Effect of 635 nm Light-Emitting Diode Irradiation on Intracellular Superoxide Anion Scavenging Independent of the Cellular Enzymatic Antioxidant System

Abstract

Objective: The aim of this study was to examine the reactive oxygen species (ROS) that are dissipated by 635 nm irradiation, and the effect of 635 nm irradiation on ROS scavenging system. Background data : Intracellular ROS are produced in the form of superoxide anion by either nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase in response to a number of stimuli. Low-level light irradiation decreases the intracellular ROS level and has been used in clinical situations for reducing the level of oxidative stress. Methods: Human epithelial cells were exposed to exogenous and endogenous oxidizing agents that promote the generation of harmful ROS. These were then irradiated with 635 nm LED light, 5 mW/cm² for 1 h, 18 J/cm² or by 470 nm LED light, also 5 mW/cm² for 1 h, 18 J/cm² on a 9 cm cell culture dish. After irradiation, the MTT reduction method and malondialdehyde (MDA) colorimetric assay were performed in xanthine/xanthine oxidase (XXO)- or hydrogen peroxide (H₂O₂)-treated HaCaT cells. The superoxide anion was detected by an electron spin resonance (ESR) spectrometer using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap and H₂O₂ was assayed by flow cytometry using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA). Results: Irradiation at 635 nm enhanced cell viability in the XXO-treated HaCaT cells. Also, irradiation had a much lesser effect on cell viability in the HaCaT cells treated with exogenous H₂O₂ as compared with that in cells treated with N-acetyl- L-cysteine. The level of the superoxide anion increased in response to XXO treatment, and then decreased after 635 nm irradiation. Irradiation with 635 nm led to a decrease in superoxide anion and lipid peroxidation levels in the presence or absence of diethyldithiocarbamate. Conclusions: These results highlight the potential role of 635 nm irradiation in protection against oxidative stress by scavenging superoxide anions. Also, a pathway that is independent of the activities of intracellular enzymatic ROS scavengers, such as superoxide dismutase, glutathione peroxidase and catalase might be involved in its mechanism of action.

Introduction

R eactive oxygen species (ROS) are involved in many physiological and pathological conditions including atherosclerosis, hypertension, heart failure, aging, and tissue/organ damage associated with ischemia-reperfusion injury. These potent oxidizers can cause direct damage to proteins, DNA, carbohydrates, and lipids.¹

The superoxide anion (•O₂ ^-), hydrogen peroxide (H₂O₂) and the hydroxyl radical (•OH-) are major ROS in human tissues. The superoxide anion is generated within the cells by several enzyme systems including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, nitric oxide synthase, and xanthine oxidase, as well as via the mitochondrial respiratory chain. The one-electron reduction of molecular oxygen produces a relatively stable intermediate, the superoxide anion, which serves as the precursor of most ROS. The mitochondrial electron transport chain contains several redox centers that can leak electrons to molecular oxygen, which serves as the primary source of superoxide production in most tissues.

Superoxide production through these biological systems is subject to intricate regulation by a variety of hormones, cytokines, and toxins. The level of ROS generated in cells is controlled by various intracellular enzymatic antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GP), and catalase (CAT).^2,3 SOD catalytically dismutes the superoxide anion to H₂O₂, whereas CAT and GP render H₂O₂ harmless within the cells by converting it into water and oxygen.⁴ GP utilizes glutathione (GSH) to destroy H₂O₂ and other lipid hydroperoxides. Therefore, these enzymes regulate the intracellular levels of ROS like H₂O₂ and superoxide anion. Animal cells produce several different superoxide scavenger enzymes located in different areas of the cell. Cu/ZnSOD accounts for 50–80% of the total SOD and is localized primarily in the cytosol and nucleus. MnSOD is expressed in smaller quantities in the cells, more abundantly in endothelial cells (ECs), and is localized to the mitochondria. Extracellular SOD is bound to the cell membrane through its heparin-binding domain and is located extracellularly.^5,6 Oxidative stress occurs when there is a disturbance in the oxidant–antioxidant balance in favor of the former. This imbalance can cause damage at the macromolecular level including DNA strand breakage, damage to membranes and membrane transport systems, oxidation of enzymes and other proteins, and lipid peroxidation.⁷

