肺癌易感性与CYP1A1、GSTM1基因多态性及烟焦油的相关研究
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摘要
本文首先研究了内蒙古地区正常蒙、汉族人群CYP1A1基因Msp Ⅰ位点多态性,为研究肺癌易感性与CYP1A1和GSTM1基因多态性的关系奠定了基础。为阐明肺癌易感性与CYP1A1和GSTM1及烟焦油的关系,进一步研究了烟焦油对CYP1A1、AHR基因在mRNA水平上表达的影响以及烟焦油对AHH、GST的影响,从基因型、基因表达及相关酶三个层面上对肺癌的易感性进行了系统的研究。
一、内蒙古地区正常蒙族和汉族人群CYP1A1基因MspⅠ位点多态性的研究
采用PCR-RFLP技术分析了无血缘关系的80例蒙族和120例汉族人群的基因型。研究内蒙古地区正常蒙族和汉族人群CYP1A1基因Msp Ⅰ位点的多态性,结果表明内蒙古地区蒙族和汉族人群CYPlA1基因野生型纯合子(wt/wt)、杂合子(wt/vt)、突变型纯合子(vt/vt)三种基因型的频率分布分别是:蒙族35.0%、48.7%、16.3%和汉族33.3%、52.5%、14.2%。二者之间经x~2检验无显著性差异(P>0.05)。通过对内蒙古地区蒙族、汉族人群是否符合Hardy-Weinberg平衡法则
1. Polymorphisms of CYPlAl genes Msp I in the Mongolian nationality and Han nationality population of Inner MongoliaThe polymorphisms of CYP1Al genes Msp I were determined by PCR-RFLP in 80 unrelated subjects of the Mongolian nationality and 120 unrelated subjects of Han nationality. The genotype frequency distributions of CYPlAl genotypes of wild-type, heterozygote, homozygous variant in Mongolian nationality population and Han nationality population were 35.0% 48.7% 16.3% and 33.3% 52.5% 14.2% respectivily. Chi-square tests showed that there was no significant difference between the two groups. The mathematics test showed that Mongolian nationality squares with the law of Hardy-Weinberg of genetic equilibrium but Han nationality deviates from the law of Hardy-Weinberg of genetic equilibrium a little. The coincidental test showed that both Mongolian and Han nationality
square with the law of Hardy-Weinberg of genetic equilibrium. The genotype frequency distributions of CYPlAl gene of both groups did not exhibit obvious difference.2. Susceptibility to lung cancer in relation to CYPlAl and GSTM1 genetic polymorphisms in population of Inner MongoliaCYPlAl and GSTM1 genetic polymorphisms were determined by PCR-RFLP in 63 lung cancer cases and healthy controls respectively. The frequencies of CYPlAl variation type (vt/vt) and GSTMl(-) were 36.5% , 65.1% and 19.1% , 47.6% in lung cancer cases and healthy controls respectively. Statistical tests showed significant difference between the two groups. Combined analysis of the polymorphisms showed that CYP1Al (vt/vt)/ GSTMl(-) and CYPlAl(vtM) / GSTM1(+) were 28.6% and 7.9% respectively among the lung cancer patients who were tested. Statistical tests showed significant difference between the two groups. Pearson Chi-Square of sex differential showed that there was no significance between the homozygous variation genotype of CYPlAl / GSTMl(-) and the other genotypes of CYPlAl / GSTMl(-). Statistical tests showed that susceptibility to lung cancer was related to smoking. There may be a synergetic interaction between CYPlAl variation type (vt/vt) and smoking on the elevated susceptibility to lung cancer, but
GSTMl(-) and smoking may not. The polymorphisms of CYPlAl variation type (vt/vt) and GSTMl(-) were the risk factors of lung cancer of Inner Mongolia. There may be a synergetic interaction between CYPlAl variation type (vt/vt) and GSTMl(-) on the elevated susceptibility to lung cancer, So is the CYPlAl variation type (vt/vt) and smoking. Smoking may also relate to the susceptibility to lung cancer.3. Influence of tar of cigarettes on the expression of aromatic hydrocarbon receptor genes and CYPlAl genes of mice lungsTo study the influence of tar of cigarettes on the expression of AHR genes and CYPlAl genes of mice lungs and their time and dose-response relationships, the mice were injected by intra-abdominally with 1%LD5O (5.29mg/kg), 2%LD50 (10.58mg/kg) and 3%LD50 (15.87mg/kg) cigarettes tar for 24, 48 and 72 hours once a day respectively. Total RNA of mice lungs extracted was purified by RNA kit. RT-PCR technique was used to determine AHR and CYPlAl genetic expression. The expression of AHR and CYPlAl genes of mice lungs at different doses of tar of cigarettes and at different times were analyzed with P -actin as control. AHR genetic expression increased after 72 hours treated with 1%LD5O cigarettes tar. AHR and CYPlAl genes increased after 48 and 72 hours treated with 2%LD50 and 3%LD50 cigarettes tar.
