Fini JB et al. (2017), Human amniotic fluid contaminants alter thyroid...

XB-ART-53134

Sci Rep 2017 Mar 07;7:43786. doi: 10.1007/s00467-016-3372-y.

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Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos.

Fini JB , Mughal BB , Le Mével S , Leemans M , Lettmann M , Spirhanzlova P , Affaticati P , Jenett A , Demeneix BA .

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Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.

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Species referenced: Xenopus laevis
Genes referenced: bdnf ctrl dbp dio1 dio2 dio3 eef1a2 h3-3a ilf2 mbp ntsr1 odc1 pc.1 sox2 thra thrb tubb2b

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	Figure 1. Thyroid disrupting activity of individual chemicals assessed with XETA.Screening of thyroid disrupting activity of molecules measured in humans with the Xenopus Embryonic Thyroid Assay (XETA), based on the quantification of fluorescence-using the transgenic TH/bZip-eGFP e.g. [(Tg(thibz:eGFP)] line. Fifteen compounds were tested at different concentrations in presence of T3 5 × 10−9 M for 72 h. Scattered plots are shown with mean +/− SD of three to five independent experiments pooled (normalised on T3 to 100%). The GFP fluorescence in whole tadpoles (mainly heads) was measured and quantified after 72 h exposure. (a) Phenolic compounds: BPA, Triclosan and Benzophenone-3. (b) Phthalates: DBP and DEHP. (c) Organochlorine pesticides: HCB and 4′4-DDE. (d) Perfluorinated compounds: PFOA and PFOS. (e) Polyaromatic hydrocarbon: 2-Naphtol. (f) Halogenated compounds: Sodium perchlorate, PCB-153 and BDE-209. (g) Metals: Methylmercury and Lead chloride. Red arrowheads indicate concentrations of chemicals used in mix 1x (Table S1). Statistics were done with non-parametric Kruskal-Wallis test (p < 0.05, p < 0.01, p < 0.001, **p < 0.0001). Hashes (###) represent p < 0.001, T3 vs Control using column to column comparison (non parametric Mann Whitney).
	Figure 2. Thyroid disrupting activity of mixture assessed with XETA.Screening of thyroid disrupting activity of mixture of 15 molecules at concentrations measured in human amniotic fluid (mix 1x) 10 times more concentrated (mix 10x) and 10 times less concentrated (mix 0.1x) using Tg(thibz:eGFP) tadpoles. (a) GFP fluorescence (mainly localised in heads) of whole tadpoles exposed to mixture at 0.1x, 1x, 10x or a TR antagonist NH-3 (1 μM) with (right) or without (left) a T3 spike at 5 × 10−9 M. Quantification was done on images taken at 72 h exposure. Scattered dot plots are shown with mean +/− SD of five independent pooled experiments (normalised against T3). Statistics used non-parametric Kruskal-Wallis test (p < 0.05, p < 0.01, p < 0.001, **p < 0.0001). Hashes (###) represent p < 0.001, T3 vs Control using column-to-column comparison. (b–d) Histograms represent mean (+/SEM) of relative fluorescence units (RFU) of GFP in forebrain (b), midbrain (c) and hindbrain (d) of tadpoles exposed to mixture for 72 h in the absence of T3. Regions were delimited manually on ventral brain images (see Fig. S2c). Statistics used non parametric Kruskal Wallis compared to CTRL.
