Cellular phone exposure and hair follicle cell collection
All chemicals were purchased from Sigma Aldrich Chemical Company (St. Louis, MO, USA) unless stated otherwise. Twenty male human subjects between 20 and 30 years of age participated in this study. The mean age was 25 years. All subjects were pre-screened with a questionnaire to ensure that they were healthy and had no indicators for increased DNA damage (such as drug, tobacco use, and recent ionizing radiation exposure). The study was reviewed and approved by the University of Washington’s Institution Review Board (Human Subjects Division). All subjects went through the process of informed consent and signed consent forms prior to participation in the study.
A Samsung Galaxy Note 4 cellular phone (Samsung, Seoul, South Korea) with a FCC-certified head average SAR of 0.10 W/kg operating on an AT&T GSM (Global System for Mobile) network at 850 MHz was used in the study. N8 State Software App (protection application) was installed into the phone. The phone was first connected (by calling) to another phone in the lab and then it was activated by a speaker attached to the mouth piece that played the same section of an audiobook.mp3 file . Each subject was asked to hold the phone on one side of his head for 30 min as if he was carrying out a normal conversation. However, subjects were not asked to speak. The side of the head (left or right side) was determined by the subject’s normal habit of phone use. Immediately before the phone was switched on, each subject was asked to hold the phone and five to ten hairs were plucked, using a pair of hair forceps, from a location on the subject’s head close to the base of the phone antenna. The phone was then switched on, connected, and activated by feeding the audio signal to the mouth piece. Immediately after exposure, five to ten hairs were again plucked from the same location on the subject’s head. The protection application was disabled for the ‘exposure group’ (10 subjects) and enabled for the ‘protected group’ (10 subjects).
After the hairs were plucked, they were immediately immersed in a microfuge tube containing 750 µL of RPMI-1640 media with serum (Life Technologies, Grand Island, NY, USA). In order to process the pre- and post-exposure hairs at the same time under the same electrophoresis conditions, the microfuge tube containing the pre-exposure hairs was put on ice for 30 min to prevent any DNA damage or repair in hair follicular cells. We have previously shown that 30 min storage on ice does not produce any significant change in DNA damage levels in cells . Processing both samples from the same individual at the same time enhanced the reliability of the study. This allows samples to be handled identically during processing.
Using the forceps, hairs were transferred to a microfuge tube that contained 750 µL of collagenase (1 mg/mL) to dissociate the cells from hair follicles. They were then incubated for 15 min at 37°C with 5% CO2 in air and 100% humidity. During the incubation, the tube was vortexed at the 5 and 10 min time points for more efficient action of the collagenase.
Then, 750 µL of RPMI-1640 medium with 10% fetal bovine serum was added to the tubes to inactivate the collagenase. Hairs were then taken out using a pair of forceps. The microfuge tube was then centrifuged for 5 min at 5000x g (Sorvall Microspin, Model 245, Thermo Scientific, Waltham, MA, USA). The supernatant was discarded and the cell pellet was re-suspended in 10 µL of medium. DNA damage in hair root cells was assayed by the alkaline comet assay.
DNA strand breaks assessment in hair follicular cells
Approximately 200 to 1000 hair follicular cells, mostly epithelial (>95%) in origin, can be obtained from a single hair follicle. A small number of fibroblast like cells and endothelial cells may be present. Only less than 0.1 % of the cells are melanocytes. Comet assay for assessment of DNA single-strand breaks was performed on this mixed population of cells.
Microgels were prepared on custom-made slides (MGE Slides, Mac & Sons Specialty Glass, Titusville, FL, USA). These slides provide a clear, non-frosted area in the center (1×3 cm) for low background visualization of DNA and a frosted area around for firm attachment of agarose. The first layer of microgel was made by putting 100 mL of 0.5%, 1:3 high-resolution agarose (Amresco, Solon, OH, USA) in the center of a slide and covering by a 24 x 50 mm cover glass. After cooling the slide for 1 min on ice, the cover glass was removed and the microgel was air dried at room temperature. On top of the dried layer, the first wet microgel layer was made using 250 mL of 0.7%, 1:3 high-resolution agarose. Ten microliters of the hair follicular cell suspension were mixed with 50 mL of 0.7%, 1:3 high-resolution agarose. Fifty microliters of this mixture was layered onto the pre-coated slide to make a second layer of microgel. After removing the cover glass, another layer of 250 mL of 0.7% agarose was layered on top of the cell layer, and then covered with a cover glass. This thicker third layer helped to prevent escape of DNA during lysis and electrophoresis.
After removing the cover glass, slides were incubated for 1 h in a lysing solution (pre-warmed to 37°C) having 1.25 M NaCl, 0.01% sodium lauryl sarcosine, 50 mM tetra-sodium EDTA, 10 mM Tris, at pH 10 and containing fresh reduced glutathione (1 mg/mL) and proteinase K (0.5mg/mL) (Amresco, Solon, OH, USA). They were then placed on a horizontal electrophoretic unit (Ellard Instruments, Monroe, WA, USA) modified to allow electrical input from a power supply to both ends of a positive and negative electrode. The unit was filled with a liter of a solution containing 300 mM sodium hydroxide, 1 mM EDTA (ethylene diamine tetra acetic acid), and 0.2% DMSO. After 20 min of DNA unwinding and equilibrium of the microgel in the solution, electrophoresis (18 volts, 480 mA) and solution recirculation (100 ml/min) was started simultaneously for 20 min. Slides were then immersed in a neutralizing/DNA precipitating solution of CTAB (cetyl tri-methyl ammonium bromide) for 10 min and this step was repeated once more and the slides were immersed in a mixture of 75% ethanol and 25% Tris (tris(hydroxymethyl)aminomethane hydrochloride) for 10 min. This step was repeated twice more. Slides were then air-dried.
Slide Staining and analysis:
One hundred microliters of Yellow-Orange Yellow-Orange-1 (YOYO-1, Thermo-Fischer Scientific, Grand Island, NY, USA) stain were applied in two rows of 10 small, equally spaced droplets over the clear window area of a slide (to minimize variation in the intensity of stained DNA) and covered with a cover glass.
Images of the comets at 400× magnification were captured using a charge-coupled device (CCD) camera GW 525 EX (Genwac Inc., Orangeburg, New York, USA) attached to an epifluorescence microscope (DM LB Leica Microsystems GmbH, Wetzlar, Germany) with an excitation filter of 490 nm, a 500 nm dichroic filter, and an emission filter of 515 nm. One hundred hair follicle cells from each slide were analyzed using the VisComet image analysis software (Impuls Imaging GmbH, Türkheim, Germany). Comet extent, tail moment, and integrated intensity were analyzed and used as indices of DNA strand breaks. Comet extent is the distance from the leading edge to the trailing edge of the tail of the comet. The tail moment is the product of the tail length and the percent of nuclear DNA contained in the tail. Integrated intensity is a parameter used to measure changes in length, breadth, and intensity of nuclear DNA due to damage. These parameters have been used previously and are considered acceptable and reliable assessments of DNA damage. Only cells with intact nuclei were scored and highly damaged cells (comet extent greater than 625 pixels) were excluded by setting a strict maximum limit in the image analysis program. This ensured that apoptotic, necrotic, pre-necrotic, or pre-apoptotic cells were not included in the analysis.
Average of the scores from 100 cells from a subject was calculated for each parameter and used in data analysis. Statistical analysis was performed using the GraphPad Prism 6.03 (La Jolla, CA, USA). Changes in DNA damage before and after cell phone use were compared using the paired t-test. A difference at P < 0.05 was considered statistically significant.