臺灣博碩士論文加值系統

摘 要
土震素A ( territrem A; TRA ) 為Aspergillus terreus 23-1菌株經米培養，以氯仿萃取分離的震顫性黴菌毒素之一。本實驗室在先前的研究指出土震素A在成熟大鼠以phenobarbital或dexamethasone前處理，其雄性之肝臟微粒體有三種代謝物產生，即MA1、 MAX與MA2，而雌性大鼠肝臟微粒體土震素A的代謝則僅能產生MA1，而無法產生MAX及MA2，並且在化學與免疫抑制研究推論，CYP3A1/2為主要參與土震素A代謝的P450酵素。
由於土震素A早期是在台灣穀倉黴米中被篩選出的黴菌Aspergillus terreus 23-1菌株在米培養產生的毒素，故土震素A在人肝臟微粒體中的生化轉變過程是本實驗欲探討的重點，其代謝情形是否如同本實驗室先前的研究是具有性別差異？肝臟細胞色素P450 3A次家族參與性如何？因此我們採用人肝臟微粒體及以基因工程轉殖並會表現人類細胞色素P450 3A4的中國倉鼠肺臟纖維母細胞( Chinese hamster lung fibroblast ) V79衍生的V79MZh3A4細胞株，作為探討其對於土震素A代謝研究的體外生物模式。
研究結果指出 (1) 四個人肝臟微粒體主要表現testosterone 6 -hydroxylase的活性，(2) V79MZh3A4細胞株也有testosterone 6 - hydroxylase的活性，而V79MZ細胞株則偵測不到此酵素活性， (3) 由免疫染色分析結果可知人肝臟微粒體及V79MZh3A4細胞株均有CYP3A4蛋白表現，(4) 在以土震素A為受質的人肝臟微粒體代謝研究發現，不論男性或女性肝臟微粒體均能產生MA1與微量MAX，在性別上無差異性，而在活細胞V79MZh3A4中僅能產生代謝物MA1，(5) 在V79MZh3A4細胞之酵素動力學研究中，由代謝物MA1之Vmax / Km值得知其對於土震素A有很好的代謝潛力，(6) CYP3A4的化學抑制劑ketoconazole研究分析，人肝臟微粒體中testosterone 6-hydroxylase的活性會受到抑制，同時土震素A之代謝物MA1亦會受抑制。綜合以上研究結果，推測土震素A的代謝過程中，CYP3A4只參與在土震素A至MA1的代謝步驟。

Abstract
Territrem A (TRA) is a tremogenic mycotoxin isolated from the chloroform extracts of the sub-merged rice culture of Aspergillus terreus 23-1. The previous studies indicated that three metabolites, designated as MA1, MAX and MA2, were obtained when TRA was the substrate in liver microsomes of adult male Wistar rats which was pretreated with phenobarbital or dexamethasone. However, only MA1 was formed from TRA in liver microsomes of female rats. From chemical and immuno inhibition studies, we had suggested that CYP3A1/2 mainly involved in TRA metabolism.
In the present study, I investigate the territrem A metabolism in human liver microsomes and in V79 Chinese Hamster cells, designed as V79MZh3A4, in which human cytochrome P450 3A4 were expressed. The aims of this study were to elucidate the questions as following: (1) TRA metabolism in different species. (2) TRA metabolism in sex difference of human. (3) Role of CYP3A4 in TRA metabolism.
From the studies, I have obtained the following results. (1) Both human livers and V79MZh3A4 cells could express the activities of testosterone 6hydroxylase. Otherwise, the testosterone 6hydroxylase was not being determined in V79MZ cells. (2) Inmmunobloting assay showed that CYP3A4 protein was detected in human liver microsomes and V79MZh3A4 cells, but not in V79MZ cells. (3) When TRA was used as the substrate, MA1 and trace amount MAX were formed in male and female human liver microsomes. (4) Furthermore the results obtained was using live V79MZh3A4 cells showed only MA1 was found. When MA1 was used as the substrate in the same experimental condition, no metabolites were found. (5) In enzyme kinetics study, I found that V79MZh3A4 cells could metabolize TRA judged from the value of Vmax / Km of MA1 calculatedly activity. (6) The specific cytochrome P450 inhibitor of CYP3A4, such as ketoconazole, inhibited the activities of testosterone 6 - hydroxylase as well as MA1 in human liver microsomes was increased of dose of inhibition. Therefore, it is suggested that CYP3A4 has a major role in metabolic pathway from TRA to MA 1.

