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Comparative Study
, 139 (1), 4-20

Comparison of Life-Stage-Dependent Internal Dosimetry for Bisphenol A, Ethinyl Estradiol, a Reference Estrogen, and Endogenous Estradiol to Test an Estrogenic Mode of Action in Sprague Dawley Rats

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Comparative Study

Comparison of Life-Stage-Dependent Internal Dosimetry for Bisphenol A, Ethinyl Estradiol, a Reference Estrogen, and Endogenous Estradiol to Test an Estrogenic Mode of Action in Sprague Dawley Rats

Mona I Churchwell et al. Toxicol Sci.

Abstract

Bisphenol A (BPA) was administered by gavage (2.5-300,000 μg/kg body weight (bw)/day) to pregnant Sprague Dawley dams, newborn pups, and continuing into adulthood. Aglycone (i.e., unconjugated and active) and conjugated (i.e., inactive) BPA were evaluated by liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS) in serum to better interpret toxicological endpoints measured in the study. Ethinyl estradiol (EE2, 0.5 and 5 μg/kg bw/day) and the endogenous hormones, 17β-estradiol (E2) and testosterone, were similarly evaluated. Mean BPA aglycone levels in vehicle and naïve control rat serum (0.02-0.5 ng/ml) indicated sample processing artifact, consistent with literature reports of a propensity for postexposure blood contamination by BPA. Direct measurements of BPA-glucuronide in vehicle and naïve control serum (2-10nM) indicated unintentional exposure and metabolism at levels similar to those produced by 2.5 μg/kg bw/day BPA (7-10nM), despite careful attention to potential BPA inputs (diet, drinking water, vehicle, cages, bedding, and dust) and rigorous dosing solution certification and delivery. The source of this exposure could not be identified, but interpretation of the toxicological effects, observed only at the highest BPA doses, was not compromised. Internal exposures to BPA and EE2 aglycones were highest in young rats. When maximal serum concentrations from the two highest BPA doses and both EE2 doses were compared with concurrent levels of endogenous E2, the ERα binding equivalents were similar to or above those of endogenous E2 in male and female rats of all ages tested. Such evaluations of estrogenic internal dosimetry and comprehensive evaluation of contamination impact should aid in extrapolating risks from human BPA exposures.

Keywords: bisphenol A; estrogen receptor; ethinyl estradiol; mass spectrometry; pharmacokinetics.

Figures

FIG. 1.
FIG. 1.
Serum concentration-time profiles for total and aglycone d6-BPA in PND 4 Sprague Dawley rats (mean ± SD, n = 4 pups at each time point) treated by gavage with a single dose of 100 μg d6-BPA/kg bw in 0.3% carboxymethylcellulose in water.
FIG. 2.
FIG. 2.
Aglycone and total BPA serum concentrations at Cmax across treatment groups for PND 4 rats. Values shown by treatment group are means of either total or aglycone BPA ± SD for dosed males and females, combined (n ≥ 8). Aglycone BPA was not quantified in 2.5–25 μg/kg bw dose groups.
FIG. 3.
FIG. 3.
Aglycone and total BPA/BPA-glucuronide serum concentrations at Cmax across treatment groups for PND 21 rats. Values shown by treatment group are means of either total or aglycone BPA ± SD for dosed males and females, combined (n ≥ 8), and BPA-glucuronide combined means ± SD for naïve and vehicle control groups (NC, n = 8 and VC, n = 8, respectively). Aglycone BPA was not quantified in the 25 μg/kg bw dose group.
FIG. 4.
FIG. 4.
Aglycone and total BPA/BPA-glucuronide serum concentrations at Cmax across treatment groups for PND 80 rats. Values shown by treatment group are means of either total or aglycone BPA ± SD for dosed males and females, combined (n ≥ 8), and BPA-glucuronide combined means ± SD for naïve and vehicle control groups (NC, n = 17 and VC, n = 15, respectively).
FIG. 5.
FIG. 5.
EE2 Cmax values following gavage treatment of Sprague Dawley rats at different ages. EE2 concentrations (unconjugated) were quantified using LC/MS/MS from serum collected from male and female rats approximately 30 min after gavage treatment (mean ± SD, n = 5–7/sex/group/age) of the designated ages (note: log ordinate scale; LOD 5pM, 1.5 pg/ml).
FIG. 6.
FIG. 6.
E2 levels in male and female Sprague Dawley rats at different ages. Serum concentrations of E2 (unconjugated) were quantified using LC/MS/MS (LOD 5pM, 1.5 pg/ml) from naïve and vehicle control rats (mean ± SD, n = 5–10/sex/age).
FIG. 7.
FIG. 7.
Comparison of estrogen receptor α (ERα) binding-equivalent serum concentrations of endogenous E2 with EE2- and BPA-treated rats of different ages (see the Results section for description of the process used). The combined male + female Cmax values for BPA in the 100,000 and 300,000 μg/kg bw treatment groups are shown along with combined Cmax values for both doses of EE2 and the endogenous E2 values obtained in either male or female pups at various life stages. The KD for E2 with ERα of 200pM is shown for reference.

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