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.
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    ABSTRACT: Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and tris(1-chloropropyl) phosphate (TCPP) belong to a group of chemicals collectively known as triester organophosphate flame retardants (OPFRs). OPFRs are used in a wide range of consumer products and have been detected in biota, including free-living avian species; however, data on toxicological and molecular effects of exposure are limited. An in vitro screening approach was used to compare concentration-dependent effects of TDCPP and TCPP on cytotoxicity and messenger RNA (mRNA) expression in cultured hepatocytes and neuronal cells derived from embryonic chickens. TDCPP was toxic to hepatocytes (LC₅₀ = 60.3 ± 45.8μM) and neuronal cells (LC₅₀ = 28.7 ± 19.1μM), whereas TCPP did not affect viability in either cell type up to the highest concentration administered, 300μM. Real-time reverse transcription-PCR revealed alterations in mRNA abundance of genes associated with phase I and II metabolism, the thyroid hormone (TH) pathway, lipid regulation, and growth in hepatocytes. None of the transcripts measured in neuronal cells (D2, D3, RC3, and Oct-1) varied in response to TDCPP or TCPP exposure. Exposure to ≥ 10μM TDCPP and TCPP resulted in significant upregulation of CYP2H1 (4- to 8-fold), CYP3A37 (13- to 127-fold), and UGT1A9 (3.5- to 7-fold) mRNA levels. Transthyretin was significantly downregulated more than twofold by TCPP at 100μM; however, TDCPP did not alter its expression. Liver fatty acid-binding protein, TH-responsive spot 14-α, and insulin-like growth factor-1 were all downregulated (up to 10-fold) in hepatocytes exposed to ≥ 0.01μM TDCPP and TCPP. Taken together, our results indicate that genes associated with xenobiotic metabolism, the TH pathway, lipid regulation, and growth are vulnerable to TDCPP and TCPP administration in cultured avian hepatocytes. The mRNA expression data were similar to those from a previous study with hexabromocyclododecane.
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