Tar of cigarettes could regulate up AHR and CYP1A1 genes at some doses and some times. The effects of up-regulation showed time and dose-dependent relationship. The regulations of expression of AHR genes were earlier than CYP1 Al genes and with low doses.4. Influence of tar of cigarettes and passive smoking on the activity of aromatic hydrocarbon hydryoxylase and glutathion S-transferase of mice lungsThe Influence of tar of cigarettes and passive smoking on the activity of aromatic hydrocarbon hydryoxylase (AHH) and glutathion S-transferase(GST) of mice lungs and their time and dose-response relationship were studied. The mice were intra-abdominally with 1%LD5O (5.29mg/kgX2%LD50( 10.58mg/kg)and 3%LD50( 15.87mg/kg)cigarettes tar for 24, 48 and 72 hours once a day respectively. The control groups were injected intra-abdominally with 0.5% tween-80 normal saline once a day. Then the activity of AHH and GST were tested. Passive smoking group was put in the box of 50L. The level of smog was maintained 69.41 g/m3 for 30 minute two times a day. Control group was not smoking but at the same condition. Then the activity of AHH and GST were tested. The activity of AHH increased after 72 hours treatment with 2%LD50 cigarettes tar and with 3%LD50 cigarettes tar for 48 hours, 72hours . The activity of
AHH of mice lungs treated with tar of cigarettes of 2%LD50 and 3%LD50 was different 48 hours and 72 hours later. The statistical tests showed significant difference. The effects of up-regulation showed dose-dependent and time-dependent relationship. The activity of AHH of mice lungs was also increased by passive smoking. There were no significant difference of GST activity between the treatment of three kinds of concentration of cigarettes tar and control, so was the passive smoking.
一、内蒙古地区正常蒙族和汉族人群CYP1A1基因MspⅠ位点多态性的研究
采用PCR-RFLP技术分析了无血缘关系的80例蒙族和120例汉族人群的基因型。研究内蒙古地区正常蒙族和汉族人群CYP1A1基因Msp Ⅰ位点的多态性,结果表明内蒙古地区蒙族和汉族人群CYPlA1基因野生型纯合子(wt/wt)、杂合子(wt/vt)、突变型纯合子(vt/vt)三种基因型的频率分布分别是:蒙族35.0%、48.7%、16.3%和汉族33.3%、52.5%、14.2%。二者之间经x~2检验无显著性差异(P>0.05)。通过对内蒙古地区蒙族、汉族人群是否符合Hardy-Weinberg平衡法则
1. Polymorphisms of CYPlAl genes Msp I in the Mongolian nationality and Han nationality population of Inner MongoliaThe polymorphisms of CYP1Al genes Msp I were determined by PCR-RFLP in 80 unrelated subjects of the Mongolian nationality and 120 unrelated subjects of Han nationality. The genotype frequency distributions of CYPlAl genotypes of wild-type, heterozygote, homozygous variant in Mongolian nationality population and Han nationality population were 35.0% 48.7% 16.3% and 33.3% 52.5% 14.2% respectivily. Chi-square tests showed that there was no significant difference between the two groups. The mathematics test showed that Mongolian nationality squares with the law of Hardy-Weinberg of genetic equilibrium but Han nationality deviates from the law of Hardy-Weinberg of genetic equilibrium a little. The coincidental test showed that both Mongolian and Han nationality
square with the law of Hardy-Weinberg of genetic equilibrium. The genotype frequency distributions of CYPlAl gene of both groups did not exhibit obvious difference.2. Susceptibility to lung cancer in relation to CYPlAl and GSTM1 genetic polymorphisms in population of Inner MongoliaCYPlAl and GSTM1 genetic polymorphisms were determined by PCR-RFLP in 63 lung cancer cases and healthy controls respectively. The frequencies of CYPlAl variation type (vt/vt) and GSTMl(-) were 36.5% , 65.1% and 19.1% , 47.6% in lung cancer cases and healthy controls respectively. Statistical tests showed significant difference between the two groups. Combined analysis of the polymorphisms showed that CYP1Al (vt/vt)/ GSTMl(-) and