In the current study, we report dose-dependent phenotypic and epigenetic responses following maternal dietary exposure to three levels of BPA. First, we observed a decrease in d22 wean body weight in a/a
offspring exposed to 50 ng/kg diet of BPA versus control offspring. This association persists when analysis is restricted to a/a
offspring alone, indicating that this effect is not associated with the epigenetically controlled adult onset obesity associated with Avy/a
offspring, but rather manifests as a result of perinatal BPA exposure. Both low-birth weight in humans and early BPA exposure in animal models have been correlated to adult onset obesity [Barker, 2004
; Heindel et al., 2009
]; hence, it will be of interest to further evaluate early BPA exposure as a potential obesogen in adulthood. Body weight differences were not detected in offspring exposed to either 50 μg or 50 mg BPA/kg diet indicating a nonmonotonic dose response of wean weight and, corroborating previous studies using multiple doses of BPA with nonlinear outcomes [Rubin et al., 2001
; Honma et al., 2002
]. Mechanisms of action supporting nonmonotonic effects BPA, and endocrine disruptors in general, should be further investigated.
Global methylation of the mouse genome assessed using the LUMA assay reveals a significant increase in methylation across all BPA exposure groups in comparison to controls. This assay provides a measure of methylation at CCGG sites throughout the entire genome regardless of location, representing the degree to which the genome is globally methylated. The LUMA assay has been extensively used in analysis of human cancers [Lee et al., 2008
; Deneberg et al., 2010
; Poage et al., 2011
]. There are limited studies, however, exploring environmental and/or nutritional exposures and their impact on global methylation [Gallou-Kabani et al., 2010
]. Recently, using a mouse model, Gallou-Kabani et al. 
associated maternal high fat diet with decreased placental tissue DNA methylation in female offspring. The global decrease in CCGG methylation was not associated with decreased methylation at LINE-1 or B1 repetitive elements. Gene specific methylation at the Igf2r
gene in female offspring exposed to high fat diet, on the other hand, was increased. Thus, it is important to note that the LUMA assay is restricted to methylation of CCGG sequences throughout the genome and is not necessarily representative of environmentally induced local changes at candidate genes or repetitive content derived from transposable elements such as LINE-1 and B1.
We also note dose-dependent shifts in the coat color distribution of genetically identical Avy/a
offspring exposed to a 50 mg, μg, or ng/kg diet of BPA perinatally. The coat color distribution of offspring exposed to a 50 mg/kg diet of BPA displays a shift toward the yellow obese phenotype, reproducing our 2007 single dose study results [Dolinoy et al., 2007
], whereas the μg dose offspring displays a shift toward the pseudoagouti lean coat color phenotype. Average methylation at the Avy
locus of the mg exposed offspring is significantly decreased in comparison to the control group, providing epigenetic validation of the coat color distribution shift. In contrast, average methylation at the Avy
locus of the μg exposure group was not statistically significant. An excess of categorization as slightly mottled Avy/a
offspring may have offset a hypermethylation response among the μg exposure group when compared with the control group. Increased methylation in μg offspring compared with control offspring was, however, detected at the CabpIAP
metastable epiallele, signifying that perinatal exposure to BPA at this dose increases methylation at this particular epigenetically labile locus. Taken together, these results (1) indicate that methylation at more than one locus is variable after perinatal exposure to BPA, (2) strengthen the evidence for nonmonotonic dose-dependent effects of BPA, and (3) provide evidence that variable dose levels of BPA act across different biological pathways [Vandenberg et al., 2009
]. Genome-wide methylation and transcriptomics investigation should now be considered in light of this evidence.
