The HepG2 cell line was purchased from American Type Culture Collection (ATCCHB-8065). Cell were cultured in DMEM (DMEM. Gibco, United States) supplemented with 10% fetal bovine serum (FBS. Hyclone, United States), 2 mM L-glutamine (Amresco Int. United States), and 100 U/ml penicillin streptomycin (Tianlin Hao Yang Biological Manufacture Co. LTE). Cell were maintained at 37˚C in an incubactor with 5% CO₂.

CuO NPs were purchased from Beijing Nachen S&T Ltd. Dry power of CuO NPs was suspended in DMEM-FBS medium of 1000 mg/L, and then sonicated for 30 min before aseptic addition to the tissue culture plates. The number of cell added in each well was identical (3 × 10⁴ cells/well). Then, different volumes of CuO NPs stock solution were added to the cell culture to achieve predetermined concentrations of CuO NPs (1, 5, 10, 20 and 40 mg/L) for cell incubation. The Zeta potential of CuO NPs at 8 mg/L in the DMEM-FBS medium and ultrapure water were determined using a Nanosizer (Nano S90. Malvern Instruments Ltd. United Kingdom). The particle size of CuO NPs was also evaluated by a transmission electron microscopy (TEM, JEM-2100.JEOL.Japan). The crystalline nature of CuO NPs was carried out by taking X-ray diffraction (XRD) pattern. The XRD pattern of CuO NPs was acquired at room temperature with the help of PANalytical X’Pert X-ray diffractometer equipped with a Ni filtered using Cu Kα (λ = 1.54056 Å) radiations as X-ray source.

Cell viability was determined by cell Counting Kit-8 (Beyotime Institute of Biotechnology). Briefly, 3 × 10⁴ cells were spread onto each 96-multiwell plate. Cell in each well were treated with different concentrations of CuO NPs (1, 5, 10, 20 and 40 mg/L) at 37˚C for 8, 12, 16 and 20 h. The cells were then incubated with 10 μl of cholecystokinin octapeptide (CCK-8) for 4 h, and the absorbance was measured at 450 nm. The mean absorbance of nonexposed cells served as the reference value for calculating percentage of cellular viability. Finally, the half maximal inhibitory concentration (IC₅₀) was calculated according to cell viability from various CuO NPs exposures.

ROS production was determined using 2’7’-dichlorofluorescein diacetate (DCFH-DA) (Beyotime Institute of Biotechnology) as described elsewhere [24] . Cells were seeded into 24-well plates at a density of 4 × 10³ cells/500 μL/well 24 h before treatment. Treated with different concentrations of CuO NPs for 16 h. Briefly, the cells were incubated with media containing 25 μM DCFH-DA at 37˚C for 45 min. After removing the DCFH- DA solution, the cells were washed twice with phosphate buffered saline (PBS; Invitrogen), then, the plate was monitored in a Biological fluorescence inverted microscope.

Then, characterize for the CuO nanoparticles. Figure 1(a) shows the XRD pattern of prepared CuO NPs that

clearly exhibits the crystalline nature of this material. The Peaks at 2θ = 32.62˚, 35.61˚, 38.80˚, 48.75˚, 53.68˚ and 58.42˚ were assigned to (110), (002), (111), (202), (020) and (113) of CuO NPs. The crystallite size of CuO NPs was found to be around to be 30 - 50 nm. TEM image (Figure 1(b)) showed that the majority of CuO NPs were in spherical shape. These pictures exhibit that the majority of the particles were spherical sharped with smooth surface. The Zeta potential of water by DLS (Nano-Zetasizer-HT. Malvern Instruments. Malvern. UK) was −20.4 mv and −8.6 mv, respectively. The specific surface area of CuO NPs determined by BET was 15.31 m²/g. The physicochemical characteristics of CuO NPs were listed in Table 1.

