥, while CYP3A5 ¤5 h¥ was up-regulated ¤h1.5-fold¥, but none of these differential expressions reached statistical significance. In contrast, there was a sig-
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Early Estradiol-responsive Genes in Cynomolgus Macaque Liver Table 1. Top five estradiol-responsive genes identified in cynomolgus macaque liver by microarray analysis Gene symbol
Fold change
Description
1h ASCL1 CYP2B6
7.2 3.1
DGAT2 FETUB LECT2 SORD ACE2
2.5 2.5 2.5 2.5 2.4
ENPP2
2.4
TF GSTM5 PLK2 HSDL2 PITPN KCNT2 ABCD3 CRP TPD52 VMP1
2.3 2.1 %2.2 %2.3 %2.3 %2.2 %2.3 %2.3 %2.5 %2.6
Achaete-scute complex homolog 1 ¤Drosophila¥ Cytochrome P450, family 2, subfamily B, polypeptide 6 Diacylglycerol O-acyltransferase 2 Fetuin B Leukocyte cell-derived chemotaxin 2 Sorbitol dehydrogenase Angiotensin I converting enzyme ¤peptidyl-dipeptidase A¥ 2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 ¤autotaxin¥ Transferrin Glutathione S-transferase M5 Polo-like kinase 2 Hydroxysteroid dehydrogenase like 2 Phosphotidylinositol transfer protein Potassium channel, subfamily T, member 2 ATP-binding cassette, sub-family D ¤ALD¥, member 3 C-reactive protein, pentraxin-related Tumor protein D52 Vacuole membrane protein 1
5h ASCL1 ZBTB16 PPM1B INSIG1 NARS ELL2 FGL1 SEPX1 LDHB
3.5 %2.1 %2.1 %2.1 %2.2 %2.2 %2.5 %2.7 %3.2
Achaete-scute complex homolog 1 ¤Drosophila¥ zinc finger and BTB domain containing 16 Protein phosphatase, Mg2¦/Mn2¦ dependent, 1B Insulin induced gene 1 Asparaginyl-tRNA synthetase Elongation factor, RNA polymerase II, 2 Fibrinogen-like 1 Selenoprotein X, 1 Lactate dehydrogenase B
nificant difference between estradiol-treated animals and control animals for down-regulation of CYP2A24 ¤3.1-fold¥, CYP2C76 ¤2.2-fold¥, and CYP2E1 ¤3.1-fold¥ at 1 h ¤Fig. 1¥. These results suggest that hepatic genes, including drugmetabolizing enzyme genes, are regulated in cynomolgus macaque liver by estradiol. GSTM5 belongs to the glutathione transferase ¤GST¥ family, essential in conjugation reactions for detoxification of endogenous and exogenous substrates.13¥ In a previous study, cynomolgus GSTM5 was also up-regulated after a 4-day estradiol treatment,10¥ similar to up-regulation of GSTM5 at 5 h after estradiol injection in this study ¤Fig. 1¥. The late response of estrogen-responsive genes is largely mediated by estrogen receptors, whereas the early response of estrogen-responsive genes is mediated through nongenomic actions of estrogen, including mobilization of intracellular calcium, enhancement of adenylate cyclase activity and cAMP production, and activation of MAPK and
Table 2. Biological GO categories of estradiol-responsive genes 1h Biological process catabolic process primary metabolic process macromolecule localization oxidation reduction response to chemical stimulus vesicle-mediated transport cellular metabolic process establishment of protein localization alcohol metabolic process establishment of localization negative regulation of developmental process regulation of biological quality membrane organization transport negative regulation of cellular process establishment of localization in cell cellular localization Molecular function cofactor binding oxidoreductase activity carbohydrate binding transferase activity pattern binding enzyme inhibitor activity
5h cellular metabolic process primary metabolic process cellular response to stimulus alcohol metabolic process response to stress
selenium binding transcription repressor activity carboxylic acid binding cofactor binding
Functional categories of up- and down-regulated genes were identified with level 2 GO terms using DAVID 2.0 ¤p g 0.05¥ as described in Materials and Methods.
