Effects of low-dose Bisphenol A on calcium ion influx and on genes of proliferation and differentiation in immortalized human gingival cells in vitro: The role of estrogen receptor beta

Abstract

Objectives

Relating to low-dose Bisphenol-A (BPA), there is still a lack of mechanistic studies in oral cells, representing the first targets of BPA by oral intake. The objective of this study was to investigate an assumed mechanistic interrelationship between both low-dose BPA-modulated Calcium ion (Ca 2+ ) influx and cell behavior, and the estrogen receptor β (ERβ), in oral mucosal cells.

Methods

Indirect immunofluorescence (IIF) was conducted on estrogen receptor beta (ERβ) activity after 1, 3, and 6 days in response to 39 nM BPA, 15 μM BPA, and 200 pM 17β-Estradiol (E 2 ). In addition to Ca 2+ concentration measurement, qPCR for proliferation and differentiation biomarkers was performed, to examine cell behavior. Fulvestrant-mediated ER inhibition was employed to seek for a mechanistic role of ERβ in regulating BPA-emanating effects.

Results

While both E 2 and BPA yielded ERβ activation, 39 nM BPA and 200 pM E 2 did not change MKI67 proliferation marker expression, but reduced transcription of differentiation markers. Conversely, 15 μM BPA reduced MKI67 transcription, but significantly increased differentiation gene expression and intracellular Ca 2+ levels. Fulvestrant-induced ERβ inhibition yielded complete elimination of all E 2 − and BPA-triggered modulatory effects, suggesting a mechanistic role of activated ERβ for BPA-mediated Ca 2+ influx and keratinocyte differentiation.

Significance

Concerning cell behavior, these findings provide significant evidence of a threshold-dependent transcription of proliferation and differentiation-related genes as well as Ca 2+ influx in response to 39 nM and 15 μM low-dose BPA, which identify a mechanistic role of activated ERβ in oral keratinocytes.

Introduction

Bisphenol A (BPA) is mainly used as a precursor to manufacture polycarbonates and epoxy resins, which also find application in the food packaging industry. In the dental sector, BPA is used to produce basic monomers for dental composites like Bisphenol A glycidyl methacrylate (BisGMA) or Bisphenol A dimethacrylate (BisDMA) . In this context, BPA was shown also to elute from different dental composite materials in concentrations between 0.8 and 6.14 mg BPA/L , after storage of small composite samples (4.5 mm × 2 mm), representing small fillings, in the clinical relevant solvent ethanol 75% for time periods varying from 24 h up to 28 days. Such data cause great concern about the composite materials due to the fact that BPA is a known endocrine disruptor showing similar estrogenic activity like the normal estrogens.

Due to insecurities, concerning possible subacute effects, which are caused by low doses of BPA through dietary intake, the European Food Safety Authority (EFSA) set the tolerable daily intake (TDI) of BPA to 50 μg/kg bodyweight (BW)/day in 2007 , which is one part per thousandth of the lowest observed adverse-effect-level . Since it was established, this threshold value was updated and reduced to 4 μg/kg BW/day in January 2015 , due to an updated exposure assessment report . In this report, the average daily intake of BPA amounted to 126 ng/kg BW/day among adults in Europe through diet. Nonetheless, one of the prevailing concerns is the ability of BPA to bind to the estrogen receptors α (ERα) and β (ERβ), through which this endocrine-active substance may have an effect on the human organism at doses far below the presumably safe TDI.

