Bisphenol S Disrupts Estradiol-Induced Nongenomic Signaling in a Rat Pituitary Cell Line: Effects on Cell Functions

Background: Bisphenol A (BPA) is a well-known endocrine disruptor that imperfectly mimics the effects of physiologic estrogens via membrane-bound estrogen receptors (mERα, mERβ, and GPER/GPR30), thereby initiating nongenomic signaling. Bisphenol S (BPS) is an alternative to BPA in plastic consumer products and thermal paper. Objective: To characterize the nongenomic activities of BPS, we examined signaling pathways it evoked in GH3/B6/F10 rat pituitary cells alone and together with the physiologic estrogen estradiol (E2). Extracellular signal-regulated kinase (ERK)– and c-Jun-N-terminal kinase (JNK)–specific phosphorylations were examined for their correlation to three functional responses: proliferation, caspase activation, and prolactin (PRL) release. Methods: We detected ERK and JNK phosphorylations by fixed-cell immunoassays, identified the predominant mER initiating the signaling with selective inhibitors, estimated cell numbers by crystal violet assays, measured caspase activity by cleavage of fluorescent caspase substrates, and measured PRL release by radioimmunoassay. Results: BPS phosphoactivated ERK within 2.5 min in a nonmonotonic dose-dependent manner (10–15 to 10–7 M). When combined with 10–9 M E2, the physiologic estrogen’s ERK response was attenuated. BPS could not activate JNK, but it greatly enhanced E2-induced JNK activity. BPS induced cell proliferation at low concentrations (femtomolar to nanomolar), similar to E2. Combinations of both estrogens reduced cell numbers below those of the vehicle control and also activated caspases. Earlier activation of caspase 8 versus caspase 9 demonstrated that BPS initiates apoptosis via the extrinsic pathway, consistent with activation via a membrane receptor. BPS also inhibited rapid (≤ 1 min) E2-induced PRL release. Conclusion: BPS, once considered a safe substitute for BPA, disrupts membrane-initiated E2-induced cell signaling, leading to altered cell proliferation, cell death, and PRL release.

Research Xenoestrogens (XEs) are a diverse group of synthetic agents (e.g., pesticides, surfactants, plastics monomers) that can mimic and disrupt the actions of physiologic estrogens (Colborn et al. 1993;Le and Belcher 2010;McLachlan 2001;Soto et al. 1994). Many XEs can remain in the environment for a long time, thus increasing the likelihood for human and wildlife exposure (Ahel et al. 1993;deJager et al. 1999;Dekant and Volkel 2008;Judson et al. 2010).
Bisphenol A (BPA), a leachable monomer of polymerized polycarbonate plastics, has been used commercially since 1957 (Bisphenol A Global Industry Group 2002) and is also found in food can liners and coatings on thermal cash register paper (Zalko et al. 2011). Humans are exposed to BPA primarily from food and water (H 2 O) contaminated by manufactured products, particu larly during the heating of plastic containers (Kubwabo et al. 2009). In the National Health and Nutrition Examination Survey (NHANES), BPA levels ranged from 0.4 to 149 μg/L (1.8-660 nM) in 92.6% of urine samples from U.S. residents ≥ 6 years of age (Calafat et al. 2008).
Exposure to BPA in humans has been implicated in the development of chronic diseases, including diabetes, asthma, and cancer (Alonso-Magdalena et al. 2010;Li et al. 2011;Midoro-Horiuti et al. 2010;Watson et al. 2010), and in causing decreased fecundity in wildlife via disrupted spermato genesis and ovulation (Li et al. 2011;Oehlmann et al. 2009;Sohoni et al. 2001;Zhou et al. 2011). The European Food Safety Authority has set a tolerable daily intake (TDI) for BPA of 0.05 mg/kg body weight/day, a value accepted by many regulatory agencies, including the U.S. Environmental Protection Agency (1993). Because of increased concern over the safety of BPA, Health Canada (2009), and more recently the European Union (European Commission 2011) and the U.S. Food and Drug Administration (2012), have banned its use in plastic bottles for infants.
