藥物-藥物或藥物-食品交互作用常常會引起藥物不良反應，而藥物不良反應常見的原因是個體藥物代謝酶 ( drug-metabolizing enzymes, DMEs) 的表現差異不同而引起的。肝臟是人體最大的藥物代謝器官，其中細胞色素P450 2C9 (Cytochrome P450 2C9, CYP2C9) 為重要的代謝酶，主要受到細胞核接受體 (nuclear receptors, NRs) ，例如： 類酯醇X受體 (pregnane X receptor, PXR) 和構雄甾烷受體 (constitutive androstane receptor, CAR) 所調控。異硫氰酸烯丙酯 (allyl isothiocyanates, AITC) 大量存在於許多十字花科天然食品或保健食品中，常常用來抗癌或是抗菌，到目前為止很少研究去探討 AITC 跟細胞色素 P450 之間的相關性。在本研究中，我們主要目的是探討AITC是否藉由細胞核接受體來影響CYP2C9。利用西方墨點法及 Real Time-PCR 顯示AITC 抑制CYP2C9 蛋白質和mRNA的表現量；在reporter assay 顯示AITC破壞了核內接受體與共同活化因子之間的共同調控；經由 DNA affinity precipitation assay (簡稱DAPA) 顯示 AITC 有抑制核內接受體及共同活化因子結合在 CYP2C9 啟動子上。以上結果顯示，AITC 影響到 CYP2C9 的表現，可能會造成藥物在體內的血中濃度產生變化，對藥物安全性具有重要意義。

Drug - drug or drug - food interactions often lead to adverse drug reactions. Expression of drug-metabolizing enzymes (DMEs) is a common cause of adverse drug effects in some drugs. The liver is the largest organ in human, it contains multi-biological functions. Cytochrome P450 2C9 is predominantly regulated by nuclear receptors (NR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Allyl isothiocyanate (AITC) is commonly found in cruciferous vegetables, with various biological activities, for example, anti-cancer and anti-bacterial. Until now, none of the report, study on the correlation between AITC with the cytochrome P450. In this study, we aimed to investigate the effect of AITC on the modulation of CYP2C9 through NRs. Using western blot and real time-PCR, we showed that AITC suppressed CYP2C9 protein and mRNA expression levels. AITC disrupted the coregulation effects of PXR/CAR with coactivation thus inhibited CYP2C9 reporter activities. By using DNA affinity precipitation assay, AITC inhibit nuclear receptors and related protein binding in CYP2C9 promoter. AITC will affect the CYP2C9 expression, the blood concentration of certain drugs may changed. Our results indicated that modification of CYP2C9 by consumption of AITC could have important implications on drug safety.

中文摘要 1
Abstract 2
表目錄 6
圖目錄 7
縮寫檢表 8
第一章 緒論 9
前言： 9
一、 細胞色素 P450 (Cytochrome P450, CYP450s) 9
二、 細胞色素 P450 2C9 (Cytochrome P450 2C9, CYP2C9) 10
三、 核內接受體 (Nuclear receptor, NR) 12
四、 類酯醇 X 受體 (Pregnane X receptor, PXR) 12
五、 構雄甾烷受體 (Constitutive androstane receptor, CAR) 14
六、 維甲酸 X 受體 (Retinoid X receptors α, RXRα) 15
七、 類固醇受體輔助因子 1 (Steroid receptor coactivator-1, SRC-1) 15
八、 肝細胞核因子 4 (Hepatocyte nuclear factor 4α, HNF4α) 16
九、 過氧化物酶體增殖物激活受體-γ 共激活因子 1 (Peroxisome
proliferator-activated receptor-gamma coactivator 1 alpha , PGC-1α) 16
十、 異硫氰酸烯丙酯 (Allyl isothiocyanates, AITC) 17
研究動機 20
第二章 研究方法 21
第一節 研究設計 21
第二節 實驗方法 22
細胞培養 (Cell culture) 22
細胞存活率試驗 (Assessment of cell viability) 22
西方墨點法 (Western blotting) 23
聚合酶連鎖反應 (Real Time polymerase chain reaction) 23
CYP2C9 報告基因分析 (CYP2C9 reporter assay) 24
DNA affinity precipitation assay (簡稱 DAPA) 24
統計分析 24
第三章 結果25
第四章 討論30
第五章 參考文獻33

1.Ansell, J., M. McDonough, Y. Zhao, J. S. Harmatz and D. J. Greenblatt (2009). "The absence of an interaction between warfarin and cranberry juice: a randomized, double-blind trial." J Clin Pharmacol 49(7): 824-830.
