DNA damage in wounded, hypoxic and acidotic human skin fibroblast cell cultures after low energy laser irradiation

Hawkins Evans D., Mbene A., Zungu I., Houreld N. and Abrahamse H.

Laser Research Group, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein, Johannesburg, South Africa, 2028, (Tel): +27 11 559-6406

(Fax): +27 11 559-6550, email: dhawkins@uj.ac.za

ABSTRACT

Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2. A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2, there was a significant increase in DNA damage in irradiated cells with and without the addition of FPG. These results are indicative of the importance of both cell injury model as well as fluence when assessing the effect of phototherapy on DNA integrity. Keywords: Helium-Neon (He-Ne), Comet assay, Formamidopyrimidine glycosylase (FPG), DNA repair

1. INTRODUCTION Laser therapy has gained wide acceptance to many medical disciplines. The side-effects from laser therapy involve the potential for interactions with cellular and extracellular matrix molecules to generate reactive oxygen species (ROS) and reactive nitrogen species which in turn can initiate lipid peroxidation, protein damage or DNA modification [1]. Larkö (2002) studied the undesirable effect of 600 nm laser light and reported that visible laser light can induce mutations in normal skin after repeated doses [2]. The effects of visible light and infrared radiation have not been clearly elucidated. Various biologic effects have been shown to be exerted by visible light irradiation including erythema, pigmentation, thermal damage and free radical production. It has been shown that visible light can induce indirect DNA damage through the generation of ROS [3]. Despite these concerns visible light is used for the treatment of a variety of skin diseases and esthetic conditions.

Mechanisms for Low-Light Therapy IV, edited by Michael R. Hamblin, Ronald W. Waynant, Juanita Anders,Proc. of SPIE Vol. 7165, 71650E · © 2009 SPIE

CCC code: 1605-7422/09/$18 · doi: 10.1117/12.809093

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The Comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in an organ, regardless of the extent of mitotic activity [4]. Sasaki (2000) stated that the responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by single DNA strand breaks and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts [4]. Olive (1999) defined the Comet assay as a single-cell gel electrophoresis method that can measure a variety of types of DNA damage, and repair of damage, in individual cells [5]. The assay is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells [6]. It is now in widespread use in genetic toxicology and oncology [5]. The Comet assay which detects DNA lesions is not suitable for identifying non-genotoxic carcinogens. Sasaki (2000) also stated that the Comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic compounds [4,7]. The principle of the assay is based on the ability of denatured, cleaved DNA fragments to migrate out of the cell nucleus under the influence of an electric field, whereas undamaged DNA migrates slower and remains within the confines of the nucleoid. The migration is visualized and scored using a fluorescent microscope after staining [7].

In 1999, an expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al., (1988) [8]. The pH > 13 version), is capable of detecting DNA single-strand breaks (SSB), alkaline-labile sites (ALS), DNA-DNA/DNA-protein cross-linking and SSB associated with incomplete excision repair sites [9]. Relative to other genotoxicity tests, the advantages of the single cell gel (SCG) assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, virtually any eukaryotic cell population can be used [9], its flexibility, its low cost, its ease of application, and the short time needed to complete a study. The expert panel also decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express alkaline-labile sites and single-stranded breaks, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization and data collection [9].

Alternative or related SCG methodologies that might be useful in the interpretation of positive Comet data include: (i) the use of different pH conditions during electrophoresis to discriminate between DNA strand breaks and alkaline-labile sites, (ii) the use or repair enzymes or antibodies to detect specific classes of DNA damage, (iii) the use of a neutral diffusion assay to identify apoptotic/necrotic cells and (iv) the use of acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA [9]. The specificity of the Comet assay can be enhanced to investigate specific types of DNA damage by adding DNA modifying enzymes. Broad classes of oxidative DNA damage can be detected as additional strand breaks if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III (ENDOIII) and FPG, respectively [7,10]. FPG, ENDOIII and 8-hydroxyguanine DNA glycosylase (hOGG1) recognize oxidative DNA damage and, in addition, FPG and ENDOIII also recognize alkylation damage [11]. FPG or (Mut M) is amongst the enzymes which have been successfully used in the Comet assay to investigate oxidative damage and detects cleaves oxidized bases. Alkylation-induced single strand breaks as detected by Comet assay should be regarded as an indicator of repair rate and balance more than a measure of actual DNA damage [12]. Many studies have used to SCG electrophoresis method or Comet assay to analyze DNA damage and repair. Fortini et al., (1996) reported that DNA single-strand breaks were completely repaired within 24 h after treatment [12].

Tice et al., (2000) proposed the possibility of identifying types of DNA damage specific for ionizing radiation in selected subtypes of cells [9]. While additional research is required before the SCG assay can readily be applied as a standard biomonitoring tool for exposure to ionizing radiation, the data collected thus far supports a conclusion that such research is clearly warranted [13]. Perhaps the same can be proposed for laser therapy where a specific tool or assay such as the Comet assay using human lymphocytes isolated from a blood sample will determine laser irradiation-induced DNA damage to avoid a situation where therapy may become harmful or detrimental.

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Low energy laser irradiation has been found beneficial in a wide variety of therapeutic applications [14]. However, some concerns have arisen about the possibility of DNA damage. Even when the overall integrity of a cell’s genome is seriously degraded, the damaged DNA can be repaired without detectable consequences although long-term mutational damage cannot be completely excluded [14]. This study aimed to determine the effect of He-Ne laser irradiation on DNA damage in human fibroblasts. The responses observed represent the effect of laser irradiation in combination with cell stress. Since there is a minimal effect when normal fully functional cells are irradiated, stress conditions were identified and linked to pathological conditions where laser therapy may be beneficial.

