Toxicological and metabolomic assessment of the acute and sub-chronic effects of nanoceria (≤ 50 nm) on the human alveolar cells A549

In a society focused on sustainability, green technologies expand the use of Rare Earth Elements (REEs). Cerium dioxide nanoparticles (CeO₂NPs) possess redox/catalytic activity but raise inhalation concerns. We evaluated their effects in alveolar A549 cells exposed to 25 and 60 μg/mL, equal to 37% and 89% of the IC50, respectively. Acute exposure was evaluated at 24 h, whereas sub-chronic exposure spanned 35 days, besides recovery was assessed in cell progeny grown in CeO₂NPs absence. A multiparametric strategy combined MTT viability, ROS, mitochondrial membrane potential (ΔΨm), DNA damage (Comet assay), cell replication index (WCRI), and untargeted ¹H-NMR metabolomics. MTT showed dose-dependent cytotoxicity (IC50=67.7 μg/mL). Both concentrations increased ROS and decreased ΔΨm, with partial recovery only at 25 μg/mL. DNA damage was negligible at 25 μg/mL but persisted at 60 μg/mL during sub-chronic exposure, whereas WCRI declined with dose. Metabolomics indicated adaptive responses at 25 μg/mL (elevated glycolytic/TCA/redox metabolites), and depletion in metabolite levels at 60 μg/mL, consistent with early mitochondrial dysfunction. After 35 days of exposure, both doses caused reduction of glycine, cysteine, serotonin, acetylcholine, betaine, glucose, and succinate, and accumulation of lactate, malonate, and glutamine, indicating Warburg shift and TCA disruption. Glutathione exhibited a biphasic response. Recovery of metabolism and function occurred only at the sub-toxic dose. Overall, CeO₂NPs trigger concentration- and time-dependent cytotoxic, oxidative, mitochondrial, genotoxic, and metabolic alterations in A549 cells. These mechanistic findings support the dual antioxidant/pro-oxidant role of nanoceria and its classification as an emerging inhalable pollutant of concern for human health.