Serum BPA concentrations in diet-exposed mice Because phase II enzymes are not saturable within many orders of magnitude of human exposure, it is commonly accepted that BPA concentrations in the systemic blood are linear over a wide range of doses (Doerge et al. 2010a; Taylor et al. 2011; Teeguarden et al. 2011; Vandenberg et al. 2007, 2010a), thereby negating the need to measure inter- nal concentrations after much lower doses of BPA-d6. Intake of BPA-d6 at the levels used in our study, either as a single bolus dose or through ad libitum feeding, allowed us to measure both unconjugated and total BPA-d6 in the serum of the exposed C57Bl/6J female mice using a combination of HPLC and tan- dem mass spectrometry. The inclusion of a separate, spiked, deuterated form of BPA, (BPA-d16) in the collected serum served as an internal control to estimate recovery through the analytical steps. Moreover, the BPA-d6 consumed could be distinguished from any contaminating compound and the internal control by virtue of the transitions of 233 m/z group compared with the oral bolus group (227.4 ± 41.1 and 201.0 ± 20.6 ng-hr/mL, respectively), indicating a relative bioavailabil- ity of 113% for the diet group. Rate constants (initial and terminal rates) were estimated by using compartmental anal- ysis to fitted unconjugated BPA-d6 serum concentrations after oral bolus exposure [see Supplemental Material, Figure 1 (http:// dx.doi.org/10.1289/ehp.1003385)]. The ter- minal half-life of BPA-d6 was estimated to be 6.4 ± 1.1 hr (mean ± SE, see Supplemental Material, Table 6). Deconvolution analysis for time development of the internal exposure in the diet-exposed group supported our original finding on exposure rate obtained with food consumption. When bolus administration of BPA-d6 was used as the reference, the total esti- mated bioavailable BPA-d6 dose between 0 and 11 hr after the beginning of diet exposure based on deconvolution analysis was 15.6 mg/kg, which is close to the 13 mg/kg BW estimated by actual weighing of ingested food (Figure 1; see also Supplemental Material, Figures 3 and 4). From the deconvolution analysis, it appeared that most of BPA-d6 ingestion was during the first part of the night, and 90% of the bioavailable BPA had been computed to be absorbed into the bloodstream by 5.46 hr after the beginning of the diet exposure (i.e., before 0100 hours the next day) (see Supplemental Material, Table 7). Measurement of internal exposure to total BPA‑ d6 in serum. Total BPA is the sum of the unconjugated and conjugated BPA-d6 in the serum of exposed mice. Concentrations of the conjugated form were up to 70–100 times higher than those of unconjugated BPA (Table 1), which could account for the differences in blood clear- ance between conjugated and unconjugated forms. In the oral bolus group, peak concen- trations (i.e., presumed Cmax) of conjugated BPA-d6 occurred by 1 hr (at 2000 hours) after treatment (1596.7 ± 1006.6 ng/mL, mean ± SE) (Figure 2B). In the oral bolus group, there was a second increase, which in contrast to unconjugated BPA-d6 data was statistically significant between the 4-hr collections (2300 hours) and the measure- ments at 6 hr (693.2 ± 176.9 ng/mL ver- sus 1636.5 ± 642.6 ng/mL; p < 0.0.01) and at 11 hr (1200.5 ± 252.8) (Figure 2B). By 24 hr, however, total BPA-d6 had declined markedly in both groups to about 5% of their peak concentrations noted at 1 hr. The Cmax for the diet group (802.2 ± 126.6 ng/mL) was achieved later (at around 6 hr; 0100 hr) than in the bolus group (Figure 2B), thus mirroring the data for unconjugated BPA-d6 (Figure 2A).When total