Profiling the endocrine disrupting properties of triazines, triazoles and short-chain PFAS.

Persistent, mobile and toxic (PMT) compounds released to the environment are likely to pollute drinking water sources due to their slow environmental degradation (persistency) and high water solubility (mobility). The aim of the present study was to create in vitro hazard profiles for sixteen triazoles, nine triazines and eleven PFAS based on their agonistic and antagonistic effects in estrogen receptor (ER), androgen receptor (AR) and thyroid hormone receptor (TR) reporter gene assays, their ability to bind human transthyretin (TTR), and their effects on steroidogenesis. The triazole fungicides tetraconazole, bitertanol, fenbuconazole, tebuconazole, cyproconazole, difenoconazole, propiconazole, paclobutrazol and triadimenol had agonistic or antagonistic effects on the ER and AR. Difenoconazole, propiconazole and triadimenol were also found to be TR antagonists. The triazine herbicide ametryn was an ER, AR and TR antagonist. The same nine triazole fungicides and the triazines atrazine, deethyl-atrazine and ametryn affected the secretion of steroid hormones. Furthermore, PFAS compounds PFBS, PFHxS, PFHxA, PFOS, PFOA and GenX and the triazoles bitertanol, difenoconazole and 4-methyl benzotriazole were found to displace T4 from TTR. These results are in line with earlier in vitro and in vivo studies on the endocrine disrupting properties of triazines, triazoles and PFAS. The present study demonstrates that this battery of in vitro bioassays can be used to profile compounds from different classes based on their endocrine disrupting properties as a first step to prioritize them for further research, emission reduction, environmental remediation and regulatory purposes.

Identification of polar bioactive substances in the Upper Rhine using effect-directed analysis.

Effect-Directed Analysis (EDA) was used to identify bioactive compounds in surface and well water from the Upper Rhine, and to evaluate their properties against the criteria set for Persistent, Mobile and Toxic (PMT) and very persistent and very mobile (vPvM) substances. A multi-layered solid-phase extraction was implemented to enrich a broad range of polar substances from the collected samples. The extracts were fractionated into 108 fractions and tested in the transthyretin (TTR)-binding assay measuring displacement of fluorescently labeled thyroxine (FITC-T4 TTR-binding assay) and the Aliivibrio fischeri bioluminescence (AFB) bioassay. Bioactive fractions guided the identification strategy using high-resolution mass spectrometry. Chemical features were systematically annotated using library databases and suspect lists, incorporating an automated assessment of the quality of each annotation. Based on this assessment, each chemical feature was assigned a specific identification confidence level. Identification of bioactive compounds was facilitated by using bioassay specific suspect lists that were extracted from an in-house developed database of positive and negative TTR-binding compounds and from a recently published database of active inhibitors of AFB. This resulted in the identification and confirmation of ten bioactive substances, including four evaluated as PMT and vPvM substances (diclofenac, trifloxystrobin acid, 6:2 FTSA and PFOA), and one as a potential PMT substance (4-aminoazobenzene). This study demonstrates the effectiveness of EDA in the identification of PMT/vPvM substances in the aquatic environment, facilitating their prioritization for comprehensive environmental risk assessment and possible regulation.

Two novel in vitro assays to screen chemicals for their capacity to inhibit thyroid hormone transmembrane transporter proteins OATP1C1 and OAT4.

Early brain development depends on adequate transport of thyroid hormones (THs) from the maternal circulation to the fetus. To reach the fetal brain, THs have to cross several physiological barriers, including the placenta, blood-brain-barrier and blood-cerebrospinal fluid-barrier. Transport across these barriers is facilitated by thyroid hormone transmembrane transporters (THTMTs). Some endocrine disrupting chemicals (EDCs) can interfere with the transport of THs by THTMTs. To screen chemicals for their capacity to disrupt THTMT facilitated TH transport, in vitro screening assays are required. In this study, we developed assays for two THTMTs, organic anion transporter polypeptide 1C1 (OATP1C1) and organic anion transporter 4 (OAT4), both known to play a role in the transport of THs across barriers. We used overexpressing cell models for both OATP1C1 and OAT4, which showed an increased uptake of radiolabeled T4 compared to control cell lines. Using these models, we screened various reference and environmental chemicals for their ability to inhibit T4 uptake by OATP1C1 and OAT4. Tetrabromobisphenol A (TBBPA) was identified as an OATP1C1 inhibitor, more potent than any of the reference chemicals tested. Additionally perfluorooctanesulfonic acid (PFOS), perfluoroctanic acid (PFOA), pentachlorophenol and quercetin were identified as OATP1C1 inhibitors in a similar range of potency to the reference chemicals tested. Bromosulfophthalein, TBBPA, PFOA and PFOS were identified as potent OAT4 inhibitors. These results demonstrate that EDCs commonly found in our environment can disrupt TH transport by THTMTs, and contribute to the identification of molecular mechanisms underlying TH system disruption chemicals.