With respect to the aforementioned concern, Gayrard et al. were able to show a sublingual absorption of BPA besides typical digestion, which in turn implicates that BPA is able to diffuse through oral mucosal tissues and therefore through cell membranes. Regarding the oral mucosal epithelium, Valimaa et al. found that the ERβ is the exclusive estrogen receptor in the human gingival keratinocytes, while Kuiper et al. have proven a 6.6 fold higher binding affinity of BPA to ERβ than to ERα. Generally, nuclear transgressed ERs are rated as transcription factors which are activated after ligand binding or optionally via posttranslational modifications, to bind to certain DNA sequences named estrogen-responsive elements (EREs). At these EREs, estrogen receptors interact with differential co-factors to modulate target gene transcription . In terms of murine ERβ, phosphorylation of serines (S) 106 and 124 can either lead to ERβ activation by the E 2 ligand or in case of S124 optionally mediate ligand-independent transcriptional activity, while phosphorylation of murine S124 is equivalent to S105 phosphorylation in humans . While induced expression of ERβ in breast cancer cells yielded remarkable proliferation reduction , available reports on the biological consequences of S105 phosphorylation-induced ERβ activity in human breast cancer and/or breast cancer-derived cell models revealed impairment of migration and invasion .

At doses starting at 10 nM, BPA caused a fast increase of the important cell-modulating Calcium ion (Ca 2+ ) in different cell types , which was explained with a rapid ERβ signaling . For various cell types it has been reported that E 2 induced a rapid influx-based Ca 2+ rise , which in case of neurons occurred via membrane-bound L-type calcium channel activation . This channel activation was mediated by E 2 -responsive membrane receptors, among which membrane-associated i.e. non-genomic ERα and ERβ were identified .

Combining these findings, it has to be questioned if a low dose exposure from BPA has subacute effects on human oral mucosal cells, due to the substance’s xenoestrogenic property. One possibility would be an ERβ-dependent modulation of intracellular Ca 2+ level similar to previous findings . Since the alteration of the intracellular Ca 2+ level is a main switch of differentiation regulation in oral mucosal cells, particularly epithelial keratinocytes , this could yield consecutive cell alterations, in first instance detectable on the gene expression level. Consequently, involved genes, among others, could be associated with cell behavioral features such as proliferation, and differentiation. In the context of differentiation, this would include the intermediate filament proteins keratin 1 and 10 as early markers of epithelial keratinocyte differentiation, while involucrin and filaggrin indicate late stages, i.e. terminal differentiation .

Therefore, in the present study, we examined the effects of environmentally and tissue uptake-relevant low-dose BPA and E 2 on intracellular Ca 2+ levels and gene expression, to extend the understanding of how BPA affects oral mucosal cell behavior in mechanistic terms. Here we show for the first time that various low-dose BPA concentrations modulate proliferation and differentiation-related genes in gingival oral mucosal keratinocytes in vitro in a discriminative manner, while intracellular Ca 2+ increase showed dependence on both applied BPA concentration and time. Administration of Fulvestrant identified the activated ERβ as mechanistically indispensable for keratinocyte-innate gene expression modulation and increase in intracellular Ca 2+ levels.

Materials and methods

Determination of BPA concentrations

In 2007, EFSA published a risk assessment report about the endocrine effects of BPA, which underlined the agency’s opinion on the TDI of 50 μg/kg BW/day . In 2013, an updated exposure assessment report about BPA was published, in which a daily intake of 126 ng BPA/kg BW/day for men between 18 and 45 years living in Europe was estimated . Applied on a human model with a bodyweight of 70 kg and a total saliva amount of approximately 1000 mL/day, the following calculations were carried out:

  • 1.

    50 μg BPA/kg BW/day × 70 kg BW/1000 ml/day = 3.5 mg BPA/L or 15 μM BPA

  • 2.

    126 ng BPA/kg BW/day × 70 kg BW/1000 ml/day = 8.82 μg BPA/L or 39 nM BPA

Therefore, all experiments were conducted with these BPA concentrations. Additionally, negative controls without BPA and a positive control with 17β-Estradiol (E 2 , 200 pM) were applied. To distinct the estrogenic influence of BPA from any other non ER-dependent cell reaction, all experiments were additionally conducted with the competitive non-agonistic ER-inhibitor Fulvestrant (200 nM) .

Cell cultures and cultivation media

For the use of the tissues informed consent was obtained by the patients according to the Helsinki Declaration, and the protocol was approved by the institutional ethics committee (Votum Nr. 411/08; Date: 11/20/2008). Based on the previous work of Roesch-Ely et al. , human oral mucosa-derived gingival keratinocytes, immortalized with the human papilloma virus type 16 E6/E7 oncogenes (IHGK) were used in this study. IHGK were cultured in passages between 88–99. All experiments described below were conducted with comparable passages.