More stringent global regulations on BPA production and use have led to the development of alternative, more heat-stable bisphenol compounds (Gallar-Ayala et al. 2011;Liao et al. 2012aLiao et al. , 2012b. Among these alternative compounds is 4,4´-dihydroxy diphenyl sulphone, commonly known as bisphenol S (BPS). Because of the novel nature of BPS, in vivo toxicity studies have not been reported, nor has the ability of BPS to disrupt the actions of physiologic estrogens been explored.
Several studies have tested the effects of BPS via genomic mechanisms using extremely high concentrations (concentrations unlikely to be leached from BPS-containing products). At concentrations as high as 0.1-1 mM, BPS showed only slight estrogenic activity in a 4-hr recombinant two-hybrid yeast test system (Hashimoto et al. 2001;Hashimoto and Nakamura 2000). In another such study, Chen et al. (2002) showed that 40 μM BPS had 15-fold lower genomic estrogenic activity than BPA. However, BPS was equipotent to BPA in an estrogen-response-element-driven green fluorescent protein expression system in MCF-7 breast cancer cells (Kuruto-Niwa et al. 2005). Discrepancies between these studies were attributed to species (yeast vs. mammalian) differences (Kuruto-Niwa et al. 2005). However, tissues frequently differ in responses, so this could also explain the discrepancies. No studies prior to ours have examined BPS for non genomic mechanisms of action or at the low concentration ranges likely to be present in foods, environmental samples, or humans.
BPA can potently interfere with the actions of endogenous estrogens in pituitary cells via several types of non genomic signaling [e.g., mitogen-activated protein kinases (MAPKs), Ca 2+ influx] Wozniak et al. 2005) acting via membrane estrogen receptors [mERα, mERβ, and GPER/GPR30 (G protein-coupled estrogen receptor)], and thus alter functional responses [cell proliferation, prolactin (PRL) release, and transporter function] at pico molar and sub picomolar concentrations (Alyea and Watson 2009;Jeng et al. 2010;Jeng and Watson 2011;Wozniak et al. 2005). Physiologic estrogen actions are disrupted by BPA and other XEs for both timing and magnitude of responses-enhancing or inhibiting-depending on their concentrations Jeng and Watson 2011). Introduction of a new active bisphenol compound (BPS) into the environ ment poses an unknown threat for signaling and functional disruptions.
In the present study we examined the effects of BPS on non genomic signaling at concentrations that allow full assessment of potency given the non monotonic concentration responses we expected based on our previous studies of BPA Jeng and Watson 2011). To simulate likely exposures, we tested BPS both alone and in combination with the physio logic estrogen estradiol (E 2 ). Using proto typic receptor inhibitors, we sought to identify the predominant mER through which BPS initiates non genomic signaling. Effects of BPS on associated downstream (from MAPKs) functional end points were also examined, including changes in cell number (proliferation or decline) and caspase activations or inhibitions occurring via external stimuli (caspase 8) versus internal stimuli (caspase 9). Together, these mechanisms can contribute to alterations in cell number. Finally, we examined the effect of BPS on peptide hormone release (PRL). These measure ments employed high-throughput plate immuno assays to facilitate quantitative comparisons between responses to different compounds and mixtures.

Materials and Methods
Cell culture. We selected the clonal rat prolactinoma cell line GH 3 /B 6 /F 10 on the basis of its naturally high expression of mERα (Pappas et al. 1994(Pappas et al. , 1995a, which enables it to respond robustly in tests for non genomic signaling and functional end points. Cells were routinely sub cultured with phenol red-free Dulbecco's modification of Eagle's medium (DMEM, high glucose; Mediatech, Herdon, VA) containing 12.5% horse serum (Gibco BRL, Grand Island, NY) and defined supplemented calf and fetal serum (Thermo Fisher, Waltham, MA) at 2.5% and 1.5%, respectively. Cells of passages 10-20 were used for these experiments.
