Browsing by Subject "PBDE"

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardant chemicals that were added to furniture foam, electronics, plastics, and some textiles to reduce their flammability. While PBDEs have been phased out from use in current products, huge reservoirs of products containing PBDEs still exist. It is likely exposure to PBDEs will continue as older products are discarded and recycled. PBDEs are ubiquitous contaminants in indoor and outdoor environments due to their widespread use in many products and their ability to migrate out of treated materials.

Major health effect concerns from PBDE exposure identified in laboratory studies include neurotoxicity, reproductive/developmental toxicity, and thyroid disruption. Importantly, mammals metabolize PBDEs into the hydroxylated polybrominated diphenyl ethers (OH-BDEs), which are structurally similar to endogenous thyroid hormones. Thyroid hormones are essential for metabolic processes, growth, and development, particularly brain development. Multiple studies have demonstrated enhanced potency of OH-BDEs relative to the parent PBDE chemicals, particularly for neurodevelopmental processes. Additionally, in fish species, thyroid hormones are essential for transitioning between larval, juvenile, and adult life stages. Therefore, studying the effects of both PBDEs and OH-BDEs during sensitive developmental life stages (i.e. larval and juvenile development) is warranted. The hypothesis of this thesis research is that PBDE metabolites interfere with thyroid hormone signaling (through interacting with thyroid receptor and deiodinase enzymes) which may result in decreased growth, morphological deficits, and altered neurodevelopment. The objectives of this research project were to evaluate the toxicity of PBDE metabolites and mixtures of PBDEs/OH-BDEs on larval and juvenile zebrafish development, examining both potential modes of action as well as functional consequences of exposure in developing animals.

In the first aim of this thesis research, structural relationships were examined between eleven different halogenated phenolic compounds (OH-BDEs, OH-PCBs, halogenated phenols, and TBBPA) to test developmental toxicity in zebrafish from 0-6 days post fertilization (dpf). In addition, follow up studies were performed with the most toxic compound, 6-hydroxy- 2,2’,4,4’-tetrabromodiphenyl ether (6-OH-BDE-47), to examine effects on TH-mediated morphological development and to better understand its mechanism of action in zebrafish. Thyroid disrupting agents including propylthiouracil, iopanoic acid, and native thyroid hormones were also used as positive controls for morphologic studies. Exposures to 6-OH-BDE-47 (10 nM to 100 nM) during development resulted in severe delays, similar to exposures from the T3 and thyroid disrupting agents. Lower jaw deformities and craniofacial cartilage malformations were also observed following exposure to 6-OH-BDE-47 at doses greater than 50 nM. Of interest, these developmental delays were rescued by overexpression of TRβ mRNA during the exposure period. These data indicate that OH-BDEs can adversely affect early life development of zebrafish and suggest they may be impacting thyroid hormone regulation in vivo through downregulation of the thyroid hormone receptor.

In the second aim of this dissertation research, neurobehavioral performance was monitored in larval and juvenile fish following a developmental exposure to 6-OH-BDE-47. 6-OH-BDE-47 has been identified as a neurotoxicant in previous cell based assays, and was identified as overtly toxic to zebrafish larvae in Aim 1 of this research. Developmental exposures (0-6 dpf) to 6-OH-BDE-47 resulted in decreased larval swimming activity at 6 dpf, with persisting impacts on behavior at 45 dpf. Young adult fish, when tested at 45 dpf, exhibited increased fear/anxiety response in the novel tank diving task and hyperactivity in a test of sensorimotor habituation. These data indicate that exposures to PBDE flame retardants and their metabolites during critical developmental windows can alter long term cognitive responses more than a month after the exposure has ceased.

