2.1.

Cell Lineages and Reagents

Normal oral epithelial keratinocytes (NOK-SI) cell line³⁰ was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, $100\mathrm{U}/\mathrm{ml}$ penicillin, $100\mu \text{g}/\mathrm{ml}$ streptomycin, and $250\mathrm{ng}/\mathrm{ml}$ amphotericin B, as previously reported.³¹ Cells were maintained in a 5% ${\mathrm{CO}}_2$-humidified incubator at 37°C. NOK-SI cells were kindly provided by Dr. J. Silvio Gutkind (National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland).

2.2.

Laser Phototherapy

Three groups with different LPT parameters were established: Sham ($0\mathrm{J}/{\mathrm{cm}}^2$), $4\mathrm{J}/{\mathrm{cm}}^2$, and $20\mathrm{J}/{\mathrm{cm}}^2$ laser groups (Table 1). The selected parameters used herein are based on the previous studies in which low energy densities of irradiation result in better wound healing compared with high energy densities.^32–35 Sham group received identical treatment conditions but with the laser equipment switched off. To prevent cross-irradiation between samples, each culture condition was seeded in separated culture dishes or plates.

Table 1

Irradiation patterns of LPT.

The irradiations were performed using continuous wave indium–gallium–aluminum–phosphide (InGaAlP) diode laser with an output power of 40 mW, output density of $1\mathrm{mW}/{\mathrm{cm}}^2$, and a wavelength of 660 nm (Twin Laser, MM Optics, Sao Paulo, Brazil) in a punctual (spot size of $0.04{\mathrm{cm}}^2$) mode. Laser was applied perpendicularly and in contact with the tissue-culture plates. The energy densities (fluency) used were 4 and $20\mathrm{J}/{\mathrm{cm}}^2$ corresponding to 4 and 20 s of exposure time, respectively. Each well received three sessions of irradiations with 6-h intervals (0, 6 h, and 12 h). The output power of the equipment was tested using a power meter (Laser Check; MM Optics LTDA, Sao Paulo, Brazil).

Because the distance between the laser source and the surface of application is critical, the LPT was administered through the bottom of the optically clear plates. The irradiation occurred in partially dark conditions without the influence of other light sources.

2.3.

ROS Assay

ROS assay was performed after cells received three sessions of irradiations with 6-h intervals (0, 6 h, and 12 h). Intracellular levels of ROS were detected using chloromethyl CM-H2DCFDA (Molecular Probes/Life technologies, Grand Island, New York) and measured at a wavelength of 517 to 527 nm. ROS were detected after intracellular esterases removed the acetate groups upon cellular oxidation. Briefly, cells were resuspended in phosphate-buffered saline (PBS) containing CM-H2DCFDA and incubated for 30 min at room temperature. Negative control cells received vehicle only and baseline fluorescence intensity was determined using sham irradiation. Positive controls received hydrogen peroxide (100 μM). Grayscale images were captured separately after fluorescence excitation as well as after Hoechst 33342 staining to determine the total number of cells.

2.4.

Comet Assay

NOK-SI received three sessions of LPT using the $4\mathrm{J}/{\mathrm{cm}}^2$ protocol. Comet assay was performed immediately after the third LPT application at 0 min, 5 min, 30 min, and 24 h time points (recovery period). NOK-SI cells were embedded in 0.75% low-melting point agarose and allowed to solidify on glass slides. Cells were then placed in lysis buffer (2.5 M NaCl, 100 mM ethylenediamine tetra-acetic acid (EDTA), and 10 mM Tris; pH 10.0 to 10.5) containing fresh 1% (v/v) Triton X-100 and 10% (v/v) dimethyl sulfoxide (DMSO) for 1 h. Following the exposure to alkaline buffer (300 mM NaOH and 1 mM EDTA; $\mathrm{pH}>13$) for 20 min, DNA was electrophoresed using 25 V ($0.90\mathrm{V}/\mathrm{cm}$) and 300 mA. The slides were neutralized in 400 mM Tris (pH 7.5) and stained with Hoechst 33342 (Invitrogen). Cells were analyzed using the TriTek CometScore TM software (TriTek, Sumerduck, Virginia). NOK-SI cells treated with hydrogen peroxide (100 μM) served as positive controls. Three output parameters were measured, including the percentage of tail DNA, tail length, and tail moment. Tail moment was used for statistical analysis.^24,25

2.5.