Recently, many clinical studies have reported that low-level light treatment (LLLT) at a wavelength of ∼635 nm can serve as an anti-inflammatory tool for accelerating wound healing,^8,9 and for pain reduction.^{10

–19} Many reports have suggested that scavenging of intracellular ROS by irradiation with visible light can be applied to improve a number of pathological states.^10,20,21 A specially manufactured visible light irradiation tool can be used in treatment of many degenerative diseases and to improve the electron transfer of cytochrome c oxidase in the mitochondria, as well as adenosine-5′-triphosphate (ATP) synthesis and cellular metabolism.^22,23 In a previous study, irradiation at 635 nm had an inhibitory effect on PGE₂ synthesis similar to that observed after treatment with cyclooxygenas (COX) inhibitors.¹⁰ However, unlike the mode of action of the usual nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and ibuprofen, this inhibitory effect was attributed to a decrease in the ROS levels. In another study, irradiation with 635 nm suppressed ONOO⁻ synthesis via the dissociation of ROS in SH-SY5Y neuroblastoma cells treated with SNP, an NO donor.²⁴ These phenomena were attributed to the relationship between 635 nm irradiation and scavenging of intracellular ROS.

However, the light-induced ROS scavenging mechanism is not yet completely understood. Especially, its relationship with intracellular enzymatic ROS scavengers is obscure. This study examined the type of ROS that can be scavenged by an 635 nm irradiation system in an in vitro model, and compared the result with that of an 470 nm irradiation system, which operated on a different photoacceptor. In addition, the independent or dependent action of intracellular enzymatic scavengers, such as SOD, GSH, and catalase (CAT) on ROS removal, was investigated with inhibitor studies in an immortalized oral keratinocyte cell line.

Materials and Methods

Cell culture

Immortalized human oral keratinocyte HaCaT cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, USA) supplemented with an antibiotic-antimycotic solution (Gibco BRL, USA) and 10% heat-inactivated fetal bovine serum (JR Scientific Inc., USA) in a humidified atmosphere of 5% CO₂/95% air at 37°C. The cells were cultured for 24 h at a density of 1×10⁵cells/mL in a 9 cm culture dish (Corning Inc., USA) or 96-well plates (Corning Inc., USA) according to the type of analysis to be performed.

Chemical treatments

All chemicals except for SOD (Calbiochem, USA) were purchased from Sigma (Sigma Aldrich, USA). 100 μM xanthine and a specified concentration of xanthine oxidase were added to the cell cultures for superoxide anion generation. A specified concentration of H₂O₂ was supplied exogenously directly to the cultured cells.²⁵ SOD or N-acetyl-L-cysteine (NAC) were added to the cultured cells simultaneously with xanthine/xanthine oxidase (XXO) or H₂O₂ in order to compare their effects with those of 635 nm irradiation. Specified concentrations of inhibitors of intracellular ROS scavengers (diethyldithiocarbamate [DDC], a SOD inhibitor, buthionine sulfoximine [BSO], a GSH inhibitor, and 3-amino-1,2,4-triazole [ATZ], a CAT inhibitor) were added.^26,27 At the conclusion of all chemical treatments, the cultured cells were washed three times with phosphate-buffered-saline.

Light source and irradiation

The cultured cell samples were irradiated for 1 h with a light dose of 5 mW/cm² and chemically treated simultaneously. A manufactured irradiation tool kit (Biophoton Co, Korea) was built in a 5% CO₂ humidified chamber held at 37°C, and irradiation was performed for the specified time. The light at a wavelength of 635 nm from a LED source (U-JIN LED, Korea) was used for irradiation, and the result was compared with that at a wavelength of 470 nm. The detailed information about light irradiation is represented in Table 1.²⁸

Table 1.

