Studies On The Endocrine Disrupting Effects Of Environmental Chemicals Acting Through Nuclear Receptors

There were a plenty of man-made chemicals existing in the environment,which attributed to the modem industry.Some of these chemicals might disrupt the normal function of the endocrine system of human being,causing cancer of reproduction organs,even making human lost their fertility.They belonged to the environmental endocrine disruptor(EEDs).The present study aimed to develop the in vitro nuclear receptor mediated reporter gene assay(also known as transactivation assay),and investigate the potential disrupting effects of these environmental chemicals to the function of estrogen receptor(ER), androgen receptor(AR)and thyroid hormone receptor(TR).On the basic of receptor-mediated theory,the receptor mediated reporter gene assays were designed to identify substances that might interfere with normal hormone activity by acting as an agonist or antagonist through ligand-receptor interaction.The DNA fragment containing hormone responsive element(HRE)was cloned into a reporter gene plasmid, making the reporter gene regulated by the HRE.The reconstructed reporter plasmid and the corresponding expression plasmid of the hormone receptor were co-transfected into a host cell to create an artificial gene expression system.The 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 constructed the reporter plasmids responding to the activation of ER,AR and TR,and then transfected them to the mammalian cell,getting the reporter gene assay systems,by which,the endocrine disrupting effects of some environmental chemicals were assessed.Partâ… Effects of environmental chemicals on the function of estrogen receptorObjectiveIn order to provide a model for screening the(anti)estrogen effects of chemicals,the ER reporter gene assay was developed using the luciferase (Luc)as the reporter gene,by transient and stable transfection.The assay was applied to evaluate the(anti)estrogen effects of some alkylphenol chemicals,bisphenol A,phenylphenol and some samples from water resource.Methods1.The CV-1 was 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.2.The plasmid expressed the protein of Gal4-BD fused human ERαwas constructed and co-transfected into MCF-7 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 E₂.3.MVLN cell was planted into 96 well plates and treated with various concentrations of test chemicals.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product Result1.The rERαmediated reporter gene assay could detecte the effects cause by 10^-10M E₂;at the concentration of 10^-7M,the maximal magnitude of the fold induction of 25.9-fold of control was achieved and maintained at a plateau level at the ranges of 10^-7M-10^-6M.The median effective concentration(EC₅₀)value of E₂ was 4.7 nM.The intro coefficient of variation(CV)in 24-well plate was 7.8%,and the inter CV was 12.6%.2.The hERa mediated reporter gene assay could detecte the effects cause by 0.5×10^-10M E₂;at the concentration of 10^-9M,the maximal magnitude of the fold induction of 17.9-fold of control was achieved and maintained at a plateau level at the ranges of 10^-9M-10^-6M.The median effective concentration(EC₅₀)value of E₂ was 0.16 nM.The intro coefficient of variation(CV)in 24-well plate was 6.7%,and the inter CV was 12.8%.3.The estrogenic activity of BPA,phenylphenol and alkylphenols (APs)were assessed by both hERa and rERαreporter assay.The results indicate that,the expression of luciferase increased with each additional carbon in the substituent,from 3 to 8 carbon atoms,but all test alkylphenol chemicals were much less potent than E₂.Briefly,the estrogenicity of test chemicals was 4-propylphenol＜4-tert-butylphenol＜4-pentylphenol＜4-phenylphenol＜4-octylphenol＜BPA.4.Two water samples could induce the rERαmedicated Luc expression.5.0.5pM E2 could induce the MVLN cell to express Luc,and the maximal magnitude of the fold induction of 4-fold of control was achieved at the concentration of 50pM.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.BPA,phenylphenol and APs possessed significant estrogenic activity,and their activity increased with the increase of substituent size in both hERαand rERαassays.Furthermore,the types of cell lines and the sources of ER showed little effect on the estrogenic activity of APs.3.The water sample might disturb the normal function of estrogen.4.MVLN cell had the ability to detect the agonist activity of test chemicals. Partâ…¡Effects of environmental chemicals on the function of androgen receptorObjectiveIn order to provide a model for screening the(anti)androgen effects of chemicals,the AR reporter gene assay was developed using the Luc as the reporter gene,by transient and stable transfection.The assay was applied to evaluate the(anti)androgen effects of some pesticides and industrial chemicals.Methods1.The oligonucleotides of the mouse mammary tumor virus long terminal repeat(MMTV-LTR)containing four androgen responsive element(ARE)sequences and TATA promoter were inserted into the kpnâ… and Bglâ…¡sites of pGl₃-basic to construct the new plasmid pMMTV-Luc. The CV-1 cell was co-transfected with three plasmids including the expression plasmid AR/pcDNA3.1 containing the cDNA of AR, constructed reporter plasmid pMMTV-Luc containing the Luc regulated by ARE and the control plasmid phRL-tk to create the AR reporter gene assay.The reference androgen 5α-dihydrotestosterone(5α-DHT)was used to verify the performance of the assay.AR agonist activity was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the AR by the test chemicals.AR antagonism was performed by measure the ability of a test substance to inhibit the induction of the reporter gene Luc product by 5α-DHT.2.MDA-kb2 cell was planted into 96 well plates and treated with various concentrations of test