Triple Bioaffinity Mass Spectrometry Concept for Thyroid Transporter Ligands
Aqai, P. ; Fryganas, C. ; Mizuguchi, M. ; Haasnoot, W. ; Nielen, M.W.F. - \ 2012
Analytical Chemistry 84 (2012). - ISSN 0003-2700 - p. 6488 - 6493.
brominated flame retardants - ms-binding assays - human transthyretin - in-vitro - tetrabromobisphenol-a - disrupting chemicals - surface-water - pharmaceuticals - identification - metabolites
For the analysis of thyroid transporter ligands, a triple bioaffinity mass spectrometry (BioMS) concept was developed, with the aim at three different analytical objectives: rapid screening of any ligand, confirmation of known ligands in accordance with legislative requirements, and identification of emerging yet unknown ligands. These three purposes share the same biorecognition element, recombinant thyroid transport protein transthyretin (rTTR), and dedicated modes of liquid chromatography-mass spectrometry (LC-MS). For screening, a rapid and radiolabel-free competitive inhibition MS binding assay was developed with fast ultrahigh performance-liquid chromatography-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS) as the readout system. It uses the nonradioactive stable isotopic thyroid hormone 13C6-l-thyroxine as the label of which the binding to rTTR is inhibited by any ligand such as thyroid drugs and thyroid endocrine disrupting chemicals (EDCs). To this end, rTTR is either used in solution or immobilized on paramagnetic microbeads. The concentration-dependent inhibition of the label by the natural thyroid hormone l-thyroxine (T4), as a model analyte, is demonstrated in water at part-per-trillion and in urine at part-per-billion level. For confirmation of identity of known ligands, rTTR was used for bioaffinity purification for confirmation of naturally present free T4 in urine. As a demonstrator for identification of unknown ligands, the same rTTR was used again but in combination with nano-UPLC-quadrupole time-of-flight-MS (nLC-Q-TOF-MS) and urine samples spiked with the model “unknown” EDCs triclosan and tetrabromobisphenol-A. This study highlights the potential of BioMS using one affinity system, both for rapid screening and for confirmation and identification of known and unknown emerging thyroid EDCs.
Biotransformation of brominated flame retardants into potentially endocrine-disrupting metabolites, with special attention to 2,2 ',4,4 '-tetrabromodiphenyl ether (BDE-47)
Hamers, T. ; Kamstra, J.H. ; Sonneveld, E. ; Murk, A.J. ; Visser, T.J. ; Velzen, M.J.M. van; Brouwer, A. ; Bergman, A. - \ 2008
Molecular Nutrition & Food Research 52 (2008)2. - ISSN 1613-4125 - p. 284 - 298.
polybrominated diphenyl ethers - receptor active compounds - in-vitro - estrogen sulfotransferase - human transthyretin - exposed rats - cell-line - identification - inhibition - expression
In this study, the endocrine-disrupting (ED) potency of metabolites from brominated flame retardants (BFRs) was determined. Metabolites were obtained by incubating single-parent compound BFRs with phenobarbital-induced rat liver microsomes. Incubation extracts were tested in seven in vitro bioassays for their potency to compete with thyroxine for binding to transthyretin (TTR), to inhibit estradiol-sulfotransferase (E2SULT), to interact with thyroid hormone-mediated cell proliferation, and to (in-)activate the androgen, progesterone, estrogen, or aryl hydrocarbon receptor. For most BFRs, TTR-binding potencies, and to a lesser extent E2SULT-inhibiting potencies, significantly increased after biotransformation. Microsomal incubation had less pronounced effects on other ED modes of action, due to low biotransformation efficiency and background activities determined in control incubations without BFRs. Moreover, cell-based bioassays suffered from cytotoxicity from metabolites of lower-brominated polybrominated diphenyl ethers. For the environmentally relevant 2,2 ',4,4 '-tetrabromodiphenyl ether (BDE-47), six hydroxylated metabolites were identified. Individual metabolites had TTR-binding and E2SULT-inhibiting potencies 160-1600 and 2.2-220 times higher than BDE-47 itself, whereas their combined potencies in a realistic mixture were well predicted via concentration addition. In combination with other environmentally relevant hydroxylated organohalogens acting on TTR-binding and E2SULT inhibition, internal exposure to BFR metabolites may significantly contribute to the overall risk of endocrine disruption.
