Zinc acetate hydrate (Zn (O2CCH3)2. 2H2O) was obtained from (Merck, Germany). (India). Yeast extracts were purchased from LOBA Chemical Co. (Mumbai). All aqueous solutions were prepared using milli-Q water (18 M Ω).
Preparation of ZnO nanosphere
The sol-gel method of Bao et al. 2012 with some modifications was used for the synthesis of ZnO-NS25, where three grams of yeast extract were nurtured to 100 ml ultrapure water and left for one hour. Then, added 25mmol of zinc acetate solution and mixed with 50 ml of yeast extract solution under vigorous stirring for 60 min. followed by thermal treatment at 500 C for 2 hours. The white precipitate obtained was dried and converted into powder to be ready for characterization.
Characterization of ZnO nanosphere
The ZnO nanospheres spectra were assayed by ultraviolet-visible (UV-VIS) spectrometry (JASCO V-630 spectrophotometer, Japan). A Fourier transformed infrared (FT-IR) spectrum of the ZnO nanospheres was characterized via the Nicolet 6700 apparatus (Thermo Scientific Inc., USA). The crystalline nature and grain size were analyzed by X-ray powder diffraction (XRD) at a temperature of 25–28 oC using a D8 Advance X-ray diffractometer (Bruker, Germany) with a nickel (Ni) filter and CuKα (λ = 1.54184 A0) radiations as an X-ray source. The average hydrodynamic size of the ZnO nanosphere in cell culture medium was determined by dynamic light scattering (DLS) (Nano-ZetaSizer-HT, Malvern Instruments, Malvern, UK)26. Morphology of the synthesized nanospheres was determined by Field Emission Transmission Electron Microscopy (FETEM) (JSM 2100F, Joel Inc., Tokyo, Japan) at accelerating voltages of 15 and 200 kV, respectively
Measurement of Zn (II) released from ZnO nanospheres.
Quantification a final concentration of released zinc ions from suspended ZnO-NS, the following procures occurred. Firstly, dilution the stock suspension of a concentration of 100 µg/ml ZnO nanospheres by Dulbecco’s Modified Eagle’s Medium (DMEM) to a final volume of 15ml. Then, incubate all samples at 37 C in a humidified atmosphere (with 5 % CO2) at different times (0, 3, 6,18, and 24 hrs.), .followed by centrifugation for 20 min speed 10,000 x g. then transferring the supernatant (10 ml) into a test tube containing 0.5 ml Conc HNO3. The solution was filled up to 50 ml with water, and the Zn (II) ions were quantified by using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Perkin-Elmer, USA) 27–28
Human hepatocellular carcinoma (HuH7) cells (catalog number JCRB0403) were obtained from Japan Health Science-Research Resources Bank. Green Kidney Monkey (Vero cells) was purchased from ATCC (American Type Culture Collection) (Clone CCL-81). The cells were maintained in a 95% air and 5% CO2 humidified atmosphere at 37°C. DMEM and MEM-E medium supplemented with 10% FBS and 1% PS were used for routine sub-culturing and all experiments.
Cell viability assay
Determination of the viability of HuH7 and Vero cell lines occurred via MTT, as described by Mossman29. In brief, 1×106 cells / well were seeded in 96-well plates and cultured overnight. Different concentrations of suspended Zinc Oxide Nanosphere and ZnCl2 were added to each well for different times. Next, an MTT solution (5 mg/mL) was added to each well for an additional 4 h. The resulting formazan crystals were dissolved in DMSO, and the optical density was measured at 595 nm using an ELX-800n, Multimode Detector (Biotek, USA)
DNA content analysis
The HuH7 cells (3×105/well) were seeded into 6-well plates, cultured overnight, and treated with suspended ZnO nanospheres (100 µg/ml) for 24 hrs. The cells were fixed in 75% ethanol at − 4oC overnight, then incubated with 50 ng/mL PI staining solution and 0.1 mg/mL RNase A in a dark place for 15 min at room temperature. The DNA content of the cells was quantified by flow cytometry (BD FASCCalibur-USA).
Apoptosis Assay with Annexin V-FITC/PI staining
Huh7 cells (3 × 105/well) were seeded into 6-well plates and cultured overnight before exposure to different concentrations of ZnO nanosphere for different times. The cells were then gently collected and incubated with Annexin V-FITC/PI. According to the /manufacturer’s protocol, the detection of green fluorescence from Annexin V-FITC and red fluorescence from PI was analyzed using a (BD FASCCalibur-USA30.
