L02 cells (CBCAS, Shanghai, China) were cultured in RPMI 1640 medium (Gibco BRL, MD, USA); while HEK293 cells (CBCAS, Shanghai, China) in DMEM medium (Gibco BRL, MD, USA), with fetal calf serum (10%), l-glutamine (2.9 mg·mL⁻¹), streptomycin (1 mg·mL⁻¹), and penicillin (100 units·mL⁻¹). The cells were cultured at 37°C in water-saturated air supplemented with 5% CO₂. Culture media were changed every 2 days. Cells were passaged thrice a week. At 85% confluence, the cells were harvested using 0.25% trypsin and were subcultured into 75 cm² flasks, 6-well plates, 24-well plates, or 96-well plates according to the selection of experiments.

After the monolayer of cells was placed in 6, 24, or 96-well plates, the cells were treated with a range of concentrations of nano-sized ZnO particles suspended in medium without serum for 24 h. After the 24 h treatment, the various toxicity end points were evaluated in control and ZnO particles-exposed cells.

L02 cells and HEK293 cells were exposed as mentioned above at various concentrations of ZnO NPs for 24 h. After completion of the exposure period, the cells (control and nano-ZnO exposed) were washed with phosphate buffered solution (PBS) and observed by phase contrast inverted microscopy at ×200 magnification.

Mitochondrial function was evaluated by 3-(4,5-dimethylazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT assay helps in cell viability assessment by measuring the enzymatic reduction of yellow tetrazolium MTT to a purple formazan, as measured at 570 nm using enzyme-labeled instrument (Tecan Co., Weymouth, UK).

Cells were cultured in 75 cm² culture flask and exposed to ZnO NPs (5 to 100 μg·mL⁻¹) for 24 h. After exposure, the cells were harvested in chilled PBS by scraping and washed twice with 1 × PBS at 4°C for 6 min at 1,500 rpm. The cell pellet was then sonicated at 15 W for 10 s (3 cycles) to obtain the cell lysate.

Oxidative stress markers (malondialdehyde, MDA; glutathione, GSH; superoxide dismutase, SOD) were estimated by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to manufacturer's protocol. Protein content was measured by the method of Lowry 13 using BSA as the standard.

DNA damage by ZnO NPs was further studied using comet assay. After treatment with nano-sized ZnO particles for 4, 12, and 24 h, the cells were rinsed with ice-cold 1 × PBS and trypsinized. Then the cells were washed once in ice-cold 1 × PBS and resuspended at 1 × 10⁵ cells mL⁻¹ in ice-cold 1 × PBS. An aliquot of 10-μL cell suspension was mixed with 100 μL molten agarose (at 37°C), and 75 μL of this mixture was immediately applied to a glass slide. The slide was held horizontal at 4°C for 30 min to improve adherence. Then the slide was immersed in cold lysis solution to lyse the cells. After 50 min at 4°C in the dark, the slide was immersed in an alkaline solution (300 mM NaOH, 1 mM EDTA, pH > 13) at room temperature in the dark to denature the DNA. After 30 min the slide was placed on a horizontal electrophoresis unit, and the unit was filled with fresh buffer (300 mM NaOH, 1 mM EDTA, pH > 13) to cover the slide. Electrophoresis was conducted at 27 V (300 mA) for 40 min at 4°C in the dark. The slide was then washed gently with distilled water and immersed in 70% ethanol for 5 min. After the slide was air dried, 50 μL of ethidium bromide working solution was applied to each circle of dried agarose. All steps described above were conducted under yellow light to prevent additional DNA damage.

The slides were viewed using an epifluorescence Leica DMI 4000B microscope (Leica Microsystems Ltd., Hong Kong, China) equipped with a fluorescein filter. Observations were made at a final magnification ×400. Thirty randomly selected cells per experimental point were imaged and analyzed using CASP software (download from http://www.casp.of.pl/). Results were reported as tail moment, a parameter describing the number of migrated fragments, and represented by the fluorescence intensity in the tail, expressed as the mean of the 50 cells.

The data were expressed as mean ± standard deviation of three independent experiments. The data was subjected to statistical analysis by one-way analysis of variance followed by Dunnett's method for multiple comparisons. A value of p < 0.05 was considered significant. SPSS 16.0 software was used for the statistical analysis.

The average size of ZnO NPs (Sigma-Aldrich, St. Louis, MO, USA) as measured by transmission electron microscope (TEM) (JEOL JEM-2100, JEOL Ltd., Tokyo, Japan) was approximately 50 nm. Figure  1 shows a representative TEM image recorded ZnO nanoparticles.

After L02 and HEK293 cells were incubated with 0, 10, 25, 50, and 100 μg·mL⁻¹ of 50 nm ZnO NPs for 24 h, respectively, the resulted cells were observed with 200-fold magnification by optical microscope. L02 cells (Figure  2) and HEK 293 cells (Figure  3) are the comparative morphologies of the unexposed and ZnO particles-exposed cells. Comparing with the bulk cells in the control experiments, the cells cultivated with low dose of ZnO NPs (10 μg·mL⁻¹) appear similar to the control cells with brownish particles most likely associated with the cell membranes, indicating that lower dose of ZnO NPs did not harm the L02 and HEK293 cells. With increasing doses of ZnO NPs, the cells started to shrink and became irregular in shape. When the concentration reached 100 μg·mL⁻¹, apoptosis, necrosis, and decrease in number of cells were observed. The microscopic studies demonstrated that cells at higher doses became abnormal in size, displaying cellular shrinkage, and acquisition of an irregular shape occurred due to the increase of ZnO NPs concentration.

L02 and HEK293 cells were exposed to ZnO NPs (10 to 100 μg·mL⁻¹) for 24 h, and cytotoxicity was determined with MTT assay. The MTT results demonstrated a concentration-dependent cytotoxicity after exposure to ZnO NPs (Figure  4). The percentage MTT reduction (relative to control) of L02 cells observed after ZnO exposure at concentrations 50, 75, and 100 μg·mL⁻¹ was 52.63%, 41.12%, and 36.70%, respectively; while there were percentage reductions to 68.36%, 38.74%, 19.43%, and 15.21% of HEK293 cells after ZnO NPs exposure at concentrations 25, 50, 75, and 100 μg·mL⁻¹. It is observed that there is a statistically significant difference (p < 0.05), and the ZnO NPs showed higher toxicity to human embryonic kidney cells.