Fig. 1. qPCR analysis of estradiol-responsive genes Expression of GSTM5, CYP2A24, CYP2C76, and CYP2E1 was measured by qPCR analysis using liver total RNAs from estradiol-treated (E2) and control animals as described in Materials and Methods. Values are presented as mean + S.D. of the four animals. The expression level of each gene in control animals was arbitrarily adjusted to 1, and all other expression levels were adjusted accordingly. *p g 0.05.
phosphoinositol 3-kinase signaling pathways.5¥ It is of great interest to investigate mechanisms of estrogen to activate GSTM5 in short- and long-term estradiol treatment. In this study, CYP2A24, CYP2C76, and CYP2E1 were identified as estradiol-responsive genes in cynomolgus macaque liver. Similarly, CYP3A4 was up-regulated in cynomolgus macaque liver after a 4-day estradiol treatment.10¥ CYP2A24, CYP2C76, CYP2E1, and CYP3A4
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
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belong to the P450 family, essential for the metabolism of various drugs, steroids, fatty acids, and environmental pollutants. Although the involvement of these cynomolgus enzymes in estradiol metabolism is not known, human P450s highly identical to these cynomolgus P450s, such as CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, are involved in metabolism of estradiol.14®16¥ Therefore, these P450s, together with GSTM5, may facilitate excretion of estradiol by promoting the oxidation and conjugation of estradiol. In humans, CYP3A4 is involved in not only drug metabolism, but also in hydroxylation of bile acids and steroid hormones. Previous studies showed by microarray analysis that estradiol enhanced genes relevant to lipid metabolism in rodent and cynomolgus macaque liver.10,17¥ In rodents, treatment with 17Æ-ethinyl estradiol, a synthetic estrogen, decreased bile flow and bile acid synthesis, and mildly increased bile acid in serum.7¥ In the human CYP3Atransgenic mice, expression of human CYP3A was induced by bile acid accumulation, indicating that CYP3A4 plays a role in preventing cholestasis by metabolizing bile acid.18¥ Therefore, estradiol might enhance expression of P450 genes to eliminate extra cholesterol from the body. In this study, CYP2A24, CYP2C76, and CYP2E1 were identified as estradiol-responsive genes by qPCR analysis, but differential expression was not detected using microarrays. One of the reasons might be the use of human microarrays to detect expression of cynomolgus macaque genes. CYP2C76 is not orthologous to any human P450,11¥ and thus the probe of this gene would not be contained in the human microarrays used. Cynomolgus CYP2A24, CYP2A23, and CYP2A26, predominantly expressed in liver, are highly identical to human CYP2A6 and CYP2A13,19¥ and thus human CYP2A probes might not detect expression of cynomolgus CYP2A genes in a gene-specific manner. Genome information such as genome sequences and expressed sequence tags are gradually being accumulated in cynomolgus macaques,20®22¥ which could be helpful in the manufacture of cynomolgus macaque microarrays. Such genome tools would enhance the advancement of biomedical studies in cynomolgus macaques. In conclusion, microarray analysis using cynomolgus macaque liver samples collected at 1 h and 5 h after estradiol injection identified numerous estradiol-responsive genes, including drug-metabolizing enzyme gene GSTM5. Further analysis by qPCR revealed that drug-metabolizing enzyme genes CYP2A24, CYP2C76, and CYP2E1 were also estradiolresponsive. The results presented in this paper are useful for understanding liver physiology, especially drug metabolism, in cynomolgus macaques. Acknowledgments: The authors greatly thank Dr. Ryoichi Nagata, Dr. Koichiro Fukuzaki, and Mr. Masahiro Utoh for their support of this work, and Mr. Patrick Gray for reviewing the manuscript.