For the cultivation of IHGK, keratinocyte growth medium (KGM) (PromoCell, Heidelberg, Germany) was mixed with kanamycin (100 mM, Sigma-Aldrich, Hamburg, Germany) and the provided supplements (PromoCell, Heidelberg, Germany). In order to prevent a superposition of any BPA effects by growth factors, all media were created as serum free versions for the performed experiments. The incubation was conducted in standard cell culture flasks up to size T175 (Greiner Bio-One, Frickenhausen, Germany) at 37 °C, 97% humidity and 5% CO 2 . Splitting of cells was carried out at a cell density of approximately 70% and the cell numbers were determined by using a CASY ® Model TT cell counter (OLS, Hamburg, Germany).

Preparation of cell culture media

BPA (CAS No. 80-05-7, purity ≥ 99%), E 2 (CAS No. 50-28-2, purity > 98%) and Fulvestrant (ICI 182780; CAS No. 129453-61-8, purity > 98%) were purchased from Sigma-Aldrich (Hannover, Germany). BPA was added to phosphate buffered saline (PBS) (PromoCell, Heidelberg, Germany) with 4% DMSO under sterile conditions to prepare two solutions with 150 μM and 390 nM BPA. These solutions were added at a ratio of 1:10 to the serum free cell culture media to achieve the final concentrations of 15 μM and 39 nM BPA. The same procedure was used to obtain an E 2 concentration in serum free cell culture media of 200 pM E 2 . To eliminate any failure of dilution, native PBS containing 4% DMSO was also added to the serum free cell culture media of the negative control with the ratio of 1:10. By this, the final DMSO concentration in the cell cultures amounted to 0.4%. To evaluate if any possible effects are caused by an ER dependent reaction, all solutions were additionally prepared with 200 nM Fulvestrant/ICI 182780 , which efficiently acts as a competitive non-agonistic ER inhibitor (antiestrogen) at a concentration of 100 nM or more , and has a binding affinity of 89% of E 2 .

Determination of incubation periods

With the help of an impedance-based real-time cell analyzer (RTCA) (xCelligence System, OLS, Hamburg, Germany), a preliminary test was performed in order to evaluate time points of interest for the BPA concentrations. Therefore, 1.5 × 10 4 IHGK cells were seeded in a 16 well E-plate per well (OLS, Hamburg, Germany). After 24 h of cultivation, the BPA solutions were added to the IHGK being cultured in serum-free culture media in triplicates. A repeated measures one-way ANOVA test was performed to detect any overall differences between related means of each BPA treated group and the negative control. P -values < 0.05 were considered statistically significant.

Indirect immunofluorescence assay

For the indirect immunofluorescence assay (IIF) on filaggrin and the ERβ in its non-activated and activated state, IHGK were seeded on μ-Chamber 12 well slides (ibidi, Munich, Germany) with a cell density of 6 × 10 4 cells/cm 2 for 1 day of incubation. The addition of the BPA and E 2 solutions was conducted in the same way as for the preliminary RTCA-experiment. Additionally to this native cell culture media preparation (native), the same experiment was set up with 200 nM Fulvestrant. After 1 day of incubation, the cells were fixed with 3.8% formaldehyde in PBS for 10 min, washed thrice with PBS, permeabilized with 0.2% TritonX-100 (Roche Diagnostics GmbH, Penzberg, Germany) for 5 min, rinsed thrice with PBS, and incubated with the Image-iT FX signal enhancer (Invitrogen, Life Technologies, Carlsbad, USA) for 30 min to avoid unspecific fluorescence. After another triple rinsing step with PBS, IIF was conducted for the activated and total ERβ within the same specimen by the following modus operandi. The total ERβ was stained with the anti-estrogen receptor beta antibody [14C8] (ab288, dilution = 1:500) and simultaneously, the activated ERβ was stained with the anti-estrogen receptor beta (phospho S105) antibody (ab62257, dilution = 1:500) (both Abcam, Cambridge, England, UK) for 60 min. The same procedure was conducted within the separate filaggrin staining. Here, we used the anti-filaggrin antibody [SPM181] (ab17808, dilution = 1:500, Abcam) for 60 min. Subsequently, the cells were washed thrice with PBS for 15 min and then stained with secondary antibodies for 45 min. For this step, an Alexa Fluor 488 nm anti-rabbit and an Alexa Fluor 555 nm anti-mouse antibody (both Invitrogen) were used. After rinsing thrice with PBS for 15 min, the cell nuclei were counterstained with a 300 nM DAPI solution for 10 min. Subsequently, the cells were washed twice with PBS, once rinsed with distilled water, embedded in mounting medium (SouthernBiotech, Birmingham, Alabama, USA) and finally observed by using fluorescent microscopy (BZ-9000, Keyence, Ōsaka, Japan)