目 錄
中文摘要………………………………………………… I
英文摘要…………………………………………………III
縮寫表………………………………………………………V
第一章 緒言……………………………………………… 1
第二章 實驗材料與方法…………………………………10
第三章 實驗結果…………………………………………29
第四章 討論………………………………………………43
第五章 參考文獻…………………………………………53
附表……………………………………………………… 63
附圖……………………………………………………… 67

第五章 參考文獻
Alexander, W. W., Dene E. R., Paul, E. T., and Wayne, L. (1983) Regio-
and stereoselective metabolism of two C19 steroids by five highly
purified and reconstituted rat hepatic cytochrome P450 isozymes. J.
Biol.Chem. 258: 8839-8847.
Alvares, A. P., and Mannering, G.J. (1970) Two-substrate kinetics of
drug-metabolizing enzyme systems of hepatic microsomes. Mol.
Pharmacol. 6: 206-212.
Aoyama, T., Yamano, S., Guzelian, P. S., Gelboin, H. V., and Gonzalez,
F.J. (1990) Five of 12 forms of vaccinia virus-expressed human hepatic
cytochrome p450 metabolically activate aflatoxin B1. Proc. Natl. Acad.
Sci. U.S.A. 87: 4790-4793.
Axelrod, J. (1995) The enzymatic demethylation of ephedrine. J.
Pharmacol. Exp. Ther. 114: 430-438.
Beaune, P.H., Umbenhauer, D.R., Bork, R.W., Lloyd, R.S., and
Guengerich FP. (1986) Isolation and sequence determination of a
cDNA clone related to human cytochrome P-450 nifedipine oxidase.
Proc. Natl. Acad. Sci. U.S.A. 83(21): 8064-8068.
Brodie, B., Axelrod, J., Cooper, J. R., Gaudette, L., LaDu, B. N., Mitoma,
C., and Udefriend, S. (1955) Detoxication of drugs and other foreign
compounds by liver microsomes. Science 121: 603-604.
Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., and Mitchell,
J.B. (1987) Evaluation of a tetrazolium-based semiautomated
colorimetric assay: assessment of chemosensitivity testing. Cancer
Res. 47, 936-942.
Combalbert, J., Fabre, I., Fabre, G., Dalet, I., Derancourt, J., Cano, J.P.,
and Maurel, P. (1989) Metabolism of cyclosporin A. IV. Purification
and identification of the rifampicin-inducible human liver cytochrome
P-450 (cyclosporin A oxidase) as a product of P450IIIA gene
subfamily. Drug Metabolism and Disposition 17(2): 197-207.
Doehmer, J. (1993) V79 Chinese hamster cells genetically engineered for
cytochrome P450 and their use in mutagenicity and metabolism
studies. Toxicology 82(1-3): 105-118.
Doehmer, J., Seidel, A., Oesch, F., and Glatt, H.R. (1990) Genetically
engineered V79 Chinese hamster cells metabolically activate the
cytostatic drugs cyclophosphamide and ifosfamide. Environmental
Health Perspectives. 88:63-65.
Engst, W., Landsiedel, R., Hermersdorfer, H., Doehmer, J., and Glatt, H.
(1999) Benzylic hydroxylation of 1-methylpyrene and 1-ethylpyrene
by human and rat cytochromes P450 individually expressed in V79
Chinese hamster cells. Carcinogenesis. 20(9): 1777-1785.
