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Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many industrial applications. Exposure to PFAS is associated with several health risks, including a decrease in infant birth weight, hepatoxicity, disruption of lipid metabolism, and decreased immune response. We used the in vitro cell models to screen six less studied PFAS [perfluorooctane sulfonamide (PFOSA), perfluoropentanoic acid (PFPeA), perfluoropropionic acid (PFPrA), 6:2 fluorotelomer alcohol (6:2 FTOH), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), and 8:2 fluorotelomer sulfonic acid (8:2 FTSA)] for their capacity to activate nuclear receptors and to cause differential expression of genes involved in lipid metabolism. Cytotoxicity assays were run in parallel to exclude that observed differential gene expression was due to cytotoxicity. Based on the cytotoxicity assays and gene expression studies, PFOSA was shown to be more potent than other tested PFAS. PFOSA decreased the gene expression of crucial genes involved in bile acid synthesis and detoxification, cholesterol synthesis, bile acid and cholesterol transport, and lipid metabolism regulation. Except for 6:2 FTOH and 8:2 FTSA, all tested PFAS downregulated PPARA gene expression. The reporter gene assay also showed that 8:2 FTSA transactivated the farnesoid X receptor (FXR). Based on this study, PFOSA, 6:2 FTSA, and 8:2 FTSA were prioritized for further studies to confirm and understand their possible effects on hepatic lipid metabolism.
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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.
Assuntos
Disruptores Endócrinos , Transportadores de Ânions Orgânicos Sódio-Independentes , Transportadores de Ânions Orgânicos , Humanos , Disruptores Endócrinos/toxicidade , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Fluorocarbonos/toxicidade , Hormônios Tireóideos/metabolismo , Caprilatos/toxicidade , Tiroxina/metabolismo , Transporte Biológico/efeitos dos fármacos , Células HEK293 , Ácidos Alcanossulfônicos/toxicidade , AnimaisRESUMO
Transthyretin (TTR) and thyroxine-binding globulin (TBG) are two major thyroid hormone (TH) distributor proteins in human plasma, playing important roles in stabilizing the TH levels in plasma, delivery of TH to target tissues, and trans-barrier transport. Binding of xenobiotics to these distributor proteins can potentially affect all these three important roles of distributor proteins. Therefore, fast and cost-effective experimental methods are required for both TTR and TBG to screen both existing and new chemicals for their potential binding. In the present study, the TTR-binding assay was therefore simplified, optimized and pre-validated, while a new TBG-binding assay was developed based on fluorescence polarization as a readout. Seven model compounds (including positive and negative controls) were tested in the pre-validation study of the optimized TTR-binding assay and in the newly developed TBG-binding assay. The dissociation constants of the natural ligand (thyroxine, T4) and potential competitors were determined and compared between two distributor proteins, showing striking differences for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA).
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Pharmaceuticals, such as glucocorticoids and antibiotics, are inadequately removed from wastewater and may cause unwanted toxic effects in the receiving environment. This study aimed to identify contaminants of emerging concern in wastewater effluent with antimicrobial or glucocorticoid activity by applying effect-directed analysis (EDA). Effluent samples from six wastewater treatment plants (WWTPs) in the Netherlands were collected and analyzed with unfractionated and fractionated bioassay testing. Per sample, 80 fractions were collected and in parallel high-resolution mass spectrometry (HRMS) data were recorded for suspect and nontarget screening. The antimicrobial activity of the effluents was determined with an antibiotics assay and ranged from 298 to 711 ng azithromycin equivalents·L-1. Macrolide antibiotics were identified in each effluent and found to significantly contribute to the antimicrobial activity of each sample. Agonistic glucocorticoid activity determined with the GR-CALUX assay ranged from 98.1 to 286 ng dexamethasone equivalents·L-1. Bioassay testing of several tentatively identified compounds to confirm their activity revealed inactivity in the assay or the incorrect identification of a feature. Effluent concentrations of glucocorticoid active compounds were estimated from the fractionated GR-CALUX bioassay response. Subsequently, the biological and chemical detection limits were compared and a sensitivity gap between the two monitoring approaches was identified. Overall, these results emphasize that combining sensitive effect-based testing with chemical analysis can more accurately reflect environmental exposure and risk than chemical analysis alone.