Determination of total intracellular calcium ion concentrations

To measure the cells’ total Calcium ion (Ca 2+ ) concentrations, IHGK were seeded in 96 well microplates (F-bottom/chimney well, black, Greiner Bio One) with a cell density of 6 × 10 4 cells/cm 2 for 1 day, 4 × 10 4 cells/cm 2 for 3 days and 3 × 10 4 cells/cm 2 for 6 days of incubation. After 24 h of adhesion, the addition of the BPA and E 2 solutions without (native) and with 200 nM Fulvestrant was conducted in the same way as described above for the preliminary RTCA-experiment. After the abovementioned incubation periods, a Fluo-4 Direct™ Calcium Assay Kit (Life Technologies) was used according to the manufacturer’s instructions, by which the modified media could be left in its original state. Therefore, an equal volume of 150 μL of the prepared 2-fold calcium reagent loading solution was added to the wells, respectively. After 30 minutes of incubation at 37 °C and another 30 min at room temperature, relative fluorescence was measured using an Infinite 200 PRO Microplate Reader (Tecan, Crailsheim, Germany). For the statistical analysis, a two-way ANOVA, particularly a Dunnett’s multiple comparisons test was conducted by using the software Prism (GraphPad Software, La Jolla, USA). Statistical significance was considered with p -values < 0.01.

Quantitative real-time-polymerase chain reaction analysis (qRT-PCR)

For the qRT-PCR analysis, IHGK were seeded in 24 well plates at passage 99 with the same cell densities described for the IIF. After 24 h of adhesion, the addition of the BPA and E 2 solutions without (native) and with 200 nM Fulvestrant was conducted in the same way as for the preliminary RTCA – experiment. After the respective incubation periods, cells were lysed using RLT buffer (Qiagen, Venlo, Netherlands). RNA isolation was conducted with the RNeasy mini kit in a fully automated process using a Qiacube (both Qiagen, Venlo, Netherlands). Thereafter, RNA concentration and integrity was determined with the Experion RNA analysis kit and the corresponding automated electrophoresis system (both Biorad, Muenchen, Germany) according to the manufacturer’s instructions. Reverse transcription to First-strand cDNA was performed with 100 ng total RNA using the RT 2 First Strand Kit (Qiagen, Venlo, Netherlands) following the manufacturer’s instructions. The amount of obtained cDNA was analyzed with the Quant-iT PicoGreen dsDNA Reagent Kit (Life Technologies, Carlsbad, California, USA) and an Infinite 200 PRO Microplate Reader (Tecan, Crailsheim, Germany) according to the manufacturer’s instructions. For normalization, 5 ng cDNA were used in each qRT-PCR reaction. The quantitative amplification detection was conducted with the RT 2 SYBR Green qPCR Mastermix (SABiosciences, Qiagen, Venlo, Netherlands) on the CFX96 real-time PCR detection system (BioRad, Muenchen, Germany) according to the manufacturer’s protocol. Relevant biomarkers for proliferation and differentiation were examined using the following commercially available and pre-validated primers (SABiosciences, Qiagen): Ki-67 (MKI67) for proliferation, involucrin, filaggrin, keratin 1 and 10 (IVL, FLG, KRT1, KRT10) for differentiation plus the housekeeping genes Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and Ribosomal protein L13a (RPL13A). All qRT-PCRs were performed using the following protocol: 95 °C for 10 min (initial denaturation) and then 40 cycles 15 s at 95 °C (denaturation), 30 s at 55 °C (annealing) and 30 s at 72 °C (extension).