2. MATERIALS AND METHODS Human skin fibroblast (WS1) monolayer cultures (ATCC CRL1502) were grown to 90% confluence in Eagle’s minimal essential medium (EMEM) with Earle’s balanced salt solution was modified to contain 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acids, 1% fungizone and 1% penicillin-streptomycin and supplemented with 10% (v/v) foetal bovine serum (serum rich medium). The cultures were incubated at 37°C with 5% CO2 and 85% humidity [15]. Cells were trypsinized using a low concentration of 0.25% trypsin, 0.03% EDTA solution in Hanks balanced salt solution (HBSS) for 5 min at 37°C to minimize the amount of cellular damage [16]. Approximately 6 X 105 cells (in 3 ml culture medium containing phenol red) were seeded in 3.4 cm diameter culture plates and incubated overnight to allow the cells to attach [17]. The culture plates were randomly assigned to each treatment group of normal, wounded, acidotic or hypoxic and again into irradiated and non-irradiated for the specific dose (5 or 16 J/cm2). Cells were irradiated on day 1 and day 4 and the cellular responses were measured at 1 h and 24 h post-irradiation (after the final exposure on day 4). (i) Normal human skin fibroblasts: Normal fibroblasts, which are slender star-shaped cells that grow in an

adherent single monolayer, were used as control cells. The cells are normal, metabolically viable and fully functional. Cell cultures between passage 20-35 were used to avoid changes that may be associated with ageing cultures as the cells have a limited doubling potential of 67 population doublings.

(ii) Central scratch method (in vitro wound model): To perform a wound healing assay, a wound is typically

introduced in a cell monolayer according to Cha et al. (1996) using an object such as a pipette tip or syringe needle to create a cell-free zone with clear wound margins [18]. For the simulated wound environment, 1 ml of culture medium was removed and the confluent monolayer was scratched with a sterile pipette of 2 mm diameter and each plate was incubated at 37°C with 5% CO2 and 85% humidity for 30 min prior to laser irradiation (day 1). The central scratch has been used in many in vitro studies to successfully induce a wound [18,19,20,21].

(iii) Acidosis (low pH) model: Tsubio et al., (1989) adjusted liquid medium by adding hydrochloric acid (HCl) to

obtain a pH range of 3 - 7 to verify cell growth therefore the cell culture medium was adjusted by adding 1 N HCl acid (Associated Chemical Enterprises) until the pH was reduced to 6.7 [22]. Briefly, 20 μl of HCl was added to 20 ml of complete EMEM (pH 7.4) to obtain a pH of 6.7. The pH was measured using a Thermo Orion pH meter (LABOTEC U.S.A, model 410 A+). After an overnight incubation to allow the cells to attach, the complete EMEM was discarded and replaced with 3 ml acidic medium (pH 6.7). Changes in the cell morphology were initially used to determine the incubation time in acidotic medium and in subsequent experiments cells were incubated at 37°C with 5% CO2 and 85% humidity for 30 min prior to laser irradiation (day 1).

(iv) Hypoxia model: In vitro hypoxia was induced by incubating the tissue culture dishes in an anaerobic jar

(AnaeroPackTM System, Mitsubishi gas Chemical Co. Inc., Japan) with one anaerobic (hypoxic gas mixture 95% N2 and 5% O2) gas pack, which achieves anaerobiosis in 15 min. A methylene blue anaerobiosis indicator was used to monitor oxygen depletion. In this case, cells were being deprived of oxygen, which is important for cellular oxidation. After the overnight incubation to allow the cells to attach, cells were incubated in an anaerobic chamber for 4 h and 15 min. The cells were incubated at 37°C with 5% CO2 and 85% humidity for 30 min prior to laser irradiation (day 1). An incubation of 4 h was selected as further incubation in the anaerobic chamber results in irreversible damage to the cells [23].

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Cell morphology

Helium-Neon (632.8 nm

WoundedAcidotic (pH 6.7)Hypoxia (oxygen depletion)

Membraneintegrity

LDH (A490 )

Fibroblasts were exposed to either 5 or 16 J/cm2 on day 1 and day 4 while control cells received 0 J/cm2. Cell culture dishes containing 6 X 105 cells were placed under the laser beam and irradiated with the culture dish lid off at room temperature in the dark on a dark surface. Irradiations were performed with a Helium-Neon (He-Ne, 632.8 nm) laser (Table 1). The He-Ne laser beam was transmitted through a lens and then a scanning mirror to change the direction of the beam by 90° and direct it onto the culture dish. The beam was expanded and clipped to obtain a 3.4 cm diameter spot size (area 9.1 cm2), which was equal to the area of the cell culture dish used. As the culture dish was located some distance from the laser, the power output was reduced by approximately 21.3% and the duration of laser irradiation was compensated accordingly. The laser tip was expanded so that the spot size area was the same area as the culture dish. The entire dish was irradiated for a specific duration to deliver 5 or 16 J/cm2 accordingly (Table 1). The He-Ne (632.8 nm) beam was clipped so that a homogenous beam profile was obtained. Following laser irradiation the fibroblasts were trypsinized from the 3.4 cm culture dishes and the cell suspension (1 X 105 cells/100 μl) was used to measure changes in DNA damage (Comet assay), 1 h and 24 h post-irradiation. Fibroblast behaviour was observed using an inverted microscope (Olympus S.A. CKX41) and changes were recorded daily using an Olympus Camedia C3030 digital camera (Olympus, Center Valley, PA, USA). Table 1. Summary of laser parameters and cellular responses used.

