Sub-chronic oral exposure to titanium dioxide nanoparticles induces neurotoxicity in Wistar rats: evidence from mitochondrial, Micro-CT, and behavioral analyses

Given their widespread use in food, cosmetics, pharmaceuticals, agriculture, and industry, titanium dioxide nanoparticles (TiO₂-NPs) pose a potential risk due to frequent and prolonged exposure, raising concerns about their impact on both human and environmental health. A few numbers of studies were interested to the neurotoxicity of TiO₂ NPs through the oral pathway. This study aimed to evaluate the neurotoxic effects of sub-chronic oral exposure to TiO₂-NPs in Wistar rats and investigate their toxic mechanisms using an integrated approach that links structural, cellular, behavioral, and biochemical changes, contributing to a better understanding of their toxicity and helping to mitigate potential risks to human health and the environment. In this study, animals received daily oral gavage of TiO₂-NPs (31–15 nm) at doses of 215 or 500 mg/kg for 90 days. Oxidative stress markers were assessed using brain tissue homogenates, including antioxidant enzymes (CAT, SOD, GPx, GSH, GST) and lipid peroxidation levels. Biochemical parameters Such as carbohydrate, lipid, and protein content were also analyzed. Mitochondrial function was evaluated using an oxygen electrode system, along with assessments of mitochondrial permeability and swelling. Brain structural changes were examined using the Micro-CT Skyscan 1276 system and histological analysis. Neurobehavioral functions were assessed through standardized behavioral tests evaluating memory, learning, locomotor activity, and anxiety. Results showed that high-dose exposure led to significant oxidative stress, evidenced by decreased levels of antioxidant enzyme defenses (CAT, SOD, GPx, GSH, GST) that caused a buildup of ROS and oxidative stress, which increased MDA levels and caused membrane damage and morphological brain alterations, as confirmed by the micro-CT and the histopathological changes. Micro-CT revealed structural disorganization and reduced contrast between brain regions, while histopathological analysis confirmed neuronal degeneration, cerebral edema, and inflammatory infiltration, indicating pronounced neurotoxicity and extensive cellular damage within the brain parenchyma induced by TiO₂ nanoparticles. Mitochondrial dysfunction was also observed, including impaired respiration, elevated permeability, and swelling, as assessed. Biochemical analyses revealed disruptions in brain carbohydrate, lipid, and protein content. Neurobehavioral assessments demonstrated deficits in memory, learning, locomotor activity, and increased anxiety. These findings highlight the dose-dependent neurotoxic potential of sub-chronic TiO₂-NP exposure and emphasize the need for further investigation and regulatory oversight.