Dataset Information

Bioaccumulation and effects of dietary exposure to the alternative flame retardant, bis(2-ethylhexyl) tetrabromophthalate (TBPH), in the Atlantic killifish, Fundulus heteroclitus.

ABSTRACT: Bis(2-ethylhexyl) tetrabromophthalate (TBPH), a high production volume flame retardant chemical used as a replacement for banned flame retardants, has been detected in media and human and wildlife tissues globally. We describe bioaccumulation and biological effects from dietary exposure of TBPH to an estuarine fish, Atlantic killifish, Fundulus heteroclitus. Briefly, adult fish were fed carrier control or chemically amended diets for 28 d, followed by 14 d of control diet feeding. Diets were amended with TBPH (TBPH_LO diet, 139??g/g dry wt, or TBPH_HI diet, 4360??g/g dry wt) or a polychlorinated biphenyl congener (PCB153 diet, 13??g/g dry wt), which was included as a positive control for bioaccumulation. Although bioaccumulation of either chemical correlated with fish size, only a small proportion of the TBPH offered (<0.5% total TBPH) had bioaccumulated into TBPH-treated fish by 28 d. In contrast, 24.5% of the PCB153 offered was accounted for in 28-d PCB-treated fish. Although 28-d bioaccumulated concentrations of TBPH differed by sex and treatment, sexes did not differ in their rates of TBPH bioaccumulation, and the time to achieve 50% of 28 d concentration (T1/2 ) was estimated to be 13 d. Depuration rates of TBPH did not differ by sex or treatment, and the time after exposure to achieve T1/2 was estimated to be 22 d. Independent of treatment, male fish grew faster than female fish, but for both sexes reproductive condition (gonadal somatic index) declined unexpectedly over the experimental period. Across treatments, only the TBPH_LO treatment affected growth, reducing male but increasing female growth rates by small amounts relative to respective controls. In summary, our study used very high concentrations of dietary TBPH to contaminate fish tissues above the highest levels reported to date in wild biota, yet we observed few adverse biological effects. Environ Toxicol Chem 2018;37:2350-2360. © 2018 SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

SUBMITTER: Nacci D

PROVIDER: S-EPMC6123825 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Bioaccumulation and effects of dietary exposure to the alternative flame retardant, bis(2-ethylhexyl) tetrabromophthalate (TBPH), in the Atlantic killifish, Fundulus heteroclitus.

Nacci Diane D Clark Bryan B La Guardia Mark J MJ Miller Ken K Champlin Denise D Kirby Ian I Bertrand Ashley A Jayaraman Saro S

Environmental toxicology and chemistry 20180716 9

Bis(2-ethylhexyl) tetrabromophthalate (TBPH), a high production volume flame retardant chemical used as a replacement for banned flame retardants, has been detected in media and human and wildlife tissues globally. We describe bioaccumulation and biological effects from dietary exposure of TBPH to an estuarine fish, Atlantic killifish, Fundulus heteroclitus. Briefly, adult fish were fed carrier control or chemically amended diets for 28 d, followed by 14 d of control diet feeding. Diets were ame ...[more]

PMID: 29846010

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Project description:Although alternative Flame Retardant (FR) chemicals are expected to be safer than the legacy FRs they replace, their risks to human health and the environment are often poorly characterized. This study used a small volume, fish embryo system to reveal potential mechanisms of action and diagnostic exposure patterns for TBPH (bis (2-ethylhexyl)-tetrabromophthalate), a component of several widely-used commercial products. Two different concentration of TBPH were applied to sensitive early life stages of an ecologically important test species, Fundulus heteroclitus (Atlantic killifish), with a well-annotated genome. Exposed fish embryos were sampled for transcriptomics or chemical analysis of parent compound and primary metabolite or observed for development and survival through larval stage. Global transcript profiling using RNA-seq was conducted (n?=?16 per treatment) to provide a non-targeted and statistically robust approach to characterize TBPH gene expression patterns. Transcriptomic analysis revealed a dose-response in the expression of genes associated with a surprisingly limited number of biological pathways, but included the aryl hydrocarbon receptor signal transduction pathway, which is known to respond to several toxicologically-important chemical classes. A transcriptional fingerprint using Random Forests was developed that was able to perfectly discriminate exposed vs. non-exposed individuals in test sets. These results suggest that TBPH has a relatively low potential for developmental toxicity (at least in fishes), despite concerns related to its structural similarities to endocrine disrupting chemicals and that the early life stage Fundulus system may provide a convenient test system for exposure characterization. More broadly, this study advances the usefulness of a biological testing and analysis system utilizing non-targeted transcriptomics profiling and early developmental endpoints that complements current screening methods to characterize chemicals of ecological and human health concern.

