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

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80%提示化合物引起细胞生长抑制。将实验组相对细胞活性低于溶剂对照组的30%定义为出现细胞毒性,此时将不考虑遗传毒性。在无细胞毒性前提下,活化条件下实验组Gluc化学发光强度大于溶剂对照组的1.8倍时,非活化条件下实验组Gluc化学发光强度大于溶剂对照组的1.5倍时,判定为遗传毒性阳性;反之认为无遗传毒性。与溶剂对照组相比,阴性物质DMSO所有浓度均未产生遗传毒性。非活化条件下,MMS 12.5,25.0和50.0 mg·L^(-1)产生遗传毒性;活化条件下,CTX 6.25,12.5和25.0 mg·L^(-1)产生遗传毒性。非活化条件下,MS/PBAT的95%乙醇迁移物6.10和12.20 g·L^(-1)和MS/PBAT的50%乙醇迁移物12.20 g·L^(-1)组细胞生长抑制,所有处理组均未观察到相对细胞活性低于30%的细胞毒性,且未观察到Gluc高表达,表明5种MS/PBAT多组分迁移物在非活化条件下均未产生遗传毒性。活化条件下,MS/PBAT的95%乙醇迁移物12.20 g·L^(-1)和MS/PBAT的4%乙酸迁移物6.10,12.20 g·L^(-1)组细胞表现出显著的生长抑制,所有处理组均未观察到相对细胞活性低于30%的细胞毒性,且未观察到Gluc高表达,表明5种MS/PBAT多组分迁移物在活化条件下均未产生遗传毒性。微量波动Ames试验结果表明,在活化和非活化条件下,MS/PBAT的5种多组分迁移物3.05和12.20 g·L^(-1)作用于TA98和TA1002种菌株,致突变阳性孔的数量均不超过溶剂对照组的2倍,即均未产生致突变作用;作用于CHL细胞后的细胞染色体畸变率亦均无显著变化。结论初步建立了基于GADD45α基因的遗传毒性高通量筛选方法BSHC,提示可用于食品接触材料多组分迁移物的体外遗传毒性评价,但仍需进一步探索其最低有效浓度,并应用更多种类混合物进行进一步验证。

OBJECTIVE 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-1was 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-1with 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/PBAT at concentrations of 3.05 and 12.20 g·L~(-1).The in vitro mammalian cell chromosome aberration test was performed on CHL cells with 5 multi-component migrants of MS/PBAT at concentrations of 3.05 and 12.20 g·L~(-1)to detect the chromosomal aberration The results of genotoxicity were compared with those of BSHC.RESULTS The lowest effect centration(LEC;<80%relative cell viability)and the coytotoxicity(<30%relative cell viability)was defined.A positive genotoxicity result threshold was determined at 1.8-fold relative induction.For the liver S9protocol,the same process was followed,and the decision threshold derived was 1.5-fold relative Gluc induction.It is considered as genetic substance only when a positive genotoxicity result was reached and there was no cytotoxicity.Compared with the vehicle control group,no genotoxicity was observed at all concentration of DMSO by BSHC.MMS 12.5,25.0 and 50.0 mg·L~(-1)produced genotoxicity without liver S9 while CTX 6.25,12.5 and 25.0 mg·L~(-1)produced genotoxicity with liver S9.Significant cel growth inhibition was observed in 95%ethanol migrants of MS/PBAT at concentrations of 6.10 and12.20 g·L~(-1),and in 50%ethanol migrants of MS/PBAT at a concentration of 12.20 g·L~(-1)without liver S9.No cytotoxicity with a relative cell viability below 30%was observed in any of the treatment groups and no high expression of Gluc was observed.Therefore,none of the 5 multi-component migrants produced genotoxicity without liver S9.Significant cell growth inhibition was observed in 95%ethano migrants of MS/PBAT at a concentration of 12.20 g·L~(-1),and in 4%acetic acid migrants of MS/PBAT at concentrations of 6.10 and 12.20 g·L~(-1)with liver S9.No cytotoxicity with a relative cell viability below30%was observed in any of the treatment groups.No high expression of Gluc was observed.Therefore,none of the 5 multi-component migrants produced genotoxicity with liver S9.In the micro fluctuation Ames test,when 5 multi-component migrants of MS/PBAT were treated with concentrations of 3.05and 12.20 g·L~(-1)on TA98 and TA100 strains,there was no significant difference in the number of mutagenic positive wells compared with DMSO control group with or without liver S9,indicating that no mutagenic effect was produced.When CHL cells were treated with 5 multi-component migrants of MS/PBAT at concentration of 3.05 and 12.20 g·L~(-1),compared with DMSO control group,there was no significant difference in chromosome aberration rate of CHL cells with or without liver S9.CONCLUSION BSHC based on GADD45αgene has been established,which can be used for in vitro genotoxicity evaluation of migrants mixtures of FCM,but further exploration of its minimum effective concentrations is still needed,and more types of mixtures need to be applied for further validation.