Establishment Of Two High-throughput Screening Systems For Suspicious Carcinogenic Based On P53 And GADD45a Genes Expression And The Preliminary Application In Food-related Chemical Contaminants

Objective:In this study,we intended to establish two high-throughput screening systems for suspicious carcinogenic based on p53 and GADD45 a genes to screen the common food-related chemical contaminants and to obtain the possible susceptible carcinogenicic data.The results of the two systems were compared with rodent carcinogenicicity data and Ames experiments to evaluate the screening ability and the potential for practical use.Methods:1.we selected 19 food-related chemical contaminants by literature and related information:aflatoxin B1,acrylamide,chloramphenicol,hexsan,AHD,AMOZ,AOZ,SEM,crystal violet,leucocrystal violet,malachite green,leucomalachite green,oflaxacin,orbifloxacin,difofloxacin,sparfloxacin,enrofloxacin,thiamphenicol,florfenicol were included.According to the corresponding solubility and the concentration in the daily life,combined with the solvent of DMSO in the cell systems(should not exceed 0.5%)to configure the test concentration.2.19 food-related chemical contaminants were screened by MTT test to obtain the possible acute toxicity data.3.Plasmid p GL4.38 [luc2P/p53 RE/Hygro] and plasmid p GL4.75 [h Rluc/TK] were transfected into p53-U-2 OS cell line by transient transfection.4.Plasmid GADD45 WT-luc and plasmid p GL4.75 [h Rluc/TK] were transfected into HCT 116 cell line by transient transfection.5.17 food-related chemical contaminants were screened by p53-U-2 OS system after acute toxicity test by MTT experiment.6.17 food-related chemical contaminants were screened by GADD45a-HCT 116 system after acute toxicity test by MTT experiment.7.The results of 8 food-related chemical contaminants recorded in literature were normalized.Then Ames experiments were used to test the other 9 food-related chemical contaminants which were no recorde in literatures.8.The animal carcinogenicicity experimental data of 17 food-related chemical contaminants were accessed by relevant literature and information.9.The results of Ames experiment for 17 food-related chemical contaminants were compared with those of animal carcinogenicicity experiment.The screening ability was evaluated under the experimental conditions.10.The results of p53-U-2 OS system and the GADD45a-HCT 116 system for 17food-related chemical contaminants were compared with those of Ames experiment under the experimental conditions,respectively.The results of p53-U-2 OS system and the GADD45a-HCT 116 system for 17 food-related chemical contaminants were compared with those of animal carcinogenicicity experiment under the experimental conditions,respectively.Results:1.The acute toxicity of crystal violet(10 ?M)and malachite green(10 ?M)to U-2OS cell line were still obvious at low concentration,so they were not included in the followed-up experiment.The acute toxicity of crystal violet(10 ?M)and malachite green(100 ?M)were still obvious to HCT 116 cell line at low concentration,so they were not included in the followed-up experiment.The remaining 17 chemicals were included after MTT test.2.A high throughput screening system of p53-U-2 OS for suspicious carcinogenic was constructed by transient transfection.3.A high throughput screening system of GADD45a-HCT 116 for suspicious carcinogenic was constructed by transient transfection.4.The results of p53-U-2 OS system showed that 17 food-related chemical contaminants had different induction effects on the p53 gene(except AMOZ at 1000 ?M).5.The results of GADD45a-HCT 116 system showed that:acrylamide,leucocrystal violet,leucomalachite green,ofloxacin,bisfloxacin,sparfloxacin,enrofloxacin,aflatoxin B1,these 10 chemicals had certain induction effects on the GADD45 a gene in the system.6.The results of Ames test showed 9 chemicals which were no relevant literature were negative in the experimental conditions.7.Compared with the animal carcinogenicicity test,the sensitivity and specificity were 75% and 76.9% of Ames experiment,respectively,and the consistency rate was 76.5%.8.Compared with Ames experiment,the screening ability of p53-U-2 OS system and GADD45a-HCT 116 system were similar to those of Ames experiment.At specific boundary points,the consistency rate could reach up to 88.2% and 75%,respectively.And in the application and detection range,these two new systems were better than those of Ames;When compared with the "gold standard" animal carcinogenicicity test,the consistency rate of the p53-U-2 OS system was superior than that of the Ames experiment(mainly reflected in the high boundary points);GADD45a-HCT 116 system showed high specificity(when the consistency rate was at the similar level with Ames,the specificity could up to 100%),the ability to distinguish non-susceptible carcinogenics may be better.Conclusions:The system of p53-U-2 OS was sensitive,17 food-related chemical contaminants were screened out by induced the expression of p53 in the system(except AMOZ at 1000 ?M);10 food-related chemical contaminants were screened out by GADD45a-HCT 116 system.However,the results of these two systems should combined with other screening experiments and animal carcinogenicicity test results,as a part of comprehensive references.Under the experimental conditions,the overall screening ability of U-2 OS and GADD45a-HCT 116 systems were better than that of Ames screening experiment.Compared with the results of the "gold standard" animal carcinogenicicity experiment,the screening ability of these two screening systems were better than those of the Ames(at specific boundary points).In summary,it is possible to convert into practical application.