The Information of Light Irradiation

Parameter (unit)	635 nm	470 nm
Emitter type	LED	LED
Operating mode	Continuous wave	Continuous wave
Irradiation at target	5 mW/cm²	5 mW/cm²
Beam spot size at target (area)	63.6 cm²	63.6 cm²
Exposure duration	3600 sec (1h)	3600 sec (1h)
Radiant exposure	18 J/cm²	18 J/cm²

Cell viability

The cell viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, USA). The MTT assay is based on the ability of mitochondrial dehydrogenases in viable cells to reduce MTT to formazan through cell respiration. The medium was removed 24 h after the experimental treatments, and the cells were incubated in phosphate-buffered saline (PBS) containing 30 μL of MTT at 37°C for 3 h. The formazan product was solubilized by the addition of 50 μL of dimethyl sulfoxide (DMSO, Calbiochem, USA). The optical density was measured at 570 nm using an Enzyme-linked immunosorbent assay (ELISA) reader (ELx800uv, BIO Tek Instruments Inc., USA).

Electron spin resonance (ESR) spin trapping determination of superoxide anion (•O₂ ^-)

The superoxide anion was detected by an ESR spectrometer using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, Dojindo, Japan) as the spin trap.²⁹ Briefly, 1 h after chemical treatment or immediately after irradiation for 1 h, 1.5 μL of DMSO, 10 μL of sample (1×10⁵ cells in 0.1M phosphate buffer, pH 7.4) and 40 μL of 1M DMPO were placed in a test tube and mixed. The effects of the hydroxyl radical were eliminated by adding DMSO, which is used as a hydroxyl radical scavenger. The mixture was transferred to glass capillaries (50 μL, Paul Marienfeld GmbH & Co. KG, Germany) and the DMPO-O₂ ^- spin adduct was quantified. The measurement conditions for ESR (JEOL, Japan) were as follows: field sweep, 317–337 mT; field modulation frequency, 100 kHz; field modulation width, 0.4 mT; amplitude, 300; sweep time, 30 sec; time constant, 0.01 sec; microwave frequency, 9.166 GHz; and microwave power, 10 mW. The signal intensities were evaluated from the peak area of the first signal of the DMPO- O₂ ^- spin adducts.

Flow cytometric analysis for detection of H₂O₂ formation

H₂O₂ was assayed using 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA; Sigma, USA). DCF-DA enters the cells passively, where it is enzymatically deacetylated by esterases to become the nonfluorescent 2,7-dihydrodichlorofluorescein (DCF-H). H₂O₂ can oxidize DCF-H to a highly fluorescent DCF form.³⁰ For measuring the intracellular H₂O₂ level, the cells were detached 1 h after the chemical treatment or immediately after irradiation for 1 h using a trypsin-EDTA solution, and then incubated with 10 μM of DCF-DA for 20 min. After incubation, the H₂O₂ level was analyzed by flow cytometry (Beckman Coulter, USA) with excitation at 485 nm and emission at 530 nm.

Lipid peroxidation

At 24 h after treatment, the level of lipid peroxidation was measured using a malondialdehyde (MDA) colorimetric assay kit (Oxford Biomedical Research, USA). Oxygen radicals affect the double bonds of polyunsaturated fatty acids in the cell membrane resulting in the formation of highly reactive lipid hydroperoxides. MDA is one of the most studied lipid peroxidation products.³¹ The concentration of intracellular MDA was measured as an indicator of oxidative stress. A colorimetric assay reagent was applied to the cells according to the manufacturer's instructions, and the absorbance was measured at 586 nm using a colorimetric microplate reader (Bio Tek, USA).

Statistical analysis

All experiments were carried out performed in triplicate. The data are expressed as mean±standard deviation. In order to analyze the differences, the results were subjected to an analysis of variance (ANOVA) using SPSS for Windows Version 12.0 (SPSS, USA). A p value <0.05 was considered significant.

Results

The effect of antioxidants, inhibitors of intracellular antioxidants, and ROS generation on cell viability

To determine the concentrations of SOD and NAC, the cell viability was first measured in the XXO-treated HaCaT cell line. The cells were then treated with different doses of SOD (0, 100, 200, 400, and 800 U/mL, Fig. 1A) and NAC (0, 0.01, 0.1, 1, and 10 mM, Fig. 1B). Cell viabilities were expressed as the percentage of cell viability of untreated controls. And 200 U SOD and 0.1 mM NAC were used for further study.

FIG. 1.