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Poluentes Químicos da Água , Purificação da Água , Águas Residuárias/toxicidade , Glucocorticoides , Espectrometria de Massas , Antibacterianos/análise , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/análise , Monitoramento AmbientalRESUMO
Bisphenol A (BPA) is an industrial chemical, which has raised human health and environmental concerns due to its endocrine-disrupting properties. BPA analogues are less well-studied despite their wide use in consumer products. These analogues have been detected in water and aquatic organisms around the world, with some analogues showing toxic effects in various species including fish. Here, we present novel organ-specific time-course distribution data of bisphenol Z (BPZ) in female zebrafish (Danio rerio), including concentrations in the ovaries, liver, and brain, a rarely sampled organ with high toxicological relevance. Furthermore, fish-specific in vitro biotransformation rates were determined for 11 selected bisphenols. A physiologically based toxicokinetic (PBTK) model was adapted for four of these bisphenols, which was able to predict levels in the gonads, liver, and brain as well as the whole body within a 2-5-fold error with respect to experimental data, covering several important target organs of toxicity. In particular, predicted liver concentrations improved compared to currently available PBTK models. Predicted data indicate that studied bisphenols mainly distribute to the carcass and gonads and less to the brain. Our model provides a tool to increase our understanding on the distribution and kinetics of a group of emerging pollutants.
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Poluentes Químicos da Água , Peixe-Zebra , Animais , Compostos Benzidrílicos/toxicidade , Encéfalo , Feminino , Humanos , Fígado/metabolismo , Fenóis , Toxicocinética , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismoRESUMO
Effect-directed analysis (EDA) aims at the detection of bioactive chemicals of emerging concern (CECs) by combining toxicity testing and high-resolution mass spectrometry (HRMS). However, consolidation of toxicological and chemical analysis techniques to identify bioactive CECs remains challenging and laborious. In this study, we incorporate state-of-the-art identification approaches in EDA and propose a robust workflow for the high-throughput screening of CECs in environmental and human samples. Three different sample types were extracted and chemically analyzed using a single high-performance liquid chromatography HRMS method. Chemical features were annotated by suspect screening with several reference databases. Annotation quality was assessed using an automated scoring system. In parallel, the extracts were fractionated into 80 micro-fractions each covering a couple of seconds from the chromatogram run and tested for bioactivity in two bioassays. The EDA workflow prioritized and identified chemical features related to bioactive fractions with varying levels of confidence. Confidence levels were improved with the in silico software tools MetFrag and the retention time indices platform. The toxicological and chemical data quality was comparable between the use of single and multiple technical replicates. The proposed workflow incorporating EDA for feature prioritization in suspect and nontarget screening paves the way for the routine identification of CECs in a high-throughput manner.
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Bioensaio , Testes de Toxicidade , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectrometria de Massas , Fluxo de TrabalhoRESUMO
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.
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Disruptores Endócrinos/toxicidade , Ensaios de Triagem em Larga Escala/métodos , Hormônios Tireóideos/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Descoberta de Drogas , Disruptores Endócrinos/química , Humanos , Técnicas In Vitro , InternetRESUMO
Effect-directed analysis (EDA) is a commonly used approach for effect-based identification of endocrine disruptive chemicals in complex (environmental) mixtures. However, for routine toxicity assessment of, for example, water samples, current EDA approaches are considered time-consuming and laborious. We achieved faster EDA and identification by downscaling of sensitive cell-based hormone reporter gene assays and increasing fractionation resolution to allow testing of smaller fractions with reduced complexity. The high-resolution EDA approach is demonstrated by analysis of four environmental passive sampler extracts. Downscaling of the assays to a 384-well format allowed analysis of 64 fractions in triplicate (or 192 fractions without technical replicates) without affecting sensitivity compared to the standard 96-well format. Through a parallel exposure method, agonistic and antagonistic androgen and estrogen receptor activity could be measured in a single experiment following a single fractionation. From 16 selected candidate compounds, identified through nontargeted analysis, 13 could be confirmed chemically and 10 were found to be biologically active, of which the most potent nonsteroidal estrogens were identified as oxybenzone and piperine. The increased fractionation resolution and the higher throughput that downscaling provides allow for future application in routine high-resolution screening of large numbers of samples in order to accelerate identification of (emerging) endocrine disruptors.
