Sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation will increase in lipopolysaccharide-induced inflammation in vitro model

The neonatal (1- to 3-day-old) Sprague Dawley (SD) rats were purchased from the animal center of the ChongQing Medical University. The animal center is officially qualified to raise and breed research animals. Animals were fed according to the standard protocols approved by the Statute of Laboratory Animal Management Administration of China and the procedure was approved by the Ethics Committee of the ChongQing Medical University.

Ten neonatal SD rats were treated with 75% ethanol after receiving ethyl-ether-inhaled anesthesia. The bilateral temporal bones were removed, and the spiral ganglion tissue was dissected form the stria vascularis and basilar membrane; then, the tissue was digested with 0.25% trypsin for 30 min at 37°C. After the samples were centrifuged for 8 min at 800 rpm, the cells were resuspended and placed in 35-mm cell culture plates coated with poly-L-lysine. Cells were maintained in a growth medium with DMEM/F12 (Hyclone, Logan, UT, USA), supplemented with 20% fetal bovine serum (GIBCO, Carlsbad, CA, USA), 10% neurotrophic factors B27, and 1% penicillin-streptomycin and placed in an incubator at 37°C in 5% CO2 and 95% air. We used 5 μmol/ml cytarabine to purify the SGN for 72 h. Neuronal class III anti-β-tubulin antibody (TUJ1, 1:200)(Santa Cruz, Dallas, TX, USA) was used to identify neurons. Immunofluorescence procedures were performed according to the following steps. The SGN were digested by 0.25% trypsin and mounted onto glass microscope slides, which were treated with poly-L-lysine and sterilized ultraviolet rays for 15 min. The SGN were washed with phosphate-buffered saline (PBS, pH 7.4) once. Next, the SGN were fixed in precooled 4% paraformaldehyde for 15 min at room temperature and washed once in PBS for 5 min, permeabilized in 0.3% Triton™ X-100 (Sigma, St. Louis, MO, USA) for 15 min, and then washed in PBS once. Subsequently, the specimens were blocked in a solution of 5% bovine serum albumin (BSA) for 10 min. The specimens were incubated overnight at 4°C in a solution (1:200) containing anti-β-III tubulin (TUJ1), a mouse monoclonal antibody (Santa Cruz, Dallas, TX, USA). After being washed three times in PBS, the specimens were incubated with anti-mouse IgG (diluted 1:200 with 1% BSA) for 30 min at 37°C. After processing for immunofluorescence, the slides were mounted with glycerinum and observed using a confocal laser scanning microscope (CLSM) (Nikon, Tokyo, Japan).

SGN were digested and seeded into a 96-well growth-medium plate, and after adhering to the plates after 12 h, the SGN were treated with different concentrations of LPS (0, 20, 40, 50, 100, 200, and 400 μg/ml) and incubated for 48 h. Three replicative wells were detected for each concentration. After 48 h, the SGN were washed twice with PBS and given fresh complete growth medium. Then, 20 μl of the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) test solution was added to each well, and the cells were incubated at 37°C for 1.5 h. The 0 μg/ml group was defined as the control group. The absorbance value (OD value) at the 450-nm wavelength was measured using a microplate reader. The cell activity rate (%) = (OD experimental − OD blank)/(OD control − OD blank) × 100.

The alkaline comet assay (pH > 13) is widely used to quantify DNA damage, such as single-strand breaks (SSB), double-strand breaks (DSB), and alkali labile sites (ALS), especially in SSB [19]. It has proven to be a sensitive method of measuring the induction and repair of DNA damage in individual cells [5,20-22]. After cells were exposed to LPS (0, 20, 40, 50, 100, 200, 400 μg/ml) and H₂O₂, they were washed with PBS, trypsinized, and resuspended in PBS for the comet assay. The cell suspension (10 μl: 1,000 cells) was mixed with 60 μl LMPA (0.65% low-melting-point agarose) and immediately pipetted onto Trevigen CometSlides™ (Trevigen, Gaithersburg, MD, USA). After the slides were precooled at 4°C for 30 min in the dark and agarose had solidified, they were immersed in the lysing solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris, 1% Triton X-100, 10% DMSO, pH 10) for 2 h at 4°C in the dark. After the slides were allowed to unwind for 30 min in an alkaline buffer (1 mM Na₂EDTA, 300 mM NaOH, pH 13) at room temperature, they were subjected to electrophoresis at 25 V/300 mA for 40 min. The slides were washed twice in dH2O for 5 min and dehydrated in 95% and 75% ethanol for 5 min each. Subsequently, the slides were air-dried in the dark at 4°C. Then, these slides were dyed with attenuated SYBR® Green I (Invitrogen, Waltham, MA, USA) and examined at 200× magnification with a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The tail DNA (%), tail moment (arbitrary units), and tail length (μm) were used to express the level of DNA damage [23]. Images of 200 randomly selected cells were analyzed from each slide. All of the comet parameters were assessed by a comet assay image analysis system (Comet Assay Software Project, CASP Lab, Poland).

