体内外遗传毒性实验组合评价纳米银的遗传毒性  

Evaluation of genotoxicity of nano-silver through a battery of in vitro and in vivo genotoxic assays

作　　者：于永生[1,2] 施畅[2] 马华智[2] 王全军[2] 丁日高[2] 

机构地区：[1]烟台大学药学院,山东烟台264005 [2]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,抗毒药物与毒理学国家重点实验室,北京100850

出　　处：《军事医学》2015年第12期929-933,共5页Military Medical Sciences

基　　金：国家重大新药创制科技重大专项资助项目(2013ZX09302303,2012ZX09301-001-008)

摘　　要：目的采用小鼠淋巴瘤细胞tk+/-位点突变实验、大鼠肝细胞碱性彗星电泳实验以及大鼠外周血网织红细胞微核实验观察纳米银的体内外遗传毒性。方法 (1)以2.5、5.0、10.0、20.0、40.0和80.0μg/ml浓度纳米银处理L5178Y细胞3 h,确定细胞毒性水平;再以0.63、1.25、2.50和5.00μg/ml浓度处理细胞3 h,分析纳米银致基因突变频率。(2)SD大鼠单次尾静脉注射纳米银0.63、1.25、2.50、5.00 mg/kg,24 h后分别采用碱性彗星电泳和流式细胞术检测肝细胞DNA损伤和外周网织红细胞血微核形成率。结果 (1)L5178Y细胞相对悬浮增殖率随纳米银浓度的增加而降低,在5.0μg/ml时已降至阴性对照组的11.42%;在测试浓度下,tk+/-基因位点突变频率呈浓度依赖性升高,与阴性对照组差异显著(P<0.05)。同时,小集落比例较阴性对照组均有显著提升(P<0.01)。(2)纳米银5.0 mg/kg组给药后10 min内2只大鼠死亡,至24 h时死亡数增至3只,样本数不足不能参与统计。与阴性对照组相比,1.25、2.5 mg/kg剂量组尾部DNA百分含量均有显著性差异(P<0.05),0.63 mg/kg组未见明显异常。与阴性组相比,纳米银0.63、1.25、2.5 mg/kg 3个剂量组诱发外周血网织红细胞微核发生率均有显著性差异(P<0.05),并与暴露剂量呈正相关(Pearson r=0.98,P<0.05)。结论实验条件下检测出纳米银具有诱发诸如基因突变、染色体畸变和原发性DNA损伤等体内外遗传毒性的潜在风险。Objective To systematically evaluate the genotoxicity of nano-silver through a battery of the in vitro mouse lymphoma TK assay（ MLA）,the alkaline hepatocyte comet assay and the peripheral blood reticulocyte micronuclei test in SD rats. Methods（ 1） L5178 Y cells were treated with 2. 5,5. 0,10. 0,20. 0,40. 0,and 80. 0 μg / ml nano-silver for 3h to determine cytotoxicity before nano-silver at the concentrations of 0. 63,1. 25,2. 50 and 5. 00 μg / ml was applied to detect the mutagenicity of nano-silver.（ 2） SD Rats were respectively exposed to 0. 63,1. 25,2. 50 and 5. 00 mg / kg nano-silver for 24 h through single tail intravenous injection. Alkaline comet assay and flow cytometry were employed to analyze primary DNA damage in hepatocytes and detect peripheral blood micronucleated reticulocytes. Results（ 1） A dose-dependent reduction of relative suspension growth was found in cytotoxicity tests. RSG% dropped to 11. 42% of that in the negative control group at 5. 0 μg / ml. At 0. 63,1. 25 and 2. 50 and 5. 0 μg / ml,mutation frequency went up dosedependently（ P 0. 05）. Meanwhile,the percentage of small colony（ SC%） significantly increased compared with the negative control group（ P 0. 01）.（ 2） At 5. 00 mg / kg,two SD rats were found dead 10 minutes after administration and by the end of 24 h the the death toll became three,so there were not enough data for statistical analysis. At 1. 25,2. 50 mg / kg,tail % DNA showed significant increase compared with the negative control（ P 0. 05）. At 0. 63 mg / kg,no obvious DNA damage was found. At 0. 63,1. 25 and 2. 50 mg / kg,nano-silver was able to induce micronucleated reticulocytes in peripheral blood,which was significantly different from the negative control（ P 0. 05） and positively related to the dosage（ Pearson＇s r = 0. 98,P 0. 05）. Conclusion Nano-silve has the potential to induce gene mutation,primary DNA damage and chromosome aberration under the condition mentioned in this study.

关 键 词：纳米银 小鼠淋巴瘤细胞实验 基因突变 彗星实验 微核 

分 类 号：R114[医药卫生—卫生毒理学]