Fluoride (F) and lead (Pb) are widespread pollutants in the environment. F and Pb affect the thyroid endocrine system, but the mechanism of action between F and Pb is still unclear. In this study, in order to evaluate the effects of F or/and Pb on histopathological changes, antioxidant indices, the levels of thyroid hormones (THs), and the expression of endocrine-related genes in zebrafish thyroid. One thousand and two hundred zebrafish (female:male = 1:1) were randomly divided into four groups: control group (C group), 80 mg/L F group (F group), 60 mg/L Pb group (Pb group), and 80 mg/L F + 60 mg/L Pb group (F + Pb group) for 45 d and 90 d. Histopathological sections showed a loss of glia and follicular epithelial hyperplasia in the thyroid gland after exposure to F and Pb. Oxidative stress in the thyroid was induced after F and Pb exposure. And each oxidation index was increased after F + Pb exposure. Combined F and Pb exposure aggravated the downregulation of thyroid hormones T3 and T4 compared to exposure alone. Furthermore, F and Pb exposure altered the expression of thyroid endocrine-related genes in a time-dependent manner. These results indicate that F and Pb can affect the endocrine system of thyroid by changing the tissue structure, antioxidant capacity, thyroid hormone secretion and the levels of endocrine-related genes in thyroid. F and Pb can also produce toxic effects on thyroid, but the degree of poisoning is different in different indicators, mainly for the additive effect between them. Additionally, males are more sensitive than females to F or Pb toxicity. However, the specific molecular mechanism of the effects of F and Pb on thyroid endocrine system needs to be further studied.
Fluorine (F) and lead (Pb) are two elements that exist widely in nature. F exists in the form of F? in aqueous solution, and doesn’t change the appearance, taste and odor of aqueous solution . According to the regulations of the World Health Organization (WHO) in 2011, the allowable limit of F? concentration in drinking water is 1.5 mg/L (0.789 × 10?4 M) . As report goes, the F? concentration in uncontaminated freshwater and seawater is 0.01–0.3 mg/L and 1.2–1.5 mg/L, respectively . Pb is a nonradioactive metal with the largest atomic weight and usually exists in the form of compounds in nature not easy to degrade . The average value of Pb in surface water in the world is about 0.5 ?g/L . The Pb concentration in uncontaminated groundwater is between 1 ?g/L and 60 ?g/L . When the Pb concentration reaches 0.1 mg/L, it can inhibit the self-purification of water body .
In recent years, rapid industrial development has further deteriorated the sustainable ecological environment. Studies have reported a common contamination of F and Pb in water bodies [5,6]. Geological surveys show that contamination of F and Pb in water sources in Western and Asian countries is shocking . In Michigan, artificial fluorinated water increases the acidity of the water, which increases Pb content in water because of Pb leaching from the PCBs pipeline after being corroded by acid . The pollution of F and Pb in water mainly comes from various natural and anthropogenic factors. Natural factors include geological weathering, volcanic eruption, crustal movement and so on . Anthropogenic factors include the releases of pesticides, fertilizers, sewage, and industrial waste into the environment, which enter and gather in the water environment through rainfall and dust landing, etc . It has also been shown that F can increase Pb levels in animal blood and calcified tissues . Excessive F and Pb accumulate in animal organisms, affecting the health of organism, such as movement disorders, respiratory symptoms, kidney damage, and other clinical symptoms . The co-exposure of F and Pb inhibits hepatocyte proliferation and increases hepatocyte apoptosis . Although there are some studies on toxic effects of combined exposure to F and Pb on animals, the toxic effects and underlying mechanisms on the thyroid function are still unclear.
