## 4 Discussion  
In this study, 4 HR was tested as a potential xenoestrogen. However, 4 HR showed different behavior to breast cancer cells or ovariectomized animals compared to estradiol or BPA. The application of BPA increased cellular proliferation in both MCF-7 cells and SK-BR-3 cells, which are ER-positive and -negative breast cancer cells, respectively (Figure 1a). By contrast, the application of 4 HR showed significantly decreased proliferation in both types of cells (\(p<0.05\), Figure 1b). The application of BPA increased ER\(\alpha\), ER\(\beta\), and p-ERK1/2 expressions, but the application of 4 HR did not significantly change those expressions (Figures 2 and 3). In the animal model, the application of estradiol increased the expression of ER\(\alpha\), ER\(\beta\), and prolactin in the pituitary gland (Figure 4). However, the application of 4 HR showed a similar pattern to the solvent only group (Figure 4). Based on these results, 4 HR did not behave like estradiol or BPA in the range of tested concentrations. The warning for endocrine system disruptors has been increased annually [9; 10]. Many types of endocrine disruptors have been introduced and tested for their potential biohazards [14]. These agents include pesticides, such as dichlorodiphenyltrichloroethane (DDT), its metabolites, and many common industrial chemicals, such as polychlorinated biphenyls and BPA [21; 22]. Human health effects from exposure to xenoestrogen include: precocious puberty, increased incidence of breast cancer, testicular cancer, prostate cancer, and reduced sperm count [9; 14; 21]. Long-term uptake of low-dosage xenoestrogen is deposited in adipocytes and may be released in fasting conditions and show estrogen effects [21]. Therefore, health professionals warn against xenoestrogen for both short-term high dosage uptake and long-term low dosage uptake [9; 10]. However, the effect of long-term low dosage uptake is difficult to simulate in experimental conditions. BPA is a well-known xenoestrogen [10]. BPA increases the proliferation of breast cancer cells [23], and our results also showed the same effect on MCF-7 cells (Figure 1a). However, 4 HR was cytotoxic to breast cancer cells regardless of ER expression (Figure 1). 4 HR is also cytotoxic to primary cultured oral melanoma cells [5] and SCC-9 cells [4] in the similar range of concentration to this study. However,  
Figure 4: The results of in vivo experiment. (**a**) Daily application of 1 \(\mu\)g/kg for 17-\(\beta\)-estradiol in the ovariectomized rats resulted in increased prolactin, ER\(\alpha\), and ER\(\beta\) expression level in the pituitary gland. However, daily application of 128 mg/kg for 4 HR and solvent only groups showed low levels of prolactin, ER\(\alpha\), and ER\(\beta\) expression in the pituitary gland. Interestingly, both 17-\(\beta\)-estradiol group and 4 HR group showed high levels of vascular endothelial growth factor (VEGF)-A expression in the pituitary gland. In addition, epithelial hyperplasia was only observed in the estradiol group (arrow) (bar = 20 \(\mu\)m). (**b**) Blood prolactin level. Blood prolactin levels were increased in the estradiol group compared to the 4 HR and solvent only groups (* \(p<0.05\)). (**c**) Western blot for pituitary gland tissue samples. Prolactin, ER\(\alpha\), and ER\(\beta\) expression were increased in the estradiol group (Sol: solvent only group; Est: estradiol group). Full length blots were available in Supplementary Figure S5. its inhibitory effect is not significant in the normal human dermal fibroblasts [4]. According to NIH toxicology report, 4 HR has shown anticancer effects in various types of cancers [15]. As 4 HR increases the apoptotic stress mediated by mitochondrial proteins [18], cancer cells seem to be more influenced by 4 HR administration. Based on these findings, there has been no evidence that 4 HR might increase the incidence of breast cancer. Several xenoestrogens increase the activity of ER in the absence of estradiol [24].