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ก ก ก CYP2A6 Inhibitory studies of the human cytochrome P450, CYP2A6, the nicotine metabolizing enzyme by Thai herbal and fruit juices
ก
ก ก ก ( .)
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ก ก ก CYP2A6 ก ก ก ก ก ก ( .) ก ก ก Prof. Dr. Jung Ja P.Kim ก Medical College of Wisconsin Assoc. Prof. Dr. Emily E. Scott ก University of Kansus ก ก rat CPR CYP2A6 ก Prof. Dr. Joyce A. Goldstein ก National Institute of Health (NIH) ก ก ก ก ก P450 CYP3A4 CYP2C9 . . ก ก ก ก ก ก ก ก ก ก ก ก ก ก
ก ก ก ก CYP2A6 ก
Cytochrome P450 2A6 (CYP2A6) ก cytochrome P450 (CYP P450) ก heme ก ก ก ก ก ก CYP2A6 ก ก ก ก
Km Vmax กก ก ก CYP2A6 ก ก ก ก ก ก ก ก ก CYP2A6 ก ก ก ก ก CYP2A6 ก ก ก ก CYP2A6 กก ก CYP2A6 กก ก coumarin
Km 1 uM ก 25 3% v/v ก CYP2A6 50% 95% 40% v/v ก กก ก CYP2A6 mechanism based ก ก CYP3A4 CYP2C9 ก ก ก CYP2A6 ก ก ก ก
Abstract
Project Title Inhibitory studies of the human cytochrome P450, CYP2A6, the nicotine metabolizing enzyme by Thai herbal and fruit juices Investigators Songklod Sarapusit, Ph.D, Department of Biochemistry, Faculty of Science, Burapha University Pornpimol Rongnoparut, Ph.D, Department of Biochemistry, Faculty of Science, Mahidol University
Cytochrome P450 2A6 (CYP2A6) is the heme containing protein that belongs to the cytochrome P450 (CYP or P450) super family. In human, P450 enzymes play an essential role in metabolism of various endogenous and exogenous compounds such as hormones drugs and many environmental toxicants. The human CYP2A6 enzyme could specifically metabolize nicotine, an additive compound in cigarette, with high efficiency (low Km and high Vmax value). Therefore, nicotine metabolism by the liver specific human CYP2A6 enzymes has been report as an important route in nicotine detoxification. In addition, some of deficient CYP2A6 allele smokers that drastically decrease in nicotine metabolizing activity of CYP2A6 enzyme tend to smoke few cigarettes per day and are low risk of nicotine dependence compared to the normal allele smokers. This study aims to screen for Thai herbal juices or fruit juices that could specifically inhibit CYP2A6 enzymatic activity in vitro . As the results, the bacterially expressed and purified human CYP2A6 enzyme is functionally active and is specifically metabolized the coumarin substrate with the K m value approximately 1 uM. Out of 25 herbal and fruit juices tested, an in vitro experiment indicated that only 3% v/v of the Star fruit juice could inhibit CYP2A6 mediated coumarin hydroxylation by 50%. Interestingly, almost 95% of CYP2A6 mediated coumarin hydroxylation was inhibited in the presense of 40% v/v of the Star fruit juice in vitro. Further study indicated Star fruit juice could inhibit CYP2A6 activity by mechanism based inactivaition process. However, Star fruit juice could also inhibit human CYP3A4 and CYP2C9 activity in vitro . Therefore, care must be taken in cessation smokers who orally take drugs that metabolized by CYP3A4 and CYP2C9 enzyme, a highly distribute human drug metabolizing enzymes.
1
1.1 ก ก ก ก ก กก ก ก ก ก 1,100 3,000,000 6 (WHO,2008) กก . . 2550 10.86 ก 20% ก ( 2551) ก ก ก ก ก 2 6 10 ก 5 8 ก ก ก ก ก ก ก Cytochrome P450 2A6 (CYP2A6) ก Cytochrome P450 (CYP P450) ก ก ก ก ก CYP2A6
Km Vmax (Patten et al., 1996) ก CYP2A6 ก ก ก ก ก ก (Hukkanen et al., 2002; Miksys et al., 2002; Su et al., 2000) กก ก ก CYP2A6 ก CYP2A6 ก ก ก ก ก ก ก CYP2A6 ก ก ก CYP2A6 ก ก ก 8 methoxypsoralrn (8 MOP) ก CYP2A6 ก (Hukkanen et al., 2005) ก ก 8 MOP ก ก ก ก CYP2A6 ก ก ก ก ก ก ก ก ก
ก ก CYP2A6 ก ก ก กก ก CYP2A6 กก ก XL 1 blue ก ก ก ก ก
CYP2A6 ก 50% (IC 50 ) 3% v/v ก กก ก CYP2A6 ก mechanism based ก ก CYP3A4 CYP2C9 ก ก ก CYP2A6 ก ก ก ก
1.2 ก 1. ก ก ก CYP2A6 2. ก ก ก CYP2A6 1.3 ก ก ก ก CYP2A6
1.4 กก CYP2A6 ก ก P450
1.5 ก ก (Tranformation) (Competent cell) ก ก ก ก CYP2A6 XL 1 blue rat CPR C41 (DE3) SDS PAGE ก ก ก coumarin ก ก ก CYP2A6 ก ก ก ก ( 1 1) ก ก ก กก ก ก ก ก
DNA CYP2A6 DNA CYPOR
ก ก
CYP2A6 CYPOR ก
CYP2A6 CYPOR
1 1 ก ก
2 ก
2.1 ก Cytochrome P450 (CYPs P450s) ก ก ก ก ก ก ก ก ก P450 ก ก P450s ก ก ก P450 ก ก ก ก ก ก (Family) ก ก กก 40 ก (Subfamily) ก ก ก กก 55 ก P450s ก ก ก ก ก ก P450s ก ก ก ก ก ก ก P450s ก ก กก 1 ก (Bernhardt R, 2006; Mansuy D, 1998; Nelson et al, 1996; Ortiz, 2005)
2 1 ก P450s ก ก ก ก ก ( 1 6) ก ก ก ก ( 7 8) ก ( 9) Guengerich FG, 2001
ก ก ก P450s ( 2 1) P450s ก ก NADPH cytochrome P450 reductase (CPR) ก ก ก (Fe 3+ Fe 2+) ก P450s P450s ก ก ก ก ก ก ก ก ก ก ก (iron hydroxy complex) ก ก ก ก Endoplasmic reticulum ก ก Squalene monooxygenase, Heme oxygenase ก Cytochrome P450 ก ก ก ก (Bernhardt R, 2006; Emre M et al, 2007; Guengerich FG, 2001; Ortiz, 2005) ก cytochrome c ferricyanide ก ( 2 2)
2 2 ก NADPH cytochrome P450 reducatse ก www.uky.edu/Pharmacy/ps/porter/CPR.htm PLEASE specify reference
CPR ก ก ก FAD FMN ก ก ก ก ก NADPH ก FAD FMN ก ก ก CPR ก P450s ก ก ก ก CPR CPR , ก (Shen et al., 1989; Dohr et al., 2001; Murataliev et al, 1999) กก ก
CPR CPR ก 4 NH 2 terminal, FMN binding domain, FAD binding domain, NADPH binding domain (Wang et al., 1997) FMN domain ก
ก FAD binding domain NADPH binding domain ก ( 2 3)
NADPH binding domain
FAD binding domain
FMN binding domain
2 3 Cytochrome P450s ( ) P450s Oxoidoreductase ( ) ก Endoplasmic reticulum
2.2 ก กก ก 2549 กก ก ก ก 9.86 (Leartsakulpanitch et al , 2007) ก . . 2552 10.90 20.70 ก กก 15 52.7 23.04 ( 25 59 ) ก 10.40 0.54 ก ก ก ก ก ก ก ก ก ก ก ก 58.95 6.39 17,059 84.78 ก ก ก ก ก ก ก ก กก ก 30.4 4.7 ก ก 15.6 ( 10.9 ) 1.77 ก ก ก / / ก 38.61 27.42 ก 11.44 ก ก ก ก
20 ก ก ก ก ก ก ( 2551, 2552) กก ก ก ก ก ก 4,000 กก 40 ก ก ก ก ก ก ก ก ก (WHO, 2008) ก ก ก ก ก ก ก (WHO, 2008) ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก (Hukkanen et al, 2005) ก ก ก (Benowitz, 2008) ก ก ก (Oral mucosa) 80 90% ก ก ก ก ก กก ก (Beno witz, 2008; Flammang et al, 1992) ก CYP2A6 ก ก nicotine 1(5) iminium (Hukkanen et al, 2005) ก ก ก ก hydroxylation ก trans 3 hydroxycotinine, 5 hydroxycotinine nornicotine ( 2 4) ก ก ก (glucoronation) ก ก cytochrome P450 (CYP P450) ก heme ก ก ก ก ก ก ก P450 18 ก (Family) 43 ก (Sub family) (http://drnelson.utmen.edu/CytochromeP450.html ) CYP2A6 , CYP2A13 , CYP2B6 ก , CYP2D6 CYP2E1 ก ก ก (Hukkanen et al, 2002; Miksys et al, 2002; Su et al, 2000) CYP2A6
Km Vmax (Patten et al, 1996) CYP2A13 ก ก CYP2B6 CYP2D6 ก ก (Benowitz et al, 1996) CYP2E1 ก ก ก alcohol กก (McCarver et al, 1998) กก
ก ก ก CYP2A6 ก ก ก ก ก ก
2 4 ก ก ก ก (Hukkanen et al, 2005)
