Browsing by Subject "Flame retardant"

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.

Following the phase-out of polybrominated diphenyl ethers (PBDEs) in the early 2000s, organophosphate esters (OPEs) emerged as PBDE substitutes and have been applied to furniture foam, electronics, building materials, and some plastics to reduce their flammability. Although they have been used for quite some time in hydraulic fluids, isopropylated and tert-butylated triaryl phosphate esters (ITPs & TBPPs) have been more recently introduced as flame retardant (FR) replacements for the pentaBDE mixture in polyurethane foam (PUF). In addition to their use as FRs, ITPs and TBPPs are also used as plasticizers.

ITPs and TBPPs comprise a family of aryl organophosphate esters with varying degrees of isopropylation and tert-butylation. Individual ITP and TBPP isomers have been widely detected in indoor house dust, and recent biomonitoring studies demonstrate that human exposure to these compounds is widespread. Due to concerns about their persistence, bioaccumulation, and potential toxicity (P, B, & T), the U.S. Environmental Protection Agency (EPA) listed ITPs as one of five high priority chemicals fast-tracked for expedited risk assessment under the 2016 Toxic Substances Control Act (TSCA) reform.

As such, studying the exposure, metabolism, and in vitro effects of these compounds is especially timely. The hypothesis of this research dissertation is that ITP and TBPP isomers may exhibit some of the same P, B, & T properties that motivated the phase out of PBDEs. The main objectives of this research project were to generate meaningful data to fill gaps in our knowledge of ITP and TBPP isomers, and to contribute to the ongoing risk assessment of these compounds.

In the first aim of this thesis research, the maternal transfer of Firemaster® 550 (FM 550), a commercial mixture containing ITP isomers and brominated FRs, was investigated in dosed Wistar rats. Gestational and lactational transfer were examined separately, with dams orally exposed to 300 or 1000 µg of FM 550 for 10 consecutive days during gestation (gestational day [GD] 9-18) or lactation (postnatal day [PND] 3-12). Levels of parent compounds were measured in dam and pup urine. The two brominated components of FM 500, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) and bis (2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP), underwent both gestational and lactational transfer. Triphenyl phosphate (TPHP) and ITPs were rapidly metabolized by the dams and were not detected in whole tissue homogenates. However, diphenyl phosphate (DPHP) and mono-isopropylphenyl phenyl phosphate (ip-PPP) were detected in urine from the dosed animals. This study was the first to confirm ip-PPP as a urinary metabolite of ITPs and establish a pharmacokinetic profile of FM 550 in a mammalian model.

In the second aim of this thesis research, the contribution of individual ITP and TBPP isomers was quantified in four commercial flame retardant mixtures: FM 550, Firemaster® 600 (FM 600), an ITP mixture, and a TBPP mixture. Findings suggested similarities between FM 550 and the ITP mixture, with 2-isopropylphenyl diphenyl phosphate (2IPPDPP), 2,4-diisopropylphenyl diphenyl phosphate (24DIPPDPP), and bis(2-isopropylphenyl) phenyl phosphate (B2IPPPP) being the most prevalent ITP isomer in both mixtures. FM 600 differed from FM 550 in that it contained TBPP isomers rather than ITP isomers. ITP and TBPP isomers were also detected and quantified in house dust standard reference material, SRM 2585, demonstrating their environmental relevance.

The third aim of this thesis research investigated phase I and II in vitro metabolism of TPHP, 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), 2-isopropylphenyl diphenyl phosphate (2IPPDPP), and 4-isopropylphenyl diphenyl phosphate (4IPPDPP) at 1 and 10 µM doses using human liver subcellular fractions. Parent depletion and the formation of known metabolites, including DPHP, hydroxyl-triphenyl phosphate (OH-TPHP), ip-PPP, and tert-butylphenyl phenyl phosphate (tb-PPP), were monitored via gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS). Tb-PPP and its conjugates were identified as the major in vitro metabolites of 4tBPDPP, accounting for up to 33% of the initial parent dose. While the mass balance between parents and metabolites was conserved for TPHP and 4tBPDPP, approximately 20% of the initial parent mass was unaccounted for after quantifying metabolites of 2IPPDPP and 4IPPDPP that had authentic standards available. Two novel ITP metabolites, mono-isopropenylphenyl diphenyl phosphate and hydroxy-isopropylphenyl diphenyl phosphate, were tentatively identified by high-resolution mass spectrometry (HRMS) and screened for in recently collected human urine. This study provided insight into recent human biomonitoring and epidemiological studies and contributed to our understanding of the biological fate of ITP and TBPP isomers.

