多遗传学终点的遗传毒性试验组合的建立

摘要

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. Micronucleus 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.%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2000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)。2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(χ>6.63，<0.01)，量效关系明显。CHL细胞体2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(χ>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。P