Biosensor discovery of thyroxine transport disrupting chemicals
Marchesini, G.R. ; Meimaridou, A. ; Haasnoot, W. ; Meulenberg, E. ; Albertus, F. ; Mizuguchi, M. ; Takeuchi, M. ; Irth, H. ; Murk, A.J. - \ 2008
Toxicology and Applied Pharmacology 232 (2008)1. - ISSN 0041-008X - p. 150 - 160.
brominated flame retardants - polybrominated diphenyl ethers - polychlorinated-biphenyls pcbs - thyroid-hormone homeostasis - binding globulin - human transthyretin - in-vitro - human-serum - hydroxylated metabolites - competitive interactions
Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two inhibition assays using the main thyroid hormone transport proteins, T4 binding globulin (TBG) and transthyretin (TTR), in combination with a T4-coated biosensor chip were optimized and automated for screening chemical libraries. The transport protein-based biosensor assays were rapid, high throughput and bioeffect-related. A library of 62 chemicals including the natural hormones, polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals was tested with the two assays. We discovered ten new active compounds with moderate to high affinity for TBG with the TBG assay. Strikingly, the most potent binding was observed with hydroxylated metabolites of the brominated diphenyl ethers (BDEs) BDE 47, BDE 49 and BDE 99, that are commonly found in human plasma. The TTR assay confirmed the activity of previously identified hydroxylated metabolites of PCBs and PBDEs, halogenated BPA and genistein. These results show that the hydroxylated metabolites of the ubiquitous PBDEs not only target the T4 transport at the TTR level, but also, and to a great extent, at the TBG level where most of the T4 in humans is circulating. The optimized SPR biosensor-based transport protein assay is a suitable method for high throughput screening of large libraries for potential thyroid hormone disrupting compounds
Thyroid hormone receptor isoform selectivity of thyroid hormone disrupting compounds quantified with an in vitro reporter gene assay
Schriks, M. ; Roessig, J.M. ; Murk, A.J. ; Furlow, J.D. - \ 2007
Environmental Toxicology and Pharmacology 23 (2007)3. - ISSN 1382-6689 - p. 302 - 307.
halogenated aromatic-hydrocarbons - brominated flame retardants - polychlorinated-biphenyls - hydroxylated metabolites - competitive interactions - human transthyretin - binding globulin - expression - chemicals - protein
Some compounds, including brominated diphenyl ethers (BDEs), can interfere with thyroid hormone (TH) receptor (TR)-mediated TH-signalling. In this study, the TR isoform selectivity of some TH disrupting compounds was investigated with TR alpha/beta specific reporter gene assays. For this purpose, the effects of compounds on 3,3',5-triiodothyronine (T-3)-induced TR alpha- or TR beta-activation were tested in green monkey kidney fibroblast (CV-1) cells transiently transfected with Xenopus TRs and a luciferase reporter gene. The T-3-like BDE-OH and diiodobiphenyl (DIB) increased T-3-induced TR alpha-activation, but not T-3-induced TRP-activation. BDE28 (100 nM) did not act via TR alpha, but almost tripled T-3-induced TRP-activation relative to T-3 at its EC50. BDE206 (100 nM) was antagonistic on both TRs with a maximum repression -54% relative to T-3 at its EC50. Contrary to previous results obtained with the T-screen, HBCD was inactive. The present study illustrates the importance of testing potential TH disrupting compounds in model systems that enable independent characterization of effects on both T3-induced TRs. (c) 2006 Elsevier B.V. All rights reserved.
Spreeta-based biosensor for endocrine disruptors
Marchesini, G.R. ; Koopal, K. ; Meulenberg, E. ; Haasnoot, W. ; Irth, H. - \ 2007
Biosensors and Bioelectronics 22 (2007)9-10. - ISSN 0956-5663 - p. 1908 - 1915.