2.Azmi, A. S., G. W. Bao, J. Gao, R. M. Mohammad and F. H. Sarkar (2013). "Network insights into the genes regulated by hepatocyte nuclear factor 4 in response to drug induced perturbations: a review." Curr Drug Discov Technol 10(2): 147-154.
3.Baes, M., T. Gulick, H. S. Choi, M. G. Martinoli, D. Simha and D. D. Moore (1994). "A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements." Mol Cell Biol 14(3): 1544-1552.
4.Bollard, M., S. Stribbling, S. Mitchell and J. Caldwell (1997). "The disposition of allyl isothiocyanate in the rat and mouse." Food Chem Toxicol 35(10-11): 933-943.
5.Buler, M., S. M. Aatsinki, R. Skoumal and J. Hakkola (2011). "Energy sensing factors PGC-1alpha and SIRT1 modulate PXR expression and function." Biochem Pharmacol 82(12): 2008-2015.
6.Bushra, R., N. Aslam and A. Y. Khan (2011). "Food-drug interactions." Oman Med J 26(2): 77-83.
7.Chan, W. K., L. T. Nguyen, V. P. Miller and R. Z. Harris (1998). "Mechanism-based inactivation of human cytochrome P450 3A4 by grapefruit juice and red wine." Life Sci 62(10): Pl135-142.
8.Chang, T. K. (2009). "Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) by herbal medicines." Aaps j 11(3): 590-601.
9.Chen, Y., S. S. Ferguson, M. Negishi and J. A. Goldstein (2004). "Induction of human CYP2C9 by rifampicin, hyperforin, and phenobarbital is mediated by the pregnane X receptor." J Pharmacol Exp Ther 308(2): 495-501.
10.Chen, Y. and J. A. Goldstein (2009). "The transcriptional regulation of the human CYP2C genes." Curr Drug Metab 10(6): 567-578.
11.Chen, Y., G. Kissling, M. Negishi and J. A. Goldstein (2005). "The nuclear receptors constitutive androstane receptor and pregnane X receptor cross-talk with hepatic nuclear factor 4alpha to synergistically activate the human CYP2C9 promoter." J Pharmacol Exp Ther 314(3): 1125-1133.
12.di Masi, A., E. De Marinis, P. Ascenzi and M. Marino (2009). "Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects." Mol Aspects Med 30(5): 297-343.
13.Ellard, S. and K. Colclough (2006). "Mutations in the genes encoding the transcription factors hepatocyte nuclear factor 1 alpha (HNF1A) and 4 alpha (HNF4A) in maturity-onset diabetes of the young." Hum Mutat 27(9): 854-869.
14.Ferguson, S. S., E. L. LeCluyse, M. Negishi and J. A. Goldstein (2002). "Regulation of human CYP2C9 by the constitutive androstane receptor: discovery of a new distal binding site." Mol Pharmacol 62(3): 737-746.
15.Finck, B. N. and D. P. Kelly (2006). "PGC-1 coactivators: inducible regulators of energy metabolism in health and disease." J Clin Invest 116(3): 615-622.
16.Gerbal-Chaloin, S., M. Daujat, J. M. Pascussi, L. Pichard-Garcia, M. J. Vilarem and P. Maurel (2002). "Transcriptional regulation of CYP2C9 gene. Role of glucocorticoid receptor and constitutive androstane receptor." J Biol Chem 277(1): 209-217.