Project description:BACKGROUND:Bis-(2-ethylhexyl) tetrabromophthalate (TBPH) is widely used as a replacement for polybrominated diphenyl ethers (PBDEs) in commercial flame retardant mixtures such as Firemaster 550. It is also used in a commercial mixture called DP 45. Mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) is a potentially toxic metabolite. OBJECTIVES:We used in vitro and rodent in vivo models to evaluate human exposure and the potential metabolism and toxicity of TBPH. METHODS:Dust collected from homes, offices, and cars was measured for TBPH by gas chromatography followed by mass spectrometry. Pregnant rats were gavaged with TBMEHP (200 or 500 mg/kg) or corn oil on gestational days 18 and 19, and dams and fetuses were evaluated histologically for toxicity. We also assessed TBMEHP for deiodinase inhibition using rat liver microsomes and for peroxisome proliferator-activated receptor (PPAR) ? and ? activation using murine FAO cells and NIH 3T3 L1 cells. RESULTS:TBPH concentrations in dust from office buildings (median, 410 ng/g) were higher than in main living areas in homes (median, 150 ng/g). TBPH was metabolized by purified porcine esterases to TBMEHP. Two days of TBMEHP exposure in the rat produced maternal hypothyroidism with markedly decreased serum T3 (3,3´,5-triiodo-l-thyronine), maternal hepatotoxicity, and increased multinucleated germ cells (MNGs) in fetal testes without antiandrogenic effects. In vitro, TBMEHP inhibited deiodinase activity, induced adipocyte differentiation in NIH 3T3 L1 cells, and activated PPAR?- and PPAR?-mediated gene transcription in NIH 3T3 L1 cells and FAO cells, respectively. CONCLUSIONS:TBPH a) is present in dust from indoor environments (implying human exposure) and b) can be metabolized by porcine esterases to TBMEHP, which c) elicited maternal thyrotoxic and hepatotoxic effects and d) induced MNGs in the fetal testes in a rat model. In mouse NIH 3T3 L1 preadipocyte cells, TBMEHP inhibited rat hepatic microsome deiodinase activity and was an agonist for PPARs in murine FAO and NIH 3T3 L1 cells.

Project description:Due to the phaseout of polybrominated diphenyl ether (PBDE) flame retardants, new chemicals, such as 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB) and bis(2-ethylhexyl) 2,3,4,5-tetrabromophthalate (TBPH), have been used as replacements in some commercial flame retardant mixtures. Both chemicals have been detected in indoor dust at concentrations approaching the concentrations of PBDEs; however, little is known about their fate, metabolism, or toxicity. The goal of this study was to investigate the potential metabolism of these two brominated flame retardants in human and rat tissues by conducting in vitro experiments with liver and intestinal subcellular fractions. In all the experiments, TBB was consistently metabolized to 2,3,4,5-tetrabromobenzoic acid (TBBA) via cleavage of the 2-ethylhexyl chain without requiring any added cofactors. TBBA was also formed in purified porcine carboxylesterase but at a much faster rate of 6.29 ± 0.58 nmol min(-1) mg protein(-1). The estimated K(m) and V(max) values for TBB metabolism in human microsomes were 11.1 ± 3.9 μM and 0.644 ± 0.144 nmol min(-1) mg protein(-1), respectively. A similar K(m) of 9.3 ± 2.2 μM was calculated for porcine carboxylesterase, indicating similar enzyme specificity. While the rapid formation of TBBA may reduce the bioaccumulation potential of TBB in mammals and may be useful as a biomarker of TBB exposure, the toxicity of this brominated benzoic acid is unknown and may be a concern based on its structural similarity to other toxic pollutants. In contrast to TBB, no metabolites of TBPH were detected in human or rat subcellular fractions. However, a metabolic product of TBPH, mono(2-ethylhexyl) tetrabromophthalate (TBMEHP), was formed in purified porcine carboxylesterase at an approximate rate of 1.08 pmol min(-1) mg protein(-1). No phase II metabolites of TBBA or TBMEHP were observed. More research is needed to understand the in vivo toxicokinetics and health effects of these compounds given their current ubiquitous presence in most US households and the resulting probability of chronic exposure, particularly to young children.

Project description:Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuable non-traditional model, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off-target effects in AHR paralogs. No mutations were observed in closely related AHR genes (AHR1a, AHR1b, AHR2a, AHRR) in the CRISPR-Cas9-injected embryos. Overall, our results demonstrate that targeted genome-editing methods are efficient in inducing mutations at specific loci in embryos of a non-traditional model species, without detectable off-target effects in paralogous genes.

Dataset Information

Bioaccumulation and effects of dietary exposure to the alternative flame retardant, bis(2-ethylhexyl) tetrabromophthalate (TBPH), in the Atlantic killifish, Fundulus heteroclitus.

Publications

Bioaccumulation and effects of dietary exposure to the alternative flame retardant, bis(2-ethylhexyl) tetrabromophthalate (TBPH), in the Atlantic killifish, Fundulus heteroclitus.

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