Waterborne exposure to PFOS causes disruption of the hypothalamus–pituitary–thyroid axis in zebrafish larvae
Introduction
The thyroid hormones (THs) triiodothyronine (T3) and thyroxine (T4) have a wide range of biological effects on development, growth and metabolism in vertebrates (Power et al., 2001). Thyroid function is dependent upon iodide uptake, TH synthesis, transport, tissue-specific TH deiodination and THs binding to thyroid hormone nuclear receptors (TRs) (Yen and Chin, 1994). In mammals, the hypothalamus secretes thyrotropin-releasing hormone (TRH) to stimulate thyroid-stimulating hormone (TSH) secretion and regulation of TH synthesis within the hypothalamic–pituitary–thyroid (HPT) axis. In amphibians and fish, corticotropin-releasing factor (CRF) appears to stimulate TSH secretion (De Groef et al., 2006) and might therefore function as a common regulator of both the thyroidal and interrenal axes. Given the key role of THs in normal development and physiological functions in vertebrates, it is important to identify environmental chemicals that may adversely affect thyroid function and/or TH signaling, and to evaluate their risk to animals and humans (Brucker-Davis, 1998). As such, several groups of chemicals, including polychlorinated biphenyls (PCBs) and their metabolites, dioxins, brominated flame retardants, phenols, phthalates and pesticides, have the potential for disturbing thyroid hormone homeostasis in fish, amphibians and mammals (reviewed by Boas et al., 2006, Miller et al., 2009).
The use of perfluorinated chemicals (PFCs) in surfactants for commercial and industrial applications over the past 50 years has resulted in global contamination (Giesy and Kannan, 2001; reviewed by Houde et al., 2006, Lau et al., 2007). Perfluorooctane sulfonate (PFOS) is the most dominant PFC detected worldwide in the aquatic environment and biota (Giesy and Kannan, 2001). Although the concentrations of PFOS at the surface are generally low (<0.1 μg L−1), the compound has the potential to biomagnify in aquatic food chains, such as in fish and marine mammals (Martin et al., 2004, Kannan et al., 2005). For example, PFOS in the liver was measured to be up to 7760 μg kg−1 wet weight for plaice (Pleuronectes platessa) (Hoff et al., 2003) and 9031 μg kg−1 wet weight for feral gibel carp (Carassius auratus gibelio) in Belgium (Hoff et al., 2005). PFOS was also transferred by maternal exposure to the offspring (145–381 μg kg−1 in ovary) in lake whitefish (Coregonus clupeaformis) from Michigan, USA, waters (Kannan et al., 2005), suggesting oviparous transfer of this compound. The properties of high persistence, worldwide distribution and bioaccumulation of PFOS have received great concern over its toxicity.
PFOS has been shown to be developmentally toxic in rodents (reviewed by Lau et al., 2007). For example, exposure to the chemical in utero delayed development and reduced postnatal survival and growth (Lau et al., 2003, Thibodeaux et al., 2003, Luebker et al., 2005). In addition, PFOS exposure significantly reduced total and free THs in rats (e.g., Seacat et al., 2002, Lau et al., 2003, Thibodeaux et al., 2003, Luebker et al., 2005, Chang et al., 2008, Yu et al., 2009), suggesting interference with thyroid function. In fish, PFOS also causes reproductive and developmental toxicity in fathead minnows (Pimephales promelas) (Ankley et al., 2005). PFOS exposure to zebrafish embryos resulted in developmental toxicity and altered certain gene expressions, including the marker genes related to early thyroid differentiation (hhex and pax8) (Shi et al., 2008). A chronic exposure of PFOS to zebrafish embryos/larvae led to a trend of increased triiodothyronine (T3) levels, but the increases were not statistically significant (Du et al., 2009).
Due to the new European Chemical Policy REACH (registration, evaluation and authorization of chemicals), a dramatic increase in the number of experimental animals is expected. In addition to a high public demand for the reduction or replacement of animal tests for ethical and political reasons, there are also scientific and cost-effective reasons to embrace a more differentiated testing approach (Lammer et al., 2009). In this regard, a toxicity test in fish embryos provides an alternative approach for toxicity assessment (Scholz and Mayer, 2008, Lammer et al., 2009). Since THs play important roles in development and growth, particularly in the early life stages of fish (Walpita et al., 2007, Kawakami et al., 2008), thyroid disruption could severely compromise fitness and survival. The potential thyroid-disrupting toxicity of PFOS has not been fully elaborated in fish, particularly in the early developmental stage. Therefore, in the present study, zebrafish embryos were selected as a model and exposed to different concentrations of PFOS. Functionally relevant genes associated with the pathways of concern (HPT) were quantitatively examined. Meanwhile, the levels of THs (T3 and thyroxine, T4) were also measured. The aim of the present study was to investigate the mechanisms of the HPT axis responses to PFOS exposure and identify potential mechanisms of TH disruption in zebrafish.
Section snippets
Chemicals
Heptadecafluorooctanesulfonic acid potassium salt (PFOS,>99%) was obtained from Tokyo Kasei Kogyo Co. Ltd., (Tokyo, Japan) and a stock solution was prepared by dissolving the crystal in HPLC-grade dimethyl sulfoxide (DMSO) and stored at 4 °C. The 3-aminobenzoic acid ethyl ester, methanesulfonate salt (MS-222) was obtained from Sigma (St. Louis, MO, USA). All other chemicals used in the present study were analytical grade.
Zebrafish maintenance and embryo exposure
Adult zebrafish (Danio rerio) (AB strain) maintenance and embryo exposure
Developmental toxicity
There were no significant effects on malformation or survival after exposure to 100, 200 and 400 μg L−1 PFOS relative to the control until 15 dpf. There were no significant effects on growth in the 100 and 200 μg L−1 PFOS exposure groups. The measured body length and average weight (control: 5.15 ± 0.08 mm and 0.44 ± 0.01 mg per larvae) were significantly reduced (exposure: 4.20 ± 0.07 mm and 0.39 ± 0.01 mg per larvae) compared with those at the 400 μg L−1 PFOS exposure group (Fig. 1).
Gene expression profile
The expression of genes in
Discussion
Endocrine disrupting chemicals (EDCs) may have a direct impact on the synthesis of THs via iodide uptake, TPO activity, and TG synthesis. EDCs can also interfere with the thyroid system by feedback mechanisms triggered by changes in circulating THs, resulting in changes in TSH expression and affecting TH bioavailability by interfering with binding proteins such as TTR and deiodinases (Kloas and Lutz, 2006). Therefore, given the almost complete absence of antisera for hormone detection in the
Acknowledgements
The present study was supported by the National Nature Science Foundation of China (20890113 and 20877094) and the State Key Laboratory of Freshwater Ecology and Biotechnology (2008FBZ10).
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