Co-exposure to fluoride and sulfur dioxide induces abnormal enamel mineralization in rats via the FGF9-mediated MAPK signaling pathway

https://doi.org/10.1016/j.envres.2024.118527Get rights and content

Highlights

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    Co-exposure to fluoride and SO2 causes abnormal enamel mineralization in rats.
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    Fluoride and SO2-induced mineralization abnormalities are associated with AMBN.
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    FGF9 regulates AMBN through JNK and ERK pathways and promotes enamel damage.
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    We provide a scientific basis for studying the mechanism of enamel damage.

Abstract

Fluoride (F) and sulfur dioxide (SO2) contamination is recognized as a public health concern worldwide. Our previous research has shown that Co-exposure to F and SO2 can cause abnormal enamel mineralization. Ameloblastin (AMBN) plays a crucial role in the process of enamel mineralization. However, the process by which simultaneous exposure to F and SO2 influences enamel formation by regulating AMBN expression still needs to be understood. This study aimed to establish in vivo and in vitro models of F–SO2 Co-exposure and investigate the relationship between AMBN and abnormal enamel mineralization. By overexpressing/knocking out the Fibroblast Growth Factor 9 (FGF9) gene, we investigated the impact of FGF9-mediated Mitogen-Activated Protein Kinase (MAPK) signaling on AMBN synthesis to elucidate the mechanism underlying the induction of abnormal enamel mineralization by F–SO2 Co-exposure in rats. The results showed that F–SO2 exposure damaged the structure of rat enamel and ameloblasts. When exposed to F or SO2, gradual increases in the protein expression of FGF9 and phosphorylated p38 mitogen-activated protein kinase (p-P38) were observed. Conversely, the protein levels of AMBN, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) were decreased. AMBN expression was significantly correlated with FGF9, p-ERK, and p-JNK expression in ameloblasts. Interestingly, FGF9 overexpression reduced the levels of p-ERK and p-JNK, worsening the inhibitory effect of F–SO2 on AMBN. Conversely, FGF9 knockout increased the phosphorylation of ERK and JNK, partially reversing the F–SO2-induced downregulation of AMBN. Taken together, these findings strongly demonstrate that FGF9 plays a critical role in F–SO2-induced abnormal enamel mineralization by regulating AMBN synthesis through the JNK and ERK pathways.