References 1¥ Blair, R. M., Fang, H., Branham, W. S., Hass, B. S., Dial, S. L., Moland, C. L., Tong, W., Shi, L., Perkins, R. and Sheehan, D. M.: The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol. Sci., 54: 138®153 ¤2000¥. 2¥ Smith, C. L., Oñate, S. A., Tsai, M. J. and OöMalley, B. W.: CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription. Proc. Natl. Acad. Sci. USA, 93: 8884®8888 ¤1996¥. 3¥ Pan, Y. F., Wansa, K. D., Liu, M. H., Zhao, B., Hong, S. Z., Tan, P. Y., Lim, K. S., Bourque, G., Liu, E. T. and Cheung, E.: Regulation of estrogen receptor-mediated long range transcription via evolutionarily conserved distal response elements. J. Biol. Chem., 283: 32977®32988 ¤2008¥. 4¥ Sadovsky, Y., Webb, P., Lopez, G., Baxter, J. D., Fitzpatrick, P. M., Gizang-Ginsberg, E., Cavailles, V., Parker, M. G. and Kushner, P. J.: Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein. Mol. Cell. Biol., 15: 1554®1563 ¤1995¥. 5¥ Björnström, L. and Sjöberg, M.: Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Mol. Endocrinol., 19: 833®842 ¤2005¥. 6¥ Windler, E. E., Kovanen, P. T., Chao, Y. S., Brown, M. S., Havel, R. J. and Goldstein, J. L.: The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E. J. Biol. Chem., 255: 10464®10471 ¤1980¥. 7¥ Rodríguez-Garay, E. A.: Cholestasis: human disease and experimental animal models. Ann. Hepatol., 2: 150®158 ¤2003¥. 8¥ Farsetti, A., Misiti, S., Citarella, F., Felici, A., Andreoli, M., Fantoni, A., Sacchi, A. and Pontecorvi, A.: Molecular basis of estrogen regulation of Hageman factor XII gene expression. Endocrinology, 136: 5076®5083 ¤1995¥. 9¥ Kawamoto, T., Kakizaki, S., Yoshinari, K. and Negishi, M.: Estrogen activation of the nuclear orphan receptor CAR ¤constitutive active receptor¥ in induction of the mouse Cyp2b10 gene. Mol. Endocrinol., 14: 1897®1905 ¤2000¥. 10¥ Uno, Y. and Kito, G.: Effect of estradiol on gene expression profile in cynomolgus macaque liver: implications for drug-metabolizing enzymes. Drug Metab. Dispos., 39: 2003®2007 ¤2011¥. 11¥ Uno, Y., Fujino, H., Kito, G., Kamataki, T. and Nagata, R.: CYP2C76, a novel cytochrome P450 in cynomolgus monkey, is a major CYP2C in liver, metabolizing tolbutamide and testosterone. Mol. Pharmacol., 70: 477®486 ¤2006¥. 12¥ Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M., Davis, A. P., Dolinski, K., Dwight, S. S., Eppig, J. T., Harris, M. A., Hill, D. P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J. C., Richardson, J. E., Ringwald, M., Rubin, G. M. and Sherlock, G.: Gene ontology: tool for the unification of biology. Nat. Genet., 25: 25®29 ¤2000¥. 13¥ Hayes, J. D., Flanagan, J. U. and Jowsey, I. R.: Glutathione transferases. Annu. Rev. Pharmacol. Toxicol., 45: 51®88 ¤2005¥. 14¥ Satoh, T., Fujita, K. I., Munakata, H., Itoh, S., Nakamura, K., Kamataki, T., Itoh, S. and Yoshizawa, I.: Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase. Anal. Biochem., 286: 179®186 ¤2000¥. 15¥ Shou, M., Korzekwa, K. R., Brooks, E. N., Krausz, K. W., Gonzalez, F. J. and Gelboin, H. V.: Role of human hepatic cytochrome P450 1A2 and 3A4 in the metabolic activation of estrone. Carcinogenesis, 18: 207®214 ¤1997¥.
