A Human In Vitro Study To Identify Estrogenic-like And(Anti)Progestin-like Effects Of Chemicals On Endometrial Biomarkers

Part1The expression of endometrial biomarkers in Ishikawa cellObjectiveThe Ishikawa cell model was established to detect the expression of eight endometrial receptivity target genes(PR、ER、EGF、LIF、COX-2、IGFBP-1、VEGF-R1、IL-1β)and eight reference genes (G6PDH、ALAS1、PGK-1、b-2M、b-actin、HPRT、PBGD、 RPⅡ)expressing stably in different cells in order to verify the endometrial specificity and stability of this cell model. The expression of PR、ALPPL2and GOS2incubated with17β-estradiol was detected with the purpose of determining the stability of gene expression under the intervention of17β-estradiol, especially that of PR in this Ishikawa cell model.Methods1. Target and reference genes(eight,each) expression in human endometrial Ishikawa cellIshikawa Cells were seeded at a density of50000cells/ml and grown for72h to subconfluency in the absence of hormones. RT-qPCR was used to detect the mRNA expression of the pre-selected target(PR、ER、EGF、LIF、COX-2、IGFBP-1、VEGF-R1、 IL-1β)and reference genes(G6PH、ALAS1、PGK-1、b-2M、b-actin、HPR、PBGD、 RPⅡ).The relative quantification of gene mRNA expression was characterized relative quanimcation cycle value (Oq).2. Effects of17β-estradiol on the mRNA expression of PR, ALPPL2and GOS2Ishikawa Cells were seeded at a density of50000cells/ml and grown for72h to be subconfluency in the absence of hormones. Then cells were incubated with17β-estradiol in different density for48h. The PRmRNA expression was tested by RT-qPCR. Ethonal (0.1%) was applied as vehicles for compounds with poor water solubility. EC50was used to demonstrate the test compound concentration that produces50%of the maximal fold change. Sigmoidal Dose-response curves were made according to the results of RT-qPCR.Results1. All the Pre-selected target and reference genes were expressed in our Ishikawa cell model after72h cell culture.2. Up-regulation of PR、GOS、ALPPL2mRNA by17β-estradiol was found by RT-qPCR. The mRNA expression level of ALPPL2and G0S2mRNA was higher than of the PR, but the fold change of PRmRNA levels induced by17β-estradiol was more obviously than ALPPL2and G0S2. No significant differences of the normalized ratios were found when PR mRNA was normalized to the three different reference genes ALAS1, G6PDH and B2M(P>0.05).Conclusion1. The endometrial specificity and stability of this Ishikawa cell model was verified on the mRNA level.2. The stability of the up-regulation of PRmRNA induced by17β-estradiol was confirmed in Ishikawa cell model which provides a theoretical basis for using this model to carry out the research on PR.Part2Effects of estrogenic-like chemicals on PR in Ishikawa cellObjectiveThe Ishikawa cell model was used to test the PR mRNA expression under the incubation of eleven toxic chemicals listed in the "Feasibility study" together with the known estrogen substances17β-estradiol and DES as well as estrogen negative substance RU486to confirm whether these eleven chemicals have estrogen-like effects and to tell the difference between strong and weak effect. Methods1. Effects of estrogen and estrogen-like chemicals on the PR mRNA expression and protein secretionIshikawa Cells were incubated with eleven toxic chemicals listed in the "Feasibility study" together with the known estrogen substances17β-estradiol and DES as well as estrogen negative substance R.U486in different density for48h. The PRmRNA expression was tested by RT-qPCR. EC50and Sigmoidal Dose-response curves were demonstrated as above. The protein secretion of PR was tested by WesternBlotting.2. AlamarBlue assayAfter being subconflency, Cells were treated with17(3-estradiol,4-nonylphenol, DES and bisphenol A in different density for48h. For control, Untreated cells was used for control and then Cytotoxicity in Ishikawa cells was checked by the AlamarBlue assay.Results1. Up-regulation of PRmRNA by17β-estradiol. DES as well as4-nonylphenol and bisphenol A(Feasibility study) was found by RT-qPCR. A clear sigmoidal dose-response curve about these substances was established. RU486and the left ten substances from feasibility study had no up-regulation effect on PRmRNA expression. The EC50of17β-estradiol、DES and bisphenol A was2.44×10-11M、2.61×10-11M、2.98×10-7M respectively. On the protein level, compared to the0.1%Ethonal