Concentration ranges selected. All concentrations for time courses and dose responses were chosen based on our previous studies Jeng and Watson 2011;Kochukov et al. 2009) that demonstrated expected potencies, efficacies, and rapidity of the responses. The chosen concentrations of BPS reflect the range of concentrations likely to be found in the environ ment, centering on urinary levels (0.299 ng/mL or 1.2 nM), observed in Albany, New York, residents (Liao et al. 2012a(Liao et al. , 2012b. Lower concentrations are of interest to determine how sensitive biological systems are to presumably more widespread exposure concentrations. These concentrations of other XEs were able to activate MAPKs and caspases and disrupt PRL secretion. Quantitative ERK (extra cellular signal regulated kinase) and JNK (c-Jun-N-terminal kinase) phosphorylation assays. To quantify phospho activation of ERK (pERK) and JNK (pJNK), we used a fixed cell-based immunoassay, as previously described . Cells (10 4 /well) were plated in 96-well plates (Corning Incorporated, Corning, NY) and allowed to attach for 24 hr. The origi nal plating medium was then replaced with DMEM containing 1% charcoal-stripped (4×) serum for 48 hr to deprive cells of serum hormones. Medium was then removed, and cells were exposed to BPS (10 -15 to 10 -7 M), BPA (10 -15 M), or E 2 (10 -9 M) (all from Sigma-Aldrich, St. Louis, MO) to assess time-(0-60 min) and concentration-dependent changes. Test compounds were dissolved in ethanol (EtOH), then diluted in DMEM containing 1% charcoal-stripped serum. The vehicle control was 0.001% EtOH in DMEM. To stop mER-initiated signaling, cells were fixed with a 2% paraformaldehyde/0.2% picric acid solution (Fisher Scientific, Pittsburgh, PA) at 4°C for 48 hr. Once fixed, cells were incubated with phosphate-buffered saline (PBS) containing 0.2% fish gelatin and 0.1% Triton X-100 (Sigma-Aldrich) for 1 hr at room temperature (RT) and then incubated with primary anti bodies against pERK or pJNK (1:500 in PBS/0.2% fish gelatin/0.1% Triton X-100; Cell Signaling Technology, Beverly, MA) overnight at 4°C. After washing 3 times with PBS, cells were incubated with a biotin-conjugated secondary antibody (1:500 in PBS/0.2% fish gelatin; Vector Laboratories, Burlingame, CA) for 1 hr at RT. Cells were again washed 3 times in PBS, and incubated with Vectastain ABC-AP solution (50 μL/well; Vector Laboratories) for 1 hr at RT, followed by Vectastain alkaline phosphatase substrate (pNpp solution; 50 μL/well). Plates were incubated in the dark for 30 min at 37°C, and the signal for the product para-nitrophenol (pNp) was read at A 405 (absorbance of 405 nm) in a model 1420 Wallac microplate reader (PerkinElmer, Boston, MA).
Crystal violet (CV) assays. The pNp signal was normalized to cell number, as determined by the crystal violet assay (Campbell et al. 2002). After washing 2 times with H 2 O to remove the alkaline phosphatase reaction reagents, the plates were dried at RT for 1 hr. CV solution (0.1% in H 2 O, filtered) was added (50 μL/well), incubated for 1hr at RT, and washed 4 times with H 2 O. The dye was released from the cells with acetic acid (10% in H 2 O; 50 μL/well) at RT for 30 min, and the A 590 signal was read in the Wallac microplate reader.
Determination of cell proliferation. As described previously, ), sub confluent cells were seeded into 96-well plates coated with poly-d-lysine (5,000 cells/well) and allowed to attach overnight. Plating medium was replaced with DMEM containing 1% 4× charcoal-stripped serum for 48 hr, and then with treatment medium containing increasing concentrations of BPS or E 2 (10 -15 to 10 -7 M) alone or BPS concentrations plus 10 -9 M E 2 . After 3 days, cells were fixed (2% paraformaldehyde/0.1% glutaraldehyde in PBS; 50 μL/well) for 20 min at RT. Cell numbers were assessed by CV assay to compare the proliferative effects of BPS at different concentrations.