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16¥ Yamazaki, H., Shaw, P. M., Guengerich, F. P. and Shimada, T.: Roles of cytochromes P450 1A2 and 3A4 in the oxidation of estradiol and estrone in human liver microsomes. Chem. Res. Toxicol., 11: 659®665 ¤1998¥. 17¥ Gao, H., Bryzgalova, G., Hedman, E., Khan, A., Efendic, S., Gustafsson, J. !. and Dahlman-Wright, K.: Long-term administration of estradiol decreases expression of hepatic lipogenic genes and improves insulin sensitivity in ob/ob mice: a possible mechanism is through direct regulation of signal transducer and activator of transcription 3. Mol. Endocrinol., 20: 1287®1299 ¤2006¥. 18¥ Stedman, C., Robertson, G., Coulter, S. and Liddle, C.: Feedforward regulation of bile acid detoxification by CYP3A4: studies in humanized transgenic mice. J. Biol. Chem., 279: 11336®11343 ¤2004¥. 19¥ Uehara, S., Murayama, N., Yamazaki, H. and Uno, Y.: A novel CYP2A26 identified in cynomolgus monkey liver metabolizes
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coumarin. Xenobiotica, 40: 621®629 ¤2010¥. 20¥ Ebeling, M., Küng, E., See, A., Broger, C., Steiner, G., Berrera, M., Heckel, T., Iniguez, L., Albert, T., Schmucki, R., Biller, H., Singer, T. and Certa, U.: Genome-based analysis of the nonhuman primate Macaca fascicularis as a model for drug safety assessment. Genome Res., 21: 1746®1756 ¤2011¥. 21¥ Osada, N., Hashimoto, K., Kameoka, Y., Hirata, M., Tanuma, R., Uno, Y., Inoue, I., Hida, M., Suzuki, Y., Sugano, S., Terao, K., Kusuda, J. and Takahashi, I.: Large-scale analysis of Macaca fascicularis transcripts and inference of genetic divergence between M. fascicularis and M. mulatta. BMC Genomics, 9: 90 ¤2008¥. 22¥ Uno, Y., Suzuki, Y., Wakaguri, H., Sakamoto, Y., Sano, H., Osada, N., Hashimoto, K., Sugano, S. and Inoue, I.: Expressed sequence tags from cynomolgus monkey ¤Macaca fascicularis¥ liver: A systematic identification of drug-metabolizing enzymes. FEBS Lett., 582: 351®358 ¤2008¥.
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
Note Expression Profile of Early Estradiol-responsive Genes in Cynomolgus Macaque Liver: Implications for Drug-metabolizing Enzymes Ryota I SE 1 , Go K ITO 2 and Yasuhiro U NO 2,3, * 1
Drug Safety Research Center, Shin Nippon Biomedical Laboratories, Ltd., Kagoshima, Japan 2 Laboratory of Translational Research, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 3 Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., Kainan, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Estrogen plays important roles in estrogen-responsive tissues, such as mammary glands, ovaries, and the uterus. In the liver, the major drug metabolizing organ, estrogen is known to regulate expression of some drug-metabolizing enzymes. Due to the lack of information on the role of estrogen in hepatic gene expression in primate species, we previously investigated the late response of hepatic gene expression to estradiol in cynomolgus macaques. To understand the early response of hepatic gene expression to estradiol, in this study, microarray analysis was conducted using cynomolgus macaque liver samples collected at 1 h and 5 h after estradiol injection. Comparison of expression profiles in estradiol and solvent (control)-treated ovariectomized cynomolgus macaques revealed 27 differentially expressed genes (>2.0-fold), including 18 at 1 h and 9 at 5 h after estradiol injection. As indicated by Gene Ontology analysis, these genes were related to oxidoreductase activity and transferase activity, partly representing important aspects of drug-metabolizing enzymes. Further analysis by quantitative polymerase chain reaction revealed that estradiol down-regulated CYP2A24, CYP2C76, and CYP2E1 (>2.0-fold) at 1 h and up-regulated GSTM5 (>2.0-fold) at 5 h after estradiol injection. These results suggest that the short-term estradiol treatment influenced expression of hepatic genes, including drug-metabolizing enzyme genes, in cynomolgus macaque liver. Keywords: cynomolgus macaque; cytochrome P450; liver; gene expression; metabolic activity
