Modulation of iodide uptake by dialkyl phthalate plasticisers in FRTL-5 rat thyroid follicular cells

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Abstract

Plasticisers imparting flexibility to plastics are man-made chemicals abundantly present in the environment. Effects of six different dialkyl phthalates were studied in vitro in the rat thyroid cell line FRTL-5 on their ability to modulate basal iodide uptake mediated by the sodium/iodide symporter (NIS). The present study shows that diisodecyl phthalate (DIDP), dioctyl phthalate (DOP), diisononyl phthalate (DINP) and bis (2-ethylhexyl) phthalate (DEHP) significantly enhance iodide uptake when concentrations in the magnitude between 10−4 M and 10−3 M were applied. In this range, these phthalates do not assess toxicity on the cells. Specific inhibiton of NIS demonstrated that enhancement of iodide uptake is due to NIS. In contrast, benzyl butyl phthalate (BBP) also augments iodide uptake at 1 mM but this concentration has just exceeded the toxicity threshold and dibutyl phthalate (DBP), the most toxic compound did not modulate iodide uptake at any concentration applied. As we can deduce from our results, plasticisers are capable of significantly modulating NIS mediated iodide uptake activity.

Introduction

Plasticisers like dialkyl phthalates (phthalic acid diesters) and phenol derivatives indespensable for flexibility and durability of plastics, are man-made chemicals which are widely distributed environmental contaminants due to their use in consumer products (e.g. PVC, cosmetics, perfumes), food packaging as well as in medical devices. These chemicals are not covalently bound to the plastic matrix. They leach out of, e.g. PVC when they come into contact with lipophilic substances. Due to the permanently increasing presence in the environment and their putative interference with human health, the study of effects of these environmental chemicals on humans are of significant scientific interest. Especially since an interfering role or endocrine disrupting activity of these compounds with endocrine systems, e.g. estrogenic and anti-estrogenic activities are discussed (Cooper and Kavlock, 1997, McLachlan, 2001, Okubo et al., 2003). Environmental chemicals, to do this do so most often with receptors derived from the steroid/thyroid/retinoid gene family (McLachlan, 2001). According to the capability of these compounds to alter the endocrine system they were termed endocrine disrupters (ED).

Major phthalate plasticisers are diisodecyl phthalate (DIDP), benzyl butyl phthalate (BBP), bis (2-ethylhexyl) phthalate (DEHP), dioctyl phthalate (DOP), diisononyl phthalate (DINP) and dibutyl phthalate (DBP). The ubiquity of these compounds is well known and their presence has been reported in river water (μg/l), drinking water, outdoor and indoor air (Huber et al., 1996). In the EU and North America, DEHP is produced in huge amounts and is according to this one of the most abundant phthalates in the environment. Levels of exposure in the general population are estimated to be in the order of tens of micrograms per kilogram per day (Shea, 2003). Carcinogenic and tumor promoting activity of phthalates in rodents has also been reported (Diwan et al., 1985, Ward et al., 1983, Kohno et al., 2004). Phthalates bioaccumulate in invertebrates, fish and plants but do not biomagnify, because higher animals efficiently metabolize and excrete phthalates. DEHP and DINP and their metabolites are best studied and have received considerable attention recently because of specific concerns about pediatric exposures (Shea, 2003). These are either dietary or medical exposures. It is estimated that the total intake of, e.g. DEHP, excluding non-dietary ingestion is higher in all children younger than 19 years than in adults (Meek and Chan, 1994). Environmental investigations of wildlife populations have suggested that synthetic chemicals including plasticisers could lead to an imbalance of thyroid function but it is still unclear whether the human thyroid is also a target (Brucker-Davis, 1998). In other studies, phthalates as well as phenol derivatives plasticisers were used to elucidate effects on the thyroid. As effects of DEHP on the thyroid metabolism and morphology in rats, the induction of hyperactivity and changes in the colloid were described (Hinton et al., 1986). Furthermore, BBP inhibited the T3 binding to quail transthyretin (Ishihara et al., 2003). Moreover, it is thought that due to a perpetual increasing exposure of humans, especially young children and adolescents to plasticisers, leached out of plastics and as a consequence of this, existing as contaminants in the environment, beside other organs, the development and function of the thyroid can be disturbed leading to dysfunction (Hinton et al., 1986, Price et al., 1988, Poon et al., 1997, Brucker-Davis, 1998, Howarth et al., 2001, Shea, 2003).

The key molecules of iodide metabolism in follicullar cells of the thyroid are the sodium/iodide symporter (NIS), the thyroid peroxidase (TPO) and thyroglobulin (TG). Iodide is actively transported by NIS via the basolateral membrane and transported via pendrin or by other still unknown molecules through the apical membrane into the lumen of a follicle, where it is covalently attached to tyrosyl residues in TG by TPO by a mechanism known as iodination (Dunn and Dunn, 2001). NIS is an intrinsic plasma membrane glycoprotein located at the basolateral site of thyroid follicular cells. It concentrates iodide about 40-fold in an ATP-dependent manner. The expression of the NIS gene is also observed in several other tissues (De La Vieja et al., 2000, Dohan and Carrasco, 2003). Since cloning of NIS many different strategies were invented to modulate NIS function. Especially for cancer therapeutic purpose, it was thought to augment or to induce NIS expression in tumors using different compounds and gene therapeutic approaches (Spitzweg and Morris, 2002, Dadachova and Carrasco, 2004).

The aim of the present in vitro study is to elucidate whether the six different dialkyl phthalates exert effects on a key step of thyroid hormone biosynthesis like the uptake of iodide in thyroid cells mediated by the sodium/iodide symporter (NIS). Because of the non-availability of an adequate thyroid follicular cell line of human origin, we used in our study the well characterized rat thyroid cell line FRTL-5 (Ambesi-Impiombato et al., 1980) which represents the differentiated characteristics as well as exhibits the essential key steps in the metabolism of a thyroid follicular cell.

Section snippets

Cell culture

FRTL-5 rat cells were grown under defined conditions as described by Behr et al. (1998) with minor modifications. These consist of growing the cells in a 1:1 mixture of Click's Medium (EHAA) (Sigma, Germany) and RPMI 1640 (Gibco, Germany). The cells were cultured under basal conditions in this medium, when not otherwise stated. Only under these basal culture conditions, FRTL-5 cells represent the biology and characteristics of a fully metabolic active thyroid cell suited for testing effects of

Effect of phthalates on cell viability

In initial experiments, we examined whether the FRTL-5 cells are accessible for cytotoxicity of plasticisers using the MTS cell proliferation assay. This was necessary to determine in which concentration ranges phthalates should be applied for iodide uptake. Approximately 3 × 104 cells were grown in wells of a 96 well plate for 72 h in the presence of dilutions made of each chemical in the range between 10−1 M and 10−10 M. The detection solution containing MTS was added directly into the medium and

Discussion

Several recent lines of evidence mainly based on in vivo studies in laboratory animals indicate that the function of the thyroid is also a target of phthalate plasticisers (Hinton et al., 1986, Price et al., 1988, Poon et al., 1997, Brucker-Davis, 1998, Howarth et al., 2001). In the present functional study on iodide uptake mediated by NIS, the effects of six different dialkyl phthalates on iodide uptake in FRTL-5 rat thyroid cells were examined. We found that diisodecyl phthalate, dioctyl

Acknowledgement

This study (EU QLK4-CT-2002-02637 “ENDOMET”) is part of ENDOMET which is a European Commission Research Directorate supported project (QLRT-2001-02637) in the Fifth Framework Programme.

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