CYPlAl(vtM) / GSTM1(+) were 28.6% and 7.9% respectively among the lung cancer patients who were tested. Statistical tests showed significant difference between the two groups. Pearson Chi-Square of sex differential showed that there was no significance between the homozygous variation genotype of CYPlAl / GSTMl(-) and the other genotypes of CYPlAl / GSTMl(-). Statistical tests showed that susceptibility to lung cancer was related to smoking. There may be a synergetic interaction between CYPlAl variation type (vt/vt) and smoking on the elevated susceptibility to lung cancer, but
GSTMl(-) and smoking may not. The polymorphisms of CYPlAl variation type (vt/vt) and GSTMl(-) were the risk factors of lung cancer of Inner Mongolia. There may be a synergetic interaction between CYPlAl variation type (vt/vt) and GSTMl(-) on the elevated susceptibility to lung cancer, So is the CYPlAl variation type (vt/vt) and smoking. Smoking may also relate to the susceptibility to lung cancer.3. Influence of tar of cigarettes on the expression of aromatic hydrocarbon receptor genes and CYPlAl genes of mice lungsTo study the influence of tar of cigarettes on the expression of AHR genes and CYPlAl genes of mice lungs and their time and dose-response relationships, the mice were injected by intra-abdominally with 1%LD5O (5.29mg/kg), 2%LD50 (10.58mg/kg) and 3%LD50 (15.87mg/kg) cigarettes tar for 24, 48 and 72 hours once a day respectively. Total RNA of mice lungs extracted was purified by RNA kit. RT-PCR technique was used to determine AHR and CYPlAl genetic expression. The expression of AHR and CYPlAl genes of mice lungs at different doses of tar of cigarettes and at different times were analyzed with P -actin as control. AHR genetic expression increased after 72 hours treated with 1%LD5O cigarettes tar. AHR and CYPlAl genes increased after 48 and 72 hours treated with 2%LD50 and 3%LD50 cigarettes tar.
Tar of cigarettes could regulate up AHR and CYP1A1 genes at some doses and some times. The effects of up-regulation showed time and dose-dependent relationship. The regulations of expression of AHR genes were earlier than CYP1 Al genes and with low doses.4. Influence of tar of cigarettes and passive smoking on the activity of aromatic hydrocarbon hydryoxylase and glutathion S-transferase of mice lungsThe Influence of tar of cigarettes and passive smoking on the activity of aromatic hydrocarbon hydryoxylase (AHH) and glutathion S-transferase(GST) of mice lungs and their time and dose-response relationship were studied. The mice were intra-abdominally with 1%LD5O (5.29mg/kgX2%LD50( 10.58mg/kg)and 3%LD50( 15.87mg/kg)cigarettes tar for 24, 48 and 72 hours once a day respectively. The control groups were injected intra-abdominally with 0.5% tween-80 normal saline once a day. Then the activity of AHH and GST were tested. Passive smoking group was put in the box of 50L. The level of smog was maintained 69.41 g/m3 for 30 minute two times a day. Control group was not smoking but at the same condition. Then the activity of AHH and GST were tested. The activity of AHH increased after 72 hours treatment with 2%LD50 cigarettes tar and with 3%LD50 cigarettes tar for 48 hours, 72hours . The activity of
AHH of mice lungs treated with tar of cigarettes of 2%LD50 and 3%LD50 was different 48 hours and 72 hours later. The statistical tests showed significant difference. The effects of up-regulation showed dose-dependent and time-dependent relationship. The activity of AHH of mice lungs was also increased by passive smoking. There were no significant difference of GST activity between the treatment of three kinds of concentration of cigarettes tar and control, so was the passive smoking.