17.Gerbal-Chaloin, S., J. M. Pascussi, L. Pichard-Garcia, M. Daujat, F. Waechter, J. M. Fabre, N. Carrere and P. Maurel (2001). "Induction of CYP2C genes in human hepatocytes in primary culture." Drug Metab Dispos 29(3): 242-251.
18.Germain, P., B. Staels, C. Dacquet, M. Spedding and V. Laudet (2006). "Overview of nomenclature of nuclear receptors." Pharmacol Rev 58(4): 685-704.
19.Girennavar, B., G. K. Jayaprakasha and B. S. Patil (2007). "Potent inhibition of human cytochrome P450 3A4, 2D6, and 2C9 isoenzymes by grapefruit juice and its furocoumarins." J Food Sci 72(8): C417-421.
20.Gotoh, O. (1992). "Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences." J Biol Chem 267(1): 83-90.
21.Handschin, C. and U. A. Meyer (2003). "Induction of drug metabolism: the role of nuclear receptors." Pharmacol Rev 55(4): 649-673.
22.Hwang, E. S. and H. J. Lee (2006). "Allyl isothiocyanate and its N-acetylcysteine conjugate suppress metastasis via inhibition of invasion, migration, and matrix metalloproteinase-2/-9 activities in SK-Hep 1 human hepatoma cells." Exp Biol Med (Maywood) 231(4): 421-430.
23.Jiao, D., C. T. Ho, P. Foiles and F. L. Chung (1994). "Identification and quantification of the N-acetylcysteine conjugate of allyl isothiocyanate in human urine after ingestion of mustard." Cancer Epidemiol Biomarkers Prev 3(6): 487-492.
24.Joffe, H. V., R. Xu, F. B. Johnson, J. Longtine, N. Kucher and S. Z. Goldhaber (2004). "Warfarin dosing and cytochrome P450 2C9 polymorphisms." Thromb Haemost 91(6): 1123-1128.
25.Kamiyama, Y., T. Matsubara, K. Yoshinari, K. Nagata, H. Kamimura and Y. Yamazoe (2007). "Role of human hepatocyte nuclear factor 4alpha in the expression of drug-metabolizing enzymes and transporters in human hepatocytes assessed by use of small interfering RNA." Drug Metab Pharmacokinet 22(4): 287-298.
26.Kanebratt, K. P., U. Diczfalusy, T. Backstrom, E. Sparve, E. Bredberg, Y. Bottiger, T. B. Andersson and L. Bertilsson (2008). "Cytochrome P450 induction by rifampicin in healthy subjects: determination using the Karolinska cocktail and the endogenous CYP3A4 marker 4beta-hydroxycholesterol." Clin Pharmacol Ther 84(5): 589-594.
27.Kim, J. Y., S. Lee, D. H. Kim, B. R. Kim, R. Park and B. M. Lee (2002). "Effects of flavonoids isolated from Scutellariae radix on cytochrome P-450 activities in human liver microsomes." J Toxicol Environ Health A 65(5-6): 373-381.
28.Krul, C., C. Humblot, C. Philippe, M. Vermeulen, M. van Nuenen, R. Havenaar and S. Rabot (2002). "Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model." Carcinogenesis 23(6): 1009-1016.
29.Kushad, M. M., A. F. Brown, A. C. Kurilich, J. A. Juvik, B. P. Klein, M. A. Wallig and E. H. Jeffery (1999). "Variation of glucosinolates in vegetable crops of Brassica oleracea." J Agric Food Chem 47(4): 1541-1548.
30.Kyrklund, C., J. T. Backman, M. Neuvonen and P. J. Neuvonen (2004). "Effect of rifampicin on pravastatin pharmacokinetics in healthy subjects." Br J Clin Pharmacol 57(2): 181-187.
31.Lown, K. S., D. G. Bailey, R. J. Fontana, S. K. Janardan, C. H. Adair, L. A. Fortlage, M. B. Brown, W. Guo and P. B. Watkins (1997). "Grapefruit juice increases felodipine oral availability in humans by decreasing intestinal CYP3A protein expression." J Clin Invest 99(10): 2545-2553.