Introduction

Fluoride (F) and sulfur dioxide (SO2) are significant environmental contaminants, and many geographic areas are contaminated by F and SO2 (Han et al., 2021; Liu et al., 2022). More than 100 million people worldwide are at risk of fluorosis due to exposure to high concentrations (>1.2 ppm) of F (Gu et al., 2020). From 2013 to 2017, the daily average concentration of SO2 in most cities in India and China exceeded 18 μg/m3 (Kuttippurath et al., 2022; Yao et al., 2022). According to the 2016 Air Quality Guidelines from the World Health Organization (WHO), the maximum short-term exposure (24-h average) to SO2 is 20 μg/m3 (WHO Regional Office for Europe, 2006); however, authoritative research has demonstrated that only 24 h of exposure to 9.0 μg/m³ SO2 can increase the risk of an acute coronary syndrome (ACS) incident by 0.67% (Chen et al., 2022). This suggests that even SO2 concentrations below the WHO's threshold may still pose a risk to certain sensitive populations. Epidemiological studies have reported that chronic exposure to high fluoride levels can lead to dental fluorosis (DF), which is characterized by reduced enamel mineral content and increased porosity (Revelo-Mejia et al., 2021). In contrast, chronic exposure to SO2 fumes leads to dental erosion, which is characterized by enamel demineralization, dentin loss, and pulp exposure (Shirlene et al., 2018). Our previous research has shown that Co-exposure to F–SO2 causes abnormal mineralization of rat tooth enamel to varying degrees, but the exact mechanism is unclear.
Human epidemiology and animal experiments have demonstrated that fluoride-induced under- or over-mineralization of enamel is associated with abnormal secretion of enamel matrix proteins (Suzuki et al., 2014; Wong et al., 2022). Enamel matrix proteins provide an organic microstructure during the enamel formation stage, guiding the initiation and directional growth of hydroxyapatite crystals in enamel and ensuring their elongation and directed growth. Among these proteins, ameloblastin (AMBN) is the most prevalent non-amelogenin enamel matrix protein (Moradian-Oldak and George, 2021). It interacts with hydroxyapatite crystals and regulates the formation and mineralization of enamel by preserving the shape and size of these (Su et al., 2022). Genetic polymorphism analysis has revealed that AMBN is associated with an increased risk of dental fluorosis, and mutations in the AMBN gene can increase the likelihood of developing this condition (Charone et al., 2019; Gonzalez-Casamada et al., 2022). However, the mechanism by which F–SO2 Co-exposure regulates enamel development by regulating AMBN expression has yet to be fully elucidated.
The development of dental enamel involves the regulation of many signaling molecules, including Fibroblast Growth Factor 9 (FGF9). FGF9, a fibroblast growth factor, significantly regulates enamel development by promoting the multiplication and differentiation of dental epithelial cells while also regulating the expression of signaling molecules to ensure the standard establishment and development of enamel (Kamiunten et al., 2017). Our previous studies have shown that Co-exposure to F–SO2 can lead to abnormal enamel mineralization by affecting the FGF9-regulated synthesis of enamel matrix proteins (Lv et al., 2023a, Lv et al., 2023b). However, the downstream regulatory mechanism still needs further research.
The Mitogen-Activated Protein Kinase (MAPK) signaling pathway is primarily composed of Extracellular Signal-Regulated Kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. This pathway regulates enamel structure and morphology by controlling ameloblast proliferation and differentiation, enamel matrix protein synthesis, and the formation and arrangement of enamel prisms (Chaweewannakorn et al., 2017). There is evidence that FGF9 can inhibit the osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) and reduce the deposition and mineralization of bone by regulating the MAPK signaling pathway; thus, FGF9 regulates bone formation (Tang et al., 2021a). However, it is unclear whether Co-exposure to F–SO2 may participate in enamel damage by affecting ANBN synthesis through the FGF9-regulated MAPK signaling pathway.
This study aimed to establish in vivo and in vitro models of F–SO2 Co-exposure to investigate the relationship between AMBN and enamel damage. Additionally, the impact of FGF9-mediated MAPK signaling pathway regulation on ANBN synthesis was studied by overexpressing and knocking out FGF9 to reveal the mechanisms underlying the abnormal enamel mineralization caused by Co-exposure to F–SO2 in rats. Through these experiments, we aimed to provide scientific evidence for future research on the toxic effect of simultaneous F–SO2 exposure on enamel.

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Section snippets

Animal model

Sodium fluoride (NaF) is commonly used as a test substance in toxicological experiments on fluoride exposure. There are two standard approaches for toxicological experiments on SO2 exposure: inhalation of SO2 gas and oral or intraperitoneal injection of SO2 derivatives. Due to the difficulty in controlling dose during SO2 gas poisoning, this study used exposed animals to SO2 derivatives by adding these derivates to drinking water. The dose was chosen based on the daily total fluoride intake by

F–SO2 exposure leads to abnormal enamel development in rats

To investigate the impact of F–SO2 exposure on the development of rat enamel, we treated SD rats with varying concentrations of NaF and SO2 for 12 weeks. The findings revealed that after 4, 8, and 12 weeks of treatment, the rats in the control group exhibited robust incisor growth, and the dentition was glossy brownish-yellow with good transparency; however, the group exposed solely to SO2 had poor tooth transparency. The groups exposed solely to NaF and to both F–SO2 began showing signs of

Discussion

Our previous research has shown that F–SO2 exposure can lead to abnormal enamel mineralization (Lv et al., 2023a, Lv et al., 2023b). Nevertheless, the involvement of the FGF9 and MAPK pathways in enamel development has yet to be documented. In this report, we initially examined the significant correlation of the enamel matrix protein AMBN with FGF9 and critical signaling molecules in the MAPK pathway in rat incisor ameloblasts. Subsequently, based on the model in which FGF9 was overexpressed

CRediT authorship contribution statement

Ying Lv: Writing – review & editing, Writing – original draft, Conceptualization. Jin Yao: Methodology, Data curation. Yang Wang: Software, Formal analysis. Guohui Bai: Visualization, Validation. Changhu Lin: Supervision, Resources. Chenglong Tu: Visualization, Project administration, Funding acquisition.

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.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant Nos. 41967051 and 82360632) and School Bohe J character [2022]045.

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