Aromatase is a crucial enzyme for the biosynthesis of estrogens and is involved in the process of breast carcinogenesis. Concerns have been raised regarding the effects of environmental estrogens as potential regulators of aromatase expression in human breast cells. Zeranol is a non-steroidal agent with potent estrogenic activity, which is widely used as a growth promoter for cattle in certain countries. The present study hypothesized that aromatase expression and activity may be elevated by low dose zeranol exposure, providing a source of estrogens that may stimulate cell proliferation. In the present study, primary cultured human breast preadipocytes were used as an
Breast cancer is currently the most common cancer among women in the United States (US), and is the second leading cause of cancer-associated mortality in women worldwide (
Estrogen, and various growth factors in the breast adipose microenvironment that affect tumor behavior, are increasingly been recognized. Although the major site of estrogen production is reproductive tissue, peripheral estrogen synthesis in adipose or fat tissue is thought to be a major source of estrogen in postmenopausal women (
Zeranol is a non-steroidal estrogen agonist that is approved for use as a growth promoter in livestock, including beef cattle, in various countries. However, previous studies have suggested that it may not be as safe as previously demonstrated (
The long-term goal of the present study is to investigate the effects of zeranol residues in beef and their potential adverse effects on human breast health. The present study hypothesized that aromatase expression and activity may be elevated in response to low dose zeranol exposure, providing a source of estrogens, which may promote the proliferation of carcinoma clones derived from breast cells. The aim of the present study was to investigate this hypothesis and to improve understanding regarding the effects of zeranol on breast carcinogenesis.
Human breast preadipocytes were isolated from normal adipose tissue during breast reduction surgery (36-year-old female) by collagenase and hyaluronidase digestion, and cultured as previously described (
Primary cultured human breast preadipocytes were cultured in 100
The cells were grown on slides overnight, and were then fixed with 4% paraformaldehyde at room temperature for 10 min, washed in 10 mM phosphate-buffered 150 mM saline (PBS, pH 7.4) with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min, and blocked in 10% normal donkey serum (Sigma-Aldrich) in 0.1% BSA in PBS for 60 min. The slides were incubated with monoclonal mouse anti-human aromatase antibody (MCA2077S; AbD Serotec, Raleigh, NC, USA; 1:50 dilution in 3% BSA) overnight at 4°C. Subsequently, the slides were incubated with goat anti-mouse Texas Red-labeled antibody (sc-2781; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200 dilution in PBS) for 45 min at room temperature. Prior to mounting, the nuclei were stained for 5 min with 5
Preadipocytes were cultured for 24 and 48 h in DMEM/F12 medium with or without various doses (0, 2 and 50 nM) of zeranol and 50 nM zeranol plus 1
All experiments were carried out on cells that exhibited >95% viability, as measured using the trypan blue dye exclusion method (
cDNA synthesis was performed as described previously (
The newly synthesized cDNA was used as a template for PCR. cDNA (2
Cells were seeded in 6-well plates at 1×105 cells/well in 5 ml high-calcium DMEM/F12 containing 10% FBS, and were cultured overnight at 37°C. The medium was replaced with DMEM/F12 supplemented with 5% DCC-treated FBS for a further 24 h, and the preadipocytes were then treated with the indicated doses of zeranol (2, 10 and 30 nM) for 48 h. Following treatment, 200
Statistical analysis was performed using Minitab 15 (Minitab Inc., State College, PA, USA). Differences between groups were evaluated by one-way analysis of variance, followed by Dunnett's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
Following 48 h of treatment, zeranol increased the proliferation of preadipocytes in a dose-dependent manner, as determined by an MTS assay (
Positive aromatase immunofluorescent staining was observed in >80% of cells in a random field, whereas DAPI staining was only observed in the nucleus (
The effects of zeranol on the mRNA expression levels of aromatase in human breast preadipocytes were investigated by RT-PCR. The results demonstrated that treatment with 2, 10 and 50 nM zeranol for 48 h induced a significant increase in the mRNA expression levels of aromatase compared with the control (P=0.039, 0.022 and 0.028, respectively;
Preadipocytes were incubated in the presence of various concentrations of zeranol (2 and 50 nM) for 24 and 48 h. Subsequently, the aromatase activity was measured by tritiated water assay. As demonstrated in
To determine whether the increase in aromatase gene expression and activity resulted in increased E2 levels, the levels of E2 were detected in the medium of preadipocytes treated with 2, 10 and 30 nM zeranol for 48 h using a commercially available ELISA kit. E2 concentrations were significantly increased in the medium of zeranol-treated cells, as compared with in the control cells in a dose-dependent manner (P=0.037, 0.026 and 0.024, respectively;
Cumulative exposure to estrogen is known to be a risk factor for the development and mitogenic stimulation of breast cancer (
Zeranol, which is a nonsteroidal agent with potent estrogenic activity, is used in the US beef industry as an anabolic growth promoter. Bioactive zeranol and its metabolites present in the meat of zeranol-implanted beef cattle may be considered an endocrine disruptor for human consumers. It has previously been demonstrated that zeranol enhances cell proliferation and increases the ERα content of ER-positive breast cancer cells (
In the present study, primary preadipocytes isolated from human breast adipose tissues were used as a cell model. The results indicated that zeranol (2–50 nM) increased proliferation of preadipocytes in a dose-dependent manner, as determined by an MTS assay, and this effect was blocked by the ER antagonist, ICI (
In conclusion, the results of the present study indicated that exposure to low doses of zeranol may increase the risk of breast cancer by increasing estrogen levels in adipose tissue. Estrogen generated from preadipocytes acts as a functional signal linking adipose to epithelial tissue, and
This research project was supported by grants from the National Natural Science Foundation of China (grant no. 31201424) and the Natural Science Foundation of Guangdong Province (grant no. S2012040006790), and the Science and Technology Department of Guangdong Province (grant no. 2015A020209166).
Effects of zeranol on proliferation of human breast preadipocytes. Cells were incubated with various concentrations of zeranol, or were co-incubated with 100 nM ICI for 48 h. Dimethyl sulfoxide (0.1%) was used as a control. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. CT. OD, optical density; CT, control; ICI, ICI 182,780.
Aromatase protein expression in primary cultured human breast preadipocytes, as detected by immunocytochemistry. (A) Aromatase was widely expressed in the cytoplasm only. (B) Nuclei were stained with DAPI. (C) Merged aromatase and DAPI fluorescence. Magnification, ×430. DAPI, 4,6-diamidino-2-phenylindole.
Effects of zeranol on aromatase mRNA expression in primary cultured human breast preadipocytes. Cells were treated with 2, 10 or 50 nM zeranol for 48 h. The CT group was treated with dimethyl sulfoxide (0.1%). The upper panel is a representative image of gel electrophoresis following reverse transcription-polymerase chain reaction. In the lower panel, each bar represents the mean ± standard deviation of three experiments semi-quantified by MultiGauge software. *P<0.05 vs. CT. CT, control; CYP19A1, aromatase; 36B4, ribosomal protein lateral stalk subunit P0.
Effects of zeranol on aromatase activity in primary cultured human breast preadipocytes. Cells were cultured for 24 and 48 h in the absence or presence of 2 and 50 nM zeranol. Cells were also treated with the aromatase inhibitor letrozole (1
Effects of zeranol on estradiol production in primary cultured human breast preadipocytes. Cells were treated with various doses of zeranol for 48 h. Data are presented as the mean ± standard deviation (n=3) *P<0.05 vs. CT. CT, control.