Immunofluorescence

Cells were placed on glass coverslips in 12-well plates and submitted to three sessions of LPT using the $4\mathrm{J}/{\mathrm{cm}}^2$ protocol. At the end of the last LPT session, the immunofluorescences were performed at 0 min, 5 min, 30 min, and 24 h time points (recovery period). The cells were fixed with absolute methanol at $-20°\mathrm{C}$ for 5 min. Cells were blocked with 0.5% (v/v) Triton X-100 in PBS and 3% (w/v) bovine serum albumin and incubated with anti-phospho-Histone H2A.X (Ser139) (Millipore, Billerica, California), anti-BRCA1 (C-20) (Santa Cruz Biotechnology, Dallas, Texas) and anti-phospho-BRCA1 (Ser1524, Cell Signaling Technology, Danvers, Massachusetts) antibodies as indicated. Cells were then washed three times, incubated with FITC or TRITC-conjugated secondary antibodies, and stained with Hoechst 33342 for visualization of DNA content. Images were captured using a QImaging ExiAqua monochrome digital camera attached to a Nikon Eclipse 80i Microscope (Nikon, Melville, New York) and visualized with QCapturePro software, as previously described.³¹ Grayscale images were captured separately after fluorescence excitation using FITC_HYQ and TRITC_HYQ filters. The number of cells was quantified using grayscale images captured following Hoechst 33342, γ-H2AX, or BRCA1 staining.

2.6.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, California). Statistical analyses of the Comet assay, γ-H2AX, and BRCA1 stains were performed by one-way analysis of variance followed by Tukey’s multiple comparison tests. Asterisks denote statistical significance (*$p<0.05$, **$p<0.01$, ***$p<0.001$, and ns $p>0.05$).

3.1.

LPT Induces Intracellular Accumulation of ROS in Human Oral Epithelial Cells

Emerging clinical studies have revealed the benefits of using LPT for healing diseases and wounds.^6,36–38 Further dissection of the molecular signaling associated with laser therapy-induced accelerated healing revealed the involvement of signaling networks, including the activation and accumulation of intracellular ROS that was identified by our group.^8,12 Interestingly, although accumulation of ROS has long been associated with toxic buildup of byproducts derived from anaerobic respiration, emerging evidence suggests a role for physiological levels of ROS in signal transduction.^28,29,39,40 It remains unclear how LPT induces accumulation of ROS in oral epithelial cells and whether there are safety concerns with using LPT. To evaluate the effects of LPT on ROS accumulation, genomic instability, and DNA damage in oral epithelial cells, we exposed NOK-SI cells to 4 and $20\mathrm{J}/{\mathrm{cm}}^2$ irradiation doses of laser. Unexpectedly, we found that cells irradiated with $4\mathrm{J}/{\mathrm{cm}}^2$ generated higher levels of ROS (***$p<0.001$) compared to cells receiving $20\mathrm{J}/{\mathrm{cm}}^2$ (ns $p>0.05$) [Figs. 1(a) and 1(b)]. Because accumulation of ROS is associated with increased genomic instability, which may cause chromosome rearrangements, and accumulation of DNA breaks^41,42 (reviewed in Ref. 24), we examined whether LPT ($4\mathrm{J}/{\mathrm{cm}}^2$) induced increased DNA fragmentation (T0). We also monitored these cells for an additional 24 h (T5 min to 24 h) [Figs. 2(a) and 2(b)] using the Comet assay. LPT did not induce DNA fragmentation (NS $p>0.05$) even when used at optimal conditions that promote accumulation of ROS ($4\mathrm{J}/{\mathrm{cm}}^2$) (Fig. 1). Hydrogen peroxide was used as a positive control (***$p<0.001$). These findings suggest that LPT activates physiological levels of ROS in oral epithelial cells without interfering with genomic stability.