PRL release. Our study design and conditions were based on previous studies from our laboratory Wozniak et al. 2005). After cells (0.5-0.7 × 10 6 ) were plated into poly-d-lysine-coated 6-well plates overnight, they were hormone deprived in DMEM containing 1% 4× charcoal-stripped serum for 48 hr. Cells were then incubated for 30 min in DMEM/0.1% BSA and exposed to different concentrations of BPS alone (10 -15 to 10 -7 M) or in combination with E 2 (10 -9 ) for 1 min. Cells were then centrifuged at 350 × g for 5 min at 4°C; the supernatant was collected and stored at -20°C until radio immunoassay (RIA) for PRL. Cells were then fixed with 1 mL of 2% paraformaldehyde/0.1% glutaraldehyde in PBS, and cell numbers were determined via the CV assay.
Concentrations of PRL secreted into the media were determined using components of the rat PRL RIA kit from the National Institute of Diabetes and Digestive and Kidney Disease and the National Hormone and Pituitary Program (Baltimore, MD). We combined 100 μL of cold standard (rat PRL-RP-3) or unknown sample with 500 μL rPRL-s-9 antiserum (final dilution of 1:437,500 in RIA buffer containing 80% PBS, 20% DMEM, and 2% normal rabbit serum) and 200 μL of 125 I-labeled rat PRL (15,000 counts/tube diluted in RIA buffer; PerkinElmer, Wellesley, MA). The samples were then incubated and shaken overnight at 4°C. Anti-rabbit IgG was then added (200 μL of 1:9 final dilution in RIA buffer) and the samples were incubated and shaken for 2 hr at RT. After polyethylene glycol solution (1 mL; 1.2 M polyethylene glycol, 50 mM Tris, pH 8.6) was added, samples were incubated and then shaken at RT for 15 min. The samples were centrifuged at 4,000 × g for 10 min at 4°C, the supernatants decanted, and the pellets counted in a Wizard 1470 Gamma Counter (Perkin Elmer). PRL concentrations were calculated and normalized to CV values representing cell number.
Statistical analysis. Statistical analysis was performed using SigmaPlot, version 12 (Systat Software Inc., Chicago, IL). We applied oneway analysis of variance (ANOVA) to the dose-and time-dependent studies to assess the statistical significance of mean values produced by varying XE exposures. A Holm-Sidak comparison against vehicle control or against E 2 treatment was used after the ANOVA to evaluate significance. We considered the overall α level of 0.05 to be statistically significant. In addition, we ran a Student's t-test where the significance between values was borderline by one-way ANOVA, as noted in the figures.

Results
Exposure of GH 3 /B 6 /F 10 cells to BPS for 5 min caused ERK activation ( Figure 1A) simi lar to that caused by E 2 both here and previously Jeng and Watson 2011). The lowest tested BPS concentrations evoked a higher pERK response than did 10 -9 M E 2 ; the response steadily decreased with increasing BPS, indicating a nonmonotonic dose response (Vandenberg et al. 2012). The combination of increasing concentrations of BPS with constant 10 -9 M E 2 caused a lower pERK activity than did BPS alone, and was significantly lower than the nanomolar E 2 response at the highest BPS concentrations (10-100 nM). In contrast, BPS did not produce significant pJNK activation ( Figure 1B); instead the highest BPS concentration (10 -7 M) caused deactivation significantly below vehicle levels. However, when BPS and E 2 were adminis tered together, JNK was strongly activated-above the level seen with E 2 alone-and again featured a non monotonic dose-response curve, with the lowest concentrations evoking the largest responses.