引文
1. Lautmite S, Musonada AC, Doehmer J, etc. Flavonoids inhibit genetic toxicity produced by carcinogens in cells espressing CYP1A1 and CYP1A1. Mutagenesis, 2002,17(1):45-53
2. Wong JM, Okey AB, Harper PA. Human aryl hydrocarbon receptor polymorphisms that result in loss of CYP1A1 induction. Biochem. Biophys. Res. Commun., 2001,288(4): 990-996
3. Smith G, Stubbins MJ, Harries LW, etc . Molecular genetics of the human cytochrome P450 monooxygenase superfamily. Xenobiotica, 1998, 28(12): 1129- 1165
4. Rendic S, Di Carlo FJ. Human cytochrome P450 enzymes: a status report summarizing their reaction, substrates,inducers and inhibitors. Drug Metab Rev, 1997, 29(l-2):413-580
5. Wang HW, Chen TL, Yang PC, etc. Induction of cytochromes P4501A1 and 1B1 in human lung adenocarcinoma CL5 cells by frying-meat emission particulate. Food Chem Toxicol, 2002, 40(5): 653-661
6. Shinkyo R, Sakaki T, Ohta M, etc. Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYPIA subfamily in the metabolism of dioxins. Arch. Biochem. Biophys., 2003, 409(1): 180-187
7. Oeky AB. Enzyme induction in the cytochrome P450 system. Pergamon Press Inc(New York), 1992, 549-608
8. Kawajiri K, Nakachi K, Imai K, etc. Identification of genetically high risk individuals to lung cancer by DNA polymorphisms of the cytochrome P450 1A1 gene. FEBS LETT, 1990,263(1): 131-135.
9. Hayashi SI, Watanabe J, Nakachi K, etc. Gene linkage of lung cancer associated Msp I polymorphisms with amino acid replacement in the heme binding region of human cytochrome P4501A1 gene. J Biochem, 1991, 110(3): 407-411
10. Eric J. Duell, Elizabeth A. Holly, Paige M. Bracci, etc. A population-based, case- control study of polymorphisms in carcinogen-metabolizing genes, smoking, and pancreatic adenocarcinoma risk. J Natl Cancer Inst, 2002, 94(4):297-306
11. Maja Krajinovic, Damian Labuda, Chantal Richer, etc. Susceptibility to childhood acute lymphoblastic leukemia: Influence of CYPIA 1,CYP2D6,GSTM1,and GSTT1 genetic polymorphisms. Blood, 1999, 93(5):1496-1501
12. Ingolf Cascorbi, Jurgen Brockmoller, Ivar Roots. A C4887A Polymorphism in exon 7 of human CYP1A1: Population frequency, mutation linkages, and impact on lung cancer susceptibility. Cancer Research, 1996, 56: 4965-4969
13. C Sobti R, Sharma S, Joshi A, etc. CYP1A1 and CYP2D6 polymorphism and risk
of lung cancer in a North Indian population. Biomarkers, 2003, 8:415-428
14. Nakachi K, Imai K, Hayashi SI, etc. Genetic susceptibility to squamous cell carcinoma of the lung in relation to cigarette smoking dose. Cancer Res, 1991,51:5177-5180
15. Acevedo C, Opazo JL, Huidobro C, etc. Postive correlation between single or combined genotypes of CYP1A1 and GSTM1 in relation to prostate cancer in Chilean people. Prostate, 2003, 57(3): 111-117
16. Balta G, Yuksek N, Ozyurek E, etc. Characterization of MTHFR,GSTM1,GSTT1,GSTP1, and CYP1A1 genotypes in childhood acute leukemia. Am. J. Hematol. 2003, 73(3): 154-160
