Detectable levels of BPA-like immunoreactivity were observed in all room temperature samples of water following incubation in polycarbonate water bottles, regardless of whether or not the bottle was new or previously used by a consumer. By contrast, water samples collected after identical exposure to negative control water bottles made of HDPE, a polymer consisting of long chains of the monomer ethylene, contained much lower levels of BPA-like immunoreactivity. The water samples from two of HDPE bottles were estimated to contained concentrations of BPA significantly above the theoretical mean for the 0.05 ng/ml detection limit of the assay (bottle no.1 p=0.049; bottle no. 2 p=0.046 as calculated using a one sample t-test). The differences between the mean values for untreated water and water samples incubated in the HDPE bottles were reproducible and highly significant (p=0.0025). Neither the HDPE polymers, nor the polypropylene plastic loop-tops common to tested HDPE and PC bottles, are expected to contain BPA which could account for the observed elevation in BPA-like immunoreactivity. However, the presence of non-polymer additives or coating can not be ruled out as a source of the apparently elevated levels of BPA relative to untreated water.
The bioequivalence of water samples from the PC drinking bottles and BPA was demonstrated using a rapid and sensitive in vitro assay that detects “non-genomic” rapid estrogenic signaling effects in developing neurons (Wong et al., 2001
; Wong et al., 2003
). Specifically, the endpoint of this assay (LDH release due to oncotic cell death) is dependent on activation of a defined estrogen signaling mechanism that requires activation of two signaling pathways; activation of extracellular-signal regulated kinase (ERK) dependant signaling at cell surface and a distinct signaling pathway dependant on intracellular activation of protein phosphatase 2A (PP2A) activity (Belcher et al., 2005
; Wong et al., 2003
). Rather than using the traditional MCF7 growth assay to demonstrate estrogen-like activity of BPA and the BPA-like immunoreactivity present in the collected water samples, we employed this non-genomic assay because rapid non-genomic actions are typically more sensitive for detection of estrogen-like EDC activity (Wetherill et al., 2007
). Additionally this assay benefits from being more rapid than the MCF7 growth assay. Using the granule cell/LDH system, results from multiple samples can be treated, collected and analyzed in <36 hours, avoiding the much longer incubation periods required to detect estrogen-like increases in MCF7 cell population growth. The use of primary cerebellar neurons under defined conditions also avoids many of the complexities associated with using transformed MCF7 cells. Potentially confounding factors with the MCF7 growth assay include more complicated culture conditions, requirements of serum/hormone withdrawal following culture expansion, and variability resulting from population heterogeneity of MCF7 sub-lines which results in differential ER expression and variable estrogen growth response characteristics.
The results presented here confirm and extend previous studies that demonstrated migration of BPA from PC plastics. In the seminal study of Krishnan and coworkers, biologically active and environmentally relevant levels of BPA (minimal concentration of ~2.85 ng/ml) were shown to leach into water from PC flasks used for growth of bakers yeast (Saccharomyces cerevisiae) upon autoclaving at 120-125°C. While those studies established clearly that biologically active BPA could migrate from PC into water at elevated temperatures, a comparison of the presented results with subsequent studies analyzing migration of BPA under less extreme temperature conditions could be considered more relevant.
The studies of Howdershell et al (2003) evaluated whether BPA was released from plastic animal caging at room temperature and found that BPA was released into water incubated in new or used PC cages. From a surface area of PC caging comparable to that of the water bottles analyzed here (478 cm2
) low concentrations (~0.27 ng/ml) of BPA were detected in the 250 ml water samples following a 7 day exposure period. In contrast, exposed samples collected from used cages contained large amounts of BPA which resulted in BPA concentrations up to 310 ng/ml of water (Howdeshell et al., 2003
). This stands in apparent contradiction to the findings reported here that demonstrate no difference in BPA-liberation between new and used PC bottles. While a number of factors might account for those apparently different findings, differences in previous treatment of polycarbonate animal caging and the used water bottles studied here are a likely explanation for the observed differences in BPA migration. The used PC cages were acquired following use in an animal care facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (Howdeshell et al., 2003
). As a result, sanitization of cages would have followed the minimal standards outline in the Guide for the Care and Use of Laboratory Animals
(Institute of Laboratory Animal Research, 1996
), which would entail frequent washing with hot water of at least 180° F (82.2 °C) and soap or detergent. While not stated explicitly, these mouse cages were likely used in a pathogen-free barrier facility and would have also undergone repeated rounds of autoclaving at temperatures of 120° to 122°C. In comparison to the used PC drinking bottles, which would have been washed by consumers at a typical household hot water temperature of ~50°C, it is likely that the elevated temperatures of autoclaving would result in a greater extent of PC polymer hydrolysis, which could account for increased migration of monomeric BPA. This suggestion is supported by the finding that brief exposure to 100°C water greatly increased the rate of BPA migration from the water bottles analyzed here.