Effect-Directed Analysis Based on Transthyretin Binding Activity of Per- and Polyfluoroalkyl Substances in a Contaminated Sediment Extract.

Only a fraction of the total number of per- and polyfluoroalkyl substances (PFAS) are monitored on a routine basis using targeted chemical analyses. We report on an approach toward identifying bioactive substances in environmental samples using effect-directed analysis by combining toxicity testing, targeted chemical analyses, and suspect screening. PFAS compete with the thyroid hormone thyroxin (T4 ) for binding to its distributor protein transthyretin (TTR). Therefore, a TTR-binding bioassay was used to prioritize unknown features for chemical identification in a PFAS-contaminated sediment sample collected downstream of a factory producing PFAS-coated paper. First, the TTR-binding potencies of 31 analytical PFAS standards were determined. Potencies varied between PFAS depending on carbon chain length, functional group, and, for precursors to perfluoroalkyl sulfonic acids (PFSA), the size or number of atoms in the group(s) attached to the nitrogen. The most potent PFAS were the seven- and eight-carbon PFSA, perfluoroheptane sulfonic acid (PFHpS) and perfluorooctane sulfonic acid (PFOS), and the eight-carbon perfluoroalkyl carboxylic acid (PFCA), perfluorooctanoic acid (PFOA), which showed approximately four- and five-times weaker potencies, respectively, compared with the native ligand T4 . For some of the other PFAS tested, TTR-binding potencies were weak or not observed at all. For the environmental sediment sample, not all of the bioactivity observed in the TTR-binding assay could be assigned to the PFAS quantified using targeted chemical analyses. Therefore, suspect screening was applied to the retention times corresponding to observed TTR binding, and five candidates were identified. Targeted analyses showed that the sediment was dominated by the di-substituted phosphate ester of N-ethyl perfluorooctane sulfonamido ethanol (SAmPAP diester), whereas it was not bioactive in the assay. SAmPAP diester has the potential for (bio)transformation into smaller PFAS, including PFOS. Therefore, when it comes to TTR binding, the hazard associated with this substance is likely through (bio)transformation into more potent transformation products. Environ Toxicol Chem 2024;43:245-258. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Screening for endocrine disrupting chemicals inhibiting monocarboxylate 8 (MCT8) transporter facilitated thyroid hormone transport using a modified nonradioactive assay.

Early neurodevelopmental processes are strictly dependent on spatial and temporally modulated of thyroid hormone (TH) availability and action. Thyroid hormone transmembrane transporters (THTMT) are critical for regulating the local concentrations of TH, namely thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3), in the brain. Monocarboxylate transporter 8 (MCT8) is one of the most prominent THTMT. Genetically induced deficiencies in expression, function or localization of MCT8 are associated with irreversible and severe neurodevelopmental adversities. Due to the importance of MCT8 in brain development, studies addressing chemical interferences of MCT8 facilitated T3 uptake are a crucial step to identify TH system disrupting chemicals with this specific mode of action. Recently a non-radioactive in vitro assay has been developed to rapidly screen for endocrine disrupting chemicals (EDCs) acting upon MCT8 mediated transport. This study explored the use of an UV-light digestion step as an alternative for the original ammonium persulfate (APS) digestion step. The non-radioactive TH uptake assay, with the incorporated UV-light digestion step of TH, was then used to screen a set of 31 reference chemicals and environmentally relevant substances to detect inhibition of MCT8-depending T3 uptake. This alternative assay identified three novel MCT8 inhibitors: methylmercury, bisphenol-AF and bisphenol-Z and confirmed previously known MCT8 inhibitors.

Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals-The ATHENA Project.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

Transthyretin-Binding Activity of Complex Mixtures Representing the Composition of Thyroid-Hormone Disrupting Contaminants in House Dust and Human Serum.

BACKGROUND: House dust contains many organic contaminants that can compete with the thyroid hormone (TH) thyroxine (T4) for binding to transthyretin (TTR). How these contaminants work together at levels found in humans and how displacement from TTR in vitro relates to in vivo T4-TTR binding is unknown. OBJECTIVES: Our aims were to determine the TTR-binding potency for contaminant mixtures as found in house dust, maternal serum, and infant serum; to study whether the TTR-binding potency of the mixtures follows the principle of concentration addition; and to extrapolate the in vitro TTR-binding potency to in vivo inhibition levels of T4-TTR binding in maternal and infant serum. METHODS: Twenty-five contaminants were tested for their in vitro capacity to compete for TTR-binding with a fluorescent FITC-T4 probe. Three mixtures were reconstituted proportionally to median concentrations for these chemicals in house dust, maternal serum, or infant serum from Nordic countries. Measured concentration-response curves were compared with concentration-response curves predicted by concentration addition. For each reconstituted serum mixture, its inhibitor-TTR dissociation constant (Ki) was used to estimate inhibition levels of T4-TTR binding in human blood. RESULTS: The TTR-binding potency of the mixtures was well predicted by concentration addition. The ∼20% inhibition in FITC-T4 binding observed for the mixtures reflecting median concentrations in maternal and infant serum was extrapolated to 1.3% inhibition of T4-TTR binding in maternal and 1.5% in infant blood. For nontested mixtures reflecting high-end serum concentrations, these estimates were 6.2% and 4.9%, respectively. DISCUSSION: The relatively low estimated inhibition levels at median exposure levels may explain why no relationship between exposure to TTR-binding compounds and circulating T4 levels in humans has been reported, so far. We hypothesize, however, that 1.3% inhibition of T4-TTR binding may ultimately be decisive for reaching a status of maternal hypothyroidism or hypothyroxinemia associated with impaired neurodevelopment in children. https://doi.org/10.1289/EHP5911.

Effects of environmental pollutants on calcium release and uptake by rat cortical microsomes.

Dysregulation of neuronal intracellular Ca2+ homeostasis can play a crucial role in many neurotoxic effects, including impaired brain development and behavioral dysfunctions. This study examined 40 suspected neurotoxicants from different chemical classes for their capacity to alter Ca2+ release and uptake from rat cortical microsomes. First, ten suspected neurotoxicants have been tested using a well-established cuvette-based Ca2+ flux assay. Five out of ten compounds (TOCP, endosulfan, PCB-95, chlorpyrifos and BDE-49) showed a significant, concentration-dependent alteration of Ca2+ release and uptake in adult rat cortical microsomes. The original cuvette assay was downscaled and customized to a fast, higher throughput microplate method and the 40 suspected neurotoxicants were screened for their effects on intracellular Ca2+homeostasis. In decreasing order of potency, the 15 test compounds that showed the strongest alteration of Ca2+ levels in adult rat microsomes were TOCP, endosulfan, BDE-49, 6-OH-BDE-47, PCB-95, permethrin, alpha-cypermethrin, chlorpyrifos, bioallethrin, cypermethrin, RDP, DEHP, DBP, BDE-47, and PFOS. Results from co-exposure experiments with selective inhibitors suggested that for some compounds Ca2+ releasing effects could be attributed to RyR activation (PFOS, DBP, and DEHP) or to SERCA inhibition (a potential novel mechanism of action for all four tested pyrethroid insecticides). The effects of the two most potent compounds, endosulfan and TOCP, were not blocked by any of the inhibitors tested, indicating other possible mechanism of action. For all other potent test compounds, a combined effect on RyR, IP3R, and/or SERCA has been observed. PFOS and 6-OH-BDE-47 caused increased Ca2+ release from adult but not from neonatal rat brain microsomes, indicating age-dependent difference in susceptibility to these test compounds. The current study suggests that the neurotoxic potential of compounds belonging to different chemical classes could partly be attributed to the effects on intracellular Ca2+ release and uptake. Although further validation is required, the downscaled method developed in this study presents technical advance that could be used for the future screening of suspected intracellular Ca2+ disruptors.