Finally, for the third aim of this dissertation research, zebrafish undergoing larval-juvenile metamorphosis were exposed to a mixture of PBDEs (30-600 µg/L DE-71) and OH-BDEs (1-300 nM) from 9-23 dpf. Metamorphosis is a unique developmental period in fishes which is partially mediated by thyroid hormones. Juvenile animals, like larval animals, represent a sensitive and unique subpopulation of animals. At the end of the exposure period (23 dpf), a subset of fish were reared in clean water until 45 dpf for neurobehavioral testing. Fish samples were collected at 3 time points throughout the experiments, Days 12, 23, and 45. Tissue accumulation of test chemicals was monitored, and juvenile fish treated with the High Mixture were found to accumulate over 100 µg/g ww ∑PentaBDEs. The highest mixture treatment was found to be acutely toxic to zebrafish juveniles, resulting in >85% mortality within 14 days of exposure. Fish treated with 30 nM 6-OH-BDE-47 or the lower mixture exhibited reduced morphology scores relating to fin, pigmentation, and swim bladder maturation. In addition, reduced skeletal ossification and caudal area was observed at earlier time points with treatment to 6-OH-BDE-47. These alterations were accompanied by increases in chondrogenic gene expression, declines in osteogenic gene expression, and increases in thyroid receptor expression. Approximately 3.5 weeks after the exposure period, juvenile fish were tested on neurobehavioral tasks of novel tank exploration and sensorimotor habituation, however, no significant treatment related effects on task performance were observed. Collectively, these data suggested that the larval/juvenile development stage is a sensitive developmental window which can be adversely impacted by PBDE/OH-BDE exposure.

Polybrominated diphenyl ether (PBDE) flame retardants are ubiquitous contaminants in the environment due to their heavy usage in plastics, foam, and textiles to comply with flammability standards from the 1970s through the late 2000s. Due to their toxicity and persistence in the environment, two of the three PBDE commercial mixtures (PentaBDE and OctaBDE) were banned by the Stockholm Convention on Persistent Organic Pollutants in 2009. The DecaBDE commercial mixture, which consists primarily of the fully brominated congener BDE-209, has been banned or phased out in the United States and Europe but is still in use in other parts of the world. Human exposure to PBDEs persists via environmental reservoirs of PBDEs and products produced before the bans/phase-outs. PBDEs disrupt thyroid hormone levels and neurodevelopment in fish and rodents and are associated with altered thyroid hormone levels and neurodevelopmental impairments in humans. However, the mechanism by which PBDEs alter neurodevelopment remains unclear. Knowledge of the mechanisms and molecular targets of PBDEs is necessary for a causal link to be established between PBDEs and neurodevelopmental impairments. The hypothesis of this thesis research is that PBDEs alter thyroid hormone levels in the brain by interfering with the activity of PBDE-metabolizing deiodinase enzymes in brain cells, which may result in decreased levels of thyroid hormones in the brain and impaired neurodevelopment.

In the first aim of this thesis research, the biotransformation of PBDEs was examined to determine whether hydroxylated PBDEs (OH-BDEs) are formed in the human brain. In biotransformation assays performed with human astrocytes, which are cells located at the blood brain barrier, no debrominated or OH-BDE metabolites were identified. The results indicate that the enzyme responsible for PBDE hydroxylation (CYP2B6) was not expressed in sufficient quantities to metabolize PBDEs in the astrocyte cells used in this study, but future studies should analyze the potential for PBDE hydroxylation in other brain cells.

In the second aim of this thesis research, the effects of PBDEs on the thyroid-activating enzyme Type 2 deiodinase (DIO2) were determined in human astrocyte cells. DIO2 converts thyroxine (T4) into triiodothyronine (T3), which is the primary ligand that binds to the thyroid nuclear receptors, and is a very important signaling molecule during neurodevelopment. Cultured primary astrocytes and a human glioma cell line (H4 cells) were exposed to PBDEs and OH-BDEs, and changes in DIO2 activity were measured using liquid chromatography with tandem mass spectrometry (LC/MS/MS). Exposure to BDE-99, -153, and -209, 3-OH-BDE-47, and 5'-OH-BDE-99 all resulted in significant decreases in DIO2 activity in the H4 cells by up to 80% at doses of 500-1,000 nM. Further experiments deduced that the primary mechanism responsible for this decrease in activity was attributed to decreased DIO2 mRNA expression, increased post-translational degradation of DIO2, and competitive inhibition of DIO2. The reduction in DIO2 activity by PBDE and OH-BDE exposures could potentially reduce the concentration of T3 in the brain, which may be responsible for the neurodevelopmental impairments produced by exposure to this class of compounds and needs to be further explored.