Because of BPA’s ubiquitous existence in the environment and the ongoing debate about whether human internal BPA levels pose a health concern [Volkel et al., 2002
; Ginsberg et al., 2009
], it is of significance that animal studies capture human physiologically relevant exposure levels to determine BPA’s impact on human health outcomes. In the present study, we aimed to achieve physiologically relevant levels by including a high (mg), medium (μg), and low (ng) dose of BPA in the maternal diet. Calafat et al. 
reported a range of urinary total BPA (free and conjugated) of 0.4–149 ng/mL representative of individuals 6 years of age or older (n
= 2,517) in subjects measured as a part of the 2003–2004 National Health and Nutrition Examination Survey (NHANES). Lang et al. 
reported urinary total BPA levels ranging from 3.34 to 4.48 ng/mL in individuals aged 18 years or older who have normal BMI (n
= 469) from the 2003–2004 NHANES. Additionally, Padmanabhan et al. 
measured a range of 0.5–22.3 ng/mL of circulating free BPA (unconjugated) in maternal blood collected upon delivery.
We report d22 mouse liver BPA measurements ranging from below the limit of quantitation (LOQ) to 870 ng/g across all exposure groups comparable to human fetal liver measurements ranging from below LOQ to 96.8 ng/g. Ideally, liver BPA levels would be measured in fetal mouse tissues and compared with developmentally matched human fetal tissues; however, study design and current analytical requirements preclude this direct comparison. Nonetheless, mouse liver total and free BPA levels among Avy/a
offspring exposed to 50 mg BPA/kg diet in our study range from 9.46 to 870 and 2.68 to 390 ng/g, respectively, () and are comparable to mouse circulating serum BPA concentrations recently reported in adult mice exposed to 100 mg BPA-d
6/kg diet ad libitum
for a 24-hr period [Sieli et al., 2011
]. Sieli et al. 
show that in comparison to mice receiving a single oral bolus exposure of 20 mg BPA/kg body weight, animals fed BPA in the diet reach a maximum serum concentration of total and unconjugated (free) BPA at 6 hr of 802 and 18.8 ng/mL, respectively compared with 1 hr in the bolus group. Moreover, the observed serum concentrations following BPA administration in the diet are within the range of human exposure. Within the current study, we also observe a high degree of interindividual variation in mouse liver BPA concentrations within a particular dose group as well as the controls, perhaps reflecting time and metabolism effects associated with recent feeding bouts and/or continued nursing of pups. Unlike single bolus ingestion or injection routes of exposure, dietary exposure through feed results in inherent interindividual variability. It is important to note that we do not see a profound difference in mean or median mouse liver BPA concentrations among control and the two low dose groups; in fact, BPA is detected in some control animals despite housing in BPA-free caging and receiving BPA-free water. A limitation of this study is possible BPA cross-contamination via air given that animals were housed in a single room to minimize environmental heterogeneity that contributes to underlying epigenetic lability.
To our knowledge, this is the first study conducted using the viable yellow agouti epigenetic biosensor to analyze offspring phenotypic and epigenetic effects following multiple dose levels of either an environmental exposure or nutritional agent. Isogenic Avy
mice allow for reproducible experiments as seen here with the coat color shift toward yellow in the Avy/a
offspring exposed to the 50 mg BPA/kg diet [Dolinoy et al., 2007
]. We also took a candidate gene approach limited to metastable epialleles unique to murine models and a global CCGG sequence assay. In order to conduct an unbiased epigenetic analysis, genome-wide methylation experiments must be applied in animal models. Epigenome-wide approaches will generate a template useful for the foundation and understanding of the full effect of BPA on the mouse epigenome. Concurrent studies are needed to assess BPA’s effect on the human epigenome, and whether labile loci between the mouse and human display significant overlap. In understanding the epigenome as a whole, one must also consider other epigenetic mechanisms such as histone modifications and microRNA interference separately and in conjunction with each other, and their sensitivity to environmental disruptions. Recently, DNA methylation and histone modifications have been observed to act in concert with one another at the Avy
metastable epiallele [Dolinoy et al., 2010
]. Increasing studies focusing on multiple epigenetic mechanisms will strengthen the understanding of environmentally induced alterations to the epigenome.