Serum BPA concentrations in diet-exposed mice conclude that unconjugated BPA is so rapidly metabolized and/or cleared that it is relatively harmless to humans (Dekant and Volkel 2008; Willhite et al. 2008). Rodent models have been criticized as inappropriate to calculate human BPA expo- sures based on the prediction of species dif- ferences in metabolism of BPA (Dekant and Volkel 2008), although this conclusion was disputed by an expert panel of the Food and Agriculture Organization of the United Nations and the World Health Organization (2010). Although both adult rodents and pri- mates use glucuronidation of BPA through uridine 5´-diphospho-glucuronosyltransferases as part of their phase II metabolism system, the primary mechanism through which BPA is cleared from blood differs, with urinary excre- tion being the primary route in primates ver- sus the biliary–fecal route in rodents (Inoue et al. 2005; Sakamoto et al. 2002). BPA might also be metabolized by sulfonation, but this form accounts for only a very minor com- ponent across various adult animal species (Balakrishnan et al. 2010; Kurebayashi et al. 2003; Pottenger et al. 2000). Enterohepatic recirculation of BPA is less prominent in pri- mates than in rodents, but even in rats and mice, enterohepatic recirculation is not a major factor (Doerge et al. 2010a; Taylor et al. 2011). A recent study that performed a side-by-side analysis of the serum concentrations of BPA in CD1 mice and rhesus monkeys (Macaca mulatta) that had, in each case, received an oral bolus of the chemical concluded that the clearance of unconjugated BPA over 24 hr was comparable in the two species (Taylor et al. 2011) and similar to what had been observed in a second macaque species, Macaca fascicu- laris (Tominaga et al. 2006). Thus, by these criteria, mice appear to be an acceptable ani- mal model to predict the pharmacokinetic of BPA in nonhuman primates and potentially, by extrapolation, in humans (Gies et al. 2009; Taylor et al. 2011). Both in the single human study by Volkel et al. (2002) and in the various tri- als performed on nonhuman primates and rodents, serum concentrations of conjugated and unconjugated BPA have invariably been measured after administering a single dose of the chemical as either an oral bolus or via subcutaneous injection (Doerge et al. 2010a, 2010b; Taylor et al. 2011; Vandenberg et al. 2009), whereas continuous exposure through the diet seems more likely to mimic exposures outside the laboratory. Moreover, dietary BPA exposure would be a more appropriate and convenient route than bolus exposure for studies of the developmental effects of in utero BPA exposure on offspring and adults. Although pregnant mice fed a diet containing BPA have given birth to offspring with epi- genetic (Dolinoy et al. 2007) and behavioral abnormalities (Cox et al. 2010), neither study measured serum concentrations of BPA dur- ing pregnancy. Finally, it remains controver- sial whether the quantities of BPA supplied to mice in these studies reflect exposure levels that might be expected to occur outside of the laboratory. In the present study, we measured serum concentrations of BPA in mice exposed through natural feeding behavior to more pre- cisely characterize circulating concentrations resulting from dietary exposure and compared concentrations with those in mice exposed through single oral bolus exposure, as in pre- vious studies (Doerge et al. 2010a, 2010b; Taylor et al. 2011). Instead of using BPA, we employed the isotopically tagged form, [dimethyl-d6]-BPA (BPA-d6; C/D/N Isotopes Inc., Quebec, Canada), to ensure that only the BPA provided experimentally was assayed. Materials and Methods Animals. All animal experiments were approved by the University of Missouri Animal Care and Use Committee and per- formed in accordance with National Institutes of Health animal care and use guidelines (Institute of Laboratory Animal Resources 1996). All animals were treated humanely and with regard for alleviation of suffering. Adult (10–12 weeks of age) C57Bl/6J female mice were purchased from Jackson Labs (Bar Harbor, ME, USA). On arrival, animals were placed on AIN93G