ASD was collected from the atmosphere on March 16, 2009 outside the Gachon University building in Korea, using a high volume air sampler (HV500F; Sibata Scientific Technology, Ltd., Saitama, Japan) at a flow rate of 500 L/min. These ASD samples were prefiltered into filter packs (Prefilter AP, 124 mm; EMD Millipore, Bedford, MA, USA) and sieved through a filter with a 10-μm pore size, after mixing with phosphate buffered saline (PBS) in a 15-mL tube. The filtered ASD particles were sterilized in an autoclave at 121°C for 15 minutes; subsequently, these particles were weighed. Each sample was transferred to a 1.5-mL tube. The filtered PM was stored at –20°C until further use. The particle diameter of the samples was measured (a total of 600 particles) under a scanning electron microscope (JSM-5800; JEOL Ltd., Tokyo, Japan). The mean distribution peak of the ASD particle diameter was observed at 6 μm. Chemical components of the ASD particles were analyzed via X-ray fluorescence spectrometry (PHILIPS pw2404; Philips, Eindhoven, The Netherlands) at the Korea Basic Science Institute. The chemical composition of ASD was determined to be as follows: 78.4% SiO₂, 9.35% Al₂O₃, 2.52% K₂O, 2.41% Na₂O, 2.06% Fe₂O₃, and 1.74% CaO. MaO, TiO₂, P₂O₅, and MnO made up less than 0.1% of the total composition.

HMEECs (kindly provided by Dr. David J. Lim, House Ear Institutes, Los Angeles, CA, USA), immortalized with the E6/E7 genes of human papilloma virus type [9] were maintained in a mixture of Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) and bronchial epithelial basal medium (BEBM) (Lonza, Walkersville, MD, USA) (1:1). The cells were maintained at subconfluence in a carbon dioxide-enriched (95% air, 5% CO₂) humidified atmosphere, at 37°C. In order to study the effects of ASD, the cells were grown to 60% confluence in six-well culture plates at 37°C in a carbon dioxide-enriched (95% air, 5% CO₂) humidified atmosphere. These cells were starved for 2 hours; these were exposed to ASD and subsequently incubated for 24 hours. The experimental group received various concentrations of ASD (50–400 μg/mL) while the control group did not receive ASD.

Cell viability was measured using the cell counting kit (CCK-8, Dojindo Laboratories, Kumamoto, Japan). HMEECs were seeded in 96-well plates, with each well containing 1×10⁴ cells. The following day, cells were treated with 0, 6.25, 12.5, 25, 50, 100, 200, 300, or 400 μg/mL of ASD. CCK-8 solution was added to each well after 24 hours, and the plates incubated for 150 minutes at 37°C. The contents of the plates were mixed using a shaker (at room temperature for 5 minutes), and the optical density was measured at 450 nm using a microplate reader (Spectra Max plus 384; Molecular devices, Sunnyvale, CA, USA).

Real-time polymerase chain reaction (RT-PCR) was performed using a LightCycler 480 Real-Time PCR System (Hoffmann-LaRoche, Basel, Switzerland). Each reaction mixture contained 10 μL of LightCycler 480SYBR Green I Master (Hoffmann-LaRo che), 1 μL of 4 pmol sense and antisense primers, and 0.4 μL of cDNA, in a final volume of 20 μL. Reaction mixtures were incubated at 95°C for 5 minutes to activate the FastStart Taq DNA Polymerase; this was followed by amplification for 50 cycles (one cycle: 15 seconds at 95°C, 30 seconds at 60°C, and 30 seconds 72°C). The data was analyzed using the LightCycler 480 software 1.5 (Hoffmann-LaRoche). Target mRNA expression was normalized to that of GAPDH, and calculated using the comparative Ct method. The specific primers of genes used for realtime PCR are listed in Table 1.

HMEECs were stimulated with 0, 50, 100, 200, or 400 μg/mL of ASD for 24 hours. After treatment of HMEECs with ASD, the medium was removed and the cells were washed twice in PBS (10 mM, pH 7.4). Lysis buffer (0.2 mL, PRO-PREP; INtRON, Seongnam, Korea) was added, and the cells were incubated for 1 hour at –20°C. The cells were then centrifuged at 13,000 ×g for 10 minutes at 4°C. The supernatant containing the total cell lysate was collected and stored at –80°C. Protein concentration of the lysates was measured using Quick Start Bradford 1 dye reagent (Bio-Rad, Hercules, CA, USA). Equal quantities of protein were analyzed using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein content of the gels was transferred to a polyvinyl difluoride membrane (Hybond P; Amersham Biosciences Corp., GE Healthcare, Buckinghamshire, UK), and the membranes blocked with 25 mM Tris and 120 mM NaCl and 0.05% Tween-20 containing 5% (w/v) skim milk for 1 hour at room temperature. Membranes were probed with antibodies against COX-2 (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA) and β-Actin (1:3,000; Sigma-Aldrich, St. Louis, MO, USA), followed by peroxidase-conjugated antimouse IgG (1:5,000; Vector Laboratories, Burlingame, CA, USA). The membranes were developed using an ECL detection kit (Pierce, Rockford, IL, USA) and the signal captured using an image reader (LAS3000; Fuji Photo Film, Tokyo, Japan).

