The Interaction Test of Binary Mixtures of Endocrine-Disrupting Chemicals Using In Vitro Bioassays

Typical environmental endocrine-disrupting chemicals (EDCs) such as estradiol valerate (EV), diethylstilbestrol (DES), di-2-ethylhexyl phthalate (DEHP), mono-2-ethylhexyl phthalate (MEHP), and bisphenol A (BPA) have a strong reproductive and developmental toxicity at low concentrations. However, information on their joint toxicity is scarce. In this study, we evaluated the combined effects of EV and other four EDCs (DES, DEHP, MEHP, and BPA) on the human breast MCF-7 cells by detecting the cell proliferation, intracellular reactive oxygen species (ROS) levels, and estrogen receptor alpha (ERα) protein expression using equal concentration ratio method. The results showed that, after exposure for 24, 48, and 72 h, single EV, DES, and BPA can promote the proliferation of MCF-7 human breast cancer cells, and EV has the strongest effect in inducing cell proliferation. DEHP and MEHP cannot induce MCF-7 cell proliferation for all exposure time, while cell proliferation induced by EV was significantly attenuated by DES, BPA, DEHP, and MEHP when they mixed with EV. For intracellular ROS, single EV, BPA, DES, DEHP, and MEHP elevated intracellular ROS levels for different exposure time. Similar to the cell proliferation, DES and BPA decreased intracellular ROS levels induced by EV when they mixed with EV for 24 h. EV, DES, and BPA exposed alone or combined with EV upregulated the ERα protein expression. However, DEHP and MEHP exposed alone or combined with EV had no effect on ERα protein expression, indicating that DEHP or MEHP could attenuate ERα protein expression upregulated by EV. These results showed that the joint toxicity of binary mixtures of EV and other EDCs do not interact in a synergistic fashion in inducing cell proliferation, intracellular ROS levels, and ERα protein expression. These findings have important implications in the human risk assessments of EV mixed with other EDCs in the environment.

EV is one of the most common sources of free E2 in hormone replacement therapy [3]. In animal farming, EV is mainly applied as a growth promoter [4] and for developing a single-sex population of fish in aquaculture [5,6]. EV may act as an endocrine disruptor and adversely affect reproductive outcome [7]. EV has been detected in natural waters in China, and its concentration range is approximately 1-10 ng/L [8,9]. DES was the first synthetic estrogen originally used for clinical therapy to prevent miscarriages. However, exposure to DES can cause adverse effects on male and female reproductive tracts in humans, even causing developmental anomalies in subsequent generations [10].
ough the use of DES has been banned in humans, it is still employed in livestock and aquaculture operations as a growth promoter or to produce monosex (female) populations of fish in some parts of the world [11]. DES is also quite potent in aquatic organisms such as fish and may be comparable to, or even exceed, that of 17α-ethinylestradiol (EE2) with a predicted no-effect concentration (PNEC) in fish lower than 1 ng/L [12]. Detectable concentrations of DES in surface water and effluents from different locations have been reported to range from slightly below 1 to greater than 10 ng/L [13][14][15][16]. BPA, as one of the high-volume chemicals produced worldwide, has wide applications in organic synthesis industry as raw material in the manufacture of plastics and epoxy resins [17]. BPA has been widely detected in various environmental media, foodstuffs, and human samples [17]. Exposure to BPA has shown lowdose effects including disturbed mammary gland development, changes in normal behavioral development, and changes in obesity associated parameters in rodents [17]. DEHP and MEHP belong to phthalates, and MEHP is metabolite of DEHP. DEHP is used in raw materials of clothing, food packaging, building, and children's products [18]. DEHP pollution has raised public concern, especially after the plasticizer incident in Taiwan in May 2011. High levels of DEHP are detected in municipal domestic waste, soil, rivers, fruits, vegetables, plastic-wrapped food, and medical devices to which people are closely related. A recent study indicated that the concentration of DEHP was 78 μg/L in Bohai Sea (in Tianjin, China), and MEHP was also found at a relatively high concentration [19]. DEHP has been reported to have carcinogenic, mutagenic, and hepatotoxic effects [20]. ese compounds can affect the reproductive health of organisms at low concentrations. However, the estrogen activity and reproductive toxicity have been extensively studied mainly based on individual EDCs. Few studies have addressed the joint effects of mixtures of these compounds on estrogen-dependent responses. Estrogen activity of these individual EDCs is relatively weak; even DEHP and MEHP present antiestrogenic activity [21,22]; however, the "real-life" mixtures of EDCs may carry significant biological potency [18]. erefore, the joint toxicity of complex EDCs should not be ignored and potential interactions of weakly anti-or estrogenic substances could have important implications for public health. e estrogen-sensitive tumor cell proliferation is an ideal method for evaluating estrogenic activity of compounds [23]. Occurrence of reactive oxygen species (ROS) in estrogensensitive cells has been a common response of chemical estrogenic activity [24]. For environmental estrogen, it is widely accepted that a moderate increase of ROS can stimulate proliferation of estrogen-sensitive tumor cells [25,26]. Besides, environmental estrogen can induce estrogen-sensitive cell proliferation and may increase mitochondrial ROS production by estrogen receptor alpha/βeta (ERα/β) pathway [27]. In this study, we evaluate the effects of single EV, DES, BPA, DEHP, and MEHP on proliferation of MCF-7 cells to assess their estrogen activities. Among these EDCs, EV induced the highest MCF-7 cell proliferation. Its estrogenic potency is similar to estradiol (E2). To evaluate influence of other EDCs on cellular biological effects induced by EV, we further assess joint toxicity of the binary mixtures of EV and other four EDCs by measuring the cell proliferation, ROS generation, and ERα expression in MCF-7 cells. In order to simplify the joint toxicity experimental method, the equal concentration ratio mixing method was widely recommended in joint toxicity mechanism research of binary mixtures [28,29]. In this study, we also used equal concentration ratio mixing method to study joint toxicity of binary mixtures of EV and the other four EDCs.
e results can provide a comprehensive understanding of binary mixture of EV and other EDCs toxicity effects.