Fig. 1

Reactive oxygen species (ROS) levels in human oral keratinocytes receiving laser phototherapy (LPT). Representative immunofluorescence microphotographs of CM-H2DCFDA-positive oral epithelial cells. (a) Cells that received $4\mathrm{J}/{\mathrm{cm}}^2$ of laser energy density show accumulation of ROS compared to cells receiving $20\mathrm{J}/{\mathrm{cm}}^2$ of laser energy density or sham irradiation. (b) Quantification of the intracellular levels of ROS (CM-H2DCFDA) following administration of 4 or $20\mathrm{J}/{\mathrm{cm}}^2$ of laser. Administration of $4\mathrm{J}/{\mathrm{cm}}^2$ results in significant accumulation of ROS (***$p<0.001$) compared to administration of $20\mathrm{J}/{\mathrm{cm}}^2$ that is not statistically different from sham irradiation (NS $p>0.05$). ${\mathrm{H}}_2{\mathrm{O}}_2$ was used as a positive reaction for the experiment (*$p<0.05$). Scale bars represent 50 μm.

Fig. 2

LPT does not induce genomic instability. (a) Representative microphotographs of the alkaline comet assay depict deoxyribose nucleic acid (DNA) fragmentation in response to sham irradiation, hydrogen peroxide, and $4\mathrm{J}/{\mathrm{cm}}^2$ of laser irradiation in NOK-SI cells. The DNA damage recovery phase was established after LPT irradiation and followed for 24 h. Note that the undamaged NOK-SI cells following sham irradiation and $4\mathrm{J}/{\mathrm{cm}}^2$ (comet head only) and fragmentation of DNA in positive control cells (${\mathrm{H}}_2{\mathrm{O}}_2$—100 μM; tail formation of the comet). (b) Comet assay quantification shows DNA damage exclusively in cells that received ${\mathrm{H}}_2{\mathrm{O}}_2$ (***$p<0.001$). Note that the lack of DNA damage in NOK-SI cells that received $4\mathrm{J}/{\mathrm{cm}}^2$ of laser (no comet tail) (NS $p>0.05$) ($n=150\text{cell}/\text{condition}$; error bar: $\text{mean}\pm \mathrm{SEM}$).

3.2.

Low Doses of LPT do not Induce DNA Double-Strand Breaks in Normal Oral Epithelial Cells

Application of different types of laser irradiation results in the generation and accumulation of a wide variety of DNA damage in various tissues.^43,44 Because laser therapy involves low doses of irradiation, we investigated whether LPT induces DNA strand breaks. We used the γ-H2AX DNA double-strand break marker as a tool for identifying DNA breaks. The histone H2AX is involved in assembling the DNA damage response complex following genomic injury. γ-H2AX is phosphorylated at serine 139 by Ataxia Telangiectasia Mutated (ATM) in response to DNA double-strand breaks. Phosphorylation of γ-H2AX results in recruitment of several components of the DNA damage response machinery, including BRCA1, BRCA2, Rad51, Mre11, NBS1, FANCD2, and p53.^45,46 Furthermore, the continuous presence of phosphorylated γ-H2AX in the chromatin denotes continuous double-strand break repair.^45,46 To access the effect of LPT on genomic material in epithelial cells, we analyzed the number of phosphorylated γ-H2AX foci at different time points (0 min, 5 min, 30 min, and 24 h) [Fig. 3(a)]. Surprisingly, we found that LPT did not induce double-strand breaks at any time points, similar to results observed in the sham irradiation group (NS $p>0.005$) [Fig. 3(b)]. The hydrogen peroxide positive control cells showed greater accumulation of DNA double-strand break foci compared to sham irradiated cells (***$p<0.001$) [Fig. 3(a), arrow].

