Interaction of environmental fluoride exposure and gut microbes: potential implication in the development of fluorosis in human subjects.

Fluoride, the 13th most abundant element in the earth’s crust, affects around 200 million people in 25 countries through fluoride-contaminated groundwater, with India and China being the most affected (Chen at al. 2022a). In India, over 62 million people across 20 states are facing this issue, with Rajasthan, Telangana, and Karnataka being the most affected (Faith et al. 2013). Geogenic sources, industrial activities, coal burning and volcanic activity contribute significantly to fluoride in water (Liu et al. 2021). The fluoride concentrations in different environmental components vary due to geological and chemical factors (Chen at al. 2021a). Groundwater from calcium-poor aquifers and regions rich in common fluoride-bearing minerals often exhibit high fluoride levels (Rasool et al. 2017). The distribution and mobility of fluoride in the environment are governed by diverse geochemical processes (Liu et al. 2021). Fluoride, while beneficial in low concentrations, can be toxic in excess, leading to dental and skeletal manifestations (Daillère et al. 2016). Dental fluorosis, a condition caused by excessive fluoride intake during tooth development, can progress to skeletal fluorosis, a more severe condition characterized by joint pain and stiffness, as well as bone and ligament calcification (Meazzi et al. 2022). This progression is particularly concerning in regions with high fluoride levels in drinking water, where skeletal fluorosis is prevalent (Pramanik and Saha 2017). Moreover, fluoride toxicity extends beyond skeletal system, affecting other organs and tissues, including the digestive tract, liver, kidneys, and brain (Rowland et al. 2012). Therefore, investigation of potential mitigation and prevention strategies for fluorosis is very important.

Drinking water is the major route of exposure to fluoride. Following ingestion, plasma fluoride levels rise rapidly due to pH-dependent absorption in the stomach. Fluoride that is not absorbed in the stomach is absorbed in the small intestine, where absorption occurs independently of pH. Peak plasma fluoride concentrations are reached within 20 to 60 minutes after ingestion and subsequently decrease as fluoride is sequestered by calcified tissues and excreted via the urine (Andoh 2016, Buzalafa & Whitfordb, 2011). About 75% of fluoride is absorbed through intestine (Jeerasak et al. 1989), which is inhabited by gut microbiota, indicating that they may play important role by interacting with the fluoride. The human gastrointestinal tract hosts a vast and diverse microbial community comprising an estimated 10ˆ¹⁴ microbial cells and over 1,000 species (Srivastava and Lohani 2015, Krishnan et al. 2015). The gut microbiota profoundly influences human health through its metabolic activities, by synthesizing bioactive molecules that interact with the host (Scott et al. 2006). The SCFAs are one of the significant bacterial metabolites that regulate the host’s metabolic functions, immune homeostasis, calcium metabolism, osteogenesis, and osteoclastogenesis (Madhogaria et al. 2022, Arpaia et al. 2013, Choubisa 2012, Buzalafa and Whitfordb 2011, Jansson et al. 2019). Numerous studies have demonstrated the production of SCFAs, like acetic, butyric, propionic, and valeric acid, by common gut bacterial phyla, like Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Verrucomicrobia (Klingseisen and Lyons 2018, Coryell et al. 2018, Shetty et al. 2013, Singh et al. 2018, Manges et al. 2010). The intricate interplay between the gut microbiota and bone homeostasis is gaining recognition as a significant factor in skeletal well-being. SCFAs, such as acetate, butyrate, and propionate, promote bone formation indirectly by regulating insulin-like growth factor-1 (IGF-1). Specific bacterial strains, such as segmented filamentous bacteria, can negatively impact skeletal maturation, while dysbiosis in the gut microbiota is associated with bone diseases like osteoporosis (Van den et al. 2013, Tyagi et al. 2021). The alterations in gut microbiota, such as lower abundance of Prevotella and higher abundance of Lactobacillus, are associated with bone diseases (Tyagi et al. 2021, Kho and Lal 2018). These findings underscore the importance of investigating the association of gut microbiota with the pathophysiology of bone diseases such as skeletal fluorosis, which may present a new target for preventive and therapeutic interventions.

The limited accessibility and high cost of defluoridated water in fluoride-affected regions highlight the urgent need for alternative strategies to mitigate fluorosis. The gut microbiota may present a potential target for such interventions. While metagenomic studies offer rapid and comprehensive insight into the overall structural and functional aspects of the total gut microbial community, it is unable to assign and establish the strain specific functions experimentally (Amrane et al. 2018). In contrast, culture-based methods allow for the isolation of specific bacterial strains, enabling detailed phenotypic characterization and functional studies (Lee et al. 2019). For instance, culture-based approaches successfully isolated distinct strains of Bacteroides and Fusobacterium in colorectal cancer patients, facilitating in-depth analyses of their functional roles (Lee et al. 2019). Secondly, several animal studies have reported fluoride-induced alterations in gut microbiota (Gupta et al. 2015; Xu et al. 2014), but such studies in human are yet to be reported. Also, the association of gut microbiota with the clinical outcome of fluorosis in fluoride-exposed population remains entirely unexplored.

To address the above limitations, our study aimed to isolate cultivable gut bacteria from fluoride-exposed population to facilitate detailed characterization and functional analysis of gut microbial community. A control population without any exposure to fluoride was also investigated to determine the effect of fluoride on cultivable gut microbial diversity. Variability in individual susceptibility to fluorosis has been attributed to genetic predispositions (Pramanik and Saha 2017); however, interactions between gut microbiota and fluoride exposure may also significantly contribute to this variability. Investigating the cultivable gut microbiota from fluoride-exposed individuals with different clinical outcomes of fluorosis is critical to elucidating the potential roles of these microorganisms in the progression and prognosis of fluorosis, thereby paving the way for microbiota-targeted therapeutic strategies. Therefore, the primary aim of this study was to isolate and characterize microorganisms that exhibited differential abundance across study groups with varying fluoride exposure levels and fluorosis symptoms. Additionally, we identified and quantified the potential metabolites produced by these isolates that may affect the host physiology, including fluoride-associated pathology, followed by correlation of these findings with the clinical manifestations of fluorosis in the study groups. In our knowledge, it is the first study to characterize the association of gut microbiota with clinical outcome of fluorosis in human population. Also, the culture-based approach followed in this study led to the isolation of microorganisms that may present promising candidates for future research to explore their interactions with fluoride and assess their potential therapeutic or diagnostic applications.