There are several possible sites on the Hypothalamus-Pituitary-Thyroid (HPT) axis for analysed environmental contaminants to act. In MiSSE, several approaches were investigated to identify and evaluate the potency of the thyroid hormone disruptors of human relevance. The major conclusion of the project was that there is reason for concern regarding exposure to thyroid hormone-disrupting compounds (THDCs) for both humans and pets. Several studies pointed to potential risks posed by chemical mixtures. All referensen can be found in Publications.
In silico –
We have used in silico modelling to predict the binding of dust associated compounds to bind to the transport protein transthyretin (TTR) and the thyroid receptor (Zhang et al 2015, 2016a, 2016b). Results showed that that the developed virtual screening protocols for the TTR and thyroid receptor binding were able to successfully identify potentials THDCs, and it was suggested that they can be used to propose THDCs for future toxicological evaluations. The studies also highlighted the importance of metabolic activation for TTR binding potency.
Graphical abstract in Zhang et al 2016
In vitro-
Based on literature review (Weiss et al 2015) and chemical analysis we created three complex mixtures of 25 human relevant THDCs, in molar ratios representing the internal blood levels of adults and newborns and found in normal house dust (Hamers et al 2020). These mixtures were tested in the newly established transthyretin binding assay using a fluorescent probe for high throughput screening developed in this project (Quyang et al 2017).
In concordance with previous studies, the TTR binding potency of the complex mixtures were well predicted by the concentration addition model (Hamers et al 2020). Using a 20% inhibition level as the threshold, the median concentrations in maternal and infant serum was extrapolated to correspond to a 1.3% inhibition of T4-TTR binding in maternal and 1.5% in infant blood. These levels were further predicted to correspond to 6.2% and 4.9%, respectively, for high-end serum concentrations. Based on these results we hypothesized, that these potential inhibitions of T4-TTR binding may ultimately be decisive for reaching a status of maternal hypothyroidism or hypothyroxinemia associated with impaired neurodevelopment in children. It is important to stress that only 25 THDCs were tested here, whereas we know that the exposure to synthetic chemicals is so much larger.
Figure 2. Concentration–response curves for the reconstituted mixtures based on the ratio of median concentrations reported in infant serum from Nordic countries. The vertical gray bar in plots B and C indicates the mixture concentration reflecting the median concentration of the individual compounds actually detected in maternal or infant blood samples from Nordic countries. Note: CA, concentration addition; CI, confidence interval; FITC, fluorescein 5-isothiocyanate; T4, thyroxine; TTR, transthyretin; TU, Toxic Unit. (Hamers et al 2020)
In vivo-
We performed in vivo testing on the AMA (amphibian metamorphosis assay) on a selection of THDCs from the MiSSE mixture. Frogs have an established TH dependent development and the assay is recommended by the OECD guideline for testing EDCs. It was shown that the halogenated organophosphorus flame retardant TDCiPP acts as a THDCs in metamorphosing tadpoles by causing increased epithelial cell height in thyroid glands at levels of 10 ng TDCiPP/L (Carlsson et al 2019). It is not straightforward to extrapolate the exposure in an in vivo experiment to the real-life situation, but levels of around 1 µg TDCiPP/g dust were found in this project in Swedish household dust (Li et al 2019).
We have also tested individual compounds and mixtures in the zebrafish embryo toxicity test and conducted a review to identify all genes, proteins and compounds related to the thyroid system that have been altered after exposure to one of the 25 THDCs (Table 2 and Fig 4. in Spaan et al 2019).
Fig. 4. Visualization of the affiliations between genes, proteins and compounds related to the thyroid system that have been altered after exposure to one of the 25 selected compounds within 7 dpf. The circles around the genes/proteins show whether the gene transcript was up- and/or downregulated according to the gathered literature. Made on ConsensusPathDB. N.B. this is the interaction network in human, not zebrafish necessarily. (Spaan et al 2019.)
One of the conclusions were that authors considering thyroid hormone disrupting traits monitor behavioral effects significantly more commonly, and that the lack of standardized methods complicated the comparison of the results.
Table 2. Overview on summarized results from the gathered literature showing which endpoints showed adverse effects or not per compound and specific concentration ranges (lowest observed adverse effect concentrations). From the top down: non-halogenated, chlorinated, brominated, fluorinated. Reference numbers are specified in Table S1. Meta-analysis of effects is given in the lower rows. Percentage of compounds that were tested per endpoint and the percentage of positive responses are calculated. Values above 50% are indicated in bold. (Spaan et al 2019)