The free Cu²⁺ ions concentration dissolved from CuO NPs into the cell medium was determined using a modified procedure by Navarro et al. The CuO NPs (40 mg/L) in the DMEM medium were allowed to equilibrate for 2, 4, 6, 8, 10, 12, 14 and 16 h (5% CO₂, 37˚C). CuO NPs suspensions were then centrifuged for 30 min at 1500 g using ultrafiltrate centrifugal tubes (3 kDa, Millipore). The Cu concentration in the filtrate was determined using flame atomic absorption. No Cu²⁺ ions in the medium were observed from 2 h until 16 h. These date suggest only a neglectable role of copper ions in our experiment design, even during the longest incubation times.

HepG2 cells were exposed to CuO NPs at the concentrations of 1, 5, 10, 20 and 40 mg/L for 8, 12, 16 and 20 h and cytotoxicity was determined using CCK-8 assay. Results showed that CuO NPs significantly decreased the cell viability in dosedependent manner. CuO NPs at low concentrations (1 and 5 mg/L) has insignificant toxicity to HepG2 cells after exposure for 8 h, which is likely the results of Cu being a required micronutrient. The toxicity of CuO NPs at moderate concentrations (10 and 20 mg/L) began to appear after exposure for 12 h become significantly greater in the subsequent 20 h. For high concentration of CuO NPs (40 mg/L) toxicity to HepG2 cells was the greatest at 20 h, and the cell viability dropped to 4.5% (Figure 2(a)). The toxicity of CuO NPs

was concentration-dependent (Figure 2(b)), and the 16 h IC₅₀ was calculated as 8 mg/L. Thus, 8 mg/L was chosen in the following toxicity studies, and chose the low concentration (1 mg/L) and moderate concentration (20 mg/L) for comparison. Thus, CuO NPs had a much higher toxicity potential, a finding supported by Karlsson et al. [23] , who observed that CuO NPs were more toxic to A549 cells than ZnO and TiO₂ NPs.

NAC act as a normal antioxidant, played an important role in clean the extra free radical to protect the body. We examined the effect of CuO NPs on ROS generation in the presence or absence of anti-oxidant N-acetyl-cystein (NAC). Fluorescent microscopy data revealed that CuO NPs (1, 8, 20 mg/L) induced the intracellular production of ROS in dose-dependent (Figure 3). We further observed that coexposure of NAC effectively prevented the ROS generation induced by 20 mg/L of CuO NPs. ROS level was reduced up to control level for CuO NPs in the presence of NAC.

Oxidative stress has been suggested to play an important role in the toxicity mechanisms of nanoparticles [24] [25] . We proposed that CuO NPs were able to induce ROS mediated cytotoxicity in HepG2 cells. Thus, we detected the effect of CuO NPs on cell viability in the presence or absence of the NAC (Figure 2(b)). Figure 2(b) shows that NAC abolished almost fully the harmful effect of CuO NPs at all concentrations studied.

In summary, this study has demonstrated the relationship between the oxidative stress and the cytotoxicity of the CuO NPs to HepG2 cells, therefore CuO NPs cause significantly reduced viability of HepG2 cells in a concentration-dependent manner. The presence of the antioxidant NAC significantly reduces the cytotoxic effects induced by the CuO NPs. In addition, the CuO NPs can generate intracellular ROS in a concentration-dependent manner and cause oxidative damage to DNA and proteins in HepG2 cells. These results all suggest that an oxidative stress mechanism is involved in the cytotoxic effects determined in HepG2 cells after 16 h CuO NPs.

These results suggest that an ROS-related mechanism at least partially contributes to the CuO NPs-induced cytotoxicity and genotoxicity. These results are useful for developing a better understanding of the potential cellular mechanisms of toxicity of CuO NPs. These data clearly show that increased attention and careful investigations are needed regarding potential exposure, toxicity, and ultimate risk from metal oxide NPs such as CuO. ROS such as superoxide anion (), hydroxyl radical () and hydrogen peroxide (H₂O₂) elicit a variety of physiological and cellular eventa including inflammation, DNA damage and apoptosis [26] - [28] . ROS can affect many signal transduction pathways by directly reacting with and modifying all major classes of biomolecules, resulting in changes in their structures and functions [29] . In the recent study, we found that with the increasing of ROS induce various diseases including cancer [30] [31] .