## 4 Discussion
In this study, BPA increased ER\(\alpha\) and ER\(\beta\) expression in MCF-7 cells (Figure 2a). However, 4 HR did not change ER\(\alpha\) and ER\(\beta\) expression (Figure 2b). A previous study demonstrated that 4 HR increased luciferase activity, which was localized in the estrogen-responsive promoter region [14]. As the function of estrogen is broad, many genes are regulated by estrogen [25]. Some genes influenced by estrogen may also be influenced by other hormones, cytokines, or chemicals. Activation of ER\(\alpha\) results in the increased phosphorylation of ERK [20,26]. In this study, BPA application increased p-ERK1/2 expression, but 4 HR did not (Figure 3). When animals receive an ovariectomy, blood estrogen levels decrease [27]. Accordingly, the sensitivity of parenteral administration of pseudoestrogen is increased [22,28]. Many types of estrogen-like chemicals increase the expression of ER\(\alpha\) and the production of prolactin in the anterior pituitary gland [29,30]. In this study, the administration of estradiol showed significantly higher levels of blood prolactin compared to the 4 HR and solvent only groups (\(p<0.05\), Figure 4b). The expression level of ER\(\alpha\), ER\(\beta\), and prolactin in the pituitary glands were higher in the estradiol group than those in the 4 HR and solvent only groups (Figure 4c). The BPA-injected group (128 mg/kg) also showed similar findings to those of estradiol group (data not shown). The uterine epithelium was thicker in the estradiol groups than those of the 4 HR and solvent only groups (Figure 4a). The thickness of uterine epithelium increased in the ovariectomized rats after the administration of BPA [29]. There have been several reports on the estrogen-like behavior of 4 HR [14,15]. Lethal dosage of 4 HR shows the inhibition of spermatogenesis in some animals [15]. In addition, 4 HR showed spermicidal activity in in vitro tests [15]. 4 HR has anticancer effects via inducing cellular apoptosis [4,5]. As a complication of anticancer drugs, the inhibition of spermatogenesis is frequent [19]. Thus, the inhibition of spermatogenesis by lethal dosage of 4 HR might be due to its apoptosis-inducing effect, not due to endocrine system disruption. 4 HR increases the estrogen-responsive promoter region [14]. However, too many genes are included in the estrogen-responsive promoter region [25]. For example, the expression of vascular endothelial growth factor (VEGF) is modulated by xenoestrogens through the ER-dependent pathway [31]. 4 HR also increases VEGF expression, but through the hypoxia-inducible factor-independent pathway [8]. ER blocker could not change VEGF expression induced by 4 HR (data not shown). In the same report, 4 HR showed low affinity to ER [14]. In this study, 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). However, the level of their expression was lower in the 4 HR group than those in the BPA group (Figures 2 and 3). The anticancer effect of 4 HR has been demonstrated in different types of oral cancer cells [4,5] and in the animal models [4]. Several mechanisms have been clarified. 4 HR suppresses calcium oscillation in oral melanoma cells [4]. The suppression of calcium oscillation is important for mitochondrial function [32]. Accordingly, 4 HR might suppress the mitochondrial function. Mitochondria are main source of ATP production and cancer cells have higher energy demands. Therefore, 4 HR application might have a higher impact on cellular proliferation in the cancer cells rather than in the normal human dermal fibroblast (see supplementary data). The signaling pathways of ER\(\alpha\), ER\(\beta\), and ERK1/2 are associated with the carcinogenesis of breast cancer [33]. As the application of 4 HR might not be associated with carcinogenesis of breast cancer, its application could not change the expression level of these proteins. The limitation of this study was the design of the animal study.