Using a protocol of repeated washing and exposure to 100°C water for 1 hr Brede et al. (2003)
demonstrated that BPA from new PC baby bottles leached into boiling water (0.23 ± 0.12 ng/ml). Following simulated use that included dishwashing 51 times, brushing and boiling, the concentrations of BPA liberated was elevated to 8.4 ± 4 ng/ml, a 36.5-fold increase. However, an additional 118 wash cycles did not change the amount of BPA released which was reported to be 6.7 ± 4 ng/ml. From these results it was concluded that there was a link between elevated temperatures and increased BPA migration. It is notable that the concentration of BPA liberated from the PC baby bottles, and the increases in migration rate resulting from exposure to boiling water were similar to the values reported here. However, because the additional variables of washing and brushing were included in the treatments of the baby bottles (Brede et al., 2003
), it is only possible to link increased BPA migration with simulated use. In contrast, the data presented here clearly uncouple the independent variables of new and used, from exposure to elevated water temperature. As a result, in the presented analysis, the study’s treatment design has isolated elevated temperature of water as a single variable. Thus, the linkage of BPA liberation and elevated temperatures is clearly established. While the exposure to elevated temperatures of boiling water is much higher than the typical home wash water temperatures used by consumers, the storage of higher temperature (boiling) water or beverages in these PC bottles is a common practice in cold weather outdoor activities such as alpine snow sports, climbing, and mountaineering. Such practices would likely result in increased levels of BPA in beverages stored and consumed from those bottles.
The studies investigating BPA migration from scientific-grade PC labware (Krishnan et al., 1993
) and animal caging (Howdeshell et al., 2003
) directly address potential confounding factors in the laboratory environment. Those studies also suggest that BPA released from PC plastic could be one point source of BPA contamination in humans. Studies investigating migration of BPA from infant feeding bottles have been especially influential and raised much concern within the general public (Biles et al., 1997
; Brede et al., 2003
; Sun Y, 2000
; Wong et al., 2005
). To date, however, few epidemiological studies have been conducted to investigate the relationship between BPA exposure and human health. Although, as reviewed by Vandenberg and colleagues, a fair number of studies have been conducted that have identified sources or levels of BPA in humans (Vandenberg et al., 2007
). The aim of the majority of those studies has been to estimate levels of BPA migrating from packaging into foods, with most studies focusing on BPA leaching from epoxy resin lining into canned food. As mentioned above, a subset of studies more directly relevant to those presented here, investigated the release of BPA from commercial infant formula bottles. In most studies, significant release of BPA from PC food and beverage containers was detected, yet based on current acceptable exposure levels the potential adverse heath effects were concluded to be minimal (Brede et al., 2003
; D’Antuono et al., 2001
; Wong et al., 2005
). However, there is evidence demonstrating that mixtures of different estrogenic chemicals can have much greater disruptive effects than those predicted from exposure to low concentrations of an individual compound (Rajapakse et al., 2002
). Studies of rapid actions of BPA in developing neurons of the rat cerebellum have also demonstrated that BPA alone acts as a highly potent and efficacious estradiol-mimetic; yet, when combine at very low concentrations (1 pM) with estradiol, BPA acts as a potent antagonist of estrogen signaling (Zsarnovszky et al., 2005
). Those finding and others have highlighted the complex, and often unanticipated nature of EDC action. It is now clear that conclusions from single-compound studies, or association of health-risks with a single EDC, may not fully reflect the effects of environmental exposures to EDCs. Thus, the contribution of the concentrations of BPA that contaminate drinking water and foods stored in PC bottles should be considered as a single, though important component of the total mixture of EDCs to which humans are acutely and chronically exposed through out their life-time.
In summary, in this study it was found that bioactive BPA was liberated from both new and used PC drinking bottles and migrated at a similar rate into water at room temperature. Exposure to elevated temperatures above those typically used for washing by consumers, but not outside normal household practice (e.g. boiling to sterilize infant feeding bottles), or outdoor applications (addition of very hot or boiling water or beverages to drinking bottles) greatly elevated the rate of BPA migration. The exposures anticipated from the BPA drinking bottles are likely one of many sources of BPA contamination that contributes to the total amount of endocrine disrupting compounds to which some individuals are exposed.