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Disruptores Endócrinos , Poluentes Químicos da Água , Bioensaio , Genes Reporter , ÁguaRESUMO
Hexabromocyclododecane (HBCD) has been recognized as an indoor pollutant. HBCD is added as a flame retardant to many consumer products and leaches from the products into house dust. HBCD might be potentially hazardous to the airways because of inhalation of house dust. Sensitization to house dust mite (HDM) is a risk factor for the development of allergic asthma. In this study, we examined whether HBCD can affect the immune response to HDM allergens. Bone-marrow-derived dendritic cells (BMDCs) were exposed simultaneously to HBCD and HDM in vitro. HBCD enhanced oxidative stress in HDM-pulsed BMDCs, which was accompanied by a higher production of Interleukin (IL)-6 and -10. Adoptive transfer of HDM/HBCD-exposed BMDCs into naiÌve mice resulted in enhanced levels of IL-17A after inhalational challenge with HDM. Direct mucosal exposure to HBCD during HDM inhalation enhanced IL-4 or IL-17A production, depending on the HDM extract used, but did not aggravate the eosinophilic airway inflammation or airway hyper-reactivity. Our results indicate that exposure to HBCD can have a mild immune-modulating effect by enhancing the inflammatory cytokine production in response to inhaled HDM in mice.
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Poluentes Atmosféricos/farmacologia , Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Asma/imunologia , Hidrocarbonetos Bromados/farmacologia , Imunidade Celular/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Animais , CamundongosRESUMO
The chemical safety of consumer products is an issue of emerging concern. Plastics are widely used, e.g. as casings of consumer electronics (TVs, computers, routers, etc.), which are present in houses and offices in continuously increasing numbers. In this study, we investigate the estrogenic activity of components of plastics coming from electronics' casings. A recently developed fractionation platform for effect-directed analysis (EDA) was used. This platform combines reversed-phase liquid chromatography in parallel with bioassay detection via nanofractionation and with online high-resolution time-of-flight mass spectrometry (TOFMS) for the identification of bioactives. Four out of eight of the analyzed plastics samples showed the presence of estrogenic compounds. Based on the MS results these were assigned to bisphenol A (BPA), 2,4-di-tert-butylphenol, and a possible bisphenol A analog. All samples contained flame retardants, but these did not show any estrogenicity. The observed BPA, however, could be an impurity of tetrabromo-BPA (TBBPA) or TBBPA-based flame retardants. Due to the plausible migration of additives from plastics into the environment, plastics from consumer electronics likely constitute a source of estrogenic compound contamination in the indoor environment.
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Plásticos/química , Cromatografia Líquida , Estrogênios , Retardadores de Chama , Espectrometria de MassasRESUMO
Thyroid hormone disrupting chemicals (THDCs) interfere with the thyroid hormone system and may induce multiple severe physiological disorders. Indoor dust ingestion is a major route of THDCs exposure in humans, and one of the molecular targets of these chemicals is the hormone transporter transthyretin (TTR). To virtually screen indoor dust contaminants and their metabolites for THDCs targeting TTR, we developed a quantitative structure-activity relationship (QSAR) classification model. The QSAR model was applied to an in-house database including 485 organic dust contaminants reported from literature data and their 433 in silico derived metabolites. The model predicted 37 (7.6%) dust contaminants and 230 (53.1%) metabolites as potential TTR binders. Four new THDCs were identified after testing 23 selected parent dust contaminants in a radio-ligand TTR binding assay; 2,2',4,4'-tetrahydroxybenzophenone, perfluoroheptanesulfonic acid, 3,5,6-trichloro-2-pyridinol, and 2,4,5-trichlorophenoxyacetic acid. These chemicals competitively bind to TTR with 50% inhibition (IC50) values at or below 10 µM. Molecular docking studies suggested that these THDCs interacted similarly with TTR via the residue Ser117A, but their binding poses were dissimilar to the endogenous ligand T4. This study identified new THDCs using an in silico approach in combination with bioassay testing and highlighted the importance of metabolic activation for TTR binding.