Estabrook, R. W. (1996) The remarkable P450s: a historical overview of
these versatile hemeprotein catalysts. FASEB J. 10: 202-205.
Gallagher, E.P., Kunze, K.L., Stapleton, P.L., and Eaton, D.L. (1996) The
kinetics of aflatoxin B1 oxidation by human cDNA-expressed and
human liver microsomal cytochromes P450 1A2 and 3A4. Toxicology
& Applied Pharmacology. 141(2): 595-606.
Grant, M.H., Burke, M.D., Hawksworth, G.M., Duthie, S.J., Engeset, J.
and Petrie, J.C. (1987) Human adult hepatocytes in primary monolayer
culture. Maintenance of mixed function oxidase and conjugation
pathways of drug metabolism. Biochemical Pharmacology 36(14):
2311-2316.
Guillouzo, A., Beaune, P., Gascoin, M.N., Begue, J.M., Campion, J.P.,
Guengerich, F.P., and Guguen-Guillouzo, C. (1985) Maintenance of
cytochrome P-450 in cultured adult human hepatocytes. Biochemical
Pharmacology 34(16): 2991-2995.
Hunt, C.M., Westerkam, W.R., and Stave, G.M. (1992) Effect of age and
gender on the activity of human hepatic CYP3A. Biochemical
Pharmacology 44(2): 275-283.
Kariya K., Okumura H., Lee E. and Hirata M. (1992) Stereoselective
conjugation of a uricosuric diuretic with glutathione by glutathione
transferase 3-3. Xenobiotica 22(3): 319-323.
Kato, R., and Yamazoe, Y. (1992) Sex-specific cytochrome P450 as a
cause of sex- and species-related differences in drug toxicity. Toxicol.
Lett. 64/65: 661-667.
Laemmil, U.K. (1970) Cleavage of structural proteins during the
assembly of head bacteriophage T4. Nature 227, 680-685.
Li, A.P., Kaminski, D.L., and Rasmussen, A. (1995) Substrates of human
hepatic cytochrome P450 3A4. Toxicology. 104(1-3): 1-8.
Lin, R. L. (1997) Study on sexual difference of biotransformation of
territrem A by liver microsome from Wistar rat. Master dissertation,
Institute of Toxicology. National Taiwan University.
Ling, K. H., Yang, C. K., and Peng, F. T. (1979) Territrems, tremorgenic
mycotoxins of Aspergillus Terreus. Appl. Enviro. Microbiol. 37: 355-
357.
Ling, K. H., Yang, C. K., and Kau, C. A. (1982) Solvent system for
improved isolation and separation of territrem A and B. Appl. Enviro.
Microbiol.44: 806-863.
Ling, K. H., Liou, H. H., Fu, T. C., Tsai, M. C., and Lin, M. Y. (1986)
Mechanism of action of territrem, tremogenic mycotoxin isolated from
Aspergillus Terreus. IUPAC 6th International symposium on
mycotoxin and phycotoxin in 1985, pp.387-398.
Ling, K. H., Tsai, S. D., and Liou, H. H. (1988) Disposition of
radiolabelled territrem B in mice. J. Biomed. Lab. Sci. (ROC) 1: 28-34.
Ling, K. H., Chiou, C. M., and Tseng, Y. L. (1991) Biotransformation of
territrems by S9 fraction from rat liver. Drug Metab. Dispos. 19: 587-
595.
Lin Wu, S. W. (1999) Study on metabolism of territrem A by cytochrome
P450 from liver microsomes of Wistar Rat. Master dissertation,
Institute of Toxicology. National Taiwan University.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)
Protein measurement with the Folin phenol reagent. J. Biol. Chem.
193: 265-275.
Luch, A., Schober, W., Soballa, V.J., Raab, G., Greim, H., Jacob, J.,
Doehmer, J., and Seidel, A. (1999) Metabolic activation of
dibenzo[a,l]pyrene by human cytochrome P450 1A1 and P450 1B1
expressed in V79 Chinese hamster cells. Chemical Research in
Toxicology 12(4): 353-364.
