Salivary Proteomic Signatures Reveal Novel Mechanistic Insights into Moderate Dental Fluorosis Pathogenesis and Oral Homeostasis Disruption

Moderate dental fluorosis (MF) represents a pivotal yet understudied stage of enamel fluorosis, characterized by distinct opacities indicating substantial but incomplete enamel disruption. Despite its prevalence, the molecular basis underlying this transitional phenotype remains unclear. This study employed a system proteomic approach to delineate salivary proteomic alterations associated with MF and to elucidate their biological and mechanistic implications. Unstimulated whole saliva was collected from school-aged children with MF (n = 10) and age-matched controls without fluorosis (CF, n = 21) for comparative proteomic analysis using liquid chromatography-tandem mass spectrometry. Urinary and drinking water fluoride levels were assessed to confirm comparable exposure between groups. Differentially expressed proteins (DEPs) were identified and subjected to Gene Ontology enrichment analysis, pathway analysis, and protein-protein interaction network analysis. Among 101 shared salivary proteins, 12 DEPs were significantly different between MF and CF subjects. Up-regulated proteins (e.g., neutrophil defensin 3, protein LEG1 homolog, immunoglobulin kappa constant, pyruvate kinase, mucin-7, and alpha-enolase) converged on pathways related to immune activation and altered glycolytic metabolism. Conversely, down-regulated proteins (e.g., superoxide dismutase [Cu-Zn], neutrophil gelatinase-associated lipocalin, thymidine phosphorylase, metalloproteinase inhibitor 1, galectin-3-binding protein, and alpha-1B-glycoprotein) reflected compromised antioxidant defense and perturbed extracellular matrix remodeling. Network topology analysis revealed distinct MF-specific interactomes dominated by immune-epithelial clusters. Notably, the cystic fibrosis transmembrane conductance regulator emerged as a central hub connecting multiple DEPs and MF-exclusive proteins, suggesting that fluoride-mediated ion transport perturbation may underlie enamel hypomineralization and broader oral homeostatic imbalance. Collectively, these findings provide novel molecular insights into MF pathogenesis and establish salivary proteomic profiles as a promising noninvasive platform for biomarker discovery, disease monitoring, and understanding of fluorosis progression.