Epigenetic toxicity and cytotoxicity of perfluorooctanoic acid and its effects on gene expression in embryonic mouse hypothalamus cells

The mHypoE-N46 (Cellutions Biosystems, Burlington, Canada) cell line, derived from the mouse embryonic hypothalamus was immortalised by retroviral transfer of SV40 T-antigen into embryonic mouse hypothalamic primary cell cultures (days 15, 17, and 18) (26). The cells were cultured as a monolayer in Dulbecco’s modified Eagle medium (DMEM) with 25 mmol/L glucose (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2 % foetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1 % penicillin/ streptomycin (Welgene, Daegu, Korea). The cells were incubated at 37 °C in 5 % CO₂.

PFOA (Sigma-Aldrich) at final concentrations with a 10-fold difference from 0.25 to 250 μmol/L was dissolved in dimethyl sulphoxide (final concentration 0.1 %; Sigma-Aldrich). Dimethyl sulphoxide (0.1 %) was not toxic to the mHypoE-N46 cells and had no effect on cell survival or ability to divide (data not shown). To eliminate the effects of steroids contained in DMEM and FBS, PFOA treatment was performed in phenol red-free DMEM with 25 mmol/L glucose (Gibco), supplemented with 1 % charcoal-stripped FBS and 1 % penicillin/streptomycin.

The cells were seeded at 5x10³ cells per well in 96-well plates with 200 μL of fresh complete medium and incubated at 37 °C in 5 % CO₂ for 24 h. Cell culture medium (200 μL) was then replaced, and PFOA (at final concentrations of 0, 0.25, 2.5, 25, and 250 μmol/L) was added and incubated for 24 or 48 h. The CellTiter 96^® non-radioactive cell proliferation assay kit (Promega, Madison, WI, USA) was used according to the manufacturer’s protocol. The absorbance was read at 570 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Devices, San Jose, CA, USA). The measured optical density values were normalised and cell viability analysed with the GraphPad Prism Software 7.0 (GraphPad Software, San Diego, CA, USA).

Neurons were harvested and total RNA extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA purity and concentration were quantified with the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed using the iTaq^TM Universal SYBR^® Green One-Step kit (Bio-Rad, Foster, CA, USA) with gene-specific primers (Table 1). Data were analysed using the 2^-ΔΔCT method and transcript levels were normalised to those of the standardisation gene (β-actin).

Genomic DNA from mHypoE-N46 was extracted immediately after PFOA treatment using the G-spin^TM Total DNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). Total DNA quantity and quality were tested with the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). The whole-genome DNA methylation profile was determined using the EpiGentek MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (Epigentek, Farmingdale, NY, USA) following the manufacturer’s protocols.

Results are presented as mean ± SEM (standard error of the mean) and analysed with the GraphPad Prism Software 7.0. Statistical significance at p<0.05 was determined with one-way ANOVA, followed by a Bonferroni post hoc test. For the calculation of the half maximal inhibitory concentration (IC₅₀), we relied on the Quest Graph™ IC₅₀ Calculator program from the AAT Bioquest website (27).

The MTT analysis showed a drop in cell viability in a dose-dependent manner in both 24 and 48-hour PFOA treatment (Figure 1). No statistically significant differences were found between the PFOA concentrations of 0.25 μmol/L and 2.5 μmol/L, but they started to show with 25 μmol/L (p<0.01) and 250 μmol/L (p<0.001). As 48-hour PFOA treatment showed little difference from the 24-hour treatment, all subsequent experiments were done in cells cultured with PFOA for 24 h. IC₅₀ for 24-hour exposure to PFOA was 27.5 μmol/L. Gene expression experiments were therefore performed on cells treated with 2.5 μmol/L and 25 μmol/L of PFOA, whose viability was higher than the IC₅₀.

Figure 1

Because of the previously reported effects of PFOA on cell cycle and proliferation (25, 28, 29, 30) and the strong effect on cell viability found in this study, we decided to continue by testing several genes related to apoptosis, cell cycle, and proliferation.

Figure 2A shows the expression levels of the apoptotic genes Bax, Casp3, and Trp73. The expression of the Bax gene, which acts as a pro-apoptotic regulator, increased significantly (p<0.001) upon PFOA exposure in a dose-dependent manner, while the Casp3 gene expression in apoptotic cells significantly decreased at 2.5 μmol/L (p<0.01) and 25 μmol/L (p<0.05). The mRNA level of the Trp73 gene, which encodes for the p73 protein involved in cell-cycle regulation and induction of apoptosis, significantly decreased after exposure to 2.5 μmol/L (p<0.05) and 25 μmol/L (p<0.01) of PFOA.

Figure 2

Figure 2B shows the expression patterns of three cell cycle-related genes, Ccna2, Ccnb1, and Ccne1. PFOA had no significant effect on Ccna2, but the expression of Ccnb1, which encodes for G2/mitotic-specific cyclin-B1, increased significantly in dose-dependent manner upon exposure to both 2.5 μmol/L (p<0.01) and 25 μmol/L (p<0.001) of PFOA. Ccne1 gene expression, on the other hand, was downregulated at both PFOA concentrations (p<0.01).

Figure 2C shows the expression levels of Cdkn1a, Cdk4, Rps6, and Kit1, which are genes related to cell proliferation. The expression of the cyclin-dependent kinase (CDK) inhibitor 1-encoding Cdkn1a significantly increased in a dose-dependent manner (p<0.05 at 2.5 μmol/L and p<0.001 at 25 μmol/L), but other proliferation gene expressions were not significantly affected.

In the nerve growth factor – neurotrophic tyrosine kinase receptor type 1 (NGF-NTRK1) neurotrophin system, the gene expression level of the ligand Ngf significantly increased (p<0.05) with exposure to 25 μmol/L of PFOA, unlike the gene expression of receptor Ntrk1 (Figure 3A).

Figure 3

In the BDNF-NTRK2 neurotrophin system, 25 μmol/L of PFOA significantly (p<0.01) increased the expression of the ligand Bdnf gene, while both concentrations significantly increased the expression of Ntrk2, a high-affinity catalytic receptor for several neurotrophins and BDNF (p<0.001) (Figure 3B).

As PFOA showed dose-dependent effects on some genes, we decided to assess global DNA methylation to better understand its effect on genome-wide gene expression. Genome-wide 5-methylcytosine content showed a significant dose-dependent increase compared to control at both PFOA concentrations (p<0.01 at 2.5 μmol/L and p<0.001 at 25 μmol/L) (Figure 4A).

Figure 4

To better understand the mechanism of how methylation levels change, we also analysed relative gene expression of Tet1, Tet2, Tet3, Dnmt1, Dnmt3a, and Dnmt3b. Tet1 and Tet3 increased significantly with both PFOA concentrations (p<0.001), whereas the mRNA expression of Tet2 significantly decreased at PFOA concentration of 2.5 μmol/L (p<0.05) (Figure 4B). The expression of Dnmt1 (p<0.05), Dnmt3a, and Dnmt3b (p<0.001) significantly decreased at 25 μmol/L of PFOA, while the Dnmt3b gene expression also significantly decreased at 2.5 μmol/L (p<0.01) (Figure 4C). Finally, in order to investigate the effect of PFOA on CpG-DNA-mediated gene transcription and the involvement of methyl-CpG binding protein, we analysed the changes in gene expression of Mecp2 isoforms e1 and e2, which are important readers of DNA methylation. The expression of both isoforms, significantly decreased at 2.5 μmol/L (p<0.01 and p<0.05, respectively; Figure 4D).