Laser Helium-Neon Wavelength 632.8 nm Wave emission Continuous Percentage output loss 21.3% Power output 18.8 mW Power density 2.07 mW/cm2 Energy density 5 J/cm2 or 16 J/cm2 Spot size/area 3.4 cm/9.1 cm2 Irradiation condition Room temperature,

lid off, dark surface Duration:

5 J/cm2 16 J/cm2

40 min 15s 128 min 49s

Exposure 1 on day 1 and 4 Cellular response 1 h and 24 h

Cell morphology: The fibroblast behaviour was observed using an inverted microscope. The number and intensity of colony formation, the haptotaxis (direction or orientation) of the edge fibroblasts, the numbers of fibroblasts present in the centre of the scratch, chemotaxis-chemokinesis (migration) and degree of confluency were evaluated to determine the activity of fibroblasts [21,24,25]. Changes in cell number, lysis of cells and cellular debris were evaluated to determine the extent of cellular damage in hypoxic and acidotic cells. The morphology results presented in this study reflect changes observed 1 h post-irradiation (day 4). Comet assay for DNA damage and repair: DNA damage was assessed by measuring changes in DNA damage using the Comet assay, also known as single cell gel electrophoresis (SCGE). Cells are embedded in an electrophoresis gel; their nuclei are chemically perforated and subsequently an electric field is applied. DNA migrates towards the positive side of the electrophoresis chamber because it is negatively charged. In a given time, damaged (small) DNA fragments migrate long distances (10-20 µm), large molecules migrate a correspondingly shorter distance. Undamaged DNA molecules cannot leave the cell nucleus and after staining with 4’6 diamidine-2-phenylindol dihydrochloride (DAPI), (Scientific Group SA, 32804803) a fluorescent dye, it appears as a sphere or circle. When DNA is damaged, broken strands migrate out of the nucleus and after staining, such cells appear like a comet with a bright head and a tail whose length is a measure of the degree of damage (Figure 1) [26].

A cell suspension (2 X 104 cells/ml) was embedded in 1% low melting point agarose then lysed in lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris pH 10 and 1% Triton X-100) for 1 h at 4°C. Damaged DNA underwent alkaline unwinding in electrophoresis solution (0.3 M NaOH and 1 mM EDTA) for 40 min at 4°C in the

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electrophoresis tank. During electrophoresis at 300 mA for 30 min at 4°C, relaxed coils were pulled towards the anode forming the tail of a comet like image. Cells were then washed in three changes of neutralizing buffer (0.4 mM Tris, pH 7.5) for 5 min each. Slides were then allowed to dry over night and stained with 20 µl DAPI solution (1 µg/ml). The stained cells were viewed on a fluorescent microscope (Olympus BX41/BX51) at 400 X magnification. One hundred comets per gel were analysed at random. Scoring was done according to the five recognizable classes ranging from Class 0 (comets with a large, dense nucleus with no halo) to Class 4 (comets with a small nucleus surrounded by a wide halo of diffused DNA) which is the most damaged class (Figure 1). To quantify the damage, arbitrary units per gel were calculated by multiplying the number of cells in each class by the value of the class and the total number of arbitrary units was the sum of these products. Throughout the experiment, cell suspensions were placed on ice between centrifugations to minimize DNA degradation.

Modified Comet assay: The Comet assay without FPG (conventional) detects single strand breaks while the Comet assay with FPG (modified) detects and cleaves oxidized bases, thereby inducing additional strand breaks [26]. The specificity of the Comet assay can be enhanced to investigate specific types of DNA damage by adding DNA modifying enzymes. FPG (Mut M) is amongst the enzymes which have been successfully used in the Comet assay to investigate oxidative damage. After the lysis step, slides were rinsed in three changes of enzyme reaction buffer at 4°C for 5 min each (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8). Thereafter, one set of slides was incubated with 50 µl of FPG (Sigma-Aldrich S.A., F3174), diluted 1:3000 in enzyme reaction buffer at 37°C for 30 min in a humidified chamber to allow the enzyme to react with the DNA strands. The other set served as a control and was incubated with 50 µl enzyme reaction buffer only. Following the enzyme incubation step, the normal protocol for DNA unwinding, electrophoresis, neutralization and cell counting was followed [26].

Figure 1. Cell DNA migration pattern produced by the Comet assay as a result of strand breakage. Damaged DNA

is pulled towards the anode during electrophoresis - the more the damage the longer the tail. Classes of comets (0-4) stained with DAPI (1 µg/ml). Images were visualized under the Olympus BH2-RFCA Epifluorescent microscope. Class 0 does not have a tail; Class 1 has a short almost indistinguishable tail and a large head; Class 2 has a big head and short tail; Class 3 has a slightly bigger head than Class 4 but shorter tail, while Class 4 has the smallest head and the longest tail. Severe DNA damage is indicated by Class 4 (400 X magnification).

All experiments were performed six times and the biochemical assays were done in duplicate, the average calculated and used for statistical analysis (n=6). The mean, standard deviation and standard error was calculated and the results were analyzed using the Student t-test with SigmaPlot version 8.2 (SYSTAT software). Statistical significance was accepted at the 0.05 level (95%) confidence interval).

3. RESULTS AND DISCUSSION The effect of He-Ne laser irradiation using 5 J/cm2 or 16 J/cm2 on DNA damage was assessed. Changes in cell morphology were used to determine the effect of laser irradiation on cellular damage. Changes such as a decrease in cell number or an increase in the amount of lysis or debris were used as indicators of cellular damage.

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Cell morphology: Normal human skin fibroblasts showed typical cell morphology where cells were slender, elongated or star-shaped. The cells grew in a single monolayer attached to the surface of the culture plate. Normal irradiated (5 or 16 J/cm2) or non-irradiated (0 J/cm2) did not display any evidence of cellular damage as there were no dead or detached cells floating in the culture medium and there was no evidence of lysis, debris or fragmented cells (Figure 2).