17. Kao SY, Wu CH, Lin SC, etc. Genetic polymorphism of cytochrome P4501A1 and susceptibility to oral squamous cell carcinoma and oral precancer lesions associated with smoking/betel use. J. Oral. Pathol. Med., 2002, 31 (9):505-511
18. Ye z, Parry JM. Genetic polymorphisms in the cytochrome P4501A1,glutathione Stransferasw M1 and T1, and susceptibility to colon cancer. Teratog. Carcinog. Mutagen., 2002, 22(5): 385-392
19. Tsuchiya Y, Sato T, Kiyohara C, etc. Genetic polymorphisms of cytochrome P4501A1 and risk of gallbadder cancer. J. Exp. Clin. Cancer Res. 2002, 21 (1): 119-124
20. Firozi PF, Bondy ML, Sahin AA, etc. Aromatec DNA adducts and polymorphisms of CYP1A1,NAT2,and GSTM1 in breast cancer. Carcinogenesis, 2002,23(2):301-306
21. Chan DK, Mellick GD, Buchanan DD, etc. Lack of association between CYP1A1 polymorphism and Parkinson's disease in a chinese population. J. Neural Transm., 2002,109(1):35-39
22. Kawajiri K, Eguchi H, Nakschi K, etc. Association of CYP1A1 gene line polymorphisms with mutations of the p53 gene in lung cancer. Cancer Res., 1996, 56(1):72-76
23.王安辉,孙长生,李良寿,等.CYP1A1与GSTM1基因的多态性与西安地区食管癌的关系.第四军医大学学报,2002,23(1):53-55
24.刘平,刘振华,邵明,等.中国北方汉族人细胞色素P4501A1基因MspⅠ多 态性与早发性帕金森病关系的研究.中国医学遗传学杂志,2001,18(4):283-285
25.尹立红,浦跃朴,林嫔嫔.南京市正常人群NQO1、CYP1A1、mEH基因的多态性研究.遗传,2001,23(3):199-202
26.胡毅玲,高杨,张桥.细胞色素P4501A1、2D6和谷胱甘肽硫转移酶基因多态性与肺癌易感性的研究.肿瘤,1998,18(4):269-271
27. Tefre T, Ryberg D, Haugen A,etc. Human CYP1A1(cytochrome P450) gene: lack of association between the Msp Ⅰ restriction fragment length polymorphism and incidence of lung cancer in a Norwegian population.Pharmacogenetics, 1991,1:20-25
28.胡云珍,姚彤炜.细胞色素P4501A1的研究进展.中国药学杂志,2003,38(4):246-250
2. Wong JM, Okey AB, Harper PA. Human aryl hydrocarbon receptor polymorphisms that result in loss of CYP1A1 induction. Biochem. Biophys. Res. Commun., 2001,288(4): 990-996
3. Smith G, Stubbins MJ, Harries LW, etc . Molecular genetics of the human cytochrome P450 monooxygenase superfamily. Xenobiotica, 1998, 28(12): 1129- 1165
4. Rendic S, Di Carlo FJ. Human cytochrome P450 enzymes: a status report summarizing their reaction, substrates,inducers and inhibitors. Drug Metab Rev, 1997, 29(l-2):413-580
5. Wang HW, Chen TL, Yang PC, etc. Induction of cytochromes P4501A1 and 1B1 in human lung adenocarcinoma CL5 cells by frying-meat emission particulate. Food Chem Toxicol, 2002, 40(5): 653-661
6. Shinkyo R, Sakaki T, Ohta M, etc. Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYPIA subfamily in the metabolism of dioxins. Arch. Biochem. Biophys., 2003, 409(1): 180-187
7. Oeky AB. Enzyme induction in the cytochrome P450 system. Pergamon Press Inc(New York), 1992, 549-608
8. Kawajiri K, Nakachi K, Imai K, etc. Identification of genetically high risk individuals to lung cancer by DNA polymorphisms of the cytochrome P450 1A1 gene. FEBS LETT, 1990,263(1): 131-135.
