Sub-chronic Pulmonary Toxicity Of Ceria Nanoparticles In Rat

Purposes:Nano-ceria has been widely applied in biomedical and industrial activities.Domestic and foreign researches have reported the lung toxicity of nano-ceria,but there have long been controversies on the nano-ceria toxicity.Based on this,our study focused on the lung toxicity induced by subchronic exposure of nano and micro-ceria,in which Sprague-Dawley(SD)rats were exposed to nano-and micro-ceria suspensions by intratracheal instillation every other day for 25 times.We explore the subchronic toxicity of nano-and micro-ceria on rat lung by detecting the weight,lung index,lung pathological changes,bronchoalveolar lavage fluid,alterations of inflammatory mediators in lung,oxidative stress,apoptosis of alveolar macrophages and pulmonary fibrosis.Through the comparison of two particles,we aim to clarify the relationship between particle properties and toxicological characteristic of cerium oxide particles,thus to provide experimental evidence for the evaluation of nano-ceria induced lung toxicity after inhalation.Methods:A total of 50 male SD rats were randomly divided into control group(normal saline,n=10),low dose nano group(2mg/kg nano-ceria,n=10),high dose nano group(10mg/kg nano-ceria,n=10),low dose micro group(2mg/kg micro-ceria,n=10),high dose micro group(10mg/kg micro-ceria,n=10),and rats were exposed to nano and micro-ceria suspensions by intratracheal instillation every other day for 25 times.Scanning electron microscopy(SEM)was used to characterize the particle size and shape,after which dynamic light scattering was performed to detect the hydrodynamic sizes and Zeta potentials of particles.Weight and lung index were measured to evaluate the general toxicity of ceria particles to rats,and HE staining was used to observe the morphological changes of lung.Proportion of white blood cells in bronchoalveolar lavage fluid were detected.We evaluate the changes of collagen fibers in lung tissue by using Van-Gieson staining,and then biochemical assay was performed to detect the hydroxyproline content,total protein in BALF(TP),total anti-Oxidant(T-AOC),superoxide dismutase(SOD),glutathione(GSH)and malondialdehyde(MDA)content.ELISA assay was used to detect acid phosphatase(ACP)in BALF,alkaline phosphatase(AKP),TNF-?,TGF-? and IL-10.Cell apoptosis of pulmonary macrophages was detected by flow cytometry.Results:1.Characterization of nano and micro cerium oxide particlesThe initial particle size of nano-ceria was 48.7±5.1nm.While dispersed in medium,the hydrodynamic size was 345.9±12.3nm with the Zeta potential value of 17.65 mV.The micro-ceria exhibited an initial particle size of 1143.3±241.3nm and a hydrodynamic size of 1374.9±39.7nm,and the Zeta potential value was at 25.75 mV.2.Change of rat’s general conditionWeight of rats decreased in nano and micro-cerium treated groups,but there was no significant difference compared with control group.No significant changes were detected in lung coefficient.There was no statistical difference between the nano-ceria and micro-ceria groups in same dose.3.Observation of pulmonary histopathologic changesIn low dose nano group,different degree of congestion was observed in alveolar interval,and there was slight inflammatory cells aggregation in peribronchiolar area.Moreover,red blood cells and inflammatory cells infiltration was found in endovascular.In high dose nano group,inflammatory cells aggregation exacerbated and hyaline degeneration of small vessels was obviously observed,following with granuloma generation.While in micro groups,evident alterations including compensatory emphysema,bronchial mucous membrane swelling and vessel wall thickening with Inflammatory cel s exudation were observed.4.Alterations of white blood cel s and total protein in BALFWith the increasing doses of nano-ceria and micro-ceria,neutrophil granulocyte ratios increased significantly.Between the same dosage of nano-ceria and micro-ceria,the differences of neutrophil granulocyte ratios were not obvious.The total protein in BALF of high dosage nano-ceria increased evidently,while other groups showed insignificant changes.Between the same dosage of nano-ceria and micro-ceria,the alterations of total protein in BALF were not significant.5.Alterations of oxidative stress injuryHigh dose nano-ceria decreased the level of T-AOC and increased MDA level.There were no significant differences in the alterations of SOD and GSH levels.No significant changes were detected in T-AOC,MDA,SOD and GSH levels in low dose nano-ceria group.High dose micro-ceria decreased the level of T-AOC.There were no significant alterations in MDA,SOD and GSH levels of micro-ceria.6.Changes in the level of AM apoptosisBoth low and high dose nano-ceria increased AM apoptosis rate in a dose dependent manner.Different doses of micro-ceria induced the increase of AM apoptosis rate in a same way with the nano particles.There were no significant differences between same doses of nano and micro-ceria.7.Nano and micro-ceria induced fibrosisCompared with the control group,only high dose nano-ceria caused the increase of hydroxyproline content,in which the hydroxyproline content was higher than high dose micro-ceria group.VG staining showed that nano-ceria induced more serious pulmonary fibrosis than micro-ceria.Conclusion:Nano-ceria induced pulmonary inflammation,oxidative stress,cell apoptosis and obvious pulmonary fibrosis in a dose dependent manner.Micro-ceria induced pulmonary inflammation and cell apoptosis were also increased with the increase of dosage.Compared micro-sized cerium oxide particles,cerium oxides nanoparticles can cause more pronounced pulmonary fibrosis.