ก ก ก ก CYP2A6 ก coumarin 7 hydroxycoumarin ( ก coumarin 7 hydroxylase) coumarin ก CYP2A6 (Miles et al, 1990; Yamano et al, 1990) ก ก CYP2A6 chlormethiazole ก ก nitrosamine NNK [4 (methylnitrosamino) 1 (3 pyridyl) 1 butanone] NNAL [4 methylnitrosamino) 1 (3 pyridyl) 1 butanol] NNN [ N9 nitrosonornicotine] (Patten et al, 1996; Pelkonen et al, 2000;Peterson et al, 1990) ก (Hecht, 1998; Hecht, 1999a, 1999b)
กก ก ก ก (Polymorphism) CYP2A6 ก ก ก 37 ก ก (http://www.imm.ki.se/cypalleles Koudsi et al, 2009) ก ก (Kamataki et al, 2005) ก ก ก ก CYP2A6 ก ก ก ก ก กก ก ก CYP2A6 50% ก ก CYP2A6 ก (Wild type) ก ก ก CYP2A6 ก กก ก (Schoedel et al, 2004; Sellers et al 2000; Tyndale & Sellers 2002) ก ก CYP2A6 (CYP2A6*4C) ก ก ก ก (Kamatiki et al, 2005; Miyamoto et al, 1999) CYP2A6*1A ก (Wild type) CYP2A6*1B ก (Extensive metabolite) ก 32 52% 27 40% CYP2A6*4C ก CYP2A6 8 9 % (Mahavorasirikul et al, 2009; Peamkrasatam et al, 2006; Ujjin et al, 2002) ก ก ก (Kamataki et al, 2005; Yoshida et al, 2002; Kwon et al, 2001; Xi, 2009) ก ก CYP2A6 ก ก (Extensive metabolizer) ก ก ก CYP2A6 ก ก ก CYP2A6 coumarin ก ก CYP2A6 ก ก ก (Apinan et al, 2010) ก ก ก ก ก ก ก กก ก ก ก ก ก ก ก ก ก ก ( 2546;Sellers et al, 2003) ก ก ก ก ก (First line drug) Bupropion HCl Varenicline ก (Second line drug) Nortriptyline Clonidine ก ก ก ก (Carrozzi et al, 2008) ก ก ก (Gonzalez et al, 2006; Jorenhy et al, 2006) ก ก ก ก (Carrozzi et al, 2008) ก CYP2A6 ก
ก ก CYP2A6 ก ก ก (Sellers et al, 2003; Xi, 2009) ก ก ก CYP2A6 ก ก CYP2A6 ก ก ก ก ก (Sellers et al, 2003) ก ก ก 1 ก ก coumarin methoxsalen (8 methoxypsoralen; 8 MOP) ก ก ก ก (psoriasis) ก CYP2A6 coumarin (Siu and Tyndale, 2007; von Weymarn et al, 2005) ก ก ก ก ก (Damaj et al, 2007; Miyazaki et al, 2005) ก ก 8 MOP ก ก ก ก ก ก (Sellers et al, 2000, 2003a) methoxsalen ก ก ก ก ก (Sellers et al, 2000) ก 2 ก ก Cyclopropylbene Tranylcypromine (Synthetic compounds) N1 (4 fluorophenyl) cyclopropane 1 carboxamide ก CYP2A6 Coumarin (Rahnasto et al, 2008; Taavitsalnen, 2001) ก ก ก N1 (4 fluorophenyl) cyclopropane 1 caboxamide ก กก ก tranylcypromine ก ก ก ก methoxsalen (Sellers et al, 2000) ก 8 MOP Tranylcypromine ก ก (Siu and Tyndale, 2007) ก 3 ก ก 3 heteroaromatic analog 3 heteroaromatic 3 aliphatic pyridine ก ก CYP2A6 Coumarin (Yano et al, 2006) ก ก ก ก Selegiline ก ก ก ก Monoamine oxidase ก ก metabolism dopamine Selegiline ก CYP2A6 (Siu and Tyndale, 2008) ก ก ก ก ก ก ก ก (Natural products) กก ก ก Menthol ( Alcohol ก ก) ก ก (Ahijevych et al, 2002) Menthol ก ก CYP2A6 (MacDougall et al, 2003)
ก ก ก กก Menthol ก ก ก ก ก ก ก ก CYP2A6 Coumarin ก ก ก ก Kava (Piper methysticum ) ก ก ก Kavalactone ก CYP2A6 ก CYP3A4 CYP2C9 ก ก ก ก ก ก (Mathews et al, 2002) ก ก ก ก Isothiocyanate ก กก ก CYP2A6 ก ก ก ก ก (von Weymarn et al, 2007) Grapefruit ก ก CYP3A4 ก
CYP2A6 ก Nootkatone ( ก Naphthalene derivatives ก coumarin) ก ก Grapefruit ก CYP2A6 ก P450 (Merket et al, 1994; Runkel et al, 1997; Tassaneeyakul et al, 2000) ก ก Decursinol angelate ก Pyranocoumarin ก ก ก Angellica gigas ( ก Coumarin CYP2A6 ) ก CYP2A6 ก (Yoo et al, 2007) ก ก กก ก ก ก ก Coumarin ก ก CYP2A6 ก ก ก ก CYP2A6 ก ก ก ก ก ก ก ก Asteraceae (Compositae), Gramineae, Leguminoceae, Rutaceae, Umbelliferae ก coumarin ก (Gou et al, 2004; Matern et al, 1999; Weinmann, 1997) ก ก ก ก CYP2A6 ก ก ก ก ก ก ก ก ก (Wongwiwatthananukit et al, 2009) ก ก ก ก ก ก ก ก ก ก ก ก ก ก
3 ก ก
3.1 ก 1. ก ก (Graduated cylinder) 10, 100, 1000, 2000 2. (Erlenmeyer flask) 150,250,500 3. (Autoclave) 4. (Centrifuge) J.P.selecta Bio Active 5. PH (pH meter) 713 pH Meter Metrohm 6. ก (Spectrophotometer) UV 2501PC SHIMADZU 7. (Hot box) 8. (Sonicator) VCX 500 sonic and materials ก 9. ก ( ก 850 ) 10. ก ก (Electrophoresis) 11. SDS PAGE Bio rad ก 12. (Laminar flow) Clean ก 13. 37 (Incubator 37 C) BC 500 Memmert 14. (Shaking incubator) Inova 4230 ก 15. 20, 200, 1000 Quality scientific plastic ก 16. ก ก 50,100,250,1000 17. (Parafilm) Pechiney ก 18. (Microtube) 1.5 19. (Micropiprtte) 1 10, 2 200, 100 1,000 Bio lab Gilson 20. 15, 50 21. (Aluminimium foil)
3.2 1. Agarose FMC bioproducts Genetic technology grade ก 2. Alcohol MW 32.042 Carlo erba analytical grade ก 3. Buffer EB Elution buffer QIAGEN
4. Calcium chloride (CaCl 2) MW 147.02 Fluka analytical grade ก 5. Glycerol MW 92.09 USB Corporation analytical grade ก
6. Human liver microsome (CYP2C9) Invitrogen ก 7. Human liver microsome (CYP3A4) Invitrogen ก 8. Hydrochloric acid (HCl) MW36.46 Carlo erba analytical grade ก 9. Imidazole MW 82.068 Fluka analytical grade ก 10. Isopropyl ß D thiogalactopyranoside (IPTG) MW 238.31 promega ก 11. Laemmli sample Buffer Bio RAD ก 12. Sodium choloride (NaCl) MW 58.443 Carlo erbo analytical grade ก 13. Phenylmethylssulphonyl fluoride (PMSF) Fluka HPLC grade ก 14. Protein marker Bio rad ก 14. Sodium hydroxide (NaOH) MW 39.997 Carlo erbo analytical grade ก 15. Tryptone Becto ก 16. Triton X 100 Fluka ก
17. Tris (hydroxymethy) aminomethane NH 2C(CH 2OH) 3 MW 121.14 USB Corporation ก 18. Yeast extract Scharlau chemie
3.3 ก 3.3.1 pKK232 8 CYP2A6 ก ( 23human CYP2A6) Bam H I Hind III ( ก Assoc. Prof. Dr. Emily E. Scott, Dept. of Medicinal Chemistry, Kansus University, http://www.medchem.ku.edu/faculty_scott.shtml )
3 1 pKK232 8 ก ก ( : http://edoc.hu berlin.de/dissertationen/jacob daniela 2003 07 15/HTML/jacob_html_6dfb7b09.jpg )
3.3.2 pINIIIompA3 CPR (ratCPR) XbaI Hind III ( ก Prof. Dr. Jung Ja Kim, Dept. of Biochemistry, Medical College of Wisconsin, http://www.mcw.edu/biochemistry/Jung_Ja_Kim.htm )
3 2 pINIIIompA3 ก ก ก ( : http://www.uky.edu/Pharmacy/ps/porter/Plasmids/pINIIIompA3_map.htm )
3.4 ก 3.4.1 ก (Competent cell) E.coli BL21 (DE3), C41 (DE3) XL 1 blue Modified LB media (MLB) 15 ก 37 16 18 ก ก 0.5 MLB 5 ก (1% starter) ก 37 ก ก 600 (
OD 600 ) 0.4 0.6 15 3,000 rpm 4 2 ก ก
50 mM Tris pH 7.5, 50 mM CaCl 2 ก ก ก ก ก 2000 rpm 4 5
50 mM Tris pH 7.5, 50 mM CaCl 2 ก 15 ก 2000 rpm 4 5 50 mM Tris pH 7.5, 50 mM CaCl 2 ก ก ก (Competent cell) glycerol 20% ก 80 ก
3.4.2 ก (Tranformation) Competent cell ก 3.4.1 ก CYP2A6 rat CPR 1 (50 ng/ml) ก 200 15 30 ก ก ก (Heat shock) 42 90 ก 2 ก ก ก ก 0.8 15 37 1 200 spread plate ก 37 18 3.4.3 ก ก ก CYP2A6 rat CPR 4 15 LB Amplicilin (100 ug/ml) 16 37 ก ก ก ก 5,000 rpm 3 ก Resuspension buffer 200 (1 M Tris HCl PH 8.8 ,0.5 M EDTA) ก Lysis buffer 400 (4M NaOH ,20% SDS) ก Neutralization buffer 300 (3.0 M potassium acetate, pH 5.5 ) ก 5 ก ก ก 12,000 rpm 4 10 ก ก ก Absolute Ethanol 1000 20 1 2 10,000 rpm 10 ก 70% Ethanol 500 ก EB buffer (10 mM Tris pH7.5) RNase 25 30 Agarose elctrophorasis 3.4.4 ก ก ก (Polymerase Chain Reaction; PCR) ก (Specific region) CYP2A6 ( 3 3) ก (Conserved region) CPR ( 3 4) ก