Altered Adipogenesis in Zebrafish Larvae Following High Fat Diet and Chemical Exposure Is Visualised by Stimulated Raman Scattering Microscopy.

Early life stage exposure to environmental chemicals may play a role in obesity by altering adipogenesis; however, robust in vivo methods to quantify these effects are lacking. The goal of this study was to analyze the effects of developmental exposure to chemicals on adipogenesis in the zebrafish (Danio rerio). We used label-free Stimulated Raman Scattering (SRS) microscopy for the first time to image zebrafish adipogenesis at 15 days post fertilization (dpf) and compared standard feed conditions (StF) to a high fat diet (HFD) or high glucose diet (HGD). We also exposed zebrafish embryos to a non-toxic concentration of tributyltin (TBT, 1 nM) or Tris(1,3-dichloroisopropyl)phosphate (TDCiPP, 0.5 µM) from 0-6 dpf and reared larvae to 15 dpf under StF. Potential molecular mechanisms of altered adipogenesis were examined by qPCR. Diet-dependent modulation of adipogenesis was observed, with HFD resulting in a threefold increase in larvae with adipocytes, compared to StF and HGD. Developmental exposure to TBT but not TDCiPP significantly increased adipocyte differentiation. The expression of adipogenic genes such as pparda, lxr and lepa was altered in response to HFD or chemicals. This study shows that SRS microscopy can be successfully applied to zebrafish to visualize and quantify adipogenesis, and is a powerful approach for identifying obesogenic chemicals in vivo.

Disruption of oxidative phosphorylation (OXPHOS) by hydroxylated polybrominated diphenyl ethers (OH-PBDEs) present in the marine environment.

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are of growing concern, as they have been detected in both humans and wildlife and have been shown to be toxic. Recent studies have indicated that OH-PBDEs can be more toxic than PBDEs, partly due to their ability to disrupt oxidative phosphorylation (OXPHOS), an essential process in energy metabolism. In this study, we determined the OXPHOS disruption potential of 18 OH-PBDE congeners reported in marine wildlife using two in vitro bioassays, namely the classic rat mitochondrial respiration assay, and a mitochondrial membrane potential assay using zebrafish PAC2 cells. Single OH-PBDE congeners as well as mixtures were tested to study potential additive or synergistic effects. An environmental mixture composed of seven OH-PBDE congeners mimicking the concentrations reported in Baltic blue mussels were also studied. We report that all OH-PBDEs tested were able to disrupt OXPHOS via either protonophoric uncoupling and/or inhibition of the electron transport chain. Additionally we show that OH-PBDEs tested in combinations as found in the environment have the potential to disrupt OXPHOS. Importantly, mixtures of OH-PBDEs may show very strong synergistic effects, stressing the importance of further research on the in vivo impacts of these compounds in the environment.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Effect-directed analysis of municipal landfill soil reveals novel developmental toxicants in the zebrafish Danio rerio.

Effect-directed analysis (EDA) is an approach used to identify (unknown) contaminants in complex samples which cause toxicity, using a combination of biology and chemistry. The goal of this work was to apply EDA to identify developmental toxicants in soil samples collected from a former municipal landfill site. Soil samples were extracted, fractionated, and tested for developmental effects with an embryotoxicity assay in the zebrafish Danio rerio. Gas chromatograph mass selective detection (GC-MSD) chemical screening was used to reveal candidate developmental toxicants in fractions showing effects. In a parallel study, liquid chromatography-hybrid linear ion trap Orbitrap mass spectrometry was also applied to one polar subfraction (Hoogenboom et al. J. Chromatogr. A2009, 1216, 510-519). EDA resulted in the identification of a number of previously unknown developmental toxicants, which were confirmed to be present in soil by GC-MS. These included 11H-benzo[b]fluorene, 9-methylacridine, 4-azapyrene, and 2-phenylquinoline, as well as one known developmental toxicant (retene). This work revealed the presence of novel contaminants in the environment that may affect vertebrate development, which are not subject to monitoring or regulation under current soil quality assessment guidelines.

In vitro toxicity profiling of ultrapure non-dioxin-like polychlorinated biphenyl congeners and their relative toxic contribution to PCB mixtures in humans.

