Development and validation of in vitro bioassays for thyroid hormone receptor mediated endocrine disruption.
Projectomschrijving
Onderzoekers hebben een nieuwe test gemaakt om zonder proefdieren te testen of chemische stoffen de werking van de schildklierhormoonreceptor kunnen verstoren. Hiervoor is een cellijn voorzien van een waarschuwingssysteem dat licht geeft als de receptor wordt geactiveerd. Normaal gebeurt dat door het natuurlijke schildklierhormoon, maar soms ook door chemische stoffen. De komende jaren moeten voor het Europese stoffen programma REACH vele tienduizenden stoffen getest worden op schildklierhormoon-verstorende werking. De nieuw ontwikkelde test kan helpen te voorkomen dat dit allemaal met proefdieren gebeurt. In de Verenigde Staten is de cellijn met succes in de praktijk getest in een robotsysteem, waarin het doormeten van ruim 5000 stoffen op activatie en remming van de schildklierhormoon receptor minder dan drie dagen duurde. Samen met andere onderzoekers wordt er aan een plan gewerkt om met een geselecteerde batterij van diervrije (in vitro) testen de belangrijkste risico’s op schildklierhormonverstroing te kunnen uitsluiten. De nieuw ontwikkelde TR-GH3.Luc test is één van de geadviseerde testen.
Producten
Titel: Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay.
Auteur: Freitas J, Cano P, Craig-Veit C, Goodson ML, David Furlow J, Murk AJ.
Auteur: Freitas J, Cano P, Craig-Veit C, Goodson ML, David Furlow J, Murk AJ.
Titel: Detection of thyroid hormone receptor disruptors by a novel stable in vitro
reporter gene assay
Auteur: Jaime Freitas, Patricia Cano, Christina Craig-Veit, Michael L. Goodson, J. David Furlow, Albertinka J. Murk.
Magazine: Toxicology in Vitro
Auteur: Jaime Freitas, Patricia Cano, Christina Craig-Veit, Michael L. Goodson, J. David Furlow, Albertinka J. Murk.
Magazine: Toxicology in Vitro
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Samenvatting van de aanvraag
Thyroid hormones regulate crucial processes in vertebrates such as reproduction, development and energy metabolism. At least two thyroid hormone receptors with a specific function exist: TRa and TRb. Endocrine disruption via the thyroid hormone system is gaining more attention, both from scientists because of the increasing incidence of hormone-related cancers and developmental defects, and from legislators demanding that newly marketed compounds are tested for thyroid hormone disruption. For mechanistic studies of the human thyroid system mostly rodents are used. For regulatory purposes the Endocrine Disruptor Screening and Testing Advisory Committe (EDSTAC) recommended to include in Tier 1 toxicity testing of chemicals a screening battery to identify chemicals that may affect the estrogen, androgen and thyroid hormone system. However, in contrast to the other endpoints, for screening of possible thyroid hormone disrupting effects no in vitro tests are available yet enabling rapid and high throughput in vitro screening of new and existing chemicals. Therefore a frog metamorphosis test is proposed in the Tier 1 screening battery. Due to the high natural variability associated with metamorphosis, at least 400-600 animals are needed per test, which means up to 60.000 animals may be required to test 100 selected chemicals according to the proposed tiered approach. To reduce the number of experimental animals used and to increase the insight into the mechanisms of toxic interference with thyroid hormone receptor (TR)-function, the aim of this project is to develop and validate functional in vitro bioassays for thyroid hormone receptor-mediated toxicity. These assays will be based on cell lines which, when transiently transfected with TRa or TRb-specific reporter gene expression systems, have recently been shown to detect TR-mediated toxic effects. We propose to develop stable mammalian cell lines based on these constructs to allow easy, fast and comprehensive testing. As several toxicants only interfere with the TRs after hydroxylation by CYP1A2 or 2B1, cell line variants also stably overexpressing these metabolizing enzymes will be constructed as well. The cell lines will be validated with standards known for their TR-mediated agonistic, antagonistic and potentiating effects. In addition, the assay will be implemented in a GLP environment, and the protocol will be standardized and applied accordingly.