Cell apoptotic mechanisms for RNA extraction and quantitative RT-PCR
The HuH7 cells were cultured in six-well plates and exposed to ZnO nanospheres (100 µg/ml) for 24 hours. At the end of the exposure process, according to manufacture protocol, the extracted RNA concentration was quantitated by using the RNeasy Mini Kit (Qiagen, Valencia CA, USA) 31. Expressions of p53, Bax, Bcl-2, and cytochrome C genes were quantified using 10 ng of the total RNAs from each sample for cDNA synthesis by reverse transcription using the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, USA). The cDNA was subsequently amplified with the Syber Green I PCR Master Kit (Fermentas) in a 48-well plate using the Step One instrument (Applied Biosystems, USA), as a following: 10 min at 95 ºC for enzyme activation followed by 40 cycles of 15 sec at a temperature of 95 ºC, 20 sec at 55 ºC and 30 sec at 72 ºC for the amplification step. Changes in each target gene expression were normalized relative to the mean critical threshold (CT) values of β-actin as a housekeeping gene by the ΔCt method, one µl of both primers specific for each target gene. The specific primer sets for p53, Bax, BCL-2, and cytochrome C are given in table(1). The mRNA levels were quantified using the 2ΔΔCq method. βactin was used as the internal control. Experiments for each gene were conducted in triplicate.
Table (1). the sequence of the oligonucleotide primers used for real-time PCR
The sequences of the specific sets of primer
F: 5ˋ-TCA GAT CCT AGC GTC GAG CCC-3ˋ
R: 5ˋ-GGG TGT GGA ATC AAC CCA CAG-3ˋ
F: 5ˋ-ATG GAC GGG TCC GGG GAG CA-3ˋ
R: 5ˋ-CCC AGT TGA AGT TGC CGT CA-3ˋ
F: 5ˋ-GTG AAC TGG GGG AGG ATT GT-3ˋ
R: 5ˋ-GGA GAA ATC AAA CAG AGG CC-3ˋ
R: 5'GAC CTC ACA GAC TAC CTC AT3'
F: 5'AGA CAG CAC TGT GTT GGC TA3'
Gene expression by flow cytometry
All flow cytometric analyses were performed on a FACSCalibur flow cytometer (BD, Biosciences, CA, USA). The instrument was aligned and calibrated daily with the use of a 4-color mixture of CaliBRITE beads (BD, Biosciences) with FACSComp Sofware (BD, Biosciences), according to the manufacturer’s instructions32.
The flow cytometry technique evaluated the Bcl-2, Bax, P53, and Cytochrome C oncoproteins. Briefly, after HuH7 Cells were treated with ZnO nanospheres (100 µg/ml) for 24 hrs, cells collected by cold centrifugation at approximately 5000 x g for 10 min then washed twice and re-suspended in 500 µl of cold (+ 4 oC) 1X PBS buffer containing Triton X-100 (permeabilization step). After centrifugation as previously described, the supernatant was removed, and the pellet has been re-suspended again in PBS containing BSA (1%) and diluted primary antibody rabbit monoclonal antibody (1:100) (Oncogene, Cambridge, MA, USA) for P53, Bax, Bcl2 and cytochrome C. followed by incubation at room temperature for 1hr. After centrifugation, the pellet has been washed three times using PBS, and the cells were incubated with secondary antibodies, anti-rabbit (all from Santa Cruz Biotechnology, USA) in dilution of 1:100 followed by incubation in the dark for 30 min at RT. Finally, the cells were centrifuged, and the supernatant was removed, then the cells have been washed as previously described. The pellet was finally re-suspended in 500 µl PBS. The cells were immediately analyzed by flow cytometry (BD FASCCalibur-USA) 32.
A standard fluorometric microplate assay determined the activity of the caspase-3 enzyme33. Briefly, HuH7 cells (1×104 cell/well) were cultured in a 96-well plate and exposed to ZnO nanosphere at the concentrations of 50,100, and 150 µg/mL for 24 hrs. After the exposure was complete, cells were harvested in ice-cold PBS for preparing cell lysate. Further, a reaction mixture containing 30 µL of cell lysate, 20 µL of Ac-DEVDAFC (caspase-3 substrate), and 150 µL of protease reaction buffer (50 mM HEPES, 1 mM EDTA, and 1 mM DTT) (pH 7.2) was incubated for 15 min. Fluorescence of the reaction mixture was measured at 5-minute intervals for 15 minutes at excitation/emission wavelengths of 430/535 nm using an ELISA reader apparatus (ELX-800n, Biotek, USA). The 7-amido-4- tri-fluoro-methyl coumarin (AFC) standard ranging from 5 Μm to 15 µM was prepared, and its fluorescence was recorded to calculate caspase-3 activity in terms of pmol AFC released/minute/mg protein.