Cell viability after treatment at indicated concentration of superoxide dismutase (SOD) (0, 100, 200, 400, 800 U/mL (A) and N-acetyl-L-cysteine (NAC) (0, 0.01, 0.1, 1, 10 mM (B). SOD (200 U) and NAC (0.1 mM) were chosen for further study. Cell viability after treatment with the inhibitors of intracellular reactive oxygen species (ROS) at indicated concentration (C). The vertical bars indicate the means±SD (n=3). *p<0.05 was considered significant.

To determine the effect of the inhibitors of intracellular antioxidants on cell viability, different concentrations (0, 10, 50, 100, 500 μM, 1, 10 mM) of DDC, BSO, and ATZ were added to the cultured cells and incubated for 24 h. Incubation with DDC, a known inhibitor of Cu/ZnSODs, led to a dose-dependent decrease in cell viability (Fig. 1C). However, BSO or ATZ did not individually cause a decrease in cell viability, not even at a concentration of 10 mM. However, simultaneous treatment with BSO and ATZ decreased cell viability in a dose-dependent manner. Therefore, 1 mM DDC and BSO+ATZ were used in further studies.

The addition of increasing amounts of XXO or H₂O₂ to the cultured cells led to a dose-dependent decrease in cell viability to <40% of the control and eventually to cell death (Fig. 2A and B). SOD (200 U) treatment and 635 nm irradiation prevented cell death. Especially at a concentration of 40 mU/mL XXO, 635 nm irradiation contributed to the recovery of cell viability to 80% of the control. On the other hand, 635 nm irradiation was assumed to be an ineffective tool for preventing loss of cell viability in the cells exposed to exogenous H₂O₂.

FIG. 2.

HaCaT cell viability after exposure to indicated concentration of xanthine/xanthine oxidase (XXO) (A), and H₂O₂ (B). Superoxide dismutase (SOD) (200 U), N-acetyl-L-cysteine (NAC) (0.1mM) treatment or irradiation at 635 or 470 nm was performed for 1 h simultaneously with the XXO or H₂O₂ treatment. At 1 h after treatment, the cells were washed and cultured for 24 h in FBS-free Dulbecco's modified Eagle's medium (DMEM), and the cell viability was analyzed. The vertical bars indicate the means±SD (n=3). *p<0.05 was considered significant.

Level of superoxide anion/H₂O₂ and inhibitory effect of lipid peroxidation

The superoxide anions were measured by ESR analysis in the XXO (40 U)-treated cells (Fig. 3). The ESR signal intensity (peak area) increased to 4662 (arbitrary unit) by XXO treatment, and then decreased to 2210 by 635 nm irradiation. The level of superoxide anion production was decreased to 2689 by SOD treatment. However, there was no change in the level of superoxide anion production in the DDC (1 mM)-treated cells. Irradiation at 635 nm caused a decrease in the level of superoxide anion production in the presence or absence of DDC. The peak area at 470 nm irradiation was always higher than that observed at 635 nm irradiation.

FIG. 3.

DMPO-O₂ ^- peak and first peak area of DMPO-O₂ ^- in a HaCaT cell line by electron spin resonance (ESR) spectroscopic measurement after a xanthine/xanthine oxidase (XXO) (40 mU/mL) treatment. Superoxide dismutase (SOD) (200 U), N-acetyl-L-cysteine (NAC) (0.1mM) treatment or 635 or 470 nm irradiation was performed for 1 h simultaneously with the XXO treatment. In addition, diethyldithiocarbamate (DDC) (1 mM) or buthionine sulfoximine and 3-amino-1,2,4-triazole (BSO+ATZ) (1 mM) were added together with XXO. The DMPO-O₂ ^- peak was measured 1h after initiating the treatment. The values indicate the means±SD (n=3).

In the cells treated with H₂O₂ (0.8 mM), the level of superoxide anion production was decreased by 635 nm irradiation in all the groups (Fig. 4). The addition of NAC, a GSH precursor, led to a slight decrease in the level of superoxide anion production to 3726, but it could not reduce the level of superoxide anion production in the BSO+ATZ (1 mM)-treated cells. The peak area at 470 nm irradiation was slightly decreased to 2801 when the cells were treated with DDC in the presence of, H₂O_2, however it was always higher than that observed at 635 nm irradiation.

FIG. 4.

