The Mechanism Of Cell Cycle Arrest, Apoptosis And Autophagy Induced By Four Black Carbon Particles In A549 Cells

Objective:A number of epidemiological and toxicological studies have demonstrated that short-term and long-term exposure to ambient fine particulate matter(PM_2.5)is associated with a variety of adverse health effects,including acute and chronic cardiopulmonary diseases.Black carbon（BC）,produced by the incomplete combustion of carbonaceous material such as biomasses,biofuel and fossil fuels,is an important carbonaceous component of PM_2.5.5 and traffic-related particles.Vehicle emissions account for over 70%of ambient PM_2.5.5 in urban settings,including that emitted by gasolineand diesel engines.In fact,BC is mainly related to diesel and vehicle emissions.Source apportionments have demonstrated that the BC fraction of total fine carbon particulate matters is higher in diesel exhaust than that in gasoline engine exhaust.Furthermore,anthropogenic combustion-derived trace metals（e.g,Pb,Fe,Zn,Ni,As）such as vehicle emissions,industrial emissions,fossil fuel combustion,industrial emissions,and long-range transportation contribute to the composition of ambient PM_2.5.In fact,trace metal lead（Pb）is also a typical component of trace metals in PM or traffic-related particles,although trace metals make up only a small part of PM mass.Recent many studies have also demonstrated that BC or oxidized-BC could induce toxic effects in cells and animals.However,the combined interactions of BC and heavy metal lead on adverse biological effects are still poorly understood.Methods:In this study,we used black carbon（BC）and lead ions as model materials.The BC was successfully modified into carboxylated carbon black（c-BC）and then carboxylated black carbon-Pb complex（c-BC-Pb）was prepared and characterized by elemental analyzer（CEA）,inductively coupled plasma emission spectroscopy（ICP-AES）and transmission electron microscope（TEM）.In this work,the different treatment groups of experimental design and were randomly assigned into 4 groups.（1）unmodified BC exposure group;（2）c-BC exposure group;（3）c-BC-Pb exposure group;（4）c-BC+Pb co-exposure group,Cells exposed to c-BC+Lead acetate combined group;（5）control group:Cells exposed to equal volume of serum-free medium.For this purpose,exposure to four different black carbon particles in A549 cells chosen to understand the possible mechanisms of the toxicity.Human lung adenocarcinoma cell line（A549 cells）was donated from the School of Life Sciences,University of Science and Technology of China.The cells were cultured in Dulbecco’s modified Eagle’s medium（DMEM;Corning,USA）supplemented with 10%（v/v）fetal bovine serum（FBS）and 100 U/mL penicillin and streptomycin.A549 cells were routinely maintained a fully humidified incubator with5%CO₂ at 37°C and were used for experimental procedures when they were in logarithmic growth phase.Cells were assigned to different groups,as described above.To minimize the aggregation of oxidized-BC particles,they were sonicated at 20 kHz,160 W for 5 min before being added into the culture medium.Logarithmically growing cells were incubated in sterile T-flasks or 6-well and 96-wellculture plates containing oxidized-BC and/or Pb.The flasks or culture plates were placed in an incubator for a specified treatment groups and time intervals.Cell viability was measured by MTT assay.Lactate dehydrogenase（LDH）leakage was used to determine the effect of each group on the integrity of cell membrane.To assess the oxidative stress induced by each treatment group,and to determine ROS generation in BEAS-2B cells,a fluorescent probe,2′7′-dichlorofluorescin diacetate（DCFH-DA）was used as a sensitive indicator of intracellular oxidation.Oxidative damage is determined by the content of malondialdehyde（MDA）,the activity of superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）.Commercially available kits（Jiancheng Bioeng Inst.,Nanjing,China）were used for the measurements.Interleukin-6（IL-6）,Interleukin-8（IL-8）and tumor necrosis factor-alpha（TNF-α）in A549 cells were measured using a commercial immunoassay kit（Beyotime Company,China）after 24 h exposure to each treatment group.Apoptosis in A549 cells was detected by an Annexin V and PI assay kit（Key Gen,Nanjing,China）.The cells were immersed in 500μL binding buffer and stained with 5μL Annexin V-FITC for 15 min,before being treated with5μL PI at room temperature.The cells were later loaded on a flow cytometer（Becton Dickinson,USA）,and data from approximately 10000 cells were analyzed at 488 nm excitation and 525 nm emission wavelengths.Western Blot（WB）was performed on the total proteins of the four kinds of the BC particles after 16h exposure.The primary antibody was the autophagy key protein LC3B and the lysosomal membrane protein lamp2,and the secondary antibody was specific goat anti-rabbit LgG.Results:The features and characteristics of c-BC and c-BC-Pb particles were also analyzed by our previous studies.Briefly,the hydrodynamic size range of oxidized-BC particles was 190 to 232 nm（average 218.3±8.7 nm）by dynamic light scattering（DLS）.After oxidation treatment,the oxygen content in the BC particles increased significantly from 1.31%of raw carbon black to 17.11%by X-ray photoelectron spectroscopy（XPS）.The cell viability changes were determined in various concentrations of four exposure groups for 24 h,suggesting that dose-dependent cell viability declined in cells induced by the BC materials.Similarly,the change of LDH was consistent with the cell activity.The increase of the level of oxidative stress was consistent with the change of the level of antioxidant markers,indicating the imbalance of the clearance of ROS in cells.The expression levels of cellular inflammation markers were significantly different after lead-containing black carbon exposure,suggesting that lead may play a key role in inducing the inflammatory response.Apoptosis results showed that the four kinds of BC particles induced apoptosis occurred in a dose-dependent manner,and c-BC-Pb group was the most significant.The induced cell cycle arrest effects of the four BC treatment groups all appeared in the S phase and showed a dose effect.As a result of western blotting,all four kinds of BC particles would induce the production of autophagy in A549 cells,but Pb ions treatment group alone could not induce the occurrence of autophagy.The reduced autophagy flux and lysosomal membrane protein expression at high doses in the c-BC-Pb group indicate that the lysosomal membrane structure on which autophagy depends was damaged,and plasmid transfection and TEM results are consistent with it.Conclusion:Compared with the other three BC particles,the results showed that the c-BC-Pb would induce more severe apoptosis and S-phase arrest in the cell cycle,and significantly up-regulation of LC3-Ⅱprotein levels showed that the particles were first enriched in the lysosome by the autophagy-lysosomal pathway after entering the cells.With the increase of exposure dose and time,the lysosomal membrane protein lamp2 was significantly down-regulation indicated that the damage of its membrane is intensified,causing the release of lysosome contents（such as hydrolase,lysosome-iron,acidic substances,etc.）,and then oxidative stress.Therefore,autophagy and lysosomal dysfunction are synergistic cytotoxic effects after c-BC-Pb exposure,and provide new insights into the toxicological and health effects of atmospheric PM_2.5.