Maintenance and Treatments of MCF-7 Cells.
In our previous study, cell maintenance and treatment of MCF-7 cells are described in detail [30]. e specific process is that stock cultures of MCF-7 cells were maintained in regular RPMI 1640 medium (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) and supplemented with 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen, Burlington, ON, Canada) in a humidified atmosphere at 37°C with 5% CO 2 . e medium was changed every 2-3 d. e exponential growth phase cells were used for the experiments. Before each experiment, cells were starved in steroid-free (SF) medium for 24 h to minimize the basal hormonal levels during assays and allow cell adhesion. SF medium consists of phenol red-free RPMI 1640 (HyClone, Logan, UT, USA) and 5% charcoal-stripped FBS (Biological Industries, Israel) supplemented with 100 U/mL streptomycin/penicillin. 0.1% DMSO was used as the solvent control in each experiment. For ROS experiment, 400 μM tBHP was used as the positive control. In this study, we followed the methods described by Lei et al. 2017 [30]. e tBHP significantly increased intracellular ROS production and the ROS level (compared to control × 100) of MCF-7 cells treated with 400 μM tBHP was 280 ± 35.6%.

Cell Viability Assay.
In order to study the proliferation effect of tested compounds on MCF-7 cells, CCK-8 was used to measure the viability of MCF-7 cells. e specific experiment methods were provided elsewhere [23,30]. Briefly, the treated cells were added with the CCK-8 reagents (10 μL in 90 μL phenol red-free RPMI 1640 per well), and optical density (OD) was read with a Multiskan MK3 plate reader ( ermo Electron Corporation, Waltham, MA, USA) at a wavelength of 490 nm. e OD value can reflect the number of living cells indirectly, and cell viability (% of control) was calculated according to the OD values of exposure group and control group.
For joint toxicity, the cells were exposed to binary mixtures of EV and the other four EDCs according to equal concentration ratio method. e corresponding cell viability was detected.

ROS Detection.
Intracellular ROS were assayed using a fluorescent dye DCFH-DA. e specific experiment methods were provided elsewhere [30]. Briefly, the treated cells were incubated with 50 μL DCFH-DA (2 × 10 −4 M) at 37°C for 30 min in the dark, and the fluorescence was evaluated under a fluorescence microscope (Olympus BX-51; Olympus, Japan). e intensity of fluorescence was analyzed by Image-Pro Plus 6.
For joint toxicity, the cells were exposed to binary mixtures of EV and the other four EDCs according to the equal concentration ratio method, and the corresponding ROS levels were detected according to the above method.
To study the joint effect of binary mixtures of EV and the other four EDCs on ERα protein expression, the cells were coexposed to binary mixtures of EV and the four EDCs according to the equal concentration ratio method for 24 h. ereafter, ERα protein samples were collected, and protein expression was analyzed.
2.6. Data Analysis. All results are expressed as the mean-± standard deviation (SD). e differences between exposure groups and the negative control were tested using one-way analysis of variance with specific mean comparisons by Dunnett's test. Student's t-test was used to analyze the difference between two different exposure groups. Difference was considered significant at a p value <0.05. For E2 and EV, the concentration-response analyses were performed with fourparameter logistic curve regression analysis, and EC50 was calculated, which is the concentration that induces half of the maximum proliferation effect.

