Comparison of Serum Bisphenol A Concentrations in Mice Exposed to Bisphenol A through the Diet versus Oral Bolus Exposure

Background: Bisphenol A (BPA) is a widely produced endocrine-disrupting chemical. Diet is a primary route of exposure, but internal exposure (serum concentrations) in animals and humans has been measured only after single oral bolus administration. Objective: We compared serum concentrations of BPA over a 24-hr period after oral bolus administration or ad libitum feeding in mice and assessed for buildup with dietary exposure. Methods: Adult female mice were administered [dimethyl-d6]-BPA (BPA-d6) as a single oral bolus (20 mg/kg body weight) or fed a diet containing 100 mg BPA-d6/kg feed weight ad libitum for 1 week. Serum concentrations were analyzed using isotope dilution liquid chromatography coupled with electrospray tandem mass spectrometry and compared between exposure groups over the first 23 hr and after 7 days of dietary exposure. Results: Maximum concentration (Cmax) for BPA-d6 during the first 24 hr was reached at 1 hr and 6 hr for oral bolus and diet groups, respectively. Relative BPA-d6 bioavailability (unconjugated BPA-d6) was higher in diet-exposed mice than in the bolus group despite a relative lower absorption, a phenomenon consistent with an inhibitory effect of food on first-pass hepatic metabolism. In mice with ongoing dietary exposure, unconjugated BPA-d6 was higher on day 7 than on day 1. Conclusions: This is the first report of serum BPA concentrations in an animal model exposed to this chemical via the diet. Although bolus administration of BPA-d6 led to peak concentrations within 1 hr, Cmax for diet-exposed mice was delayed for several hours. However, bolus administration underestimates bioavailable serum BPA concentrations in animals—and presumably humans—than would result from dietary exposure. Exposure via diet is a more natural continuous exposure route than oral bolus exposure and is thus a better predictor of BPA concentrations in chronically exposed animals and humans.

Components of AIN-93G control diet with 7% corn oil from Harlan-Teklad 9 2 Components of BPA-d 6 diet (100mg/kg feed weight) from Harlan-Teklad 10 3 Definition of the different pharmacokinetic parameters computed by using a non compartmental analysis 11 4 Pharmacokinetic parameters of unconjugated and total BPA-d 6 obtained by using a non compartmental data analysis in mice, BPA-d 6 dose of 20 mg/kg by oral route of exposure 12 5 Pharmacokinetic parameters of unconjugated and total BPA-d 6 obtained by a non compartmental data analysis in mice exposed to BPA-d 6 through the diet Body weight was measured and food consumption from the beginning of the dosing period was calculated at each timepoints. Blood samples were collected by cardiac puncture in polypropylene (PP) syringes, placed in PP microcentrifuge tubes, and centrifuged twice (15 min at 13,800 g) (Mikro 22R Microcentrifuge, Hettich Zentrifugen, Germany) for serum collection.
The serum samples were stored at -20°C. Individual samples within the same timepoint and treatment group that provided less than the minimum 400 μL needed for the BPA-d 6 analysis were pooled to yield 9 negative control samples.

Analysis of Unconjugated and Conjugated BPA-d 6 in Mouse Serum Samples
To assess "freely available" BPA, i.e. that fraction that had not been metabolized to its watersoluble, glucuronyl derivative, each sample was allowed to thaw at room temperature for 30 min and transferred into a 15 mL PP tube. An internal standard (5 ng of deuterated 16-bisphenol A (BPA-d 16 ) was added. Five mL of ethyl acetate was then added, and the mixture shaken in an orbital shaker for 30 min. After centrifugation at 4500 g for 3 min (Eppendorf Centrifuge 5804, Hamburg, Germany), the ethyl acetate layer was transferred to a clean PP tube, the residue extracted two more times with 3.5 mL of ethyl acetate by shaking, and the extracts were combined. The extracts were mixed with 3.5 mL of milli-Q water and centrifuged at 5000 rpm for 3 min (Eppendorf Centrifuge 5804). The ethyl acetate layer was collected, dried under a gentle stream of nitrogen and reconstituted in 0.5 mL of methanol. This fraction that was soluble in ethyl acetate was considered freely available BPA-d 6 .
After addition of the internal control, the second aliquot of thawed serum was digested with 1 mL of 2 μL/mL β-glucuronidase that also has aryl-sulfatase activity (from Helix pomatia, 145700 unit/mL, Sigma, St Louis, MO) at 37ºC for 12 h. The digested sample was processed as described above and referred to as total BPA-d 6 , which includes the free, i.e. ethyl acetate soluble, plus the conjugated BPA-d 6 .
The BPA-d 6 serum concentrations in samples were measured by a procedure similar to that described earlier, but with some modifications (Padmanabhan et al. 2008 Quality assurance and control parameters included validation of the method by spiking BPA-d 16 into the sample matrices and passing through the entire analytical procedure to calculate recoveries of BPA-d 16 through the analytical method. The matrix spike recovery was 109 % (106-111%) with a standard deviation of 3.5%. A procedural blank, containing milli-Q water in place of serum was analyzed in parallel with the samples to check for interferences that would correlate with the target compound or laboratory contamination. Trace concentrations of BPA present in such blanks (<0.01 ng) were subtracted from sample values for determining the concentrations in samples. The limit of detection, i.e. sensitivity of the assay, was 0.1 ng/mL, which was calculated as twice that of the valid "lowest acceptable calibration standard". The reported concentrations of BPA-d 6 for each sample were corrected, based on the recovery value of the surrogate standard, BPA-d 16 (i.e., isotope dilution). The BPA-d 6 standard spiked into sample matrices and passed through the entire analytical procedure yielded a mean recovery of 117%. An external calibration curve prepared by injecting 10 μL of 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 ng/mL standards of BPA-d 6 and BPA-d 16 yielded a calibration coefficient of 0.99.

Non-compartmental analysis of total and unconjugated BPA-d 6 concentrations after oral and diet exposure
Extrapolation to infinity to obtain AUC (0-infinity) was calculated by dividing the last observed quantifiable serum concentrations by the slope of the terminal phase as estimated by linear regression by using the best fit option of WinNonlin.
Mean Residence Time (MRT), which refers to the average total time BPA-d 6 molecules of a given BPA-d 6 dose spend in the body, was obtained with and without extrapolation to infinity by using statistical moments (Gibaldi and Perrier, 1982). MRT can be viewed as the arithmetic mean of times that each BPA-d 6 molecule spends in the body, and it is a metric of persistency of BPA in the body because it is a stochastic view of BPA-d 6 pharmacokinetics in the body.
The apparent oral clearance (Cl/F) was obtained by dividing the administered BPA-d 6 dose by the corresponding AUC (0-Clast) , C last being the 24h serum BPA-d 6 concentration.

Deconvolution analysis
The curve representing mean unconjugated BPA-d 6 serum concentrations over time (0-11 h) was presented with automatic smoothing procedure in Supplemental Material, Figure 3, and these are the data that were analyzed by deconvolution.
Deconvolution computed for each discretization step (here 0.08h or 5.04 min, Supplemental Material,