Maenpaa, J., Pelkonen, O., Cresteil, T., and Rane, A. (1993) The role of
cytochrome P450 3A (CYP3A) isoform(s) in oxidative metabolism of
testosterone and benzphetamine in human adult and fetal liver. Journal
of Steroid Biochemistry & Molecular Biology. 44(1): 61-67.
Maron, D.M. and Ames, B.N. (1983) Revised methods for the Salmonella
mutagenicity test. Mutat. Res. 113, 173-215.
Maurel, P. (1995) Use of human hepatocytes to characterize the
metabolism of drugs (lecture), in Short Course I, ISSX Meeting,
Seattle, WA, August 27-September 1.
Namkung, M.J., Yang, H.L., Hulla, J.E., and Juchau, M.R. (1988) On the
substrate specificity of cytochrome P450IIIA1. Molecular
Pharmacology 34(5): 628-637.
Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P.,
Estabrook, R. W., Feyereisen, R., Gonzalez, F. J., Coon, M. J.,
Gunsalus, I. C., Gotoh, O., Okuda, K., and Nebert, D. W. (1993) The
P450 superfamily: update on new sequences, gene mapping, accession
numbers, early trivial names of enzymes and nomenclature. DNA and
Cell Biology 12: 1-51.
Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen,
R.,Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J.,
Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) P450
superfamily: update on new sequences, gene mapping, accession
numbers and nomenclature. Pharmacogenetics 6: 1-42.
Newton, DJ., Wang, RW. and Lu, AY. (1995) Cytochrome P450
inhibitors: evaluation of specificities in the in vitrometabolism of
therapeutic agents by human liver microsomes. Drug Metabolism and
Disposition. 23(1):154-158.
Oguri, K., Yamada, H., and Yoshimura, H. (1994) Regiochemistry of
cytochrome P450 isozymes. Annual Review of Pharmacology &
Toxicology 34: 251-279.
Omura T. (1999) Forty years of cytochrome P450. Biochemical &
Biophysical Research Communications. 266(3): 690-698.
Omura, T., and Sato, R. (1964) The carbon monoxide-binding pigment of
liver microsomes. I. Evidence for its hemoprotein nature. J. Biol.
Chem. 239: 2370-2378.
Paine, A.J. (1990) The maintenance of cytochrome P-450 in rat
hepatocyte culture : some applications of liver cell cultures to the study
of drug metabolism, toxicity and the induction of the P-450 system.
Chem Biol Interact. 74(1-2): 1-31.
Phillips, A. H., and Langdon, R. G. (1962) Hepatic triphosphopyridine
nucleotide-cytochrome c reductase: isolation, characterization and
kinetic studies. J. Biol. Chem. 237: 2652-2660.
Porter TD. And Coon MJ. (1991) Cytochrome P-450. Multiplicity of
isoforms, substrates, and catalytic and regulatory mechanisms. J. Biol.
Chem 266(21): 13469-13472.
Rauschenbach, R., Gieschen, H., Husemann, M., Salomon, B.,
Hildebrand, M. (1995) Stable expression of human cytochrome P450
3A4 in V79 cells and its application for metabolic profiling of ergot
derivatives. European Journal of Pharmacology 293(3): 183-190.
Schenkman, J. B. and Griem, H. (Eds), 1993, Cytochrome P450,
Heidelberg: Springer-Verlag.
Schmalix, W.A., Maser, H., Kiefer, F., Reen, R., Wiebel, F.J., Gonzalez,
F., Seidel, A., Glatt, H., Greim, H., and Doehmer, J. (1993) Stable
expression of human cytochrome P450 1A1 cDNA in V79 Chinese
hamster cells and metabolic activation of benzo[a]pyrene. European
Journal of Pharmacology 248(3): 251-261.