e Effects of Tested Compounds on the Viability of MCF-7
Cells.
e CCK-8 assay showed that E2 at all exposure concentrations and all exposure time significantly induced cell viability except for the lowest concentration at 24 h (Figure 1(a)). e increases of cell viability presented very well dose-dependent, for the concentration range of 0.0001-1000 nM, and time-dependent manner.
e EC50 values of E2 for 24, 48, and 72 h were 0.20, 0.16, and 0.059 nM, respectively. e cell viability reached the maximum value at about 1 nM and then reached a platform period. EV at all exposure concentrations and all exposure time significantly induced the viability of MCF-7 cells and presented a concentration-dependent manner at the concentration range of 0.0001-1000 nM (Figure 1 Figures 4(a)-4(c). DEHP cannot induce significant cell proliferation for three exposure periods. e binary mixtures of DEHP and EV induced significant cell viability; however, cell viability induced by the binary mixtures of DEHP and EV was significantly lower than that induced by single EV. is indicated that DEHP addition decreased cell viability induced by EV. Similarly, single MEHP or the binary mixtures of MEHP and EV cannot all induce significant cell viability for all exposure concentrations ( Figure 5). is result indicated that MEHP addition completely inhibited cell viability induced by single EV. Figure 6, the dose response for ROS formation is demonstrated with MCF-7 cells for individual chemicals alone or combined with EV. With the prolongation of exposure time, EV induced more ROS production. At 24 h, EV induced the highest ROS generation in MCF-7 cells. e binary mixtures of EV and other EDCs presented different change trend in inducing intracellular ROS generation for different concentrations and different exposure time.

ROS Levels. As shown in
At 24 h, the binary mixtures of EV and DES did not elevate intracellular ROS levels, while single EV significantly increased intracellular ROS levels, indicating that DES addition inhibited ROS generation induced by EV. At 1 or 3 h, the binary mixtures of EV and DES increased intracellular ROS levels, but the ROS levels induced by EV and DES mixtures were lower than those induced by single EV or DES. Similarly, at 24 h, the binary mixtures of EV and BPA cannot significantly elevate intracellular ROS levels. At 1 and 3 h, EV and BPA binary mixtures significantly increased intracellular ROS, and ROS levels induced by them were higher than those induced by single EV. e results indicated that BPA addition increased ROS levels induced by single EV for 1 and 3 h and decreased intracellular ROS levels induced by single EV for 24 h. Similarly, at 1 and 3 h, EV and DEHP mixtures significantly increased intracellular ROS levels, but the ROS levels induced by them had no significant difference between EV and DEHP binary mixtures and single EV. From the above results, at 24 h, antigenic effects of binary mixtures of EV and DES or BPA can be found in inducing intracellular ROS generation.
However, single DEHP and MEHP, and the binary mixtures of EV and DEHP or MEHP cannot all upregulate ERα protein expression, indicating that DEHP or MEHP attenuated ERα protein expression upregulated by EV.

