Cells and cell culture
HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France), 100 U/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Inc.) at 37°C in a humidified atmosphere containing 5% CO2.
TLR-encoding genes were purchased from InvivoGen (San Diego, CA, USA). The pUNO1-mcs expression vector was used as an “empty” control vector. Since pUNO1-mcs does not contain a therapeutic gene, it can be used in conjunction with other vectors of the pUNO1 family to serve as an experimental control. Overproduction of TLR3 and TLR4 was provided by transfection with pUNO-hTLR3 (which encodes the human TLR3 protein) and pUNO1-hTLR04a (CD284a) (which harbors the human TLR04a (CD284a) encoding open reading frame), respectively. HepG2 cells were seeded in 6-well plates. After overnight incubation, the cells were co-transfected with TLR3 or TLR4 expression vectors and control plasmid (pUNO1-mcs) using Lipofectamine™ LTX Reagent (Invitrogen, Carlsbad, CA, USA) according to the supplier’s protocol. Transfection efficiency of at least 50% was obtained.
Preparation and exposure to TiO2 NPs
The preparation and characterization of TiO2 NPs were described in previous studies . Briefly, nano-TiO2 (AeroxideR P25; Sigma-Aldrich, St Louis, MO, USA) was dispersed in distilled water and autoclaved at 120°C for 20 min. The suspension was cooled to room temperature and then sonicated for 10 min at 200 kHz using a high-frequency ultrasonic sonicator (MidSonic 600, Kaijo Corp., Tokyo, Japan). The resulting nano-TiO2 suspension was designated “TiO2 NPs”. The concentration of TiO2 NPs was determined using a UV–vis spectrophotometer at 370 nm (UV-1600, Shimadzu, Kyoto, Japan). The suspension was adjusted to the desired concentration by the addition of distilled water and stored at 4°C until use. The particle size distribution was measured by dynamic light scattering (Zetasizer Nano-ZS, Malvern Instruments, Malvern, UK). The aggregated particle size of the TiO2 NPs was determined to be 216 ± 70 nm. The size of the aggregated TiO2 NPs remained stable for several weeks under the indicated storage conditions. Prior to addition to the cell cultures, the suspension of TiO2 NPs was diluted with supplemented medium and used as described above. For the reporter gene (transfected cell) assays, the culture medium was replaced (1 day after transfection) with medium containing the TiO2 NPs at the indicated concentration. Specifically, TiO2 NPs were added to the culture medium immediately before the medium was applied to the cells. After 48 h, the cells were harvested and assayed.
DCF assay for oxidative stress determination
The accumulation of intracellular free radicals was quantified using a ROS assay kit (OxiSelect, Cell Biolabs, Inc., San Diego, CA, USA), which employs the cell-permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA can cross cell membranes and be deacetylated by intracellular esterases to non-fluorescent 2′, 7′-dichlorodihydrofluorescein (DCFH). In the presence of ROS, DCFH is rapidly oxidized to the highly fluorescent DCF, which is readily detectable. The fluorescence intensity is proportional to the ROS levels in the cell cytosol. HepG2 cells were cultured in 96-well black plates; after overnight incubation, the cells were co-transfected with TLR3, TLR4 or control plasmid (pUNO1-mcs). After 24 h, the cells were exposed to TiO2 NPs for 48h, and were then incubated with DCHF-DA for 30 min in the dark. Parallel sets of wells containing freshly cultured cells, which were not treated with NPs or plasmids, and were suspended in the same concentration ratio of DPBS and DMEM, were regarded as negative controls. The fluorescence emission of DCF was monitored at regular intervals at an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a fluorescence plate reader (Twinkle LB 970 Microplate Fluorometer, BERTHOLD TECHNOLOGIES GmbH & Co. KG, Calmbacher, Bad Wildbad Germany). The amount of DCF formed was calculated from a calibration curve constructed using an authentic DCF standard.
Measurement of H2O2
The levels of hydrogen peroxide (H2O2) were measured using a hydrogen peroxide assay kit (ab102500, Abcam, Tokyo, Japan). In the presence of horseradish peroxidase (HRP), the OxiRed Probe reacts with H2O2 to produce a colored product. Following the experiment, the cells were collected in H2O2 assay buffer and then centrifuged for 15 min at 1000 × g. A total of 50 μl of the supernatant was mixed with 50 μl of the reaction mix (assay buffer: 46 μl; OxiRed Probe: 2 μl; HRP: 2 μl) and then incubated at room temperature for 10 min. The optical density at 570 nm was read with a microplate reader (Benckmark Plus microplate spectrophotometer, BioRad, [city?] CA, USA), and the H2O2 concentration was calculated according to a standard concentration curve.
Measurement of GPX
Glutathione peroxidase activity was measured using a glutathione peroxidase assay kit provided by Cayman Chemical Company (Ann Arbor, MI, USA). Cells were washed in phosphate buffer, pH 7.4, collected by centrifugation (2000 × g for 10 min at 4°C), then homogenized in cold assay buffer (50 mM Tris-HCL, pH 7.5, 5 mM EDTA, 1 mM DTT). Following centrifugation at 10,000 × g for 15 min at 4°C, the supernatant was removed for assay. Sample (20 μl of supernatant) was added to the desired well of a 96-well plate, then 100 μL of assay buffer and 50 μl of co-substrate mixture was added. The reaction was initiated by adding 20 μl of cumene hydroperoxide to each reaction well, then mixed by shaking for second. The absorbance was read at 340 nm using a plate reader. At least 5 time points were obtained.
