Gene expression profiles in human keratinocytes exposure to ultrafine, fine, and submicron TiO2 particles

Gene expression profiling of the human keratinocytes cell line (HaCaT) exposure to ultrafine, fine, and submicron TiO2 particles were employed to gain insights into the molecular events. Three types of bulk TiO2 anatase particles, ST-01(average primary particle size= 7nm, specific surface area = 316m2/g), ST-21(20nm and 66m2/g), and ST-41(200nm and 10m2/g) used in this study were purchased from Ishihara Sangyo Kaisha Ltd., Japan. TiO2 particles were dispersed and size-fractionated in Dulbecco’s modified Eagle’s medium (DMEM) . TiO2 particles was dispersed in heat-inactivated fetal bovine serum (FBS) and centrifuged at 16,000 x g for 20 min. Removing the supernatant, the pellet was washed with DMEM, and centrifuged at 16,000 x g for 20 min. The supernatant was removed and dispersed in fresh DMEM with 10 % FBS medium and antibiotic/antifungal-agent (100 units/ml of penicillin, 100 μg/ml of streptomycin, and 250 ng/ml of amphotericin B) defined as DMEM-FBS medium, and centrifuged at 8,000 x g for 20 min. Removing the supernatant, the pellet was vortexed with fresh DMEM-FBS medium and centrifuged at 4,000 x g for 20min. The supernatant of 4,000 x g fraction was collected. Continuously, the centrifugal force was reduced to 2,000 x g, 1,000 x g and 500 x g. Each supernatant of 2,000 x g, 1,000 x g, and 500 x g fraction was collected. The 500 x g fraction supernatant was 0.8 µm membrane-filtered. The concentrations and the secondary average diameter of TiO2 particles in the supernatant fractions were individually characterized using X-ray diffraction (XRD), dynamic light scattering (DLS), and transmission electron microscopy (TEM) at 120 kV. The samples were defined as T-7 (ST-01, 4,000 x g fraction), T-20 (ST-21, 1,000 x g fraction), and T-200 (ST-41, 500 x g fraction), and were utilized for the following DNA microarray analysis. Human keratinocyte cells (HaCaT) purchased from the German Cancer Research Center (DKFZ) were cultured in DMEM-FBS medium at 37 °C in a humidified atmosphere of 5% CO2 and 95% air The passage numbers 2–10 were used in the experiments, and cells were seeded at a density of approximately 2 x 10^5 cells/ml. All experiments were performed in triplicate (n=3).