Browsing by Subject "Flame retardants"

Accumulating evidence is suggesting that exposure to some environmental contaminants may alter adipogenesis, resulting in accumulation of adipocytes, and often significant weight gain. Thus these types of contaminants are often referred to as obesogens. Many of these contaminants act via the activation (i.e. agonism) of the peroxisome proliferator activated receptor γ (PPARγ) nuclear receptor. To date, very few chemicals have been identified as possible PPAR ligands. In the thesis, our goal was to determine the PPARγ ligand binding potency and activation of several groups of major semi-volatile organic compounds (SVOCs) that are ubiquitously detected in indoor environments, including flame retardants such as polybrominated diphenyl ethers (PBDEs) and Firemaster 550 (FM550), and other SVOCs such as phthalates, organotins, halogenated phenols and bisphenols. Additional attention was also given to the potential activity of the major metabolites of several of these compounds. Since the primary sink for many of these SVOCs is dust, and dust ingestion has been confirmed as an important pathway for SVOCs accumulation in humans, the potential PPAR binding and activation in extracts from environmentally relevant dust samples was also investigated.

Previous studies have also shown that SVOCs sorbed to organic matrices (e.g., soil and sediment), were only partially bioaccessible (bioavailable), but it was unclear how bioaccessible these compounds are from indoor dust matrices. In addition, bioactivation of SVOCs (via metabolism) could exacerbate their PPAR potency. Therefore, to adequately assess the potential risk of PPARγ activation from exposure to SVOC mixtures in house dust, it is essential that one also investigates the bioaccessibility and bioactivation of these chemicals following ingestion.

In the first research aim of this thesis, the bioaccessibility and bioactivation of several important SVOCs in house dust was investigated. To accomplish this, Tenax beads (TA) encapsulated within a stainless steel insert were used as an infinite adsorption sink to estimate the dynamic absorption of a suite of flame retardants (FRs) commonly detected in indoor dust samples, and from a few polyurethane foam samples for comparison. Experimental results demonstrate that the bioaccessibility and stability of FRs following ingestion varies both by chemical and by matrix. Organophosphate flame retardants (OPFRs) had the highest estimated bioaccessibility (~80%) compared to brominated compounds (e.g. PBDEs), and values generally decreased with increasing Log Kow, with <30% bioaccessibility measured for the most hydrophobic compound tested, BDE209. In addition, the stability of the more labile SVOCs that contained ester groups (e.g. OPFRs and 2-ethylhexyl-tetrabromo-benzoate (TBB)) were examined in a simulated digestive fluid matrix. No significant changes in the OPFR concentrations were observed in this fluid; however, TBB was found to readily hydrolyze to tetrabromobenzoic acid (TBBA) in the intestinal fluid in the presence of lipases.

In research aims 2 and 3, two commercially available high-throughput bioassays, a fluorescence polarization PPAR ligand binding assay (PolarScreenTM PPARγ-competitor assay kit, Invitrogen, Aim 2) and a PPAR reporter gene assay (GeneBLAzer PPARγ non-DA Assay, Invitrogen, Aim 3) were used to investigate the binding potency and activation of several groups of SVOCs and dust extracts with human PPARγ LBD; respectively. In the PPAR binding assay (Aim 2), most of the tested compounds exhibited dose-dependent binding to PPARγ. Mono(2-ethylhexyl) tetrabromophthalate (TB-MEHP), halogenated bisphenol/phenols, triphenyl phosphate and hydroxylated PBDEs were found to be potent or moderate PPARγ ligands, based on the measured ligand binding dissociation constant (Kd). The most potent compound was 3-OH-BDE47, with an IC50 of 0.24 μM. The extent of halogenation and the position of the hydroxyl group strongly affected binding. Of the dust samples tested, 21 of 24 samples showed significant PPAR binding potency at a concentration of 3 mg dust equivalents (DEQ)/mL. In the PPAR reporter assay (Aim 3), many SVOCs or their metabolites were either confirmed (based on previous reports) or for the first time were found to be potential PPARγ agonists with various potency and efficacy. We also observed that 15 of 25 dust extracts examined showed an activation percentage more than 8% (calculated activation threshold) of the maximal activation induced by rosiglitazone (positive control). In some cases, activation was as high as 50% of the rosiglitazone activation for the dust extracts with the highest efficacy. Furthermore, the correlation between the reporter assay and the ligand binding assay among the house dust extracts was significant and positive (r = 0.7, p < 0.003), suggesting the binding potency was predicting activation. In research aim 2, the effect of bioactivation on the PPARγ binding potency was also investigated. In vitro bioactivation of house dust extracts incubated with rat and human hepatic S9 fractions was used to investigate the role of in vivo biotransformation on PPAR gamma activity. The result showed that metabolism may lead to an increased binding affinity, as a 3-16% increase in PPARγ binding activity was observed following bioactivation of the dust extracts.

In research aim 4, an effect-directed analysis (EDA) was used to identify compounds likely contributing to the observed PPAR activity among the dust extract. Three dust extracts which showed significant PPAR activity with approximately 25, 30, and 50% of the maximal response induced by rosiglitazone at the highest efficacy were fractionated using normal phase high-performance liquid chromatography (NP-HPLC) and each fraction was individually tested for PPAR activity. Active fractions were then analyzed using gas-chromatography mass spectrometry (GC-MS) and possible compounds identified. Three dust extracts showed a similar PPAR activity distribution among the NP-HPLC fractions. In the most active fractions, fatty acids (FAs) were identified as the most active chemicals. The concentrations of four FAs were measured in the house dust extracts, and the concentrations were found to be highly correlated with the observed PPAR activity. These four FAs were also tested for PPAR activity and found to be partial PPAR agonists, particularly oleic and myristic acid. To tentatively identify sources of FAs, FAs in human/animal hair, dead skin cells, and two brands of cooking oil were analyzed. We found the same FAs in those samples and there concentrations were relatively abundant, ranging from 186 to 14,868 µg/g. Therefore, these results suggest that FAs are likely responsible for the observed PPAR activity in indoor dust. Also, this is the first study reporting on the level of FAs in dust samples. The source of these FAs in dust may be either from the cooking or accumulation of human/animal cells in indoor dust.

In conclusion, this research demonstrates that many SVOCs ubiqutiously detected in house dust, and/or their metabolites, can be weak or moderate PPAR ligands. In addition, chemical mixtures in house dust can effectively bind to and activate PPAR. However, our results suggest FAs are probably responsible for these observations, and likely outcompeting the synthetic environmental contaminants present in the dust extract. Furthermore, bioactivation of contaminants present in house dust can potentially increase their affinity for PPAR. And lastly, the bioaccessibility and stability of SVOCs in house dust after ingestion are likely to modulate the PPAR activity in the environmental mixtures and should be considered in future risk assessments.

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.