PosterPDF Available

Adverse outcome pathway network-based testing strategy for thyroid disruption

Poster

Adverse outcome pathway network-based testing strategy for thyroid disruption

Figures

Content may be subject to copyright.
Adverse outcome pathway network-based
testing strategy for thyroid disruption
Jiří Novák, Klára Hilscherová
RECETOX, Masaryk University, Kamenice 753/5, 625
00 Brno, Czech Republic
Adverse outcome pathway (AOP) concept5,6 -
a model identifying sequence of biochemical
events required to produce a toxic effect
when an organism is exposed to a substance.
AOP can provide f or:
detailed mechanistic characteristics of
an adverse effect
design of testing strategy by
identification of key events in the
pathway
linking biomarkers assessed
in vitro
with
toxic outcome
in vivo
for risk
assessment
Fig. 2 Cross-class thyroid hormone disruption AOP network
covering mammalian, amphibian and teleost endpoints
ERGO - Breaking down the wall between human health and environmental testing
of endocrine disrupters (EU Horizon2020 Topic-SC1-BHC-27-2018;
15 partners across Europe).
Main project hypothesis:
For conserved endocrine systems such as Hypothalamic
Pituitary Thyroid (HPT) axis it is feasible to extrapolate
effects of EDCs across vertebrate classes
Fig.1Overview of Adverse
Outcome Pathway (AOP)concept
Fig. 1 The three key societal impact areas of ERGOproject, which
are affected by improved ED testing and screening methods
Fig. 3 Cross-class thyroid hormone
disruption AOP network covering
mammalian, amphibian and teleost
endpoints
Project aims for development of a battery of
in vitro
assays (Tab. 1) and
evaluation of HPT disruption-related biomarkers suitable for extrapolation
of effects from fish and amphibian tests to humans and other mammals
(Fig. 2).
Project outcome is based on data from
in silico, in vitro
and cross-
vertebrate class
in vivo
endpoints.
Project objectives:
1. Investigation, development and pre-validation of new thyroid-related
biomarkers for inclusion in OECD test guidelines for improved
identification of thyroid system disrupting compounds.
2. Development an adverse outcome pathway (AOP) network across
vertebrate classes for identification of thyroid B/E applicable for
assessment of cross-class thyroid disrupting key events.
3. An Integrated Approach to Testing and Assessment (IATA) of chemicals
for TD based on a multi-class vertebrate AOPs (Fig.2, 3) network
connecting endocrine mechanisms in one vertebrate class to adverse
outcomes in another class.
4. A tool for test guidelines end users, such as regulators and industry, to
extrapolate effects on thyroid regulation between vertebrate classes.
Implementation of the ERGO IATA strategy in regulations of EDC will
make hazard and risk assessment faster, cheaper, simpler and safer.
Tab. 1 Prioritized endpoints to include into
in vitro
bioassay battery addressing specific
molecular initiation events (MIE) with corresponding Adverse OutcomePathways(AOP)
Ref e r en c e s :
1. Wu, Y., Beland, F. A. & Fang, J. L. Effect oftriclosan, triclocarban, 2,2’,4,4’-tetrabromodiphenyl ether, and bisphenolA on theiodide upt ake , t hyr oid p eroxi dase act iv it y, and e xpres sion of ge nes i nvol ved i n t hyr oid h ormone syn thes is.
Toxicol. Vitr.
32, 310–31 9 (2016).
2.St in cken s, E.
et al .
An AOP-based a lt erna ti ve t esti ng st rat egy t o pred ict t he impa ct of t hyroi d hor mone disr upti on on swi m blad der i nfl at ion i n zeb raf ish.
Aqua t. T oxi col .
200, 1 –12 (201 8).
3. Ren, X. - M.
et al .
Bindinginteractionsofperfluoroalkyl substanceswiththyroidhormone transport proteinsand potential toxicological implications.
Toxicology
366–367, 32–42 (201 6).
4.Illés, P., Brtko, J. & Dvořák, Z. Development and Characterization of a Human Reporter Cell Line f or the Assessment of Thyroi d Receptor Transcript ional Acti vit y: A Case of Organotin Endocrine Disruptors.
J. Agr ic. Food Chem.
63, 7074–83 (2015).
5.Aopwiki. Availableat: https://aopwiki.org/. (Accessed: 18th March2019)
6.Villeneuve, D. L.
et al .
Adve rse Out come Pa th way ( AOP) Deve lopme nt I : S tr ate gi es and Pri nci pl es.
Toxicol. Sci.
142, 312–320 (2014).
level of interaction/function of target MIE included in AOP No. model origin
Iodide transmembrane transporter Na+/I- symporter (NIS)
1
AOP 65, 54, 134, 176 human, rat
TH synthesis thyroperoxidase (TPO) inhibition
2
AOP 42,119, 159, 175, 271 human, rat, pig
TH activation/inactivation Iodothyronine deiodinase 1 (DIO1) inhibition
3
AOP 157, 158, 189, rat, pig
Iodothyronine deiodinase 2 (DIO2) inhibition
3
AOP 155, 156, 190 rat, pig
serum TH binding protein T4-TTR displacement
4
AOP 152 in chemico
T4-TBG displacement
4
AOP 152 in chemico
TH responses Thyroid hormone receptor antagonism
5
AOP 300 human
www.ergo‐project.eu
jiri.novak@recetox.muni.cz
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
An adverse outcome pathway (AOP) is a conceptual framework that organizes existing knowledge concerning biologically plausible, and empirically supported, links between molecular-level perturbation of a biological system and an adverse outcome at a level of biological organization of regulatory relevance. Systematic organization of information into AOP frameworks has potential to improve regulatory decision-making through greater integration and more meaningful use of mechanistic data. However, for the scientific community to collectively develop a useful AOP knowledgebase that encompasses toxicological contexts of concern to human health and ecological risk assessment, it is critical that AOPs be developed in accordance with a consistent set of core principles. Based on the experiences and scientific discourse among a group of AOP practitioners, we propose a set of five fundamental principles that