Cytotoxic Effects Of Multi-wall Carbon Nanotubes On Human Umbilical Vein Endothelial Cells

[Objective] Carbon nanomaterials have multiple applications in various areas due to its unique physical, chemical and biological characterization. However, there is increasing evidence suggesting that exposure to ultrafine particulate air pollution (<100 nm) is linked to increased incidence of cardiovascular diseases. It has been suggested that exposure to nanoparticles may be a risk factor for the development of vascular diseases due to injury and dysfunction of the vascular endothelium. Thus the cardiovascular effects of nanoparticles have widely attracted people's interests.Nanoparticles generated mRNA level changes including changes in mRNA levels from genes involved in metabolism, apoptosis, cell cycle arrest, stress response, cellular transport, and inflammatory response. There are varies hypothesis about the mechanism in which the most popular explanation is about oxidative stress.Endothelial cells line the entire circulatory system, and their impairments can result in a variety of cardiovascular diseases, therefore, it is important to investigate the adverse effects of multiwall carbon nanotubles (MWCNTs), one of the most popular used materials, on endothelial cells. In the present study, we are intended to evaluate the potential safety risks of MWCNTs in cardiovascular system by investigating their influence on cell viability, morphological changes, ROS generation, DNA damage and cellular redox status in HUVECs. In order to simulate the complicated circumstance of human body, we cocultured HUVECs with THP-1, then investigated the changes of ROS and TNF-α.[Methods]1. Cytotoxicity, apoptosis and DNA damageTrypan Blue exclusion test of cell viability was used to study the cytotoxicity ofdifferent concentration (0.2, 1, 2, 5, 10, 20, 50,100μg/ml) of MWCNTs on HUVECs.Investigate the morphological changes by optical microscope and transmission electronmicroscope (TEM).Apoptosis kit and flow cytometry were used to detect the apoptosis rate of HUVECsafter treatment with MWCNTs (0.5, 5, 20μg/ml).Immunofluorescence was employed to examine HUVECsγH2AX foci formation aftertreatment with MWCNT s (0.5, 5, 20μg/ml).2. Cellular redox statusDCFH-DA was applied to detect ROS generation by HUVECs after treatment with MWCNTs (0.5, 5, 20μg/ml).Measurements of the activities of malondialdehyde (MDA) superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) contents after treatment with MWCNTs (0.5, 5, 20μg/ml) using commercially available kits following the manufacture's instructions.3. TNF-αgeneration by HUVECsELISA kit was used for detecting TNF-αgeneration by HUVECs after treatment with MWCNT s(0.5, 5, 20μg/ml).4. Changes of HUVECs after pre-treated with NACPre-treated with NAC, and then 1) apoptosis kit were used to detect the changes of apoptosis rate. 2) DCFH-DA was applied to assess ROS generation by HUVECs after treatment with MWCNTs (0.5, 5, 20μg/ml). 3) Immunofluorescence was used to detect formation ofγH2AX foci by the cells after treatment with MWCNTs (5μg/ml). All the results were compared with those without NAC treatment.5. Changes of HUVECs after cocultured with THP-1 Cocultured HUVECs with THP-1, and then DCFH-DA was applied to assess ROSgeneration after treatment with MWCNTs (0.5, 5, 20μg/ml).[Results]1. Cytotoxicity, apoptosis and DNA damageTrypan blue exclusion assay revealed that viabilities of HUVECs treated with 0, 0.5, 2,5, 10, and 20μg/ml MWCNTs were all beyond 70%. MWCNTs reduced the viability ofHUVECs in dose and time dependent manner.Optical microscope and TEM study showed that MWCNTs were able to enter cellsrapidly, distribute in the cytoplasm and intracellular vesicles and induce morphologicalchanges, such as cytosolic vacuole formation, cell disorientation, and decreased densityin a dose-dependent manner.MWCNTs induced apoptosis in HUVECs, which was significantly different comparedwith the control.MWCNTs could cause DNA damage as indicated by the formation ofγH2AX foci in adose dependent manner, which was significantly different from the control.2. Cellular redox statusMWCNTs increased intracellular ROS level. The difference in 20μg/ml group was significant compared with the control group.MWCNTs increasd malondialdehyde (MDA) levels, and altered superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) levels: treatment with 20μg/ml MWCNTs reduced activities of GSH-Px and SOD, while the activities of GSH-Px and SOD were increased in HUVECs after treatment with 0.5 and 5μg/ml MWCNTs.3. MWCNTs has no significant influence on TNF-αgeneration.4. Changes of HUVECs after pre-treated with NACFree radical scavenger N-acetyl-L-cysteine (NAC) preincubation inhibited the apoptosis rate, reduced theγH2AX foci formation and intracellar ROS level induced by MWCNTs. 5. Changes of HUVECs after cocultured with THP-1Intracellar ROS was increased in HUVECs cocultured with THP-1 compared with thosewithout coculture.[Conclusions]MWCNTs can enter into HUVECs, reduced cell viability, induce apotosis and genotoxic effects, increased intracellar ROS level and affected cellular redox status. The presence of neutrophils could increase ROS generation and aggravate MWCNTs induced oxidative damage to MWCNTs.