DMPO-O₂ ^- peak and first peak area of DMPO-O₂ ^- by electron spin resonance (ESR) spectroscopic measurement on H₂O₂ (0.8 mM) induced in HaCaT cell line. Superoxide dismutase (SOD) (200 U), N-acetyl-L-cysteine (NAC) (0.1 mM) treatment or 635nm, 470nm irradiation was performed for 1 h simultaneous with H₂O₂. Diethyldithiocarbamate (DDC) (1mM) or buthionine sulfoximine and 3-amino-1,2,4-triazole (BSO+ATZ) (1 mM) were also added with H₂O₂ at the same time in each group. At 1 h after the treatment, the DMPO-O₂ ^- peak was measured. The values indicate the means±SD (n=3).

The concentration of intracellular H₂O₂ was decreased to 1.33 (arbitrary unit) by 635 nm irradiation or to 1.21 by 470 nm irradiation in the XXO-treated cells (Fig. 5). DDC treatment led to a slight decrease or increase in the intracellular H₂O₂ level. However, none of the treatments including SOD, NAC, or 635 or 470 nm irradiation decreased the H₂O₂ concentration in the BSO+ATZ (1 mM)-treated cells.

FIG. 5.

Flow cytometry analysis of 2′,7′-dichlorodihydrofluorescein (DCF) on xanthine/xanthine oxidase (XXO) (40 mU/mL) induced in the HaCaT cell line. Superoxide dismutase (SOD) (200 U), N-acetyl-L-cysteine (NAC) (0.1 mM) treatment or 635 or 470 nm irradiation was performed for 1 h simultaneous with XXO. Diethyldithiocarbamate (DDC) (1 mM) or buthionine sulfoximine and 3-amino-1,2,4-triazole (BSO+ATZ) (1 mM) were also added with XXO at the same time in each group. At 1 h after treatment, flow cytometry was performed. The value indicate the means±SD (n=3).

An exogenous treatment with H₂O₂ increased the intracellular H₂O₂ level to 11.14 (Fig. 6). NAC scavenged H₂O₂ to some extent. In the BSO+ATZ (1 mM)-treated cells, only 470 nm irradiation resulted in a decrease in the intracellular H₂O₂ level.

FIG. 6.

Flow cytometry analysis of 2′,7′-dichlorodihydrofluorescein (DCF) on H₂O₂ (0.8 mM) induced in the HaCaT cell line. Superoxide dismutase (SOD) (200U), N-acetyl-L-cysteine (NAC) (0.1 mM) treatment or 635 or 470 nm irradiation was performed for 1 h simultaneous with H₂O₂. Diethyldithiocarbamate (DDC) (1 mM) or buthionine sulfoximine and 3-amino-1,2,4-triazole (BSO+ATZ) (1 mM) were also added with H₂O₂ at the same time in each group. At 1 h after the treatment, flow cytometry was performed. The value indicate the means±SD (n=3).

ROS initiates the lipid peroxidation reactions that lead to loss of membrane integrity and cell death. Therefore, lipid peroxidation can represent oxidative stress caused by an imbalance between oxidants and antioxidants. MDA, a highly reactive bifunctional molecule, is an end product of membrane lipid peroxidation. The MDA level was increased by XXO treatment (Fig. 7). DDC treatment led to an increase in the MDA levels in the SOD- or NAC-treated groups, whereas the MDA levels in the 635 and 470 nm irradiated groups were reduced.

FIG. 7.

Lipid peroxidation assay by the malondialdehyde (MDA) level on xanthine/xanthine oxidase (XXO) (A, 40 mU/mL) or H₂O₂ (B, 0.8 mM) induced in the HaCaT cell line. A superoxide dismutase (SOD) (200 U), N-acetyl-L-cysteine (NAC) (0.1 mM) treatment or 635 or 470 nm irradiation was performed for 1 h simultaneous with H₂O₂. Diethyldithiocarbamate (DDC) (1 mM) or buthionine sulfoximine and 3-amino-1,2,4-triazole (BSO+ATZ) (1 mM) were also added with XXO or H₂O₂ at the same time in each group. At 1 h after treatment, the cells were washed and cultured for 24 h in FBS-free Dulbecco's modified Eagle's medium (DMEM), and the MDA level was then measured. The vertical bars indicate the means±SD (n=3).

