Sex-Specific Mechanisms of Fluoride-Induced Gonadal Injury: A Multi-Omics Investigation into Reproductive Toxicity and Gut Microbiota Disruption

Fluoride, a common agricultural additive used to enhance plant resilience and pest control, poses toxicity risks when exposure surpasses safe thresholds, affecting ecosystems and human health. While its reproductive toxicity is recognized, the sex-specific and cross-generational effects remain underexplored. To address this gap, we employed an integrative approach combining transcriptomics (next-generation sequencing (NGS)), bioinformatic network analysis, gut microbiota sequencing, and <i>in vivo</i> functional assays. ICR mice (F0 generation), both male and female, were exposed to fluoride (100 mg/L in drinking water) for 35 days, continuing through gestation and offspring weaning. Our transcriptomic analysis revealed significant upregulation of autophagy (via the PI3K-AKT-mTOR pathway) and oxidative stress-induced mitochondrial dysfunction in gonadal tissue, with more pronounced effects observed in males. Further integrated analyses of transcriptomic and metabolomic data, supported by <i>in vivo</i> experiments, highlighted oxidative stress, mitochondrial dysfunction, and PI3K-AKT-mTOR pathway activation with stronger effects in males. The principal component analysis confirmed sex-specific transcriptome alterations, with males showing more substantial disruption. Additionally, 16S rRNA sequencing identified significant gut dysbiosis, particularly in males, with an increased <i>Firmicutes/Bacteroidetes</i> ratio and higher abundances of <i>Oscillospirales</i> and <i>Anaerovoracaceae</i>. Moreover, our study identified significant correlations between specific gut microbiota (e.g., <i>Firmicutes</i>, <i>Proteobacteria</i>) and autophagy, oxidative stress, and mitochondrial dysfunction pathways, with notable sex-dependent differences. These findings suggest that gut microbiota may play a critical role in modulating fluoride-induced reproductive toxicity, particularly through their effects on oxidative stress and cellular homeostasis. The breakdown of the gut barrier and elevated serum/gonadal lipopolysaccharide (LPS) levels in fluoride-treated mice further established a link between gut dysbiosis and fluoride-induced reproductive toxicity. These findings underscore the importance of considering sex differences in xenobiotic-induced reproductive and developmental toxicity.