We also examined the time dependence of these responses at optimal response concentrations (10 -14 M BPS, 10 -9 M E 2 ; Figure 2A,B). E 2 produced a typical oscillating two-peak ERK response, with the first peak within 5 min, followed by a second peak at 30 min Jeng and Watson 2011). BPS phospho activated ERK within 2.5 min but did not show significant oscillation. Responses induced by BPS and E 2 were significantly different from each other. The combination of 10 -14 M BPS and E 2 showed a slightly oscillating pattern, although differences between stimulated points were not significant. We have previously observed rephasing of responses due to XE combined with E 2 Jeng and Watson 2011;Kochukov et al. 2009). Therefore, even at this very low concentration (10 -14 M), BPS was able to disrupt the timing of the response to a physiologic estrogen. Figure 1. pERK (A) and pJNK (B) responses to 10 -15 to 10 -7 M BPS, 10 -9 M E 2 , 10 -9 M BPA, or 10 -9 M E 2 plus BPS at varied concentrations in GH 3 /B 6 /F 10 cells. pERK (A) and pJNK (B) pNp signals measured by plate immuno assay after 5 min exposure were normalized to cell number estimates. Mean absolute absorbance values (normalized to cell number) of the vehicle control are 0.834 for ERK and 0.395 for JNK. The width of shaded areas represents means ± SEs for vehicle (gray) and E 2 (blue); n = 24 over three experiments. *p < 0.05 compared with vehicle. # p < 0.05 compared with 10 -9 M E 2 . † p < 0.05 compared with E 2 using Student's t-test.

BPS concentration (log M) BPS concentration (log M)
-15 -14 -13 -12 -11 -10 -9 -8 -7 Vehicle Vehicle Even though 10 -14 M BPS alone could not activate JNK at any time point tested, its combination with E 2 dramatically enhanced the early and sustained pJNK response to E 2 ( Figure 2B). A prototypic chemical inhibitor for ERα (MPP, 10 -8 M) was the most effective antagonist of E 2 and BPS-induced responses (Figure 3). In comparison, inhibitors for ERβ (PHTTP, 10 -7 M) and GPER/GPR30 (G15, 10 -7 M) were much less effective in reducing the phospho activation of ERK by E 2 and BPS. Therefore, mERα appears to be the predominant receptor that mediates this nongenomic response to BPS.

pERK (% of vehicle) pJNK (% of vehicle)
After a 3-day exposure, 10 -9 M E 2 and BPS had similar effects on cell proliferation, causing a non monotonic stimulation, as we observed previously with E 2 Kochukov et al. 2009). The combination of BPS and E 2 did not stimulate cell proliferation, but instead suppressed cell numbers to levels below those of cells exposed to vehicle (Figure 4).
Because decreases in cell number can be caused by apoptosis, we assayed caspases 8 and 9 to determine whether the extrinsic or intrinsic apoptotic pathways were activated. Caspase 8 was activated by both BPS and BPS plus E 2 (10 -9 M) at all time points tested (4-24 hr), regardless of the BPS concentration used ( Figure 5A). In contrast, caspase 9 was significantly activated only at 24 hr, and by low concentrations of BPS (10 -14 M) or its combination with E 2 ( Figure 5B). The positive control (staurosporine) was active at all times and on all caspases, as expected. Interestingly, nano molar E 2 alone suppressed caspase 9 activity below the level of vehicle controls at all time points, whereas inhibition below vehicle levels was observed only at the 8-hr time point for caspase 8, with some timing differences from our previous studies ).
The GH 3 /B 6 /F 10 cell line secretes PRL in response to E 2 and a variety of estrogenic compounds, thus making this model an excellent tool for evaluating functional responses to estrogens (Dufy et al. 1979;Jeng et al. , 2010Kochukov et al. 2009;Pappas et al. 1995b;Wozniak et al. 2005). After a typical exposure time of 1 min, BPS did not significantly increase PRL secretion but E 2 did ( Figure 6). In cells treated with BPS plus E 2 , the E 2 -induced PRL release was severely inhibited in a non monotonic pattern, well below that in nanomolar-E 2 -treated cells; at most concentrations of BPS, the PRL release was well below that of vehicle. The PRL release after treatment with BPS (10 -10 M) plus E 2 was not statistically different from the level of release caused by E 2 alone, nor was it statistically different from vehicle because of errors for that measurement.