plasmon resonance sensors - spr sensor - estrogenic compounds - human transthyretin - thyroxine-binding - bisphenol-a - xenoestrogens - chemicals - globulin - system
The construction and performance of an automated low-cost Spreeta¿-based prototype biosensor system for the detection of endocrine disrupting chemicals (EDCs) is described. The system consists primarily of a Spreeta miniature liquid sensor incorporated into an aluminum flow cell holder, dedicated to support a Biacore chip frame, in combination with a simple pressurized air-driven fluid system. During the optimization, a monoclonal antibody (MAb)-based immunoassay for the estrogenic compound bisphenol A (BPA) was used as a model. After the optimization two thyroxine transport protein inhibition assays for thyroid endocrine disruptors were implemented. The average noise of the system for 1 min of baseline was 1.1 ¿RIU (refractive index units) and it could be operated in the range of 18-22 °C with a minimum baseline drift (5-10 ¿RIU/100 min). Optimum signal to noise ratio (S/N R) was obtained using a flow cell height of 100 ¿m and a flow rate of 180 ¿l/min. The sensitivity of the Spreeta-based biosensor inhibition assays implemented (50% inhibition concentration (IC50) of 30.2 nM for BPA using MAb12 and 12.3 and 11.6 nM for thyroxine (T4) using thyroxine-binding globulin (TBG) and recombinant transthyretin (rTTR), respectively) was comparable to the sensitivity previously obtained using a Biacore 3000 (IC50 of 39.9 nM for BPA and 8.6 and 13.7 nM, respectively, for T4). The results indicate that the alternative prototype system can be used in combination with ready-to-use biosensor chip surfaces and it is potentially a useful tool for the bioeffect-related screening of EDCs
Biosensor recognition of thyroid-disrupting chemicals using transport proteins
Marchesini, G.R. ; Meulenberg, E. ; Haasnoot, W. ; Mizuguchi, M. ; Irth, H. - \ 2006
Analytical Chemistry 78 (2006)4. - ISSN 0003-2700 - p. 1107 - 1114.
human transthyretin - competitive interactions - thyroxine-binding - pre-albumin - biphenyls - metabolism - mechanisms - globulin - toxicity - estrogen
Novel surface plasmon resonance-based biosensor assays for the bioeffect-related screening of chemicals with thyroid-disrupting activity are described. Two thyroid transport proteins (TPs), thyroxine binding globulin (TBG) and recombinant transthyretin (rTTR), were applied in an inhibition assay format in a Biacore 3000 using CM5 biosensor chips coated with L-thyroxine (T4), the main hormone of the thyroid system. Assay conditions were optimized for the natural thyroid hormones, and known thyroid disruptors and structurally related compounds were selected as model compounds to be tested in both assays for their relative potency (RP) compared to T4. The chosen compounds were halogenated phenols, halogenated bisphenols, bisphenol A, 3,5-dichlorobiphenyl, and its hydroxylated metabolite 4-hydroxy-3,5-dichlorobiphenyl (4-OH PCB 14). The TBG-based assay was highly specific for T4, and the rTTR-based assay was sensitive toward several compounds, the highest sensitivity (RP = 4.4) being obtained with 4-OH PCB 14, followed by tetrabromobisphenol A (RP = 1.5) and tetrachlorobisphenol A (RP = 0.75). For the bioeffect-related screening of known and identification of possible new thyroid disruptors, the TPs-based biosensor assays were more sensitive (IC50 of 13.7 ± 1.3 and 8.6 ± 0.7 nM for the rTTR and the TBG-based assay, respectively), easier to perform, and faster alternatives (10 min/sample) than the currently used methods such as radioligand binding assays and immunoprecipitation-HPLC
In vitro profiling of the endocrine-disrupting potency of brominated flame retardants
Hamers, T.H.M. ; Kamstra, J.H. ; Sonneveld, E. ; Murk, A.J. ; Kester, M.H.A. ; Andersson, P.L. ; Legler, J. ; Brouwer, A. - \ 2006
Toxicological sciences 92 (2006)1. - ISSN 1096-6080 - p. 157 - 173.
