Effect And Mechanism Of Multiwall Carbon Nano-Onions On DNA Damage In HUVEC Cells

[Objective] With the rapid development of nanotechnology, the problem of safety of nanotechnology has been concerned greatly. Carbon nanomaterials, including carbon nanoparticles and nanotubes, have been one of the most extensively used nanoparticles, because of their unique and superior properties. Multiwall carbon nano-onions (MWCNOs), which will be the focus of this study, represent a relatively recently discovered allotrope of carbon derived from the more intensively studied fullerene (C₆₀). Giant, nested fiillerenes, generally called nano-onions, are usually produced by an underwater carbon-arc discharge. Our arc-produced MWCNOs are typically about 30nm in diameter. Recent investigations provide evidence that nanoparticles can be translocated directly from the lungs into blood circulation and extrapulmonary organs. Someone speculates that MWCNO will be effective cancer killing agents and they can directly affect blood circulation. Thus it is necessary to evaluate the biological safety of MWCNO. Frank chen et found that MWCNOs generated mRNA level changes in exposed skin fibroblasts, including changes in mRNA levels from genes involved in metabolism, apoptosis, cell cycle, stress response, cellular transport, and inflammatory response. So far, there are no relative research reports of MWCNO on the cardiovascular system.Recently the phosphorylation of histone H2AX denoted γH2AX has gained attention for its relationship with DNA damage. The phosphorylated form of histone variant H2AX plays an important role in the recruitment of DNA repair andcheckpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Many relation proteins of maintaining, protecting, repairing of chromosome structure can form a foci organically, when they combine with the site of DNA damage, to check and repair the DNA damage. In this process, it can induce cell cycle to stop and cell to apoptosis according to the degree of DNA damage.Excessive generation of ROS that overwhelms the antioxidant defense system can oxidize cellular biomolecules. Free radicals generate a large number of oxidative modifications in DNA, including SB and base oxidations. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from particles. Oxidative stress has been implicated in many diseases, including cardiovascular disease, macular degeneration, pancreatitis, and cancer. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. DNA is considered to be an important target for reactive oxygen species (ROS) generated as a consequence to air pollution exposure.We investigated the influence of MWCNO on the cell cycle, apoptosis, DNA damage and its mechanism of HUVECs.[Methods] In vitro experiment, the HUVEC cells were cultured in complete 1640 medium. Trypan Blue Exclusion Test of Cell Viability was used to study the cytotoxicity of different concentration(0.2,1, 5, 25, 50, 100, 200μg/ml) of Multiwall Carbon Nano-Onions on HUVEC cells. Apoptosis and cell cycle of HUVEC were detected by flow cytometry. And immunofluorescencer was used to observe γH2AX foci formation in HUVEC cells after treated with different concentration (0.2 , 1, 5μg/ml) at different time point (6, 12, 24h). The fluorescent intensity of reactive oxygen species (ROS) was determined by Microplate Spectrophotometer.[Results] The results showed that MWCNO failed to induce the change of cell cycle on HUVEC. The percentage of apoptosis increased with the increase of dose after the administration of MWCNO. The result from immunofluorescence microscope shown that MWCNO induced γH2AX foci formation exhibited a time-and dose-dependent manner, as the highest concentration (5μg/ml) and 24h induced the most foci. ROS levels were significantly higher than that of the controlgroup after the administration of MWCNO for 12h.[Conclussion] MWCNO induced apoptosis in HUVEC. A dose-dependent increasein the percentage of apoptosis cells was observed. Exposure to Multiwall carbonnano-onions induces DNA damage in HUVEC cells. In the meantime, the level ofROS in HUVEC cell increased after treated with MWCNO.