Effects Of PLK1 Signal On Genetic Damages Induced By Carbon Black Nanoparticles

Objective: To investigate the genetoxicity induced by carbon black nanoparticles and the possible molecular mechanism of PLK1 signal pathway.Methods: The surface morphology of CBNPs was observed by scanning electron microscopy,the size was measured by transmission electron microscopy,and the specific surface area of carbon black nanoparticles was calculated by BET adsorption isotherm,the zeta potential was performed on nano particle analysis instrument.Rat models of CBNPs inhalation were established.The concentrations of the CBNPs were 5 mg/m3 and 30 mg/m3,respectively,6 hours per day for 28 days.The 16 HBE cells were treated with CBNPs for 24 hours at the dose of 0,50,100,200 μg/m L,respectively.Lipofectamine TM 2000 was used to transfect pc DNA3-PLK1 into 16 HBE.800μg/m L G418 was used to screen the positive cells.16 HBE cells and pc DNA3.1 transfection cells were used as negative control and transfection control respectively,PLK1 protein levels were detected by Western blot.The16 HBE cells line with high PLK1 expression was constructed.The contents of CBNPs in 16 HBE cells were determined by SDS-PAGE electrophoresis.Apoptosis and cell cycle were detected by flow cytometry.Pathological changes of lung tissues were observed by HE.DNA damages were detected by the single cell gel electrophoresis.Chromosome damages were detected by micronucleus test.The DNA repair ability was determined by host cell reactivation assay.Western blot was used to detect the proteins’ levels.Results:1.The characteristics of CBNPsCBNPs were spherical particles and the size was 30~50 nm,which were composed of aggregates and the diameters were tens to hundreds of nanometers.The specific surface area of CBNPs determined by BET was74.85 m2/g and the zeta potential was-15.37 MV.2.The contents of CBNPs in 16 HBE cellsThe contents of CBNPs in 16 HBE cells treated by the 50 μg/m L,100μg/m L and 200 μg/m L were 6.57±1.99 μg,11.67±1.06 μg,18.26±4.96 μg,respectively.The CBNPs in 16 HBE cells of the 200 μg/m L group was significantly higher than that of the 50 μg/m L group(P<0.05).3.Histopathology of the lung tissue induced by CBNPs in ratsCompared with the control,inflammatory cell infiltration and alveolar wall thickening were observed in CBNPs groups,and hemorrhage and macrophage shedding were observed in the 30 mg/m3 CBNPs.4.Genetic damage induced by CBNPsAfter 5 mg/m3 and 30 mg/m3 CBNPs inhalation for 28 days,Tail DNA%of lung cells in rats were significantly increased compared with the control(P<0.05).After 30 mg/m3 CBNPs inhalation for 28 days,OTM of lung cells in rats were significantly increased compared with the control(P<0.05).Compared with the control,the micronucleus rates in rats were significantly increased in the CBNPs treatment groups(P<0.05).Compared with control,the OTM value and Tail DNA% of 16 HBE cells were significantly increased after CBNPs exposure in a dose dependent manner(P<0.05).The cytokinesis block proliferation index was significantly lower after CBNPs exposure than control(P<0.05).The micronucleus rates were significantly increased in a dose dependent manner(P<0.05).Compared with the control,the ratio of firefly-luciferase and renilla-luciferase activities was significantly increased in the 50 μg/m L and 100 μg/m L groups(P<0.05),which indicated that DNA repair ability was enhanced.Whereas there was reduced in the 200 μg/m L group compared with the 50 μg/m L and 100 μg/m L groups(P<0.05).5.Effects of CBNPs on apoptosis and cell cycleCompared with the control,16 HBE cells in G0/G1 phase after CBNPs treatment were significantly decreased,whereas cells in G2/M phase were significantly increased in a dose dependent manner(P<0.05).The cell survivalrates were statistically decreased,whereas the late apoptosis rates and necrosis rates were statistically increased(P<0.05).However,the early apoptosis rates in 16 HBE cells after CBNPs treatment had no statistical difference(P>0.05).6.Effects of PLK1 signal on genetic damages induced by CBNPsCompared with the control,the expression of PLK1 protein in the lung tissue of rats decreased after Inhalation CBNPs in a dose dependent manner(P<0.05).The expression levels of PLK1 protein in 16 HBE cells were significantly decrease in 100 μg/m L and 200 μg/m L groups,compared with the control(P<0.05).The GADD45α and Ch K2 protein expression were significantly increased in a dose dependent manner(P<0.05).The levels of XRCC1 protein were significantly increased in 50 μg/m L and 100 μg/m L groups(P<0.05),but there was reduced in 200 μg/m L group compared with 50 μg/m L and 100μg/m L groups(P<0.05).The PLK1 protein expression in pc DNA3-PLK1 plasmid transfected cells was about twice-fold higher than that of 16 HBE cells and pc DNA3.1transfected cells.The constructed cells line with PLK1 high expression has been established successfully.After 100 μg/m L CBNPs exposure,there was no G2/M phase retardation in the PLK1 high expression cell lines compared with16 HBE cells and pc DNA3.1 transfected cells.The cell survival rates were significantly increased whereas the late apoptosis rate,mortality,Tail DNA %and OTM value were significantly decreased in the PLK1 high expression cell lines after 100 μg/m L CBNPs treatment compared with 16 HBE cells and pc DNA3.1 transfected cells(P<0.05).The cytokinesis block proliferation index,replicative index were increased whereas micronucleus rates were decreased in the PLK1 high expression cell lines after 100 μg/m L CBNPs treatment compared with 16 HBE cells and pc DNA3.1 transfected cells(P<0.05).The Ch K2,GADD45α and XRCC1 protein expression were decreased in the PLK1 high expression cell lines after 100 μg/m L CBNPs treatment compared with 16 HBE cells and pc DNA3.1 transfected cells(P<0.05).Conclusions:1.CBNPs could induce genetic damages in a dose dependent manner both in vitro and in vivo.2.PLK1 played important roles in the genetic damages induced by CBNPs.