体外微核高内涵筛选法在食品毒理学遗传毒性评价中应用研究  

Genotoxicity assessment of food ingredients using high-content screening in vitro micronucleus assay

作　　者：孙静秋[1] 洪新宇[1] 肖萍[1] 帅怡[1] 王彦琴[1] 郑卫东[1] 李晨[1] 陶功华[1] SUN Jingqiu;HONG Xinyu;XIAO Ping;SHUAI Yi;WANG Yanqin;ZHENG Weidong;LI Chen;TAO Gonghua(Shanghai Municipal Center for Disease Control and Prevention,Shanghai 200336,China)

机构地区：[1]上海市疾病预防控制中心,上海200336

出　　处：《中国食品卫生杂志》2020年第2期124-129,共6页Chinese Journal of Food Hygiene

基　　金：上海市卫生和计划生育委员会科研课题资助项目(201540208);上海市制修订地方标准项目(DB31-E4-16082);上海市第四轮公共卫生三年行动计划“高端海外研修团队培养计划”(GWTD2015S03);上海市卫生和计划生育委员会地方标准预研制项目。

摘　　要：目的探讨基于高内涵筛选(high-content screening, HCS)技术的体外微核(in vitro micronucleus, IVMN)检测方法应用于食品毒理学遗传毒性评价的可行性。方法采用IVMN和HCS法,分别对10种化合物进行遗传毒性评价,其中5种已知遗传毒性化合物(染色体断裂剂和非整倍体诱发剂)、1种已知非遗传毒性化合物以及4种食品原料,每个受试物至少设置3个剂量组,每个剂量组设2个复孔,同时设置阴性对照组(无血清最小必需培养基)和阳性对照组(+S9为环磷酰胺20μg/ml、-S9为丝裂霉素C 1.0μg/ml)。以中国仓鼠肺细胞为细胞模型,在有和/或无代谢活化系统条件下,依次对上述受试物采用短时处理(4 h)后进行微核检测,并分析微核细胞率。结果经IVMN和HCS法得到的IVMN试验结果显示:2.5~10μg/ml苯并芘[B(a)P]、5~20μg/ml甲磺酸甲酯(MMS)、0.01~0.04μg/ml 4-硝基喹啉-N-氧化物(4NQO)、0.25~1.0μg/ml秋水仙碱(COL)和0.5~2.0μg/ml硫酸长春碱(VB)在各自浓度范围内,在有或无代谢活化系统的条件下,诱导产生的微核细胞率随着受试物浓度的升高而增加,呈明显剂量-反应关系,且微核细胞率与阴性对照组比较,差异均有统计学意义(P<0.05),试验结果为阳性;1 250~5 000μg/ml氯化钠(NaCl)、1 250~5 000μg/ml食品原料A、1 250~5 000μg/ml食品原料B、312.5~1 250μg/ml食品原料C和156.25~625μg/ml食品原料D在各自浓度范围内,在有和无代谢活化系统的条件下,微核细胞率虽呈现一定的剂量依赖性增加趋势,但均维持在较低的水平,且与阴性对照组比较,差异均无统计学意义(P>0.05),试验结果为阴性。结论本试验条件下,两种方法对10种物质的检测结果均一致,提示将IVMN HCS法应用于食品毒理学遗传毒性评价具有一定的可行性。Objective To validate the feasibility of genotoxicity assessment of food ingredients using high-content screening(HCS) in vitro micronucleus(IVMN) assays. Methods Two IVMN methods were used to evaluate the genotoxicity of ten compounds, including five known genotoxic compounds(clastogens and aneugens), one known non-genotoxic compound and four food ingredients. At least three concentrations of each compound and two parallels were set. No serum minimum essential medium(MEM)was treated as negative control. 20 μg/ml cyclophosphamide and 1.0 μg/ml mitomycin C was treated as positive controls. With and without metabolic activation, Chinese hamster lung(CHL)cells were treated with ten test compounds for 4 h. And the frequency of micronuclei was analyzed. Results The frequencies of micronuclei, which were obtained by conventional IVMN and HCS IVMN assay, induced by benzo(a)pyrene(dose range from 2.5 to 10 μg/ml), methyl methanesulphonate(dose range from 5 to 20 μg/ml), 4-nitroquinoline-N-oxide(dose range from 0.01 to 0.04 μg/ml), colchicine(dose range from 0.25 to 1.0 μg/ml), and vinblastine sulfate(dose range from 0.5 to 2.0 μg/ml) were significantly different with negative controls(P<0.05), and there was a obvious dose-response relationship. The frequencies of micronuclei induced by sodium chloride(dose range from 1 250 to 5 000 μg/ml), food ingredient A(dose range from 1 250 to 5 000 μg/ml), food ingredient B(dose range from 1 250 to 5 000 μg/ml), food ingredient C(dose range from 312.5 to 1 250 μg/ml), and food ingredient D(dose range from 156.25 to 625 μg/ml) showed a gradual upward trend in dose-response curve. Compared with negative controls, the differences of the result were not statistically significant(P>0.05). Conclusion In this study, the result of HCS IVMN assays were accordance to that of traditional IVMN assay. It was revealed that the genotoxicity assessment of food ingredients using HCS IVMN method was feasible.

关 键 词：食品毒理 遗传毒性 体外微核 高内涵筛选 

分 类 号：R155[医药卫生—营养与食品卫生学]