| The estrogen receptor mediated reporter gene assay,also named receptor transcriptional activation assay,is an in vitro methods to screen the environmental estrogens proposed by the US EPA.On the basic of receptor-mediated theory,the estrogen receptor mediated reporter gene assays were designed to identify substances that might interfere with normal estrogenic activity by acting as an agonist or antagonist through ligand-receptor interaction.The DNA fragment containing estrogen responsive element(ERE)was cloned into a reporter gene plasmid, making the reporter gene regulated by the ERE.The reconstructed reporter plasmid and the corresponding expression plasmid of the estrogen receptor were co-transfected into a host cell to create an artificial gene expression system.The estrogen receptor mediated reporter gene assay provided a relatively simple way to indirectly reveal whether a substance could activate or inhibit the transcriptional activation of receptor-regulated genes by the measurement of the reporter gene product, typically an enzyme or a protein.The assays possessed some advantages over the receptor binding assays because they provided the information on both the ability of receptor binding and the biological response after binding(i.e.,RNA transcription).In addition,unlike receptor binding assays,the assays could distinguish a receptor agonist from an antagonist. In this study,we conducted two different types of ERαmediated reporter gene assays in African green monkey kidney cell line CV-1 to clarify the relationship between these two assays for three pyrethroids.Partâ… Development of estrogen receptor mediated reporter gene assaysObjectiveTwo ER reporter gene assays employing the human ERαand rat ERαwas developed in order to evaluate the(anti)estrogen effects of chemicals,and the assays was applied to clarify the relationship between these two different types of ERα.Methods1.The plasmid expression Gal4-BD fused human ERαwas constructed and transiently co-transfected into CV-1 cell with Gal4 responsed reporter plasmid pUAS-tk-luc and the control plasmid phRL-tk to develop the hERαmediated reporter gene assays.The reference estrogen E2 was used to verify the performance of the assays.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ERαby the test chemicals.ER antagonist assay was performed by measure the ability of a test substance to inhibit the induction of the reporter gene Luc product by E22.The CV-1 was transiently co-transfected with three plasmids including rat ERαexpression plasmid rERα/pCI,reporter plasmid pERE-aug-Luc containing the reporter gene Luc regulated by the estrogen responsive element(ERE)and the control plasmid phRL-tk to develop the rERαmediated reporter gene assays.The reference estrogen 17β-estradiol(E2)was used to verify the performance of the assays.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ERαby the test chemicals.Result1.Following a 24-h exposure to E2,the luciferase activity in these two reporter gene assays was stimulated in a dose-dependent manner at concentrations of more than 10-11M.The maximal induction of 19.6-fold and 21.6-fold of vehicle controls were obtained at 10-7M,with EC50 values of 2.4nM and 0.9nM respectively.2.In hERαreporter assay,the potent synthetic estrogen,DES was evaluated at concentrations from 10-10M to 10-8M.As expected,it induced luciferase activity at concentrations similar to that seen with E2.And the ER antagonist ICI182,780 could completely inhibit the response to E2 in this assay.Conclusion1.The assays showed high sensitivity and acceptable repeatability to E2.The reference estrogen E2 induced Luc activity in an ER-depended and dose-response manner.The assays were useful in identifying ER agonists from a large number of chemicals.2.The results revealed that hERαreporter assay could be adapted for evaluation of the antiestrogenic activity of diverse synthetic chemicals.3.The two assays showed the similar reliability and responsiveness.Partâ…¡Application of estrogen receptor mediated reporter gene assaysObjectiveTwo reporter gene assays employing the human ERαand rat ERαwas applied to evaluate the(anti)estrogen effects of three frequently encountered pyrethroid insecticides and clarify the relationship between these two different types of ERα. Methods1.The reporter plasmid pUAS-tk-luc,pGal4-ERαdef and the control plasmid phRL-tk was transiently co-transfected into CV-1 cell for screening the(anti)estrogenic activity of three pyrethroid insecticides.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ER by the test chemicals.ER antagonist assay was performed by measure the ability of a test substance to inhibit the induction of the reporter gene Luc product by E2.2.The reporter plasmid rERα/pCI,pERE-aug-Luc and the control plasmid phRL-tk was transiently co-transfected into CV-1 cell to screen the(anti)estrogenic activity of three pyrethroid insecticides.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ER by the test chemicals.Result1.Three pyrethroid insecticides(cypermethrin,fenvalerate, permethrin)increased the luciferase expression in a dose-dependent manner,and the maximum induction was observed at a dose of 10-4M in both assays.2.In hERαreporter assay,the tested pesticide was added to the assay system along with 1 nM of E2 in the medium.As a result,none of the three test chemicals could reduce the E2-inducing luciferase expression in this assay. Conclusion1.All of the three pyrethroids possessed estrogenic activities,but they were much less potent than E2.2.None of the three pyrethroids exhibited statistically significant antiestrogenic activity in hERαgene assay.3.No marked differences in the estrogenic activities of pyrethroids were found between these two assays.
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