Cytotoxicity Induced By Nanoscale Carbon Black Particles In 16HBE Cells And Its Influence Factors

Objective: Carbon black(CB)is a superfine carbon particle that is a major component of the smoke produced when the fuel is not completely burned.Carbon black nanoparticles(CBNPs)are pure carbon black powders made by controlling the gas phase cracking of hydrocarbons.With the commercialization of CB and the continued increase in CB emissions to the atmosphere,CBNPs exposure is gaining more and more attention.Studies have shown that cells in different cycle phases will be different in the uptake of nanoparticles,and the content of intracellular nanoparticles and cytotoxicity is closely related.It is not clear how the CBNPs do harm to cells and the effect of different cell cycle phase on the CBNPs uptake.Therefore,it is very important to explore the effect of CBNPs on cell damage and the effect of different cycles on its uptake.In this study,the effects of CBNPs on 16 HBE cytotoxicity and its influencing factors were investigated by exploring the CBNPs uptake in human bronchial epithelial cell lines(16HBE)with different cell cycle.Methods:1 Detection of CBNPs characterizationScanning Electron Microscopy(SEM),Transmission Electron Microscopy(TEM),and Brunauer-Emmett-Teller were used to characterize CB.2 Cell culture and cell model establishment16HBE cells were cultured in MEM medium containing 10% heat inactivated fetal bovine serum at 37? with a 5% CO2 saturated humidity.The 16 HBE cells in logarithmic growth phase were exposed to CBNPs for 24 hours at a dose of 50?g/mL,100?g/mL,200?g/mL,and MEM(containing 0.04%Tween80)as a negative control.3 Cell viability and apoptosis16HBE cells seeded in 96 well plate were treated with 1,10,50,100,200,300?g /mL CBNPs.After 3,6,12,24 and 36 h incubation,MTT was added at a final concentration of 0.5mg/mL.After 4h,the supernatant was removed and the formazan product was dissolved in 150?l of dimethyl sulfoxide(DMSO).The absorbance was measured on a microplate reader at length of 495 nm.In cell apoptosis analysis,cells were exposed to different concentrations of CBNPs(0,50,100 and 200?g/mL)for 24 h.After treatment,16 HBE cells were harvested and resuspended in 500?l binding buffer.Annexin V coupled with fluoresce in isothiocyanate(FITC)was added according to the AnnexinV-FITC kit.Following 30 min of incubation at room temperature in darkness,50?g/mL PI was added for 10 min before analyzed by flow cytometry.4 Determination of oxidative damageThe 16 HBE cells were digested and incubated with 2,7-dichlorodihydrf-luorescein diacetate(DCFH-DA)fluorescent probe,and the flow cytometry was used to detect the reactive oxygen species(ROS)contents of 16 HBE cells after treatment with different doses of CBNPs.The activities of antioxidant enzymes superoxide dismutase(SOD),glutathione peroxides Glutathione peroxidase(GSH-Px)and lipid peroxidation product malonaldehyde(MDA)content were detected by commercial biochemical kits to evaluate the oxidative damages in 16 HBE cells after CBNPs exposure.5 DNA damage in 16 HBE cellsDNA damage was detected by the single cell gel electrophoresis(SCGE)assay after CBNPs treatment.6 Cell cycle analysisThe distribution of DNA in the cell cycle was detected by flow cytometer.Cells were exposed to different concentrations of CBNPs(0,50,100 and 200?g/mL)for 0,3,6,12,18,24,30,and 36 h.The treatment was removed and cells were harvested and after centrifuged at 1000 rpm for 10 min at 37°C.The pellet was fixed in ice-cold ethanol(80% in PBS)and incubated at-20°C for 18 h.Cells harvested were further centrifuged and resuspended in 20?g/mL RNAseA prepared in PBS and then incubated at 37°C for 30 min.Further PI was added and the final concentration was 50?g/ml.After staining for 30 min,samples were analyzed by flow cytometer at emission wavelength of 633 nm and excitation wavelength of 488 nm.7 Cellular uptakesThe cellular uptake assay was performed according to the literature.16 HBE were treated with CBNPs for 6h and 24 h,respectively.After exposure,Cells were harvested and resuspended in 200?l of PBS.The uptake of particles was determined using flow cytometer.For imaging flow cytometry analysis,cells were cultured and treated by CBNPs.Cells were trypsinized,fixed with 4% formaldehyde in PBS and centrifuged to obtain a pellet of about 106 cells in 50?L.Cell images were acquired using the ImageStreamX multispectral imaging flow cytometer(Amnis Corporation),collecting 20,000 events per sample at 40 × magnification.Cell images were analyzed using IDEAS? image-analysis software(Amnis).8 Cell synchronization in different cell cycle phasesG0/G1 phase(mimosine block)Cells seeded in 6 well plate were incubated with 500?M mimosine prepared in MEM(10% FBS)for 24 h.After incubation,cells were stained with PI and the cell cycle was analyzed by flow cytometer.S phase(double thymidine block)Cells seeded