diet (Harlan Teklad, Madison, WI, USA) [see Supplemental Material, Table 1 (http://dx.doi.org/10.1289/ ehp.1003385)], and their food consump- tion and body weights (BWs) (mean ± SE, 19.1 ± 0.5 g) were measured daily. Mice were maintained on a 12-hr dark:12-hr light cycle with lights out at 1900 hours. To minimize background BPA exposure, mice were housed in polypropylene cages and provided glass water bottles. Their water was stringently puri- fied by a reverse osmosis and carbon filtration system and did not contain detectable BPA. BPA‑ d6 treatments. Based on the widely accepted studies demonstrating that BPA dis- position is linear over a wide range of doses, including in humans (Doerge et al. 2010a; Taylor et al. 2011; Teeguarden et al. 2011; Vandenberg et al. 2007), our experiments were confined to a single test dose that would provide detectable and accurate measure- ments for mice exposed to BPA-d6 through diet or oral bolus exposure. At 1900 hours (the beginning of the dark cycle), one group of nonfasted mice that had not yet initi- ated their nocturnal feeding received a single oral bolus of 20 mg/kg BW BPA-d6 in an exceedingly small volume (40 Lμ) of tocoph - erol-stripped corn oil (MP Biomedicals, LLC, Solon, OH, USA). These mice continued to receive AIN93G diet after exposure. This oral bolus dose was chosen to approximate Environmental Health Perspectives • volume 119 | number 9 | September 2011 the amount of BPA-d6 that mice on a diet containing 100 mg BPA-d6/kg feed weight [see Supplemental Material, Table 2 (http:// dx.doi.org/10.1289/ehp.1003385)] had been predicted to consume in a 24-hr period based on preliminary data and data from another published study using the same strain of mice (Cox et al. 2010). For the BPA-d6 diet–exposed group, the food placed at the beginning and remaining at the end of each time point was weighed to calculate the amount consumed. Each mouse was weighed immediately before it was killed for blood collection. Two to four mice for each time point and group were caged together to reduce space. To account for the varying numbers of mice in the cage, each cage was considered a unit, and the total con- sumption for each cage was divided by the average weight of the mice in this cage. Serum samples from mice that had not been administered BPA-d6 were col- lected from females (n = 12) maintained on AIN93G diet beginning at 1900 hours, which is when mice normally begin nocturnal feed- ing at the beginning of the dark phase of the dark:light cycle (Kavaliers et al. 1985). Representative mice from the diet and oral bolus groups (n = 8 for each time point) were culled and cardiac blood was collected 1 hr, 4 hr, 6 hr, 11 hr, 24 hr, and 7 days (168 hr) after initiating exposure to BPA-d6 at 1900 hours. For additional information on BPA-d6 treatments, see Supplemental Material, p. 3 (http://dx.doi.org/10.1289/ehp.1003385). Analysis of unconjugated and conjugated BPAd‑ 6 in mouse serum samples. BPA-d6 serum concentrations were measured as described previously (Padmanabhan et al. 2008) but with some modifications. The mouse serum samples were divided into two aliquots (each of 150–200 μL) for the analysis of free and total BPA-d6, respectively. A reference stan- dard [5 ng deuterated 16-BPA (BPA-d16)] was included as a quality control to validate the method, as the inclusion of BPA-d16 into the test serum samples served as an internal con- trol to estimate recovery through the analytical steps. Analyte separation and detection were carried out by using an Agilent 1100 series HPLC interfaced with an Applied Biosystems API 2000 electrospray MS/mS mass spectrom- eter (Applied Biosystems, Foster City, CA, USA). Additional information is provided in Supplemental Material, pp. 3–5 (http://dx.doi. org/10.1289/ehp.1003385). Statistical analysis. We used analysis of variance and SAS software (version 9.2; SAS Institute Inc., Cary, NC, USA) to analyze differences in serum concentrations of uncon- jugated and total BPA-d6 within diet and oral bolus groups. However, because there were heterogeneous variances, tests for significance were performed after log10 transformation. 1261