Figure 2. Cell morphology of irradiated (5 or 16 J/cm2) and non-irradiated normal, wounded, acidotic and hypoxic WS1 fibroblasts. A confluent monolayer was scratched with a sterile 1 ml pipette to simulate a wound. Non-irradiated (0 J/cm2) cells were used as controls. The central scratch (CS) of cells irradiated with 16 J/cm2 and non-irradiated cells showed incomplete closure whereas cells irradiated with 5 J/cm2 showed complete closure and more haptotaxis. Non-irradiated cells showed colony formation (C) and migration (M) along the CS in an attempt to close the wound. Non-irradiated acidotic cells showed a decrease in cell number and increase in areas without cell growth however 5 J/cm2 appeared to stimulate cell growth. Hypoxic cells showed an increase in cellular damage with a decrease in cell number and an increase in non-viable cells that had rounded and detached from the culture plate. All three stress models maintained a large proportion of viable cells (200 X magnification).

According to Kornyei et al., (2000) a central scratch wounds approximately 5-10% of the cultured monolayer cells as the periphery of the culture plate still contains un-wounded cells [27]. Wounded cells demonstrated a clear wound margin on both sides of the central scratch. Non-irradiated wounded (0 J/cm2) cells demonstrated a clear wound

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margin with some evidence of haptotaxis but with few fibroblasts present in the central scratch (CS). Non-irradiated wounded cells showed a limited amount of colony formation (C) and migration (M) along the CS in an attempt to close the wound (Figure 2). Haptotaxis and cell migration was observed in all the non-irradiated wounded cultures but was more evident in cells irradiated with 5 J/cm2. Wounded cells irradiated with 5 J/cm2 showed complete wound closure. There was an increase in the number of fibroblasts in the central scratch of wounded cells irradiated with 5 J/cm2 when compared to non-irradiated (0 J/cm2) cells or cells irradiated with 16 J/cm2. Wounded cells irradiated with 16 J/cm2 did not show complete wound closure. There was no evidence of cellular damage in wounded cells as there were no dead or detached cells floating in the culture medium and there was no evidence of lysis, debris or fragmented cells. The morphology results indicate that laser irradiation with 5 J/cm2 has a stimulatory effect on wounded cells which resulted in complete wound closure whereas irradiation with 16 J/cm2 does not demonstrate the same stimulatory effect and as a result there was incomplete wound closure.

Since all the cells in the culture plate were subjected to the same acidic growth media, they were homogenously (100%) injured or stressed. The non-irradiated plates showed non-viable (round and opaque) cells still attached to the surface of the cell culture plate. Acidotic cells irradiated with 5 J/cm2 appeared more confluent than non-irradiated cells and also showed a decrease in the number of non-viable detached cells. A fluence of 16 J/cm2 did not improve the cell number or growth rate indicating that the fluence did not promote cell proliferation. There was a decrease in cell number as indicated by the increase in the areas without cell growth compared to cells irradiated with 5 J/cm2.

Since all the cells in the culture plate were subjected to anaerobic conditions (hypoxia), they were homogenously (100%) injured or stressed. Viable and non-viable cells were noted in hypoxic cultures however many of the non-viable cells had detached from the surface of the culture plate. The non-irradiated plates showed non-viable (round and opaque) cells still attached to the surface of the cell culture plate. Hypoxic cultures irradiated with 5 J/cm2 showed a decrease in cell number and a decrease in viability as non-viable cells had completely detached from culture plates. Cells irradiated with 16 J/cm2 showed a higher proportion of non-viable cells with an increase in the number of free floating or detached cells in the culture medium. Cultures irradiated with 16 J/cm2 did contain a number of viable attached cells despite the increase in the number of free floating or detached cells. Cell fragments and debris were also noted in the background and culture medium indicating an increase in cell lysis. Cells irradiated with 16 J/cm2 showed the highest proportion of non-viable cells when compared to normal cells and hypoxic cultures irradiated with 5 J/cm2. Hypoxic and acidotic injuries were assessed by changes in cell number, cell lysis and the number of cells detached from the culture plate. All three stress models maintained a large proportion of viable cells. The morphology results indicate that laser irradiation with 5 J/cm2 has a stimulatory effect on the viability of acidotic and hypoxic cells whereas irradiation with 16 J/cm2 contributed to additional cellular damage with a decrease in cell number and an increase in the number of non-viable free floating or detached cells.

Comet assay for DNA damage and repair: DNA damage was assessed using the Comet assay only on cells irradiated with 5 J/cm2 and the non-irradiated control (0 J/cm2). Studies have shown that high doses (10 and 16 J/cm2) are characterized by a decrease in cell viability and cell proliferation with a significant amount of damage to the cell membrane and DNA [28,29,30,31]. Damage caused by 16 J/cm2 appears to be irreversible so this study only focused on the effect of 5 J/cm2 to determine if laser irradiation reverses and repairs or contributes to causing additional DNA damage. When comparing non-irradiated cells at 1 h, there was significant DNA damage caused to the injured cells when (i) hypoxic cells were compared to normal cells (P<0.001), (ii) acidotic cells compared to normal cells (P<0.05), (iii) wounded cells compared to hypoxic cells (P<0.001), and (iv) hypoxic cells compared to acidotic cells (P<0.001). No significant damage was noted between normal and wounded cells (P=0.83).