In the liver, estrogen plays roles in the regulation of lipid metabolism, bile acid excretion, and blood coagulation.6®8¥ Estrogen also regulates drug metabolism by affecting expression of drug-metabolizing enzyme genes in rodents9¥. Similarly, in the cynomolgus macaque, a primate species widely used in drug metabolism studies due to its evolutionary closeness to humans, we previously identified several estradiol-responsive genes for drug-metabolizing enzymes in cynomolgus macaque liver after a 4-day estradiol treatment, including CYP3A4, CYP4F12, and GSTM5.10¥ However, the early response of estradiol to hepatic genes remained to be investigated in cynomolgus macaques. In this study, therefore, expression profiles of hepatic genes were analyzed in ovariectomized cynomolgus macaques by microarray analysis at 1 h and 5 h after estradiol
Introduction Estrogen, including 17Ç-estradiol, estriol, and estrone, plays an important role in the biological events in many organs such as mammary glands, ovaries, and the uterus. Most estrogenic chemicals are biological response modulators mediated by direct interaction with estrogen receptors.1¥ Estrogen receptors form a transcriptional complex by recruiting coactivators such as CREB-binding protein, RNA polymerase II, steroid receptor coactivator 1, and TATA element binding protein, which in turn bind to estrogen responsive elements in the upstream region of the target genes ¤late estrogen-responsive genes¥.2®4¥ In addition, rapid non-genomic responses can be mediated by membrane estrogen receptors, stimulating signal transduction.5¥
Received December 5, 2011; Accepted February 3, 2012 J-STAGE Advance Published Date: February 21, 2012, doi:10.2133/dmpk.DMPK-11-NT-147 *To whom correspondence should be addressed: Yasuhiro UNO, D.V.M., Ph.D., Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., 16-1 Minami Akasaka, Kainan 642-0017, Japan. Tel. +81-73-483-8881, Fax. +81-73-483-7377, E-mail: uno-yasuhiro@snbl. co.jp 451
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injection. The estradiol-responsive genes identified were classified by Gene Ontology ¤GO¥ analysis. Further analysis by quantitative polymerase chain reaction ¤qPCR¥ was conducted for drug-metabolizing enzyme genes identified to be estradiol-responsive in addition to 11 cytochrome P450 ¤P450¥ genes relevant to drug metabolism, including CYP2A23, CYP2A24, CYP2B6, CYP2C20, CYP2C43, CYP2C75, CYP2C76, CYP2D17, CYP2E1, CYP3A4, and CYP3A5. Materials and Methods Animals, treatments, tissues, and RNA extraction: Twelve female cynomolgus macaques ¤from Indochina, 14®16 years of age, 3®6 kg¥ were ovariectomized according to the standard procedure established at Shin Nippon Biomedical Laboratories, Ltd. ¤Kagoshima, Japan¥. Treatment was carried out at least 30 days after the ovariectomy, using subcutaneous injections of estradiol benzoate ¤50 µg/ kg body weight, 8 animals¥ or sesame oil ¤4 animals as control¥, accompanied by injection of 17Ç-estradiol ¤5 µg/kg body weight, i.v.¥ for a rapid increase in blood estrogen levels, as described previously.10¥ Liver samples were collected from 4 treated and 2 control animals at 1 h or 5 h, and flash-frozen in liquid nitrogen. The study was reviewed and approved by the institutional ethics committee. Total RNA was extracted from these 12 liver samples and treated with DNase I as described previously.11¥ The extracted RNAs were subsequently purified using RNeasy Mini Kit ¤Qiagen, Valencia, CA¥, according to the manufacturerös protocols. Microarray analysis: Total RNA from 8 liver samples ¤2 treated and 2 control animals at 1 h and 5 h¥ were used for microarray analysis, which was performed using the Human Genome U130 