guide AOP development: (1) AOPs are not chemical specific; (2) AOPs are modular and composed of reusable components-notably key events (KEs) and key event relationships (KERs); (3) an individual AOP, composed of a single sequence of KEs and KERs, is a pragmatic unit of AOP development and evaluation; (4) networks composed of multiple AOPs that share common KEs and KERs are likely to be the functional unit of prediction for most real-world scenarios; and (5) AOPs are living documents that will evolve over time as new knowledge is generated. The goal of the present article was to introduce some strategies for AOP development and detail the rationale behind these 5 key principles. Consideration of these principles addresses many of the current uncertainties regarding the AOP framework and its application and is intended to foster greater consistency in AOP development. Published by Oxford University Press on behalf of the Society of Toxicological 2014. This work is written by US Government employees and is in the public domain in the US.
Article
The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecu- lar initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and pri- oritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.
Article
Perfluoroalkyl substances (PFASs) have been shown to cause abnormal levels of thyroid hormones (THs) in experimental animals, but the molecular mechanism is poorly understood. Here, a fluorescence displacement assay was used to determine the binding affinities of 16 PFASs with two major TH transport proteins, transthyretin (TTR) and thyroxine-binding globulin (TBG). Most of the tested PFASs bound TTR with relative potency (RP) values of 3 × 10−4 to 0.24 when compared with that of the natural ligand thyroxine, whereas fluorotelomer alcohols did not bind. Only perfluorotridecanoic acid and perfluorotetradecanoic acid bound TBG, with RP values of 2 × 10−4 when compared with that of thyroxine. Based on these results, it was estimated that displacement of T4 from TTR by perfluorooctane sulfonate and perfluorooctanoic acids would be significant for the occupationally exposed workers but not the general population. Structure-binding analysis revealed that PFASs with a medium chain length and a sulfonate acid group are optimal for TTR binding, and PFASs with lengths longer than 12 carbons are optimal for TBG binding. Three mutant proteins were prepared to examine crucial residues involved in the binding of PFASs to TH transport proteins. TTR with a K15G mutation and TBG with either a R378G or R381G mutation showed decreased binding affinity to PFASs, indicating that these residues play key roles in the interaction with the compounds. Molecular docking showed that the PFASs bind to TTR with their acid group forming a hydrogen bond with K15 and the hydrophobic chain towards the interior. PFASs were modeled to bind TBG with their acid group forming a hydrogen bond with R381 and the hydrophobic chain extending towards R378. The findings aid our understanding of the behavior and toxicity of PFASs on the thyroid hormone system.
Article
Triclosan, triclocarban, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), and bisphenol A (BPA) have been reported to disturb thyroid hormone (TH) homeostasis. We have examined the effects of these chemicals on sodium/iodide symporter (NIS)-mediated iodide uptake and the expression of genes involved in TH synthesis in rat thyroid follicular FRTL-5 cells, and on the activity of thyroid peroxidase (TPO) using rat thyroid microsomes. All four chemicals inhibited NIS-mediated iodide uptake in a concentration-dependent manner. A decrease in the iodide uptake was also observed in the absence of sodium iodide. Kinetic studies showed that all four chemicals were non-competitive inhibitors of NIS, with the order of Ki values being triclosan < triclocarban < BDE-47 < BPA. The transcriptional expression of three genes involved in TH synthesis, Slc5a5, Tpo, and Tgo, and three thyroid transcription factor genes, Pax8, Foxe1, and Nkx2-1, was examined using quantitative real-time PCR. No significant changes in the expression of any genes were observed with triclosan or triclocarban. BDE-47 decreased the level of Tpo, while BPA altered the expression of all six genes. Triclosan and triclocarban inhibited the activity of TPO at 166 and > 300 μM, respectively. Neither BDE-47 nor BPA affected TPO activity. In conclusion, triclosan, triclocarban, BDE-47, and BPA inhibited iodide uptake, but had differential effects on the expression of TH synthesis-related genes and the activity of TPO.
Article
We developed and characterized the human luciferase reporter cell line PZ-TR for the assessment of thyroid receptor (TR) transcriptional activity. Triiodothyronine (T3) induced luciferase activity in a dose-dependent manner, and the sensitivity of assay allowed for the detection of nanomolar T3 concentrations. The luciferase activity was induced by a maximum of (2.42 ± 0.14)-(2.73 ± 0.23)-fold after 24 h of exposure to 10 nM T3. We did not observe a nonspecific induction of luciferase activity by other steroid hormones and VDR ligands, with the exception of partial activation by retinoic acids. Cryopreservation of PZ-TR cells did not influence their functionality, responsivity to T3, or cell morphology, even after long-term cultivation. PZ-TR cells were used to evaluate the effects of organic tin compounds on TR. We found that the tributyltin and triphenyltin derivatives induced luciferase activity and that application of organotins in combination with T3 enhanced the effect of T3. These findings indicate that organic tin compounds have potential to interfere with TR-mediated regulation of gene expression and influence the physiological activity of thyroid hormones.