The relative expression levels of each mRNA were calculated using a modified ΔΔC T equation, which allows the counting for differences in efficiencies between the PCRs . The data were normalized to the index C T of the non-modulated housekeeping genes GAPDH and RPL13A and referred to the relative gene expression values of the cells’ RNA of the negative control. After RNA collection, reverse transcription to cDNA and conduction of the qRT-PCR, the relative gene expression data of all biomarkers and periods of time were summarized in gene studies using the manufacturer’s software CFX Manager™ Software v3.1 (Bio-Rad Laboratories, Inc., Hercules, USA) for evaluation. By performing the software’s Student’s t -test for unequal variance, all changes of gene expression values with a cut off of p < 0.05 were considered statistically significant.

Materials and methods

Determination of BPA concentrations

In 2007, EFSA published a risk assessment report about the endocrine effects of BPA, which underlined the agency’s opinion on the TDI of 50 μg/kg BW/day . In 2013, an updated exposure assessment report about BPA was published, in which a daily intake of 126 ng BPA/kg BW/day for men between 18 and 45 years living in Europe was estimated . Applied on a human model with a bodyweight of 70 kg and a total saliva amount of approximately 1000 mL/day, the following calculations were carried out:

  • 1.

    50 μg BPA/kg BW/day × 70 kg BW/1000 ml/day = 3.5 mg BPA/L or 15 μM BPA

  • 2.

    126 ng BPA/kg BW/day × 70 kg BW/1000 ml/day = 8.82 μg BPA/L or 39 nM BPA

Therefore, all experiments were conducted with these BPA concentrations. Additionally, negative controls without BPA and a positive control with 17β-Estradiol (E 2 , 200 pM) were applied. To distinct the estrogenic influence of BPA from any other non ER-dependent cell reaction, all experiments were additionally conducted with the competitive non-agonistic ER-inhibitor Fulvestrant (200 nM) .

Cell cultures and cultivation media

For the use of the tissues informed consent was obtained by the patients according to the Helsinki Declaration, and the protocol was approved by the institutional ethics committee (Votum Nr. 411/08; Date: 11/20/2008). Based on the previous work of Roesch-Ely et al. , human oral mucosa-derived gingival keratinocytes, immortalized with the human papilloma virus type 16 E6/E7 oncogenes (IHGK) were used in this study. IHGK were cultured in passages between 88–99. All experiments described below were conducted with comparable passages.

For the cultivation of IHGK, keratinocyte growth medium (KGM) (PromoCell, Heidelberg, Germany) was mixed with kanamycin (100 mM, Sigma-Aldrich, Hamburg, Germany) and the provided supplements (PromoCell, Heidelberg, Germany). In order to prevent a superposition of any BPA effects by growth factors, all media were created as serum free versions for the performed experiments. The incubation was conducted in standard cell culture flasks up to size T175 (Greiner Bio-One, Frickenhausen, Germany) at 37 °C, 97% humidity and 5% CO 2 . Splitting of cells was carried out at a cell density of approximately 70% and the cell numbers were determined by using a CASY ® Model TT cell counter (OLS, Hamburg, Germany).