	Figure 3. Mixture exposure modifies thyroid hormone and neuronal development related gene expression in brain.Wild type NF45 X. laevis tadpoles were exposed to mixture for 72 h in the absence of T3. For each concentration tested between 7 to 12 pools of three brains were used from at least four independent experiments. Total brain mRNA transcripts levels were quantified using RT-qPCR: (a), dio1 (b), dio2 (c), dio3 (d), thra (e), thrb (f), sox2 (g), tubb2b (h), mbp (i), bdnf. Relative fold changes were calculated using geometric mean of ef1a and odc as normalizers. Results are presented as fold changes using a log2 scale and DMSO-treated animals (CTRL) values for the 1.0 reference. Statistics were done on dCts and used Kruskal-Wallis tests (Box plots median and quartiles), p < 0.05, p < 0.01, p < 0.001, **p < 0.0001.
	Figure 4. Mixture enhances proliferation in brain, modifies neural cell populations and behaviour.(a) Dorsal views of brains of mixture exposed wild type NF45 X. laevis tadpoles. Immunohistochemistry used the anti-PH3 antibody (red, mitosis) and DAPI (blue, nucleus). Scale bar, 200 μm. (b) Numbers of proliferating cells in (PH3 + cells) in tadpole brains following mixture exposure. n = 13 brains per condition, 5 independent experiments pooled (representative number of positive cells in each brain). Statistics used 2-way ANOVA and Dunnett’s post-test (Medians ± SDs, p < 0.05,**p < 0.0001) (c) CLARITY imaging illustrating the region of interest delimited for analysis in (d–g) on hind brain. Examples of control (left) and mix 1x exposed (right) double transgenic tadpoles Tg(nβt:DSRED) (neurons, red) and Tg(Mmu.mbp:NTR-eGFP) (oligodendrocyte, green). Scale bar, 200 μm. (d–g) Quantification of CLARITY signals obtained for each fluorescent signal in hindbrain. Neuron (d) and oligodendrocyte (e) numbers and cell volumes (f,g), n = 3 to 5 brains, Statistics used non parametric Kruskal Wallis ANOVA and Dunn’s post-test (Means ± SDs, p < 0.05, p < 0.01) (h) Wild type NF45 X. laevis tadpoles were exposed to DMSO (CTRL), or mixture (0.1x, 1x, 10x), T3 5 × 10−9 M for 72 h for mobility analysis. Example of total distance covered in 10 mins under 30 secs/30 secs light (blue lines)/dark (grey lines) cycles by one tadpole per condition (i) Mean distance covered during 10 mins under different conditions. Distance is normalized versus controls for 4 independent experiments with n = 12 per experiment. Representation uses scattered dot plots Mean +/− SD. Statistics used meta-analysis with Kruskal-Wallis. Note that stars directly over a group indicates significant difference with CTRL group (Error bars indicate s.e.m, p < 0.01, ****p < 0.0001).