group, the protein secretion of PR was increased obviously by17β-estradiol,DES (10-9M, each) as well as by bisphenol A and4-nonylphenol (10-6M and5×10-6M, respectively)(P<0.05).2. None of the tested compounds displayed cytotoxicity in the AlamarBlue assay at different concentrations(10-16M-10-6M) when effects on PR mRNA up-regulation were observed in48h(P>0.05).Conclusion1. The Ishikawa cell model was verified not only can detect whether compounds with estrogenic effects or not but also distinguish between the strong and weak estrogenic effect.2. Bisphenol A and4-nonylphenol from the "Feasibility study" were tested to have estrogenic effect and methylacetoacetate(MAA) was found to be estrogen negative effect chemical. Part3Effects of (anti)progestin-like chemicals on PR and SULT1E1in Ishikawa cellObjectiveSome chemicals and natural substances interacted with PR were collected through the literature retrieval. The most sensitive gene under the intervention of RU486was determined by the gene chip. The Ishikawa cell model was used to test the expression of PR and the most sensitive gene found in order to confirm whether the collected compounds have (anti)Progestin like effect and to distinguish between strong and weak effect.Methods1. Affymetrix Genechip microarrayWhen being subconflency, the cells were treated with P4+RU486(10-8M, each) or P4alone (10-8M) for48h. Accepting a maximum false discovery rate of10%(i.e. q=0.1) and a minimal absolute median fold change of1.5. The most responsive genes under RU486treatment were identified by Affymetrix GeneChip microarray.2. Effects of P4and P4+RU486on the PR、most responsive gene mRNA expressionand protein secretionWhen being sub-confluence, Ishikawa cells were grown with17p-estradiol (10-8M) for72h and then the cells were incubated with Progesterone(10-8M) alone and combined with RU486in different density(10-4-10-12M) for48h. DMSO (0.1%) was tested as negative control. The PR and most responsive gene mRNA expression was tested by RT-qPCR. EC50and Sigmoidal Dose-response curves were demonstrated as above. The protein secretion of PR、SULT1E1was tested by WesternBlotting.3. The effect of collected compounds on the mRNA expression of most responsive geneWhen being sub-confluence. Ishikawa cells were incubated with P4combined with ZK137316、UPA、4-NP、bisphenol A as well as Apigenin in different intensity for48h respectively. DMSO (0.1%) was tested as negative control. The most responsive gene mRNA expression was tested by RT-qPCR. EC50and Sigmoidal Dose-response curves were demonstrated as above.Results 1. Sixty-nine genes out of28,869genes were considered differentially regulated in P4+RU486treated compared to P4-treated cells. The most responsive out of these sixty-nine genes headed by the estrogen sulfotransferase SULT1E1is along with computed median fold change and q-values.2. A up-regulation of SULT1E1and a down-regulation of PR mRNA expression only treated with P4were demonstrated by Sigmoidal dose-response curves. The P4affects SULT1E1mRNA expression much stronger than PR mRNA. The effects of P4mentioned above on PR、SULT1E1mRNA was concentration-dependently antagonized in combinations with RU486.0.1%DMSO as a negative control in all tested concentrations had no effect on relative SULT1E1and PR mRNA levels. On the protein level, compared to the0.1%DMSO group, the protein secretion of PR was decreased by P4and that of SULT1E1was increased by P4; compared with P4treated, the P4combined with RU486increase the protein secretion of PR and decrease that of SULT1E1.3. A up-regulation of SULT1E1mRNA by P4was concentration-dependently antagonized in combinations with ZK137316、UPA、4-NP、BPA、API. The strong antagonistic effect of UPA. ZK137316on SULT1E1was found with EC509.0×10-10M、6.5×10-9M respectively. The relative weak antagonistic effect of4-NP、BPA、API on SULT1E1was found with EC503.4×10-6M、7.5×10-6M,9.8×10-7M respectively.Conclusion1. The Ishikawa cell model was verified not only can detect whether compounds with (anti)progestin effects or not but also distinguish between the strong and weak (anti) progestin effect.2. SULT1E1is the most sensitive gene under the treatment of anti-progestin compounds and can be used as a biomarker to detect the compounds with anti-progestin effects.3. Bisphenol A and4-nonylphenol from the "Feasibility study" were tested to have anti-progestin effect.