9. Hayashi SI, Watanabe J, Nakachi K, etc. Gene linkage of lung cancer associated Msp I polymorphisms with amino acid replacement in the heme binding region of human cytochrome P4501A1 gene. J Biochem, 1991, 110(3): 407-411
10. Eric J. Duell, Elizabeth A. Holly, Paige M. Bracci, etc. A population-based, case- control study of polymorphisms in carcinogen-metabolizing genes, smoking, and pancreatic adenocarcinoma risk. J Natl Cancer Inst, 2002, 94(4):297-306
11. Maja Krajinovic, Damian Labuda, Chantal Richer, etc. Susceptibility to childhood acute lymphoblastic leukemia: Influence of CYPIA 1,CYP2D6,GSTM1,and GSTT1 genetic polymorphisms. Blood, 1999, 93(5):1496-1501
12. Ingolf Cascorbi, Jurgen Brockmoller, Ivar Roots. A C4887A Polymorphism in exon 7 of human CYP1A1: Population frequency, mutation linkages, and impact on lung cancer susceptibility. Cancer Research, 1996, 56: 4965-4969
13. C Sobti R, Sharma S, Joshi A, etc. CYP1A1 and CYP2D6 polymorphism and risk
of lung cancer in a North Indian population. Biomarkers, 2003, 8:415-428
14. Nakachi K, Imai K, Hayashi SI, etc. Genetic susceptibility to squamous cell carcinoma of the lung in relation to cigarette smoking dose. Cancer Res, 1991,51:5177-5180
15. Acevedo C, Opazo JL, Huidobro C, etc. Postive correlation between single or combined genotypes of CYP1A1 and GSTM1 in relation to prostate cancer in Chilean people. Prostate, 2003, 57(3): 111-117
16. Balta G, Yuksek N, Ozyurek E, etc. Characterization of MTHFR,GSTM1,GSTT1,GSTP1, and CYP1A1 genotypes in childhood acute leukemia. Am. J. Hematol. 2003, 73(3): 154-160
17. Kao SY, Wu CH, Lin SC, etc. Genetic polymorphism of cytochrome P4501A1 and susceptibility to oral squamous cell carcinoma and oral precancer lesions associated with smoking/betel use. J. Oral. Pathol. Med., 2002, 31 (9):505-511
18. Ye z, Parry JM. Genetic polymorphisms in the cytochrome P4501A1,glutathione Stransferasw M1 and T1, and susceptibility to colon cancer. Teratog. Carcinog. Mutagen., 2002, 22(5): 385-392
19. Tsuchiya Y, Sato T, Kiyohara C, etc. Genetic polymorphisms of cytochrome P4501A1 and risk of gallbadder cancer. J. Exp. Clin. Cancer Res. 2002, 21 (1): 119-124
20. Firozi PF, Bondy ML, Sahin AA, etc. Aromatec DNA adducts and polymorphisms of CYP1A1,NAT2,and GSTM1 in breast cancer. Carcinogenesis, 2002,23(2):301-306
21. Chan DK, Mellick GD, Buchanan DD, etc. Lack of association between CYP1A1 polymorphism and Parkinson's disease in a chinese population. J. Neural Transm., 2002,109(1):35-39
22. Kawajiri K, Eguchi H, Nakschi K, etc. Association of CYP1A1 gene line polymorphisms with mutations of the p53 gene in lung cancer. Cancer Res., 1996, 56(1):72-76
23.王安辉,孙长生,李良寿,等.CYP1A1与GSTM1基因的多态性与西安地区食管癌的关系.第四军医大学学报,2002,23(1):53-55
24.刘平,刘振华,邵明,等.中国北方汉族人细胞色素P4501A1基因MspⅠ多 态性与早发性帕金森病关系的研究.中国医学遗传学杂志,2001,18(4):283-285
25.尹立红,浦跃朴,林嫔嫔.南京市正常人群NQO1、CYP1A1、mEH基因的多态性研究.遗传,2001,23(3):199-202
26.胡毅玲,高杨,张桥.细胞色素P4501A1、2D6和谷胱甘肽硫转移酶基因多态性与肺癌易感性的研究.肿瘤,1998,18(4):269-271
27. Tefre T, Ryberg D, Haugen A,etc. Human CYP1A1(cytochrome P450) gene: lack of association between the Msp Ⅰ restriction fragment length polymorphism and incidence of lung cancer in a Norwegian population.Pharmacogenetics, 1991,1:20-25
28.胡云珍,姚彤炜.细胞色素P4501A1的研究进展.中国药学杂志,2003,38(4):246-250