polybrominated diphenyl ethers - receptor active compounds - bisphenol-a - estrogen sulfotransferase - developmental exposure - tetrabromobisphenol-a - human transthyretin - brain-development - gene-expression - cell-line
Over the last years, increasing evidence has become available that some brominated flame retardants (BFRs) may have endocrine disrupting (ED) potencies. The goal of the current study was to perform a systematic in vitro screening of the ED potencies of BFRs (1) to elucidate possible modes of action of BFRs in man and wildlife, and (2) to classify BFRs with similar profiles of ED potencies. A test set of twenty-seven individual BFRs was selected, consisting of nineteen polybrominated diphenylethers (PBDE) congeners, tetrabromobisphenol-A (TBBPA), hexabromocyclododecane (HBCD), 2,4,6-tribromophenol (246-TBP), ortho-hydroxylated BDE-47 (6OH-BDE-47), and TBBPA-bis(2,3)dibromopropylether (TBBPA-DBPE). All BFRs were tested for their potency to interact with the arylhydrocarbon receptor (AhR), androgen receptor (AR), progesterone receptor (PR), and estrogen receptor (ER). In addition, all BFRs were tested for their potency to inhibit estradiol (E2) sulfation by E2-sulfotransferase (E2SULT), to interfere with thyroid hormone 3,3`,5-triiodothyronine (T3) mediated cell proliferation, and to compete with T3-precursor thyroxine (T4) for binding to the plasma transport protein transthyretin (TTR). The results of the in vitro screening indicated that BFRs have ED potencies, some of which had not or only marginally been described before (AR-antagonism, PR-antagonism, E2SULT inhibition, and potentiation of T3-mediated effects). For some BFRs, the potency to induce AR-antagonism, E2SULT inhibition and TTR competition was higher than for natural ligands or clinical drugs used as positive controls. Based on their similarity in ED profiles, BFRs were classified into five different clusters. These findings support further investigation of the potential endocrine disrupting effects of these environmentally relevant BFRs in man and wildlife.
T-screen to quantify functional potentiating, antagonistic and thyroid hormone-like activities of poly halogenated aromatic hydrocarbons (PHAHs)
Schriks, M. ; Vrabie, C.M. ; Gutleb, A.C. ; Faassen, E.J. ; Rietjens, I.M.C.M. ; Murk, A.J. - \ 2006
Toxicology in Vitro 20 (2006)4. - ISSN 0887-2333 - p. 490 - 498.
pituitary-tumor cells - 3,3',4,4'-tetrachlorobiphenyl cb-77 - 5,5'-diphenylhydantoin decreases - tetrabromobisphenol-a - human transthyretin - thyroxine-binding - flame retardants - in-vitro - rat - growth
The present study investigates chemical thyroid hormone disruption at the level of thyroid hormone receptor (TR) functioning. To this end the (ant)agonistic action of a series of xenobiotics was tested in the newly developed T-screen. This assay makes use of a GH3 rat pituitary cell line, that specifically proliferates when exposed to 3,3¿,5-triiodo-l-thyronine (T3). The growth stimulatory effect is mediated via T3-receptors. (Ant)agonistic and potentiating action of compounds was studied in absence and presence of T3 at its EC50 level (0.25 nM). The compounds tested included the specific TR-antagonist amiodarone, as well as a series of brominated diphenyl ethers (BDEs), including specifically synthesized BDEs with a structural resemblance to 3,5-diiodo-l-thyronine (T2), T3 and T4 (3,3¿,5,5¿-tetraiodo-l-thyronine). The results obtained reveal that only BDE206 and amiodarone are specific antagonists. Interestingly some compounds which did not respond in the T-screen in absence of T3, potentiated effects when tested in combination with T3. This points at possibilities for disruption at the TR in vivo, where exposure generally occurs in presence of T3. Altogether the results of the present study show that the newly developed T-screen can be used as a valuable tool for identification and quantification of compounds active in disturbing thyroid hormone homeostasis at the level of TR-functioning.
Toxicological profiling of sediments with in vitro mechanisms-based bioassays for endocrine disruption
Houtman, C.J. ; Cenijn, P.H. ; Hamers, T. ; Lamoree, M.H. ; Legler, J. ; Murk, A.J. ; Brouwer, A. - \ 2004
Environmental Toxicology and Chemistry 23 (2004)1. - ISSN 0730-7268 - p. 32 - 40.
biotesten - sediment - toxiciteit - hormonen - estuaria - rivieren - nederland - hormoonverstoorders - waterbodems - ecotoxicologie - rijn - maas - bioassays - sediment - toxicity - hormones - estuaries - rivers - netherlands - endocrine disruptors - water bottoms - ecotoxicology - river rhine - river meuse - reporter gene assays - estrogenic activity - aromatic-hydrocarbons - human transthyretin - expression assays - toxic potency - extracts - chemicals - exposure - wildlife
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.