多遗传学终点的遗传毒性试验组合的建立

doi:10.3969/j.issn.1004-616x.2013.06.014

癌变·畸变·突变

多遗传学终点的遗传毒性试验组合的建立

马华智,施畅^#，*,石富江,孙艳丽,于永生,吴纯启,王全军,丁日高

( 军事医学科学院毒物药物研究所，国家北京药物安全评价研究中心，北京 100850 )

收稿日期:2013-09-03 修回日期:2013-09-16 出版日期:2013-11-30 发布日期:2013-11-30
通讯作者: 施畅，E-mail：shich08@hotmail.com
作者简介:马华智 (1962- )，男，山东人，博士，高级实验师，研究方向：药物毒理。E-mail：huazhima@aliyun.com。#：并列第一作者。
基金资助:
重大新药创制科技重大专项 ( 2012ZX09301-003-008，2013ZX09 302303)，国家自然科学基金项目 (81001254)。

Establishment of a new battery of toxicity tests for multiple genetic endpoints

MA Hua-zhi，SHI Chang^#，*，SHI Fu-jiang，SUN Yan-li，YU Yong-sheng，WU Chun-qi，WANG Quan-jun，DING Ri-gao

(Beijing Institute of Pharmacology and Toxicology, National Beijing Center for Drug Safety Evaluation and Research, Beijing 100850, China)

Received:2013-09-03 Revised:2013-09-16 Online:2013-11-30 Published:2013-11-30
Contact: SHI Chang，E-mail：shich08@hotmail.com

摘要/Abstract

摘要：

目的： 建立Ames波动试验、中国仓鼠肺成纤维(CHL)细胞体外微核试验和小鼠淋巴瘤细胞基因突变试验 (MLA)方法，探讨其作为一套新的遗传毒性试验组合检测化合物诱变性的可能性。方法：在Ames波动试验中，用TA100沙门氏菌进行96孔板的Ames波动试验，非活化条件下以叠氮钠 (SA)、活化条件下以环磷酰胺 (CP)染毒处理，计数阳性孔数并进行卡方检验。在CHL细胞体外微核试验中，非活化条件下用丝裂霉素C (MMC)分别染毒处理4和24 h，活化条件下用CP染毒4 h，计数2 000个细胞中含微核的细胞数，并进行卡方检验。在MLA中，小鼠淋巴瘤细胞经清除自发突变后，非活化条件下用MMC、活化条件下用CP分别染毒处理3 h，计算相对存活率 (RS)、相对悬浮增长率 (RSG)、相对总增长率 (RTG)、平板效率PE0、PE2、突变率 (MF)、小集落比例 (SC)。结果：Ames波动试验中SA和CP在各自试验条件下阳性孔数均较对照组显著增加 (χ2 >6.63，P<0.01)，量效关系明显。CHL细胞体外微核试验中，MMC和CP在各自试验条件下均引起微核率显著升高 (χ2 >6.63，P<0.01)，量效关系明显。MLA中MMC和CP在各自试验条件下均引起L5178Y 3.7.2C-tk+/-细胞的PE、RS、RSG和RTG呈剂量依赖性下降，MF呈剂量依赖性升高，高出溶剂对照的2倍以上。结论：此3种短期遗传毒性试验方法可互为补充、相互验证，其组合应用可提高检出化合物遗传毒性的准确性，有望得到进一步推广应用。

关键词: Ames波动试验, 体外微核试验, 小鼠淋巴瘤细胞基因突变试验

Abstract:

OBJECTIVE: To establish Ames fluctuation test，in vitro micronucleus assay with Chinese hamster lung fibroblast cells (CHL) and mouse lymphoma thymidine kinase (tk+/-) locus mutation assay (MLA) as a new battery of methods to evaluate genotoxicity. METHODS：Ames fluctuation test：TA100 Salmonella strain was exposed to sodium azide (SA) without S9 and cyclophosphamide (CP) with S9 in 96-well plate. The number of positive wells was recorded and Chi-square test was performed to analyze statistical significance. In vitro micronucleus test：CHL cells were exposed to mitomycin C (MMC) without S9 for 4 h or 24 h，and CP with S9 for 4 h. The number of micronucleated cells in 2 000 cells was counted under oil lens to calculate micronucleus rate. Chi-square test was performed to analyze statistical significance. MLA：After spontaneous mutation having been eliminated，L5178Y 3.7.2C cells were exposed to MMC without S9 and CP with S9 for 3 h. Relative survival rate (RS)，relative suspension growth rate (RSG)，relative total growth rate (RTG)，plate efficiency (PE0，PE2)，mutation rate (MF) and the proportion of small colony (SC) were calculated. RESULTS：Both SA and CP showed statistically significant positive dose-dependently results without and with S9，respectively. Micronucleu s rates increased dose-dependently both in MMC (4 h，24 h，-S9) and CP (4 h，+S9) treated groups at different concentrations. MFs of L5178Y 3.7.2C cells exposed to different concentrations of MMC (4 h， 24 h，-S9) and CP (4 h，+S9) increased dose-dependently and more than twice compared to the vehicle control， while PE，RS，RSG and RTG decreased dose-dependently. CONCLUSION：These three new short-term assays could be used to detect genotoxicity more accurately and more extensively.

Key words: Ames fluctuation test, in vitro micronucleus assay, mouse lymphoma thymidine kinase locus mutation assay

马华智,施畅,石富江,孙艳丽,于永生,吴纯启,王全军,丁日高. 多遗传学终点的遗传毒性试验组合的建立[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2013.06.014.

MA Hua-zhi，SHI Chang，SHI Fu-jiang，SUN Yan-li，YU Yong-sheng，WU Chun-qi，WANG Quan-jun，DING Ri-gao. Establishment of a new battery of toxicity tests for multiple genetic endpoints[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2013.06.014.

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多遗传学终点的遗传毒性试验组合的建立

Establishment of a new battery of toxicity tests for multiple genetic endpoints

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