致突变性检测方法—微量波动法的建立及应用研究
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摘要
随着工业化进程的加快,由化学物质和辐射等因子带来的环境问题日益突出,这些因子导致生物体的致突变效应,改变生物体的遗传物质,对生物体及其后代造成遗传性伤害。因此准确、快速地评价这些因子造成的生物危害是当代环境评价的一项重要工作。而传统的致突变性检测主要采用Ames法,该法手续繁琐,检测周期长,在实际应用中有一定的局限性,亟待进行改良。
本研究根据Ames法的基本原理,通过对培养、分析方法的改良,提出了Ames-微量波动法的最佳反应条件及其具体应用情况。微量波动法用液体培养基代替了传统Ames法的固体培养基,以观察培养液浊度或pH指示剂颜色的变化代替菌落计数,提高了实验的简便性和灵敏性。采用鼠伤寒沙门氏菌TA98菌株为测试菌株,二氨基芴为诱变物,通过分析菌液浓度,S9浓度,诱变物浓度的配比关系,确定了微量波动法的最佳反应条件,即微量波动法反应体系中的菌浓度和S9浓度分别为10ul/ml、20u1/ml。应用建立的方法,以TA98、TA97、TA100、TA102为测试菌株,对采自昆明市内的八条入滇河流的水质进行了验证性检测。并通过GC-MS检测和分析了八条入滇河流的复杂有机物组分,并从生物学角度初步揭示了水体污染物的致突变效应。
同时根据应用需要,用已建立的Ames-微量波动法对标准烟3R4F卷烟烟气冷凝物(TPM)进行了致突变性测试,以传统Ames平板测试法为对照对实验结果做了对比分析,结果表明以TA98、TA100为测试菌株微量波动法在传统Ames测试浓度下都呈较好线性关系,且好于传统的Ames平板法,但实验中也发现微量波动法过于灵敏,对于烟气TPM检测来讲,结果不够稳定。
由于烟草全烟气暴露能够更好的反应烟草烟气致突变性,通过跟踪国内外全烟气暴露研究报导,对全烟气暴露法的暴露体系做了全面的分析,应用自行开发的全烟气暴露装置,结合微量波动法,测试烟草全烟气致突变性,得到了适用于微量波动法的全烟气暴露的技术参数。
With the development of industrialization, the environmental problems resulted from chemical and radiation factors have become increasingly prominent. These factors lead to the mutagenesis effects on organisms and alteration of genetic materials of organisms, causing genetic damage to the organisms and their offspring. Therefore, accurate and rapid evaluation of biological hazards caused by these factors is very important for contemporary environmental assessment. Traditional mutagenicity detection is mostly carried out by Ames test, which is complicated, time-consuming and has some limitations in the practical application. Much improvement is needed.
Based on the principle of Ames test, this study proposed the best reaction conditions and their specific application of "microtitre" fluctuation test by improving the culture and analysis methods. "Microtitre" fluctuation test was carried out with liquid medium to observe the medium turbidity and the color changes of pH indicator instead of solid medium used for colony count in traditional Ame test, with the aim to improve the experimental simplicity and sensitivity.
Salmonella typhimurium strain TA98 and mutation inducing substance diamino fluorene were first used to determine the optimal reaction conditions of "microtitre" fluctuation test. By analyzing the ratio of the bacterial concentration, S9 concentration and the amount of mutation-inducing substance, 10ul/ml of bacterial concentration and 20ul/ml S9 concentration were determined. Based on this, TA98, TA97, TA100, TA102 were further used as test strains, water samples collected from eight rivers inflowing to the Dianchi Lake in Kunming were used for a confirmatory test. GC-MS analysis was used to detect the complex organic components in water samples collected from the eight rivers and preliminarily reveal the mutagenic effects of polluted water from the biological point of view.
Using the above established method, "microtitre" fluctuation test was used to detect the mutagenicity of tobacco smoke condensate (TPM) of the standard cigarette 3R4F. The resultant experimental results were further analyzed and verified by the traditional Ames plate test. Results revealed that "microtitre" fluctuation test using the strains TA98 and TA100 showed a good linear relationship in concentration and even a better result, compared to the traditional Ames test, However, "microtitre" fluctuation test was too sensitive and not stable enough for the TPM analysis of smoke.