Schneider, A., Schmalix, W.A., Siruguri, V., DeGroene, E.M., Horbach,
G.J., Kleingeist, B., Lang, D., Bocker, R., Belloc, C., Beaune, P.,
Greim, H., and Doehmer, J. (1996) Stable expression of human
cytochrome P450 3A4 in conjunction with human NADPH-
cytochrome P450 oxidoreductase in V79 Chinese hamster cells.
Archives of Biochemistry & Biophysics. 332(2): 295-304.
Sesardic, D., Boobis, AR., Murray, BP., Murray, S., Segura, J., De La
Torre, R. and Davies, DS. (1990) Furafylline is a potent and selective
inhibitor of cytochrome P450IA2 in man. British Journal of Clinical
Pharmacology. 29(6): 651-663.
Shimada, T., and Guengerich, F.P. (1989) Evidence for cytochrome P-
450NF, the nifedipine oxidase, being the principal enzyme involved in
the bioactivation of aflatoxins in human liver. Proc. Natl. Acad. Sci.
U.S.A 86(2): 462-465.
Shimada, M., Nagata, K., Murayama, N., Yamazoe, Y., and Kato, R.
(1989) Role of growth hormone in modulating the constitutive and
phenobarbital-induced levels of two P-4506β(testosterone 6β-
hydroxylase) mRNAs in rat livers. J. Biochem. 106: 1030-1034.
Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F.P.
(1994) Interindividual variations in human liver cytochrome P-450
enzymes involved in the oxidation of drugs, carcinogens and toxic
chemicals: studies with liver microsomes of 30 Japanese and 30
Caucasians. Journal of Pharmacology & Experimental Therapeutics.
270(1): 414-423.
Soucek, P., and Gut, I. (1992) Cytochromes P-450 in rats: structures,
functions, properties and relevant human forms. Xenobiotica. 22(1):
83-103.
Szklarz, G.D, and Halpert, J.R. (1997) Molecular modeling of
cytochrome P450 3A4. J Comput Aided Mol Des 11(3): 265-272
Thummel, K.E., Lee, C.A., Kunze, K.L., Nelson, S.D., and Slattery, J.T.,
(1993) Oxidation of acetaminophen to N-acetyl-p-aminobenzoquinone
imine by human CYP3A4. Biochemical Pharmacology. 45(8): 1563-
1569.
Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer
of protein from polyacrylamide gels to nitrocellulose sheets: procedure
and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354.
Trivia, J.M., Libersa, C., Belloc, C. and Lhermitte, M. (1993)
Amiodarone N-deethylation in human liver microsomes：Involvement
of cytochrome P450 3A enzymes (first report) Life Sciences 52, 91-96.
Tseng, Y. L. (1990) I. Biotransformation of territrems in rat, II.The
stability of territrems. Master dissertation, Institute of Biochemistry,
National Taiwan University.
Wang, B. L. (1995) Biotransformation of territrem A by liver microsome
of male rat. Master dissertation, Institute of Toxicology. National
Taiwan University.
Waxman, D.J., Lapenson, D.P., Aoyama, T., Gelboin, H.V., Gonzalez,
F.J., and Korzekwa, K. (1991) Steroid hormone hydroxylase
specificities of eleven cDNA-expressed human cytochrome P450s.
Archives of Biochemistry & Biophysics. 290(1): 160-166.
Waxman, D.J., Lapenson, D.P., Morrissey, J.J., Park, S.S., Gelboin, H.V.,
Doehmer, J., and Oesch, F. (1989) Androgen hydroxylation catalysed
by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450
PB-4 (IIB1). Biochemical Journal 260(1): 81-85.
Wrighton, S.A., and Stevens, J.C. (1992) The human hepatic cytochromes
P450 involved in drug metabolism. Critical Reviews in Toxicology
22(1):1-21.
Yun, C.H., Shimada, T., and Guengerich, F.P. (1992) Roles of human liver
cytochrome P4502C and 3A enzymes in the 3-hydroxylation of
benzo(a)pyrene. Cancer Research. 52(7): 1868-1874.