Discussion
In the environment, EDCs cannot exist alone. erefore, humans and other creatures are rarely exposed to only a single EDC, but large amounts of chemicals through a variety of ways at the same time [31]. Joint toxicity may occur when two toxic chemicals are presented in environment simultaneously [32]. When two or more pollutants act on organism, the combined effects tend to be completely different compared with a single pollutant acting on the organism [18,33]. Usually, joint toxicity presents four forms depending on different interactions of the two compounds. Additive effect and independent effect are the result of the two compounds having no interaction or reaction, while synergistic or antagonistic effect pertains to the interaction of the two compounds, making their toxicity increscent or reductive [34].
EV, DES, BPA, DEHP, and MEHP are typical EDCs; these high production volume chemicals could be released into the ambient environment during production, storage, and transport and coexist in environment simultaneously. Although many researches were done, they focused on single EDC or a series of EDCs at a high dosage. In these EDCs, EV, DES, and BPA have estrogenic activity, while DEHP and MEHP have antiestrogenic activity [21,22]. Generally, estrogen compounds can induce the proliferation of estrogensensitive tumor cells and increase the intracellular ROS levels through ERα pathway [25,26]. erefore, to evaluate the combined health risk of these different types of EDCs in environment, in this study, we investigated the effect of different types of EDCs on estrogenic activity of EV by detecting MCF-7 cell viability, intracellular ROS level change, and ERα protein expression. After MCF-7 cells were exposed to a series of concentration equal proportion binary mixtures of DES, BPA, DEHP and MEHP with EV for 24, 48, or 72 h, the binary mixtures of EV and the other four EDCs can significantly attenuate cell viability induced by single EV. e antagonistic effects of the binary mixtures of EV and the other four EDCs can be found by detecting cell viability. e joint effects of the binary mixtures of EV and the other four EDCs on intracellular ROS level change are totally different. At 24 h, similar to the cell viability, DES and BPA decreased intracellular ROS levels induced by EV when they mixed with EV. While at 1 and 3 h, the influence of DES, BPA, and DEHP on intracellular ROS level change induced by EV presented inconsistent results. at is to say, at some exposure concentrations, they increased intracellular ROS levels induced by EV, while, at other exposure concentrations, they decreased intracellular ROS levels. However, in general, the joint toxicity of binary mixture of EV and the other EDCs did not interact in a synergistic fashion in inducing cell proliferation and intracellular ROS levels. Ramirez et al. [34] also found that estrogenic binary mixtures of endosulfan and dieldrin did not interact synergistically in binding to ER or activating ER-mediated responses in vitro or in vivo.
Generally, the cell proliferation of estrogen-sensitive cells can give insight into estrogenic effects of environmental contaminants [35]. ERα is an estrogen nuclear receptor.
Estrogen, through binding to or activating ERα, can activate downstream signals that facilitate breast cancer cell proliferation. Both of them are generally found to be upregulated in many types of cancer and are related to cell proliferation. Numerous studies also showed that ERα signals are critical regulators of estrogen-sensitive tumor cell proliferation induced by environmental estrogen. For example, bisphenol F (BPF) can induce MCF-7 cells to proliferate by enhancing expression of proteins such as Cyclin D1 and Cyclin E1 via the ERα-dependent pathway [36]. Low concentration of BPA (1 μM) has been shown to increase proliferation of both breast cancer cells and cancer-associated fibroblasts by an estrogen receptor-dependent pathway [37]. García et al. [38] found that environmental estrogen such as hexachlorobenzene (HCB) at low concentrations (5 and 50 nM) can increase MCF-7 cell proliferation by activating the ERα receptor. In the present study, we also found that single E2, EV, DES, and BPA can upregulate ERα protein expressions increase cell viability. In addition, the binary mixtures of EV and DES or BPA also upregulated ERα protein expression at 24 h, and their binary mixtures also increased cell viability at this exposure time. However, the binary mixtures of EV and DEHP or MEHP cannot increase the expression of ERα protein and cell viability. Some other studies also showed that DEHP and MEHP cannot activate ERα in estrogen receptor-positive cells [22,39] and have antiestrogenic activity [22,40]. ese results showed that ERα protein expression is consistent with the results of the cell viability, which indicated that these EDCs might increase proliferation of MCF-7 breast cancer cells by activating the ERα.
However, except for ERα, EDCs can also induce estrogen-sensitive cancer cell proliferation by estrogen member receptor G protein coupled receptor 30 (GPR30) pathway [41,42]. Moreover, there may be a crosstalk between membrane receptor such as GPR30 (nongenomic) and nuclear receptor ER (genomic) signaling pathways [43][44][45]. In our previous studies, we found that bisphenols such as BPF, TDP, and BPAF can induce proliferation of MCF-7 cells via interaction of ERα-and GPR30-mediated signaling pathways [23,30,45]. erefore, the specific mechanism needs to further study more proteins associated with the cell proliferation such as GPR30 and its downstream signals.
Occurrence of ROS in cells has been a common response of chemical toxicity, which can damage active cellular substance, such as DNA, lipids, and proteins [46]. e redox system may be especially important during estrogen-induced cell proliferation, and different cellular signals may operate in response to varying levels of estrogen-induced ROS [47]. Many studies have found that natural or synthetic estrogen can induce ROS generation in breast cancer cells by estrogen receptor pathway, and increased ROS has been involved in both cell proliferation and carcinogenesis [25][26][27]47]. However, cells have various antioxidant defense systems, consisting of both enzymatic and nonenzymatic substances, to reduce ROS levels and thus protect the cells from ROS damage [46]. When the balance between ROS and antioxidant defense system is broken, oxidative damage, or so-called oxidative stress, occurs. However, the balance between ROS and antioxidant defense system is a dynamic process.
e levels of generated ROS in intracellular are closely related to exposure concentrations and exposure time of the exposed chemicals [48]. In this study, we also found that, at different exposure concentrations and different exposure time, the intracellular ROS levels are different. e ROS generation in the binary mixtures of EV and the other four EDCs also presented different change trends at different exposure concentrations and different exposure time. e deeper reasons need further study.

Conclusions
Single EV, DES, and BPA can promote the proliferation of MCF-7 cells, and EV has the strongest potency in inducing cell proliferation. Cell viability induced by EV was significantly attenuated by DES, BPA, DEHP, and MEHP when they were mixed with EV. ERα signals play a key role in MCF-7 cell proliferation induced by low-concentration EDCs like EV, DES, or BPA. Single EV, BPA, DES, DEHP, and MEHP elevated intracellular ROS levels for different exposure durations. DES and BPA decreased intracellular ROS levels induced by EV when they mixed with EV for 24 h. ese results indicated that the joint effects of binary mixtures of EV and other EDCs do not interact synergistically in inducing cell proliferation, intracellular ROS generation, and ERα protein expression. e results have important implications in the human risk assessments of EV coexposed with other EDCs in the environment.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.