Discussion

Human epithelial cells are exposed to exogenous and endogenous oxidizing agents that promote the generation of harmful ROS.^32,33 The intracellular concentration of ROS, in turn, is widely regarded as the critical factor in aging and in a number of degenerative diseases.¹

ROS are very short-lived molecules, and their production and removal is tightly regulated by enzymatic or nonenzymatic antioxidants. Oxidative stress can be defined as an increase in the reduction potential by the reactions described previoiusly, or as a decrease in the activities of the enzymes that prevent oxidative denaturation, including SOD, CAT, GP, GSH reductase (which regenerates GSH), and NADH-metHgb reductase.² An imbalance in these enzyme activities causes oxidative stress that can lead to cell death.⁷

In the present study, superoxide anion generation in vitro by XXO led to a dose-dependent decrease in cell viability/proliferation and cell death. The addition of SOD and intracellular superoxide anion scavenging led to an increase in cell viability in the XXO-treated HaCaT cells. SOD, a ubiquitous metal-containing enzyme, converts superoxide anion to H₂O₂ according to the reaction shown below:

Irradiation at 635 nm also led to an increase in cell viability in the XXO-treated cells in a manner similar to that observed in the SOD-treated cells. Therefore, 635 nm irradiation appears to act as a superoxide anion scavenger. The superoxide anion can be reduced during electron transitions, in which elevated electron are induced to drop from higher energy levels to lower energy levels by UV irradiation.^34
–36 However, the superoxide anion exhibits absorption at 245 nm and does not have absorption band in the visible range of the spectrum. This means that the dissociation or detachment of the superoxide anion depends upon the intracellular antioxidants including the enzymatic or nonenzymatic antioxidant systems.

In the present study, after adding the SOD inhibitor, DDC to the XXO-treated cells and measuring the resultant intracellular superoxide anion levels, it was observed that the effects of superoxide anion were decreased by 635 nm light irradiation independent of the intracellular enzymatic antioxidant SOD. As a result, the amount of superoxide anion was decreased by SOD treatment and SOD did not have any effect on the intracellular superoxide anion levels in the DDC (1 mM)-treated cells. Irradiation at 635 nm led to a decrease in the superoxide anion levels regardless of the presence or absence of DDC, which suggests that 635 nm irradiation removes the superoxide anions in a SOD-independent manner. The scavenging of superoxide anion by 635 nm irradiation led to a decrease in oxidative stress and lipid peroxidation in the presence or absence of DDC.

In the cells treated with H₂O₂, the level of superoxide anions was decreased by 635 nm irradiation. NAC treatment resulted in a slight decrease in the level of superoxide anions but it could not reduce the level of superoxide anions in the BSO+ATZ-treated cells (BSO, ATZ are the inhibitors of GSH and CAT, respectively). The concentration of intracellular H₂O₂ was decreased by 635 nm irradiation in the XXO-treated cells, and, consequently, the superoxide anion was scavenged by 635 nm irradiation, even though H₂O₂ could not be removed via the same pathway that was involved in superoxide anion scavenging. Treatment of cells with H₂O₂ also led to a dose-dependent decrease in cell viability. H₂O₂ is frequently used as the oxidative stimulus in order to examine the various effects of oxidative stress. Treatment with exogenous H₂O₂ led to a significant increase in the intracellular hydrogen peroxide levels. NAC treatment and the subsequent decrease in the intracellular H₂O₂ levels led to an increase in cell viability in the H₂O₂-treated cells. NAC is a GSH precursor, which is a H₂O₂ scavenger causing a decrease in the cytostatic and cytotoxic effects.¹⁸ The effect of H₂O₂ on the viability of cells was attributed to the finely regulated detoxification of intracellular H₂O₂: NAC can increase the cell viability. However, 635 nm irradiation did not alter cell viability compared with that by NAC. It means that 635 nm irradiation led to scavenging of superoxide anion rather than that of H₂O₂.

