Effects of water fluoridation on early embryonic development of zebrafish.

4. Discussion

The effects of water fluoridation on the environment and human health are generally regarded as chronic and long-term. Multiple studies have affirmed that prolonged exposure to fluoride adversely affects the neurological, metabolic, immune, and reproductive systems of adult zebrafish (Table. S1). In reality, water fluoridation also exhibits significant acute toxic effects on early embryonic development. Exposure to certain doses of fluoride can lead to embryonic mortality and severe developmental abnormalities (Table. S1). In this study, the influence of water fluoridation on zebrafish embryonic development manifests primarily in three aspects: the accumulation of peroxides in the liver and intestines, leading to cellular apoptosis and yolk sac edema. Furthermore, abnormal vascular formation reduces systemic blood circulation. Besides, the edematous yolk sac restricts the development of the skeletal system, cranial nerves, retina, and cardiac looping.

During the early stages of development, typically within 24–48 hpf, zebrafish’s liver assumes its vital role in functioning and eliminating waste (Goessling and Sadler, 2015). This critical period is also susceptible to programmed cell apoptosis. As a defense mechanism, exogenous stimuli often delay the hatching of zebrafish embryos. Surprisingly, our study found that fluoride exposure did not significantly impede hatching but rather led to the accumulation of abnormal coagulants within the embryonic cytoplasm. Moreover, following fluoride exposure, the cytoplasm of hepatocytes exhibited occasional vacuoles, indicating the presence of lipid accumulation and oxidative stress in the abdomen (Wu et al., 2022). We speculate that the cytotoxin associated with fluorosis may persist within the yolk sac, hindering nutrient utilization and promoting lipid peroxidation (Sant and Timme-Laragy, 2018), ultimately leading to severe edema. These impaired liver functions could potentially correlate with oxidative stress and inflammatory gastrointestinal tract disease.

The accumulation of ROS inflicts oxidative damage upon DNA, fatty acids, and various cellular components, leading to afflictions like atherosclerosis, neurodegeneration, and ischemia-related ailments (Karaman et al., 2023, Panieri and Santoro, 2016, Siddiqui et al., 2016). Notably, individuals residing in fluorosis-prone regions are susceptible to developing atherosclerosis (Liu et al., 2014). The earliest report showed that acute fluorosis in children induced severe hypocalcemia, ventricular arrhythmias, and respiratory failure (Yolken et al., 1976). Rodents with fluorosis experience pulmonary and esophagus enlargement, moreover, the impaired lungs show the congestion of alveolar septal vessels and thick-walled vessels in the parenchyma (Mathur et al., 2022, Oncu et al., 2006). Therefore, fluorosis is closely associated with angiogenesis and blood circulation. Here, we provide evidence that this association endures, irrespective of the concentration of fluoride treatment.

Besides, the cytotoxic metabolism of F^? also regulates different membrane F^?/H⁺ exportation based on the intake of fluoride concentration (Baker et al., 2012). The urinary fluoride concentrations caused by water and pill taking might be different and ultimately result in different malformations (Liu et al., 2019). Trace elements are complex and have competitive interaction in the environment. The cytotoxicity of the coexisting elements is not the same as that treated separately (Liu et al., 2021, Mondal et al., 2019). In chronic fluorosis zebrafish models, most studies detected cytotoxicity in a dose-dependent way (Cao et al., 2020, Wu et al., 2022, Zhang et al., 2022). However, the dose threshold of fluoride shows no linear effects on larval deformities in our results. Most abnormalities were more frequent in the 184??M exposed group. We suspect that low-dose of fluoride exposure is more likely to induce embryonic malformations in zebrafish, primarily manifesting in the development of bone and neural pathways.

Fluorosis affects bone morphology and commonly results in spinal curvature and osteoporosis. In rats, it thickens bone trabeculae, reduces gaps, and causes chromatin condensation as well as cytoplasmic vacuoles in osteoblasts and chondrocytes (Meng et al., 2014, Yan et al., 2015). Fluorapatite formation weakens bone lattices, risking skeletal structure (Johnston and Strobel, 2020). Fluorosis also reduces the skull bone size and anophthalmia, with widened cranial sutures (Mathur et al., 2022). Interestingly, a minute dosage of fluoride stimulates cell proliferation and increases bone mass in osteoblast cell models cultured in vitro (Li et al., 2014, Qu et al., 2008). It validates our speculation that low-dose fluoride stimulation is more prone to induce developmental alterations in bone, and such changes may manifest opposite results in different tested cells/animals.

Besides, in our study, water fluorination triggers a negative swimming behavior and responsiveness in zebrafish larvae. Previous studies demonstrated that prenatal fluoride exposure is associated with a high risk of miscarriage and worse neurobehavioral outcomes, especially in male offspring. The neurotoxicity of fluorosis attenuates the functional hippocampus by altering the BDNF-TrkB pathway, which increases the incidence of attention deficit hyperactivity disorder in these children (Ge et al., 2018, Griebel-Thompson et al., 2023, Li et al., 2022). Hence, fluoride could penetrate the blood-fetal barrier and exert an influence on the neurodevelopment of future generations, leading to behavioral disorders in offspring. Our findings highlight that the manifestation of neurotoxicity caused by low-concentration fluoride is particularly prominent. This discovery holds great significance in deepening our understanding of fluoride’s potential hazards and ensuring the well-being of children and expectant mothers.

5. Conclusions

Water fluoridation disrupts the oxidative balance of zebrafish embryonic nutritive organs, affecting their circulatory and behavioral responses. However, the teratogenic effects on organ development exhibit a dose-dependent relationship, wherein subtle fluoride exposure is more prone to inducing anomalous development in skeletal, cerebral, and cardiac structures within non-lethal concentration levels. Despite fluoride stimulation alters embryogenesis by freely crossing through and accumulating in all types of tissues and cells, the effects of fluoride toxicity could have various outcomes for different doses, ages, genders, durations, and exposure windows. The developmental anomalies in multiple organs of zebrafish embryos resulting from acute and short-term fluoride exposure provide crucial insights for anticipating potential toxicity in humans and evaluating environmental toxicological monitoring. This discovery holds significant implications for water quality surveillance in high-fluoride regions and the diagnosis/ treatment of fluorosis. Such environmental stress factors on the next generations are important in terms of the sustainability of ecosystems.