Toxicology. 2016 Aug 10;368-369:69-79. doi: 10.1016/j.tox.2016.08.012.

Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants.




Endocrine-disrupting chemicals (EDCs) are chemicals that interfere with normal function of the endocrine system which governs the development and function of virtually all tissues and organs. Exposure to EDCs have been implicated in developmental anomalies, neuro-behavioral and learning deficits, as well as in recent increase in metabolic syndrome, diabetes, infertility, and endocrine-related cancers (De Coster and van Larebeke, 2012; Diamanti-Kandarakis et al., 2009; Diamanti-Kandarakis et al., 2010; Gore et al., 2015). EDCs can be found in food, water, household items, personal care products, and industrial waste (Yang et al., 2015). In United States, the health burden associated with exposure to these contaminants is estimated to cost over $340 Million annually (Attina et al., 2016).

Even though the dangers of EDC exposure are well recognized, and the economic and social burden are substantial, biologically-relevant assays to rapidly analyze large numbers of chemicals affecting endocrine system have been lacking (Roy and Pereira, 2005). Standard detection of EDCs in the environment relies on expensive and laborious analysis of chemical structures using HPLC, MS/GS and related technologies. These methods are time consuming and frequently fail to identify a specific chemical structure because many biologically-active compounds are subjected to bio-modifications in the environment which are not present in the currently existing libraries. The existing mammalian cells-based assays, such as the CALUX reporter gene assays, are based on transcriptional response (Simon et al., 2010; Steinberg, 2013) and even though sensitive, are time-consuming and difficult to interpret when screening for antagonistic activities. Therefore, there is yet a significant unmet need for rapid, sensitive, and biologically-relevant methods for detecting EDCs.

To address this need, we developed novel, high-throughput assay for biological testing of EDCs using mammalian cell lines that express GFP-tagged nuclear steroid receptor constructs (patent number: 9040248). High-throughput, high-content imaging is applied for an automatic detection and quantification of GFP-tagged nuclear receptor translocation from the cytoplasm to the nucleus. An algorithm for nuclear segmentation is then used to calculate the efficiency of receptor activation by compounds interacting with the hormone-binding domain of the receptor. We have previously described the use of this assay for detection of GFP-tagged glucocorticoid and androgen receptors translocation in response to natural and synthetic ligands. Using this approach, we screened more than 100 samples from US water sources and detected androgen activity in 35% and a previously unrecognized glucocorticoid activity in 27% of the samples (Stavreva et al., 2012).

The current manuscript describes the development and implementation of this assay for detection of thyroid receptor (TR)-interacting contaminants. Because TR isoforms are mostly nuclear in the absence of hormone (Baumann et al., 2001) and thus cannot be used directly for a nuclear translocation assay, we generated a fusion construct of ligand-binding domain (LBD) of TR beta and GFP-tagged N terminal domain of glucocorticoid receptor (GR). Similar approach was used previously to generate fluorescent chimeric molecules by fusion of GR and the retinoic acid receptor as well as GR and estrogen receptor (Mackem et al., 2001; Martinez et al., 2005). We developed a novel mammalian cell line expressing cytoplasmic GFP-GR-TR beta chimeric construct capable of translocating in response to thyroid hormones and known EDCs such as BPA and TBBPA. We also screened over 100 concentrated water samples from US water sources and detected a low, but reproducible contamination in 53 % of the samples. This method can be readily extended to cell lines expressing other nuclear receptors and will greatly facilitate detection of various classes of EDCs in the environment. This information is much needed for the development of comprehensive policy on EDCs in the environment.



Figure : Principles of the translocation assay for detection of the EDCs. (A) Schematic representation of the GFP-GR-TR beta chimeric construct. (B) Schematic representation of GFP-GR-TR beta translocation in response to hormonal treatment. (C) Cell micrographs showing GFP-GR-TR beta translocation from the cytoplasm to the nucleus upon stimulation with thyroid hormone. Control (DMSO) treatment failed to induce GFP-GR-TR beta translocation. Nuclei are stained with DAPI. Scale bar, 10 μm.




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