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Simulação por Computador , Poeira/análise , Disruptores Endócrinos/análise , Metaboloma , Hormônios Tireóideos/metabolismo , Ácido 2,4,5-Triclorofenoxiacético , Poluição do Ar em Ambientes Fechados/análise , Sítios de Ligação , Bases de Dados como Assunto , Análise Discriminante , Humanos , Concentração Inibidora 50 , Análise dos Mínimos Quadrados , Simulação de Acoplamento Molecular , Pré-Albumina/metabolismo , Multimerização Proteica , Relação Quantitativa Estrutura-Atividade , Máquina de Vetores de SuporteRESUMO
A variety of anthropogenic compounds has been found to be capable of disrupting the endocrine systems of organisms, in laboratory studies as well as in wildlife. The most widely described endpoint is estrogenicity, but other hormonal disturbances, e.g., thyroid hormone disruption, are gaining more and more attention. Here, we present a review and chemical characterization, using principal component analysis, of organic compounds that have been tested for their capacity to bind competitively to the thyroid hormone transport protein transthyretin (TTR). The database contains 250 individual compounds and technical mixtures, of which 144 compounds are defined as TTR binders. Almost one third of these compounds (n = 52) were even more potent than the natural hormone thyroxine (T4). The database was used as a tool to assist in the identification of thyroid hormone-disrupting compounds (THDCs) in an effect-directed analysis (EDA) study of a sediment sample. Two compounds could be confirmed to contribute to the detected TTR-binding potency in the sediment sample, i.e., triclosan and nonylphenol technical mixture. They constituted less than 1% of the TTR-binding potency of the unfractionated extract. The low rate of explained activity may be attributed to the challenges related to identification of unknown contaminants in combination with the limited knowledge about THDCs in general. This study demonstrates the need for databases containing compound-specific toxicological properties. In the framework of EDA, such a database could be used to assist in the identification and confirmation of causative compounds focusing on thyroid hormone disruption.
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Bases de Dados Factuais , Disruptores Endócrinos/análise , Sedimentos Geológicos/química , Hormônios Tireóideos , Humanos , Relação Estrutura-AtividadeRESUMO
As a consequence of climate change, increased precipitation in winter and longer periods of decreased precipitation in summer are expected to cause more frequent episodes of very high or very low river discharge in the Netherlands. To study the impact of such extreme river discharge conditions on water quality, toxicity profiles and pollutant profiles were determined of suspended particulate matter (SPM) collected from Rivers Meuse and Rhine. Archived (1993-2003) and fresh (2009-2011) SPM samples were selected from the Dutch annual monitoring program of the national water bodies (MWTL), representing episodes with river discharge conditions ranging from very low to regular to very high. SPM extracts were tested in a battery of in vitro bioassays for their potency to interact with the androgen receptor (AR), the estrogen receptor (ER), the arylhydrocarbon receptor (AhR), and the thyroid hormone transporter protein transthyretin (TTR). SPM extracts were further tested for their mutagenic potency (Ames assay) and their potency to inhibit bacterial respiration (Vibrio fischeri bioluminescence assay). Target-analyzed pollutant concentrations of the SPM samples and additional sample information were retrieved from a public database of MWTL results. In vitro toxicity profiles and pollutant profiles were analyzed in relation to discharge conditions and in relation to each other using correlation analysis and multivariate statistics. Compared to regular discharge conditions, composition of SPM during very high River Meuse and Rhine discharges shifted to more coarse, sandy, organic carbon (OC) poor particles. On the contrary, very low discharge led to a shift to more fine, OC rich material, probably dominated by algae. This shift was most evident in River Meuse, which is characterized by almost stagnant water conditions during episodes of drought. During such episodes, SPM extracts from River Meuse demonstrated increased potencies to inhibit bacterial respiration and to compete with thyroid hormone to bind to TTR, possibly due to the presence of fycotoxins. Meanwhile concentrations of polychlorobiphenyls (PCBs) in SPM were also increased. Very high River Meuse discharges on the other hand corresponded to increased androgenic and AhR agoniztic responses, which coincided with increased PAH levels and PAH-related in vivo risk estimates (i.e. multi-substance potentially affected fraction of species; msPAF). In River Rhine, very high discharges also corresponded to increasing androgenic potencies in SPM. Concentrations and corresponding msPAF values of PAHs (and metals), however, decreased with very high discharges in River Rhine in contrast to River Meuse. Mutagenicity was observed for SPM extracts from River Rhine collected during all discharge conditions, except during regular discharge. Aggregated toxicity index values, which were useful to identify toxicity profiles deviating from the generally observed pattern, did not correlate with river discharges, probably due to opposite effects of discharge conditions on different bioassay responses. In conclusion, SPM quality and related in vivo risk estimates changed during very low or very high discharge conditions but the changes were specific for the different toxic endpoints and pollutants in the different rivers. Moreover, bioassay responses to a series of consecutively collected samples from River Rhine during the Christmas flood of 1993 indicated that SPM quality is variable within a single episode of extreme discharge.