Discussion
Increased scrutiny and concern by government agencies and environmental advocacy groups led to the development of potential chemical replacements for BPA, such as BPS. Although BPS is less likely to leach from plastic containers with heat and sunlight, it does still escape Our results show that BPS is active at femtomolar to picomolar concentrations, and can alter a variety of E 2 -induced non genomic responses in pituitary cells, including pERK and pJNK signaling and functions (e.g., cell number, PRL release). BPS had the same capability as E 2 for initiating phospho activation of ERK across concentrations and times Watson 2009, 2011;Kochukov et al. 2009;Wozniak et al. 2005), with lower concentrations of BPS being more effective. BPS was also equi potent to BPA in the phosphoactivation of ERK. Such non monotonic dose responses are controversial and have been heavily examined recently (Vandenberg et al. 2012). The fluctuation of MAPK activities with concentration and time could involve several mechanisms (Conolly and Lutz 2004;Vandenberg et al. 2012;Watson et al. 2010;Weltje et al. 2005), including receptor desensitization due to over stimulation, activation of phosphatases, and simultaneous activation of multiple signaling pathways, thereby activating proteins at different rates (Vandenberg et al. 2012;Watson et al. 2011). MAPK down-regulation is critical for preventing adverse effects of extended pathway stimulation (Hunter 1995). In our mixture studies (BPS plus E 2 ), attenuation of the ERK response may protect the cell against unnecessary and perhaps dangerous estrogenic stimulation caused by the increased over all estrogenic concentration of two estrogenic compounds.
Non genomic and functional actions initiated in this cell line were mediated predominantly by mERα. In previous studies, chemical inhibitors effective for both mERα and mERβ (ICI 187,634) also blocked ERK responses . In contrast to the GH 3 /B 6 / F 10 cells used here, GH 3 /B 6 /D 9 pituitary cells expressing low mERα levels were unable to respond via E 2 -induced activation of MAPK signaling . In the present study, our experiments with subtype-selective antagonists also demon strated that mERα was the predominant membrane receptor mediating these responses, as we reported previously (Alyea et al. 2008;Jeng and Watson 2011), although, as in our past studies, ERβ and GPR30 also contributed to this ERK response to estrogens.
Phospho activation of ERK and JNK has been closely associated with opposing functional end points. For example, ERK signaling (RAF→MEK1,2→ERK1,2) is often associated with cell differentiation and growth, whereas JNK signaling is usually thought to accompany the initiation of apoptosis (Junttila et al. 2008;Meloche and Pouyssegur 2007;Nordstrom et al. 2009;Xia et al. 1995). Simultaneous phospho activation of ERK and inactivation of JNK by BPS, as our data show, could simultaneously stimulate proliferation Figure 4. Cell proliferation in GH 3 /B 6 /F 10 cells after 3-day exposure to increasing concentrations of BPS or E 2 alone, or to BPS plus a physiologically relevant concentration of E 2 (10 -9 M). Cell number was estimated by the CV assay (n = 24 over three experiments). The mean absolute absorbance value of the vehicle control is 0.299. The width of the shaded area represents the means ± SEs of vehicle-treated cells. *p < 0.05 compared with vehicle. # p < 0.05 compared with the corresponding concentration of E 2 alone.

Concentration (log M)
-15 -14 -13 -12 -11 -10 -9 -8 -7 Cell number (% of vehicle) and inactivate cell death, magnifying the increase in cell number (Junttila et al. 2008). Our BPS/E 2 mixture activated both ERK and JNK, perhaps correlating with a decline we saw in cell number, if the balance of these two activities is important for the outcome. Earlier studies reported that BPS alone is capable of inducing cell proliferation in the MCF-7 cell line (Hashimoto and Nakamura 2000;Hashimoto et al. 2001;Kuruto-Niwa et al. 2005) but noted that BPS began to show cytotoxic effects at concentrations > 10 -4 M (well above the highest concentration we tested). Therefore, the proliferative/anti proliferative responses caused by BPS can happen in multiple responsive tissues. This is the first study to explore the ability of BPS to activate caspases. Early activation of caspase 8 (compared with caspase 9) indicates that the extrinsic pathway, which involves extra cellular stimuli acting on cell-surface receptors, is the primary apoptotic pathway. The reason for later and weaker activation of caspase 9 can be explained by cross over to that pathway via a lengthy process initiated by the cleavage of Bcl2-interacting protein (BID) in the caspase 8 pathway; this results in the translocation of BID to mitochondria, where it causes later release of cytochrome c and subsequent activation of caspase 9 pathways (Kruidering and Evan 2000;Medema et al. 1997). We previously showed increased activation of caspase 8-but not caspase 9-in phytoestrogen-treated GH 3 /B 6 /F 10 cells after 24 hr of treatment .