Preparation of cell culture media

BPA (CAS No. 80-05-7, purity ≥ 99%), E 2 (CAS No. 50-28-2, purity > 98%) and Fulvestrant (ICI 182780; CAS No. 129453-61-8, purity > 98%) were purchased from Sigma-Aldrich (Hannover, Germany). BPA was added to phosphate buffered saline (PBS) (PromoCell, Heidelberg, Germany) with 4% DMSO under sterile conditions to prepare two solutions with 150 μM and 390 nM BPA. These solutions were added at a ratio of 1:10 to the serum free cell culture media to achieve the final concentrations of 15 μM and 39 nM BPA. The same procedure was used to obtain an E 2 concentration in serum free cell culture media of 200 pM E 2 . To eliminate any failure of dilution, native PBS containing 4% DMSO was also added to the serum free cell culture media of the negative control with the ratio of 1:10. By this, the final DMSO concentration in the cell cultures amounted to 0.4%. To evaluate if any possible effects are caused by an ER dependent reaction, all solutions were additionally prepared with 200 nM Fulvestrant/ICI 182780 , which efficiently acts as a competitive non-agonistic ER inhibitor (antiestrogen) at a concentration of 100 nM or more , and has a binding affinity of 89% of E 2 .

Determination of incubation periods

With the help of an impedance-based real-time cell analyzer (RTCA) (xCelligence System, OLS, Hamburg, Germany), a preliminary test was performed in order to evaluate time points of interest for the BPA concentrations. Therefore, 1.5 × 10 4 IHGK cells were seeded in a 16 well E-plate per well (OLS, Hamburg, Germany). After 24 h of cultivation, the BPA solutions were added to the IHGK being cultured in serum-free culture media in triplicates. A repeated measures one-way ANOVA test was performed to detect any overall differences between related means of each BPA treated group and the negative control. P -values < 0.05 were considered statistically significant.

Indirect immunofluorescence assay

For the indirect immunofluorescence assay (IIF) on filaggrin and the ERβ in its non-activated and activated state, IHGK were seeded on μ-Chamber 12 well slides (ibidi, Munich, Germany) with a cell density of 6 × 10 4 cells/cm 2 for 1 day of incubation. The addition of the BPA and E 2 solutions was conducted in the same way as for the preliminary RTCA-experiment. Additionally to this native cell culture media preparation (native), the same experiment was set up with 200 nM Fulvestrant. After 1 day of incubation, the cells were fixed with 3.8% formaldehyde in PBS for 10 min, washed thrice with PBS, permeabilized with 0.2% TritonX-100 (Roche Diagnostics GmbH, Penzberg, Germany) for 5 min, rinsed thrice with PBS, and incubated with the Image-iT FX signal enhancer (Invitrogen, Life Technologies, Carlsbad, USA) for 30 min to avoid unspecific fluorescence. After another triple rinsing step with PBS, IIF was conducted for the activated and total ERβ within the same specimen by the following modus operandi. The total ERβ was stained with the anti-estrogen receptor beta antibody [14C8] (ab288, dilution = 1:500) and simultaneously, the activated ERβ was stained with the anti-estrogen receptor beta (phospho S105) antibody (ab62257, dilution = 1:500) (both Abcam, Cambridge, England, UK) for 60 min. The same procedure was conducted within the separate filaggrin staining. Here, we used the anti-filaggrin antibody [SPM181] (ab17808, dilution = 1:500, Abcam) for 60 min. Subsequently, the cells were washed thrice with PBS for 15 min and then stained with secondary antibodies for 45 min. For this step, an Alexa Fluor 488 nm anti-rabbit and an Alexa Fluor 555 nm anti-mouse antibody (both Invitrogen) were used. After rinsing thrice with PBS for 15 min, the cell nuclei were counterstained with a 300 nM DAPI solution for 10 min. Subsequently, the cells were washed twice with PBS, once rinsed with distilled water, embedded in mounting medium (SouthernBiotech, Birmingham, Alabama, USA) and finally observed by using fluorescent microscopy (BZ-9000, Keyence, Ōsaka, Japan)

