Study On Pulmonary Toxicity Of Nanoparticles Of Calcium Carbonate

Objective: The aim of the present study is to investigate the potential pulmonary toxicity of nano calcium carbonate in rats and explore for its possible mechanism.Methods: 1 Animal experiment: Forty healthy male Wistar rats were divided into five groups randomly, they are blank control, micron calcium carbonate control, and there different doses of nano calcium carbonate (0.8, 4, 20 mg/ml). They were exposed nano calcium crabonate, micron calcium carbonate and NS respectively by intratracheally instillation once a week for five weeks. The bronchoalveolar lavage fluids (BALF) were collected, and lung morphological change was observed under light microscopic. The indices in BALF and lung tissue were determined, including albumin, TP, LDH, AKP, ACP, SOD, MDA and hydroxyproline. Comet assay was performed to detect peripheral blood lymphocytes DNA damages.2 Cell experiment: NR8383 cell were selected for in vitro study. We studied the cytotoxicity, oxidative stress and cell apoptosis of NR8383. NR8383 cells were respectively exposure to different doses of nano calcium carbonate (6.25, 12.5, 25, 50, 100, 200μg/ml), micron calcium carbonate (200μg/ml), and PBS for 24h. Cells morphological changes observed by optical microscopy. MTT colormetric assay was used to detect the cell viability of NR8383 cells. Indicatirs of cytotoxicity and oxidative stress, including TP, LDH, SOD, GSH, MDA in supernatant as well as in cultured cells, were quantitatively assessed by kit methods. The cells apoptosis induced by nano calcium carbonate was characterized by annexin V-FITC/PI staining assay. Using ELISA detected content of NO, TNF-αin the culture medium after exposure to calcium carbonate 24h. The ultrastructural changes of NR8383 cells were observed by TEM.Results: 1 Animal experiment: The level of LDH and ACP activitives increased as the dose rises, and the different was statistically significant(P<0.05). The activities of AKP increased with exposure dose increased, but the difference was not statistically significant. The contents of SOD, hydroxyproline and MDA of the lung have not significantly difference among the groups. Pathological results showed that the bronchus and alveoli of rats in three nano calcium carbonate groups were damaged in different degree. We also observed infiltration of lympholeukocyte and heterophil granulocyte, the proliferation of interstitial fibrocyte. The results also showed that serious DNA damage of peripheral blood lymphocytes was induced by nano calcium carbonate was highter than that of the negative control groups(P<0.05).2 Cell experiment: After 24h exposure to nano calcium carbonate, the viability of NR8383 cells was decreased significantly comparied with control group (P<0.05), the LDH levels in the supernatants of exposed groups increased with the dose upward trend, which was consistent changes in cell morphology. The levels of NO and TNF-αin exposed groups were higher than control group, there is significantly different in NO levels (P<0.05), but there is not significantly different in TNF-α. The contents of SOD and GSH in cultured cells showed a decreasing trend with the doses increasing, but the MDA contents increaaed. AS well as there is significantly different in groups. After 24h exposure, the apoptosis rate of NR8383 cells in nano-doses were higher than control group, but there is not significantly different among groups. The cell ultrastructure shown that mitochondria swelled, crest disorders, and a series of changes in chromosome condensation in higher nano calcium carbonate groups.Conclusion: 1 Contents of albumin, LDH and ACP in BALF change after exposed to nano calcium carbonate five weeks. It is showed that nano calcium carbonate can cause inflammation of lung tissue, lead to substantial and membrane of the lung tissue damage. Pathological changes in lung of rats appeared congestion, edema, inflammatory cell infiltration. At the same time the injuries more serious in nano calcium carbonate group than micron calcium carbonate of same dose.2 Exposed to 4mg/ml, 20mg/ml nano calcium carbonate can cause DNA damage in peripheral blood lymphocytes.3 Exposed to nano calcium carbonate 24h, survival rate, anti-oxidation and phagocytosis of NR8383 was decreased, but cell membrane permeability was increased. In addition cell ultrastructure was changed.4 The cytotoxicity of calcium carbonate was more serious in nano than micron in the same dose.