Finally, the fourth aim of this thesis research evaluated ITPs, TBPPs, and related commercial mixtures for their effect on the activity of purified human liver carboxylesterase (hCE1). Four of the 15 OPEs tested had IC50 values lower than 100 nM, including TPHP, 2-ethylhexyl diphenyl phosphate (EHDPP), 4-isopropylphenyl diphenyl phosphate (4IPPDPP), and 4-tert-butylphenyl diphenyl phosphate (4tBPDPP), as did four commercial flame retardant mixtures tested. Because hCE1 is critical for the activation of imidapril, an ACE-inhibitor prodrug prescribed to treat hypertension, the most potent inhibitors, TPHP and 4tBPDPP, and an environmentally relevant mixture (house dust) were further evaluated for their effect on imidapril bioactivation in vitro. TPHP and 4tBPDPP were potent inhibitors of hCE1-mediated imidapril activation (Ki = 49.0 and 17.9 nM, respectively), as were extracts of house dust (100 µg/ml), which caused significant reductions in imidapril activation. Combined, these data suggest that exposure to OPEs can affect pharmacotherapy.

Collectively and in context of other recently published findings, this thesis research suggests that ITPs and TBPPs may be regrettable substitutes for PBDEs.

Following the phase out of polybrominated diphenyl ethers (PBDEs), manufacturers turned to several alternative flame retardants (FRs) to meet flammability standards. Organophosphate FRs (OPFRs), and in particular tris (1,3-dichloropropyl) phosphate (TDCPP), have been increasingly detected in textiles and foam padding used in a variety of consumer products including camping equipment, upholstered furniture, and baby products. Like PBDEs, OPFRs are additive, meaning that they are not chemically bound to the treated material and can more readily leach out into the surrounding environment. Indeed, OPFRs have been detected in numerous environmental and biological matrices, often at concentrations similar to or exceeding that of PBDEs.

Although OPFRs have been in use for several decades, relatively little is known regarding their potential for adverse human and environmental health consequences. However, based on their structural similarity to OP pesticides, they may have analogous mechanisms of toxicity. OP pesticide toxicity is classically associated with cholinesterase inhibition, resulting in cholinergic intoxication syndrome. OPFRs have been shown to be ineffective cholinesterase inhibitors, however chlorpyrifos (CPF) and other OP pesticides have been shown to elicit adverse effects on developing organisms through other mechanisms.

The main objective of this research project was to evaluate the toxicity of four structurally similar OPFRs (TDCPP; tris (2,3-dibromopropyl) phosphate, (TDBPP); tris (1-chloropropyl) phosphate (TCPP) and tris (2-chloroethyl) phosphate (TCEP)) in comparison to chlorpyrifos (CPF), a well-studied OP pesticide. A combination of in vitro and in vivo models was used to elucidate potential mechanisms as well as functional consequences of exposure in developing organisms.

In the first research aim, a series of in vitro experiments with neurotypic PC12 cells was used to evaluate the effects of four structurally similar OPFRs (TDCPP, TDBPP, TCEP, or TCPP) and CPF on neurodevelopment. The effects of TDCPP were also compared to that of BDE-47, a major component of the commercial PentaBDE mixture. In general, TDCPP elicited similar or greater effects when compared to an equimolar concentration of CPF. All OPFRs tested produced similar decrements in cell number and altered phenotypic differentiation, while BDE-47 had no effect on cell number, cell growth, or neurite growth.

