摘要
目的建立基于GADD45α基因的遗传毒性高通量筛选体系(Green Screen human cells,GSHC),并将其应用于新型可生物降解的食品接触材料改性淀粉/聚对苯二甲酸-己二酸丁二酯(MS/PBAT)的遗传毒性评价。方法将克隆有GADD45α基因启动子和增强型绿色荧光蛋白(EGFP)的质粒用慢病毒转染的方法感染p53野生型人淋巴母细胞系TK6,构建稳转细胞系。用已知遗传毒性阳性和阴性的物质对构建的GSHC系统进行验证。在此基础上,以MS/PBAT为研究材料,对使用4%乙酸、20%乙醇、50%乙醇和95%乙醇作为食品模拟物进行迁移实验获得的多组分迁移物开展遗传毒性评价,并采用鼠伤寒沙门氏菌回复突变试验(Ames试验)对遗传毒性阳性迁移物进行验证。结果在GSHC筛选体系下,遗传毒性阳性物质甲基磺酸甲酯(MMS)、丝裂霉素C(MMC)和环磷酰胺(CTX)和遗传毒性阴性物质DMSO,均得到了预期结果。GSHC筛选体系下的MA/PBAT的4%乙酸、50%乙醇和95%乙醇3种阳性迁移物的Ames实验结果为阴性。结论GSHC方法可用于多组分迁移物的遗传毒性评价。需要进一步确定GSHC的生物检测限(LOBD),并与成熟的体外测试法进行对比分析。
Objective To establish a high-throughput assay(GSHC)for genotoxicity based on GADD45αgene and apply it on the genotoxicity evaluation of modified starch/polybutylene terephthalate adipate(MS/PBAT),a novel biodegradable food contact material.Methods The plasmid cloned with GADD45αgene promoter and enhanced green fluorescent protein(EGFP)was transfected with p53 wild-type human lymphoblastic cell line TK6 by lentivirus transfection to construct a stable expression cell line.The GSHC assay was validated with substances known to be genotoxic positive and negative.Using MS/PBAT as research material,the genotoxicity of multi-component migrants with 4%acid,20%alcohol,50%alcohol as well as 95%alcohol as food simulants respectively were evaluated,and the genotoxic positive migrations were verified by Ames test.Results The sensitivity of GSHC was verified by genotoxic methyl methylate(MMS),mitomycin C(MMC)and cyclophosphamide(CTX),and the specificity of GSHC was verified by non-genotoxic DMSO,and the expected result were obtained.The positive migrants from GSHC assay was negative when using Ames test.Conclusion The GSHC assay can be used to evaluate the genotoxicity of migrants mixtures.The biological detection limit(LOBD)of GSHC needs to be further determined and compared with mature in vitro assays.
作者
李敏
吴超
林珺
吴炜亮
隋海霞
杨杏芬
LI Min;WU Chao;LIN Jun;WU Wei-liang;SUI Hai-xia;YANG Xing-fen(Tianjin Centers for Disease Control and Prevention,Tianjin 300011,China;Department of Pancreatic Cancer,Tianjin Medical University Cancer Institute and Hospital,Tianjin 300022,China;Huadu Center for Disease Control and Prevention,Guangzhou,Guangdong 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,Guangdong 510515,China;NHC Key laboratory of food safety risk assessment,China National Center for Food Safety Risk Assessment,Beijing 100022,China)
出处
《毒理学杂志》
CAS
2023年第6期499-506,513,共9页
Journal of Toxicology
基金
广东省重点领域研发计划(2019B020210002)。