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Mudança Climática , Material Particulado/análise , Rios/química , Movimentos da Água , Poluentes Químicos da Água/análise , Qualidade da Água , Aliivibrio fischeri/efeitos dos fármacos , Países Baixos , Material Particulado/toxicidade , Testes de Toxicidade , Poluentes Químicos da Água/toxicidadeRESUMO
Recent studies suggest that exposure to endocrine-disrupting compounds (EDCs) may play a role in the development of obesity. EDCs such as the flame retardant 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) have been shown to enhance adipocyte differentiation in the murine 3T3-L1 model. The mechanisms by which EDCs direct preadipocytes to form adipocytes are poorly understood. Here, we examined transcriptional and epigenetic mechanisms underlying the induction of in vitro adipocyte differentiation by BDE-47. Quantitative high content microscopy revealed concentration-dependent enhanced adipocyte differentiation following exposure to BDE-47 or the antidiabetic drug troglitazone (TROG). BDE-47 modestly activated the key adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) in COS7 cells, transiently transfected with a GAL4 reporter construct. Increased gene expression was observed for Pparγ2, leptin (Lep), and glucose-6-phophatase catalytic subunit (G6pc) in differentiated 3T3-L1 cells after BDE-47 exposure compared to TROG. Methylation-sensitive high resolution melting (MS-HRM) revealed significant demethylation of three CpG sites in the Pparγ2 promoter after exposure to both BDE-47 and TROG in differentiated 3T3-L1 cells. This study shows the potential of BDE-47 to induce adipocyte differentiation through various mechanisms that include Pparγ2 gene induction and promoter demethylation accompanied by activation of PPARγ, and possible disruption of glucose homeostasis and IGF1 signaling.
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Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular/genética , Epigênese Genética/efeitos dos fármacos , Retardadores de Chama/toxicidade , Éteres Difenil Halogenados/toxicidade , Transcrição Gênica/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Chlorocebus aethiops , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Humanos , Camundongos , Desnaturação de Ácido Nucleico/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
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.
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Disruptores Endócrinos , Transportadores de Ácidos Monocarboxílicos , Simportadores , Transporte Biológico/efeitos dos fármacos , Disruptores Endócrinos/isolamento & purificação , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Tiroxina , Humanos , Animais , Cães , Células Madin Darby de Rim Canino , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Simportadores/antagonistas & inibidores , Testes de ToxicidadeRESUMO
Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.
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Barreira Hematoencefálica , Transportadores de Ácidos Monocarboxílicos , Simportadores , Tri-Iodotironina , Animais , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Humanos , Simportadores/metabolismo , Simportadores/genética , Barreira Hematoencefálica/metabolismo , Camundongos , Tri-Iodotironina/metabolismo , Tri-Iodotironina/análogos & derivados , Chlorocebus aethiops , Células COS , Peixe-Zebra , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/genética , Transporte Biológico , Hipotonia Muscular/metabolismo , Hipotonia Muscular/genética , Camundongos Knockout , Atrofia Muscular , Deficiência Intelectual Ligada ao Cromossomo XRESUMO
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.
Assuntos
Ácidos Alcanossulfônicos , Fluorocarbonos , Pré-Albumina , Ácidos Alcanossulfônicos/análise , Ácidos Sulfônicos , Fluorocarbonos/toxicidade , CarbonoRESUMO
Per- and polyfluoroalkyl substances (PFAS) receive global attention due to their adverse effects on human health and the environment. Fish consumption is a major source of human PFAS exposure. The aim of this work was to address the lack of harmonization within legislations (in the EU and the USA) and highlight the level of PFAS in fish exposed to pollution from diffuse sources in the context of current safety thresholds. A non-exhaustive literature review was carried out to obtain PFAS concentrations in wild fish from the Norwegian mainland, Svalbard, the Netherlands, the USA, as well as sea regions (North Sea, English Channel, Atlantic Ocean), and farmed fish on the Dutch market. Median sum wet weight concentrations of PFOA, PFNA, PFHxS, and PFOS ranged between 0.1 µg kg-1 (farmed fish) and 22 µg kg-1 (Netherlands eel). Most concentrations fell below the EU environmental quality standard (EQSbiota) for PFOS (9.1 µg kg-1) and would not be defined as polluted in the EU. However, using recent tolerable intake or reference dose values in the EU and the USA revealed that even limited fish consumption would lead to exceedance of these thresholds - possibly posing a challenge for risk communication.
