| Objective: Carbon black is an amorphous carbon,with a very large surface area,up to 3000m2/g.Due to the stability of carbon black,carbon black is often used in negative controls to explain the toxicity of some nanomaterials.There is evidence that nanoscale carbon black particles can induce pulmonary inflammation and histopathological damage.The International Cancer Research Institute(IARC)then classifies carbon black as a possible carcinogen.However,the lung inflammation and histopathological damage caused by carbon black are still controversial because there is no literature on carbon black-induced lung disease clinically.Therefore,we investigated the mechanism of pulmonary injury in rats after 28-day nano-carbon black particles inhaltion.The previous studies suggested that the oxidative stress might play an important role on the damages induced by nanoparticles.The FOXO family of transcription factors,served as the oxidative stess factors,involved in cell cycle arrest,cell death and protection of stress to stimulate multiple processes.In this study,the nanoscale carbon black particles inhalation rat model was established.We evaluated the deposition and clearance of carbon black in lung after inhalation and the damages of the lung.The oxidative stess,DNA dmages,and the expression of FOXO3 a,FOXO1 protein were detected to explore the mechanism of pulmonary injury induced by carbon black.Method:1 The size and morphology of nanoscale carbon black particles were measured by Tecnai G220 transmission electron microscopy;the surface morphology was observed by scanning electron microscopy.The specific surface area of nano-sized carbon black particles was calculated using BET adsorption isotherm.2 48 healthy SD male rats,weighing 150-180 g,were randomly divided into four groups,the control group,14 d exposure group,28 d exposure group and recoverry group.Rats in 14 d and 28 d exposure groups were inhalated nano-sized carbon black particles for 6 hours at a concentration of 30mg/m3 for 14 d and 28 d,respectively.Rats in the recovery group were exposed to CB for 28 d and recovered for another 14 d.At the same time,the control animals were exposed to filtered air for 6 h/day.The rats in 14 and 28 d exposure groups were sacrificed 24 h after the last exposure.The rats in the recovery group were sacrificed 14 d after the last exposure.Each of the 8 rats in the control group were randomly selected and sacrificed at the same time as the three exposure groups.3 The body weight of the rats was recorded every 3 days during the exposure.At the end of the exposure,the animals were anesthetized and then blood samples were collected.Serum was harvested by centrifugation at 700 ×g for 10 min.The trachea,lungs,liver,kidneys,and spleen tissues were removed,weighed,and stored in liquid nitrogen for further analysis.4 Hematoxylin-Eosin(HE)staining: The lungs were separated and maintained in a fixative solution(4% paraformaldehyde)until they were embedded with paraffin.Paraffin sections were prepared with routine methods.For pathological changes of lung tissue,the slice was stained with HE using standard procedures.5 The deposition and clearance of carbon black in lung tissue: Estimated lung deposition of CB nanoparticles was calculated using the following equation: Lung burden=(MV)×(T)×(CON)×(DF)=(7.3ml/min)(360min)(30mg/m3)(33.8%)where(MV)is the minute ventilation of the exposed animal(mL/min);(T)is the total exposure time(min);(CON)is the average exposure concentration(mg/m3);and(DF)is the pulmonary deposition fraction for the alveolar region of the particles inhaled.6 Determination of oxidative stress in lung tissue of rats: Changes of SOD,GSH-Px activity and MDA contents in rat lung tissue after exposure to nano-scale carbon black were detected with commercially available kits according to manufactureers’ recommendation.the cell suspension of lung from rats were isolated and incubated at a final concentration of 10 mM 2,7-dichlorodihydrf-luorescein diacetate(DCFH-DA)fluorescent probe.The forma-tion of the fluorescent-oxidized derivative of DCF was monitored using a flow cytometry at emission wavelength of 525 nm and excitation wavelength of 488 nm.Finally,ROS generation was quantified by the median fluorescenceintensity of 10,000 cells.7 Determination of Apoptosis in Lung: The lungs were embedded in paraffin-embedded sections,and the whole apoptotic nuclei or apoptotic bodies were in situ stained to detect the number of apoptotic cells in the lung tissue after exposure to nano-scale carbon black particles.8 DNA damage determination: The comet assay(SCGE)was used to detect the changes of OTM and TailDNA% in rat tissue after exposure to nano-scale carbon black particles.9 FOXO3 a and FOXO1 expression in lungImmunohistochemistry: paraffin sections were dewaxed in xylene,rehydrated with distilled water,and then subjected to antigen retrieval for 30 min at 95°C.The slides were subsequently incubated overnight at 4°C with the following antibodies: FOXO3 a,P-FOXO3 a,FOXO1 and P-FOXO1.Slides were then treated with an anti-rabbit secondary antibody and developed using avidin-conjugated HRP with diaminobenzidine as a substrate,followed by hematoxylin counterstaining.Images were observed under a light microscope.Western blot: Cell lysates were prepared using modified RIPA buffer with HaltTM protease and phosphatase inhibitor cocktail.Western blotting was performed by standardmethods using following antibodies: FOXO3 a,P-FOXO3 a,FOXO1 and P-FOXO1.Primary antibody was detected withperoxidase-conjugated anti-rabbit Ig G secondary antibody followed by ECL detection.Result:1 Characteristics of Nanoscale Carbon Black ParticlesThe expected three-dimensional nanostructure of CB is clearly confirmed by the Scanning Electron Microscopy(SEM)and Transmission Electron Microscopy(TEM)CB consists of globular shaped particles and aggregates of tens to hundreds of nanometers were formed from spherical particles of size30 to 50 nm without intermediates The Brunauer-Emmett-Teller(BET)results show that the CB particle surface area is about 74.85 m2/g.2 The general condition of the ratsDuring the course of the experiment,all of the rats survived and were generally in good condition;no significant toxic effects were observed during the exposure and recovery.The eating and drinking water of rats is normal and