Bisphenol A is released from polycarbonate drinking bottles and mimics the neurotoxic actions of estrogen in developing cerebellar neurons☆
Introduction
Bisphenol A (BPA, 2,2-bis (4-hydroxyphenyl) propane; CAS RN 80-05-7) is a high-production chemical used in the manufacture of numerous consumer goods and products. Bisphenol A has well characterized estrogenic and other endocrine disrupting activities that are mediated via multiple molecular mechanisms (Wetherill et al., 2007). In 2004, the estimated production volume of BPA in the United States was ∼2.3 billion pounds (CERHR, 2007). Of the 1.9 billion pounds of BPA used in the US in 2003, nearly 3/4 was used to manufacture polycarbonate resins that were in turn used to manufacture various consumer products including polycarbonate containers for storage of foods and beverages (CERHR, 2007).
Because of BPA's high volume production and extensive use in plastics, there is widespread environmental contamination and well-documented human exposure to BPA. While not diminishing the importance of BPA pollutants in marine, aquatic, and soil ecosystems (for review see Crain et al., 2007), recent studies have demonstrated that there is wide-spread BPA contamination of most individuals in industrialized human populations (Calafat et al., 2005, Vandenberg et al., 2007). The detection of adverse health effects in a number of laboratory animal models upon exposure to environmentally relevant doses of BPA that correspond to those observed in humans, strongly supports the idea that the endocrine disrupting activities of BPA contribute to adverse effects on human health (reviewed in Richter et al., 2007).
In the laboratory setting, biologically active and environmentally relevant levels of BPA were shown to leach from polycarbonate (PC) flasks upon autoclaving (Krishnan et al., 1993), from used PC rodent-housing containers (Howdeshell et al., 2003), and from various other forms of polycarbonate plastics and BPA-containing resins (Kang et al., 2006, Vandenberg et al., 2007). Because the major source of human exposure to BPA in the general population is likely through consumption of contaminated foods and beverages that have contacted epoxy or PC resins (Kang et al., 2006), we evaluated whether BPA migrated from new or used high-quality PC bottles that are commonly used by consumers for storage of water and other beverages. The exposures and mild treatments used in this study were designed to mimic conditions representative of normal consumer usage, including typical use in outdoor recreation settings. Along with characterizing the rate of BPA liberation into water at room temperature, the effect of short-term exposure to hot (100 °C) water was determined. A sensitive and quantitative competitive ELISA employing a BPA-specific monoclonal antibody was used to determine relative concentrations of BPA that were leached into water samples from newly purchased polycarbonate bottles and those subjected to normal use by consumers. The estrogenic bioactivity of the BPA-like immunoreactivity released into the water samples was demonstrated with an in vitro assay of rapid estrogen-signaling and neurotoxicity in immature cerebellar neurons (Belcher et al., 2005, Wong et al., 2003).
Section snippets
Reagents
Bisphenol A (CAS RN 80-05-7; purity grade >99%; 23,965-8; lot no. Cl 03105ES) and dimethyl sulfoxide (Chromasolv Plus, for HPLC ≤99.7%; batch no. 00451HE) were purchased from Sigma–Aldrich (St. Louis, MO). HPLC-grade water (W5sk, lot no. 056618) and methanol (A452sk lot no. 061495) were purchased from Fisher Scientific (Fairlawn, NJ) and used for all washes, dilutions, and sample preparations. New PC and high-density polyethylene (HDPE) bottles (32 ounce loop-top; Nalgene, Rochester, NY) were
Results
Over the course of these experiments, results of the BPA-specific ELISA were highly reproducible. Demonstrating the reproducibility of the assay, mean values and variance (S.E.M.) for the combined standard curves generated from 17 different experiments are shown in Fig. 1A. Within that large data set, ANOVA analysis showed significant differences between mean values for BPA concentration standards at 0.005 ng/ml and 0.05 ng/ml even though they were outside the detection range of a typical assay.
Discussion
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 the
Conflict of interests
The author's have no conflicts of interest that would have inappropriately influenced the work presented in this manuscript.
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These studies were supported by NIH grant R01-ES015145 awarded to SMB; Emily Carlson, and Jason Chua were fellows of the University of Cincinnati College of Medicine's Summer Undergraduate Research Program. The study sponsors had no involvement in the design of this study, the collection, analysis or interpretation of data, the writing of this report, nor the decision to submit this manuscript for publication.