Bisphenol A is released from polycarbonate drinking bottles and mimics the neurotoxic actions of estrogen in developing cerebellar neurons

Abstract

The impact of endocrine disrupting chemical (EDC) exposure on human health is receiving increasingly focused attention. The prototypical EDC bisphenol A (BPA) is an estrogenic high-production chemical used primarily as a monomer for the production of polycarbonate and epoxy resins. It is now well established that there is ubiquitous human exposure to BPA. In the general population, exposure to BPA occurs mainly by consumption of contaminated foods and beverages that have contacted epoxy resins or polycarbonate plastics. To test the hypothesis that bioactive BPA was released from polycarbonate bottles used for consumption of water and other beverages, we evaluated whether BPA migrated into water stored in new or used high-quality polycarbonate bottles used by consumers. Using a sensitive and quantitative competitive enzyme-linked immunosorbent assay, BPA was found to migrate from polycarbonate water bottles at rates ranging from 0.20 ng/h to 0.79 ng/h. At room temperature the migration of BPA was independent of whether or not the bottle had been previously used. Exposure to boiling water (100 degrees C) increased the rate of BPA migration by up to 55-fold. The estrogenic bioactivity of the BPA-like immunoreactivity released into the water samples was confirmed using an in vitro assay of rapid estrogen signaling and neurotoxicity in developing cerebellar neurons. The amounts of BPA found to migrate from polycarbonate drinking bottles should be considered as a contributing source to the total "EDC-burden" to which some individuals are exposed.

Figure 1. Bisphenol A (BPA) ELISA

(A) The combined standard curve resulting from a non-linear sigmoidal curve fit from all BPA standard curves used in the 17 experiments presented in this study were averaged and are shown graphically. Data points are mean optical density values ± SEM at each concentration. (B) Shown are the results for a single replicate dilution analysis of sample NPC2 in which the concentration of undiluted and diluted sample NPC2 was calculated independently from the results shown in Table 1 (i.e. this result was not included in Table 1). Standard curve data points for this assay are indicated with open circles and NPC2 sample values are indicated with filled circles (mean ± SEM; n=3). The standard curve used to calculate the indicated BPA concentrations is shown as a gray dashed curve.

Figure 2. Comparison of bisphenol A (BPA) migration into water from new and used polycarbonate bottles

Individual values calculated following room temperature incubation for 7 days in new or used polycarbonate bottles are show. The mean values calculate are indicated and graphically represented with a horizontal dashed line. Error bars represent the standard deviation. * indicates that values are significantly different from the 0.05 ng/ml detection limit as calculated with a one sample t-test using a theoretical mean of 0.05. Values from new and used samples were not significantly different from one another (unpaired t test; p = 0.1688).

Figure 3. Comparative analysis of BPA-like bioactivity of water samples in developing cerebellar neurons

(A) Concentration response analysis of LDH-released from granule cells for known concentrations of 17β-estradiol and BPA. Results are expressed as means ± SEM (n=8). (B) Dilutions of water samples from two polycarbonate bottles stimulate BPA-like increases in LDH release following brief exposure. After 15′ exposure to 17β-estradiol, BPA, or concentrations of diluted water samples corresponding to approximately 10^-11, 10^-10, or 10^-9 M BPA-like immunoreactivity, the amount of LDH-released from granule cells into culture media at 24 hrs was determined and compared to LDH levels released from vehicle treated controls cultures. Levels of LDH released were normalized to the maximal estrogenic effect induced by 10^-10 M 17β-estradiol (E2) and compared to the effect induced by known concentrations of BPA. Results are expressed as means ± SEM (n=4 for BPA and water samples n=7 for vehicle and n=6 for estradiol controls). Levels of significance difference between values calculated fo reach group was assessed with a one-way ANOVA. In panel B, * indicates a significant difference in the values between treatment groups exposed to the same concentration of BPA (0.1 nM) and the estimated concentration of BPA in the experimental water sample.