Study On The Toxicological Effects And Apoptosis Induced By Dynamic Inhalation Nanoscale Carbon Black Aerosol For 28 D In Mice

Objective:1.The purpose of this study was to investigate health effects induced by dynamic inhalation nanoscale carbon black(CB)aerosol for 28 d in mouse,and further provide the toxicological data for the risk assessment of carbon black nanoparticles;2.To investigate the lung apoptotic injury mechanism induced by nanoscale carbon black particles by inhalation particle aerosol exposure to mice.Methods:1.96 male C57BL/6 eight weeks old mice were random Ly divided into three groups,including the negative control group(filtered air),the low dose exposed group(15mg/m3)and the high dose exposed group(30 mg/m3).Carbon black aerosols were blown into the inhalation chambers by aerosol generator.The mice were exposed to carbon black particles for 6 h/d per d for a continuous exposure of 28 d.During the exposure,the aerodynamic diameter,the spatial distribution and the concentrations of carbon black particles were assessed.Water and feed consumptions as well as body weight were observed every week.After 24 h of the last exposure,blood samples were collected for clinical examinations.The lungs,heart,kidneys,spleen,liver were removed and weighed.For pathological changes of these abovem entioned organ tissues,the slice was stained with hematoxylin and eosin(HE)using standard procedures.The pu Lmonary fibrous protein and apoptosis were assessed by immunohistochemistry(IHC)andtransferase-mediated d UTP nick end-labeling(Tunel)method.The u Ltrastructure of lung tissue was observed using transmission electron microscopy(TEM).Differential cell counts in bronchoalveolar lavage fluid(BALF)were conducted in the microscope.Besides,the protein concentration in BALF was detected by the method of coomassie brilliant blue.2.At the end of exposure,preparation of single cell suspension of lung tissue by cutting the proper size of lung tissue.The intracellu Lar levels of reactive oxygen species(ROS)in lung were assayed by DCFH-DA staining described previously.In addition,superoxide dismutase(SOD)and catalase(CAT)activity as well as glutathione peroxidase(GSH-px)levels in lung were measured by the standard ELISA protocols.RIPA and TRIZOL were used to extract total protein and m RNA from mice lung tissue.The levels of apoptosis related proteins Bcl-2,Bax,Caspase-3,Caspase-7,Caspase-8,Caspase-9,PARP-1,Fas in the pu Lmonary were tested by Western blot,IHC,Real-Time PCR,respectively.The expression levels of apoptosis related proteins in lung tissues were evaluated from three aspects: protein quantification,localization and gene expression.Results:1.The concentrations of carbon black particles in the low and high dose groups were(15.31 ± 3.30)mg/m3and(30.05 ± 14.20)mg/m3,respectively.No significant differences of body weight and the consumptions of water and feed were observed between the control and treatment groups.The blood biochemical indexes had no obvious changes between the control and exposed groups.Compared with the control group,red blood cells,hemoglobin concentration and hematocrit were slightly increased in the exposed groups.In comparison with the control group,there was a remarkable increase in the and lung organ coefficients in the high dose group(P =0.016),but there were no significant changes in the low dose group.The kidney and heart organ coefficients in low and high dose groups were significantly decreased(P = 0.01,P<0.01)compared with the control group.Other organ coefficients had no distinct changes.The appearance of lung tissue in the exposed groups indicated gray-black while the lung in the control group was white.Histopathology displayed that carbon black particles were deposited in the pu Lmonary alveoli or bronchiole in the exposed groups,especially in high dose exposed group.Carbon black particles swallowed by macrophages were seen in pu Lmonary interstitial.There were other pathological changes in lung tissue,including disordered alveolar wall,thickened pu Lmonary septal,and inflammatory cells infiltration.The pathological score of lung injury in the low and high dose groups were 6.63±1.13 and 9.8±0.38,which was higher than that in the control group(1.66±0.55).The spleen lymphocytes had a slight increase in the high dose group.The pathological changes were not found in other organs.In the high dose group,a large number of fibrin was deposited in pu Lmonary interstitial and the periphery of bronchiole.Pu Lmonary apoptosis cells were mostly observed at the basal lamina of epithelial cells and in close to the airways.TEM indicated that there was an increase of primary and secondary lysosome and broken mitochondrial cristae in the exposure group in contrast to the control.Some insoluble particles,that had the same electron density as the CB particles,were observed in the secondary lysosome in the exposure groups.2.Compared with the control group,in the low and high exposure groups ROS levels had 2.12-and 3.69-fold increases(P<0.01),and CAT activity had significant decreased of 33.66% and 30.94%(P<0.01).In the high exposure group,remarkable reductions of SOD activity and GSH-px levels were found(P<0.05).No significant changes were observed in SOD activity and GSH-px contents in the low exposure group.28 days after inhalation of carbon black particles in mice,the levels of Bax m RNA in exposure groups were significantly higher than that of the control group(P<0.05).The levels of Caspase-3,Caspase-7,PARP-1 m RNA in the high dose exposed group were obviously higher than that of the control and low dose exposed groups(P<0.05).Compared with the control group,in the high exposure group Caspase-8 and Caspase-9m RNA levels were also evidently increased.There were no significant differences in the expression of Bcl-2 and Fas m RNA in the three groups.The expression of Bax,Caspase3,Caspase7,Caspase8,Caspase9,PARP-1 proteins in the lung tissue of each group increased with the increase of the dose,and the exposure group was significantly higher than that of the control group(P<0.05).Immunohistochemical staining of lung tissue in mice showed that there were a lot of Caspase3,Caspase7,Caspase8,Caspase9 proteins positive apoptosis cells in exposure groups,especially in the deposition of carbon black particles.Conclusions:1.The 28 days repeated inhalation of carbon black particles primarily resulted in lung tissue injury,providing an ideal model and a reliable analytical method for the study of the pu Lmonary toxicological effects induced by carbon black nanoparticles.2.Inhalation exposure to CB nanoparticles cou Ld induce lung tissue oxidative stress and apoptosis injuries in mice.The activating oxidative stress with decreased anti-oxidative capacity and increased oxidative capacity may involve in the process of CB adverse health effects.