Assuntos
Peixes , Fluorocarbonos , Poluentes Químicos da Água , Animais , Humanos , Fluorocarbonos/análise , Poluentes Químicos da Água/análise , Contaminação de Alimentos/análise , Estados Unidos , Exposição Dietética/estatística & dados numéricos , Exposição Dietética/análise , Países Baixos , Noruega , Alimentos Marinhos/análise , Exposição Ambiental , Monitoramento Ambiental , Medição de Risco , União EuropeiaRESUMO
Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in various industrial and consumer products. They have gained attention due to their ubiquitous occurrence in the environment and potential for adverse effects on human health, often linked to immune suppression, hepatotoxicity, and altered cholesterol metabolism. This study aimed to explore the impact of ten individual PFAS, 3â¯H-perfluoro-3-[(3-methoxypropoxy) propanoic acid] (PMPP/Adona), ammonium perfluoro-(2-methyl-3-oxahexanoate) (HFPO-DA/GenX), perfluorobutanoic acid (PFBA), perfluorobutanesulfonic acid (PFBS), perfluorodecanoic acid (PFDA), perfluorohexanoic acid (PFHxA), perfluorohexanesulfonate (PFHxS), perfluorononanoic acid (PFNA), perfluorooctanoic acid (PFOA), and perfluorooctanesulfonic acid (PFOS) on the lipid metabolism in human hepatocyte-like cells (HepaRG). These cells were exposed to different concentrations of PFAS ranging from 10⯵M to 5000⯵M. Lipids were extracted and analyzed using liquid chromatography coupled with mass spectrometry (LC- MS-QTOF). PFOS at 10⯵M and PFOA at 25⯵M increased the levels of ceramide (Cer), diacylglycerol (DAG), N-acylethanolamine (NAE), phosphatidylcholine (PC), and triacylglycerol (TAG) lipids, while PMPP/Adona, HFPO-DA/GenX, PFBA, PFBS, PFHxA, and PFHxS decreased the levels of these lipids. Furthermore, PFOA and PFOS markedly reduced the levels of palmitic acid (FA 16.0). The present study shows distinct concentration-dependent effects of PFAS on various lipid species, shedding light on the implications of PFAS for essential cellular functions. Our study revealed that the investigated legacy PFAS (PFOS, PFOA, PFBA, PFDA, PFHxA, PFHxS, and PFNA) and alternative PFAS (PMPP/Adona, HFPO-DA/GenX and PFBS) can potentially disrupt lipid homeostasis and metabolism in hepatic cells. This research offers a comprehensive insight into the impacts of legacy and alternative PFAS on lipid composition in HepaRG cells.
Assuntos
Fluorocarbonos , Hepatócitos , Metabolismo dos Lipídeos , Humanos , Fluorocarbonos/toxicidade , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Linhagem Celular , Poluentes Ambientais/toxicidade , Ácidos Alcanossulfônicos/toxicidadeRESUMO
This research presents an analytical technology for highly efficient, high-resolution, and high-yield fractionation of compounds after gas chromatography (GC) separations. The technology is straightforward, does not require sophisticated cold traps or adsorbent traps, and allows collecting large numbers of fractions during a GC run. The technology is based on direct infusion of a carrier solvent at the end of the GC column, where infusion takes place in the GC oven. Pentane and hexane used as carrier solvent showed good results. Acetonitrile also showed good results as a more polar carrier solvent. Development and optimization of the technology is described, followed by demonstration in a high-throughput effect directed analysis setting toward dioxin receptor bioactivity. The GC fractionation setup was capable of collecting fractions in the second range. As a result, fractionated compounds could be collected into one or two fractions when 6.5 s resolution fractionation was performed. Subsequently, mixtures containing polycyclic aromatic hydrocarbons, of which some are bioactive toward the dioxin receptor, were profiled with a mammalian gene reporter assay. After fractionation into 96-well plates, we used our new approach for direct cell seeding onto the fractions prior to assaying which allowed dioxin receptor bioactivity to be measured directly after fractionation. The current technology represents a great advance in effect directed analysis for environmental screening worldwide as it allows combining the preferred analytical separation technology for often non-polar environmental pollutants with environmentally relevant bioassays, in high resolution.