Oxidative stress and antioxidant biomarkers
The HuH7 cells were exposed to different ZnO nanospheres and free Zn+ 2 ion (release from 100 µg/ml ZnO-nanosphere) for 24 hrs. After the exposure, the cells were washed and harvested in cold PBS at 4°C. The harvested cell pellets were lysed using a cell lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, Triton X 100 (1% v/v) and 2.5 mM sodium pyrophosphate]. After cold centrifugation at 15,000 g for 10 min, the supernatant (cell extract) was maintained on ice until assayed for oxidative-stress biomarkers. The extent of membrane lipid peroxidation (LPO) was estimated by quantifying malondialdehyde (MDA) 34. MDA is one of the final products of membrane LPO. In brief, 0.1 ml cell extract mixed with 1.9 ml of 0.1 M sodium phosphate buffer (pH 7.4) then incubated at a temperature of 37°C for 1hr. After precipitation with trichloroacetic acid (TCA) (5% v/v), then incubated mixture was centrifuged (2300 g for 15 min at room temperature). The supernatant was collected and to which 1 ml of TBA (1% v/v) was added and placed in boiling water for 15 min. After cooling to room temperature, the absorbance of the mixture was taken at a wavelength of 532 nm and converted to nmol/mg protein using the molar extinction coefficient of 1.56×105 M− 1 Cm− 1.
The level of the reduced glutathione (GSH) was estimated using Ellman's reagent35. The reaction was monitored at a wavelength of 412 nm, and the amount of GSH was expressed in nmol/mg protein. Also, nitric oxide (NO) level was measured by determining total nitrate and nitrite concentrations in a sample using the method reported by Green et al. (1982)36.
After exposure of HuH7 cells to ZnO nanospheres (100 µg/ml), the sample is collected and centrifuged at 1,000 g for 20 min to remove particulates, followed by collecting pellet and consecutive washing with PBS (0.02 mol/l at pH 7.0 ± 0.2) to be used for determination of Reactive Oxygen Species (ROS) by kit (Life Span Bioscience Inc., Seattle, WA, USA). All reagents were prepared by adding 100 µl of a sample, standard and blank for each well, followed by incubation at 37°C for 90 min. All samples were then aspirated, and 100 µl of biotinylated detection antibody was added to the pellet and incubated for 1 hr (37°C). After centrifugation (3,000 g), the supernatants were aspirated, and the pellets were washed three times by adding 100 µl of HRP conjugate, followed by incubation at 37°C for 30 min. The supernatants were removed by aspiration and washed five times before adding 90 µl of TMB substrate solution and incubating at 37°C for 15 min. The reaction was stopped by adding 50 µl of stop solution, and then the absorbance of the medium was measured immediately at a wavelength of 450 nm.
Superoxide dismutase (SOD) assay is a mixture containing sodium pyrophosphate buffer, nitro blue tetrazolium (NBT), phenazine methosulphate (PMS), reduced nicotinamide adenine dinucleotide (NADH), and the required volume of cell extract. One unit of SOD enzyme activity is defined as the amount of enzyme required to inhibit chromogen production (optical density at 560 nm) by 50% in 1 minute under assay conditions and expressed as specific activity in units/mg protein37.
Morphological of apoptosis
To investigation the total fraction of cell with a fragment nucleus among the total count of cell population was taken place by using hematoxylin-stained slides. Also, study the apoptotic process for Huh7 occurred18.
Ultrastructure of HuH7 using a transmission electron microscope
The HuH7 cells were remedied to ZnO nanosphere at concentration 100 µg/ml for different time intervals. After the end of exposure, cells were harvested and washed with PBS then fixed in ice-cold glutaraldehyde (2.5%) for one hour. The cells were washed with PBS three times for 15 min and post-fixed in OsO4 (1%) for one hour, then stained with uranyl acetate (2%) for 30 min at room temperature. The cells were dehydrated through serial dilutions of ethanol (50, 70, and 90%) for 15 min each. The dehydration followed this in ethanol (100%) for 20 min and acetone (100%) for 20 min, respectively, and then embedded in Epon812. Ultrathin sections (120 nm) were obtained and stained with uranyl acetate (2%) for 20 min, and lead citrate for 5 min then examined using Field Emission Transmission Electron Microscopy (FETEM) (JSM 2100F, Joel Inc., Tokyo, Japan) at accelerating voltages of 15 kV and 200 kV, respectively 18.