BPA-d6 concentra- tions for diet exposure were scaled to the oral bolus dose [i.e., (20 mg/kg ÷ 6.5 mg/kg) × 802.2 ng/mL], the estimated peak con- centration for the diet-exposed group was 2468.3 ng/mL. Total BPA-d6 concentra- tions declined after 6 hr in the diet-exposed group (Figure 2B). Concentrations of total serum BPA-d6 at 24 hr and 7 days in the diet-exposed group were not significantly different (193.9 ± 44.4 ng/mL vs. 359.6 ± 64.23 ng/mL; p > 0.05). Assuming that total BPA-d6 is formed only by a hepatic first-pass effect (Dekant and Volkel 2008), the AUC for total BPA-d6 reflects the extent of BPA-d6 absorption, whereas the amount of unconjugated BPA-d6 that reaches systemic circulation after escaping hepatic first-pass metabolism reflects the extent of BPA-d6 bioavailability. The AUC0–24hr for diet exposure and bolus administration at the same external dose were 11547.3 ± 1219.5 and 21979.3 ± 3813.5 ng-hr/mL, respectively, indicating a relative absorption of 81% for the diet group. Thus, less total BPA-d6 was absorbed after diet exposure than after oral bolus administration. Consequently, the higher bioavailability associated with diet exposure (113%) is presumably explained by postabsorption events rather than by increased BPA-d6 absorption. Discussion Previous studies have demonstrated that exposure to BPA through diet can induce epigenetic and behavioral changes in mice (Cox et al. 2010; Dolinoy et al. 2007), sug- gesting that exposure to BPA has measurable biochemical and phenotypic effects in ani- mals. However, to our knowledge, the pres- ent study is the first to quantify serum BPA concentrations in any species after exposure through the diet. We chose an external expo- sure of BPA that was lower than the lowest observed adverse effect level of 50 mg/kg BW/ day reported in rodents (Cox et al. 2010) but still within the detection limit of the assay that would yield accurate measurements. Table 1. Pharmacokinetics parameters of BPA‑ d6 obtained after NCA of unconjugated and total BPA‑ d6 serum concentrations in mice over a 24‑hr period after exposure via oral bolus or diet. Oral bolus (20 mg/kg BW) Pharmacokinetic parameter Unconjugated Total Cmax (ng/mL) 21.0 ± 3.9 1636.5 ± 642.6 AUC0–24hr (ng.hr/mL) 201.0 ± 20.6 21979.3 ± 3813.5 Average 24-hr concentration (ng/mL) 8.3 915.8 AUC0–24hr total/unconjugated BPA-d6 109 aEstimated ingested dose based on food consumption. Diet (13 mg/kg BW)a Unconjugated Total 18.8 ± 4.4 802.2 ± 126.6 147.8 ± 26.7 11547.3 ± 1219.5 6.1 481.1 79 Environmental Health Perspectives • volume 119 | number 9 | September 2011 1263 8 12 Time (hr) 16 20 c a,b b,c b Oral bolus Diet exposure c a 24 a,b a 24 25 b 6 -PBAd /)Lg 20 m rum (sn 15 e n tsdae o titra 10 n jnconug conce 5a a U 0 0 3,000 ) -d6 /Lm PA g (n2,000 B s rum tion a e r s tn1,000 ltaTo enoc c b,c 0 a a 0 c c b,c c a,b 4 b 4 b a,b Dark 8 12 Time (hr) 16 20 Light 1900 2300 0300 0700 1100 1500 1900 Dark/light cycle Figure 2. Arithmetic plot of the mean (± SE) serum concentrations of unconjugated BPA‑ d6 (A) (and the total BPA‑ d6 serum concentrations (B) ver‑ sus time and versus night/day cycle after a sin‑ gle oral bolus of BPA‑ d6 at 20 mg/kg BW or after exposure to BPA‑ d6 at 100 mg/kg feed weight (13 mg/kg BW in the first 24 hr; see Figure 1) in mice. For the oral bolus, Cmax for unconjugated BPA‑d 6 and total BPA‑ d6 occurred 1 hr after the treatment (2000 hours). In contrast, Cmax was not obtained until 6 hr after treatment (0100 hours) in the diet‑ exposed group. Within each treatment group, values with different superscripts are sig‑ nificantly different (p < 0.05).