themental state is good.3 Effects of Carbon Black on Body Weight Changes in RatsDuring the exposure of carbon black for 28 days,the body weight growth rate of exposed group was slightly lower than that of control.But there was no significant difference in body weight between the exposure and control group(P>0.05).4 The organ coefficients of ratsAfter carbon black inhalation for 14 days,the absolute weight of the lung and lung organ coefficient was no significant difference compared with the control group(P>0.05).After 28 days of inhalation,the absolute weight of the lungs was not significantly different from that of the control group,and the organ coefficient of the lungs increased significantly(P<0.05).After 14 days of recovery,the organ coefficient of the lung was still significantly higher than that of the control group(P<0.05).Other organs of the organ coefficient were no significant changes.5 Deposition rate and clearance rate of carbon black in lung tissueThe theoretical deposition of carbon black in each rat was 373.03 mg,746.06 mg.After the 28 days exposure of carbon black,most of the carbon black particles are deposited in the lung interstitial,and a small amount of carbon black in the alveoli,after 14 days recovery carbon black can be removed to the original 82.5%.It was significantly decreased,compared with the 28 d inhalation group(P<0.05).6 Pathological changes of lung in ratsThe appearance of lung tissue in the CB exposure group became black while the lung in the control group was white.In lung tissue,CB particles were deposited in the pulmonary alveoli or bronchioli after CB exposure for14 and 28 d.In the recovery group,there were still some CB particles deposited in the bronchioli but more particles in the pulmonary alveoli and the alveolar wall tissue,whereas the weight of the lung decreased.After CB exposure for 14 and 28 d,the alveolar wall thickened significantly and a large amount of inflammatory cells,constituted mainly by lymphocytes,were infiltrated.The characteristic features of the pulmonary alveoli and the bronchiole were slightly disarranged and swollen with some of the cells showing vacuolar degeneration in the CB exposure groups.This histological change remained in the recovery group.7 Changes of apoptosis in lungThe results of TUNEL showed that the percentage of apoptotic cells in the 14 days and 28 days exposed group was significantly increased,compared with the control(P < 0.05).Compared with the 28 d inhahation group,the apoptotic cells were not significantly different from the recovery group(P>0.05).8 Effects of nano-scalecarbon black on oxidative stress in lung tissue of ratsCompared with the control group,the activities of SOD and GSH-Px were significantly decreased in the lung tissue of rats after inhalation for 14 and 28 days(P<0.05).The activity of SOD and GSH-Px in the recovery group were significantly increased,compared with the 28 d inhalation group(P<0.05).Compared with the control group,the levels of ROS were significantly increased in the lung tissue of rats after inhalation for 14 and 28 days(P<0.05).The levels of ROS in the recovery group were significantly decreased,compared with the 28 d inhalation group(P<0.05).And the levels of ROS were not significantly different from that of the control group(P>0.05).9 Effects Carbon Black on DNA Damage in lungAfter carbon black inhalation for 14 days,the TailDNA% and OTM value in the lung tissue were no significant difference compared with the control group(P>0.05).After 28 days of inhalation,the TailDNA% and OTM value in the lung tissue were significantly decreased,compared with control group(P<0.05).After 14 days of recovery,the TailDNA% and OTM value in the lung tissue were no significant changes,compared with the control group;and the TailDNA% and OTM value in the lung tissue were significantly different from that of the 28 d inhalation group(P<0.05).10 Effects of Carbon Black on expression of FOXO3 a,P-FOXO3 a,FOXO1 and P-FOXO1 Proteins in LungCompared with the control group,the expression of FOXO3 a and FOXO1 protein were significantly increased in 14 days and 28 days inhalation group(P<0.05),it was mainly expressed in the nucleus,and the protein expression increased with the exposure time.The expression of FOXO3 a and FOXO1 protein in the recovery group were significantly decreased,compared with the 28 d group(P<0.05).Compared with the control group,the expression of FOXO3 a and FOXO1 protein in the recovery group were were no significant changes.After carbon black inhalation for 14 days,the expression of P-FOXO3 a protein was not significantly different compared with the control group(P > 0.05).After 28 days of inhalation,the expression of P-FOXO3 a protein was significantly increased,compared with the control group(P<0.05),and it was expressed in the cytoplasm and nucleus.After 14 days of recovery,the expression of P-FOXO3 a was no significant changes,compared with the control group.The expression of P-FOXO1 protein were significantly increased in 14 days and 28 days inhalation group(P<0.05),and it was expressed in the cytoplasm and nucleus.After 14 days of recovery,the expression of P-FOXO1 was no significant changes,compared with the control group.Conclusion:1 The Nano scale carbon black inhalation model for 28 d in rats was established.During the inhalation,the body weight of the experimental group was slightly lower than that of the control group.2 After inhalation,most of carbon black deposited in the interstitial lung,a small amount in the alveoli.In the recovery group,there were still some CB particles deposited in the bronchioli but more particles in the pulmonary alveoli and the alveolar wall tissue,which indicated that the carbon black can not be cleared in the short term.3 Nano-scale carbon black particles could induced the pathological changes of lung and this histological change remained in the recovery group.4 Nano-scale carbon black particles could induce the ROS generation and oxidative stress.After recovery for 14 days,oxidative stress levels could alleviate partially.5 Nano-scale carbon black could induce DNA damage in lung tissue.After 14 days of recovery,DNA dmages could alleviate partially.6 Nanoscale carbon black particles could increase the expression of FOXO3 a and FOXO1 protein. |
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