10X buffer 2.5 50 mM MgCl 2 2 100 mM dNTPs 4 ก ก 14 Taq polymerase 0.5 2A6 forward 1 2A6 reverse 1 ( ก CYP2A6 ( 3 1)) CPRforward 1 CPR reverse 1 ( ก rat CPR ( 3 1)) 25
ก ก 1 (Denaturation) 95 1 2 (Annealing) 50 1 3 (Extention) 72 1 ก 30
CYP2C9 ------MDSL---VVLVLCLSCLLLLSLWRQSSGRGKLPPGPTPLP 37 CYP2E1 ------MSALGVTVALLVWAAFLLLVSMWRQVHSSWNLPPGPFPLP 40 CYP2A6 ------MVASGMLLVALLVCLTVMVLMSVWQQRKSKGKLPPGPTPLP 41 CYP2B6 ------MELSVLLFLALLTG---LLLLLVQRHPNTHDRLPPGPRPLP 38 CYP3A4 ------MALIPDLAMETWLLLAVSLVLLYLYGTHSHGLFKKLGIPGPTPLP 45 . CYP2C9 VIGNILQIGIKDISKS-LTNLSKVYGPVFTLYFGLKPIVVLHGYEAVKEALIDLGE--EF 94 CYP2E1 IIGNLFQLELKNIPKS-FTRLAQRFGPVFTLYVGSQRMVVMHGYKAVKEALLDYKD--EF 97 CYP2A6 FIGNYLQLNTEQMYNS-LMKISERYGPVFTIHLGPRRVVVLCGHDAVREALVDQAE--EF 98 CYP2B6 LLGNLLQMDRRGLLKS-FLRFREKYGDVFTVHLGPRPVVMLCGVEAIREALVDKAE--AF 95 CYP3A4 FLGNILSYHKGFCMFD--MECHKKYGKVWGFYDGQQPVLAITDPDMIKTVLVKECYS-VF 102 * . .* . : : Forward primer
CYP2C9 SGRGIFPLAERANRG---FGIVFS--NGKKWKEIRRFSLMTLRNFGMGKR------SIE 142 CYP2E1 SGRGDLP-AFHAHRD---RGIIFN--NGPTWKDIRRFSLTTLRNYGMGKQ------GNE 144 CYP2A6 SGRGEQATFDWVFKG---YGVVFS--NGERAKQLRRFSIATLRDFGVGKR------GIE 146 CYP2B6 SGRGKIAMVDPFFRG---YGVIFA--NGNRWKVLRRFSVTTMRDFGMGKR------SVE 143 CYP3A4 TNRRPFGPVGFMKSA-----ISIAE--DEEWKRLRSLLSPTFTSGKLKEM------V 146 : : . : CYP2C9 DRVQEEARCLVEELRKTKASP--CDPTFILGCAPCNVICSIIFHKRFDYKDQQFLNLMEK 200 CYP2E1 SRIQREAHFLLEALRKTQGQP--FDPTFLIGCAPCNVIADILFRKHFDYNDEKFLRLMYL 202 CYP2A6 ERIQEEAGFLIDALRGTGGAN--IDPTFFLSRTVSNVISSIVFGDRFDYKDKEFLSLLRM 204 CYP2B6 ERIQEEAQCLIEELRKSKGAL--MDPTFLFQSITANIICSIVFGKRFHYQDQEFLKMLNL 201 CYP3A4 PIIAQYGDVLVRNLRREAETGKPVTLKDVFGAYSMDVITSTSFGVNIDSLNNPQDPFVEN 206 : : . CYP2C9 LNENIKILSSPWIQICNNFSPIIDYFPGTHNKLLKNV-AFMKSYILEKVKEHQESMDMNN 259 CYP2E1 FNENFHLLSTPWLQLYNNFPSFLHYLPGSHRKVIKNV-AEVKEYVSERVKEHHQSLDPNC 261 CYP2A6 MLGIFQFTSTSTGQLYEMFSSVMKHLPGPQQQAFQLL-QGLEDFIAKKVEHNQRTLDPNS 263 CYP2B6 FYQTFSLISSVFGQLFELFSGFLKYFPGAHRQVYKNL-QEINAYIGHSVEKHRETLDPSA 260 CYP3A4 TKK--LLRFDFLDPFFLSITVFPFLIPILEVLNICVFPREVTNFLRKSVKRMKESRLEDT 264 . CYP2C9 P-QDFIDCFLMKMEKE--KHNQPSE---FTIESLENTAVDLFGAGTETTSTTLRYALLLL 313 CYP2E1 P-RDLTDCLLVEMEKE--KHSAERL---YTMDGITVTVADLFFAGTETTSTTLRYGLLIL 315 CYP2A6 P-RDFIDSFLIRMQEE--EKNPNTE---FYLKNLVMTTLNLFIGGTETVSTTLRYGFLLL 317 CYP2B6 P-KDLIDTYLLHMEKE--KSNAHSE---FSHQNLNLNTLSLFFAGTETTSTTLRYGFLLM 314 CYP3A4 Q-KHRVDFLQLMIDSQNSKETESHK--ALSDLELVAQSIIFIFAGYETTSSVLSFIMYEL 321 .: .. . : : : CYP2C9 LKHPEVTAKVQEEIERVIGRNRSPC------MQDRSHMPYTDAVVHEVQRYIDLLP 363 CYP2E1 MKYPEIEEKLHEEIDRVIGPSRIPA------IKDRQEMPYMDAVVHEIQRFITLVP 365 CYP2A6 MKHPEVEAKVHEEIDRVIGKNRQPK------FEDRAKMPYMEAVIHEIQRFGDVIP 367 CYP2B6 LKYPHVAERVYREIEQVIGPHRPPE------LHDRAKMPYTEAVIYEIQRFSDLLP 364 CYP3A4 ATHPDVQQKLQEEIDAVLPNKAPPT------YDTVLQMEYLDMVVNETLRLFPIA- 370 * .*:. : : : * * CYP2C9 TSLPHAVTCDIKFRN--YLIPKGTTILISLTSVLHDNKEFPNPEMFDPHHFLDEGGNFKK 421 CYP2E1 SNLPHEATRDTIFRG--YLIPKGTVVVPTLDSVLYDNQEFPDPEKFKPEHFLNENGKFKY 423 CYP2A6 MSLARRVKKDTKFRD--FFLPKGTEVYPMLGSVLRDPSFFSNPQDFNPQHFLNEKGQFKK 425 CYP2B6 MGVPHIVTQHTSFRG--YIIPKDTEVFLILSTALHDPHYFEKPDAFNPDHFLDANGALKK 422 CYP3A4 MRLERVCKKDVEING--MFIPKGVVVMIPSYALHRDPKYWTEPEKFLPERFSKKNKDNID 428 . : *. : : .* * ..: CYP2C9 ------SKYFMPFSAGKRICVGEALAGMELFLFLTSILQNFNLKSLVDPKNLD-TTP 471 CYP2E1 ------SDYFKPFSTGKRVCAGEGLARMELFLLLCAILQHFNLKPLVDPKDID-LSP 473 CYP2A6 ------SDAFVPFSIGKRNCFGEGLARMELFLFFTTVMQNFRLKSSQSPKDID-VSP 475 CYP2B6 ------TEAFIPFSLGKRICLGEGIARAELFLFFTTILQNFSMASPVAPEDID-LTP 472 CYP3A4 ------PYIYTPFGSGPRNCIGMRFALMNMKLALIRVLQNFSFK-PCKETQIP-LKL 477 *. * * * :* : : :: . . Reverse Primer
CYP2C9 VVNGFASVPPFYQLCFIPV------490 CYP2E1 IHIGFGCIPPRYKLCVIPRS------493 CYP2A6 KHVGFATIPRNYTMSFLPRHHHH------498 CYP2B6 QECGVGKIPPTYQIRFLPR------491 CYP3A4 SLGGLLQPEKPVVLKVESRDGTVSGA---- 503
3 3 ก ก CYP2A6 ก P450 Isoform ก ก ก ก
minimus ---MDAQAETEMPTGSVS---DEPFLGPLDIILLVCLLAGTAWYLLKGKKKENQASQFKS 54 musca ---MSAEHVEEVVS------EEPFLGTLDIALLVVLLVGATWYFMRSRKKEE--APIRS 48 human MINMGDSHVDTSSTVSEAVAEEVSLFSMTDMILFSLIVGLLTYWFLFRKKKEE----VPE 56 rat ---MGDSHEDTSATMPEAVAEEVSLFSTTDMVLFSLIVGVLTYWFIFRKKKEE----IPE 53 minimus YSIQPTTVNTMTMVENSFIKKLQSSGRRLVVLYGSQTGTAEEFAGRLAKEGIRYQMKGMV 114 musca YSIQPTTVSTVSTTENSFIKKLKASGRSLVVFYGSQTGTAEEFAGRLAKEGLRYRMKGMV 108 human FTKIQTLTS--SVRESSFVEKMKKTGRNIIVFYGSQTGTAEEFANRLSKDAHRYGMRGMS 114 rat FSKIQTTAP--PVKESSFVEKMKKTGRNIIVFYGSQTGTAEEFANRLSKDAHRYGMRGMS 111 minimus ADPEECNMEELLMLKDIDKSLAVFCLATYGEGDPTDNCMEFYDWIQNNDLDMTGLNYAVF 174 musca ADPEECDMEELLQMKDIPNSLAVFCLATYGEGDPTDNAMEFYEWITNGEVDLTGLNYAVF 168 human ADPEEYDLADLSSLPEIDNALVVFCMATYGEGDPTDNAQDFYDWLQETDVDLSGVKFAVF 174 rat ADPEEYDLADLSSLPEIDKSLVVFCMATYGEGDPTDNAQDFYDWLQETDVDLTGVKFAVF 171 minimus GLGNKTYEHYNKVGIYVDKRLEELGANRVFELGLGDDDANIEDYLITWKEKFWPTVCDFF 234 musca GLGNKTYEHYNKVAIYVDKRLEELGATRVFELGLGDDDANIEDDFITWKDRFWPSVCDFF 228 human GLGNKTYEHFNAMGKYVDKRLEQLGAQRIFELGLGDDDGNLEEDFITWREQFWPAVCEHF 234 rat GLGNKTYEHFNAMGKYVDQRLEQLGAQRIFELGLGDDDGNLEEDFITWREQFWPAVCEFF 231 minimus GIESTGEDVLMRQYRLLEQPEVGADRIYTGEVARLHSLQTQRPPFDAKNPFLAPIKVNRE 294 musca GIEGSGEEVLMRQFRLLEQPDVQPDRIYTGEIARLHSMQNQRPPFDAKNPFLASVIVNRE 288 human GVEATGEESSIRQYELVVHTDIDAAKVYMGEMGRLKSYENQKPPFDAKNPFLAAVTTNRK 294 rat GVEATGEESSIRQYELVVHEDMDVAKVYTGEMGRLKSYENQKPPFDAKNPFLAAVTANRK 291 minimus LHKAGGRSCMHVEFDIEGSKMRYEAGDHLAMYPVNDRDLVERLGKLCNADLETVFSLINT 354 musca LHKGGGRSCMHIELDIDGSKMRYDAGDHIAMYPINDKILVEKLGKLCDANLDTVFSLINT 348 human LNQGTERHLMHLELDISDSKIRYESGDHVAVYPANDSALVNQLGKILGADLDVVMSLNNL 354 rat LNQGTERHLMHLELDISDSKIRYESGDHVAVYPANDSALVNQIGEILGADLDVIMSLNNL 351 minimus DTDSSKKHPFPCPTTYRTALTHYLEITALPRTHILKELAEYCSEEKDKEFLRFISSTAPE 414 musca DTDSSKKHPFPCPTTYRTALTHYLEITAIPRTHILKELAEYCSDEKDKEFLRNMASITPE 408 human DEESNKKHPFPCPTSYRTALTYYLDITNPPRTNVLYELAQYASEPSEQELLRKMASSSGE 414 rat DEESNKKHPFPCPTTYRTALTYYLDITNPPRTNVLYELAQYASEPSEQEHLHKMASSSGE 411 minimus GKAKYQEWVQDSCRNVVHVLEDIPSCHPPIDHVCELLPRLQPRYYSISSSSKIHPTTVHV 474 musca GKEKYQNWIQNSSRNIVHILEDIKSCRPPIDHICELLPRLQPRYYSISSSSKLYPTNVHI 468 human GKELYLSWVVEARRHILAILQDCPSLRPPIDHLCELLPRLQARYYSIASSSKVHPNSVHI 474 rat GKELYLSWVVEARRHILAILQDYPSLRPPIDHLCELLPRLQARYYSIASSSKVHPNSVHI 471 Forward primer minimus TAVLVKYETKTGRLNKGVATTFLAEKHPNDGEP-LPRVPIFIRKSQFRLPPKPETPVIMV 533 musca TAVLVQYETPTGRVNKGVATSYMKEKNPSVG---EVKVPVFIRKSQFRLPTKSEIPIIMV 525 human CAVVVEYETKAGRINKGVATNWLRAKEPAGENGGRALVPMFVRKSQFRLPFKATTPVIMV 534 rat CAVAVEYEAKSGRVNKGVATSWLRAKEPAGENGGRALVPMFVRKSQFRLPFKSTTPVIMV 531 minimus GPGTGLAPFRGFIQERDFSKQEGKDIGQTTLYFGCRKRSEDYIYEDELEDYSKRGIIN-L 592 musca GPGTGLAPFRGFIQERQFLRDGGKVVGDTILYFGCRKKDEDFIYREELEQYVQNGTLT-L 584 human GPGTGVAPFIGFIQERAWLRQQGKEVGETLLYYGCRRSDEDYLYREELAQFHRDGALTQL 594 rat GPGTGIAPFMGFIQERAWLREQGKEVGETLLYYGCRRSDEDYLYREELARFHKDGALTQL 591 Reverse primer minimus RVAFSRDQDKKVYVTHLLEQDSDLIWNVIGENKGHFYVCGDAKNMATDVRNILLKVIRSK 652 musca KTAFSRDQQEKIYVTHLIEQDADLIWKVIGEQKGHFYICGDAKNMAVDVRNILVKILSTK 644 human NVAFSREQSHKVYVQHLLKQDREHLWKLI-EGGAHIYVCGDARNMARDVQNTFYDIVAEL 653 rat NVAFSREQAHKVYVQHLLKRDREHLWKLIHEGGAHIYVCGDARNMAKDVQNTFYDIVAEF 651 minimus GGLSETEAQQYIKKMEAQKRYSADVWS 679 musca GNMNESDAVQYIKKMEAQKRYSADVWS 671 human GAMEHAQAVDYIKKLMTKGRYSLDVWS 680 rat GPMEHTQAVDYVKKLMTKGRYSLDVWS 678