GeneChip set ¤Affymetrix, Santa Clara, CA¥ by Bio Matrix Research, Inc. ¤Nagareyama, Japan¥ in accordance with the procedures established by Affymetrix. These include verification of RNA quality, labeling, hybridization with biotin-labeled cRNAs ¤15 µg¥, scanning of the arrays, and data analysis. GeneChip Operating Software ¤GCOS; Affymetrix¥ was used for image processing and data acquisition. Differentially expressed genes were selected at a threshold of 1.5-fold difference between estradiol-treated and control animals ¤p g 0.05¥. Bioinfomatics: Estradiol-responsive genes were subjected to GO classification, to describe the roles of the genes identified using precisely defined GO terms.12¥ Overrepresented biological processes were determined using DAVID 2.0 ¤http://david.abcc.ncifcrf.gov/¥ according to the protocol. The analysis included 250 up- and 111 downregulated genes ¤h1.5-fold, p g 0.05¥ at 1 h and 5 h after estradiol injection, respectively. Significantly overrepresented Biological process and Molecular function categories were selected ¤p g 0.05¥. Quantitative PCR: Real-time reverse transcriptionPCR was performed using liver total RNAs as described previously for GSTM5, CYP2A23, CYP2A24, CYP2B6,
CYP2C20, CYP2C43, CYP2C75, CYP2C76, CYP2D17, CYP2E1, CYP3A4, CYP3A5, and PXR.10¥ Relative expression levels were determined by normalizing the raw data to the 18S ribosomal RNA level based on three independent amplifications. Statistical significance for differences in gene expression was determined using a two-tailed unpaired Studentös t test. For the statistical analysis, mean values of all 4 control animals ¤1 h and 5 h¥ were compared with those of 4 estradiol-treated animals ¤1 h or 5 h¥. Values were presented as mean + S.D. from the 4 animals and were considered significant if p was g0.05. Results and Discussion To identify early estradiol-responsive genes in cynomolgus macaque liver, gene expression was measured using DNA microarrays at 1 h and 5 h after estradiol injection. Comparison of gene expression between estradiol-treated and control liver showed that 250 genes were up- and 111 genes were down-regulated by estradiol treatment ¤h1.5-fold¥ at 1 h and 5 h after estradiol injection. The genes expressed differently ¤h2.0-fold¥ between estradiol-treated and control liver included 10 and 1 up-regulated genes; and 8 and 8 down-regulated genes at 1 h and 5 h after estradiol injection, respectively ¤Table 1¥. GO analysis of the estradiol-responsive genes ¤h1.5-fold¥ with level 2 GO annotation using DAVID 2.0 revealed that 17 and 6 functional categories were enriched for Biological process and Molecular function ¤p g 0.05¥, respectively, at 1 h after injection ¤Table 2¥. At 5 h after injection, 5 and 4 functional categories were enriched for Biological process and Molecular function ¤p g 0.05¥, respectively ¤Table 2¥. The Biological process categories included various metabolic processes and oxidation reduction, while the Molecular function categories included oxidoreductase activity and transferase activity, which included drug-metabolizing enzyme genes. Microarray data showed that expression of drugmetabolizing enzyme genes was also changed ¤h1.5-fold¥ by estradiol treatment, including GSTM5, CYP2B6, and CYP3A4. qPCR analysis showed that GSTM5 was up-regulated at 1 h ¤2.2-fold¥ and 5 h ¤3.9-fold, p g 0.05¥ ¤Fig. 1¥, but differential expression at 1 h did not reach statistical significance. qPCR analysis also showed up-regulation of CYP2B6 at 1 h ¤1.5-fold¥ and 5 h ¤2.9-fold¥ and downregulation of CYP3A4 at 5 h ¤2.5-fold¥, but none of these differential expressions reached statistical significance. Further qPCR analysis of P450 genes relevant to drug metabolism ¤CYP2A23, CYP2A24, CYP2C20, CYP2C43, CYP2C75, CYP2C76, CYP2D17, CYP2E1, and CYP3A5¥ and PXR ¤an important transcriptional regulator of P450 genes¥ revealed that CYP2A24 ¤5 h¥, CYP2C20 ¤5 h¥, CYP2C75 ¤1 h, 5 h¥, CYP2E1 ¤5 h¥, CYP4A11 ¤1 h, 5 h¥, and PXR ¤1 h¥ were downregulated ¤h1.5-fold¥, while CYP3A5 ¤5 h¥ was up-regulated ¤h1.5-fold¥, but none of these differential expressions reached statistical significance. In contrast, there was a sig-
