Estrogens from the Outside In: Alkylphenols, BPA Disrupt ERK Signaling in Vitro

The body produces estrogens—including estrone (E1), estradiol (E2), and estriol (E3)—that direct reproductive system processes and contribute to the normal function of tissues including the brain, bone, and cardiovascular system. Certain xenoestrogens (estrogenic compounds introduced from outside the body) are suspected of disrupting these activities. In a new study, xenoestrogenic alkylphenols and bisphenol A (BPA) interfered with normal estrogenic signaling in vitro, which suggests they could disrupt normal physiologic function at critical life stages [EHP 119(1):104–112; Jeng and Watson]. 
 
Different estrogen receptors control different functions: receptors in the cell nucleus direct gene transcription, whereas receptors in the cell membrane direct signaling pathways via extracellular signal–regulated kinases (ERKs). ERK-controlled pathways respond to many biochemical stimuli and integrate these signals to direct a cell toward division, differentiation, death, or malignant transformation. The structurally related alkylphenols and BPA interact weakly with nuclear estrogen receptors, but they can have pronounced effects on signaling pathways mediated by estrogen receptors in the cell membrane. 
 
In the current study, a rat pituitary cancer cell line was used to study the effect of alkylphenols and BPA on ERK1 and ERK2 activation (measured as phosphorylation), both alone and in combination with each physiologic estrogen. After treatment with each physiologic and environmental estrogen, the researchers measured time-dependent surges in ERK activation. In most cases, E1 and E2 prompted early, intermediate, and late surges in ERK activation at 5, 10–30, and >30 min, respectively; alkylphenols and E3 typically triggered early and late surges. Interestingly, a very low concentration of BPA (10−14 M) yielded a similar two-peak response, but a higher concentration (1 nM) induced a three-peak response like that of E1 and E2. Both BPA concentrations were typical of environmental exposures and, along with ineffective midrange doses, also illustrated the nonmonotonic dose–response relationship characteristic of many estrogenic compounds. 
 
When physiologic estrogens and xenoestrogens were combined, the response pattern generally shifted to a single major peak at an intermediate time. Xenoestrogens that caused a strong response when administered alone at a particular point in time or concentration tended to inhibit ERK activation in response to a physiologic estrogen. But at other times or concentrations, the same xenoestrogen might cause a weak response on its own, in which case it would tend to enhance ERK phosphorylation in response to physiologic estrogens. 
 
There were exceptions to these general patterns, however, which highlights the need to study effects of individual xenoestrogens at different points in time, at varying concentrations, and in different tissues. The effect of shifts in the patterns of ERK activation are only just beginning to be explored, although it is known that these patterns constitute an important component of information flow within a cell. The correct flow of information is likely to be especially critical during windows of vulnerability that are based in part on life stage.

at indivi duals. They base their recommendations on published studies and discussions from a 2009 workshop on this topic held in Vancouver, Canada.
The authors argue that cities can improve residents' health by considering air quality during land-use planning. For example, creating high-density, mixed-use areas would enable more people to walk or bicycle to work, school, and shops, thereby reducing emissions and encouraging more exercise; ideally, safe pedestrian and cycling greenways would be located away from traffic. For longerdistance travel, the authors suggest low-emission public transit.
And in areas where wood burning is an important heating method, woodstove exchange programs can help residents acquire cleaner-burning stoves affordably. Risk factors for heart disease include a sedentary lifestyle, obesity, and a high-sodium diet. Therefore, the authors posit that another approach to reducing a person's risk of being affected by air pollution is to minimize one's overall risk of heart disease. This could involve interventions that encourage people to eat a diet rich in omega-3 fatty acids and antioxidants and to get regular exercise. However, because pollution levels vary even within cities, exercise should be planned to minimize exposure. Variations occur by season, with ozone being higher in the summer and particulates from woodstoves higher in the winter, for example. Traffic-related pollutants also spike during rush hour and are higher in heavily traveled areas.