3 4 ก ก CPR (R. norvegicus ) ก CPR isoform ก (An . minimus ) ( M. domestica ) ( H. sapiens ) ก ก ก ก
3 1 ก CYP2A6 rat CPR 2A6Forward 5 TTCGACTGGGTCTTCAAAGGC 3 2A6Reverse 5 GCGGGGCAGGAAGCTCATG 3 CPR Forward 5 CGC AA(AG) (TA) (CG) (GTC) CA (AG) TT (TC) (CA) G (GT) TTG 3 CPR Reverse 5 CAT C (CA) CC (AG)C A(ATG)A (TC) (GA)T A (AT) (GA) TG 3
3.4.5 ก ก ก (Expression of interested Protein) E. coli (LB media, modified LB media TB media) 250 rpm 37 16 18 ก 0.5 15 4.5 ( Amplicilin 100 ug/ml) 25 30 37
ก 250 rpm ก ก ก 600 (OD 600 ) 0.6 0.8 ก ก IPTG 0.2 0.6 1.0 mM ( 5 mM Imidazold CYP2A6) ก ก 25 30 37 24 (rat CPR) 48 (CYP2A6)
ก ก OD 600 ก ก protein buffer 400 ( PMSF (Phenylmethylsulfonyl fluoride) 0.2 mM) ก ก ก sonication (Crude extract) ก ก (Cell lysate) ก
Bradford ก ก 595 (OD 595 ) กก ก ก ก ( ก / ) ก ก ก SDS PAGE 3.4.6 ก ก ก CYP2A6 rat CPR ก ก ก ก ก CYP2A6 rat CPR Devore et al, 2009; DeVore et al, 2008; Hubbard PA, 2001 E. coli XL 1 blue ( CYP2A6) C41(DE3) ( rat CPR) modified LB 250 rpm 37 16 18 ก 4 (Erlenmeyer flask) 1,000 400 ( Amplicilin 100 ug/ml) 37
ก 250 rpm ก ก ก 600 (OD 600 ) 0.6
0.8 ก ก IPTG 0.2 mM ( 5 mM Imidazold CYP2A6) ก ก 160 rpm 25 48 (CYP2A6) 37 24 (rat CPR)
ก ก OD 600 ก ก protein buffer A (100 mM Kpi buffer pH 7.4, 200 mM NaCl, 0.1% Triton X 100, 10% glycerol, 0.2 mM PMSF) 2 ก ก ก ก sonication (Pulse on: off ก 9:5 90 8 ) (Crude extract) ก 17,000 rpm 15 ก ก ก 17,000 rpm 45 ก (Cell lysate) ก ก ก protein buffer A 10 mM Imidazole ก protein buffer A ก 10 mM Imidazole 20 mM Imidazole ก ก (Elude) ก protein buffer A 50 mM Imidazole 100 mM Imidazole ก SDS PAGE ก ก 80 20% glycerol ก 3.4.7 ก ก ก CYP2A6 rat CPR ก ก ก ก CPR ก (Cytocheome c ก ) rat CPR ก 3.3.5 ก Cytocheome c (40 uM) 300 mM Kpi buffer pH 7.7 1 ก NADPH (50 uM) ก ก ก 550 (ก cytocheome c ) specific activity ( nmol of cytochrome c reduced/min/mg protein) ก ก ก ก ( ε λmax) ก 21.4 mM 1cm 1 (Shen et al., 1989) (Unit nmol) CPR ก ก ก ก cytochrome c 3 umol (Lamb et al., 2001) ก ก ก ก ก CYP2A6 ก ก Coumarin 7 hydroxycoumarin (Reconstitution assay system) ก Donato et al (2004) CYP2A6 (100 ug ) ก rat CPR (10 unit) 50 mM Tris Cl pH 7.4 10 ก Coumarin ( 0.1 20 uM) ก (NADPH regenerating system: 5 mM + MgCl 2, 1 mM NADP , 10 mM glucose 6 phosphate 0.3 U/ml glucose 6 phosphate dehydrogenase) 37 ก ก ก (7 hydroxycoumarin) Fluorescence spectroscopy (Emission wavelength) 460 ก (Excitation wavelength) 355 15 3.4.8 ก 25 ก ก ก ก ก . . ก (ก 2545 2549 ก ก 2552 ก 2550 2551) ก ก (Filter) ก 0.45 ก 20 ก ก ก ก ก 1 ก coumarin ก phenolic compound ก ก ก phenol gallic acid ก gallic acid ก ก ก Folin Ciocalteu Sodium bicarbonate ก ก 765 nm ก phenol ก ก mg/ml gallic acid (Shui et al, 2006) ก flavonoid ก quercetin quercetin ก
ก NaNO 2 AlCl3 6H 2O NaOH ก ก ก ก 510 nm mg/ml quercetin (Liu et al, 2002) ก ก phenolic ก 3.4.9 ก ก ก CYP2A6 ก ก CYP2A6 CYP2A6 rat CPR ก 10% v/v
Coumarin ( Km) ก (NADPH regenerating system) 37 ก ก ก 3.4.7 ก ก ก (Remaining activity) ก ก ก CYP2A6 ( ก ก ) Student s t test analysis of variances ก SPSS ก ก ก ก 8 MOP CYP2A6 (ก )
ก ก ก IC 50 ( ก ก ) P450 (100 ug ) ก CPR (10 unit) 50 mM Tris Cl pH 7.4 10
Coumarin ( Km) ก ก
(NADPH regenerating system) 37 ก ก ก IC 50
ก ก (Logarithm scale) ก ก ก ก Graph pad prism (La Jolla, CA) 3.4.10 ก ก ก CYP2A6 ก ก CYP2A6 ก CYP2A6 ก ก n butanol ethyl acetate ก ก ก ก Ethyl acetate Sephadex LH20 Hosoi (2008) ก ก ก ก ก ก ก ก ก 3.4.9 3.4.11 ก ก ก CYP2A6 ก ก ก ก cytochrome P450 ก ก ก CYP3A4 CYP2C9 human liver microsome (Invitrogen, USA) ก ก ก ก ก ก ก ก ก ก ก ก 3.4.9
4 ก
4.1 ก ก ก CYP2A6 rat CPR ( ก Assist. Prof. Emily Scott Prof. Jung Ja Kim ) E. coli (XL 1 blue, BL 21(DE3) C41(DE3)) ก ก ก 23human CYP2A6 ก 4,500, 7,000, 10,000 กก 23,000 bp ก DNA ( 4 1A) rat CPR ก 7,000, 12,000 กก 23,000 bp ก DNA ( 4 1B)
A B kb M 1 2 3 kb M 1 2 3
23.13 9.42 6.56 4.31
2.32 2.03
4 1 CYP2A6 ( 4 1A) rat CPR ( 4 1B) 1% Gel electrophoresis M λ Hind III DNA marker ก ก ก XL 1 blue (lane 1) BL 21 (DE3) (lane 2) C41 (DE3) (lane 3)
4.2 ก ก ก 4.1 ก CYP2A6 rat CPR ก ก CYP2A6 ( 3 3) rat CPR ( 3 4) ก DNA ก PCR ก DNA กก PCR CYP2A6 1,100 bp rat CPR 360 bp ก ก ก ก ก ( BioDesign Co., Ltd, Science Park, Thailand) ( ก ก) ก ก E. coli Transformed CYP2A6 rat CPR ก ก ก A M 1 2 3 B M 1 2 3
1,000
500 500 300
4 2 กก ก ก CYP2A6 transformed cells ( 4 4A) rat CPR transformed cells ( 4 4B) 1% agarose gel electrophoresis M DNA lane 1 3 PCR product ก ก ก XL 1 blue BL 21 (DE3) C41 (DE3)
4.3 ก ก ก CYP2A6 rat CPR ก ก ก ก ก ก CYP2A6 SDS PAGE ก ก ก (crude) ก ก ก CYP2A6 rat
CPR bioinformatics ก www. http://www.ncbi.nlm.nih.gov/protein http://expasy.org/cgi bin/protparam CYP2A6 ก 476 ( ก ก ก C terminal 4 ) ก 54806.9 Da pH 0 (pI) ก 9.29 CPR ก 77785.7 Da pI ก 5.48 4.3.1 ก ก CYP2A6 rat CPR E.coli 3 (XL 1 blue, BL 21 (DE3), C41 (DE3)) ก 3 (Luria Bertani media, Modified Luria Bertani media Terrific media (LB media, Modified LB, TB)) ก ก ก (Crude extract) ก ก ก ก ก crude extract กก (Crude lysate) SDS PAGE CYP2A6 ( ก 55 kDa) ก ก XL 1 blue Modified LB ( 4 3 4 1) rat CPR ( ก 78 kDa) ( ก ก ก 0.3% Triton X 100) ก ก C41 (DE3) Modified LB ( 4 4 4 2)
4 3 ก ก CYP2A6 ( ก 55 kDa; ก 2) Modifed LB ก C ก (Crude) Ly (Lysate) ก XL 1 blue BL 21 (DE3) C41 (DE3) M protein marker T0 ก ก ก ก
4 1 ก ก CYP2A6 ก ก Luria Bertani (LB) Modified LB (MLB) Terrific Broth (TB) XL 1 blue + +++ +++ BL 21 + ++ + C41 + + ++ + ก ก ก
XL-1 blue BL21 (DE3) C41 (DE3) M = Marker M M C = Crude
kDa Ly = Lysate
158
66.4
55.6
42.7
27.0
T0 Ly T0 Ly T0 Ly 4 4 ก ก CPR ( ก 78 kDa; ก ) Modifed LB ก C Ly ก (Crude) Ly (Lysate) ก XL 1 blue BL 21 (DE3) C41 (DE3) M protein marker T0 ก ก ก ก
4.2 ก ก rat CPR ก ก Luria Bertani (LB) Modified LB (MLB) Terrific Broth (TB) XL 1 blue + +++ +++ BL 21 (DE3) ++ ++ ++ C41 (DE3) ++ +++ +++ + ก ก ก
