ABSTRACT
Objectives:
Bisphenol A (BPA) is a synthetic monomer used in the production of polycarbonate and an environmental contaminant with endocrine disrupting properties. BPA release from plastic carriers is thought to cause high amounts of exposure, which result in high risk to human and environment health.
Materials and Methods:
The study examined the possible changes in global DNA methylation, CpG promoter DNA methylation, and gene expressions of Rassf1a and c-myc after BPA exposure in rat kidney epithelial cells (NRK-52E).
Results:
The IC50 values of BPA in NRK-52E cells were 133.42 and 101.74 μM in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake tests, respectively. The cells were treated with BPA at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM concentrations for 24 h and at 100 nM concentration for 24, 48, 72, 96 h, and 6 days. Decreased global 5-methylcytosine levels were observed after 48, 72, 96 h, and 6 days at the concentration of 100 nM BPA. Changes in CpG promoter DNA methylation were detected in the genes of Rassf1a and c-myc in BPA-treated NRK-52E cells. Expression levels of Rassf1a and c-myc changed in response to BPA at the high concentrations after 24 h treatment, whereas 100 nM exposure to BPA altered gene expression after 48, 72, and 96 h.
Conclusion:
These results indicate that changes in global and gene-specific DNA methylation may play an important role in the mechanism of BPA toxicity in kidney cells.
INTRODUCTION
Bisphenol A [(BPA); 4,4’-dihydroxy-2,2-diphenyl propane] is an organic compound used in the production of polycarbonate. BPA is found in the composition of many products such as medical supplies, the inner coating of food and beverage packaging, the coating of electrical materials, and protective clothing.1 It was determined that BPA can pass into food and beverages from many materials in very small amounts. Even in normal conditions, BPA is indicated to be released from plastic carriers. Various studies have shown that BPA affects enzyme activities, metabolism, and gene activity in hormone receptors in target tissues, and deteriorates the endocrine system due to estrogenic and androgenic effects.2 It was underlined that BPA may play a role in the etiology of many diseases such as diabetes, obesity, reproductive disorders, cardiovascular disorders, congenital anomalies, renal disorders, and breast cancer.3
Many studies have been performed to evaluate the toxicity mechanisms of BPA, especially epigenetic mechanisms in various systems.3,4 Therefore, epigenetic events such as DNA methylation may play an important role in the toxicity of BPA as an endocrine disrupting chemical.2,3,5,6 In the present study, we aimed to examine the effects of BPA on global and gene specific DNA methylation as epigenetic mechanisms in BPA toxicity in rat kidney epithelial cells (NRK-52E) cells. The results obtained are expected to contribute to a better understanding of the key molecular mechanisms of BPA toxicity.
MATERIALS AND METHODS
Chemicals
Stock solution (100 mmol/L) of BPA (99% purity, Sigma-Aldrich, St Louis, MO, USA) was prepared by dissolving it in dimethyl sulfoxide [(DMSO), Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada] and kept at -20 °C. Cell culture supplements were obtained from Wisent Bioproducts (Saint-Jean-Baptiste, QC, Canada) and sterile plastic materials were obtained from Greiner (Frickenhausen, Germany).
Cell culture and Bisphenol A treatment
The rat kidney proximal tubular epithelial cell line (NRK-52E) was used from the American type culture collection and cultured in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12, supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 U-100 µg/mL) under standard cell culture conditions (37 °C, humidified atmosphere with 5% CO2). Subculturing was performed every 2-3 days (70-80% confluence) using trypsinization. Cells were counted by a LUNA automated cell counter (Logos Biosystems, Annandale, VA, USA).
For the cytotoxicity assays, cells (1x104 in 200 µL of medium in a 96 well plate) were exposed to BPA in the concentrations range of 31.25-1000 µM and DMSO (1%) as solvent control for 24 h.
For the DNA methylation and gene expression analysis, cells (1.5x106 in 10 mL of medium in a 25-cm2 culture flask) were exposed to BPA at the concentrations of 1 nM, 10 nM, 100 nM, 1 µM, and 10 µM and DMSO (1%) as solvent control for 24 h, and BPA at the concentration of 100 nM and DMSO (1%) as control for 48 h, 72 h, 96 h, and 6 days. For all concentrations, it was tested in triplicate. Treated cells were performed for DNA isolation by High Pure PCR Template Preparation Kit (Roche Applied Science, Mannheim, Germany), and for total RNA isolation by High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany) according to the procedure provided by the manufacturer.
