BlueScreen HC在食品接触材料多组分迁移物遗传毒性检测中的适用性  

Applicability of BlueScreen HC in genotoxicity detection of migrants mixtures of food contact materials

作　　者：李敏[1] 林珺 吴炜亮 隋海霞 杨杏芬 LI Min;LIN Jun;WU Weiliang;SUI Haixia;YANG Xingfen(Tianjin Centers for Disease Control and Prevention,NHC Specialty Laboratory of Food Safety Risk Assessment and Standard Development,Tianjin Key Laboratory of Pathogenic Microbiology of Infec-tious Disease,Tianjin 300011,China;Huadu Center for Disease Control and Prevention,Guangzhou 510880,China;School of Public Health,Food Safety and Health Research Center,Key Laboratory of Cosmetics Safety Evaluation of National Medical Products Administration,Guangdong Provincial Key Laboratory of Tropical Disease Research,Southern Medical University,Guangzhou 510515,China;NHC Key Laboratory of Food Safety Risk Assessment,China National Center for Food Safety Risk Assessment,Beijing 100022,China)

机构地区：[1]天津市疾病预防控制中心,国家卫生健康委员会食品安全风险评估与标准研制特色实验室,天津市传染病病原微生物重点实验室,天津300011 [2]广州市花都区疾病预防控制中心卫生检验部,广东广州510880 [3]南方医科大学公共卫生学院,食物安全与健康研究中心,国家药监局化妆品安全评价重点实验室,广东省热带病研究重点实验室,广东广州510515 [4]国家卫生健康委食品安全风险评估重点实验室,国家食品安全风险评估中心,北京100022

出　　处：《中国药理学与毒理学杂志》2024年第10期796-806,共11页Chinese Journal of Pharmacology and Toxicology

基　　金：广东省重点领域研发计划(2019B020210002)。

摘　　要：目的探讨基于人生长阻滞和DNA损伤诱导45α(GADD45α)基因的遗传毒性高通量筛选体系BlueScreen HC(BSHC)在食品接触材料多组分迁移物遗传毒性检测中的适用性。方法将人GADD45α基因开放阅读框上游2000 bp序列作为启动子,采用分子克隆构建入嘌呤霉素和高斯荧光素酶(Gluc)双标记的慢病毒质粒pEZX-LvPG04中,并用慢病毒感染人淋巴母细胞TK6,获得稳转细胞系TK6-Gluc。以甲基磺酸甲酯(MMS,终浓度0,1.56,3.13,6.25,12.5,25.0和50.0 mg·L^(-1))为非代谢活化条件下的阳性物质,环磷酰胺(CTX,终浓度0,0.78,1.56,3.13,6.25,12.5和25.0 mg·L^(-1))为代谢活化条件下的阳性物质,二甲基亚砜(DMSO,终浓度0,0.35,0.69,1.38,2.75,5.5和11.0 g·L^(-1))为阴性物质,分别在非活化和活化条件下验证构建的BSHC。将改性淀粉/聚对苯二甲酸-己二酸丁二醇酯(MS/PBAT)作为研究对象,采用体积分数4%乙酸及10%,20%,50%和95%乙醇作为食品模拟物,40℃、浸泡24 h获得5份MS/PBAT多组分迁移物,并用DMSO作为溶剂复溶得到5份多组分迁移溶液作为受试物。以终浓度为0,0.38,0.76,1.53,3.05,6.10和12.20 g·L^(-1)的不同受试物在活化和非活化2种条件下处理TK6-Gluc细胞。非活化条件下作用48 h;活化条件下,在添加体积分数1%大鼠肝S9代谢活化系统的同时,作用3 h后更换新鲜培养基,继续培养至48 h。处理结束后,采用CCK-8法检测细胞活性,同时采用Secrete-Pair^(TM)Gaussia Luciferase Assay试剂盒检测培养基中Gluc化学发光强度。另外,采用终浓度为3.05和12.20 g·L^(-1)的不同受试物对鼠伤寒沙门氏菌TA98和TA100进行微量波动Ames试验以及对体外培养的中国仓鼠肺细胞CHL进行体外哺乳细胞染色体畸变试验,检测受试物的致突变和染色体畸变作用,与BSHC遗传毒性结果进行对比分析。结果采用BSHC法,将相对细胞活性80%定义为生长抑制的最低有效浓度阈值,实验组相对细胞活性低于溶剂对照组的80OBJECTIVE To explore the applicablity of′BlueScreen HC′(BSHC),a high throughpu genotoxicity screening system based on human growth arrest and DNA damage inducible 45α(GADD45α)gene,in detecting the genotoxicity of migrants mixtures from food contact materials(FCM).METHODS The 2000 bp sequence upstream of the open reading frame of human GADD45αgene was used as the promoter to construct the lentiviral plasmid pEZX-LvPG04,which was double labeled by purinamycin and Gausluciferase(Gluc),and the lentiviral plasmid was infected with human lymphoblastocyte TK6 to obtain a stable transmutation cell line TK6-Gluc.Methyl methylate(MMS)at concentrations of 0,1.56,3.13,6.25,12.5,25.0 and 50.0 mg·L^(-1) was selected as the genotoxin without liver S9,cyclophosphamide(CTX)0,0.78,1.56,3.13,6.25,12.5,25.0 mg·L^(-1)was selected as the pre-genotoxin with liver S9,and dimethyl sulfoxide(DMSO)0,0.35,0.69,1.38,2.75,5.5 and 11.0 g·L^(-1)was selected as the non-genotoxin.The constructed BSHC was verified with the above known genetic positive and negative substance respectively.Polybutyleneadipate-co-terephthalate(MS/PBAT)was tested using 4%(V/V)acetic acid,and 10%,20%,50%and 95%(V/V)ethanol as food simulants at40℃for 24 hours to obtain 5 multi-component migrants of MS/PBAT that were obtained by using DMSO as a solvent.TK6-Gluc cells were treated with 5 multi-component migrants of MS/PBAT at concentrations of 0,0.38,0.76,1.53,3.05,6.10 and 12.20 g·L^(-1) with or without liver S9.Cells were treated without liver S9 for 48 h.Cells treated with liver S9-mix were incubated for 3 h at a final concentration of 1%(V/V)liver S9 before being washed and re-suspended in fresh recovery media for another45 h.After exposure,the cell viability was detected using the CCK-8 cell activity kit,and the Gluc Luminescence in the medium was detected with Secrete-Pair^(TM)Gaussia Luciferase Assay Kit.In addition the mutagenicity on Salmonella typhimurium TA98 and TA100 was detected by micro-fluctuation Ames test with 5 multi-component migrants of MS

关 键 词：食品接触材料 多组分迁移物 人生长阻滞和DNA损伤诱导45α基因 遗传毒性评价 

分 类 号：R99[医药卫生—毒理学]