When normal non-irradiated cells were compared to wounded non-irradiated cells there was no significant difference in DNA damage both at 1 and 24 h post-irradiation (P=0.83; P=0.61). When acidotic and hypoxic non-irradiated cells were compared to normal non-irradiated cells, the differences were significant (P<0.01) at both 1 and 24 h post-irradiation. Likewise when acidotic and hypoxic irradiated (5 J/cm2) cells were compared to normal irradiated cells,

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the differences were also significant (P<0.01). The results suggest that the acidotic and hypoxic models cause an increase in cellular damage with a considerable amount of DNA damage that was not observed in the wounded model. At 1 h post-irradiation there were insignificant differences when irradiated cells were compared to their non-irradiated control cells. (W and WI P=0.35; H and HI P=0.12; A and AI P=0.28). However at 24 h, the decrease in damage between acidotic irradiated cells and acidotic non-irradiated cells was significant (P=0.05). The results indicate the 5 J/cm2 stimulates the repair mechanisms to reverse or repair the DNA damage in acidotic cells. The effect of laser irradiation with 5 J/cm2 on repair mechanisms may be dependent on (i) the extent of the cellular damage, (ii) the extent of the DNA damage and (iii) the use of the correct laser fluence. If the extent of damage to the cells or DNA is too low (wounded model) repair mechanisms will not be activated whereas if the extent of damage is too high (hypoxic model) then repair mechanism may be damaged, inactivated or insufficient to repair. Results confirm that the effect of laser irradiation is dependent on the physiological status of the cells at the moment of irradiation and also on the correct use of laser parameter such as fluence, wavelength, power density and number of exposures. In the irradiated wounded, hypoxic, and acidotic cells the damage was reduced when compared to the non-irradiated cells both at 1 and 24 h post-irradiation. This suggested that 5 J/cm2 had either protective effect or enhanced repair mechanisms (Figure 3). The results show that there was a decrease in the DNA damage in wounded, acidotic and hypoxic irradiated and non-irradiated cells at 24 h post-irradiation. This suggests that repair mechanisms are active since many studies report that cells are capable of DNA repair between 4 – 24 h [12,32].

R e s p o n s e o f c e l l s t o i n j u r y a n d l a s e r i r r a d i a t i o n u s i n g 5 J / c m 2

Com

et a

ssay

for D

NA

dam

age

in a

bitra

ry u

nits

(AU

)

1 0

2 0

3 0

4 0

5 0

6 0

7 0

N o n - i r r a d i a t e dI r r a d i a t e d ( 5 J / c m 2 )

N W H A N W H A

N = N o r m a lW = W o u n d e dH = H y p o x i aA = A c i d o s i s

1 h 2 4 h

*

Figure 3. Comet assay was used to assess DNA damage. Normal and injured WS1 cells were irradiated using a He-Ne (632.8 nm) laser with 5 J/cm2 and the DNA damage was assessed at 1 h and 24 h post-irradiation. An increase in arbitrary units indicates an increase in DNA damage. Non-irradiated (0 J/cm2) cells were used as controls. In the irradiated wounded, hypoxic, and acidotic cells the DNA damage was reduced when compared to the non-irradiated cells both at 1 and 24 h post-irradiation. At 24 h, the decrease in damage between acidotic irradiated cells and acidotic non-irradiated cells was significant. (*P≤0.05; n=6)

Comet assay without FPG: The conventional Comet assay was used to detect single strand breaks. Normal cells irradiated with 5 or 16 J/cm2 and incubated for 1 h or 24 h post-irradiation showed an increase in DNA damage when compared to non-irradiated control (0 J/cm2) cells, however this difference was not statistically significant. At 1 h and 24 h post-irradiation, normal cells irradiated with 5 J/cm2 showed less DNA damage than normal cells irradiated

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with 16 J/cm2. Normal cells irradiated with 0, 5 or 16 J/cm2 did not show any significant change when incubation times of 1 h and 24 h post-irradiation were compared (P=0.68, P=0.34 and P=0.57 respectively). (Figure 4).

Wounded cells (1 and 24 h) irradiated with 5 J/cm2 showed no significant change compared to their respective non-irradiated wounded controls (P=0.33 and P=0.36 respectively), while cells irradiated with 16 J/cm2 showed a significant increase at both repair times (P<0.01). Wounded cells (1 h) irradiated with 5 or 16 J/cm2 showed a significant difference when compared to each other (P<0.01), with cells irradiated with 5 J/cm2 showing a decrease in DNA damage. Equally, the same observation was noted when wounded cells (24 h) irradiated with 5 or 16 J/cm2 were compared (P=0.02). Non-irradiated wounded cells incubated for 24 h showed a decrease in DNA damage when compared to the same cells incubated for 1 h (P=0.06). Wounded cells irradiated with 5 J/cm2 did not show any significant difference when 1 and 24 h incubation times were compared (P=0.11). Wounded cells irradiated with 16 J/cm2 showed a significant decrease in DNA damage at 24 h (P=0.04).

Non-irradiated wounded cells (1 h) showed an increase in DNA damage when compared to non-irradiated normal cells at 1 h post-irradiation, confirming that the central scratch method successfully stimulates a wounded environment with damage to the cells. Non-irradiated wounded cells (24 h) did not show an increase in DNA damage when compared to non-irradiated normal cells at 24 h post-irradiation, indicating active repair mechanisms that reduce the DNA damage within 24 h. Wounded cells (1 and 24 h) irradiated with 5 J/cm2 showed an increase in DNA damage when compared to normal cells (1 and 24 h) irradiated with the same fluence (P=0.19 and P=0.52 respectively). Wounded cells (1 h) irradiated with 16 J/cm2 showed a significant increase when compared to normal cells (1 h) irradiated with the same fluence (P<0.01), while the increase at 24 h was not significant (P=0.06).

Modified Comet assay with FPG: The modified Comet assay was used to detect and cleave oxidized bases in addition to single strand breaks. At 1 h and 24 h post-irradiation, normal cells irradiated with 5 J/cm2 showed an increase in DNA damage when compared to non-irradiated control cells while normal cells irradiated with 16 J/cm2 showed a decrease. At 1 h and 24 h post-irradiation, normal cells irradiated with 5 J/cm2 showed an increase in DNA damage when compared to normal cells irradiated with 16 J/cm2. Normal cells irradiated with 0, 5 or 16 J/cm2 did not show any significant change when incubation times of 1 h and 24 h post-irradiation were compared (P=0.06, P=0.09 and P=0.13 respectively) (Figure 4).