Fig. 3

LPT does not induce DNA strand breaks. (a) Microphotographs of representative examples of immunofluorescence staining for γ-H2AX at different time points in NOK-SI cells. (b) Graphic representations of time course quantification of γ-H2AX foci per cell following irradiation with $4\mathrm{J}/{\mathrm{cm}}^2$ of energy density. Note that the amount of γ-H2AX foci formation does not change following LPT and remains similar to basal levels observed in the sham irradiation control group (NS $p>0.05$). The positive control group (${\mathrm{H}}_2{\mathrm{O}}_2$) shows the significant accumulation of γ-H2AX foci (***$p<0.001$) ($n=50\text{cells}/\text{time}$ point; error bar: mean±SEM). Scale bars represent 25 μm.

3.3.

Low Energy of LPT does not Trigger DNA Damage Repair (DDR) Machinery

The maintenance of chromatin integrity requires constant repair of DNA damage that is mediated by several molecules, including the breast cancer type 1 susceptibility protein (BRCA1). The protein encoded by the BRCA1 tumor suppressor gene protects the genome by initiating cell cycle checkpoints and actively participating in DNA repair by interacting with RAD51 following DNA damage.⁴⁷ ATM-dependent phosphorylation of serine 1524 causes BRCA1 nuclear translocation.⁴⁸ Administration of the genotoxic hydrogen peroxide resulted in increased phosphorylation of BRCA1 at serine 1524 and nuclear translocation [Fig. 4(a)]. Additionally, hydrogen peroxide caused high levels of nuclear foci formation and colocalization of phospho-BRCA1 and phospho-γ-H2AX, as observed in Fig. 4(b) (bright foci). In agreement with our previous findings, irradiation of NOK-SI cells with $4\mathrm{J}/{\mathrm{cm}}^2$ did not trigger nuclear accumulation of BRCA1. LPT resulted in low levels of phospho-γ-H2AX, similar to results from the sham irradiated control group [Fig. 4(a)] but phospho-γ-H2AX failed to colocalize with phospho-BRCA1 [Fig. 4(b)]. LPT did not induce DNA damage, as revealed by the number of cells with nuclear-localized BRCA1 and the level of phosphorilated BRCA1 at serine 1524 phosphorylation (p-BRCA1) following irradiation (ns $p>0.05$) [Fig. 4(c)]. These findings suggest that LPT may be a safe therapeutic strategy for lesions and ulcers from the oral mucosa. Furthermore, our analyses that used two independent molecular markers for genomic integrity showed that LPT did not trigger accumulation of DNA double-strand breaks or activate DDR.

Fig. 4

DNA damage repair machinery is not activated by LPT. (a) Immunofluorescent staining localization of p-BRCA1 and γ-H2AX in NOK-SI cells that received $4\mathrm{J}/{\mathrm{cm}}^2$ at different time points (5 min to 24 h). Note that the nuclear accumulation of p-BRCA1 and γ-H2AX exclusively in the ${\mathrm{H}}_2{\mathrm{O}}_2$ positive control group. Similar to the sham irradiation group, cells that received $4\mathrm{J}/{\mathrm{cm}}^2$ do not activate DDR. Nuclei were stained with Hoechst 33342. (b) Double staining of p-BRCA1 and γ-H2AX show colocalization of both markers in foci. LPT does not increase the accumulation of p-BRCA1 and γ-H2AX above basal levels. Note that the two markers do not colocalize in the LPT group. (c) BRCA1 and phospho-BRCA1 quantification. LPT did not increase BRCA1 or phospho-BRCA1 levels compared to sham irradiation (NS $p>0.05$). Treatment with ${\mathrm{H}}_2{\mathrm{O}}_2$ induces the accumulation of BRCA1 and phospho-BRCA1 compared to sham irradiation (***$p<0.001$). Scale bars represent 25 μm.