Effects of the anti-thyroidal compound potassium-perchlorate on the thyroid system of the zebrafish
Highlights
► We investigated the effects of perchlorate on the thyroid system of the zebrafish. ► In the thyroid, the colloid reacted most sensitive to the exposure to perchlorate. ► The pituitary displayed statistically significant increases of TSH-producing cells. ► A histologically observable increase of adenohypophyseal tissue was observed. ► Glycogen depletion could be observed in the liver.
Introduction
The thyroid system in vertebrates is essential for growth, development, and aspects of reproduction (Brown et al., 2004, Cyr and Eales, 1988, Leatherland, 1994, Power et al., 2001). Whereas it is well-studied in mammals (Momotani et al., 2000, Schreiber, 2002, York et al., 2003) and amphibians (Grim et al., 2009, Huang et al., 2001, Regard and Mauchamp, 1971, Tata, 2006), information on the thyroid system in fish is scarce and only several studies about effects of industrial chemical compounds exist (Bradford et al., 2005, Grau, 1988, Schmidt and Braunbeck, 2011, van der Ven et al., 2006). Nevertheless, for today's chemical regulatory purposes the zebrafish is one of the most important test organisms and thus its possible usability in screening tests for goitrogens is highly appreciated.
The present study was designed to elucidate the thyroid-disrupting effects of potassium-perchlorate, a known thyroid function inhibitor (Leung et al., 2010). Perchloric acid and its salts are strong oxidizers and used in pyrotechnics, explosives, and jet or rocket fuels (von Burg, 1995). Furthermore, perchlorate salts have been used in the medical treatment of specific hyperthyroid conditions and as a provocative test for the release of thyroid hormones (Martino et al., 1986, Wenzel and Lente, 1984). Naturally occurring perchlorate is formed in the atmosphere leading to trace levels in precipitation, which can concentrate geologically in some locations such as northern Chile (Urbansky et al., 2001) or West Texas (Dasgupta et al., 2005), but the majority of environmental perchlorate is of anthropogenic origin.
Environmental perchlorate pollution of ground and surface waters has mainly been documented in the United States. Urbansky (1998) reports of perchlorate concentrations of 8 μg/L up to 3.7 g/L in several areas in the United States. The US EPA conducted one nationwide perchlorate sampling, between 2001 and 2005, and detected perchlorate at or above 4 μg/L in 160 of the 3865 public water systems tested (about 4.1%). In 31 of these 160 systems, perchlorate was found above 15 μg/L (GAO, 2010). Data from the Department of Defense revealed perchlorate concentrations ranging from less than 1 μg/L to 2.6 g/L during the years 1997 through 2009 (GAO, 2010). All these data are well in the range of the US EPA (2002) Tier II acute and chronic effects screening levels for ecotoxicological effects (5 and 0.6 mg/L, respectively) and necessitate additional data on adverse effects in freshwater fish. The perchlorate concentrations used in the present study are covering the range reported by the abovementioned studies to allow conclusions on the environmental situation.
Perchlorates together with thiocyanates and nitrates are known to affect thyroid function by competitive inhibition of the sodium iodide symporter, which is responsible for the uptake of iodide in the thyrocytes, with perchlorate being the most potent inhibitor of the sodium iodide symporter followed by thiocyanate and nitrate (Leung et al., 2010). In Chinese hamster ovary cells transfected with human sodium-iodide symporter, Tonacchera et al. (2004) reported that concentrations of ClO4−, SCN−, I−, and NO3− required for 50% inhibition of radioiodine uptake were in the ratios 1:15:30:240.
Thyroid hormone activity is exceptionally important during early development in amphibians and fish being responsible for the completion of metamorphosis (Einarsdottir et al., 2006, Miwa and Inui, 1987, Shi, 2000). The importance of thyroid hormones in this phase of development of fish is exceptionally evident in flatfish, which are dependent on thyroid hormones to metamorphosize to the asymmetrical juvenile (Einarsdottir et al., 2006, Miwa and Inui, 1987). In zebrafish, the first thyroid follicle differentiates around 55 h post-fertilization and thyroxin (T4) production starts around 72 h post-fertilization (Alt et al., 2006a, Elsalini and Rohr, 2003). The first follicle corresponds to the anterior-most follicle in the adult (Alt et al., 2006b), which is important for evaluating histopathological samples, since follicle size is a major endpoint.
Although the thyroid gland acts as the downstream hormone-producing gland, the key organ for regulation of, e.g., growth, development, reproduction or adaption to environmental challenges along hormonal axes is the pituitary (Kasper et al., 2006). Regulatory pathways of the thyroid system start with the reception of external and internal sensory information reaching the brain and the hypothalamus. In contrast to higher vertebrates, the role of the thyrotropin-releasing hormone in the regulation of thyroid-stimulating hormone (TSH) release in fish is less well established (Janz, 2000). Unlike mammals, teleost fish lack a portal system between the hypothalamus and the pituitary gland. Instead, there is a direct neuronal connection to endocrine cells through the hypophyseal stalk (Peter et al., 1990). The hypothalamus thus directly innervates the pituitary exerting control through secretion of several hormones – in this case, via TSH (Kime, 1998). The functional significance of TSH is limited to the regulation of T4 release and iodide uptake by the thyroid follicles (Eales et al., 1999).