Cytotoxicity
After 24 h of BPA treatment, cytotoxicity tests were performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake (NRU) tests. Briefly, in the MTT test, 20 µL of MTT solution was added to each well followed by gentle mixing, and then the plate was incubated for 3 h at 37 °C, 5% CO2. After the incubation, 100 µL of DMSO (100%) was added to each well followed by gentle mixing again. The resulting purple solution was spectrophotometrically measured at 560 nm with a reference wavelength of 670 nm using a microplate spectrophotometer system (Biotek-Epoch, Winooski, VT, USA). In the NRU test, in a 96 well plate cells were washed with 150 µL of Phosphate buffered saline (PBS) solution and 100 µL of neutral red vital dye was added to the cells; then the plate was incubated for 3 h at 37 °C, 5% CO2. The cells were washed with PBS and then 100 µL of neutral red vital dye dissolution was added to each well followed by gentle mixing. Then the optical density of the plate was read at 540 nm using a microplate spectrophotometer system (Biotek-Epoch, Winooski, VT, USA). The cytotoxicity results were expressed as 50% of inhibitory concentration (IC50) of the BPA that caused 50% inhibition of the enzyme activity in the cells. Based on our cytotoxicity results and previous studies in various cells,7,8,9,10,11,12 in the present study we chose BPA concentrations of 1 nM, 10 nM, 100 nM, 1 µM, and 10 µM for 24 h treatment and a concentration of 100 nM for 24, 48, 72, 96 h, and 6 day treatments for the analysis of DNA methylation and gene expression.
Global DNA methylation
Levels of global 5-methylcytosine (5-mC) were determined by using a 5-mC DNA ELISA kit (Zymo Research, Irvine, CA, USA) according to the instructions provided by the manufacturer. For this, 100 ng of each DNA was denatured at 98 °C for 5 min in a thermal cycler. The denatured DNA was added to the wells and incubated at 37 °C for 1 h. Then samples were incubated with antibody mix consisting of anti-5-mC and secondary antibody. After the washing steps, HRP developer was added to each well and absorbance was measured at 405 nm using a microplate spectrophotometer system (Biotek-Epoch, Winooski, VT, USA). The methylation ratio of DNA samples was calculated as a percentage using calibration standard curves by preparing mixtures of negative control (100 ng/µL) and positive control (100 ng/µL) standards.
Gene-specific DNA methylation
Determination of DNA methylation on CpG islands in promoter regions of Rassf1a and c-myc genes was performed using methylation specific (MSP) PCR. In our previous study we described the study protocol in detail.13 In MSP, genomic DNA is modified by treatment with sodium bisulfite, which converts all methylated cytosines to uracil and then to thymidine during the subsequent PCR step.14,15 Bisulfite DNA modification was performed by using an EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manual’s instructions. Methylated and unmethylated primer pairs were used to amplify each region of interest. The primer sequences are listed in Table 1.16,17 After the PCR reaction, MSP products were analyzed by agarose gel electrophoresis, stained with ethidium bromide, and visualized under ultraviolet light (Quantum ST4-Vilber Lourmat, Torcy, France).
Gene expression
First-strand cDNA synthesis was performed using 1000 ng of total RNA by using a Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science, Mannheim, Germany) with a mixture of anchored-oligo (dT) and random hexamer primers according to the manual’s instructions. Expression analysis of Rassf1a and c-myc genes was performed by using real-time quantitative PCR employing Light Cycler 480 Probes Master with Real Time ready Custom Single Assays (Universal ProbeLibrary Probes, Roche Applied Science, Mannheim, Germany) containing target specific primers for Rassf1a and c-myc according to our previous study.9 Cycle threshold (Ct) values of Rassf1a and c-myc and the reference gene (β-actin) were determined and evaluation of the relative expression was performed by comparative Ct method.
Statistical analysis
Global methylation levels and cytotoxicity results were represented as mean ± standard deviation. Statistical analysis was performed by ANOVA (Tukey’s multiple comparison procedure) using SPSS version 21.0 for Windows (IBM Analytics, New York, NY, USA). P values <0.05 and <0.001 were considered statistically significant.