As whole smoke exposure can reflect the mutagenicity of tobaccothe system of whole smoke exposure was analyzed comprehensively in this study after referring to similar research carried out by other labs. Based on the self-developed devices of whole smoke exposure and the above mentioned "microtitre" fluctuation test, analysis of whole smoke mutagenicity was carried out. Some corresponding technical parameters of whole smoke exposure applicable to "microtitre" fluctuation test were obtained.
本研究根据Ames法的基本原理,通过对培养、分析方法的改良,提出了Ames-微量波动法的最佳反应条件及其具体应用情况。微量波动法用液体培养基代替了传统Ames法的固体培养基,以观察培养液浊度或pH指示剂颜色的变化代替菌落计数,提高了实验的简便性和灵敏性。采用鼠伤寒沙门氏菌TA98菌株为测试菌株,二氨基芴为诱变物,通过分析菌液浓度,S9浓度,诱变物浓度的配比关系,确定了微量波动法的最佳反应条件,即微量波动法反应体系中的菌浓度和S9浓度分别为10ul/ml、20u1/ml。应用建立的方法,以TA98、TA97、TA100、TA102为测试菌株,对采自昆明市内的八条入滇河流的水质进行了验证性检测。并通过GC-MS检测和分析了八条入滇河流的复杂有机物组分,并从生物学角度初步揭示了水体污染物的致突变效应。
同时根据应用需要,用已建立的Ames-微量波动法对标准烟3R4F卷烟烟气冷凝物(TPM)进行了致突变性测试,以传统Ames平板测试法为对照对实验结果做了对比分析,结果表明以TA98、TA100为测试菌株微量波动法在传统Ames测试浓度下都呈较好线性关系,且好于传统的Ames平板法,但实验中也发现微量波动法过于灵敏,对于烟气TPM检测来讲,结果不够稳定。
由于烟草全烟气暴露能够更好的反应烟草烟气致突变性,通过跟踪国内外全烟气暴露研究报导,对全烟气暴露法的暴露体系做了全面的分析,应用自行开发的全烟气暴露装置,结合微量波动法,测试烟草全烟气致突变性,得到了适用于微量波动法的全烟气暴露的技术参数。
With the development of industrialization, the environmental problems resulted from chemical and radiation factors have become increasingly prominent. These factors lead to the mutagenesis effects on organisms and alteration of genetic materials of organisms, causing genetic damage to the organisms and their offspring. Therefore, accurate and rapid evaluation of biological hazards caused by these factors is very important for contemporary environmental assessment. Traditional mutagenicity detection is mostly carried out by Ames test, which is complicated, time-consuming and has some limitations in the practical application. Much improvement is needed.
Based on the principle of Ames test, this study proposed the best reaction conditions and their specific application of "microtitre" fluctuation test by improving the culture and analysis methods. "Microtitre" fluctuation test was carried out with liquid medium to observe the medium turbidity and the color changes of pH indicator instead of solid medium used for colony count in traditional Ame test, with the aim to improve the experimental simplicity and sensitivity.
Salmonella typhimurium strain TA98 and mutation inducing substance diamino fluorene were first used to determine the optimal reaction conditions of "microtitre" fluctuation test. By analyzing the ratio of the bacterial concentration, S9 concentration and the amount of mutation-inducing substance, 10ul/ml of bacterial concentration and 20ul/ml S9 concentration were determined. Based on this, TA98, TA97, TA100, TA102 were further used as test strains, water samples collected from eight rivers inflowing to the Dianchi Lake in Kunming were used for a confirmatory test. GC-MS analysis was used to detect the complex organic components in water samples collected from the eight rivers and preliminarily reveal the mutagenic effects of polluted water from the biological point of view.
Using the above established method, "microtitre" fluctuation test was used to detect the mutagenicity of tobacco smoke condensate (TPM) of the standard cigarette 3R4F. The resultant experimental results were further analyzed and verified by the traditional Ames plate test. Results revealed that "microtitre" fluctuation test using the strains TA98 and TA100 showed a good linear relationship in concentration and even a better result, compared to the traditional Ames test, However, "microtitre" fluctuation test was too sensitive and not stable enough for the TPM analysis of smoke.