The toxic equivalency concept used for the risk assessment of polychlorinated biphenyls (PCBs) is based on the aryl hydrocarbon receptor (AhR)-mediated toxicity of coplanar dioxin-like (DL) PCBs. Most PCBs in the environment, however, are non-dioxin-like (NDL) PCBs that cannot adopt a coplanar structure required for AhR activation. For NDL-PCBs, no generally accepted risk concept is available because their toxicity is insufficiently characterized. Here, we systematically determined in vitro toxicity profiles for 24 PCBs regarding 10 different mechanisms of action. Prior to testing, NDL-PCB standards were purified to remove traces of DL compounds. All NDL-PCBs antagonized androgen receptor activation and inhibited gap junctional intercellular communication (GJIC). Lower chlorinated NDL-PCBs were weak estrogen receptor (ER) agonists, whereas higher chlorinated NDL-PCBs were weak ER antagonists. Several NDL-PCBs inhibited estradiol-sulfotransferase activity and bound to transthyretin (TTR) but with much weaker potencies than reported for hydroxylated PCB metabolites. AhR-mediated expression of uridine-glucuronyl transferase isozyme UGT1A6 was induced by DL-PCBs only. Hierarchical cluster analysis of the toxicity profiles yielded three separate clusters of NDL-PCBs and a fourth cluster of reference DL-PCBs. Due to small differences in relative potency among congeners, the highly abundant indicator PCBs 28, 52, 101, 118, 138, 153, and 180 also contributed most to the antiandrogenic, (anti)estrogenic, antithyroidal, tumor-promoting, and neurotoxic potencies calculated for PCB mixtures reported in human samples, whereas the most potent AhR-activating DL-PCB-126 contributed at maximum 0.2% to any of these calculated potencies. PCB-168 is recommended as an additional indicator congener, given its relatively high abundance and antiandrogenic, TTR-binding, and GJIC-inhibiting potencies.

Development of a polydimethylsiloxane film-based passive dosing method in the in vitro DR-CALUX® assay.

In bioassays, exposure concentrations of test compounds are usually expressed as nominal concentrations. As a result of various processes, such as adsorption, degradation, or uptake, the actual freely dissolved concentration of the test compound may differ from the nominal concentration. The goal of the present study was to develop a method to dose passively the freely dissolved fraction of organic chemicals in an in vitro bioassay with adherent cells. To this end, a polydimethylsiloxane (PDMS) film-based method was developed for a reporter gene assay for dioxin-like compounds in a rat liver cell line. Polydimethylsiloxane films loaded with test compounds ensure that the concentration during exposure is in equilibrium and that the ratio between the concentration on the film and the concentration in medium is constant. Benzo[k]fluoranthene (BkF) was used as a model compound to develop the passive dosing method in transwell plates, which was further tested with a complex mixture, i.e., an extract prepared from a contaminated sediment. A higher dioxin-like activity was found when extracts were dosed by passive dosing with PDMS than when directly added to medium. Comparison with analysis of the concentration of BkF in medium shows that passive dosing of individual chemicals may not be necessary if freely dissolved concentrations are known. Use of PDMS for passive dosing of complex samples may represent a more realistic method for exposure in in vitro bioassays.

Dithiocarbamates induce craniofacial abnormalities and downregulate sox9a during zebrafish development.

Dithiocarbamates (DTCs) have a wide variety of applications in diverse fields ranging from agriculture to medicine. DTCs are teratogenic to vertebrates but the mechanisms by which they exert these effects are poorly understood. Here, we show that low nanomolar exposure to three DTCs, tetraethylthiuram (thiram), tetramethylthiuram (disulfiram), and sodium metam (metam), leads to craniofacial abnormalities in developing zebrafish embryos that are reminiscent of DTC-induced abnormalities found in higher vertebrates. In order to better understand the molecular events underlying DTC teratogenesis, we exposed embryonic zebrafish (PAC2) cells to thiram and disulfiram and measured changes in gene expression with microarrays. We found differential expression of 166 genes that were specific for exposure to DTCs and identified a network of genes related to connective tissue development and function. Additionally, we found eight downregulated genes related to transforming growth factor beta-1 (TGF-beta1) signaling, including an essential transcription factor for zebrafish craniofacial development, SRY-box-containing gene 9a (sox9a). Finally, we show that sox9a expression is perturbed in the ceratobranchial arches of DTC-exposed zebrafish, suggesting that this is an important event in the development of DTC-induced craniofacial abnormalities. Together, we provide evidence for a novel teratogenic endpoint and a molecular basis for a better understanding of DTC-induced teratogenesis in vertebrates.

