Sieli et al. > 215 m/z for BPA-d6 and 241 m/z > 223 m/z for BPA-d16. Hence, the method was not only sensitive but specific and accurate for the compounds analyzed. Although the spacing of sampling times did not permit precise pharmacokinetic pro- files to be assessed, it was possible to com- pare the consequences of ingestion of BPA-d6 as a bolus versus ad libitum consumption in the food, which was the primary goal of the study. Most studies on the effects of BPA in rodents or in nonhuman primates and humans have used a single bolus adminis- tration (Doerge et al. 2010a, 2010b; Taylor et al. 2011). We have shown that BPA-d6 is rapidly metabolized to one or more conju- gated forms and that both free and conjugated forms are cleared fairly rapidly from serum after exposure through diet as well as after oral bolus administration. The terminal half- life value for disposition of the unconjugated form of BPA-d6 after bolus administration was approximately 6 hr, and thus by 24 hr this group had low circulating concentrations of the substance. An important finding from our experi- ments is that the bioavailability is higher after diet administration than after bolus adminis- tration of BPA-d6, despite evidence of lower relative absorption after diet administration. This finding may be tentatively explained by a “food-effect,” which has been previously described for several highly extracted drugs (Tam 1993; Wilkinson 1997). Specifically, it has been hypothesized that food transiently inhibits the intrinsic ability of the liver to metabolize highly extracted substances (i.e., chemicals that are preferentially metabolized by the liver), particularly during the absorption phase (Tam 1993; Wilkinson 1997). Thus, consumption of BPA-d6 in food increased its internal bioavailable concentrations, despite lower BPA-d6 absorption, relative to oral bolus exposure. When the treatments were initiated at the beginning of the dark phase (1900 hours), neither oral bolus nor diet- exposed groups had commenced their noc- turnal feeding. After treatments were initi- ated, both groups were fed the same amount of a diet that was identical except for the addi- tion of BPA-d6 [see Supplemental Material, Tables 1 and 2 (http://dx.doi.org/10.1289/ ehp.1003385)]. It is unlikely that the very small volume of corn oil used for oral bolus administration affected the results, particu- larly because maximum concentrations (Cmax) also occurred 1 hr postadministration in mice provided an oral bolus of BPA-d6 during the light phase (versus the dark phase) of the cycle (Taylor et al. 2011). Moreover, although the scaled Cmax (57.9 ng/mL) after administration of BPA-d6 in the diet was almost three times higher than the Cmax after oral bolus expo- sure (21.0 ng/mL), the scaled AUC for diet 1264 versus oral bolus exposure was only increased by about 13%. This difference between Cmax and AUC is consistent with a transient food effect increasing Cmax but not AUC, which was calculated over a 24-hr period (i.e., before and after the Cmax period). These findings of the effect of food on BPA absorption might account for the spurious or dramatic varia- tions in peak plasma concentrations observed in human biomonitoring studies (Vandenberg et al. 2010b). Our finding that diet exposure resulted in increased serum concentrations of active BPA-d6 is relevant to animals and humans, where a significant portion of the total expo- sure to BPA is believed to occur through diet (Matsumoto et al. 2003). Recently, Teeguarden et al. (2011) demonstrated that in humans there is considerable inter- and intrameal vari- ability in BPA urinary excretion, consistent with an estimated range of exposure from 3.29 to 73.29 μg. However, these findings might also be explained by a food effect, the inhibi- tory effect of food on first-pass hepatic metabo- lism. In addition, BPA exposure through food consumption is experimentally more conve- nient; because it is less stressful than other routes of administration, it may provide a more relevant pharmacokinetic profile by reducing stress-related confounds. Another potential noteworthy finding is possible bioaccumulation of free BPA when BPA-d6 is provided through the diet, which has not been observed when BPA is admin- istered as a single bolus (Doerge et al. 2010a; Taylor et al. 2011). For example, in the 24-hr experiment, concentrations of free BPA-d6 were significantly higher in the diet-exposed group than in mice given BPA-d6 as a single bolus. In addition, concentrations of unconju- gated or active BPA-d6 in the diet group were higher after 7 days of dietary exposure than at 24 hr after exposure. One untested explana- tion is that chronic exposure to BPA might eventually compromise metabolizing capacity of the liver, as suggested previously (Hanioka et al. 2008), leading to progressively elevated concentrations of active BPA. Variation in animal feeding habits might also account for differences between diet compared with bolus exposure, although there is no reason to pre- sume that mice would exhibit different feeding patterns after 7 days versus 1 day of BPA-d6 exposure, particularly as the samples were col- lected at the same time of day (1900 hours). Another consideration is that the stage of the estrous cycle might influence BPA metabolism and accumulation. However, in a previous study Nepomnaschy et al. (2009) suggested that menstrual cycle stage did not influence urinary BPA concentrations in samples from 60 women taken 2 and 4 weeks apart. In short, the reason why circulating unconjugated BPA-d6 increased over time after diet exposure eludes us, but studies are currently under way with radioactive BPA to determine where the ingested BPA becomes concentrated. Conclusions Our data highlight possible limitations of single oral bolus administration of BPA, the experimental design used for the major- ity of studies examining the pharmacokinetics of BPA exposure in both animal models and humans (Doerge et al. 2010a, 2010b; Taylor et al. 2011; Vandenberg et al. 2009). Results of the present study suggest that the presence of food may increase internal exposure to bio- active BPA, possibly by an inhibitory effect on first-pass (presystemic) elimination, and thus diet exposure is presumably the more relevant way of modeling the natural route of contact to BPA that occurs in humans. In contrast, experiments using single oral bolus exposure may not only underestimate exposure to bio- active BPA in serum but also lead to inaccurate conclusions concerning long-term concentra- tions of active BPA in serum or plasma of ani- mals and humans. Our data may explain how although humans can rapidly eliminate BPA when it is provided as a single bolus (Volkel et al. 2002), continuous external BPA exposure appears to lead to sustained concentrations that are detectable in serum or plasma of humans who have not been knowingly exposed to this endocrine-disrupting chemical (FDA 2008; Vandenberg et al. 2007; vom Saal et al. 2007). We conclude that exposure through the diet provides a better approach for assessing the impact of BPA on internal organ systems than delivery as a single bolus. Ref Rencs Bailin PS, Byrne M, Lewis S, Liroff R. 2008. Public Awareness Drives Market for Safer Alternatives: Bisphenol A Market Analysis Report. 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