Cytotoxicity And Mechanism Of CuO Nanoparticles In Human Hepatoma G2(HepG2) Cells

With the development of nanotechnology, many kinds of nanoparticles were used gradually in energy, biological and medicine fields. However, disposal of NPs inevitably result in their release into environment, so the biological safety of nanomaterials and the unexpected health impacts to humans become one of the interesting points of researches. Cu O NPs were widly used in many fields, such as catalysis, desulfidation, antibacterial action and medical because of the unique character istics on electricity, magnetism and catalysis. As the basic unit of life activities, cell line is recommended as an appropriate model in vitro to monitor the molecular mechanism leading to cytotoxic and genotoxic of xenobiotics. The current study was designed to clarificate the cytotoxicity and genotoxicity profile of Cu O NPs to metabolically competetent human hepatoma G2(Hep G2) cells and the main results are summarized as follows:1. Colorimetric CCK-8 assays demonstrated that Cu O NPs inhibited the growth of Hep G2 cells in different hours. The IC50 value for 16 h was 8mg/L. This initial cytotoxicity revealed that Cu O NPs was toxic to mammalian cells, and the inhibition increase with increased exposure time as well as Cu O NPs concentration.2. To comprehensively understand the acute cytotoxicity of Cu O NPs in Hep G2 cells, a systematic study involving the assessment of Cu O NPs-induced malondialdehyde(MDA), protein carbonyls, DNA damage and ROS generation were performed. Our results showed that within the exposure concentrations ranging from 1 to 20 mg/L, the content of MDA and protein carbonyls were significant increased compared with control cells. With the increased of the concentration of Cu O NPs, the percentages of tail DNA were significant increased compared with control cells, and a statistically significant increase of 8-OHd G was observed after Hep G2 cells treated with Cu O NPs, which preliminary indicated that Cu O NPs was a potent agent to induce extensive DNA damage in Hep G2 cells. According to the measurement of intracellular ROS, the elevated level of ROS was an early event in Cu O NPs induced cytotoxicity. To further confirm that genotoxicity of induced by Cu O NPs is due to oxidative stress, the assays of antioxidant N-Acetylcysteine(NAC)were performed. Our data demonstrated that the DNA damage, MDA contents, protein carbonyl adducts and ROS generation in Cu O NPs treated cells were effectively countered by NAC pretreatment, which clearly corroborated that oxidative stress was the main mechanism of Cu O NPs-induced cytotoxicity in Hep G2 cells.3. Using the flow cytometry as the quantitative detection method for cell cycle, the results showed that the increasing concentration of Cu O NPs aggravate the block of the Hep G2 cell cycle, the expression of CDK genes were increased accompanied by increasing Cu O NPs concentration, whereas the cyclin expression quantity decreased, which proves that the Hep G2 cells were blocked at the S phase after Cu O NPs treatment. Our data obtained from FCM study showed a dramatic increase in the proportion of apoptotic cells. Increased oxidative stress evoked a concentration-dependent collapse of m, which indicated that mitochondrial membrane depolarization was closely relevant to generation of intracellular ROS. Caspase-3 and Caspase-9 are two cell apoptosis related genes, these two gene expressions were increased when treated by Cu O NPs. The increase of these genes expression revealed that the apoptosis induced by Cu O NPs required the activation of the initators(Caspase-9) and executors(Caspase-3).In summary, Cu O NPs can inhibit the Hep G2 cell proliferation and cause the oxidative damage to the cells; at the same time Cu O NPs can cause cell cycle arrest and induce apoptosis. Cu O NPs can cause the cell cycle arrest and induce the cell apoptosis, and one predominant mechanism of apoptosis was found to be largely mediated through mitochondrial pathway in Hep G2 cells.