In vitro assay shows that PCB metabolites completely saturate thyroid hormone transport capacity in blood of wild polar bears (Ursus maritimus).

Persistent chemicals accumulate in the arctic environment due to their chemical reactivity and physicochemical properties and polychlorinated biphenyls (PCBs) are the most concentrated pollutant class in polar bears (Ursus maritimus). Metabolism of PCB and polybrominated biphenyl ether (PBDE) flame-retardants alter their toxicological properties and these metabolites are known to interfere with the binding of thyroid hormone (TH) to transthyretin (TTR) in rodents and humans. In polar bear plasma samples no binding of [125I]-T(4) to TTR was observed after incubation and PAGE separation. Incubation of the plasma samples with [14C]-4-OH-CB107, a compound with a higher binding affinity to TTR than the endogenous ligand T(4) resulted in competitive binding as proven by the appearance of a radio labeled TTR peak in the gel. Plasma incubation with T(4) up to 1 mM, a concentration that is not physiologically relevant anymore did not result in any visible competition. These results give evidence that the binding sites on TTR for T(4) in wild living polar bears are completely saturated. Such saturation of binding sites can explain observed lowered levels of THs and could lead to contaminant transport into the developing fetus.

Microarray analysis reveals a mechanism of phenolic polybrominated diphenylether toxicity in zebrafish.

Polybrominated diphenylethers (PBDEs) are ubiquitous in the environment, with the lower brominated congener 2,2',4,4'-tetrabromodiphenylether (BDE47) among the most prevalent. The phenolic PBDE, 6-hydroxy-BDE47 (6-OH-BDE47) is both an important metabolite formed by in vivo metabolism of BDE47 and a natural product produced by marine organisms such as algae. Although this compound has been detected in humans and wildlife, including fish, virtually nothing is known of its in vivo toxicity. Here we report that 6-OH-BDE47 is acutely toxic in developing and adult zebrafish at concentrations in the nanomolar (nM) range. To identify possible mechanisms of toxicity, we used microarray analysis as a diagnostic tool. Zebrafish embryonic fibroblast (PAC2) cells were exposed to 6-OH-BDE47, BDE47, and the methoxylated metabolite 6-MeO-BDE47. These experiments revealed that 6-OH-BDE47 alters the expression of genes involved in proton transport and carbohydrate metabolism. These findings, combined with the acute toxicity, suggested that 6-OH-BDE47 causes disruption of oxidative phosphorylation (OXPHOS).Therefore, we further investigated the effect of 6-OH-BDE47 on OXPHOS in zebrafish mitochondria. Results show unequivocally that this compound is a potent uncoupler of OXPHOS and is an inhibitor of complex II of the electron transport chain. This study provides the first evidence of the in vivo toxicity and an important potential mechanism of toxicity of an environmentally relevant phenolic PBDE of both anthropogenic and natural origin. The results of this study emphasize the need for further investigation on the presence and toxicity of this class of polybrominated compounds.

Recombinational micro-evolution of functionally different metallothionein promoter alleles from Orchesella cincta.

