Effects of Perfluoroalkyl Compounds on mRNA Expression Levels of Thyroid Hormone-Responsive Genes in Primary Cultures of Avian Neuronal Cells

Department of Biology, Centre for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5.
Toxicological Sciences (Impact Factor: 3.85). 03/2011; 120(2):392-402. DOI: 10.1093/toxsci/kfq395
Source: PubMed


There is growing interest in assessing the neurotoxic and endocrine disrupting potential of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, prenatal and postnatal development and behavior. PFC exposure altered hormone levels and the expression of hormone-responsive genes in mammalian and aquatic species. This study is the first to assess the effects of PFCs on messenger RNA (mRNA) expression in primary cultures of neuronal cells in two avian species: the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following thyroid hormone (TH)-responsive genes were examined using real-time reverse transcription-PCR: type II iodothyronine 5'-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif-binding factor (Oct-1), and myelin basic protein. Several PFCs altered the mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. Short-chained PFCs (less than eight carbons) altered the expression of TH-responsive genes (D2, D3, TTR, and RC3) in chicken embryonic neuronal cells to a greater extent than long-chained PFCs (more than or equal to eight carbons). Variable transcriptional changes were observed in herring gull embryonic neuronal cells exposed to short-chained PFCs; mRNA levels of Oct-1 and RC3 were upregulated. This is the first study to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and may provide insight into the possible mechanisms of action of PFCs in the avian brain.

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    • "PFCs significantly antagonized the T3-induced cell proliferation . Vongphachan et al. (2011) investigated the effect of seven PFAAs and four PFASs on the TH-responsive gene expression in primary cultures of chicken embryonic neuronal and found several PFCs including PFOS-altered gene expression levels in the same manner as T3 did. In another study, PFOS was found to exhibit TR antagonistic activity by the reporter gene assay (Du et al. 2013). "
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    ABSTRACT: Perfluoroalkyl compounds (PFCs) have been shown to disrupt thyroid functions through thyroid hormone receptor (TR)-mediated pathways, but direct binding of PFCs with TR has not been demonstrated. We investigated the binding interactions of 16 structurally diverse PFCs with human TR, their activities on TR in cells, and the activity of perfluorooctane sulfonate (PFOS) in vivo. In fluorescence competitive binding assays, most of the 16 PFCs were found to bind to TR with relative binding potency in the range of 0.0003-0.05 compared with triiodothyronine (T3). A structure-binding relationship for PFCs was observed, where fluorinated alkyl chain length longer than ten, and an acid end group were optimal for TR binding. In thyroid hormone (TH)-responsive cell proliferation assays, PFOS, perfluorohexadecanoic acid, and perfluorooctadecanoic acid exhibited agonistic activity by promoting cell growth. Furthermore, similar to T3, PFOS exposure promoted expression of three TH upregulated genes and inhibited three TH downregulated genes in amphibians. Molecular docking analysis revealed that most of the tested PFCs efficiently fit into the T3-binding pocket in TR and formed a hydrogen bond with arginine 228 in a manner similar to T3. The combined in vitro, in vivo, and computational data strongly suggest that some PFCs disrupt the normal activity of TR pathways by directly binding to TR.
    Archives of Toxicology 05/2014; 89(2). DOI:10.1007/s00204-014-1258-y · 5.98 Impact Factor
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    • "Effects on the TH pathway were assessed because previous studies reported reduced TH levels (i.e., triiodothyronine [T3] and thyroxine [T4]) in rodents exposed to PFAAs (Chang et al., 2008; Martin et al., 2007). Vongphachan et al. (2011) demonstrated that short-chained PFAAs (C < 8) altered the expression of TH-responsive genes, including type II and III 5´-deiodinases (d2 and d3), transthyretin (ttr), and neurogranin (rc3), in chicken embryonic neuronal cells to a greater extent than long-chained PFAAs (C ≥ 8). Furthermore, among 24 PFAAs examined, PFHxS had the strongest binding potency for ttr and was able to displace T4 from binding to ttr (Weiss et al., 2009). "
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    ABSTRACT: In a recent egg injection study, we showed that in ovo exposure to perfluorohexane sulfonate (PFHxS) affects the pipping success of developing chicken (Gallus gallus domesticus) embryos. We also found evidence of thyroid hormone (TH) pathway interference at multiple levels of biological organization (i.e., somatic growth, messenger RNA expression, and circulating free thyroxine levels). Based on these findings, we hypothesize that PFHxS exposure interferes with TH-dependent neurodevelopmental pathways. This study investigates global transcriptional profiles in cerebral hemispheres of chicken embryos following exposure to a solvent control, 890 or 38,000ng PFHxS/g egg (n = 4-5 per group); doses that lead to the adverse effects indicated above. PFHxS significantly alters the expression (≥ 1.5-fold, p ≤ 0.001) of 11 transcripts at the low dose (890ng/g) and 101 transcripts at the high dose (38,000ng/g). Functional enrichment analysis shows that PFHxS affects genes involved in tissue development and morphology, cellular assembly and organization, and cell-to-cell signaling. Pathway and interactome analyses suggest that genes may be affected through several potential regulatory molecules, including integrin receptors, myelocytomatosis viral oncogene, and CCAAT/enhancer-binding protein. This study identifies key functional and regulatory modes of PFHxS action involving TH-dependent and -independent neurodevelopmental pathways. Some of these TH-dependent mechanisms that occur during embryonic development include tight junction formation, signal transduction, and integrin signaling, whereas TH-independent mechanisms include gap junction intercellular communication.
    Toxicological Sciences 07/2012; 129(2):380-91. DOI:10.1093/toxsci/kfs219 · 3.85 Impact Factor
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    ABSTRACT: Perfluoroalkyl acids (PFAAs), specifically perfluorinated sulfonates and carboxylates, are synthetic substances known for their chemical stability, resistance to degradation, and potential to biomagnify in food chains. The toxicological and biological effects of PFAAs in avian species are not well characterized, although there is some evidence to suggest that they can impact neurodevelopment and hatching success. Our laboratory recently reported significant effects of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) on messenger RNA (mRNA) levels of thyroid hormone (TH)-responsive genes in chicken embryonic neuronal cells. In this study, we determined in ovo effects of PFHxS and PFHxA exposure (maximum dose = 38,000 and 9700 ng/g egg, respectively) on embryonic death, developmental endpoints, tissue accumulation, mRNA expression in liver and cerebral cortex, and plasma TH levels. Pipping success was reduced to 63% at the highest dose of PFHxS; no effects were observed for PFHxA. PFHxS exposure (38,000 ng/g) decreased tarsus length and embryo mass. PFHxS and PFHxA accumulated in the three tissue compartments analyzed as follows: yolk sac > liver > cerebral cortex. Type II and type III 5'-deiodinases (D2 and D3) and cytochrome P450 3A37 mRNA levels were induced in liver tissue of chicken embryos exposed to PFHxS, whereas D2, neurogranin (RC3), and octamer motif binding factor 1 mRNA levels were upregulated in cerebral cortex. Plasma TH levels were reduced in a concentration-dependent manner following PFHxS exposure; PFHxA had no effect. This in ovo study successfully validated previous in vitro results concerning the modulation of TH-responsive genes and identified adverse effects associated with TH homeostasis in response to PFHxS treatment.
    Toxicological Sciences 02/2012; 127(1):216-24. DOI:10.1093/toxsci/kfs072 · 3.85 Impact Factor
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