Study Of The Genotoxicity Of Bisphenol A And 4-Nonylphenol

Both the rapid screening of mutagenesis and accurate assessment of genotoxicity on exnobiotics have been the key research issues in toxicology area all along. This study has two purposes. One is attempting to establish a set of based-on-human-cell-line test systems consisting of TK gene mutation assay, in vitro micronuclei test and comet assay, which can test genotoxicity rapidly and sensitively in vitro, and the other is evaluating comprehensively the genotoxicity of Bisphenol A(BPA) and 4-Nonylphenol(NP) by using these three assays, to provide toxicology data for the risk assessment of BPA and NP and to accumulate data for the further use of TK gene mutation assay, in vitro micronuclei test and comet assay in rapid screening of genotoxicity of chemicals, and raw materials for newly-synthesized drug and newly-developed food.In TK gene mutation assay, TK6 cells were treated with BPA (100μM, 150μM, 200μM, 250μM) and NP(50μM, 60μM, 70μM, 80μM) for 4h, respectively. After treatment, a sample of cell cultures was used for the determination of plating efficiency (PEO), and then the rest of treated cultures were maintained in flasks for 3 days to permit expression of the TK deficient phenotype. On completion of the 3-day expression time, both the plating efficiency(PE3) and the trifluorothymidine-resistant mutation frequency(MF) of TK6 cell cultures were determined. The results showed that MF induced by BPA at a concentration range of from 100μM to 250μM had no significant difference compared with solvent control, and that MF of TK6 cells treated with NP at concentrations of 70μM and 80μM increased significantly (P<0.05) and were 2.46- and 3.83-times the solvent control respectively, but there was no dose-response relationship among all dosage groups. Besides, cytotoxicity was observed at NP concentrations of 70μM and 80μM and the relative survival ofcells was 13.63% and 4.97% respectively. It suggested that the increase of MF induced by NP was probably associated with high cytotoxicity.In in vitro micronuclei test, TK6 cells were received continuous 48-h treatment with BPA(50~125Wvl) and NP(40~70MM) respectively, as well as 4-h treatment with BPA(100~250MM) and NP(50~80l^M) respectively. On completion of continuous 48-h treatment or 48-h maintenance after 4-h treatment, the micronucleated cell(MNC) frequency of TK6 cells was determined, during which both Giemsa staining and acridine orange(A.O) staining were used. The same treated cell cultures were used for TK gene mutation assay and micronuclei assay with 4-h treatment. The findings showed that no obvious effect of both BPA and NP was observed on MNC frequency of TK6 cells, but that MNC frequency using A.O staining increased significantly compared with that using Giemsa staining(P<0.05). It suggested that A.O staining had higher sensitivity in micronuclei assay.In comet assay, no obvious effect was observed on the percentage of comet cells and the length of comet tail in TK6 cells treated for lh with BPA at concentrations of 50MM, 100MM and 200MM. But both the percentage of comet cells and the length of comet tail induced by NP at concentrations of 2.5, 5.0 and 10.0WV1 after 1-h treatment increased significantly compared with solvent control(P<0.05), with a good dose-response relationship. It suggested that NP could induce DNA single strand breaks in TK6 cells.The results of this study showed that BPA had no direct genotoxicity effects while NP had some genotoxicity effects as far as induction of DNA single strand breaks was concerned. The study also indicated that the same treated culture of TK6 cells could be used for testing multiple end-points including gene mutation, chromosome aberration and DNA damage, which was not only convenient and feasible but also more convictive.So it was well worth popularizing and applying to rapid screening of mutagen and carcinogen.