Effects of Low-Dose Bisphenol A on DNA Damage and Proliferation of Breast Cells: The Role of c-Myc

Abstract

Background: Humans are exposed to low-dose bisphenol A (BPA) through plastic consumer products and dental sealants containing BPA. Although a number of studies have investigated the mammary gland effects after high-dose BPA exposure, the study findings differ. Furthermore, there has been a lack of mechanistic studies.

Objective: The objective of this study was to investigate the effect and the mechanism of low-dose BPA in mammary gland cells.

Methods: We evaluated DNA damage following BPA exposure using the comet assay and immunofluorescence staining, and used cell counting and three-dimensional cultures to evaluate effects on proliferation. We examined the expressions of markers of DNA damage and cell-cycle regulators by immunoblotting and performed siRNA-mediated gene silencing to determine the role of c-Myc in regulating BPA's effects.

Results: Low-dose BPA significantly promoted DNA damage, up-regulated c-Myc and other cell-cycle regulatory proteins, and induced proliferation in parallel in estrogen receptor-α (ERα)-negative mammary cells. Silencing c-Myc diminished these BPA-induced cellular events, suggesting that c-Myc is essential for regulating effects of BPA on DNA damage and proliferation in mammary cells.

Conclusions: Low-dose BPA exerted c-Myc-dependent genotoxic and mitogenic effects on ERα-negative mammary cells. These findings provide significant evidence of adverse effects of low-dose BPA on mammary cells.

Figure 1

Low-dose BPA induces nuclear γ-H2AX levels through c-Myc. (A,B) MCF10A cells were treated with EtOH or BPA (10 or 100 nM) for 24 hr, and the levels and subcellular localization of endogenous γ-H2AX were detected using an anti-γ-H2AX antibody, followed by an Alexa-Fluor-488-conjugated secondary antibody; DAPI was used to visualize the nuclei. (B) An average of 150 of the stained cells was analyzed, and a histogram shows the percentage of cells with nuclei positive for γ-H2AX. (C,D) 184A1 cells were treated with EtOH or BPA for 24 hr, and the levels of γ-H2AX were detected; the histogram is displayed (D). MCF10A (E) and 184A1 (F) cells were treated with BPA (10 nM) for up to 24 hr, and total lysates from these cells were subjected to Western blotting using c‑Myc, cyclin D1, cyclin E, E2F‑1, and β-actin antibodies. MCF10A (G,H) and 184A1 (I,J) cells were transfected with c‑Myc–siRNA or control-siRNA for 48 hr, and the c‑Myc–knockdown and control cells were treated with EtOH or BPA for 24 hr; the levels of γ-H2AX were detected (G,I), and the histograms are shown (H,J). Bars = 20 μm (A,C,G,I). **p < 0.0005.

Figure 2

Low-dose BPA induces DNA DSB in normal mammary cells through c‑Myc. (A,B) 184A1 cells were treated with BPA or EtOH for 3 hr, and the comet assay was performed under neutral conditions. (B) The relative comet tail moments represent the averages of 100 cells in three independent experiments. (C,D) 184A1 cells were transfected with c‑Myc siRNA or control siRNA for 48 hr, the c‑Myc–knockdown and control cells were treated with EtOH or BPA for 24 hr, the comet assay was performed, and the relative comet tail moments are displayed (B). Bars = 40 μm.

Figure 3

Low-dose BPA induces production of ROS in breast cells through c‑Myc. 184A1 (A) and MCF7 (B) cells were treated with BPA or EtOH for 2 hr; cells were stained with DCF-DA (10 μM) to show ROS-induced oxidation (green) and MitoTracker-Red (50 nM), a mitochondrial dye, and DAPI was used to visualize the nuclei. Merged images show the colocalization of mitochondria and ROS production. (C,D) MCF10A cells were transfected with control siRNA (C) or c‑Myc siRNA (D) for 48 hr, and c‑Myc–knockdown and control cells were treated with EtOH or BPA for 24 hr; ROS production was detected as described for (A,B). Bars = 20 μm.

Figure 4

Low-dose BPA induces proliferation in wt-p53 breast cells through c‑Myc. Wt-p53 MCF10A (A), 184A1 (B), MCF-7 (C), and mutant-p53 MDA-MB-231 (D) cells were treated with EtOH (–), estradiol (E2), or BPA for 4 days; trypsinized; mixed with trypan blue; and counted in five replicates. The histograms show the relative number of cells (mean ± SD) in each sample compared with the control. (E) To show that the proliferation was not caused by an effect of BPA on up‑regulating ERα, Western blotting was performed using an anti-ERα antibody. Cells were treated with EtOH or 10 nM BPA for 72 hr, and the blot was stripped and reprobed for total proteins and β-actin. MCF10A (F) and 184A1 (G) cells were transfected with c‑Myc siRNA or control siRNA for 24 hr, and c‑Myc–knockdown and control cells were treated with EtOH or BPA for 48 hr; the relative number of cells are shown in the histograms.

Figure 5

Low-dose BPA promotes spheroid proliferation in 3D cultures through c‑Myc. Cells were incubated for 10 days in 3D ECM-gel overlay cultures under treatment with EtOH, E2, or BPA. Cells were analyzed in a bright-field microscope and the area of each spheroid above a cutoff size was measured. (A) Phase-contrast/brightfield images at day 8; all images at the same magnification, resolution, and size. (B) The histogram shows the average size of spheroids (mean ± SD; n = 100; p = 0.004). (C) Percentage of spheroids that were two, three, and four times larger than the cutoff size is shown (mean ± SD of triplicate) as shown in (B). (D–F) MCF10A cells were transfected with c‑Myc siRNA or control siRNA for 24 hr, and c‑Myc–knockdown and control cells were treated with EtOH or BPA for 10 days in 3D cultures; images for spheroids are displayed in (D), and the histograms are presented in (E,F). Bars = 100 μm.

Figure 6

Silencing c‑Myc reduces the BPA-promoted expression of cell-cycle regulatory proteins. MCF10A (A) and 184A1 (B) cells were transfected with control siRNA or c‑Myc siRNA for 48 hr, and c‑Myc–knockdown and control cells were treated with BPA for 24 hr; whole lysates from these cells were subjected to Western blotting with ATM-pS1981, c‑Myc, cyclin D1, cyclin E, γ-H2AX, and β-actin antibodies in (A) and BRCA1, BRCA2, c‑Myc, cyclin D1, cyclin E, γ-H2AX, and β-actin antibodies in (B).