At 1 h post-irradiation, wounded cells irradiated with 5 J/cm2 showed a decrease in DNA damage when compared to non-irradiated wounded cells (P=0.32) while at 24 h post-irradiation wounded cells irradiated with 5 J/cm2 showed an increase in DNA damage when compared to non-irradiated wounded cells (P=0.35). At 1 h and 24 h post-irradiation, wounded cells irradiated with 16 J/cm2 showed a significant increase in DNA damage when compared to non-irradiated wounded cells (P<0.01). Wounded cells irradiated with 16 J/cm2 and incubated for 1 or 24 h post irradiation showed a significant increase in oxidized DNA damage compared to the same cells irradiated with 5 J/cm2 (P<0.01 and P=0.01 respectively). Wounded cells irradiated with 0 or 5 J/cm2 did not show any significant change when incubation times of 1 h and 24 h post-irradiation were compared (P=0.2 and P=0.12 respectively). Wounded cells irradiated with 16 J/cm2 and incubated for 24 h showed a significant decrease in DNA damage when compared to cells incubated for 1 h (P=0.02).

At 1 h and 24 h post-irradiation, non-irradiated wounded cells showed an increase in DNA damage when compared to normal non-irradiated cells. These results again confirm that the central scratch method successfully stimulates a wounded environment with damage to the cells. At 1 h post-irradiation, wounded cells irradiated with 5 J/cm2 showed a decrease in DNA damage when compared to normal cells irradiated with the same fluence. This result suggests that laser irradiation with 5 J/cm2 has a stimulatory or beneficial effect. At 1 h and 24 h post-irradiation, wounded cells irradiated with 16 J/cm2 showed a significant increase when compared to normal cells irradiated with the same fluence (P<0.01).

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N1 h

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ified

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et a

ssay

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rbitr

ary

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)

0

1 0 0

2 0 0

3 0 0

4 0 0

0 J / c m 2 ( n = 1 2 )5 J / c m 2 ( n = 6 )1 6 J / c m 2 ( n = 6 )

N2 4 h

W1 h

W 2 4 h

C o m e t w i t h o u t F P GC

omet assay w

ithout FPG in arbitrary units (A

U)

0

5 0

1 0 0

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0 J / c m 2 ( n = 1 2 )

5 J / c m 2 ( n = 6 )

1 6 J / c m 2 ( n = 6 )

C o m e t w i t h F P G

**

*

*

Figure 4. DNA damage was assessed at 1 or 24 h post-irradiation on day 4 using the Comet assay with and without FPG. Normal (N) and wounded (W) cells were irradiated with a He-Ne laser using 5 or 16 J/cm2 on day 1 and 4. An increase in arbitrary units indicates an increase in DNA damage. Non-irradiated cells (0 J/cm2) were used as controls. Normal cells irradiated with 5 or 16 J/cm2 showed no significant change in DNA damage at 1 and 24 h when compared to their respective controls. Wounded cells (1 and 24 h) irradiated with 16 J/cm2 showed a significant increase in damage with and without FPG compared to their respective controls. Wounded cells irradiated with 16 J/cm2 showed a significant increase in arbitrary units with FPG when incubation times at 1 h and 24 h were compared (P=0.02), and similarly without FPG (P=0.04). (*P≤0.05; n=6)

The modified (FPG) Comet assay detected more arbitrary units and was more sensitive than the conventional Comet assay. It can be explained that the modified Comet assay detected and cleaved oxidized bases in addition to single strand breaks, which the conventional Comet assay detected. Therefore, it could be said that the additional strand breaks could be attributed to irradiation with 16 J/cm2, however care must be taken in this interpretation since FPG is multifunctional; it is not specific to oxidized bases due to irradiation only. According to literature oxidized bases are caused by several factors, namely: ROS, laser irradiation and diseases just to mention a few [33]. ROS is also known to induce single strand breaks [34]. Molecular oxygen is consumed during respiration in mitochondria and thereby converted to ROS as byproducts. It is known that continued irradiation increases production of ROS possibly due to an increase in cellular metabolism and so increases the rate of their scavenging [35]. If there is dysfunction, there is increased ROS leakage and hence an increase in oxidative DNA damage. Therefore, it can be postulated that with a higher irradiation fluence of 16 J/cm2 more oxidized bases would be formed as this would disrupt the normal biochemical functions of cells, while irradiation with 5 J/cm2 would produce an insignificant number of oxidized bases as this fluence has been proved to be stimulatory and not DNA damaging. This rationale was proved by the Comet assay, where more damage was observed in wounded cells irradiated with 16 J/cm2 compared to wounded cells irradiated with 5 J/cm2. However, the degree of damage became less when cells were left to incubate for 24 h post-irradiation. It is apparent that DNA repair took place.