Using the procedures described above, we treated the SGN with LPS (40 μg/ml) for 24 h before exposure to RF-EMR. The cellular activity and DNA exhibited no obvious changes after treatment with LPS (40 μg/ml). Therefore, 40 μg/ml was used to pretreat the concentration before exposure to RF-EMR.

The RF-EMR exposure system was built by the Foundation for Information Technologies in Society (IT’IS Foundation, Zurich, Switzerland), as described in detail in previous studies [24-26]. This exposure system consists of four primary parts: two rectangular waveguides, an RF generator, a narrow-band amplifier, and an arbitrary function generator. One waveguide is used for the radiation-exposure groups, whereas the other waveguide is used for the sham-exposure group. Both of these waveguides were placed inside a CO₂ incubator. The exposure system was connected to a computer that controlled the specific absorption rate (SAR) value during the exposure process and maintained the temperature of the incubator at 37°C, 5% CO₂/95%. The SAR variability of this exposure system was controlled below 6%, with a temperature rise of 0.03°C/(W/kg) of the average SAR value following exposure of monolayer cells. This system operated under the stable frequency of 1,800 MHz. Six 35-mm Petri dishes can be placed in the H-field maxima and exposed to polarized E field (an electric field that is perpendicular to the H field) in the same manner. The temperature change is uniformly distributed in monolayer cells, and the temperature difference between the RF-exposed and sham-exposed chambers did not exceed 0.1°C [27]. All of the exposure parameters and monitor data (for example, temperature, SAR value) were encoded and recorded in a data file, which was transmitted to the IT’IS Foundation by e-mail and decoded by the foundation for the data analysis.

After SGN were treated with 40 μg/ml LPS for 24 h, the culture medium was renewed, and cells were subjected to a 24-h exposure to 1,800 MHz GSM ‘talk mode’ signals with an intermittent cycle of 5 min on and 10 min off. To explore the SAR-related effects of RF radiation exposure, dishes were randomly divided into the following groups: (1) RF radiation control, (2) 2 W/kg (either sham-exposure or exposure), (3) 4 W/kg (either sham-exposure or exposure), (4) 2 W/kg combined with LPS (40 μg/ml) (either sham-exposure or exposure), and (5) 4 W/kg combined with LPS (40 μg/ml) (either sham-exposure or exposure).

Following exposure to RF-EMR and LPS (40 μg/ml), we applied alkaline comet assay to all groups to detect DNA damage, using the steps as described in the above comet assay procedure.

After exposure to RF-EMR and LPS (40 μg/ml), cells were detached by 0.25% trypsin digestion, and samples were centrifuged for 10 min at 1,200 rpm. Using a straw to draw supernatant after centrifugation, we mixed the precooled 4% glutaraldehyde stationary liquid slowly, after 1- to 1.5-h incubation at 4°C; the samples were fixed, dehydrated, embedded, and sectioned. Ultrathin sections were observed under H-7500 transmission electron microscope (TEM).

We examined the expression of autophagy indicators in the above cell-exposure groups. Using an immunofluorescence method and CLSM (Nikon, Tokyo, Japan), we detected the distribution of LC3-II and Beclin1 in SGN. The same steps as described in the above immunofluorescence procedure were used with primary antibody LC3-II and Beclin1 (1:100, abcam, Cambridge, MA, USA). After the samples were incubated with the green and red secondary antibody (1:200) for 60 min, the slides were treated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Then, the samples were mounted and observed by CLSM.

Dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime Company, Shanghai, China) was applied to detected intracellular oxidant production. Eleven experimental or control groups were assigned to assay ROS: (1) control group, (2) LPS (40 μg/ml) group, (3) 2-W/kg (either sham-exposure or exposure), (4) 4 W/kg (either sham-exposure or exposure), (5) 2-W/kg combined with LPS (40 μg/ml) (either sham-exposure or exposure), (6) 4 W/kg combined with LPS (40 μg/ml) (either sham-exposure or exposure), and (7) H₂O₂ groups. Briefly, after exposed to LPS (40 μg/ml) and RF-EMR, the SGN were placed in the fluorescent probe then incubated with DCFH-DA (1:1,000) 1 ml at 37°C for 30 min; the control group was incubated with DMEM/F12 medium. Then, the SGN were washed three times with the medium. The amount of emitted fluorescence was correlated with the quality of ROS in the SGN. The fluorescence value was acquired with a fluorescence photometer.

All the results were expressed as means ± standard error of the mean (SEM). SPSS 17.0 was used to analyze the results. One-way ANOVA and two-tailed independent-sample t-test were used to evaluate the differences in activity among the different concentrations of LPS-treated groups and the control group at the same time, respectively. Based on the findings in previous studies, median values of DNA damage were selected to represent the amount of DNA damage in our findings. Therefore, comparisons of the averages of the median DNA damages for the different concentrations of LPS and radiation-exposed and sham-exposed control groups were compared using the two-tailed Student t-test, as described in previous studies [21,28]. Differences exhibiting P < 0.05 were considered to be statistically significant.