BACKGROUND: Metallothionein (mt) transcription is elevated in heavy metal tolerant field populations of Orchesella cincta (Collembola). This suggests that natural selection acts on transcriptional regulation of mt in springtails at sites where cadmium (Cd) levels in soil reach toxic values This study investigates the nature and the evolutionary origin of polymorphisms in the metallothionein promoter (pmt) and their functional significance for mt expression. RESULTS: We sequenced approximately 1600 bp upstream the mt coding region by genome walking. Nine pmt alleles were discovered in NW-European populations. They differ in the number of some indels, consensus transcription factor binding sites and core promoter elements. Extensive recombination events between some of the alleles can be inferred from the alignment. A deviation from neutral expectations was detected in a cadmium tolerant population, pointing towards balancing selection on some promoter stretches. Luciferase constructs were made from the most abundant alleles, and responses to Cd, paraquat (oxidative stress inducer) and moulting hormone were studied in cell lines. By using paraquat we were able to dissect the effect of oxidative stress from the Cd specific effect, and extensive differences in mt induction levels between these two stressors were observed. CONCLUSION: The pmt alleles evolved by a number of recombination events, and exhibited differential inducibilities by Cd, paraquat and molting hormone. In a tolerant population from a metal contaminated site, promoter allele frequencies differed significantly from a reference site and nucleotide polymorphisms in some promoter stretches deviated from neutral expectations, revealing a signature of balancing selection. Our results suggest that the structural differences in the Orchesella cincta metallothionein promoter alleles contribute to the metallothionein -over-expresser phenotype in cadmium tolerant populations.

Toxicological profiling of sediments using in vitro bioassays, with emphasis on endocrine disruption.

In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds. They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches. In the present study, five in vitro bioassays were used to profile toxic potencies in sediments, with emphasis on endocrine disruption. Nonpolar total and acid-treated stable extracts of sediments from 15 locations in the Rhine Meuse estuary area in The Netherlands were assessed. Dioxin-like and estrogenic activities (using dioxin-responsive chemical-activated luciferase gene expression [DR-CALUX] and estrogen-responsive chemical-activated luciferase gene expression [ER-CALUX] assays) as well as genotoxicity (UMU test) and nonspecific toxic potency (Vibrio fischeri assay) were observed in sediment extracts. For the first time, to our knowledge, in vitro displacement of thyroid hormone thyroxine (T4) from the thyroid hormone transport protein thransthyretin by sediment extracts was observed, indicating the presence of compounds potentially able to disrupt T4 plasma transport processes. Antiestrogenic activity was also observed in sediment. The present study showed the occurrence of endocrine-disrupting potencies in sediments from the Dutch delta and the suitability of the ER- and DR-CALUX bioassays to direct endocrine-disruption TIE studies.

Placental transfer of a hydroxylated polychlorinated biphenyl and effects on fetal and maternal thyroid hormone homeostasis in the rat.

Earlier studies at our laboratory indicated that several hydroxylated polychlorinated biphenyls (OH-PCBs) detected in human blood could specifically inhibit thyroxine (T(4)) transport by competitive binding to the thyroid hormone transport protein transthyretin (TTR) in vitro. In the present study we investigated the effects of prenatal exposure to 5 mg/kg body weight of [14C]-labeled or unlabeled 4-OH-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107), one of the major metabolites of PCBs detected in human blood, from gestation days (GD) 10 to 16 on thyroid hormone status and metabolism in pregnant rats and their fetuses at GD 17 and GD 20. 4-OH-CB107 is a metabolite of both 2,3,3',4,4'-pentachlorobiphenyl (CB-105) and 2,3',4,4',5-pentachlorobiphenyl (CB-118). We were able to show the accumulation of 4-OH-CB107 in the fetal compartment. The fetal/maternal ratios at GD 20 in liver, cerebellum, and plasma were 11.0, 2.6, and 1.2, respectively. The 14C-4-OH-CB107-derived radioactivity in plasma was bound to TTR in both dams and fetuses. Fetal plasma TT(4) and FT(4) levels were significantly decreased at GD 17 and GD 20 (89% and 41% respectively at GD 20). Fetal thyroid stimulating hormone levels were increased by 124% at GD 20. The T(4) concentrations in fetal forebrain homogenates at GD20 were reduced by 35%, but no effects could be detected on brain T(3) concentrations. The deiodination of T(4) to T(3) was significantly increased in fetal forebrain homogenates at GD 17, and unaltered at GD 20. In addition, no alterations were observed in maternal and fetal hepatic T(4)-UDP-glucuronosyltransferase activity, type I deiodinase activity, and EROD activity. In conclusion, exposure of pregnant rats to 4-OH-CB107 results in the distribution of the compound in the maternal and fetal compartment, which is probably caused by the binding of the PCB metabolite to TTR. Consequently, TT(4) levels in fetal plasma and brain samples were reduced. Despite reductions in fetal brain T(4) levels, the active hormone (T(3)) in fetal brains remained unaffected.