4.3.2 ก IPTG ก ก ก ก (Native form) XL 1 blue CYP2A6 C41 (DE3) rat CPR ก ก ก IPTG ก ก (0.2 0.6 1 mM) (25 30 37 ) ก ก ก ก ก SDS PAGE CYP2A6 ( ก 55 kDa) ก ก ก (Cell lysate) XL 1 blue 25 ก IPTG ( 4 5 4 3) rat CPR ( ก 78 kDa) ก ก ก (Cell lysate) C41(DE3) 30 IPTG ก 0.6 mM ( 4 6 4 4)
0.2 mM 0.6 mM 1.0 mM M kDa M = Marker 158 C = Crude Ly = Lysate 66.4 55.6 42.7
37.6
T0 T0 C Ly C Ly C Ly
4 5 ก ก CYP2A6 ( ก 55 kDa; ก ) XL 1 blue Modifed LB 25 ก C ก (Crude) Ly (Lysate) ก XL 1 blue IPTG M protein marker T0 ก ก ก ก
4 3 ก ก CYP2A6 IPTG IPTG 0.2mM 0.6mM 1.0mM 25ºC +++ +++ +++ 30 ºC +++ +++ ++ 37 ºC +++ +++ ++ + ก ก ก
0.2 mM 0.6 mM 1.0 mM M kDa M = Marker
C = Crude 158 Ly = Lysate 66.4 55.6 42.7 37. 6
T0 C Ly T0 C Ly T0 C Ly
4 6 ก ก rat CPR ( ก 78 kDa; ก ) C41 (DE3) Modifed LB 37 ก C ก (crude) Ly (lysate) ก C41 (DE3) IPTG M protein marker T0 ก ก ก ก
4 4 ก ก rat CPR IPTG IPTG 0.2mM 0.6mM 1.0mM 25ºC ++ ++ ++ 30 ºC ++ ++++ ++ 37 ºC +++ +++ + + ก ก ก
4.4 ก ก ก XL 1 blue C41 (DE3) ก ก CYP2A6 rat CPR ก ก Ni 2+ NTA (Ni 2+ Affinity chromatography) CYP2A6 ก 55 kDa ( 4 7) rat CPR ก 78 kDa ( 4 8) ก ก ก 80 ก
66.4 Kda
2A6 54 Kda 55.6 Kda
M C M Ly FT W1 W2 E 4 7 ก CYP2A6 Ni 2+ Affinity chromatography ก C ก (crude) Ly (lysate) ก FT (Flow through) W1 W2 ก ก ก (Wash) E CYP2A6 ก ก ก M protein marker
kDa
116.3 97.4
66.2
45.0 31.0
14.4 6.5
M C Ly FT W1 W2 E1 E2 M
4 7 ก rat CPR Ni 2+ Affinity chromatography ก C ก (crude) Ly (lysate) ก FT (Flow through) W1 W2 ก ก ก (Wash) E rat CPR ก ก ก M protein marker
4.5 ก ก CYP2A6 rat CPR 4.5.1 ก ก ก Coumarin Cytochrome c rat CPR ก ก NADPH ก ก ก Cytochrome c rat CPR ก ก 58.69 ± 1.58 umol cyt c reduced/min/mg protein ก ก cytochrome c (cyt c K m) ก 16.44 ± 1.37 ( 4 8A) CYP2A6 ก ก rat CPR 10 unit CYP2A6 ก Coumarin 7 hydroxylase ก ก 0.054 ± 0.001 umol coumarin/min/mg protein ก ก coumarin (coumarin Km) ก 1.25 ± 0.14 ( 4 8B)
A cyt c reduction activity B coumarin-7-hydroxylation 60 0.06
40 0.04
20 0.02 umol/min/mgprotein umol/min/mgprotein
0 0.00 0 10 20 30 40 50 60 70 80 90 0 5 10 15 20 Cyt c (uM) Coumarin (uM)
4 8 ก ก rat CPR ก Cytochrome c ( A) CYP2A6 Coumarin ( B) ก ก 37 ก (NADPH) ก 50 uM 5 (rat CPR) 30 (CYP2A6) ก ก GraphPad Prism 5
4.5.2 ก ก CYP2A6 rat CPR 55 ก ก ก 5 ก 250 ( 1 50) ก 15 20 ก ก ก 1 ก 50 ก ก ก ก ก ก ก Gallic acid Quercetin ก ก ก ก 20 ก ( 1 ) 4.5.2.1 ก ก ก ก ก ก 1,393.78 ± 5.58 % mg gallic acid equivalent ก ก ก ก ก ก ก 300 800 mg gallic acid equivalent ก ( 4 9A) . ก 312.51 ± 1.45 % mg qurecetin equivalent ก ก ก ก 150 280 mg quercetin equivalent ก ( 4 9B)
4 9 ก ก (A) ก ก ก ก gallic acid (B) ก ก ก ก qurecetin ก ก GraphPad Prism 5
4.5.2.2 ก ก ก ก ก ก 10% v/v ก ก CYP2A6 ก ก CYP2A6 ก ก ก CYP2A6 ก ก ( ก ก 100) 2 8 MOP CYP2A6 ( ก ก 2 ก ก ) กก ก 10% v/v ก
CYP2A6 90% ก ก ก ก ก ก CYP2A6 60 80 % ก CYP2A6 40 55 % ก ก CYP2A6 10 35 % ( 4 10)
4 10 ก ก CYP2A6 Coumarin 10% v/v ก ก 37 ก 2 8 MOP ก ก ก ก CYP2A6 ( ) ก ก GraphPad Prism 5
4.5.2.3 ก ก ก 50 (IC 50 ) ก ก CYP2A6 10% v/v ( 4 1) ก ก ก ก ก ก 40% v/v ก CYP2A6 95% 65% ก CYP2A6 50
(IC 50 ) ก ก CYP2A6 ก ก ก ก 3.19 ± 0.09 % v/v 8.14 ± 0.82 % v/v ก ก ( 4 11) ก ก ก ก CYP2A6 ก ก (Pre incubation) ก ก ก CYP2A6 ก mechanism based inhibition IC 50 ก 0.26 ± 0.03 % v/v ก mechanism based inhibition IC 50 ก 7.61 ± 0.14 % v/v
4 1 ก ก CYP2A6 10% v/v
IC 50 IC 50 ก ก 22.67 ± 3.37 ก 20.70 ± 5.30 ก 36.75 ± 11.01 8.14 ± 0.82 45.41 ± 11.34 3.19 ± 0.09 50.88 ± 12.93 41.06 ± 12.62 54.87 ± 15.51 48.53 ± 15.55 35.18 ± 7.90 15.55 ± 3.56 27.31 ± 8.06 ก 76.12 ± 4.34 48.27 ± 8.98 15.94 ± 4.28 51.53 ± 9.13 59.05 ± 15.57 54.15 ± 10.19 16.09 ± 4.17 23.77 ± 4.45 50.11 ± 9.07 45.11 ± 12.69 62.72 ± 19.58 ก 23.01 ± 4.95
100 [star fruit juice] [Ginger juice] 75
50
25
0 0 5 10 15 20 25 30 35 40 Remaining activity (% of control) of (% activity Remaining [juice] (%v/v) 4 11 ก ก CYP2A6 Coumarin ก ก 37 ก ก ก ก CYP2A6 ( ) ก ก GraphPad Prism 5
4.5.2.4 ก ก ก ก CYP2C9 CYP3A4 ก ก ก ก P450 ก ก ก CYP3A4
CYP2C9 ก CYP2A6 ก ก IC 50 ก ก 12.72 ± 1.24 % v/v 16.39 ± 3.04 % v/v 3.19 ± 0.09 % v/v ( 4 12)
100 CYP2A6 CYP3A4 75 CYP2C9
50
25
0 0 5 10 15 20 25 30 35 40 Remaining activity (% activity control) of Remaining [star fruit juice] (%v/v) 4 12 ก ก CYP2A6 CYP3A4 CYP2C9 ก ก 37 ก ก GraphPad Prism 5
4.5.2.5 ก ก ก CYP2A6 ก ก n butanol ethyl acetate ก ก ( n butanol ethyl acetate ) ก ก ก ก ก ก ก ก ก CYP2A6 ก ethyl acetate ก ก CYP2A6 ก ก ก n butanol ( ) ก ก ก ก Sephadex LH20 ก 4 กก ก ก ก ก CYP2A6 ( 4 2) ก ก ( ก ก proposal) ก HPLC (High Performane Liquid Chromatography) NMR (Nuclear Magnetic Resonance) ก ก ก ก ก
4 2 ก ก CYP2A6 ก ก กก ก ก ก ก ก ก ก 1 96.97% 3 114.06% 2 76.44% 4 68.58% ก 100%
5 ก
5.1 ก Cytochrome P450 2A6 (CYP2A6) ก cytochrome P450 ก ก ก ก ก ก ก ก ก (Valproic acid Losigamone) ก (Efavirenz) ก (Cyclophosphamine) ก ก Retinoic acid, Testosterone Estradiol ก P450 CYP2A6 ก ก ก ก ก ก ( 80 90
ก ) CYP2A6 Km Vmax ก ก ก ก CYP2A6 ก ก ก ก ก ก ก ก กก ก (Di et al , 2009; Hukkanen et al., 2002; Ortiz de Montellano, 2005; Patten et al., 1996) ก ก ก ก ก ก ก ก ก ก (Di et al., 2009; Hukkanen et al., 2002; Patten et al., 1996) ก ก ก CYP2A6 ก ก ก ก ก ก ก CYP2A6 CYP2A6 Coumarin ก ก ก CYP2A6 ก ก ก CYP2A6 ก ก ก ก ก ก ก ก กก ก ก ก ก ก CYP2A6 ก 8 MOP ก CYP2A6 ก (Hukkanen et al., 2005) ก ก CYP2A6 ก ก NADPH rat CPR ก ก ก 8 MOP ก (Transformation) CYP2A6 ( ก Assoc. Prof. Emily E. Scott) CPR ( ก Prof. Jung Ja P.Kim) E. coli 3 ก ก ก ก ก ก E. coli
ก (PCR) CYP2A6 ก ก P450 ( 4 2) ก CPR ก NADPH (Shen et al., 1989; Kaewpa et al., 2007) กก E. coli กก CYP2A6 rat CPR ก ก ก ก (CYP2A6 rat CPR) ก ก ก (Inclusion Bodies) ก ก ก ก ก ก rat CPR OmpA (Omp A Signal sequence) rat CPR periplasmic membrane space (DeVore et al., 2009; Shen et al., 1989) ก ก ก ก ก CYP2A6 rat CPR E. coli 3 (XL 1 blue, BL 21 (DE3) C41 (DE3)) 3 (LB, Modifed LB TB) E. coli ก ก CYP2A6 LB ก pH ก ก CYP2A6 ก E. coli ก ก Modified LB TB ก pH E. coli XL 1 blue ก ก CYP2A6 E. coli 3 ( 4 1) ก ก ก ก XL 1 blue ก BL 21 (DE3) C41 (DE3) (http://www.promega.com/techserv/techref/genetic.markers.ecoli.htm) Modified LB TB ก ก rat CPR ก ก ก ก Modified LB ก ก ก ก ก CYP2A6 ก ก ก ก ก 3 (25 30 37 ) 0.2 0.6 ก ก CYP2A6 1 ก ก CYP2A6 ก ก ก ก ก ก ก ก ก ก CYP2A6 IPTG 0.2 mM ก 25 E. coli XL 1 blue Modified LB ก ก ก CYP2A6 กก ก ก ก กก ก CYP2A6 E.coli DH5 α ( ก ก XL 1 blue) LB ก 32° ก ก IPTG 1 (Soucek et al., 1999) ก ก CYP2A6 ก TOPP 3 ( ก ก XL 1 blue) TB 28 IPTG 1
(DeVore et al., 2009; Smith et al., 2007) ก ก ก ก กก E. coli ก ก 5.1
5 1 ก ก CYP2A6 IPTG DH5 α LB 1 mM 32 °C Soucek etal., 1999 TOPP 3 TB 1 mM 28 °C Smith et al., 2007 DeVore et al., 2009; XL 1 blue Modified LB 0.2 mM 25 °C ก ก