Copyright © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)
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Early Estradiol-responsive Genes in Cynomolgus Macaque Liver Table 1. Top five estradiol-responsive genes identified in cynomolgus macaque liver by microarray analysis Gene symbol
Fold change
Description
1h ASCL1 CYP2B6
7.2 3.1
DGAT2 FETUB LECT2 SORD ACE2
2.5 2.5 2.5 2.5 2.4
ENPP2
2.4
TF GSTM5 PLK2 HSDL2 PITPN KCNT2 ABCD3 CRP TPD52 VMP1
2.3 2.1 %2.2 %2.3 %2.3 %2.2 %2.3 %2.3 %2.5 %2.6
Achaete-scute complex homolog 1 ¤Drosophila¥ Cytochrome P450, family 2, subfamily B, polypeptide 6 Diacylglycerol O-acyltransferase 2 Fetuin B Leukocyte cell-derived chemotaxin 2 Sorbitol dehydrogenase Angiotensin I converting enzyme ¤peptidyl-dipeptidase A¥ 2 Ectonucleotide pyrophosphatase/phosphodiesterase 2 ¤autotaxin¥ Transferrin Glutathione S-transferase M5 Polo-like kinase 2 Hydroxysteroid dehydrogenase like 2 Phosphotidylinositol transfer protein Potassium channel, subfamily T, member 2 ATP-binding cassette, sub-family D ¤ALD¥, member 3 C-reactive protein, pentraxin-related Tumor protein D52 Vacuole membrane protein 1
5h ASCL1 ZBTB16 PPM1B INSIG1 NARS ELL2 FGL1 SEPX1 LDHB
3.5 %2.1 %2.1 %2.1 %2.2 %2.2 %2.5 %2.7 %3.2
Achaete-scute complex homolog 1 ¤Drosophila¥ zinc finger and BTB domain containing 16 Protein phosphatase, Mg2¦/Mn2¦ dependent, 1B Insulin induced gene 1 Asparaginyl-tRNA synthetase Elongation factor, RNA polymerase II, 2 Fibrinogen-like 1 Selenoprotein X, 1 Lactate dehydrogenase B
nificant difference between estradiol-treated animals and control animals for down-regulation of CYP2A24 ¤3.1-fold¥, CYP2C76 ¤2.2-fold¥, and CYP2E1 ¤3.1-fold¥ at 1 h ¤Fig. 1¥. These results suggest that hepatic genes, including drugmetabolizing enzyme genes, are regulated in cynomolgus macaque liver by estradiol. GSTM5 belongs to the glutathione transferase ¤GST¥ family, essential in conjugation reactions for detoxification of endogenous and exogenous substrates.13¥ In a previous study, cynomolgus GSTM5 was also up-regulated after a 4-day estradiol treatment,10¥ similar to up-regulation of GSTM5 at 5 h after estradiol injection in this study ¤Fig. 1¥. The late response of estrogen-responsive genes is largely mediated by estrogen receptors, whereas the early response of estrogen-responsive genes is mediated through nongenomic actions of estrogen, including mobilization of intracellular calcium, enhancement of adenylate cyclase activity and cAMP production, and activation of MAPK and
Table 2. Biological GO categories of estradiol-responsive genes 1h Biological process catabolic process primary metabolic process macromolecule localization oxidation reduction response to chemical stimulus vesicle-mediated transport cellular metabolic process establishment of protein localization alcohol metabolic process establishment of localization negative regulation of developmental process regulation of biological quality membrane organization transport negative regulation of cellular process establishment of localization in cell cellular localization Molecular function cofactor binding oxidoreductase activity carbohydrate binding transferase activity pattern binding enzyme inhibitor activity
5h cellular metabolic process primary metabolic process cellular response to stimulus alcohol metabolic process response to stress
selenium binding transcription repressor activity carboxylic acid binding cofactor binding
Functional categories of up- and down-regulated genes were identified with level 2 GO terms using DAVID 2.0 ¤p g 0.05¥ as described in Materials and Methods.