As whole smoke exposure can reflect the mutagenicity of tobaccothe system of whole smoke exposure was analyzed comprehensively in this study after referring to similar research carried out by other labs. Based on the self-developed devices of whole smoke exposure and the above mentioned "microtitre" fluctuation test, analysis of whole smoke mutagenicity was carried out. Some corresponding technical parameters of whole smoke exposure applicable to "microtitre" fluctuation test were obtained.
引文
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[2]印木泉,余应年,郑怡年.我国遗传毒理学发展的回顾[J].卫生毒理学杂志,1999,13(4)：229-233.
[3]S 1. Achard. Assessment of cigarette whole smoke biological activity [J]. 2006 CORESTA CONGRESS IN PARIS, FRANCE SMOKE SCIENCE AND PRODUCT TECHNOLOGY,2006.
[4]张天宝.遗传毒理学的若干进展[J].毒理学杂志,2007,21(4)：279.
[5]H Tokiwa. detection of mutagenic activity in particulate air pollutants [J]. mut res,1977,48(237).
[6]李学明.中国五大城市大气飘尘的致突变性[J].卫生研究,1985,14(2)：23.
[7]林朝晖.Ames试验在水质检测方面的应用[J].微生物学通报,2002,29(3)：66-70.
[8]阮萃才.Ames试验的改进及其应用进展[J].广西医学院学报,1987,4(2)：51-54.
[9]犹学筠.尿致突变性检测的意义及其进展[J].国外医学卫生学分册,1983,1(12).
[10]K Falck. mutagenicity in urine of workers in rubber industry [J]. mutation, 1980,79(45).
[11]曹群立.沙门氏菌致变试验的应用进展[J].遗传与疾病,1986,8(1)：41-44.
[12]M Ehrlich. mutagens in the faces of 8 south-african population at different levels of risk for colon cancer [J]. mut res,1979,64(231).
[13]Aufderheide Michaela. An efficient approach to study the toxicological effects of complex mixtures [J]. Experimental and Toxicologic Pathology, 2008,60:163-180.
[14]和智君,李绍臣,陈章玉.遗传毒理学在低危害卷烟研究中的应用综述[J].烟草科技,2004(10)：24-26.
[15]Aufderheide Michaela, Helga Gressmann. A modified Ames assay reveals the mutagenicity of native cigarette mainstream smoke and its gas vapour phase [J]. Experimental and Toxicologic Pathology,2007,58(6):383-392
[16]Aufderheide Michaela, J. W. Knebel, D. Ritter. Novel approaches for studying pulmonary toxicity in vitro [J]. Toxicology Letters,2003, 140-141:205-211
[17]Fukano Yasuo, Maiko Ogura, Kentaro Eguchi, Makoto Shibagaki, Mutsuaki Suzuki. Modified procedure of a direct in vitro exposure system for mammalian cells to whole cigarette smoke [J]. Exp Toxic Pathol,2004, 55:317-323.
[18]衡正昌,杨在昌.微量波动试验在水样检测中的应用[J].环境与健康杂志,1990,7(1)：22-25.
[19]胡长灯,詹卓玲.微量波动试验的影响因素及灵敏度研究[J].卫生毒理学, 1990,4(2)：115-116.
[20]肖贤明,林耀军,潘海祥,刘祖发,傅家谟.饮用水Ames致突变性与主要有机污染指标的关系[J].中国科学院研究生院学报,2002,19(2).
[21]苗江丽齐宝宁唐国慧李劲松易建华郭剑锋.用彗星实验和微核实验检测地面水污染的遗传毒性效应[J].中国工业医学杂志,2006：3.
[22]Jh Park, Lee Bj, Lee Sk. Genotoxicity of drinking water from three Korean cities [J]. Mutat Res,2000,466(2):173-178.
[23]In vitro exposure of cells to Smoke at the Air Liquid Interface [J]. CORESTA In vitro Toxicology Task Force.
[24]卢斌斌,Ryanmeng, Lynnew Isse,焦延福.3种不同焦油卷烟烟气的细胞毒性比较[J]. Tobacco Science & Technology,2007(12):38-41.
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