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4. CONCLUSION Wounded cells irradiated with a high fluence of 16 J/cm2 showed a decrease in cell migration, incomplete wound closure at both 1 and 24 h, and more DNA damage. This fluence appears to be too much to stimulate the behavior of cells. Karu (1988) observed that high fluences cause destruction of photoreceptors which is accompanied by growth inhibition and cell lethality [36]. Studies have demonstrated that irradiation with fluences higher than 10 J/cm2 results in DNA damage DNA [28,29,30,31]. The behavior of the two fluences used in this study can be explained well using the Arndt-Schultz law which states that small fluences (5 J/cm2) stimulate biological activity whereas higher fluences (16 J/cm2) are inhibitory or even detrimental to the biological activity of the cells [37]. The cell morphology results indicate that He-Ne laser irradiation with 5 J/cm2 stimulates the biological function of injured fibroblasts and does not cause additional cellular damage. Laser irradiation with 5 J/cm2 had a stimulatory effect on wounded cells which resulted in an increase in haptotaxis, migration and complete wound closure while it stimulated cell growth and reduced the number of non-viable cells in acidotic and hypoxic models. In wounded, acidotic and hypoxic models laser irradiation with 5 J/cm2 reduced the amount of DNA damage when compared to non-irradiated controls at 1 h and 24 h post-irradiation. At 24 h post-irradiation, the decrease in damage between acidotic irradiated cells and acidotic non-irradiated confirmed that 5 J/cm2 had a protective effect or enhanced repair mechanisms. The results confirm that the activation of DNA repair mechanisms since wounded, acidotic and hypoxic cells showed less DNA damage at 24 h when compared to the same cells at 1 h. The Comet assay with FPG (modified Comet assay) showed more DNA damage compared to the Comet assay without the enzyme (conventional Comet assay). It can be explained that the modified Comet assay detected and cleaved oxidized bases in addition to single strand breaks, which the conventional Comet assay detected, suggesting that the modified Comet assay is more sensitive than the conventional Comet assay. The Comet assay does not give the certainty that mutations, photoadducts or oxidative damage has not developed or occurred. This would have to be verified with other mutagenicity assays [32]. Generation of DNA damage is considered to be an important initial event in carcinogenesis [13]. Literature reports show that red light or phototherapy up or down regulates genes involved in DNA repair, but that DNA repair is vital to cells to avoid mutation [39]. Chan et al., (2007) performed an in vitro study to examine the effect of sub-lethal 755 nm laser irradiation on the expression of p16INK4a on melanoma cell lines and found that laser damage could increase DNA damage, which led to an increase in p16 expression [38]. Chan et al., (2007) also reported that reported that use of high-energy (585 nm pulsed dye laser or 1320 nm Nd:YAG laser) and intense pulsed light (IPL) source did not cause any toxicity in mice. A significant increase in two potential markers of DNA damage namely the expression of p16 and proliferating cell nuclear antigen (PCNA) imply that further long-term studies are necessary to consider the implications of repeated exposure to laser irradiation in human skin. If left un-repaired DNA damage may result in a mutation or a block in DNA replication. Future long-term studies may investigate the effect of repeated exposures of laser irradiation on DNA damage and cell proliferation, cell cycle regulation or apoptosis since if damage is extensive it may induce apoptosis. Finally, Greulich (2003) suggests that irradiation activates enzymes of the DNA repair machinery which immediately (within 24 h) repairs possible damages [14]. In addition, these results confirm that low energy laser irradiation indeed can cause beneficial effects. In conclusion, the Comet assay results reveal possible therapeutic effects of laser irradiation but do not indicate a risk or irreparable DNA damage that may lead to carcinogenesis.

REFERENCES [1] Kim, Y. “Laser mediated production of reactive oxygen and nitrogen species: implications for therapy,” Free

Radical Research 36(12), 1243-1250 (2002) [2] Larkö, O. “Normal doses of visible light can cause mutations in skin,” Lakartidningen 99(18), 2036-3037

(2002). [3] Mahmoud, B.H., Hexsel, C.L., Hamzavi, I.H. and Lim, H.W. “Effects of visible light on the skin,” Photochem

Photobiol 84(2), 450-462 (2008).

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[4] Sasaki, Y.F., Sekihash, K., Izumiyama, F., Nishidate, E., Saga, A., Ishida, K. and Tsuda, S. “The Comet assay with multiple mouse organs: comparison of Comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP carcinogenicity database,” Crit Rev Toxicol 30(6), 629-799 (2000).

[5] Olive, P.L. “DNA damage and repair in individual cells: applications of the Comet assay in radiobiology,” Int J Radiat Biol 75(4), 395-405 (1999).

[6] McKelvey-Martin, V.J., Green, M.H., Schmezer, P., Pool-Zobel, B.L., De Méo, M.P. and Collins, A. “The single cell gel electrophoresis assay (Comet assay): an European review,” Mutat Res 288(1), 47-63 (1993).

[7] Møller, P. “Genotoxicity of environmental agents assessed by the alkaline Comet assay,” Basic Clin Pharmacol Toxicol 96(1), 1-42 (2005).

[8] Singh, N.P., McCoy, M.T., Tice, R.R. and Schneider, E.L. “A simple technique for quantitation of low levels of DNA damage in individual cells,” Exp Cell Res 175(1), 184-191 (1988).

[9] Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C. and Sasaki, Y.F. “Single cell gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing,” Environ Mol Mutagen 35(3), 206-221 (2000).

[10] Speit, G., Schütz, P., Bonzheim, I., Trenz, K. and Hoffmann, H. “Sensitivity of the FPG protein towards alkylation damage in the Comet assay,” Toxicol Lett 146(2), 151-158 (2004).

[11] Smith, C.C., O’Donovan, M.R. and Martin, E.A. “hOGG1 recognizes oxidative damage using the Comet assay with greater specificity than FPG or ENDOIII,” Mutagenesis 21(3), 185-190 (2006).

[12] Fortini, P., Raspaglio, G., Falchi, M. and Dogliotti, E. “Analysis of DNA alkylation damage and repair in mammalian cells by the Comet assay,” Mutagenesis 11(2), 169-175 (1996).

[13] Møller, P. “The alkaline Comet assay: towards validation in biomonitoring of DNA damaging exposures,” Basic Clin Pharmacol Toxicol 98(4), 336-345 (2006).

[14] Greulich, K.O. “Low level laser therapy (LLLT) – Does it damage DNA?” Clinixperience Laser Partner 64, 1-3 (2003).

[15] Ausubel, R., Brent, R., Kingston, R.E., Moore, D.D., Seidan, J.G., Smith, J.A. and Struhl, K. [Short protocols in molecular cloning], 4th Edition. Wiley and Sons, Inc. New York and Brisbane.1-14, 1-39, 6-12, A3-20 (1994).

[16] Zungu, I., Hawkins Evans, D., Houreld, N. and Abrahamse, H. “Biological responses of injured human skin fibroblasts to assess the efficacy of in vitro models for cell stress studies,” African Journal of Biochemistry Research 1(4), 060-071 (2007)

[17] Sambrook. J., Fritsch, E.F. and Maniatis, T. [Molecular Cloning: A laboratory Manual], 2nd Edition. Cold Spring Laboratory, Cold Spring Harbour, New York (1989).