References [+] :

Abdelouahab, Maternal and cord-blood thyroid hormone levels and exposure to polybrominated diphenyl ethers and polychlorinated biphenyls during early pregnancy. 2013, Pubmed

	Figure 1. Thyroid disrupting activity of individual chemicals assessed with XETA.Screening of thyroid disrupting activity of molecules measured in humans with the Xenopus Embryonic Thyroid Assay (XETA), based on the quantification of fluorescence-using the transgenic TH/bZip-eGFP e.g. [(Tg(thibz:eGFP)] line. Fifteen compounds were tested at different concentrations in presence of T3 5 × 10−9 M for 72 h. Scattered plots are shown with mean +/− SD of three to five independent experiments pooled (normalised on T3 to 100%). The GFP fluorescence in whole tadpoles (mainly heads) was measured and quantified after 72 h exposure. (a) Phenolic compounds: BPA, Triclosan and Benzophenone-3. (b) Phthalates: DBP and DEHP. (c) Organochlorine pesticides: HCB and 4′4-DDE. (d) Perfluorinated compounds: PFOA and PFOS. (e) Polyaromatic hydrocarbon: 2-Naphtol. (f) Halogenated compounds: Sodium perchlorate, PCB-153 and BDE-209. (g) Metals: Methylmercury and Lead chloride. Red arrowheads indicate concentrations of chemicals used in mix 1x (Table S1). Statistics were done with non-parametric Kruskal-Wallis test (p < 0.05, p < 0.01, p < 0.001, **p < 0.0001). Hashes (###) represent p < 0.001, T3 vs Control using column to column comparison (non parametric Mann Whitney).
	Figure 2. Thyroid disrupting activity of mixture assessed with XETA.Screening of thyroid disrupting activity of mixture of 15 molecules at concentrations measured in human amniotic fluid (mix 1x) 10 times more concentrated (mix 10x) and 10 times less concentrated (mix 0.1x) using Tg(thibz:eGFP) tadpoles. (a) GFP fluorescence (mainly localised in heads) of whole tadpoles exposed to mixture at 0.1x, 1x, 10x or a TR antagonist NH-3 (1 μM) with (right) or without (left) a T3 spike at 5 × 10−9 M. Quantification was done on images taken at 72 h exposure. Scattered dot plots are shown with mean +/− SD of five independent pooled experiments (normalised against T3). Statistics used non-parametric Kruskal-Wallis test (p < 0.05, p < 0.01, p < 0.001, **p < 0.0001). Hashes (###) represent p < 0.001, T3 vs Control using column-to-column comparison. (b–d) Histograms represent mean (+/SEM) of relative fluorescence units (RFU) of GFP in forebrain (b), midbrain (c) and hindbrain (d) of tadpoles exposed to mixture for 72 h in the absence of T3. Regions were delimited manually on ventral brain images (see Fig. S2c). Statistics used non parametric Kruskal Wallis compared to CTRL.
	Figure 3. Mixture exposure modifies thyroid hormone and neuronal development related gene expression in brain.Wild type NF45 X. laevis tadpoles were exposed to mixture for 72 h in the absence of T3. For each concentration tested between 7 to 12 pools of three brains were used from at least four independent experiments. Total brain mRNA transcripts levels were quantified using RT-qPCR: (a), dio1 (b), dio2 (c), dio3 (d), thra (e), thrb (f), sox2 (g), tubb2b (h), mbp (i), bdnf. Relative fold changes were calculated using geometric mean of ef1a and odc as normalizers. Results are presented as fold changes using a log2 scale and DMSO-treated animals (CTRL) values for the 1.0 reference. Statistics were done on dCts and used Kruskal-Wallis tests (Box plots median and quartiles), p < 0.05, p < 0.01, p < 0.001, **p < 0.0001.
	Figure 4. Mixture enhances proliferation in brain, modifies neural cell populations and behaviour.(a) Dorsal views of brains of mixture exposed wild type NF45 X. laevis tadpoles. Immunohistochemistry used the anti-PH3 antibody (red, mitosis) and DAPI (blue, nucleus). Scale bar, 200 μm. (b) Numbers of proliferating cells in (PH3 + cells) in tadpole brains following mixture exposure. n = 13 brains per condition, 5 independent experiments pooled (representative number of positive cells in each brain). Statistics used 2-way ANOVA and Dunnett’s post-test (Medians ± SDs, p < 0.05,**p < 0.0001) (c) CLARITY imaging illustrating the region of interest delimited for analysis in (d–g) on hind brain. Examples of control (left) and mix 1x exposed (right) double transgenic tadpoles Tg(nβt:DSRED) (neurons, red) and Tg(Mmu.mbp:NTR-eGFP) (oligodendrocyte, green). Scale bar, 200 μm. (d–g) Quantification of CLARITY signals obtained for each fluorescent signal in hindbrain. Neuron (d) and oligodendrocyte (e) numbers and cell volumes (f,g), n = 3 to 5 brains, Statistics used non parametric Kruskal Wallis ANOVA and Dunn’s post-test (Means ± SDs, p < 0.05, p < 0.01) (h) Wild type NF45 X. laevis tadpoles were exposed to DMSO (CTRL), or mixture (0.1x, 1x, 10x), T3 5 × 10−9 M for 72 h for mobility analysis. Example of total distance covered in 10 mins under 30 secs/30 secs light (blue lines)/dark (grey lines) cycles by one tadpole per condition (i) Mean distance covered during 10 mins under different conditions. Distance is normalized versus controls for 4 independent experiments with n = 12 per experiment. Representation uses scattered dot plots Mean +/− SD. Statistics used meta-analysis with Kruskal-Wallis. Note that stars directly over a group indicates significant difference with CTRL group (Error bars indicate s.e.m, p < 0.01, ****p < 0.0001).