Applicability of BlueScreen HC in genotoxicity detection of migrants mixtures of food contact materials
10.3867/j.issn.1000-3002.2024.10.10
- VernacularTitle: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
- Author:
Min LI
1
;
Jun LIN
;
Weiliang WU
;
Haixia SUI
;
Xingfen YANG
Author Information
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
- Keywords:
food contact material;
migrants mixtures;
growth arrest and DNA damage-inducible 45α;
genotoxicity evaluation
- From:
Chinese Journal of Pharmacology and Toxicology
2024;38(10):796-806
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To explore the applicablity of 'BlueScreen HC'(BSHC),a high throughput 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 GADD45α gene 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 at 40℃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 concen-tration of 1% (V/V)liver S9 before being washed and re-suspended in fresh recovery media for another 45 h.After exposure,the cell viability was detected using the CCK-8 cell activity kit,and the Gluc Lumi-nescence in the medium was detected with Secrete-PairTM 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 centra-tion(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 S9 protocol,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 cell growth inhibition was observed in 95% ethanol migrants of MS/PBAT at concentrations of 6.10 and 12.20 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% ethanol 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 below 30% was observed in any of the treatment groups.No high expression of Gluc was observed.There-fore,none of the 5 multi-component migrants produced genotoxicity with liver S9.In the micro fluctua-tion Ames test,when 5 multi-component migrants of MS/PBAT were treated with concentrations of 3.05 and 12.20 g·L-1 on TA98 and TA100 strains,there was no significant difference in the number of muta-genic 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 signifi-cant difference in chromosome aberration rate of CHL cells with or without liver S9.CONCLUSION BSHC based on GADD45α gene has been established,which can be used for in vitro genotoxicity eval-uation 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.
