Using in vitro and in vivo Placental Models to Investigate the Localization of Brominated Flame Retardants and Implications for Thyroid Hormone Homeostasis

Thyroid hormones (TH) are essential for fetal growth, neural development, tissue differentiation, metabolism and overall maintenance during pregnancy (Haddow 1999). When circulating levels of TH in maternal serum are disrupted, there is an increased risk for adverse pregnancy outcomes, such as intrauterine growth restriction (IUGR) (Kilby et al. 1998; Loubière et al. 2010; S. Y. Chan et al. 2006). Previous studies have observed significant differences in TH levels and regulation with IUGR, suggesting TH disruption may be a causative factor for IUGR. Although it is not clear what may be driving TH dysregulation, it is possible that exposure to contaminants including persistent organic pollutants (POPs) may be involved. Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (FR) that were introduced in the 1970s, and which have been heavily used to meet flammability requirements in furniture, electronics, and building materials. Although Penta- and OctaBDEs were voluntarily phased out in 2005, they are highly persistent, bioaccumulate and are susceptible to global transport, resulting in ubiquitous human exposure (Tay et al. 2017). The third and last PBDE mixture, DecaBDE, was phased out in 2013, and is also persistent and ubiquitous in the environment. These POPs are of toxicological concern because of their neurotoxicity and ability to disrupt the endocrine system (Linares, Bellés, and Domingo 2015). There are several epidemiological studies that have found significant associations between prenatal PBDE exposure and lower birth weight; however, the mechanisms are largely unexplored (Lopez-Espinosa et al. 2015; Harley et al. 2011). To better understand potential mechanisms of PBDE mediated thyroid hormone dysfunction, in vivo animal models, in vitro cell culture systems and archived human placental tissue were used. In Aim 1, pregnant Wistar rats were orally dosed with a mixture of PBDEs for 10 days. Dams were sacked and maternal serum, placenta and fetal tissue was collected and quantified for PBDEs. The placenta samples were dissected to isolate fetal and maternal placental tissue to understand tissue-specific accumulation. Furthermore, we investigated tissue-specific mRNA expression of several transporters in each portion of the placenta. We found PBDE concentrations in the fetal portion of the placenta to be approximately two times greater than concentrations quantified in the maternal portion of the placenta, despite no differences in lipid or protein content. We observed a significant upregulation of ATP-binding cassette transporter proteins ABCC1 and ABCC5 in the maternal portion of the placenta in dosed female fetuses relative to controls. We observed significant upregulation of ABCC4 and nearly significant upregulation of the thyroid hormone solute carrier transporter MCT8 in the fetal portion of the placentas in dosed male fetuses relative to controls. This is the first study to investigate tissue-specific accumulation of pollutants in the placenta and the first study to observe sex-specific differences in placental transporter gene expression. This aim also investigated the tissue-specific accumulation of POPs in crudely isolated maternal and fetal placental tissue to evaluate whether the tissue-specific accumulation of POPs in the rat model were also reflected in human tissue. We observed a significantly greater accumulation of PBDEs and pesticides in the fetal portion of the placenta relative to the maternal portion, and at ratios similar to those observed in the rat model. These results suggest the rat may be a relevant model for future investigations related to placental transfer and accumulation of POPs. In Aim 2, dam serum, maternal and fetal placental tissue and fetal tissue from the animal study in Aim 1 were used to investigate the effect of PBDEs on birth weight and TH levels in each matrix. Additionally, this aim sought to understand how placental tissue-type and gestational age influence TH levels in the Wistar rat model. We observed a significant increase in T3 levels in maternal serum from dosed dams harvested on gestational day (GD) 14/15, but not GD 12/13. Although we did not detect a significant effect of dose on TH levels in the placenta, we did observe significantly higher concentrations of T3 and T4 in the maternal portion of the placenta, relative the fetal portion. Furthermore, control and dosed maternal placental tissue from GD 14/15 had significantly higher levels of T3 relative to control and dosed maternal placental tissue from GD 12/13. Our data suggested an increase in T3 levels in fetal tissue from dosed females and a decrease in T3 levels in fetal tissue from dosed males, however, these differences were not statistically significant. In Aim 3, BeWo b30 cells, a model for placental barrier function, were cultured on Transwell inserts to assess the placental transport of two commonly detected flame retardants, BDE-47 & -99. Cells were incubated with and without inhibitors of efflux transporters to understand the transporters’ role in the placental transfer of BDEs. We observed a significant decrease in the transport of BDE-99 across the placental barrier in the apical to basal direction when efflux transporters were inhibited. We saw a weakened and non-significant effect on BDE transport in the basal to apical direction when efflux transporters were inhibited. We hypothesize that BDEs are not primarily transported across the placental barrier via ATP-binding cassette transporters. Instead, the inhibition of efflux transporters may indirectly affect the function and activity of solute-like carrier transport proteins that share binding substrates. Future research is needed to understand the role of solute-like carrier transport proteins in the placental transfer of xenobiotics. In Aim 4, we evaluated temporal changes in placental POP accumulation and associations with birthweight and health of the infant at delivery. Placental tissues (n=99) previously collected and archived at the Duke University Medical Center from 2009-2015 were analyzed for 22 POPs using gas chromatography mass spectrometry. The mean age of mothers was 30.6 years, 8% of newborns were characterized as low birthweight (<2500 g), and 25.3% of deliveries suffered from an adverse event, such as preeclampsia, fetal growth restriction, placenta previa, and premature rupture of membranes. Of the 22 POPs targeted in the analysis, only p,p’-DDE, BDE-47 and BDE-100 were detected in more than 50% of the samples, with median concentrations of 0.110, 0.310, and 0.033 ng/g wet weight, respectively. Temporal analyses indicated that concentrations of BDE-47 significantly decreased over these years. Placental tissue from female infants had significantly higher levels of BDE-100 than males. And among female infants, higher levels of placental POP accumulation were associated with adverse deliveries, suggesting gestational exposure to POPs may play a role in adverse pregnancy outcomes. Placental POP concentrations were not associated with birthweight. Our data indicate placental PBDE concentrations are generally decreasing over time; furthermore, these data suggest that there may be a sex-specific association between gestational exposure to POPs and adverse pregnancy outcomes. This was the first study to (i) quantify tissue-specific accumulation of PBDEs in the placenta; (ii) quantify tissue-specific thyroid hormone levels in the placenta; (iii) quantify thyroid hormone levels in matched maternal serum, placenta, and fetus; (iv) detect sex-specific differences in placental transporter gene expression; (v) observe an increase in BDE-47 and -99 transport when MRP transport proteins were inhibited; and (vi) quantify TBB and TBPH concentrations in human placental tissue. Future research is needed to understand the mechanisms driving tissue-specific POP accumulation and sex-specific differences in adverse pregnancy outcomes.