In the third aim of this thesis research, the effects of PBDEs and OH-BDEs were examined in the H4 cells and in a mixed culture containing a human neuroblastoma cell line (SK-N-AS cells). The SK-N-AS cells express the thyroid hormone-inactivating enzyme Type 3 deiodinase (DIO3), which works in concert with DIO2 to buffer the concentration of T3 in the brain. Exposure to BDE-99 decreased the concentration of T3 and the inactive thyroid hormone rT3 in the cell culture medium of co-cultured cells by 59-76%. 3-OH-BDE-47 competitively inhibited DIO3 with an IC50 of 19 uM. 5'-OH-BDE-99 increased the rT3 concentrations in cell culture medium by 400%, increased DIO3 activity in exposed cells by 50%, and increased DIO3 catalytic activity in cellular homogenates by over 500%. Further effects on the mRNA expression of several thyroid-regulated genes (DIO3, TR-a, TR-b, MCT8, and ENPP2) and oxidative respiration were also assessed in the SK-N-AS cells. DIO3 mRNA expression increased by 9 fold in cells exposed to 400 nM BDE-99, and ENPP2 mRNA expression increased by 2 fold in cells exposed to 500 nM BDE-99 and a mixture of the three congeners, but no other significant effects on mRNA expression were observed. The basal respiration rates and other parameters of oxidative respiration were also not significantly altered by exposure to PBDEs or OH-BDEs, but proton leak was increased by over 400% in cells exposed to 2 uM 5'-OH-BDE-99.

This was the first study to examine the effects of an environmental contaminant on human DIO2 and DIO3 in cultured cells. The results indicated that BDE-99 and OH-BDEs decreased the activity of DIO2 and 5'-OH-BDE-99 increased the activity of DIO3, which combined would lead to decreased levels of T3 exported from the cells into the extracellular environment. These results provide more evidence that disruption of DIO2 and DIO3 by PBDEs during development may mediate the neurodevelopment effects associated with PBDEs.

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardant chemicals that are added to plastics, electronic components, furniture foam, and textiles to reduce their combustibility. Of the three commercial mixtures historically marketed, only DecaBDE, which is constituted almost entirely (~97%) of the fully brominated congener decabromodiphenyl ether (BDE-209), continues to be used in the U.S. today. While decaBDE is scheduled for phase-out in the U.S. at the end of 2013, exposures to BDE-209 and other PBDEs will continue into the foreseeable future as products that contain them continue to be used, recycled, and discarded. In addition, decaBDE use continues to be largely unrestricted across Asia, although restricted from use in electronic equipment in Europe.

Despite limits placed on PBDE uses, they are ubiquitous contaminants detected worldwide in humans and wildlife. Major health effect concerns for PBDEs come largely from evidence in laboratory rodents demonstrating neurotoxicity, reproductive and developmental impairments, and thyroid disruption. The potential for PBDEs, particularly BDE-209, to disrupt thyroid regulation and elicit other toxic outcomes in fish is less clear. Thus, the overall objective of this thesis research was to answer questions concerning how fish, as important indicators of overall environmental health, are metabolizing PBDEs and whether and how PBDEs are disrupting thyroid hormone regulation. The central hypothesis was that PBDE metabolism in fish is mediated by iodothyronine deiodinase (dio) enzymes, which are responsible for activating and inactivating thyroid hormones, and that PBDE exposures are causing thyroid system dysfunction across fish life stages.

Under the first research aim, in vitro experiments conducted in liver tissues isolated from common carp (Cyprinus carpio) suggested a role for dio enzymes in catalyzing the reductive debromination of PBDEs. Carp liver microsomes efficiently debrominated BDE-99 to BDE-47, and enzymes catalyzing this reaction were associated predominantly with the endoplasmic reticulum (i.e., microsomal fraction) where dio enzymes are located. Competitive substrate experiments in carp liver microsomes also demonstrated that rates of BDE-99 debromination to BDE-47 were significantly inhibited upon challenges with 3,3',5'-triiodothyronine (rT3) and thyroxine (T4). This finding supported the hypothesis that enzymes involved in the metabolism of PBDEs may have high affinities for thyroid hormones. Indeed, experiments to determine apparent enzymatic kinetics (apparent Vmax and Km values) of BDE-99 hepatic metabolism suggested that enzymes responsible for the catalytic activity appeared to have a higher affinity for native thyroid hormone than BDE-99.

