Test Methodology And Its Application Bhas42 Cell Transformation

Most of the short-term genotoxicity tests confine to the detection of genotoxiccarcinogens, but cannot detect non-genotoxic carcinogens. It’s significant to set up aquick, simple and reliable in vitro method for screening non-genotoxic carcinogens inthe early stages of research and development of drugs, and it’s the key issues in thecurrent study on safety evaluation of toxicology.We usually judge the results by focus formation method which istime-comsuming and subjective. The study is based on focus formation method toestablish Bhas42cell transformation assay using the H₂O₂treatment method, andapply in the safety evaluation of drugs. Bhas42cells are established from Balb/c3T3cells with v-Ha-ras gene and regarded as initiated. Bhas42cells are transformed bynon-genotoxic carcinogens without initiating treatment with an initiator. Bhas42cellcultures containing transformed foci ere treated hydrogen peroxide, normal cells wereselectively killed.After dying by CCK-8, we can determine non-genotoxicity based oncell survival rate by the degree of transformation of cells.Methods: The research consisted of two successive phases: establishing andapplying the method to practice.Part I: Establishment and verification of experimental method.1) Using3-MCAand TPA as positive agents, to determine the seeding concentration and theconcentration of positive agent after selecting the lot number of serum, and toestablish Bhas42cell transformation based on focus formation method.2) Todetermine the CCK-8staining time, the concentrantion and fuction point of hydrogenperoxide with Balb/c3T3and Bhas42cell transformation assay, and to verify theselected condition is appropriate.3) Choosing genotoxic carcinogen ENU andnon-genotoxic carcinogens PDD and LCA to verify Bhas42cell transformation assaybased on hydrogen peroxide treatment method.Part II: The application of the method.1) Choosing two phytoestrogen: GEN andpuerarin.2) Two food additives: ethyl gallate and cyclamate sodium. Results:1) The number of focus in the well is the largest when the cell iscultured with batch of737443FBS. In the cell growth assay, the relative growth rateis nearly to50%when seeding concentration is200cells·well^-1and the concentrationof3-MCA is1μg·ml^-1in initiation assay. The relative growth rate is nearly to100%when seeding concentration is400cells·well^-1and the concentration of TPA is50ng·ml^-1in promotion assay. The proportion of wells with transformed foci in positivecontrol was compared with that in the negative control plate by chi-square test（p<0.01）.2) In Balb/c3T3cell transformation assay, OD value achieves a smooth andthe difference between concentration groups is more visible in4h after CCK-8staining. In Group5, the OD values of positive group are higher than negative groupwhen the concentration of H₂O₂is0.0012%and0.0020%. In further optimiziton,there are significant differences between positive and negative group and the survivalrate of positive group is higher when the concentration of H₂O₂is0.0016%.3) Ininitiation assay, the OD value from cultures treated by ENU was significantlydifferent compared with that of negative control at the concentrations of ENU rangingfrom10,100,150μg·ml^-1with two consecutive concentrations. In promotion assay,there were significantly differences at the concentrations of PDD ranging from0.05、0.1、0.2、0.3、0.4μg·ml^-1and LCA ranging from0.05、0.1、0.2、0.3、0.4μg·ml^-1（Dunnett-t test, p<0.01）.4) The OD value from cultures treated by genistein andPuerarin in the initiation assay was not significantly different compared with that ofnegative control, but the differences were significant in the promotion assay at theconcentrations of genistein ranging from0.03,1,3μg·ml^-1with two consecutiveconcentrations. The OD value from cultures treated by Puerarin in the promotionassay was significantly different at3μg·ml^-1.5) The OD value from cultures treatedby ethyl gallate was significantly different at the concentration of1μg·ml^-1, but thedifferences were significant in the promotion assay at the concentrations of ethylgallate ranging from0.1,1,3μg·ml^-1. The OD value from cultures treated bycyclamate sodium in the initiation assay was different at the concentration of30μg·ml^-1, but the differences were significant in the promotion assay at the concentrations of cyclamate sodium ranging from10,30,300,1000μg·ml^-1.Conclusion:1) Choosing batch of737443FBS. Choosing the seedingconcentration is200cells·well^-1and the concentration of3-MCA is1μg·ml^-1ininitiation assay and the seeding concentration is400cells·well^-1and the concentrationof TPA is50ng·ml^-1in promotion assay. The results are positive both in3-MCA andTPA group, confirming that the method based on focus formation method in thelaboratory established successfully. Bhas42cell transformation assay based on H₂O₂method was performed with selected condition that the concentration of H₂O₂is0.0016%, measuring OD450value in4h after CCK-8staining. There are positiveresults in the verification with three known carcinogen, proved reliability andaccuracy of the method.4) According to cell transformation assay with GEN andpuerarin, GEN was positive in the promotion assay but negative in the initiation assay,suggesting that this compound may be a non-genotoxic carcinogen. Puerarin wasnegative in the initiation assay and equivocal in promotion assay, suggesting thenon-genotoxicity of puerarin is equivocal.5) According to cell transformation assay,ethyl gallate and cyclamate sodium were equivocal in the initation assay but positivein the promotion assay, suggesting that the genotoxicity of ethyl gallate and cyclamatesodium were equivocal and they may be non-genotoxic carcinogens.