32.Maglich, J. M., D. J. Parks, L. B. Moore, J. L. Collins, B. Goodwin, A. N. Billin, C. A. Stoltz, S. A. Kliewer, M. H. Lambert, T. M. Willson and J. T. Moore (2003). "Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes." J Biol Chem 278(19): 17277-17283.
33.Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon and R. M. Evans (1995). "The nuclear receptor superfamily: the second decade." Cell 83(6): 835-839.
34.Marion, M. J., O. Hantz and D. Durantel (2010). "The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies." Methods Mol Biol 640: 261-272.
35.Martinez-Jimenez, C. P., J. V. Castell, M. J. Gomez-Lechon and R. Jover (2006). "Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1." Mol Pharmacol 70(5): 1681-1692.
36.Miners, J. O. and D. J. Birkett (1998). "Cytochrome P4502C9: an enzyme of major importance in human drug metabolism." Br J Clin Pharmacol 45(6): 525-538.
37.Moore, L. B., B. Goodwin, S. A. Jones, G. B. Wisely, C. J. Serabjit-Singh, T. M. Willson, J. L. Collins and S. A. Kliewer (2000). "St. John''s wort induces hepatic drug metabolism through activation of the pregnane X receptor." Proc Natl Acad Sci U S A 97(13): 7500-7502.
38.Moore, L. B., D. J. Parks, S. A. Jones, R. K. Bledsoe, T. G. Consler, J. B. Stimmel, B. Goodwin, C. Liddle, S. G. Blanchard, T. M. Willson, J. L. Collins and S. A. Kliewer (2000). "Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands." J Biol Chem 275(20): 15122-15127.
39.Mu, Y., J. Zhang, S. Zhang, H. H. Zhou, D. Toma, S. Ren, L. Huang, M. Yaramus, A. Baum, R. Venkataramanan and W. Xie (2006). "Traditional Chinese medicines Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch) activate pregnane X receptor and increase warfarin clearance in rats." J Pharmacol Exp Ther 316(3): 1369-1377.
40.Munday, R., Y. Zhang, J. W. Fahey, H. E. Jobson, C. M. Munday, J. Li and K. K. Stephenson (2006). "Evaluation of isothiocyanates as potent inducers of carcinogen-detoxifying enzymes in the urinary bladder: critical nature of in vivo bioassay." Nutr Cancer 54(2): 223-231.
41.Musk, S. R. and I. T. Johnson (1993). "Allyl isothiocyanate is selectively toxic to transformed cells of the human colorectal tumour line HT29." Carcinogenesis 14(10): 2079-2083.
42.Nelson, D. R. (1999). "Cytochrome P450 and the individuality of species." Arch Biochem Biophys 369(1): 1-10.
43.Nielsen, P. V. and R. Rios (2000). "Inhibition of fungal growth on bread by volatile components from spices and herbs, and the possible application in active packaging, with special emphasis on mustard essential oil." Int J Food Microbiol 60(2-3): 219-229.
44.Reagan-Shaw, S., M. Nihal and N. Ahmad (2008). "Dose translation from animal to human studies revisited." Faseb j 22(3): 659-661.
45.Rhee, J., H. Ge, W. Yang, M. Fan, C. Handschin, M. Cooper, J. Lin, C. Li and B. M. Spiegelman (2006). "Partnership of PGC-1alpha and HNF4alpha in the regulation of lipoprotein metabolism." J Biol Chem 281(21): 14683-14690.
46.Schwabe, J. W. (1996). "Transcriptional control: how nuclear receptors get turned on." Curr Biol 6(4): 372-374.
47.Shepherd, G., P. Mohorn, K. Yacoub and D. W. May (2012). "Adverse drug reaction deaths reported in United States vital statistics, 1999-2006." Ann Pharmacother 46(2): 169-175.
48.Shin, I. S., H. Masuda and K. Naohide (2004). "Bactericidal activity of wasabi (Wasabia japonica) against Helicobacter pylori." Int J Food Microbiol 94(3): 255-261.
