Exposure to Titanium Dioxide Nanoparticles Leads to Specific Disorders of Spermatid Elongation via Multiple Metabolic Pathways in Testes. ACS omega Titanium dioxide nanoparticles (TiO NPs) have been extensively utilized in various applications. However, the regulatory mechanism behind the reproductive toxicity induced by TiO NP exposure remains largely elusive. In this study, we employed a model to assess potential testicular injuries during spermatogenesis and conducted bulk RNA-Seq analysis to elucidate the underlying mechanisms. Our results reveal that while prolonged exposure to lower concentrations of TiO NPs (0.45 mg/mL) for 30 days did not manifest reproductive toxicity, exposure at concentrations of 0.9 and 1.8 mg/mL significantly impaired spermatid elongation in testes. Notably, bulk RNA-seq analysis revealed that TiO NP exposure affected multiple metabolic pathways including carbohydrate metabolism and cytochrome P450. Importantly, the intervention of glutathione (GSH) significantly protected against reproductive toxicity induced by TiO NP exposure, as it restored the number of Orb-positive spermatid clusters in testes. Our study provides novel insights into the specific detrimental effects of TiO NP exposure on spermatid elongation through multiple metabolic alterations in testes and highlights the protective role of GSH in countering this toxicity. 10.1021/acsomega.4c01140