We originally embarked on studies of environmental estrogens in asthma pathogenesis [17
] based on extensive epidemiological evidence that there are major differences in asthma susceptibility between men and women during different life stages, with males predominating (male: female ratio = 3:1) from infancy to age 14 and females predominating (1:3) from ages 18 to 34 [19
]. Further, hormone replacement therapy (especially with estrogens) in women enhances both new onset [20
] and worsening of previously quiescent asthma [21
], suggesting that estrogenic agent may play a role in the gender difference in asthma prevalence. A molecular basis for BPA's effects on immune events in asthma was suggested by our and our colleagues' finding that immune cells, including mast cells and lymphocytes from humans and mice, expressed estrogen receptor (ER)α, but not ERβ [8
]. Expression of ERα was required for environmental estrogens, including nonylphenol, an alkylphenol, to enhance basophil degranulation [18
]. BPA also has a high affinity to estrogen-related receptor (ERR)γ [22
], but ERRγ has yet to be described on any immune cells. We also detected G-protein coupled receptor 30 (GPR30) on mast cells (unpublished observation from Dr. Peter Thomas). Androgen receptor (AR) was detected by others on mast cells and lymphocytes [23
]. Aryl hydrocarbon receptor (AhR) was also detected on lymphocytes, but not on mast cells, but the relationship between AHR, ERs and estrogens is quite complex.
In our previous study, we demonstrated that pups that were born to and nursed by dams that were exposed to BPA had an enhanced propensity to develop asthma after an intentionally "suboptimal" allergic sensitization during infancy [11
]. In the current study, we transferred some pups from BPA-loaded birth mothers to unexposed foster mothers and vice versa, within 48 h after their birth [14
]. These transfers created four groups of pups that allowed us to test our hypothesis that the prenatal exposures to BPA were required to promote the development of allergic asthma in our mouse model. Consistent with this hypothesis, only the pups exposed to BPA prenatally demonstrated enhanced airway inflammation and hyperreactivity. These results strongly suggest that prenatal life is a critical period, during which BPA exposure promotes the development of asthma. However, as we previously observed [11
], mice that are exposed to BPA, but not sensitized to OVA did not develop an asthma phenotype, suggesting that prenatal BPA exposure enhances the allergic component of asthma pathogenesis.
We chose the concentration of BPA to feed dams based on a previous report that this dose, fed for a similarly short period of time, produces a BPA burden (internal dose) with a mean of 10-25 ng/g tissue in brain, kidney, liver and testis, as measured by GC-MS analysis [12
]. These burdens are similar to those in human samples, which contained BPA at concentrations of up to 104.9 ng/g tissue [26
]. To confirm the relevance of this dose in our mouse model to environmental BPA exposures of humans, we quantified total BPA concentrations in the sera of pups that were exposed both pre-and postnatally to maternal BPA. The range of BPA concentrations in the sera of the mouse pups 29-39 ng/ml was only slightly greater than the upper range found in two studies of plasma BPA in pregnant women (0.5-22.3 and 0.3-18.9 ng/ml) from the US and Germany [26
]. The large variability of BPA concentrations in human cord blood, likely due to different levels of exposures of their mothers, suggests that some infants may have in utero BPA burdens similar or greater than those of our experimentally-exposed mice.
Next, we tested the hypothesis that delayed development of BPA metabolizing enzymes in the fetus enhances fetal exposure to transplacental, unconjugated (estrogenic) BPA and its immunotoxicity. Steroid hormones, including environmental estrogens like BPA, are largely metabolized by glucuronidation by UGT and then excreted through the urine [28
]. The mammalian UGT gene superfamily currently has 117 members that can be divided into four families, UGT1, UGT2, UGT3 and UGT8 [29
]. The UGT1A group share homology between species, but the UGT2B group is only homologous between rats UGT2B1 and mice Ugt2b1. In human liver, UGT2B15 showed the highest activity for BPA glucuronidation at low (1.0 μM ≈ 0.2 ng/ml) and high (20 μM ≈ 5 ng/ml) substrate concentration, while UGT1A1, UGT1A3, UGT1A9, UGT2B4 and UGT2B7 were also capable of catalyzing BPA glucuronidation, though only at higher concentration of BPA (20 μM) [30
]. In rat liver, BPA is mainly glucuronidated by UGT2B1 [31
]. Because rat UGT2B1 shares homology with mouse Ugt2b1, it is likely that BPA is mainly glucuronidated by Ugt2b1 in mouse liver. Thus, we assessed the expression of Ugt2b1 in mouse fetuses as a possible mechanism of BPA's enhancement of asthma development in mouse pups. Another isoform Ugt1a1, a major enzyme for glucuronidation of bilirubin in newborn rats, was used as a control for hepatic expression of Ugts. Our results indicated that the hepatic expression of Ugt2b1, the isoform related to BPA clearance in rats [16
] was very low throughout the fetal period in our mice, but increased rapidly after birth. It is possible that this postnatal increase in Ugt2b1 expression may have protected the pups from postnatal effects of BPA-exposure. The expression of Ugt1a1, the main bilirubin-conjugating isoforms developed more rapidly in utero and reached near adult levels at birth, indicating that the expression of some isoforms are easily detected in the fetal livers of the mice we studied. Together these results are consistent with the hypothesis that unconjugated BPA may accumulate during the prenatal period and act as an endocrine disruptor that alters the course of the rapid development of the immune system.
The relevance of these metabolic findings in our mouse model to BPA exposure of the human fetus and infants has yet to be tested directly. However, expression of Ugt2B15 [30
], which is responsible for glucuronidation of BPA in human liver was not detected at 20 weeks of gestation [32
] and glucuronidation of another estrogen, estrone, by fetal and neonatal human liver microsomes was approximately 30% of adult activity [33
]. Furthermore, high level expression of β-glucuronidase enzymes in the human fetus and placenta, which can deconjugate BPA glucuronide [34
], could also enhance free BPA concentrations in mothers and their fetuses. Sulfotransferase (SULT)1A1, a human enzyme that also metabolizes BPA [35
] was detected in human liver from early gestation [36
]. However, high level expression of sulfatases and β-glucuronidase enzymes, which deconjugate BPA glucuronide and sulfate are also present in the human fetus and placenta [34
], which could increase the concentration of free BPA in mothers and their fetuses. Understanding the dynamics of BPA in the placenta, fetus and neonate will require careful quantification of BPA and its conjugates in relevant tissues and fluids.
While the low level of prenatal expression of Ugt2b1 we describe here could allow accumulation of unconjugated BPA in the pups, and thereby, enhance BPA's effects on the development of asthma, other differences between pre-and postnatal exposures could also be involved. For instance, the route of BPA exposure (systemic for fetus vs. intestinal for infants), maturation state of the immune system and susceptibility to epigenetic alteration, alone or induced by high level exposure to maternal hormones might also enhance the impact to prenatal BPA exposures on the development of allergic asthma. Similarly, the concentration of BPA in the dam's milk may have limited the BPA exposure in the pups which did not receive maternal BPA prenatally.