The second and third research aims were focused on evaluating BDE-209 accumulation, metabolism, and thyroid toxicity in juvenile and adult life stages of fish using the fathead minnow (Pimephales promelas) as a model. BDE-209 bioaccumulated and was debrominated to several reductive metabolites ranging from penta- to octaBDEs in both juvenile and adult fish exposed to BDE-209. In addition, thyroid hormone regulation in juvenile and adult male fathead minnows was severely disrupted by BDE-209 at low, environmentally relevant exposures. In juvenile minnows, the activity of dio enzymes (T4-outer ring deiodination; T4-ORD and T4-inner ring deiodination; T4-IRD) declined by ~74% upon oral doses of 9.8 ± 0.2 µg/g wet weight (ww) food at 3% body weight (bw)/day for 28 days, compared to controls. Declines in dio activity were accompanied by thyroid follicle hypertrophy indicative of over-stimulation and injury. In addition to thyroid disruption, a distinctive liver phenotype characterized by vacuolated hepatocyte nuclei was measured in ~48% of hepatocytes from treated fish that was not observed in controls.

Under the third research aim, adult male fathead minnows received dietary treatments of BDE-209 at a low dose (95.3 ± 0.41 ng/g-food at 3% bw/day) and a high dose (10.1 ± 0.10 µg/g-food at 3% bw/day) for 28 days followed by a 14-day depuration period to evaluate recovery. Compared to negative controls, adult male fish exposed orally to BDE-209 at the low dose tested for 28 days experienced a 53% and 46% decline in circulating total T4 and T3, respectively, while fish at the high BDE-209 dose tested had total T4 and T3 deficits of 59% and 62%, respectively. Depressed levels of plasma thyroid hormones were accompanied by a 45-50% decline in the rate of T4-ORD in brains of all treatments by day 14 of the exposure. The decreased T4-ORD continued in the brain at day 28 with a ~65% decline measured at both BDE-209 doses. BDE-209 exposures also caused transient, tissue-specific upregulations of relative mRNA transcripts encoding dio enzymes (dio1, dio2), thyroid hormone receptors (TR&alpha, TR&beta), and thyroid hormone transporters (MCT8, OATP1c1) in the brain and liver in patterns that varied with time and dose, possibly as a compensatory response to hypothyroidism. In addition, thyroid perturbations at the low dose tested generally were equal to those measured at the high dose tested, suggesting non-linear relationships between PBDE exposures and thyroid dysfunction in adult fish. Thus, mechanisms for BDE-209 induced disruption of thyroid regulation can be proposed in adult male minnows that involve altered patterns of thyroid hormone signaling at several important steps in their transport and activation.

A growing body of evidence describing PBDE toxicity in biota, including data generated here, along with studies showing continued and rising PBDE body burdens, raises concern for human and wildlife health. Long delays in removing PBDEs from the market, their ongoing presence in many products still in use, and their active use outside the U.S. and European Union will leave a lasting legacy of rising contamination unless more concerted regulatory and policy actions are taken to reduce future exposures and harm.

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs) that have been heavily used in consumer products such as furniture foams, plastics, and textiles since the mid-1970’s. BFRs are added to products in order to meet state flammability standards intended to increase indoor safety in the event of a fire. The three commercial PBDE mixtures, Penta-, Octa-, and DecaBDE, have all been banned in the United States, however, limited use of DecaBDE is still permitted. PBDEs were phased out of production and added to the Stockholm Convention due to concerns over their environmental persistence and toxicity. Human exposure to PBDEs occurs primarily through the inadvertent ingestion of contaminated house dust, as well as though dietary sources. Despite the phase-out and discontinued use of PBDEs, human exposure to this class of chemicals is likely to continue for decades due to the continued use of treated products and existing environmental reservoirs of PBDEs. Extensive research over the years has shown that PBDEs disrupt thyroid hormone (TH) levels and neurodevelopmental endpoints in rodent and fish models. Additionally, there is growing epidemiological evidence linking PBDE exposure in humans to altered TH homeostasis and neurodevelopmental impairments in children. Due to the importance of THs throughout gestation, there is a great need to understand the effects of BFRs on the developing fetus. Specifically, the placenta plays a critical role in the transport, metabolism, and delivery of THs to the fetal compartment during pregnancy and is a likely target for BFR bioaccumulation and endocrine disruption. The central hypothesis of this dissertation research is that BFRs disrupt the activity of TH sulfotransferase (SULT) enzymes, thereby altering TH concentrations in the placenta.

