Sodium Fluoride Exposure Induces Developmental Toxicity and Cardiotoxicity in Zebrafish Embryos.

Epidemiological survey results show that compared with those in residents in low-fluoride areas, the rates of heart disease and mortality of residents in high-fluoride areas are significantly higher [13]. However, the molecular mechanism of fluoride-induced heart disease is unknown. We utilized the zebrafish larvae as a model to assess the toxic effects of NaF on cardiac development. Our findings suggest that NaF exposure causes cardiac developmental malformations, including pericardial edema, and cardiac structural and functional impairment.

The heart is the first organ to form and plays a critical role during the development of embryos. The zebrafish heart is mature and functional at 72 hpf [22,23,24], and its morphological structure and function are similar to the human heart during the early stage of development [25]. In recent years, zebrafish have been widely used as a vertebrate model to research human cardiovascular disease [26]. In addition, they are widely used for environmental toxicity detection and drug toxicity safety evaluation [27, 28]. The heart must undergo a series of complex processes as it matures, and any defect in these processes can lead to congenital heart disease [29]. The evaluation of NaF o is necessary for safety and zebrafish is a well-accepted animal model for researching “predictive toxicology” [30]. Early embryos are often used to assess toxicity during embryonic development [31]. Embryos (6 hpf) were distributed into six-well plates and incubated with NaF at the designated concentrations in this study. Zebrafish exposed to NaF exhibited severe heart malformation, such as pericardial edema, cardiac loop deformity, and an increased SV-BA distance. These cardiac morphological abnormalities are similar to the toxic effects of other pollutants, such as prothioconazole [32], triadimefon [33], and pyrimethanil [34]. A correctly looping heart is critical for the formation of a normal cardiac structure [35]. Previous reports indicated that HR is a key parameter in evaluating heart function [32, 36]. Our results showed that NaF exposure decreased the HR at 96 hpf and 120 hpf. These results suggested that NaF decreases the HR at the late stage of development. Overall, we hypothesized that NaF exposure leads to cardiac malformation in zebrafish.

Our results suggested that NaF might be toxic to heart development. As the NaF concentration increased, the severity of heart teratogenicity increased. However, little study has explored the mechanism of NaF-induced cardiotoxicity. To explore potential environmental health risks and mechanism induced by NaF, we conducted RNA-seq analysis in zebrafish larvae. In zebrafish, cardiac morphogenesis is regulated by critical genes, including Gata4, Tbx5a, Hand2, Tnnt2c, Nppa, and Myh6. In cardiac morphogenesis, Gata4 plays a critical role [37] and is a key gene in regulating cardiomyocyte proliferation and cardiac septal development [38]. The expression level of Gata4 and the distance of SV-BA were significantly increased in the NaF-treated groups. It is suggested that NaF-treated may lead to abnormal cardiac septal development. Transcription factor Tbx5a is vital for cardiac morphogenesis [39]. In zebrafish, the cardiac phenotype of Tbx5a deficiency was similar to Holt-Oram syndrome [40]. Interestingly, hyperactivity of Tbx5a also causes cardiac defects [41]. Transcription factor Hand2 is an important regulator of embryonic cardiac development and promotes cardiac reprogramming and ventricular cardiomyocyte expansion [42, 43]. The precise expression level of Hand2 is essential for normal heart morphogenesis and function [44, 45]. Cardiac troponin T (Tnnt2c) regulates the interaction between myosin and actin, and plays an important role in myofbrillogenesis in zebrafish [46, 47]. Atrial natriuretic peptide (Nppa) expression in the early-developing heart shows a restricted and dynamic pattern [48]. Nppa is a key and sensitive marker for the developing heart. For example, the patterning of Nppa expression is altered in congenital heart disease [49]. In addition, the upregulation of Nppa expression causes cardiac deformity [50]. In the developing heart, Myh6 is highly expressed and mutations of Myh6 lead to atrial septal defects and cardiomyopathy [51]. Our results showed that the expression level of the critical genes (Gata4, Tbx5a, Hand2, Tnnc2, Nppa, and Myh6) were significantly increased in the NaF-treated groups, indicating that NaF exposure causes cardiac dysmorphogenesis through these key genes.