chemicals with or without DHT.Result1.The AR mediated reporter gene assay could detecte the effects cause by 10^-10M 5α-DHT;at the concentration of 10^-7M,the maximal magnitude of the fold induction of 61.83-fold of control was achieved and maintained at a plateau level at the ranges of 10^-7M-10^-6M.The median effective concentration(EC₅₀)value of E₂ was 3.65 nM.The reference androgen 5α-DHT showed no significant effect in inducing Luc activity, when the CV-1 cell was not transfected with AR expression plasmid.The intro CV in 24-well plate was 8.9%,and the inter CV was 13.1%.At the concentration ranges of 10^-7M-10^-5M,nilutamide and flutamide significantly inhibited the Luc activity induced by lnM 5α-DHT.The median inhibitory concentration(IC₅₀)value was 4.05μM and 3.49μM, respectively.2.The three pyrethroid pesticides Fenvalerate(Fen),Cypermethrin (Cyp),Permethrin(Per)and their metabolite 3-PBA did not show androgenic transcriptional activity at the tested concentrations.At the concentrations of 10^-4M,Fen,Cyp,Per and 3-PBA significantly inhibited the Luc activity induced by 1nM 5α-DHT,and the IC₅₀value was 0.37 mM,0.42 mM,0.43 mM and 1.21 mM.3.BPA,tetrachlorobisphenol A(TCBPA)pentachlorophenol(PCP) didn't induce the expression of Luc in AR mediated reporter assay.PCP didn't inhibit the the Luc activity induced by 1nM 5α-DHT,while BPA and TCBPA could suppress the function of 1nM 5α-DHT,with IC₅₀of 2.14μM and 10.45μM.4.10^-10M DHT could induce the expression of Luc,and the maximal magnitude of the fold induction of 9-fold of control was achieved and maintained at a plateau level at the ranges of 10^-8M-10^-5M. Conclusion1.The assay showed high sensitivity and acceptable repeatability to 5α-DHT.The reference androgen 5α-DHT induced Luc activity in an AR-depended and dose-response manner.In addition,the assay system was highly specific to androgenic compounds without cross-talk to GR agonist.The assay is useful in identifying AR agonists and antagonists from a large number of chemicals.2.Fen,Cyp,Per and 3-PBA could not activate the AR,but all of them showed weak antiandrogenic activity.3.BPA,TCBPA and PCP could not activate the AR;BPA and TCBPA showed antiandrogenic activity while PCP didn't.4.MDA-kb2 could be used to screen the androgen disruptors.Partâ…¢Effects of environmental chemicals on the function of thyroid hormone receptorObjectiveIn order to provide a model for screening the(anti)thyroid effects of chemicals,the TR mediated reporter gene assay and GH₃ cell proliferation assay were developed.The assays were applied to evaluate the(anti)thyroid hormone effects of some pesticides and industrial chemicals.Methods1.Rat pituitary tumor cell line GH₃ cell was cultured in serum-free medium PCM.And cells were planted on 96 wells plates at the density of 2500 cells per well and treated with various concentrations of test chemicals.2.The CV-1 was co-transfected with three plasmids including human TRβexpression plasmid pCMX-hTRβ,reporter plasmid pDR4-tk-CAT containing the reporter gene CAT regulated by the thyroid hormone responsive element(TRE)and the control plasmid pSV-β-Gal to develop the hTRβmediated reporter gene assays.The reference thyroid hormone L-3,5,3'-Triiodothyronine(T₃)was used to verify the performance of the assays.TR agonist assay was generally performed by quantifying the induction of the reporter gene CAT product in response to activation of the TRβby the test chemicals.TR antagonist assay was performed by measure the ability of a test substance to inhibit the induction of CAT product inducing by T₃3.The plasmid expressed the protein of Gal4-BD fused human TRβwas constructed and co-transfected into HepG₂ cell with Gal4 responsed reporter plasmid pUAS-tk-luc and the control plasmid phRL-tk to develop the hTRβmediated reporter gene assays.The reference thyroid hormone T₃ and L-Thyroidxin(T₄)was used to verify the performance of the assays.TR agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the TRβby the test chemicals.ER antagonist assay was performed by measure the ability of a test substance to inhibit the induction of Luc product by T₃.Result1.T₃ could stimulate the proliferation of GH₃ cell at 1nM,and the proliferation fold was 3-fold of vehicle contol.When treated the cells together with 1nM T₃,OH-PCB₁₀₆(at concentration of 10^-9M or higher) could suppress the function of T₃.2.10nM T₃ could induce the expression of CAT 3-fold of vehicle control in the hTRβmediated reporter gene assay.At the concentration as low as 10^-11M,OH-PCB₁₀₆could suppress the expression of reporter gene induced by 10nM T₃.However,this kind of suppression didn't become strong when we increased the concentration of OH-PCB₁₀₆,until the concentration raised to 10^-5M.3.The TRβmediated reporter gene assay could detecte the effects cause by 10^-11M T₃;at the concentration of 10^-8M,the maximal magnitude of the fold induction of 7.85-fold of control was achieved and maintained at a plateau level at the ranges of 10^-8M-10^-6M.The median effective concentration(EC₅₀)value of T₃ was 0.46 nM.The intro CV in 48-well plate was 5.9%,and the inter CV was 11.7%.4.At the concentration below 10^-4M,carbaryl,1-naphthol(1-NAP) and 2-naphthol(2-NAP)couldn't induce the expression of Luc.However, at or above the concentration of 10^-5M,all of them could inhibit the expression of Luc induced by 5nM T₃,and the IC₅₀was 8.40×10^-5M, 7.62×10^-5M and 7.73×10^-5M for carbaryl,1-NAP and 2-NAP.Conclusion1.OH-PCB₁₀₆showed anti-thyoid hormone activity.2.The assay showed high sensitivity and acceptable repeatability to T₃ and T₄.The reference thyroid hormone T₃ induced Luc activity in a TR-depended and dose-response manner.The assay is useful in identifying TR agonists and antagonists from a large number of chemicals.3.Carbaryl,1-NAP and 2-NAP might disrupt the normal function of T₃.