CeO₂ nanoparticles induce pulmonary fibrosis via activating S1P pathway as revealed by metabolomics

CeO₂ nanoparticles (NPs) have been shown to cause lung fibrosis, however, the exact underlying molecular mechanisms are poorly understood. In this study, we have conducted a mass spectrometry-based global metabolomic analysis of human bronchial epithelial BEAS-2B cells treated by CeO₂ NPs with different aspect ratios and assessed their toxicity on the bronchial epithelial cells by various cell-based functional assays. Although CeO₂ NPs at doses ranging from 12.5 μg/mL to 25 μg/mL displayed low cytotoxicity on the bronchial epithelial cells, the metabolomic analysis revealed a number of metabolites in the cellular metabolic pathways of sphingosine-1-phosphate, fatty acid oxidation, inflammation, etc. were significantly altered by CeO₂ NPs, especially those with high aspect ratios. More importantly, the robustness of metabolomics findings was further successfully validated in mouse models upon acute and chronic exposures to CeO₂ NPs. Mechanistically, CeO₂ NPs upregulated transforming growth factor beta-1 (TGF-β1) levels in BEAS-2B cells in an aspect ratio-dependent manner through enhancing the expression of early growth response protein 1 (EGR-1). In addition, both in vitro and in vivo studies demonstrated that CeO₂ NPs significantly induced the expression of sphingosine kinase 1 (SHPK1), phosphorylated Smad2/3 and lung fibrosis markers. Moreover, targeting SPHK1, TGFβ receptor or Smad3 phosphorylation significantly attenuated the fibrosis-promoting effects of CeO₂ NPs, and SPHK1-S1P pathway exerted a greater effect on the TGF-β1-mediated lung fibrosis compared to the conventional Smad2/3 pathway. Collectively, our studies have identified the metabolomic changes in BEAS-2B cells exposed to CeO₂ NPs with different aspect ratios and revealed the subtle changes in metabolic activities that traditional approaches might have missed. More importantly, we have discovered a previously unknown molecular mechanism underlying CeO₂ NP-induced lung fibrosis with different aspect ratios, shedding new insights on the environmental hazard potential of CeO₂ NPs.