Fifty pathogen-free, male Sprague Dawley rats weighting 150–200 g (Orient Bio, Seongnam, Korea) were used for this study. The protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC); the animals were cared for according to the Guidelines of Animal Experiments of the Korea University Guro Hospital. The rats were assigned randomly to one of 5 groups: 4 groups received a unilateral tympanic membrane ASD injection (on days 1, 3, 5, and 14; each group, n=5) and the control group received a PBS vehicle injection (vehicle control group, n=5). ASD (400 μg/mL) dissolved in PBS was injected into all experimental mice (4 groups) at all time-points using a 27-gauge spinal needle. The rats were injected with ASD and subsequently sacrificed on days 1 (group 1), 3 (group 2), 5 (group 3), or 14 (group 4) postprocedure. Anesthesia was performed with an intraperitoneal injection of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg).

Five rats from each experimental group (groups 1, 2, 3, and 4) and five rats from the control group were sacrificed; the bullae (including the middle and inner ear) were isolated from the ears of all rats. Following dissection, the bullae were fixed overnight in a 4% paraformaldehyde solution, and subsequently decalcified using ethylenediaminetetraacetic acid. The specimens were embedded in paraffin, sectioned into 5-μm slices, and stained with hematoxylin and eosin (Sigma-Aldrich).

All data was expressed as mean±standard deviation. One-way analysis of variance (ANOVA) was used to determine the statistically significant differences between the control and experimental groups at each time or dose point. Scheffe F-test was used to correct for multiple comparisons when statistically significant differences were identified in the ANOVA. A P-value of <0.05 for the null hypothesis was accepted as indicating a statistically significant difference. Statistical analyses were performed using SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA).

The effect of ASD on HMEEC viability after exposure to ASD for 24 hours was initially investigated. Microscopic evaluation showed higher cell debris in the ASD-treated groups (100 and 400 μg/mL) than in the control HMEECs; in addition, there was a sharp decrease in the viable cell population in the group treated with 400 μg/mL of ASD (Fig. 1).

The CCK-8 cell viability assay demonstrated that ASD caused a dose-dependent decrease in cell viability. Cell viability was considerably decreased in groups exposed to higher concentrations of ASD (>100 μg/mL). Especially, administration of 400 μg/mL of ASD resulted in approximately 60% of viability compared to that of the control group (Fig. 2).

The expression of TNF-a, COX-2, MUC5AC, and MUC5B genes was analyzed by quantitative RT-PCR, in order to determine whether ASD induces inflammation and mucin production in HMEECs. These genes are usually activated in patients suffering from OM. Exposure to ASD at concentrations greater than 100 μg/mL led to significantly higher mRNA levels of TNF-a and COX-2 (P<0.05) compared to those in the control group (not treated with ASD) (Fig. 3A). ASD exposure also resulted in a prominent increase in the expression of genes coding for the mucin family of proteins, including MUC5AC and MUC5B (P<0.05) (Fig. 3B). In particular, ASD exposure resulted in a significantly higher TNF-a and MUC5B expression compared to that of COX-2 and MUC5AC, respectively (Fig. 3).

In addition, we investigated the possible activation of apoptosis and reactive oxygen species (ROS) reactions in HMEECs treated with ASD. Interestingly, the pro-apoptotic gene including TP53 and BAX (P<0.05) were significantly increased, whereas the BCL-2 (P<0.05) mRNA, which is antiapoptotic gene was significantly decreased in HMEECs when stimulated with 400 μg/mL of ASD (Fig. 4). Moreover, the expression of NOX4 and SOD1 genes, which are related with oxidative reaction, were also significantly increased in HMEECs treated with 400 μg/mL of ASD (P<0.05) (Fig. 4). Taken together, these results suggest that ASD induces inflammation and mucin production in HMEECs. Additionally, ASD also induces apoptosis and the production of ROS in HMEECs.

The change in COX-2 protein expression as a result of ASD exposure was also investigated. Western blot analysis demonstrated that ASD treatment significantly enhanced COX-2 expression in HMEECs (Fig. 5). This result suggests that the expression of COX-2 protein is up-regulated in HMEECs exposed to ASD.

The morphology of middle ear epithelium was observed at different time points after ASD treatment by light microscopy. Normal middle ear epithelium was thin and free of inflammatory cells as seen in the control groups (Figs. 6A, 7A). However, when rats exposed to ASD (ASD-injection), the signs of inflammation appeared. At day 1, there was a thickening of the middle ear epithelium and the infiltration of inflammatory cells in the upper region of the epithelium (Figs. 6B, 7B). At day 3, there was a further increase in inflammatory cell infiltration; these cells moved out of the tissue (Figs. 6C, 7C). In addition, we observed cilia in the epithelium of the middle ear (Fig. 7C). However, the mucosal thickness decreased gradually, and inflammatory cell infiltrating the middle ear epithelium reduced in 5 and 14 days after ASD injection (Figs. 6D, E, 7D, E).