Elsevier

Chemosphere

Volume 84, Issue 10, September 2011, Pages 1484-1488
Chemosphere

Pendrin mediates uptake of perchlorate in a mammalian in vitro system

https://doi.org/10.1016/j.chemosphere.2011.04.038Get rights and content

Abstract

Perchlorate is a known endocrine disruptor present in groundwater, vegetables and dairy food products in many regions of the United States. It interferes with the uptake of iodide into the thyrocyte by the sodium–iodide symporter at the basolateral surface, thus potentially disrupting the synthesis of thyroid hormone. Because transport of iodide from the thyroid follicular cells to the follicular lumen is mediated by the protein pendrin at the apical surface, we hypothesized that perchlorate may also interact with this protein. Therefore, HeLa cells were transfected with the human SLC26A4 gene, which encodes pendrin, to generate an in vitro mammalian system expressing the recombinant pendrin protein (HeLa-PDS). The HeLa-PDS cells, along with untransfected cells, were then cultured in presence of iodide and/or perchlorate. Intracellular levels of these two chemicals were measured by ion chromatography tandem mass spectrometry. Results from this study show that iodide and perchlorate uptake increases significantly in HeLa-PDS cells as compared to untransfected cells. Thus, recombinant HeLa cells expressing pendrin protein accumulate iodide and perchlorate intracellularly, indicating that pendrin is involved in the uptake of perchlorate. Additional results from this study suggest that iodide and perchlorate competitively inhibit each other for uptake by pendrin. The ability of perchlorate to compete with iodide for uptake by both basal and apical transporters may increase the potential of perturbation of thyroid homeostasis and therefore the estimated risk posed to susceptible human populations.

Highlights

► This study was designed to determine whether or not pendrin is able to mediate perchlorate uptake. ► A mammalian in vitro system was generated and used to assess perchlorate uptake by pendrin. ► The recombinant cells expressing pendrin accumulate iodide and perchlorate intracellularly. ► We conclude that pendrin might be involved in the uptake of perchlorate.

Introduction

The iodide-containing hormones triiodothyronine (T3) and its precursor thyroxine (T4) are synthesized in the thyroid and are essential for normal development, growth and regulation of several metabolic pathways (Zoeller, 2007). A variety of industrial chemicals reduce circulating levels of thyroid hormone, thus behaving as endocrine disruptors and resulting in adverse health outcomes. Perchlorate is one of these chemicals. It is an inorganic anion (ClO4) that behaves as endocrine disruptor by blocking iodine uptake into the thyroid. In a cross sectional study of the US population, perchlorate has been associated with decreased serum thyroxine levels in women with low iodine levels (Blount et al., 2006).

Perchlorate derives from both natural and anthropogenic sources and is a widespread, environmentally stable contaminant found in both irrigation and drinking waters and therefore in vegetables such as lettuce, tomatoes and soybeans as well as in dairy milk and breast milk (ATSDR, 2008). Contamination from anthropogenic sources results mostly from its use as an oxidant in solid rocket propellants, fireworks and airbag deployment systems. Because of its environmental stability, it can be found in many regions of the United States.

In humans, the primary concern with regard to perchlorate exposure is the potential of reduction of iodide uptake into the thyroid gland and subsequent decrease of thyroid hormone production. This effect is mediated by the ability of perchlorate to competitively inhibit the sodium-iodide symporter (NIS), which is located at the basolateral membrane of thyroid follicular cells and transports iodide into the cytoplasm (Dohán et al., 2003, Dohán et al., 2007). Indeed, perchlorate is a potent NIS inhibitor and is itself transported into the thyroid. Its potency, when compared to the NIS inhibitors thiocyanate and nitrate, has been estimated to be 15 and 240 times higher, respectively, on a molar concentration basis (Tonacchera et al., 2004). However, in addition to iodide transport by NIS, synthesis of the thyroid hormone requires several additional steps, some of these also involved in iodide transport (Scinicariello et al., 2005).

Pendrin is an iodide transporter expressed at the apical membrane of thyroid follicular cells and mediates efflux of iodide across this membrane (Yoshida et al., 2002). Structurally, it is a glycoprotein composed of 780 amino acids and is encoded by the SLC26A4 gene, which contains 21 exons with an open reading frame of 2343 bp. It consists of 12 transmembrane domains with both amino and carboxy termini located inside the cytosol (Coyle et al., 1996, Sheffield et al., 1996). Once iodide reaches the cell-colloid interface following pendrin-mediated transport, it is oxidized and rapidly organified by incorporation into selected tyrosyl residues of thyroglobulin (Scinicariello et al., 2005). It might be possible that, once inside the thyrocyte following NIS-mediated transport, perchlorate is then transported by pendrin. Therefore, the objective of this study was to ascertain whether or not pendrin is involved in the uptake of perchlorate. To accomplish this objective, the human HeLa cell line was engineered to express recombinant human pendrin by transfection with human SLC26A4 cDNA and cultured in presence of perchlorate and iodide. Intracellular levels of perchlorate and iodide were then measured by ion chromatography tandem mass spectrometry.

Section snippets

Amplification, cloning, and sequence analysis of human pendrin cDNA

Human SLC26A4 cDNA was isolated from the human mammary MCF-7 cell line grown in presence of prolactin, as previously described (Rillema and Hill, 2003). Total RNA was extracted from the cultured cells using the QIAamp RNA Blood MiniKit (Qiagen), and reverse transcribed into cDNA using oligo(dT)17 primers, followed by primer extension with the AMV reverse transcriptase (Roche Diagnostic Systems). PCR amplification of cDNA was performed with Expand High Fidelity polymerase (Roche Diagnostic

Results and discussion

A variety of industrial chemicals interfere with thyroid function and reduce circulating levels of thyroid hormone by acting at different steps of hormone synthesis, release, transport, metabolism and clearance. In many instances, the mechanisms responsible for the interference with the action of thyroid hormones are not well understood. The main function of the thyroid gland is to concentrate iodide (the form of iodine that enters the cell) and make it available for biosynthesis of thyroid

Acknowledgment

The authors thank Dr. Amy Delinsky at CDC for laboratory assistance.

This work was supported in part by the Research Program Enhancement from the Georgia State University Office of Research and Sponsored Programs, and by the Georgia Research Alliance.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Agency for Toxic Substances and Disease Registry and of the Centers for Disease Control and Prevention.

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