Measurement of GSH
The total glutathione concentration (reduced and oxidized forms) was determined in a microtitre plate assay using a glutathione assay kit (Sigma-Aldrich). After TLR transfection and nanoparticle exposure, HepG2 cells were washed twice with phosphate-buffered saline (PBS), resuspended in a 5% 5-sulfosalicylic acid solution, then centrifuged at 10,000 × g for 10 min. Supernatant (10 μl) was mixed with 150 μl of working solution, incubated for 5 min at room temperature, then 50 μl of the diluted NADPH solution was added. The absorbance of each sample was measured at 412 nm using the plate reader, as was the absorbance of the reagent blank (10 μl of 5% 5-sulfosalicylic acid); the absorbance of the blank was then subtracted from the absorbance of each sample. The final concentration of the components in the reaction mixture was 95 mM potassium phosphate buffer, pH 7.0, containing 0.95 mM ethylenediamine tetra-acetic acid (EDTA), 0.038 mg/ml (48 μM) NADPH, 0.031 mg/ml DTNB, 0.115 units/ml glutathione reductase, and 0.24% 5-sulfosalicylic acid. All measurements were performed in triplicate; the concentration (nmoles) of GSH in the samples was calculated.
Measurement of caspase-3 activity
All reagents for assessing caspase-3 activity were provided in a caspase-3 colormetric assay kit, (Sigma Aldrich). HepG2 cells (1 × 106) were cultured in 6-well plates and treated as described above. At the end of the experiment, the cells were washed and lysed in 100 μl of lysis buffer provide in the kit, then 80 μl of the sample was added to 10 μl of the 10× assay buffer and 10 μl of in a well of a 96-well plate. The reaction mix was incubated for 10 hours at 37°C, then the absorbance was read at 405 nm.
Gene expression analysis: PCR array
For polymerase chain reaction (PCR) array analysis, HepG2 cells (at 6 × 105 cells/ml) with or without TLR4 transfection were seeded in a culture dish containing culture medium with or without TiO2 NPs (suspended at 10 μg/ml). After 48 h exposure to the TiO2 NPs, the cells were detached by mechanical dissociation and total cellular RNA was extracted using an RNeasy kit (Qiagen, MD, USA). An aliquot (1 μg) of the extracted total RNA was reverse transcribed into cDNA with random hexamer primers using a RT2 First Strand kit (SABiosciences/Quiagen MD, USA) and the expression of 89 human DNA damage-related genes involved in signaling pathways were examined using a RT2 Profiler PCR array kit (SABiosciences/Quiagen) according to the manufacturer’s instructions. PCR array analysis was performed using an ABI PRISM 7000 sequence detection system (Applied Biosystems, Singapore).
Real-Time (RT) PCR
For mRNA expression analysis, 6 × 105 HepG2 cells/ml were seeded in cell culture dishes, the cells were transfected with TLR4 expression vector and exposed to a suspension of TiO2 NPs at a final concentration of 10 μg/ml for 48 h, then the cells were detached and subjected to gene expression analysis. The expression of marker genes was determined using quantitative real-time PCR (RT-PCR) as follows. Total RNA and cDNA were synthesized as described for the PCR array. The PCR primers for human APEX1, ATM, GADD45A, IP6K3, MBD4, SMC1A were purchased from SABiosciences/Qiagen. The data were normalized using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control in the same reaction as the gene of interest . The reaction mixture was composed of 12.5 μl of RT2 SYBR Green qPCR Master Mix (SABiosciences;/Qiagen), 1 μl of 10 μM gene-specific RT2 qPCR forward and reverse primers, 2 μl of cDNA, and nuclease-free water to a final volume of 25 μl. The thermocycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
Confocal microscopy observation
Confocal laser scanning microscopy was performed using a Zeiss LSM510 microscope (Carl Zeiss, Oberkochen, Germany). HepG2 cells were cultured on cover-slips (13 mm diameter; Matsunami Glass Ind., Ltd., Osaka, Japan). The following day, cultures were transfected (using the Lipofectamine™ LTX Reagent, as described above) with the expression vector encoding TLR4. At 24 h after transfection, the culture medium was replaced with medium containing 10 μg/ml TiO2 NPs. Untransfected cells and cells without NP exposure were used as controls. After 48 h incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 5 min. Fixed cells were then stained for nuclei using 1 μg/ml Hoechst33342 (Dojin Chemical, Japan) for 30 min in a 5% CO2 environment. Figures were created using NIH ImageJ software.
Data were expressed as the mean ± SD, (n ≥3). All experiments were carried out independently. The data were analyzed using Student’s t test to evaluate the significance of differences between the treated groups and control groups. Statistical significance was accepted at P < 0.05.