Assessing the health risks of environmentally relevant concentrations of fluoride and phenanthrene: Neurobehavioral toxicity induced in zebrafish through disruption of the microbiome-gut-brain axis and suppression of Wnt/β-catenin signalling
Hongmin Lu, Ruoqi Wang, Xin Zhang, Yuze Dong, Hao Liu, Yunfan Zhang, Chengxue Ma, Mingwei Xing
Abstract
Chronic exposure to fluoride and phenanthrene is recognized as a significant risk factor for neurobehavioral toxicity, and the gut-brain axis has been identified as a key mechanism in the development of anxiety behaviors following prolonged toxic insult. Nevertheless, the connections between gut microbiota disruption, serotonin (5-HT) production, and neurobehavioral toxicity induced by long-term co-exposure to fluoride and phenanthrene remain unclear. In this study, co-exposure model of phenanthrene and fluoride at environmentally relevant concentrations was established in zebrafish. Through comparative database analysis, anxiety behavior tests, 16S rRNA sequencing, metabolomics, qPCR, Western blotting, ELISA, flow cytometry, comet assay, histological analysis, and immunohistochemistry, the neurotoxic effects of fluoride or/and phenanthrene were investigated. In this study, neurobehavioral toxicity was observed in zebrafish following co-exposure to environmentally relevant concentrations of fluoride and phenanthrene, characterized by diminished swimming capacity and heightened anxiety behavior. The toxic mechanism was mediated through disruption of the microbiome–gut–brain axis homeostasis and suppression of the Wnt/β-catenin signalling pathway. Specifically, co-exposure decreased intestinal synthesis of 5-HT and impaired the blood–brain barrier. As the intestine serves as the primary source of 5-HT, its impairment (histopathological alterations, gut microbiota dysbiosis and metabolomics) led to dysregulation of the 5-HT system. Histopathological analysis of brain tissue revealed synaptic loss, nuclear pyknosis, and the presence of apoptotic bodies. Investigations into the molecular mechanisms indicated that fluoride or/and phenanthrene inhibited Wnt signalling, promoted β-catenin degradation, and induced neurological dysfunction, as evidenced by reductions in SYP and MAP2, which subsequently activated pro-apoptotic genes (Bax, Caspase9, and Caspase3) and inflammatory responses (decreased IL-10, elevated NF-κB, IL-6, TNF-α, and cleaved IL-1β). These alterations ultimately contributed to neurobehavioral deficits. Collectively, these findings indicate that fluoride and phenanthrene induce anxiety behavior and neurotoxicity, associated with perturbations in 5-HT metabolism, gut microbiota composition, oxidative stress, apoptosis, and neuroinflammation. • Combined exposure of fluoride or/and phenanthrene induces neurobehavioral toxicity in zebrafish. • This toxicity may be due to the disruption of the steady state of the 5-HT system in the microbiota-gut-brain axis. • Combined exposure of fluoride or/and phenanthrene inhibits the Wnt/β-catenin signalling pathway, leading to inflammation, apoptosis, and neuropsychiatric disorders. • Combined exposure to ecotoxicological risks for zebrafish is greater than the exposure to a single poison.