The Effects Of Single-walled Carbon Nanotubes On The Mitochondrial Electron Transport Chain

Objective:To investigate the effects of single-walled carbon nanotubes on the mitochondrial electron transport chain.Methods:SWCNTs purchased from Shenzhen nanoport were suspended in PBS through ultrasound. The morphology and size of SWCNTs were axamined through transmission electron microscopy (TEM) and atomic force microscopy (AFM). Mixed suspension of SWCNTs and FITC were prepared and adsorption was allowed under ultrasonic condition. The HepG2Cells were exposed by adding SWCNTs in the medium in concentrations of1.5～24μg/ml, HepG2cells exposed to saline were used as control, the morphology of HepG2cells were observed by AFM after being treated for24h, CCK-8assay was conducted to detect the effects on the survival rate of the cells via being exposed to SWCNTs for24、48and72h. The mitochondria of HepG2cells were obtained by density gradient centrifugation and were examined under transmission electron microscope (TEM). Four enzymes activity of the mitochondrial electron transport chain (METC), mitochondrial membrane potential (MMP) and the concentrations of ROS and malondialdehyde (MDA) in the cells were determined by confocal microscope, flow cytometer and enzyme mark instrument, respectively. Basal respiration rates of mitochondria were measured by Clark oxygen electrode. The interaction between SWCNTs and METC of mitochondria was examined after cells treated with SWCNTs and four complex inhibitor. Electron transport of NADH respiratory chain were separately or jointly intervened with Rot, DPI and Ant, while that of FADH2respiratory chain were separately or jointly intervened with Rot, TTFA and Ant, ROS content were measured by in enzyme mark instrument with multifunctional microplate reader.Results:SWCNTs can inhibit the proliferation of HepG2cells in a time dependent and dose-dependent manner. Fluorescent macroscope observation showed that fluorescently labeled SWCNTs got into HepG2 cells after incubation2h at37℃. TEM showed that normal mitochondrial structure was seen in the control groups while the SWCNTs-exposed mitochondria had abnormal appearances, such as swelling, reduction or vanish of crista. There was no significant difference tested between the1.5～3μg/ml SWCNTs groups and the control group in the enzyme activity of complex II, whereas in the6μg/ml SWCNTs-treated groups the enzyme activity of complex II was significantly lower than that in the control (P<0.05). SWCNTs had stronger inhibitive effects on the enzyme activity of complex I, complex III and complex IV, in the concentration range of3-12μg/ml, SWCNTs groups were significantly lower than that in the control groups (P<0.05). Confocal microscope showed that green fluorescent of mitochondrial gradually increased, meanwhile red fluorescent of mitochondrial gradually decreased with SWCNTs concentration. ROS increased in a dose-dependent manner in HepG2cells treated with SWCNTs. The ROS levels in SWCNTs-treated cells, were significantly higher than that in the control cells in dose range of3-24μg/ml for24h (p<0.01) and in dose range of1.5～24μg/ml for48h (p<0.01). Malondialdehyde (MDA) test showed no significant difference between the1.5～3μg/ml SWCNTs-treated groups and the controls, whereas in the6-24μg/ml SWCNTs-treated groups, the concentration of MDA were significantly higher than that in the control (p<0.05).For NADH respiration, in control groups, ROS content in DPI group decreased, while in others groups increased significantly compared with PBS group (P<0.5); in SWCNTs-treated groups, ROS content in ROT+DPI groups decreased, while ROT, ANT and ROT+ANT groups increased significantly compared with SWCNTs group (P<0.5). For FADH2respiration, in drug control groups, ROS content in ROT, DPI and ROT+DPI groups decreased, while in TTFA and ANT groups increased significantly compared with PBS group (P<0.5). In SWCNTs-treated groups, ROS content in TTFA groups increased significantly compared with single SWCNTs group (P<0.5); Compared with SWCNTs+TTFA group, ROS content in SWCNTs+ROT+TTFA and SWCNTs+DPI+ANT groups further increased significantly (P<0.5).Conclusion:SWCNTs can cause mitochondria abnormal structure appearances, significantly decreased cell proliferation of HepG2cells, significantly inhibit the three enzyme activity of METC of HepG2cells, induce increase of electron leak of NADH and FADH2respiratory chain in a dose-and time-dependent manner. For NADH respiration, SWCNTs induce increase of electron leak mainly from ubiquinone binding sites of complex Ⅰ and complex Ⅲ, while induce increase of electron leak mainly form ubiquinone binding site of complex III for FADH2respiration. Increase of electron leak induce increase of ROS content of HepG2cells, which attack membrane system of HepG2cells leading to the apoptosis of HepG2cells.