ก ก ก กก E. coli rat CPR Modifed LB TB ก pH ก ก rat CPR กก LB ก pH ก E. coli C41 (DE3) ก ก ก กก BL 21(DE3) (Miroux Walker, 1996) ก E. coli ก ก ก ก IPTG ก ก rat CPR rat CPR ก ก ก ก 30 0.6 ก ก 37 1 ก ก ก ก กก ก ก rat CPR ก ก RI90 HB101 (Hayashi et al., 2003; Sakaki et al., 1996) C 1A (Shen et al., 1989) CPR ก ก ก XL 1 blue (Kaewpa et al., 2007; et al., 2010) BL 21 (DE3) Sarapusit et al., 2008) ก ก 16 37 IPTG 0.01 mM 1 M ก ก minimal media TB media 5.2 ก ก rat CPR ก ก ก ก
5 2 ก ก rat CPR IPTG C 1A LB 0.8 mM 37 °C Shen et al., 1989 RI90 LB 0.6 mM 30 °C Sakaki et al., 1996 HB101 Minimal media 0.01 mM 37 °C Hayashi et al., 2003 XL 1 blue TB 1 mM 28 °C Kaewpa et al., 2007 Sarapusit et al., 2010 BL 21 (DE3) MLB 0.1 mM 16 °C Sarapusit et al., 2008 C41 (DE3) MLB 0.6 mM 30 °C ก ก
ก ก ก ก CYP2A6
ก coumarin 7 hydroxylase ก ก (Vmax )
ก ก coumarin (K m) ก 0.054 ± 0.1 nmol coumarin/min/mg protein 1.25 ± 0.14 ก ก กก ก ก CYP2A6
ก ( 5.3) ก ก rat CPR ก ก (Vmax ) ก 58.69 ± 1.58 umol cyt c reduced/min/mg protein ก ก cytochrome c (K m) ก 16.44 ± 1.37 ก ก กก ก ก CYP2A6 ก ( 5.4) กก ก ก ก ก ก
5 3 Vmax Km CYP2A6
CYP2A6 Vmax Km (uM) (nmol/min/mg protein) ก 0.79 2.1 Li et al., 1997 0.35 ± 0.08 9.1 ±1.3 Donato et al., 2004 ก 0.65 ± 0.16 5.6 ± 2.9 0.61 ± 0.01 0.60 ± 0.05 Fukami et al., 2004 ก 4.2 7.9 Anzenbacherová et al., 2005 1.70 ± 0.14 10.61 ± 1.58 Hung Tiong et al., 2010 0.054 ± 0.10 1.25 ± 0.14 ก ก
5 4 Vmax Km rat CPR
CPR Vmax Km (uM) (nmol/min/mg protein) (R. norvegicus ) 90.35 16.3 ± 1.7 Shen et al., 1989 Sem et al., 1993 ( H. sapiens ) 88 19 Smith et al., 1994 Döhr et al., 2001 (M. domestica ) 50.4 ± 6 4.6 ± 1.2 Murataliev et al., 1999 ก (An . minimus ) 28.99 ± 0.48 18.1 ± 0.9 Sarapusit et al., 2008 (R. norvegicus ) 58.69 ± 1.58 16.44 ± 1.37 ก ก
กก ก ก ก CYP2A6 ก ก CYP2A6 ก ก ก ก ก ก ก ก ก CYP2A6 ก 10% v/v ก ก ก ก ก CYP2A6 90% ก ก 8 MOP ( ก 95% 2 ) ก ก ก CYP2A6 ก Furanocoumarin Flavonoid ( gallic acid, catechin, ( ) epicatechain, proanthocyanin ) ก Furanocoumarin Flavonoid ( 8 MOP) ก ก P450 (Guo et al., 2000; Kim et al., 2006; Merkel et al., 1994; Tassaneeyakul et al., 2000) ก ก ก 40% (v/v)
ก CYP2A6 95% ก (IC 50 )
3.2 % (v/v) IC 50 ก ก ก P450 ก CYP2A6 ก ก ก Furanocoumarin Flavonoid ก ก ก ก Alkaloid ก ก CYP2A6 ก (Guo et al., 2000; 2539) ก Alkaloid ก ก Coumarin ก (
ก 2550) ก Alkaloid ก CYP2A6 ก Coumarin ก ก ก ก ก P450 ก CYP2A6 ก ก ก ก ก Asteaceae ก Furanocoumarin ก ก ก ก ก Furanocoumarin Flavonoid ก (Fujita et al., 2003; Guo et al., 2000) ก ก ก ก Umberiferae Rutaceae ก Furanocoumarin (Guo et al., 2000; Marten et al., 1999) ก 60 80 % ก ก ก Furanocoumarin ก ก ก ก ก ( Asteraceae) ( Graminae) ( Leguminoceae) ( : Rutaceae) ( Umbelliferae) ก CYP2A6 40 55 % ก ( Graminae) ( Leguminoceae) ก ก ก ก CYP2A6 10 35 % ก ก ก ก CYP2A6 ก ก ก กก ก ก ก ก ก P450 ก ก ก ก ก ก ก ก ก ก ก CYP2A6 mechanism based
ก ก ก กก IC 50 ก ก CYP2A6 (Pre incubation) ก ก ก CYP2A6 ก (Co incubation) (Fowler 2008; Grime et al, 2009; Zhou et al, 2005) ก ก ก ก (Di et al, 2009; Xi et al, 2009) ก CYP2A6 ก ก mechanism based inhibition ก ก ก ก ก ก ก ก ก ก ก ก CYP3A4 CYP2C9 ก ก CYP2A6 4 5 (3.19 12.72 16.39
% (v/v) ) กก ก ก ก ก Zhang (2007) ก CYP2A6 ก CYP3A4 CYP2C9 3 ก
CYP2A6 CYP3A4 CYP2C9 (IC 50 ) ก 0.9 3.2 3.0 % (v/v)
ก ก IC 50 ก ก ก ก ก ก ก ( ก ก Zhang ) CYP2A6 ก ก ก ก CYP2A6 CYPOR Zhang Human Liver microsome (HLM) ก ก HLM ก ก HLM HLM กก ก Zhang ก ก ก ก ก ก ก ก ก ก ก ก CYP2A6 ก Ethyl acetate ก ก n butanol ก ก ก ก ก proposal ก HPLC (High Performane Liquid Chromatography) NMR (Nuclear Magnetic Resonance) ก ก ก ก ก ก กก ก ก ก CYP2A6 ก ก ก ก ก ก ก ก กก ก ก ก CYP2A6 3% (v/v) ก ก CYP2A6 50% ก กก CYP3A4 CYP2C9 4 5 ก ก ก ก ก ก ก ก ก ก ก ก ก ก ก CYP2A6 ( Cyclophosphamine Efavirenz Losigamone Valproic acid ) CYP3A4 ( Alprazolam Cyclosporine Dextromethorphan Docetaxel Erythromycin Midazolam Lovastatin Tamoxifen Taxol Saquinavir Verapamil ) CYP2C9 ( Celecoxib Diclofenac Ibuprofen Glimepiride Tamoxifen Tolbutamide Torsemide S warfarin ) (Flockhart DA, 2007; Zhou et al, 2005) ก ก ก ก ก ก ก
5.2 ก กก ก CYP2A6 กก ก E. coli XL 1 blue ก ก ก ก ก CYP2A6
3% v/v ก CYP2A6 50% (IC 50 ) ก กก CYP3A4 CYP2C9 4 5 ก ก ก กก ก ก mechanism based ก ก
5.3 ก ก ก P450 ก ก CYP3A4 CYP2C9 ก ก
6 ก Ahijevych KL, Tyndale RF, Dhatt RK, Weed HG, and Browning KK (2002) Factors influencing cotinine half life during smoking abstinence in African American and Caucasian women. Nicotine Tob Res 4:423 431. Anzenbacherováa E, Veinlichováb A, Mašekb V, Anzenbacherb P. (2005). Comparison of High throughput micromethods for determination of Cytochrome P450 activities with classical methods using HPLC for product identification. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 149(2):353 5. Apinan R, Tassaneeyakul W, Mahavorasirikul W, Satarug S, Kajanawart S, Vannaprasaht S, Ruenweerayut R, Na Bangchang K. (2010). The influence of CYP2A6 polymorphism and cadmium on nicotine metabolism in Thai population. Environemntal Toxicology and Pharmacology , 28, 420 424. Benowitz,NL (2008) Clinical pharmacology of nicotine: implications for understanding, preventing and treating tobacco addiction. Clin Pharmacol Ther 83:531 541. Benowitz NL, Jacob P 3rd, and Perez Stable E (1996) CYP2D6 phenotype and the metabolism of nicotine and cotinine. Pharmacogenetics 6:239 242. Bernhardt R. (2006). Cytochrome P450 as versatile biocatalysts. Biotechnology . 124: 128 145. Carrozzi L, PistelliF and Viegi G (2008) Pharmacotherapy for smoking cessation Ther Adv Respir Dis 2008; 2; 301 317. Damaj MI, Siu ECK, Sellers EM, Tyndale RF and Martin BR (2007) Inhibition of nicotine metabolism by methoxysalen: pharmacokinetic and pharmacological studies in mice. J. Pharmacol Exp Ther 320: 250 257. Di YM, Chow VDW, Yang LP, Zhou SF. (2009). Structure, function, regulation and polymorphism of Human Cytochrome P450 2A6. Current Drug Metabolism , 10, 754 780. DeVore NM, Smith BD, Urban MJ and Scott EE (2008) Key residues controlling phenacetin metabolism by human cytochrome P450 2A enzymes. Drug Metab Disp 36:2582 2590. DeVore NM, Smith BD, Wang JL, Lushington GH and Scott EE (2008) Key residues controlling binding of diverse ligands to human cytochrome P450 2A enzymes. Drug Metab Disp 36:2582 2590 Donato MT, Jiménez N, Castell JV, Gómez Lechón J. (2004) Fluorescence based assay for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Disp 32: 699 706 Döhr O, Paine MJ, Friedberg T, Robert GCK, Wolf R. (2001) Enginerring of a functional human NADH dependent cytochrome P450 system. Proc. Natl. Acad. Sci. USA. 98: 81 86.
