Estrogen-like properties of fluorotelomer alcohols as revealed by mcf-7 breast cancer cell proliferation

Abstract

We investigated estrogen-like properties of five perfluorinated compounds using a combination of three in vitro assays. By means of an E-screen assay, we detected the proliferation-promoting capacity of the fluorotelomer alcohols 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH) and 1H,1H,2H,2H-perfluoro-decan-1-ol (8:2 FTOH). The more widely environmentally distributed compounds perfluoro-1-octane sulfonate, perfluorooctanoic acid, and perfluorononanoic acid did not seem to possess this hormone-dependent proliferation capacity. We investigated cell cycle dynamics using flow cytometric analyses of the DNA content of the nuclei of MCF-7 breast cancer cells. Exposure to both fluorotelomer alcohols stimulated resting MCF-7 cells to reenter the synthesis phase (S-phase) of the cell cycle. After only 24 hr of treatment, we observed significant increases in the percentage of cells in the S-phase. In order to further investigate the resemblance of the newly detected xenoestrogens to the reference compound 17beta-estradiol (E2), gene expression of a number of estrogen-responsive genes was analyzed by real-time polymerase chain reaction. With E2, as well as 4-nonylphenol and the fluorotelomer alcohols, we observed up-regulation of trefoil factor 1, progesterone receptor, and PDZK1 and down-regulation of ERBB2 gene expression. We observed small but relevant up-regulation of the estrogen receptor as a consequence of exposures to 6:2 FTOH or 8:2 FTOH. The latter finding suggests an alternative mode of action of the fluorotelomer alcohols compared with that of E2. This study clearly underlines the need for future in vivo testing for specific endocrine-related end points.

Figure 1

Analysis of estrogenicity of E₂ (A), 4-NP (B), 6:2 FTOH (C), 8:2 FTOH (D), PFOS (E), and PFOA (F ) by the E-screen assay in MCF-7 cells. 0.1% DMSO was the solvent control. Results are expressed as mean ± SD of three replicates for each data point.

Figure 2

Histograms of DNA content showing the effects of perfluorinated compounds on cell cycle distribution. (A) 0.1% DMSO (solvent control). Cells were cultured in DMEM plus 5% CSFBS for 72 hr before exposing them to estrogenic compounds (B, 1 nM E₂; C, 10 μM 4-NP; D, 30 μM 6:2 FTOH; and E, 10 μM 8:2 FTOH) and non-estrogenic perfluorinated compounds (F, 50 μM PFOS; G, 50 μM PFNA) for 24 hr. 4-NP (C) was the positive control, and 10 nM TCDD (H) was the negative control.

Figure 3

Effect of perfluorinated chemicals on mRNA expression of estrogen-responsive genes in MCF-7 cells were treated with 0.1% DMSO, 1 nM E₂, 10 μM 4-NP, 30 μM 6:2 FTOH, 10 μM 8:2 FTOH, 50 μM PFOS, 50 μM PFNA, 50 μM PFOA, or 10 nM TCDD. After exposure to the test compounds for 48 hr, mRNA levels of TFF1 (A), PGR (B), ESR1 (C), PDZK1 (D), and ERBB2 (E) were measured by real-time PCR and normalized using HPRT1 as an internal control. Results are means from three replicate measurements and are expressed as fold relative to 0.1% DMSO; error bars indicate SD. *p < 0.05. **p ≤ 0.001.