Effects of waterborne cadmium on thyroid hormone levels and related gene expression in Chinese rare minnow larvae

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Abstract

Cadmium is a heavy metal abundant in the environment that can induce endocrine disorder and toxicity in aquatic organisms at low levels. However, its effects on the thyroid system in fish are still unclear. In this study, the thyroid hormone (TH) levels and the expression profiles of genes related to hypothalamic– pituitary–thyroid (HPT) axis, including corticotropin-releasing hormone (crh), thyroid stimulating hormone beta (tshβ), solute carrier family 5 (sodium iodide symporter) member 5 (slc5a5), thyroglobulin (tg), thyroid hormone receptor alpha (trα) and thyroid hormone receptor beta (trβ), were determined in whole body of Chinese rare minnow (Gobiocypris rarus) larvae after exposure to different levels of Cd2 + (0, 0.5 and 2.5 mg/L) for 4 days. And the 96-h lethal concentration of Cd2 + on rare minnow larvae was determined as 2.59 mg/L. The results showed that crh, slc5a5, tg and tshβ mRNA levels were significantly up-regulated in the larvae, but the gene expression of trα and trβ was down-regulated in a concentration-dependent manner. Besides, the THs levels decreased in the whole-body of fish, especially the thyroxine (T4) level. The above results indicated that Cd2 + could alter gene expression in the HPT axis that might subsequently contribute to thyroid disruption.

Introduction

It is widely accepted that the frequency and global distribution of heavy metal contamination in aquatic system have significantly increased and become a worldwide concern. Among them, cadmium is a nonessential metal that poses a serious environmental threat to aquatic organisms, including endocrine system function (Kim et al., 2004, Li et al., 2010, Sassi et al., 2013). Dissolved cadmium levels in freshwater generally range from 10 to 500 ng/L but there have been recorded levels exceeding 17 mg/L in industrialized areas (Jones et al., 2001). This pollutant is released naturally from weathering of minerals, forest fires and volcanic emission; however most water Cd contamination results from anthropogenic sources (Garcia-Santos et al., 2013). Cd toxicity in fishes is well documented and has been found to involve several endocrine pathways important for reproduction and development, as well as for the osmoregulation and morphological structure of fish (Garcia-Santos et al., 2013, Sassi et al., 2013); such altered expression of iron metabolism related genes has also been found in the fish Pseudosciaena crocea (Chen et al., 2008).

In recent years, the impact of environmental contaminants on endocrine systems has attracted growing interest. In fish, the thyroid endocrine system is controlled primarily by the hypothalamic–pituitary–thyroid (HPT) axis, which is responsible for maintaining homeostasis of thyroid hormones by regulating their synthesis, secretion, transport and metabolism (Yan et al., 2012, Wang et al., 2013b). Based on available reports, endocrine disruption caused by Cd can occur via the hypothalamus-pituitary-interrenal (HPI) and the hypothalamus-pituitary-thyroid (HPT) axes (Lizardo-Daudt et al., 2007, Sandhu and Vijayan, 2011), however it seems that the mechanisms involved are poorly understood. The thyroid hormones (THs) triiodothyronine (T3) and thyroxine (T4) have a wide range of biological effects in physiological processes of vertebrates (Li et al., 2009b, Wang et al., 2013b). In fish, THs have been implicated as being important regulators in differentiation, growth, metabolism, and salinity adaptation (Yan et al., 2012). Cortisol is the primary circulating corticosteroid in teleosts, its release involves the coordinated activation of the HPI axis and has a vital role in maintenance of physiological homeostasis under stress conditions (Mommsen et al., 1999). Thyroid hormones and cortisol have a crucial physiological role in regulation of fish metabolism, through their effects on lipolysis, glycogenolosis and gluconeogenesis (Garcia-Santos et al., 2013).

Chinese rare minnow (Gobiocypris rarus) is considered to be an appropriate species for the assessment of endocrine disrupting chemicals due to its small size, ease of culture, short life cycle and prolific egg production with high fertilization and hatching rates, thus this species has been used in toxicological studies and environmental monitoring (Zha et al., 2007, Fang et al., 2010, Wang et al., 2013a).

Until now, the effect of heavy metals on the hypothalamic–pituitary–thyroid (HPT) axis in fish is largely unknown. In the present study, we evaluated the effects of Cd2 + on mRNA expression involved in the HPT axis using Chinese rare minnow larvae by using a Q-PCR array. Furthermore, an enzyme-linked immunosorbent assay (ELISA) was employed to measure whole-body thyroid hormone levels after Cd2 + exposure.

Section snippets

Chemicals

CdCl2·2.5H2O was purchased from Sigma-Aldrich Chemical Co. (USA). Deionized water was used as the vehicle to solve the chemical. All of the other chemicals used in this study were of analytical grade and the chemicals used for electrophoresis were purchased from Sigma-Aldrich.

Test fish and culture conditions

The rare minnow (G. rarus) larvae (7 days after hatching, 9.65 ± 0.13 mm, 1.12 ± 0.17 mg) obtained from a local hatchery (Wuhan, China), were raised in a flow-through system with dechlorinated tap water (pH 7.2–7.6; hardness

Results

On the basis of our results (Table 2 and Fig. 1), the 96 h LC50 of Cd2 + on Chinese rare minnow was determined to be 2.59 mg/L (95% CL: 1.50–4.44 mg/L).

At the end of Cd2 + treatment, the thyroid hormone levels (T4 and T3) of the whole body were measured in control and treated groups (Table 3). From the results, the whole-body T4 content in the control group was detected to be 64.51 ± 6.25 ng/mL and no significant difference was observed in the T4 content in 0.5 mg/L Cd2 + exposure group, but a

Discussion

Cadmium (Cd2 +) is a non-essential element that is potentially highly toxic to humans and other organisms, even at low doses. According to summary of the USEPA, the effects of Cd2 + on fish species were species specific and fish exhibited different ability to tolerate different levels of Cd2 +. The sensitivity to Cd2 + in fishes changes with the developmental stage and even with age within the same stage (Hallare et al., 2005). For instance, the LC50 at 96-h for common carp (Cyprinus carpio)

Acknowledgments

This study was supported by China Three Gorges Project Corporation (No. 07011034), the CENAKVA CZ.1.05/2.1.00/01.0024, the project LO1205 with a financial support from the MEYS of the CR under the NPU I program, the project P503/11/1130 of the Grant agency of Czech Republic.

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