Synergistic Effects of Vitamin C Mitigates Sodium Fluoride-Induced Dental Fluorosis and Allergic Immune Responses in Mice.

Excessive fluoride intake primarily occurs through community water sources, varying based on the concentration in drinking water (Fawell et al., 2006). Fluoride consumption at concentrations less than or equal to 0.5 mg/L in potable water shows beneficial effects in humans, such as improving dental health by reducing dental caries and strengthening tooth enamel (Hagmann, 2008). However, unwarranted fluoride consumption from the community water sources affects various tissues and organs, although the exact mechanisms are yet to be fully explored (Barbier et al., 2010; Jha et al., 2013; Perera et al., 2018). In the current scenario, other significant sources of fluoride toxicity include toothpaste, dental mouthwash, tea, seafood products such as edible bones or shells, and vitamin supplements (Aoun et al., 2018; Emekli-Alturfan et al., 2009; Krishnankutty et al., 2022; Miri et al., 2018; Shanmugam and Selvaraj, 2022). Fluoride from food, water, and other sources is readily absorbed in the small intestine and stomach, leading to calcification and storage in tissues, with some excreted through urine (Johnston and Strobel, 2020). Fluoride can replace hydroxyl ions in the hydroxyapatite to form fluorapatite or fluorohydroxyapatite in bone and teeth, resulting in the dental and skeletal fluorosis (Aoun et al., 2018; DenBesten and Li, 2011; Johnston and Strobel, 2020; Shen et al., 2019).

Reports suggest that fluoride has been linked to allergic hypersensitivity (Zhu and Wei, 2024b), although the underlying mechanism is yet to be elucidated (Strunecka and Strunecky, 2020). Our research group conducted a field study in which drinking water samples were collected in the year 2021, and fluoride levels were estimated from different rural areas of both Mysuru and Mandya districts, Karnataka, India. Interestingly, we found that the fluoride levels were above the permissible levels (> 1.5mg/L) in the collected drinking water samples (unpublished data). In addition to water analysis, the study revealed high levels of blood eosinophils and serum IgE in the population from the same study area. Additionally, we observed for food allergic symptoms along with dental fluorosis in the same affected population (Unpublished data). This indicated, there might be a linkage between fluorosis and allergic immune response in the population drinking fluoride-enriched water. Further investigation is underway at our institute to understand, how fluorinated drinking water elevates serum IgE and blood eosinophils in the population.

Limited reports are available on the immunological response developed upon the fluoride intake in drinking water and allergic response to food allergens (Guo et al., 2017; Zhu and Wei, 2024a). Hence, we developed a NaF-OVA-induced murine model of allergic response and dental fluorosis. We report that the NaF-OVA combination administered in mice developed allergic symptoms along with dental fluorosis (Fig 1d and Fig1e). The effect of fluoride exposure from various sources on tooth development and enamel formation was reported earlier (Everett, 2011). Unusually, daily drinking water intake was significantly reduced in both the NaF alone and NaF-OVA mice groups (Fig 1b). However, the reason behind the lower water consumption compared to the control was not fully understood. Additionally, fluoride accumulation was found in the liver, kidney and spleen tissues (Fig 2). Similar results were observed by Masashi Tsunoda et.al. (2005) where fluoride levels significantly increased in the liver and kidney tissues upon intake of 25 and 125 ppm NaF in drinking water (Tsunoda et al., 2005). Another study also reported that continuous exposure to fluoride from drinking water causes renal injury in humans, indicating a toxicological effect caused by fluoride (Dharmaratne, 2019; Malin et al., 2019) . Xiong et al. (2007) indicated that fluoride levels over 2.0 mg/L in the drinking water supply might damage the liver and kidney functions in children (Xiong et al., 2007).

Due to accumulation of the fluoride in tissues, we further examined their destruction and immune cell infiltration (Fig 5), followed by OVA-specific IgE and IgG1 humoral responses in mice (Fig 4). Others also investigated the role of fluoride exposure on various immune cells and the development of allergic responses in mouse models (Wang et al., 2023; Zhu and Wei, 2024b). In summary, NaF-OVA administration in mice strongly induces both the dental fluorosis and an allergic immune response.

Vitamin C is a well-known antioxidant and free radical scavenger have ability to reduce NaF-induced oxidative stress in mitochondria (Peng et al., 2019; Phan et al., 2018). Additionally, studies have explored the anti-allergic properties of vitamin C, and its reduction of allergic symptoms widely (Hagel et al., 2013; Johnston et al., 1992; Li et al., 2024; Vollbracht et al., 2018). Clinical reports suggest, the anti-allergic effects of vitamin C in individuals who exposed to allergens, which provides an insights into its therapeutic application (Ghalibaf et al., 2023). Other molecules such as oleanolic acid, caffeine, and resveratrol have shown protective effects against the NaF-induced inflammatory diseases (Atmaca et al., 2014; Inkielewicz-Stepniak and Czarnowski, 2010; Sarkar et al., 2014). Therefore, we tested the efficacy of vitamin C in reducing allergic symptoms and dental fluorosis which induced by NaF and OVA administration in mice. Interestingly, vitamin C-administered mice showed the lower clinical symptoms (Fig 1c) and OVA-specific IgG1 levels, but no changes in the total IgE and OVA-specific IgE in the serum (Fig 4). Reports suggest that vitamin C is a potential candidate as preventive and/or therapeutic treatment for allergic diseases. Abbas et al (2017) suggested that vitamin C exerts as an anti-allergic effect by inhibiting degranulation and regulating the Th1/Th2 cell polarization balance (Abbas et al., 2017). However, our study exhibits that vitamin C effectively reduces allergic symptoms (Fig 1c), but not OVA-specific IgE and total IgE responses (Fig 4). Furthermore, there is an increased basophils and eosinophils count in mice administered with the NaF-OVA, whereas vitamin C supplementation reduced both the basophils and eosinophils count (Fig 3). This suggests that vitamin C effectively reduces both the allergic immune response and dental fluorosis in mice.

Vitamin C also reduces histopathological alterations in the liver, kidney, thymus, and spleen tissues of the NaF-OVA-administered mice. Excessive fluoride intake in water induces immunosuppression by decreasing the number of immune cells and damaging the immune function of the thymus, leading to damage via various apoptotic mechanisms and signaling pathways (Deng et al., 2016; Wang et al., 2023; Wei et al., 2020). In mature rats, fluoride treatment has led to a decreased ratio of the cortex to the medulla of thymus, uneven distribution of the cortical layer, irregular ultrastructure and altered morphology of thymus lymphocytes, and enlargement or cavitation of the mitochondrial cristae and apoptotic bodies (Das et al., 2006). In addition, fluoride triggers enlargement of the thymic nuclear space, condensation, and marginalization of the chromatin (Wang et al., 2009). Report also suggest that high fluoride induces apoptosis, mitochondria-mediated, endoplasmic reticulum-mediated, and death receptor-mediated pathways (Wei et al., 2018).

5. Conclusion:

Unwarranted fluoride consumption through the food and water poses serious health complications, including the dental fluorosis and allergic immune responses. This study found that vitamin C supplementation provides protective effects by reducing tissue damage, eosinophils infiltration, dental fluorosis, and allergy symptoms in mice exposed to the sodium fluoride and OVA allergen. Thus, vitamin C might be a potential therapeutic molecule for the treatment of dental fluorosis and allergic immune responses.

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