49.Shiraki, T., N. Sakai, E. Kanaya and H. Jingami (2003). "Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state." J Biol Chem 278(13): 11344-11350.
50.Srivastava, S. K., D. Xiao, K. L. Lew, P. Hershberger, D. M. Kokkinakis, C. S. Johnson, D. L. Trump and S. V. Singh (2003). "Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits growth of PC-3 human prostate cancer xenografts in vivo." Carcinogenesis 24(10): 1665-1670.
51.Stofberg, J. and J. C. Kirschman (1985). "The consumption ratio of flavouring materials: A mechanism for setting priorities for safety evaluation." Food and Chemical Toxicology 23(9): 857-860.
52.Synold, T. W., I. Dussault and B. M. Forman (2001). "The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux." Nat Med 7(5): 584-590.
53.Tang, L. and Y. Zhang (2004). "Dietary isothiocyanates inhibit the growth of human bladder carcinoma cells." J Nutr 134(8): 2004-2010.
54.Tolson, A. H. and H. Wang (2010). "Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR." Adv Drug Deliv Rev 62(13): 1238-1249.
55.Toon, S., L. K. Low, M. Gibaldi, W. F. Trager, R. A. O''Reilly, C. H. Motley and D. A. Goulart (1986). "The warfarin-sulfinpyrazone interaction: stereochemical considerations." Clin Pharmacol Ther 39(1): 15-24.
56.Tunc, S., E. Chollet, P. Chalier, L. Preziosi-Belloy and N. Gontard (2007). "Combined effect of volatile antimicrobial agents on the growth of Penicillium notatum." Int J Food Microbiol 113(3): 263-270.
57.Van Booven, D., S. Marsh, H. McLeod, M. W. Carrillo, K. Sangkuhl, T. E. Klein and R. B. Altman (2010). "Cytochrome P450 2C9-CYP2C9." Pharmacogenet Genomics 20(4): 277-281.
58.Waxman, D. J. (1999). "P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR." Arch Biochem Biophys 369(1): 11-23.
59.Wu, R. C., C. L. Smith and B. W. O''Malley (2005). "Transcriptional regulation by steroid receptor coactivator phosphorylation." Endocr Rev 26(3): 393-399.
60.Xie, W., H. Uppal, S. P. Saini, Y. Mu, J. M. Little, A. Radominska-Pandya and M. A. Zemaitis (2004). "Orphan nuclear receptor-mediated xenobiotic regulation in drug metabolism." Drug Discov Today 9(10): 442-449.
61.Yeung, E. Y., T. Sueyoshi, M. Negishi and T. K. Chang (2008). "Identification of Ginkgo biloba as a novel activator of pregnane X receptor." Drug Metab Dispos 36(11): 2270-2276.
62.Yoon, J. C., P. Puigserver, G. Chen, J. Donovan, Z. Wu, J. Rhee, G. Adelmant, J. Stafford, C. R. Kahn, D. K. Granner, C. B. Newgard and B. M. Spiegelman (2001). "Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1." Nature 413(6852): 131-138.
63.Zhang, J., P. Kuehl, E. D. Green, J. W. Touchman, P. B. Watkins, A. Daly, S. D. Hall, P. Maurel, M. Relling, C. Brimer, K. Yasuda, S. A. Wrighton, M. Hancock, R. B. Kim, S. Strom, K. Thummel, C. G. Russell, J. R. Hudson, Jr., E. G. Schuetz and M. S. Boguski (2001). "The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants." Pharmacogenetics 11(7): 555-572.
64.Zhang, J. Y., Y. Wang and C. Prakash (2006). "Xenobiotic-metabolizing enzymes in human lung." Curr Drug Metab 7(8): 939-948.
65.Zhang, Y., L. Tang and V. Gonzalez (2003). "Selected isothiocyanates rapidly induce growth inhibition of cancer cells." Mol Cancer Ther 2(10): 1045-1052.