The thyroid gland is a very important endocrine gland in vertebrates. The main function of thyroid hormones (THs) is to maintain the normal physiological state of the body, including the osmoregulation, metabolism, growth, and somatic development . THs production is a very complex biochemical process that is regulated by receptors, transporters, enzymes, and other substances . Oxidative stress has been shown to be associated with hypothyroidism . For example, thyroid dysfunction increases the levels of thyroid stimulating hormone (TSH) and malondialdehyde (MDA), as well as the levels of antioxidants, such as superoxide dismutase (SOD) and catalase (CAT) . Thyroid dysfunction is also accompanied by the changes of glutathione peroxidase (GPx) and total antioxidant capacity (T-AOC) . Studies have shown that the expression levels of genes such as thyroid hormone receptor (tr) and deiodinase (dio) in fish exposed to environmental pollutants are sensitive molecular biomarkers of thyroid interference [1,14]. The hypothalamus secreted thyroid stimulating hormone releasing hormone (TRH) regulates pituitary synthesis and secretion of TSH, which enters thyroid cells through blood circulation system to promote the synthesis and secretion of THs . The sodium iodine transporter (NIS) transfers iodide to thyroid follicular cells to synthesize THs including triiodothyronine (T3) and thyroxine (T4). T4 forms T3 under the action of DIO, and eventually are fed back to the hypothalamus and the pituitary gland to regulate the synthesis and secretion of TRH and TSH . The expression of thyroid-related genes has been widely used to investigate the molecular mechanisms of thyroid endocrine interference caused by contaminants.
Since the early 1990s, zebrafish (Danio rerio) have been used to detect the acute and chronic toxicity of mixed chemicals and the bioaccumulative effects of heavy metals because zebrafish are small, easy to breed, highly heterogeneous, very sensitive to harmful substances, and the completed gene sequencing by the Sanger Institute in 2001 .
In this study, in order to reveal the interference effect and potential mechanism of F and Pb co-exposure on thyroid endocrine function of zebrafish, adult zebrafish were exposed to different concentrations of F or/and Pb for 45 and 90 d to observe the histopathological structure of thyroid, determine the levels of the antioxidant indicators, the levels of T3 and T4, and the expression levels of endocrine related genes by real-time fluorescent quantitative PCR (qPCR). This study will provide a scientific theoretical basis for ecotoxicological study of F and Pb combination.
Sodium fluoride (NaF) (CAS: S1540), Pb (CH3COO)2 (CAS: 215902), and MS-222 (CAS: 886-86-2) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The kits of active oxygen (ROS), SOD, CAT, GPx, MDA, and T-AOC were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA Protein Assay kit was purchased from Solarbio Technology Co., Ltd (Bejing, China). Zebrafish ethoxyresorufin-O-deethylase (EROD) kit (ELISA) was purchased from Ruixin Biotechnology Co., Ltd. (Quanzhou,
Effects of F and Pb on histopathological changes of thyroid in zebrafish
After 45 and 90 d of F or/and Pb exposure, the thyroid follicles in female and male C group were round or oval, consisting of single cubic or flat epithelial cells surrounded by uniform glial cells and surrounds (Fig. 1A, G and Fig. 2A, G).
In male zebrafish, glial loss, follicular epithelial hyperplasia, increased number of large follicles, and slight internal and external folding of large follicles were usually observed after exposure to F or Pb for 45 d (Fig. 1B, C, D). More severe
Effects of F and Pb o n thyroid microstructure in zebrafish
Thyroid is the earliest endocrine structure in the process of body development. Thyroid follicle is the structural and functional unit of thyroid . In our study, 80 mg/L F or 60 mg/L Pb exposure for 45 and 90 d resulted in a disruption of zebrafish thyroid structure, such as glial loss, follicular epithelial hyperplasia, and increased follicular number, supported by a previous study . However, 10 ?g/L Pb at Gosner stage 42 does not affect thyroid histological changes . The difference
In summary, this study reveals that F and Pb exposure in the water system can damage thyroid endocrine system in both male and female zebrafish. After treatment for 45 and 90 d, excessive F and Pb can disturb the thyroid endocrine system by impairing thyroid microstructure, disrupting the balance between oxidative stress and anti-oxidative stress, and changing thyroid hormone T3 and T4 levels and the levels of thyroid endocrine-related genes. Co-exposure to F + Pb induces stronger oxidative
CRediT authorship contribution statement
Yuting Lu: Data curation, Writing – original draft, Methodology. Xiulin Zhang: Data curation, Conceptualization, Methodology. Jianjie Chen: Formal analysis, Validation. Jinling Cao: Supervision, Project administration, Writing – review & editing. Cuiping Feng: Data curation, Methodology. Shaojun Yun: Conceptualization, Data curation. Yanfeng Cheng: Formal analysis, Methodology. Feier Cheng: Formal analysis, Methodology.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
This study was supported by the Shanxi Scholarship Council of China (2020-061), the Shanxi Provincial Key Research and Development Project (201903D221009), and the National Natural Science Foundation of China (31502141; 31440087).
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