in 6 well plate were incubated with 2mM thymidine prepared in MEM(10% FBS)for 19h(first block).After first block,the thymidine was removed and MEM(10% FBS)was added for 8h to release cells.After releasing cells,MEM(10%FBS)with 2mM thymidine were added and then further incubated for 18h(second block).Finally,thymidine was removed by washing with PBS.Cells were stained with PI and the cell cycle was analyzed by flow cytometer.G2/M phase(nocodazole block)Cells seeded in 6 well plate were incubated with 50ng/ml nocodazole in MEM(10% FBS)for 15 h.After treatment,media were removed and 16 HBE cells were washed with PBS.Cells were stained with PI and the cell cycle was analyzed by flow cytometer.9 Cellular uptakes of CBNPs in different cell cycle phasesAfter synchronization,cells were seeded and pre-treated with different cell synchronizing chemicals as described above.After removing the synchronizing chemicals,CBNPs(0,50,100 and 200?g/mL)were added into wells cells and incubated for different time intervals(6 or 24h),respectively.After exposure,particles were removed and cells were harvested.The supernatant was discarded and the pellet was resuspended in 200?L of PBS.The uptake of particles was determined using flow cytometer.Results:1 Characterization of CBNPsThe expected three-dimensional nanostructure of CB is confirmed by the Scanning Electron Microscopy(SEM)and Transmission Electron Microscopy(TEM).CB has a high carbon purity(>99.8%)and consists of globular shaped particles.Agglomerates of tens to hundreds of nanometer were formed,but no intermediates structure was observed.SEM examinations indicated that particles ranged from 30 to 50 nm in size.The larger particles exhibited numerous spherical protuberances on the surfaces,suggesting that they were formed during the spray drying process through fusion of the much smaller particles.TEM confirmed that the CBNPs contained clusters comprised of smaller particles,30 to 50 nm in size.Brunauer-Emmett-Teller(BET)measurements of the CBNPs resulted in a surface area of 74.85 m2/g.2 Cytotoxicity induced by CBNPsThe survivals of cells were performed to assess the short-term,nanocytotoxic concentration of CBNPs for 16 HBE cells.MTT results demonstrated a statistical significant(P<0.05)reduction in the mitochondrial succinate dehydrogenase enzymatic activity in cells exposed to CBNPs at concentration 100,200 and 300?g/mL,respectively for 3h.The reduction was significant at 50,100,200 and 300?g/mL concentrations respectively after 6h of exposure in 16 HBE and was further significant reduced at 1,10,50,100,200 and 300?g/mL,respectively after 12 h,24h and 36 h exposure.These results indicated that CBNPs inhibited 16 HBE cells proliferation in a dose and time dependent behavior.3 CBNPs cause 16 HBE cell apoptosisEffects of CBNPS on apoptosis and necrosis in 16 HBE cells were detected by flow cytometry.Results indicated that there were slightly more early apoptotic cells in 16 HBE treated with 50?g/mL and 100?g/mL of CBNPs compared with the negative control.Only 200?g/mL of CBNPs induced significant early apoptosis.Treatment with CBNPs of 50,100 and 200?g/mL resulted in an increase in the percentage of late apoptotic cells and necrotic cells respectively.4 Determination of Oxidative Damage in 16 HBE Cells by CBNPsCBNPS induced oxidative stress in 16 HBE cells as evident from the increased intracellular ROS.A significant(P<0.05)concentration dependent increase of ROS generation was observed The activity of SOD and GSH-Px decreased gradually compared with the control group,and the difference was statistically significant(P<0.05).MDA contents increased,copared with the control(P<0.05).Indicating that CBNPs can induce 16 HBE cell oxidative damage.5 DNA damage induced by CBNPsWith the increased concentration of CBNPs,the Olive tail moment significantly increased,compared with the control(P<0.05).This shows that CBNPs can induce DNA damage in 16 HBE cells.6 S and G2/M phase arrest induced by CBNPsThe results showed that CBNPs could induce S and G2/M phase arrest in 16 HBE cells,and the difference was statistically significant(P<0.05)compared with the control group.7 Dose dependent cellular uptakeThere was a significant increase of cellular uptake after treatment with CBNPs compared with the control group(P<0.05).At different stages of cell synchronization,both 6 and 24 hours later that S after CBNPs treatment,cellular uptake in S phase and G2/M phase were higher than that in G0/G1 phase.Compared with the control group,the difference was statistically significant(P<0.05).This indicated that CBNPs uptake in 16 HBE cells related to cell cycle phase and the CBNPs uptake in G2/M>S>G0/G1.Conclusions:1 CBNPs could induce apoptosis,viability decrese,oxidative stress and DNA damage decrease in 16 HBE cells and these might due to the CBNPs uptake.2 S and G2/M phases are important phases during CBNPs uptake.