RESULTS AND DISCUSSION
BPA has been frequently exposed in daily life and has been shown to be harmful in living organisms due to its endocrine disrupting properties.2,18,19,20 Various studies have revealed that BPA affects enzyme activity and metabolism in various tissues, causing change in the number of hormone receptors and hormone receptor gene activity in target tissues, and deteriorates the endocrine system.2 It has been revealed that BPA might induce cancer development and various studies have explained its relations with several cancer types.21 BPA can induce epigenetic changes in early life in humans and may affect several cell signaling pathways via epigenetic mechanisms.22 It has been reported in recent studies that epigenetic alterations could be useful biomarkers for the toxicity evaluation of endocrine disrupting chemicals.6,7,8,9,23,24,25 Therefore, in the present study, we aimed to investigate the effects of dose-dependent BPA exposure for 24 h and time-dependent 100 nM concentration of BPA exposure on the alteration of global and gene specific DNA methylation and gene expression in NRK-52E cells. We chose NRK-52E cells with the characteristic properties of proximal tubule epithelial cells targeted by nephrotoxic agents to show the epigenetic effects of BPA in kidney cells.
Cell viability was determined by MTT and NRU assays in the BPA treated NRK-52E cells. The relationship between the exposure concentrations of BPA treated NRK-52E cells and % reduction in cell viability is shown in Figure 1. IC50 values of BPA were determined as 105 mM and 88 mM in NRK-52E cells by the MTT and NRU tests, respectively. Decreased global DNA methylation at a concentration of 100 nM BPA was observed in the range of 19.76%-25.30% after 48, 72, 96 h, and 6 days (Figure 2). However, there was no change in the global DNA methylation at the concentrations of 1 nM-10 µM BPA for 24 h exposure (data not shown). Accordingly, in our previous studies, global levels of 5-mC% decreased after 1 and 10 µM BPA treatment for 96 h in HepG2 cells8 and 0.1 and 1 µM BPA treatments for 48 and 96 h in MCF-7 cells.7 Conversely, in another previous study, we observed no global methylation changes after 48 h exposure to BPA, whereas significant increases were seen in the global level of 5-mC% (1.3 fold) after 10 µM of BPA for 96 h exposure in neuroblastoma (SHSY-5Y) cells.9 According to the results of the present study, BPA decreased global DNA methylation in rat liver,26 human fetal liver samples,27 and porcine oocyte culture at 250 µM concentration.28
Rassf1 is a tumor suppressor gene that has a significant role in cancer and it is thought that its regulation was associated with CpG island promoter DNA methylation. c-myc, a proto-oncogene, codes a transcription factor and controls cell proliferation. It has been shown that increases in the expression of the c-myc gene were associated with hypomethylation, which could be related to cell proliferation in liver and renal cancers.29,30,31 A representative profile of MSP for the c-myc gene in BPA treated NRK-52E cells is shown in Figure 3. Increases in promoter methylation were detected in Rassf1 and c-myc genes at the BPA concentrations of 1 and 10 µM in NRK-52E cells over 24 h while no methylation was detected in control samples by using MSP following bisulfide conversion. In addition, BPA caused increases in promoter methylation of Rassf1 and c-myc genes at the concentration of 100 nM in a time-dependent manner (48 h, 72 h, 96 h, and 6 days) in NRK-52E cells. Expression levels of Rassf1a and c-myc genes are shown in Figures 4 and 5. In response to BPA, expression of Rassf1a and c-myc was decreased at 1 µM for 24 h (26.66% and 37.3%, respectively) and 10 µM for 24 h (25.11% and 22.24%, respectively). Moreover, 100 nM exposure of BPA caused decreases in expression of the Rassf1a and c-myc genes after 48, 72, and 96 h BPA treatment with regard to control samples, and there was a nonsignificant increase for 6-day BPA treatment (Figure 5). According to the results, the decrease in gene expression of Rassf1a and c-myc was correlated with DNA methylation results, which showed an increase in CpG promoter methylation of the genes. In our previous study in HepG2 cells, no change as observed in promoter methylation or gene expression of the Rassf1a gene after BPA exposure.8
CONCLUSION
Based on the findings of the present study, BPA caused alterations in expression and methylation in the promoter region of the Rassf1a and c-myc genes associated with cancer related pathways in NRK-52E cells after 24-96 h exposure periods. It has been suggested that alterations in DNA methylation in the Rassf1a and c-myc genes might be key regulators in the toxicity mechanisms of BPA. Further studies on the expression of chromatin modifying enzymes, DNA methylation, and global and gene-specific histone modifications after BPA exposure in cell cultures are needed to better understand alterations in epigenetic mechanisms in BPA-induced toxicity.