In addition, we used GO and KEGG to analyse the potential mechanisms of cardiac deformity caused by NaF exposure. The results of bioinformatic analysis offer novel insights into the underlying mechanism. After exposure to NaF, activation of the Ras signaling pathway was detected in the 120 hpf zebrafish, indicating that NaF might lead to heart development deformity through Ras signaling. Both the Ras and Rho (RhoA, Rac1, and Cdc42) subfamilies belong to the small G protein superfamily, which regulates many cellular responses, and overexpression of RhoA promotes lethal heart failure. Rac1 and Cdc42 regulate the actin/myosin cytoskeleton in many cells [52]. Our results showed that the expression level of Cdc42l was increased in the NaF exposure group at 120 hpf, further suggesting that NaF activates the Ras signaling pathway.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway can transduce numerous of cytokines, such as tumor necrosis factor (TNF), interleukin (IL) 1?, IL-6, and transforming growth factor beta (TGF?), and then regulates a series of physiological and pathophysiological processes, including cell differentiation, cell proliferation and apoptosis, immune responses, and inflammation [53]. Our results showed that the expression level of Stat3 was significantly increased in the NaF-treated groups. This suggests that NaF activates inflammatory signaling pathways. IL-6 might mediate heart failure by activating the JAK/STAT signaling pathway. Suppressor of cytokine signaling (SOCS) regulates the JAK/STAT signaling pathway, and their interaction with other inflammatory factors might lead to heart failure. IL-6-like cytokines, JAK/STAT signaling, and SOCS play key roles in the regulation of heart failure [54]. In this study, the expression level of Socs3b was significantly increased in the NaF-treated groups, it is further indicated that activation the JAK/STAT pathway play a critical role in NaF-induced cardiac deformity.

Complement cascade and blood coagulation are two major contributors to the first line of defense against infection. The complement system plays a critical role in innate and adaptive immunity [55]. In addition to the detection and removal of pathogens [56], the complement system also participates in many other life activities, such as the removal of immune complexes, the promotion of angiogenesis, tissue regeneration, and lipid metabolism [57]. Therefore, the abnormal activation and expression of the complement system in the body will unbalance homeostasis, resulting in a variety of autoimmune and inflammation-related diseases [58]. It was reported that the complement and coagulation cascade pathway is associated with the risk of heart failure, myocardial infarction, and cardiovascular-related mortality in patients with established coronary heart disease [59]. The above studies suggest that the complement and coagulation cascade pathway play a critical role in cardiovascular disease by mediating inflammation. Our results showed that the complement and coagulation cascade pathway was the most enriched pathway in the NaF-treated group. This indicated that NaF might induce cardiac malformation through the complement and coagulation cascade pathway. In the present study, the expression levels of c3a.3, c5, c6, and c7b were significantly increased in the NaF exposure group as a result of the strong immune toxicity of NaF. The expression levels of f3a, f5, f9b, and f2r were significantly increased in the NaF exposure group. The F2r gene, also known as par1, contributes to inflammatory responses. Studies indicated that Par1 activation increased inflammation [60]. To sum up, we suspected that these factors could affect the autoimmune response of zebrafish, leading to inflammation and apoptosis, and thus might initiate the sequential process of heart development.

In conclusion, this study found that NaF exposure induces numerous structural and functional malformations during zebrafish development. Our comprehensive analysis of DEGs and their associated enriched pathways revealed a complicated relationship between the effects on the immune system and the cardiac system. NaF is excessively used and frequently detected; therefore, a more comprehensive understanding of its toxic effects is important to improve the risk assessment and supervision of such environmental pollution. This study provides some clues for the clinical prevention and treatment of patients with fluorosis.