Determination of total intracellular calcium ion concentrations

To measure the cells’ total Calcium ion (Ca 2+ ) concentrations, IHGK were seeded in 96 well microplates (F-bottom/chimney well, black, Greiner Bio One) with a cell density of 6 × 10 4 cells/cm 2 for 1 day, 4 × 10 4 cells/cm 2 for 3 days and 3 × 10 4 cells/cm 2 for 6 days of incubation. After 24 h of adhesion, the addition of the BPA and E 2 solutions without (native) and with 200 nM Fulvestrant was conducted in the same way as described above for the preliminary RTCA-experiment. After the abovementioned incubation periods, a Fluo-4 Direct™ Calcium Assay Kit (Life Technologies) was used according to the manufacturer’s instructions, by which the modified media could be left in its original state. Therefore, an equal volume of 150 μL of the prepared 2-fold calcium reagent loading solution was added to the wells, respectively. After 30 minutes of incubation at 37 °C and another 30 min at room temperature, relative fluorescence was measured using an Infinite 200 PRO Microplate Reader (Tecan, Crailsheim, Germany). For the statistical analysis, a two-way ANOVA, particularly a Dunnett’s multiple comparisons test was conducted by using the software Prism (GraphPad Software, La Jolla, USA). Statistical significance was considered with p -values < 0.01.

Quantitative real-time-polymerase chain reaction analysis (qRT-PCR)

For the qRT-PCR analysis, IHGK were seeded in 24 well plates at passage 99 with the same cell densities described for the IIF. After 24 h of adhesion, the addition of the BPA and E 2 solutions without (native) and with 200 nM Fulvestrant was conducted in the same way as for the preliminary RTCA – experiment. After the respective incubation periods, cells were lysed using RLT buffer (Qiagen, Venlo, Netherlands). RNA isolation was conducted with the RNeasy mini kit in a fully automated process using a Qiacube (both Qiagen, Venlo, Netherlands). Thereafter, RNA concentration and integrity was determined with the Experion RNA analysis kit and the corresponding automated electrophoresis system (both Biorad, Muenchen, Germany) according to the manufacturer’s instructions. Reverse transcription to First-strand cDNA was performed with 100 ng total RNA using the RT 2 First Strand Kit (Qiagen, Venlo, Netherlands) following the manufacturer’s instructions. The amount of obtained cDNA was analyzed with the Quant-iT PicoGreen dsDNA Reagent Kit (Life Technologies, Carlsbad, California, USA) and an Infinite 200 PRO Microplate Reader (Tecan, Crailsheim, Germany) according to the manufacturer’s instructions. For normalization, 5 ng cDNA were used in each qRT-PCR reaction. The quantitative amplification detection was conducted with the RT 2 SYBR Green qPCR Mastermix (SABiosciences, Qiagen, Venlo, Netherlands) on the CFX96 real-time PCR detection system (BioRad, Muenchen, Germany) according to the manufacturer’s protocol. Relevant biomarkers for proliferation and differentiation were examined using the following commercially available and pre-validated primers (SABiosciences, Qiagen): Ki-67 (MKI67) for proliferation, involucrin, filaggrin, keratin 1 and 10 (IVL, FLG, KRT1, KRT10) for differentiation plus the housekeeping genes Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and Ribosomal protein L13a (RPL13A). All qRT-PCRs were performed using the following protocol: 95 °C for 10 min (initial denaturation) and then 40 cycles 15 s at 95 °C (denaturation), 30 s at 55 °C (annealing) and 30 s at 72 °C (extension).

The relative expression levels of each mRNA were calculated using a modified ΔΔC T equation, which allows the counting for differences in efficiencies between the PCRs . The data were normalized to the index C T of the non-modulated housekeeping genes GAPDH and RPL13A and referred to the relative gene expression values of the cells’ RNA of the negative control. After RNA collection, reverse transcription to cDNA and conduction of the qRT-PCR, the relative gene expression data of all biomarkers and periods of time were summarized in gene studies using the manufacturer’s software CFX Manager™ Software v3.1 (Bio-Rad Laboratories, Inc., Hercules, USA) for evaluation. By performing the software’s Student’s t -test for unequal variance, all changes of gene expression values with a cut off of p < 0.05 were considered statistically significant.

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Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Effects of low-dose Bisphenol A on calcium ion influx and on genes of proliferation and differentiation in immortalized human gingival cells in vitro: The role of estrogen receptor beta
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