In the first aim of this dissertation research, the concentrations of PBDEs and 2,4,6-TBP were measured in a cohort of 102 placenta tissue samples from an ongoing pregnancy cohort in Durham, NC. Methods were developed for the extraction and analysis of the BFR analytes. It was found that 2,4,6-TBP was significantly correlated with all PBDE analytes, indicating that 2,4,6-TBP may share common product applications with PBDEs or that 2,4,6-TBP is a metabolite of PBDE compounds. Additionally, this was the first study to measure 2,4,6-TBP in human placenta tissues.

In the second aim of this dissertation research, the placenta tissue concentrations of THs, as well as the endogenous activity of deiodinase (DI) and TH SULT enzymes were quantified using the same cohort of 102 placenta tissue samples. Enzyme activity was detected in all samples and this was the first study to measure TH DI and SULT activity in human placenta tissues. Enzyme activities and TH concentrations were compared with BFR concentrations measured in Aim 1. There were few statistically significant associations observed for the combined data, however, upon stratifying the data set based on infant sex, additional significant associations were observed. For example, among males, those with the highest concentrations of BDE-99 in placenta had T3 levels 0.80 times those with the lowest concentration of BDE-99 (95% confidence interval (CI): 0.59, 1.07). Whereas females with the highest concentrations of BDE-99 in placenta had T3 levels 1.50 times those with the lowest concentration of BDE-99 (95% CI: 1.10, 2.04). Additionally, all BFR analyte concentrations were higher in the placenta of males versus females and they were significantly higher for 2,4,6-TBP and BDE-209. 3,3’-T2 SULT activity was significantly higher in female placenta tissues, while type 3 DI activity was significantly higher in male placenta tissues. This research is the first to show sex-specific differences in the bioaccumulation of BFRs in human placenta tissue, as well as differences in TH concentrations and endogenous DI and SULT activity. The underlying mechanisms of these observed sex differences warrant further investigation.

In the third aim of this dissertation research, the effects of BFRs were examined in a human choriocarcinoma placenta cell line, BeWo. Michaelis-Menten parameters and inhibition curves were calculated for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. 2,4,6-TBP was shown to be the most potent inhibitor of 3,3’-T2 SULT activity with a calculated IC50 value of 11.6 nM. It was also shown that 2,4,6-TBP and 3-OH BDE-47 exhibit mixed inhibition of 3,3’-T2 sulfation in BeWo cell homogenates. Next, a series of cell culture exposure experiments were performed using 1, 6, 12, and 24 hour exposure durations. Once again, 2,4,6-TBP was shown to be the most potent inhibitor of basal 3,3’-T2 SULT activity by significantly decreasing activity at the high and medium dose (1 M and 0.5 M, respectively) at all measured time points. Interestingly, BDE-99 was also shown to inhibit basal 3,3’-T2 SULT activity in BeWo cells following the 24 hour exposure, despite exhibiting no inhibitory effects in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a pathway other than direct enzyme inhibition. Following exposures, the TH concentrations in the cell culture growth media and mRNA expression of TH-related genes were also examined. There was no observed effect of BFR treatment on these endpoints. Future work should focus on determining the downstream biological effects of TH SULT disruption in placental cells, as well as the underlying mechanisms of action responsible for reductions in basal TH SULT activity following BFR exposure.

This was one of the first studies to measure BFRs in a cohort of placenta tissue samples from the United States and the first study to measure THs, DI activity, and SULT activity in human placenta tissues. This research provides a novel contribution to our growing understanding of the effects of BFRs on TH homeostasis within the human placenta, and provides further evidence for sex-specific differences within this important organ. Future research should continue to investigate the effects of environmental contaminants on TH homeostasis within the placenta, as this represents the most critical and vulnerable stage of human development.