[18] Cha, D., O'Brien, P., O'Toole, E.A., Woodley, D.T. and Hudson, L. “Enhanced modulation of keratinocyte motility by TGF relative to EGF,” J Investig Dermatol 106, 590-597 (1996).

[19] Hawkins, D. and Abrahamse, H. “Biological effects of Helium-Neon (632.8 nm) laser irradiation on normal and wounded human skin fibroblasts,” Photomedicine and Laser Surgery 23(3), 251-259 (2005).

[20] Yarrow, J.C., Perlman, Z.E., Westwood, N.J. and Mitchison, T.J. “A high throughput cell migration assay using scratch wound healing, a comparison of imaged based readout methods,” Biomed Central Biotechnol 4, 21 (2004).

[21] Rigau, J., Sun, C., Trelles, M.A. and Berns, M. “Effects of the 633 nm laser on the behaviour and morphology of primary fibroblasts in culture.” In proceedings, Effects of low power light on biological systems, Barcelona, Spain, eds T. Karu and A. Young, Progress in Biomedical Optics, Barcelona Spain, 38-42 (1995).

[22] Tsubio, R., Matsuda, K., Ko, I.J. and Ogawa, H. “Correlation between culture medium pH, extracellular proteinase activity, and cell growth of Candida albicans in insoluble stratum corneum-supplemented media,” Arch Dermatol Res 281(5), 342-345 (1989).

[23] Yu, A.C.H., Lau, A.M.F., Fu, A.W.Y., Lau, L.T., Lam, P.Y., Chen, X.Q. and Xu, Z.Y. “Changes in ATP and ADP in cultured astrocytes under and after in vitro ischemia,” Neurochem Res 27(12), 1663-1668 (2002).

[24] Pinheiro, A.L.B., Nascimento, S.C., Vieira, A.L. Brugnera, A. Jr, Zanin, F.A., Barros, R.A. and Silva, P.S. “Effects of low level laser therapy on malignant cells: in vitro study.” J Clin Laser Med Surg 20, 23–26 (2002).

[25] McDermott, A.M., Kern, T.S. and Murphy, C.J. “The effect of elevated extracellular glucose on migration, adhesion and proliferation of SV40 transformed human corneal epithelial cells,” Curr Eye Res 17(9), 924–932 (1988).

[26] Collins, A.R. [Measurement of oxidative DNA damage using the Comet assay] In: Lunec J, Griffiths HR, eds. Measuring in vivo oxidative damage: a practical approach. John Wiley and Sons Ltd, UK, 83–94 (2000).

Proc. of SPIE Vol. 7165 71650E-12

[27] Kornyei, Z., Czorok, A., Vicsek, T. and Madarasz, E. “Proliferative and migratory responses of astrocytes to in vitro injury,” J Neurosci Res 61, 421-429 (2000)

[28] Hawkins, D. and Abrahamse, H. “Biological effects of Helium-Neon (632.8 nm) laser irradiation on normal and wounded human skin fibroblasts,” Photomedicine and Laser Surgery 23, 251–259 (2005).

[29] Hawkins, D. and Abrahamse, H. “The role of fluence in cell viability, proliferation and membrane integrity of wounded human skin fibroblasts following Helium-Neon laser irradiation,” Lasers in Surgery and Medicine 38, 74–83 (2006).

[30] Hawkins, D. and Abrahamse, H. “Time-dependant responses of wounded human skin fibroblasts following phototherapy,” Photochemistry Photobiology B: Biology 88, 147-155 (2007).

[31] Hawkins, D. and Abrahamse, H. “Efficacy of three different laser wavelengths for in vitro wound healing,” Photodermatology Photoimmunology and Photomedicine 24(4), 199-210 (2008).

[32] Rousset, N., Keminon, E., Eléouet, S., Le Néel, T., Auget, J.L., Vonarx, V., Carré, J., Lajat, Y. and Patrice, T. “Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT,” J Photochem Photobiol B 56(2-3), 119-131 (2000).

[33] Dale, J.W. and Park, S.F. [Mutation and variation. Molecular genetics of bacteria] 4th edition, John Wiley and Sons, England, 54-55 (2004).

[34] Takao, M. and Yasui, A. “DNA repair initiated by glycosylases in the nucleus and mitochondria of mammalian cells; how our cells respond to a flood of oxidative DNA damage,” Journal of Dermatological Science Supplement 1, 9-19 (2005).

[35] Lubart, R., Lavi, R., Friedman, H. and Rochkind, H. “Photochemistry and photobiology of light absorption by living cells,” Photomedicine and Laser Surgery 24(2), 179-185 (2006).

[36] Karu, T., [The Science of Low Power Laser Therapy], Gordon and Beach Science Publishers, Amsterdam, The Netherlands, (1998).

[37] Sommer, A.P., Pinheiro, A.L.B., Mester, A.R., Franke, R.P. and Whelan, H.T. “Biostimulatory windows in low intensity laser activation: Lasers, scanners, and NASA’s light emitting diode array system,” Journal of Clinical Medicine and Surgery 19(1), 29-33 (2001).

[38] Chan, H.H., Yang, C.H., Leung, J.C., Wei, W.I. and Lai, K.N. “An animal study of the effects on p16 and PCNA expression of repeated treatment with high-energy laser and intense pulsed light exposure,” Lasers Surg Med 39(1), 8-13 (2007).

[39] Zhang, Y., Song, S., Fong, C.C., Tsang, C.H., Yang, Z. and Yang, M. “cDNA microarray analysis of gene expression profiles in human fibroblast cells irradiated with red light,” Journal of Investigative Dermatology 120(5), 849-857 (2003).

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