Emre M, Isin F, Guengerich P. (2007). Complex reactions catalyzed by cytochrome P450 enzyme. Biochemical et Biophysica Acta . 1770: 314 329. Flammang AM, Gelboin HV, Aoyama T, Gonzalez FJ & McCoy GD (1992) Nicotine metabolism by cDNA expressed human cytochrome P 450s. Biochem Arch 8: 1 8. Flockhart DA. (2007). Drug Interactions: Cytochrome P450 Drug Interaction Table. Indiana University School of Medicine http://medicine.iupui.edu/clinpharm/ddis/table.aspx. Accessed [date]. Fowler S and Zhang H. (2008) In Vitro Evaluation of Reversible and Irreversible Cytochrome P450 Inhibition: Current Status on Methodologies and their Utility for Predicting Drug Drug Interactions. The AAPS Journa. 10 (2): 410 424 Fujita K, Hidaka M, Takamura N, Yamasaki K, Iwakiri T, Okumura M, Kodama H, Yamaguchi M, Ikenoue T, Arimori K. (2003) Inhibitory effects of citrus fruits on cytochrome P450 3A (CYP3A) activity in humans. Biol Pharm Bull, 26(9):1371 1373. Fukami T, Nakajima M, Yoshida R, Tsuchiya Y, Fujiki Y, Katoh M, McLeod HL, Yokoi T. (2004) A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo . Clin. Pharmcol Ther . 76: 519 576. Grime KH, Bird J, Ferguson D, and Riley RJ. (2009) Mechanism based inhibition of cytochrome P450 enzymes: An evaluation of early decision making in vitro approaches and drug drug interaction prediction methods. European journal of pharmaceutical sciences. 36: 175 191 Guengerich FP. (2001). Common and Uncommon Cytochrome P450 Reactions Related to Metabolism and Chemical Toxicity. Chemical Research in Toxicology . 14(6): 611 650. Guo LQ, Fukuda K, Ohta T, Yamazoe Y. (2000) Role of furanocoumarin derivatives on grapefruit juice mediated inhibition of human CYP3A activity. Drug Metab Dispos, 28(7):766 771. Hecht SS (1998) Biochemistry, biology, and carcinogenicity of tobaccospecific N nitrosamines. Chem Res Toxicol 11: 559 603. Hecht SS (1999a) DNA adduct formation from tobacco specific N nitrosamines. Mutat. Res. 424: 127 142. Hecht SS (1999b) Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst 91: 1194 1210. Hosoi S, Shimizu E, Arimori K, Okumura M, Hidaka M, Yamada M, and Sakushima A. (2008) Analysis of CYP3A inhibitory components of star fruit ( Averrhoa carambola L.) using liquid chromatography mass spectrometry. J Nat Med 62:345 348 Hubbard PA, Shen AL, Paschke R, Kasper CB, and Kim JJ P. (2001) NADPH Cytochrome P450 Oxidoreductase. J Biol Chem 276: 29163 29170.
Hukkanen JP, and Benowitz NL (2005) Metabolism and disposition kinetics of Nicotine. Pharmacol Rev 57:79 115. Hukkanen J, Pelkonen O, Hakkola J, and Raunio H (2002) Expression and regulation of xenobiotic metabolizing cytochrome P450 (CYP) enzymes in human lung. Crit Rev Toxicol 32:391 411. Kamataki T, Fujieda M, Kiyotani K, Iwano S and Kunitoh H (2005) Genetic polymorphism of CYP2A6 as one of the potential determinants of tobacco related cancer risk. Biochem Biophys Res Comm 338: 306 310. Kim H, Yoon YJ, Shon JH, Cha IJ, Shin JG, Liu KH. (2006) Inhibitory effects of fruit juices on cyp3a activity. Drug Metab Dispos . 34(4):521 523. Koudsi NA, Jasjit SA, Lin S K, Sellers EM and Tyndale RF (2009) A novel CYP2A6 allele (CYP2A6*35 ) resulting in an amino acid substitution (Asn438Tyr) is associated with lower CYP2A6 activity in vivo. Pharmacogenomics J. 1: 1 9 Kwon J, Nakajima M, Chai S et al (2001) Nicotine metabolism and CYP2A6 allele frequencies in Koreans. Pharmacogenetics 11:317 323 Lamb DC, Warrilow AGS, Venkateswarlu K, Kelly DE, Kelly SL. (2001) Activities and Kinetic Mechanisms of Native and Soluble NADPH Cytochrome P450 Reductase. Biochem. Biophy. Res. Comm. 286:48 54. Leartsakulpanitch J, Nganthavee W, Salole E (2007) The Economic Burden of Smoking Related Disease in Thailand: A Prevalence Based Analysis . J Med Assoc Thai 90 (9): 1925 9 Li Y, Li NY, Sellers EM. (1997). Comparison of CYP2A6 catalytic activity on coumarin 7 hydroxylation in human and monkey liver microsomes. Eur J Drug Metab Pharmacokinet . 22 (4):295 304. Liu M, Li XQ, Weber C, Lee CY, Brown J, Liu RH (2002) Antioxidant and antiproliferative activities of raspberries. J Agric Food Chem 50:2926 2930. MacDougall JM, Fandrick K, Zhang X, Serafin SV, and Cashman JR (2003) Inhibition of human liver microsomal ( S) nicotine oxidation by menthol and analogues. Chem Res Toxicol 16:988 993. Mahavorasirikul W, Tassaneeyakul W, Satarug S, Reungweerayut R, Na Bangchang C, and Na Bangchang K. (2009) CYP2A6 genotypes and coumarin oxidation phenotypes in a Thai population and their relationship to tobacco smoking. Eur J Clin Pharmacol 65:377 384 Mathews JM, Etheridge AS, and Black SR (2002) Inhibition of human cytochrome P450 activities by kava extract and kavalactones. Drug Metab Dispos 30: 1153 1157. Matern U, Lüer P, and Kreusch D (1999) Biosynthesis of coumarins. Pp. 623 637. In: Barton D, Nakanishi K, Meth Cohn O. and Sankawa U. (eds.): Comprehensive Natural Products Chemistry, Vol. 1,
Polyketides and Other Secondary Metabolites Including Fatty Acids and Their Derivatives. Elsevier Science Ltd., Oxford, UK. McCarver DG, Byun R, Hines RN, Hichme M, and Wegenek W (1998) A genetic polymorphism in the regulatory sequences of human CYP2E1: association with increased chlorzoxazone hydroxylation in the presence of obesity and ethanol intake. Toxicol Appl Pharmacol 152:276 281. Merkel U, Sigusch H, and Hoffmann A (1994) Grapefruit juice inhibits 7 hydroxylation of coumarin in healthy volunteers. Eur J Clin Pharmacol 46:175 177. Miksys S, Rao Y, Hoffmann E, Mash DC, and Tyndale RF (2002) Regional and cellular expression of CYP2D6 in human brain: higher levels in alcoholics. J Neurochem 82:1376 1387. Miles JS, McLaren AW, Forrester LM, Glancey MJ, Lang MA and Wolf CR (1990) Identification of the human liver cytochrome P 450 responsible for coumarin 7 hydroxylase activity. Biochem J 267:365 371. Ministry of Public Health. Thai behavioral risk factor surveillance report 2005. Bangkok: Ministry of Public Health, Bureau of Non Communicable Disease; 2005. 2008 Miyamoto M, Umetsu Y, Dosaka Akita H, Sawamura Y, Yokota J, Kunitoh H, Nemoto N, Sato K, Ariyoshi N, Kamataki T (1999) CYP2A6 gene deletion reduces susceptibility to lung cancer, Biochem Biophys Res Commun 261 658 660. Miyazaki M, Yamazaki H, Takeuchi H, Saoo K, Yokohira M, Masumura K I, Nohmi T, Funae Y, Imaida K and Kamataki T. (2005) Mechanisms of chemopreventive effects of 8 methoxypsoralen against 4 (methylnitrodamino) 1 (3 pyridyl) 1 butanone induced mouse lung carcinomas. Carcinogenesis 26: 1947 1955. Murataliev MB, Ari ňo A, Guzov VM. Feyereisen, R. (1999) Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica). Insect Biochem. Mol. Biol . 29: 233 242. Mansuy D. (1998). The great diversity of reactions catalyzed by cytochrome P450. Comparative Biochemistry and Physiology Part C . 121: 5 14. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, Waterman MR, Gotoh O, Coon MJ, Estabrook RW, Gunsalus IC, Nebert DW. (1996). P450 superfamily: update on new sequences, gene mapping, accession numbers, and nomenclature. Pharmacogenetics . 6: 1 42. Ortiz de Montellano, PR. (Ed.), (2005). Cytochrome P450: Structure, Mechanism and Biochemistry , third edition. New York : Kluwer Academic/Plenum Publishers. Patten CJ, Smith TJ, Murphy SE, Wang MH, Lee J, Tynes RE, Koch P, and Yang CS (1996) Kinetic analysis of the activation of 4 (methylnitrosamino) 1 (3 pyridyl) 1 butanone by heterologously expressed human P450 enzymes and the effect of P450 specific chemical inhibitors on this activation in human liver microsomes. Arch Biochem Biophys 333 : 127 138. Peamkrasatam S, Sriwatanakul K, Kiyotani K, Fujieda M, Yamazaki H, Kamataki T, Yoovathaworn K (2006) In vivo evaluation of coumarin and nicotine as probe drugs to predict he metabolic capacity of CYP2A6 due to genetic polymorphism in Thais. Drug Met Pharmacokinet 21:475 484. Pelkonen O, Rautio A, Raunio H and Pasanen M (2000) CYP2A6 a human coumarin 7 hydroxylase. Toxicology 144:139 147. Runkel M, Bourian M, Tegtmeier M, and Legrum W (1997) The character of inhibition of the metabolism of 1,2 benzopyrone (coumarin) by grapefruit juice in human. Eur J Clin Pharmacol 53:265 269. Schoedel KA, Hoffmann EB, Rao Y, Sellers EM, Tyndale RF. 2004. Ethnic variation in CYP2A6 and association of genetically slow nicotine metabolism and smoking in adult Caucasians. Pharmacogenetics 14:615 626. Sellers EM, Kaplan HL, and Tyndale RF (2000) Inhibition of cytochrome P450 2A6 increases nicotine s oral bioavailability and decreases smoking. Clin PharmacolTher 68:35 43. Sellers EM, Ramamoorthy Y, Zeman MV, Djordjevic MV, and Tyndale RF (2003a) The effect of methoxsalen on nicotine and 4 (methylnitrosamino) 1 (3 pyridyl) 1 butanone (NNK) metabolism in vivo. Nicotine Tob Res 5:891 899. Sellers EM, Tyndale RF and Fernandes LC (2003) Decreasing smoking behaviour and risk through CYP2A6 inhibition Drug Discov Today 8:487 493. Sem DS. and Kasper CB. (1993) Enzyme substrate binding interactions of NADPH cytochrome P450 oxidoreductase characterized with pH and alternate substrate/inhibitor studies. Biochemistry . 32: 11539 11547. Shen AL, Porter TD, Wilson TE, Kasper CB. (1989). Structural analysis of the FMN binding domain of NADPHcytochrome P450 oxidoreductase by site direct mutagenesis. Biological Chemistry . 254: 7584 7589. Shui GH, Leong LP (2006) Residue from star fruit as valuable source for functional food ingredients and antioxidant nutraceuticals. Food Chem 97:277 284. Siu ECK and Tyndale RF (2007) Non nicotinic therapies for smoking cessation. Annu Rev Pharmacol Toxicol . 47:541 64 Siu ECK and Tyndale RF (2008) Selegiline is a mechanism based inactivator of CYP2A6 inhibiting nicotine metabolism in humans and mice. J Pharmacol Exp Ther 324: 992 999.