Alkylphenols, BPA Disrupt ERK Signaling in Vitro
The body produces estrogens-including estrone (E 1 ), estradiol (E 2 ), and estriol (E 3 )-that direct reproductive system processes and contribute to the normal function of tissues including the brain, bone, and cardiovascular system. Certain xenoestrogens (estrogenic compounds introduced from outside the body) are suspected of disrupting these activities. In a new study, xenoestrogenic alkylphenols and bisphenol A (BPA) interfered with normal estrogenic signaling in vitro, which suggests they could disrupt normal physiologic function at critical life stages [EHP 119(1):104-112; Jeng and Watson].
Different estrogen receptors control different functions: receptors in the cell nucleus direct gene transcription, whereas receptors in the cell membrane direct signaling pathways via extracellular signalregulated kinases (ERKs). ERK-controlled pathways respond to many biochemical stimuli and integrate these signals to direct a cell toward division, differentiation, death, or malignant transformation. The structurally related alkylphenols and BPA interact weakly with nuclear estrogen receptors, but they can have pronounced effects on signaling pathways mediated by estrogen receptors in the cell membrane.
In the current study, a rat pituitary cancer cell line was used to study the effect of alkylphenols and BPA on ERK1 and ERK2 activation (measured as phosphorylation), both alone and in combination with each physiologic estrogen. After treatment with each physiologic and environmental estrogen, the researchers measured time-dependent surges in ERK activation. In most cases, E 1 and E 2 prompted early, intermediate, and late surges in ERK activation at 5, 10-30, and > 30 min, respectively; alkylphenols and E 3 typically triggered early and late surges. Interestingly, a very low concentration of BPA (10 −14 M) yielded a similar two-peak response, but a higher concentration (1 nM) induced a three-peak response like that of E 1 and E 2 . Both BPA concentrations were typical of environmental exposures and, along with ineffective midrange doses, also illustrated the nonmonotonic dose-response relationship characteristic of many estrogenic compounds.
When physiologic estrogens and xenoestrogens were combined, the response pattern generally shifted to a single major peak at an intermediate time. Xenoestrogens that caused a strong response when administered alone at a particular point in time or concentration tended to inhibit ERK activation in response to a physiologic estrogen. But at other times or concentrations, the same xenoestrogen might cause a weak response on its own, in which case it would tend to enhance ERK phosphorylation in response to physiologic estrogens.
There were exceptions to these general patterns, however, which highlights the need to study effects of individual xenoestrogens at different points in time, at varying concentrations, and in different tissues. The effect of shifts in the patterns of ERK activation are only just beginning to be explored, although it is known that these patterns constitute an important component of information flow within a cell. The correct flow of information is likely to be especially critical during windows of vulnerability that are based in part on life stage.  The results showed that each 1-µg/L increase of the compound in the participants' water supply was associated with a 141.5-µg/L increase in people's serum PFOA concentrations. The participants lived around DuPont's Washington Works facility in Parkersburg, West Virginia, where PFOA (also known as C8) is used in the manufacture of Teflon ® nonstick polymers. PFOA has been shown to increase risk of cancer, reproductive problems, and liver damage in laboratory animals, although human health effects are less clear. Many of the water monitoring data used in this study were collected as part of an agreement between DuPont and the U.S. Environmental Protection Agency (EPA) to conduct a human health risk assessment for PFOA.
The groundwater in the Parkersburg area had been contaminated by DuPont's releases of PFOA into the nearby Ohio River. A second source of contamination was PFOA that was released into the atmosphere and deposited onto soils, which then leached into the groundwater.
Previous research in this study area linked drinking water supplied by six local water districts and consumption of home-grown vegetables to PFOA levels in participants' serum [EHP 118(8):1100[EHP 118(8): -1108Steenland et al.]. The new study provides a quantitative estimate of the relationship between drinking water and serum PFOA levels based on exposure to a wider range of PFOA levels in drinking water from 62 wells. It also corroborates the earlier finding about consumption of home-grown vegetables.
Many of the wells in the study had PFOA concentrations that exceeded the EPA's 0.4-µg/L advisory level, although the median concentration in the well water samples was half that level. The concentrations of PFOA in participants' serum ranged from 0.9 to 4,751 µg/L, with a median of 75.7 µg/L, approximately 20 times the average level in the U.S. general population.
The association between PFOA in drinking water and serum was similar for both shorter-and longer-term residents of the area. The researchers found the associations held after excluding participants who reported drinking bottled water and those who worked at the DuPont facility. Compared with other factors (including age, sex, body weight, cigarette smoking, and alcohol consumption), drinking water was consistently the strongest predictor of serum PFOA levels.
The 141.5:1 ratio estimated for drinking water to serum PFOA concentrations is close to the 114:1 ratio predicted by a steady-state pharmacokinetic model employed by the authors. These findings may be useful in developing drinking water guidelines and studying other communities where PFOA is manufactured.
Kellyn S. Betts has written about environmental contaminants, hazards, and technology for solving environmental problems for publications including EHP and Environmental Science & Technology for more than a dozen years.

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