Fig. 1. qPCR analysis of estradiol-responsive genes Expression of GSTM5, CYP2A24, CYP2C76, and CYP2E1 was measured by qPCR analysis using liver total RNAs from estradiol-treated (E2) and control animals as described in Materials and Methods. Values are presented as mean + S.D. of the four animals. The expression level of each gene in control animals was arbitrarily adjusted to 1, and all other expression levels were adjusted accordingly. *p g 0.05.
phosphoinositol 3-kinase signaling pathways.5¥ It is of great interest to investigate mechanisms of estrogen to activate GSTM5 in short- and long-term estradiol treatment. In this study, CYP2A24, CYP2C76, and CYP2E1 were identified as estradiol-responsive genes in cynomolgus macaque liver. Similarly, CYP3A4 was up-regulated in cynomolgus macaque liver after a 4-day estradiol treatment.10¥ CYP2A24, CYP2C76, CYP2E1, and CYP3A4
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belong to the P450 family, essential for the metabolism of various drugs, steroids, fatty acids, and environmental pollutants. Although the involvement of these cynomolgus enzymes in estradiol metabolism is not known, human P450s highly identical to these cynomolgus P450s, such as CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, are involved in metabolism of estradiol.14®16¥ Therefore, these P450s, together with GSTM5, may facilitate excretion of estradiol by promoting the oxidation and conjugation of estradiol. In humans, CYP3A4 is involved in not only drug metabolism, but also in hydroxylation of bile acids and steroid hormones. Previous studies showed by microarray analysis that estradiol enhanced genes relevant to lipid metabolism in rodent and cynomolgus macaque liver.10,17¥ In rodents, treatment with 17Æ-ethinyl estradiol, a synthetic estrogen, decreased bile flow and bile acid synthesis, and mildly increased bile acid in serum.7¥ In the human CYP3Atransgenic mice, expression of human CYP3A was induced by bile acid accumulation, indicating that CYP3A4 plays a role in preventing cholestasis by metabolizing bile acid.18¥ Therefore, estradiol might enhance expression of P450 genes to eliminate extra cholesterol from the body. In this study, CYP2A24, CYP2C76, and CYP2E1 were identified as estradiol-responsive genes by qPCR analysis, but differential expression was not detected using microarrays. One of the reasons might be the use of human microarrays to detect expression of cynomolgus macaque genes. CYP2C76 is not orthologous to any human P450,11¥ and thus the probe of this gene would not be contained in the human microarrays used. Cynomolgus CYP2A24, CYP2A23, and CYP2A26, predominantly expressed in liver, are highly identical to human CYP2A6 and CYP2A13,19¥ and thus human CYP2A probes might not detect expression of cynomolgus CYP2A genes in a gene-specific manner. Genome information such as genome sequences and expressed sequence tags are gradually being accumulated in cynomolgus macaques,20®22¥ which could be helpful in the manufacture of cynomolgus macaque microarrays. Such genome tools would enhance the advancement of biomedical studies in cynomolgus macaques. In conclusion, microarray analysis using cynomolgus macaque liver samples collected at 1 h and 5 h after estradiol injection identified numerous estradiol-responsive genes, including drug-metabolizing enzyme gene GSTM5. Further analysis by qPCR revealed that drug-metabolizing enzyme genes CYP2A24, CYP2C76, and CYP2E1 were also estradiolresponsive. The results presented in this paper are useful for understanding liver physiology, especially drug metabolism, in cynomolgus macaques. Acknowledgments: The authors greatly thank Dr. Ryoichi Nagata, Dr. Koichiro Fukuzaki, and Mr. Masahiro Utoh for their support of this work, and Mr. Patrick Gray for reviewing the manuscript.
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