Smith GCM., Tew DG, Wolf CR. (1994) Dissection of NADPH cytochrome P450 oxidoreductase into distinct functional domains. Proc. Natl. Acad. Sci. USA . 91; 8710 8714. Soucek P. (1999) Expression of cytochrome P4502A6 in Escherichia coli, purification, spectral, and catalytic characterization and preparation of polyclonal antibodies. Arch Biochem Biophys . 370:190 200. Su T, Bao Z, Zhang Q Y, Smith T J, Hong J Y, and Ding X (2000) Human cytochrome P450 CYP2A13: predominant expression in the respiratory tract and its high efficiency metabolic activation of a tobacco specific carcinogen, 4 (Methylnitrosamino) 1 (3 pyridyl) 1 butanone. Cancer Res 60: 5074 5079 Taavitsainen P, Juvonen R, Pelkonen O. (2001) In vitro inhibition of cytochrome P450 enzymes in human liver microsomes by a potent CYP2A6 inhibitor, trans 2 phenylcyclopropylamine (tranylcypromine), and its nonamine analog, cyclopropylbenzene. Drug Metab Disp 29:217 222. Tassaneeyakul W, Guo LO, Fukuda K, Ohta T, Yamazoe Y. (2000) Inhibition selectivity of grapefruit juice components on human cytochromes P450. Arch Biochem Biophys 378:356 63. Tiong KH, Yiap BC, Tan EL, Ismail R, Ong CE. (2010). Functional characterization of cytochrome P450 2A6 (CYP2A6) allelic variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22 *. Drug Metab Disp . In press Tyndale RF, Sellers EM (2002) Genetic variation in CYP2A6medicated nicotine metabolism alters smoking behavior. Ther Drug Monit 24:163 171. Tyndale RF and Sellers EM (2001) Variable Cyp2a6 mediated nicotine metabolism alters smoking behavior and risk. Drug Metab Disp 29:548 552. Ujjin P, Satarug S, Vanavanitkun Y et al (2002) Variation in coumarin 7 hydroxylase activity associated with genetic polymorphism of cytochrome P450 2A6 and the body status of iron stores in adult Thai males and females. Pharmacogenetics 12:241 249 von Weymarn LB, Chun JA, Knudsen GA, and Hollenberg PF (2007) Effects of eleven isothiocyanates on P450 2A6 and 2A13 catalyzed coumarin 7 hydroxylation. Carcinogenesis 27: 782 790 von Weymarn LB, Zhang QY, Ding X, Hollenberg PF. (2005) Effects of 8 methoxypsoralen on cytochrome P450 2A13. Carcinogenesis 26: 621 629. Weinmann I (1997): History of the development and applications of coumarin and coumarin related compounds. Pp. 1 22. In: O Kennedy & Thornes. WHO Report on the Global Tobacco Epidemic, 2008: The MPOWER package. Geneva, World Health Organization. Wongwiwatthananukit S, Benjanakaskul P, Songsak T, Suwanamajo S, Verachai V. (2009). Efficacy of Vernonia cinerea for smoking cessation. Journal of Health Research , 23 (1), 31 36
Xi Z, Spiller K, Gardner EL. (2009) Mechanism based medication development for the treatment of nicotine dependence. Acta Pharmacologica Sinica , 30: 723 739. Yamano S, Tatsuno J and Gonzalez FJ (1990) The CYP2A3 gene product catalyzes coumarin 7 hydroxylation in human liver microsomes. Biochemistry 29:1322 1329. Yamazaki H, Nakamura m, Komatsu T, Ohyama K, Hatanaka N, Asahi S, Shimada N, Guengerich FP, Shimada T, Nakajima M, and Yokoi T. (2002). Roles of NADPH P450 Reductase and Apo and Holo Cytochrome b5 on Xenobiotic Oxidations Catalyzed by 12 Recombinant Human Cytochrome P450s Expressed in Membranes of Escherichia coli. Protein Expr Purif 24, 329 337 Yano JK, Denton TT, Cemy MA, Zhang X, Johnson EF, and Cashman JR (2006). Synthetic inhibitors of cytochrome P450 2A6: Inhibitory activity, difference spectra, mechanism of inhibition, and protein cocrystallization. J Med Chem 49: 6987 7001. Yoo HH, Lee MW, Kim YC, Yun C H, and Kim D H (2007) Mechanism based inactivation of cytochrome p450 2a6 by decursinol angelate isolated from Angelica gigas . Drug Metab Dispos 35:1759 1765 Yoshida R, Nakajima M, Watanabe Y, Kwon JT, Yokoi T (2002) Genetic polymorphisms in human CYP2A6 gene causing impaired nicotine metabolism. Br J Clin Pharmacol 54:511 517 Zhou S, Chan SY, Goh BC, Chan E, Duan W, Huang M, and McLeod HL (2005) Mechanism Based Inhibition of Cytochrome P450 3A4 byTherapeutic Drugs. Clin Pharmacokinet . 44 (3): 279 304 ก (2545) 108 ก ก ก ก 224 ก ก (2549) ก 2 ก ก ก ก 84 ก ก (2552) 2 ก ก ก 234 ก (2550) ก ก ก ก ก 2 ก 215 ก (2011) ก CYP2A6 : ก ก ก . ก . 16 2 (2539) 1 3 ก 2400 ก (2551) ก ก ก . . 2534 2550 ก ก 228
. (2552). ก ก ก ก . . 2552 . ก ก ก (2546) ก ก ก ก ก ก ก ก 111 (2551) 6 ก ก ก ก 84
ก ก ก
1. ก CYP2A6 1.1 ก ก 5’-TTCGACTGGGTCTTCAAAGGC TATGGCGTGGTATTCAGCAACGGGGAGCGCG CCAAGCAGCTCCGGCGCTTCTCCATCGCCACCCTGCGGGACTTCGGGGTGGGCAA GCGAGGCATCGAGGAGCGCATCCAGGAGGAGGCGGGCTTCCTCATCGACGCCCTC CGGGGCACTGGCGGCGCCAATATCGATCCCACCTTCTTCCTGAGCCGCACAGTCT CCAATGTCATCAGCTCCATTGTCTTTGGGGACCGCTTTGACTATAAGGACAAAGA GTTCCTGTCACTGTTGCGCATGATGCTAGGAATCTTCCAGTTCACGTCAACCTCC ACGGGGCAGCTCTATGAGATGTTCTCTTCGGTGATGAAACACCTGCCAGGACCAC AGCAACAGGCCTTTCAGTTGCTGCAAGGGCTGGAGGACTTCATAGCCAAGAAGGT GGAGCACAACCAGCGCACGCTGGATCCCAATTCCCCACGGGACTTCATTGACTCC TTTCTCATCCGCATGCAGGAGGAGGAGAAGAACCCCAACACGGAGTTCTACTTGA AAAACCTGGTGATGACCACGTTGAACCTCTTCATTGGGGGCACCGAGACCGTCAG CACCACCCTGCGCTATGGCTTCTTGCTGCTCATGAAGCACCCAGAGGTGGAGGCC AAGGTCCATGAGGAGATTGACAGAGTGATCGGCAAGAACCGGCAGCCCAAGTTTG AGGACCGGGCCAAGATGCCCTACATGGAGGCAGTGATCCACGAGATCCAAAGATT TGGAGACGTGATCCCCATGAGTTTGGCCCGCAGAGTCAAAAAGGACACCAAGTTT CGGGATTTCTTCCTCCCTAAGGGCACCGAAGTGTACCCTATGCTGGGCTCTGTGC TGAGAGACCCCAGTTTCTTCTCCAACCCCCAGGACTTCAATCCCCAGCACTTCCT GAATGAGAAGGGGCAGTTTAAGAAGAGTGATGCTTTTGTGCCCTTTTCCATCGGA AAGCGGAACTGTTTCGGAGAAGGCCTGGCCAGAATGGAGCTCTTTCTCTTCTTCA CCACCGTCATGCAGAACTTCCGCCTCAAGTCCTCCCAGTCACCTAAGGACATTGA CGTGTCCCCCAAACACGTGGGCTTTGCCACGATCCCACGAAACTACAC CATGAGC TTCCTGCCCCGC -3’
1 CYP2A6 กก primer ก
2. ก rat CPR 5-CGCAAATCTCAGTTCCGCTTG CCTTTCAAGTCCACCACACCTGTCATCATGG TGGGCCCCGGCACTGGGATTGCCCCTTTCATGGGCTTCATCCAGGAACGAGCTTG GCTTCGAGAGCAAGGCAAGGAGGTGGGAGAGACGCTGCTATACTATGGCTGCCGG CGCTCGGATGAGGACTATCTGTACCGTGAAGAGCTAGCCCGCTTCCACAAGGACG GTGCCCTCACGCAGCTTAATGTGGCCTTTTCCCGGGAGCAGGCCCACAAGGTCTA TGTCCAGCACCTTCTGAAGAGAGACAGGGAACACCTGTGGAAGCTGATCCACGAG GGCGGTGCC CACATCTATGTGTGCGGGGATG -3’ 2 rat CPR กก primer ก
1. ก ก ก Songklod Sarapusit, Ph.D 169 . . . . 20131 038 102 222 3058 081 914 7347 038 393 495 [email protected] ก ก . . 2551 ก . . 2543
2. ก . Pornpimol Rongnoparut, Ph.D. ก 272 . 6 ก 10400 02 201 5453 086 988 4121 02 354 7174 [email protected] ก ก . . 2533 ก ก ก . . 2523 ก