Morphologically, the pituitary in teleost fish is divided into two major parts: (1) the neurohypophysis (pars nervosa; PN), which folds down from the diencephalon and (2) the adenohypophysis, which pouches up from the roof of the oral cavity (Weltzien et al., 2004). During development, the neurohypophysis interdigitates with the adenohypophysis, which on its part can be subdivided into (1) the pars distalis (PD), which can further be divided into the rostral pars distalis (RPD) and the proximal pars distalis (PPD) and (2) the pars intermedia (PI; for further details, see (Schmidt and Braunbeck, 2011)). Multiple studies have documented the principal distribution of adenohypophyseal cells in fish (Garcia Ayala et al., 2003, Kasper et al., 2006, Leunissen et al., 1982, Quesada et al., 1988, Ueda et al., 1983) and amphibians (Garcia-Navarro et al., 1988, Miranda et al., 1996, Ogawa et al., 1995); the impact of thyroid-disrupting chemicals, however, has hardly been examined (Schmidt and Braunbeck, 2011).
The importance of the thyroid system in fish and the potency and distribution of perchlorate make further studies indispensable to properly evaluate effects of thyroid-disrupting chemicals and to identify possible environmental risks. Therefore, this study was designed to describe histological effects in the thyroid, to quantify histological and immunohistological alterations in the pituitary, and to record histological and ultrastructural effects in the liver in a modified early life-stage test with the zebrafish (Danio rerio).
Section snippets
Animals and husbandry
Fertilized eggs from zebrafish (D. rerio) were obtained from in-house breeding at the Department of Aquatic Ecology and Toxicology, Centre for Organismal Studies, University of Heidelberg. All experiments were conducted in compliance with the institutional guidelines for the care and use of animals as well as with permission by the regional animal welfare (AZ 35-9185.81/G-144/07). Fertilized eggs were initially raised in 20 cm petri dishes in a KB 115 incubator (Binder, Tuttlingen, Germany) at a
Whole body weight, whole body length, and condition factor
Overall body proportions revealed clear alterations in the higher concentration groups (Table 1). Whole body weight did not show any statistical aberrance. Concentrations ≥500 μg/L showed a slight, however not statistically relevant decrease. Instead, whole body length revealed a statistically significant increase at 125 μg/L, whereas the other exposure groups were not affected. However, the condition factor decreased throughout the concentration groups with significant alterations at
Discussion
In the present study, alterations of the thyroid system of the zebrafish by potassium perchlorate were used to determine distinct effects caused by inhibited iodide uptake. To assess effects of endocrine-disrupting chemicals, it is essential to examine appropriate endpoints since these endpoints (1) can provide indications as to the underlying mode of action of the chemical and (2) show different sensitivities in the single endpoints, which are essential to properly grade the effect, and (3) to
Acknowledgment
The first author has been supported by a grant from the Evangelisches Studienwerk e. V. Villigst.
References (104)
- et al.
The mode of action of perchlorate ions on the iodine uptake of the thyroid gland
Int. J. Appl. Radiat. Isot.
(1959) - et al.
Zebrafish hhex, nk2.1a, and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors
Dev. Biol.
(2003) - et al.
Immunohistochemical localization of thyrotropic cells during amphibian morphogenesis: a stereological study
Gen. Comp. Endocrinol.
(1988) - et al.
A comparison of physiological changes in carp, Cyprinus carpio, induced by several pollutants at sublethal concentrations. I. The dependency on exposure time
Ecotoxicol. Environ. Saf.
(1985) - et al.
Perchlorate, iodine and the thyroid. Best practice & research
Clin. Endocrinol. Metab.
(2010) - et al.
Effects of growth hormone, insulin-like growth factor I, triiodothyronine, thyroxine, and cortisol on gene expression of carbohydrate metabolic enzymes in sea bream hepatocytes
Comp. Biochem. Physiol. A: Mol. Integr. Physiol.
(2010) - et al.
Thyrotropin in teleost fish
Gen. Comp. Endocrinol.
(2009) - et al.
Detection of endocrine-modulating effects of the antithyroid acting drug 6-propyl-2-thiouracil in rats, based on the enhanced OECD Test Guideline 407
Regul. Toxicol. Pharmacol.
(2003) - et al.
Effects of various doses of thyroxine and triiodothyronine on the metamorphosis of flounder (Paralichthys olivaceus)
Gen. Comp. Endocrinol.
(1987) - et al.
Effects of larval-juvenile treatment with perchlorate and co-treatment with thyroxine on zebrafish sex ratios
Gen. Comp. Endocrinol.
(2007)