Cell survival versus cell death is determined by the balance of several cellular signaling responses, and the activation of capsases is only one of many factors. There are also discrepancies in the literature about the role of ERK and JNK activation in controlling cell number. Phosphoactivation of ERK can, for example, lead to the activation of the anti apoptotic protein Mcl-1, which binds to Bax protein and prevents its activation, thus inhibiting apoptosis (McCubrey et al. 2007). Activation of ERK has also been shown to inhibit caspase 9 upon phosphorylation Clarke 2007, 2009;Allan et al. 2003); this is perhaps a mechanism promoting the protective effects we see with E 2 both here and in past studies ). Phosphoactivation of JNK can lead to activation of several pro-apoptotic proteins such as Bax, caspase 3, cyclin D1, Fas, and interleukin 1 (Ip and Davis 1998). However, JNK has also been linked to the activation of pro survival pathways, with the final functional response dependent on the overall balance between ERK and JNK activities (Dhanasekaran and Reddy 2008;Sanchez-Perez et al. 1998). More examples of these conflicting outcomes need to be studied to resolve the composite contributions of MAPKs to the control of cell number.
BPA and other XEs are potent inducers of PRL release Kochukov et al. 2009;Wozniak et al. 2005); in contrast, BPS caused minimal PRL release on its own. However, BPS dramatically disrupted E 2 -induced PRL release, as do other XEs. Disturbances in the timing or amount of PRL released can lead to a variety of physiologic complications, including electrolyte imbalance, disruptions in growth and development, metabolic dysfunctions, behavioral disturbances, reproductive failure, or lactation failure. In all, PRL regulates > 300 biological functions (Bole-Feysot et al. 1998). The differences we have observed between these two structurally similar bisphenol compounds warrant future examination of structure-activity relationships for these responses.
Using urine samples collected for NHANES, Calafat et al. (2008) observed total BPA concentrations across various demographic groups in the United States, with a geometric mean of 2.6 μg/L (10 nM). In comparison, Liao et al. (2012a) determined the occurrence of BPS in humans in seven different countries, with the highest urinary geometric mean concentrations in Japan, followed by the United States (Albany, NY), with a geometric mean of 0.299 ng/mL (1.2 nM), a concentration much higher than those used in our studies. Because earlier studies focused entirely on genomic mecha nisms of BPS action in which BPS was active only in the micro-to milli molar range, those effects would be relevant only to high-dose exposures such as industrial accidents.
Our study is the first to demonstrate that the BPA-substitute BPS can induce rapid non genomic signaling in estrogen-responsive pituitary cells at low (femtomolar to pico molar) concentrations. Another cause for concern is that BPS also interferes with physio logic E 2 signaling that leads to several functional end points. These findings highlight the need for efficient in vitro screening methods to pretest possible substitutes for XEs before they are deployed in manufacturing. As more related compounds are tested, we can establish a list of structural features likely associated with risks in this class of chemicals, and perhaps guide future designs away from these structures that can adversely affect human and animal health. Figure 6. Effect of BPS on E 2 -induced PRL secretion in GH 3 /B 6 /F 10 cells. The amount of PRL secreted for each well (counts per minute) was normalized to the CV value for cell number, and expressed as a percentage of vehicle-treated controls. The absolute value (normalized to cell number estimates) of the vehicle control is 466. The width of shaded areas represents means ± SEs for vehicle (gray) and E 2 (blue). Values shown are means ± SEs; n = 24 over three experiments. *p < 0.05 compared with vehicle. # p < 0.05 compared with 10 -9 M E 2 .