For the second research aim, zebrafish (Danio rerio) were used to evaluate the effects of the same suite of chemicals on early development. TDCPP, TDBPP, and CPF elicited overt toxicity (e.g., malformations or death) within the concentration range tested (0.033-100 µM). TDBPP was the most potent with 100% mortality by 6 days post fertilization (dpf) at ≥3.3 µM. CPF and TDCPP showed equivalent toxicity with malformations observed in at 10 µM and significant mortality (≥75%) at ≥33 µM. There was no overt toxicity among TCEP- and TCPP-exposed fish. All test chemicals affected larval swimming behavior on 6 dpf at concentrations below the overt toxicity threshold. Parent chemical was detected in all in embryonic (1 dpf) and larval (5 dpf) tissues. TDCPP and TDBPP showed rapid and extensive metabolism.

Finally, for the third aim, juvenile (45-55 dpf) zebrafish were exposed to CPF (1 µg/g food) or TDCPP (Low TDCPP = 1 µg/g food; High TDCPP = 40 µg/g food) via diet for 28 days followed by a 7 day depuration period where all treatments received clean food. A dietary exposure was chosen to more closely recapitulate exposure in humans. Samples were collected at seven time points throughout the experiment: days 0, 7, 14, 21, 28, 30, 35. Whole tissues were collected for tissue accumulation and histopathology endpoints. Viscera and brain were dissected and flash frozen separately for DNA damage analyses.

Tissue measurements of CPF, TDCPP, and the metabolite bis (1,3-dichloropropyl) phosphate (BDCPP) were often below the method detection limit, however when present there was a trend towards increased accumulation with treatment and time. On Day 7 Low TDCPP caused a dramatic but transient increase in DNA damage in both viscera and brain that returned to control levels by Day 14. Similar results have been seen previously with other genotoxicants and may be due to CPF and High TDCPP inducing an adaptive response prior to the 7 day sampling point. All treatments shifted the neurohypophysis to adenohypophysis ratio (NH/AH; Day 7 only) and significantly increased thyroid follicle activation (Day 14). Finally High TDCPP affected gonad maturation, causing a significant increase in ovary follicle development (Day 14) and a transient but marked decrease in testes maturity (Day 7). Taken together these data suggest that dietary exposure to TDCPP and CPF elicits DNA damage in brain and viscera and alters endocrine function in juvenile zebrafish. Importantly, analyses were restricted to the first three time points (Days 0, 7, and 14) due to the emergence a disease among the experimental colony. Although these samples were collected prior to the disease becoming apparent, it remains a potential confounder of the current results.

Background: Organophosphate (OP) pesticides are well-known developmental neurotoxicants that have been linked to abnormal cognitive and behavioral endpoints through both epidemiological studies and animal models of behavioral teratology, and are implicated in the dysfunction of multiple neurotransmitters, including dopamine. Chemical similarities between OP pesticides and organophosphate flame retardants (OPFRs), a class of compounds growing in use and environmental relevance, have produced concern regarding whether developmental exposures to OPFRs and OP pesticides may share behavioral outcomes, impacts on dopaminergic systems, or both. Methods: Using the zebrafish animal model, we exposed developing fish to two OPFRs, TDCIPP and TPHP, as well as the OP pesticide chlorpyrifos, during the first 5 days following fertilization. From there, the exposed fish were assayed for behavioral abnormalities and effects on monoamine neurochemistry as both larvae and adults. An experiment conducted in parallel examined how antagonism of the dopamine system during an identical window of development could alter later life behavior in the same assays. Finally, we investigated the interaction between developmental exposure to an OPFR and acute dopamine antagonism in larval behavior. Results: Developmental exposure to all three OP compounds altered zebrafish behavior, with effects persisting into adulthood. Additionally, exposure to an OPFR decreased the behavioral response to acute D2 receptor antagonism in larvae. However, the pattern of behavioral effects diverged substantially from those seen following developmental dopamine antagonism, and the investigations into dopamine neurochemistry were too variable to be conclusive. Thus, although the results support the hypothesis that OPFRs, as with OP pesticides such as chlorpyrifos, may present a risk to normal behavioral development, we were unable to directly link these effects to any dopaminergic dysfunction.