If that was the case, how could 635 nm irradiation reduce the oxidative stress independent of SOD? In general, the elevation of antioxidants was evident in various studies after LLLT. Application of LLLT 1 h after occlusion of the femoral artery to the gastrocnemius muscle still had a similar effect on the creatine-phosphokinase activity, as did application of a laser immediately after the onset of ischemia.³⁷ LLLT enhances the production of ATP in cells, preserves the mitochondria, alleviates the oxidative stress, and elevates ATP production in an ischemic heart.²² This phenomenon may, in turn, prevent the degeneration of muscle cells under severe hypoxic conditions in an ischemia-reperfusion injury. LLLT wavelengths that have been reported to have stimulating effects in biological systems include blue (400–500 nm), green (510–550 nm) yellow (560–590 nm), red (600–700 nm) and near-infrared (710–1100 nm).²² Different chromophores have been proposed to be operating as photoacceptors depending upon the wavelengths chosen and the precise biological system under investigation. Blue and green have been proposed to be absorbed by flavins and flavoproteins. Yellow and red light have been proposed to be absorbed by porphyrins. Red and near infrared light have been proposed to be absorbed by cytochrome c oxidase (unit four in the mitochondrial respiratory chain). The absorption spectra obtained for cytochrome c oxidase in different oxidation states were recorded and found to be very similar to the action spectra for the biological responses to light.^38,39 Cytochrome c oxidase is known to be major component of the respiratory chain for producing ATP. Increases in ATP are one of the most often observed changes in vitro after LLLT, and increased cytochrome c oxidase activity would explain the increased ATP levels.⁴⁰ Particularly in the stressed or hypoxic cells, nitric oxide (NO) produced in the mitochondria can inhibit respiration by binding to cytochrome c oxidase and competitively displacing oxygen.⁴¹ In a previous research, NO donor treatment in SH-SY5Y neuroblastoma cells led to ROS generation resulting in mitochondrial-dependent apoptotic progression. However, 635 nm irradiation suppressed ONOO⁻ synthesis via the dissociation of ROS, led to a decrease in the oxidative stress, and inhibited the apoptotic pathway.²⁴ This suggests that light absorption displaced the NO and allowed the cytochrome c oxidase to recover and cellular respiration to resume, which would explain the decrease in oxidative stress in many of the observations made in LLLT. The cells or tissues damaged as a result of stress or hypoxia are likely to respond more to LLLT than normal cells and tissues, and their cytochrome c oxidase is more likely to be operating at suboptimal level because of NO inhibition.

Conclusions

In conclusion, 635 nm LED irradiation can help remove the intracellular superoxide anion in SOD-inactivated HaCaT cells, thereby resulting in a decrease in lipid peroxidation, followed by an increase in cell viability. Therefore, 635 nm irradiation can help alleviate oxidative stress that is independent of the activities of the intracellular enzymatic antioxidants, such as SOD, GSH, and CAT.

Footnotes

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0007676).

Author Disclosure Statement

No competing financial interests exist.

References

Sato

, Sakuma

, Gutterman

D.D.

2003. Mechanism of dilation to reactive oxygen species in human coronary arterioles. Am. J. Physiol. Heart Circ. Physiol., 285:H2345–2354.

Genestra

2007. Oxyl radicals, redox-sensitive signalling cascades and antioxidants. Cell Signal., 19:1807–1819.

Babior

B.M.

2000. Phagocytes and oxidative stress. Am. J. Med., 109:33–44.

Gabbita

S.P.

, Robinson

K.A.

, Stewart

C.A.

, Floyd

R.A.

, Hensley

2000. Redox regulatory mechanisms of cellular signal transduction. Arch Biochem. Biophys., 376:1–13.

Mendez

J.I.

, Nicholson

W.J.

, Taylor

W.R.

2005. SOD isoforms and signaling in blood vessels: evidence for the importance of ROS compartmentalization. Arterioscler. Thromb. Vasc. Biol., 25:887–888.

Chaudiere

, Ferrari–Iliou

1999. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem. Toxicol., 37:949–962.

Cimen

M.Y.

2008. Free radical metabolism in human erythrocytes. Clin. Chim. Acta, 390:1–11.

Walker

M.D.

, Rumpf

, Baxter

G.D.

, Hirst

D.G.

, Lowe

A.S.

2000. Effect of low-intensity laser irradiation (660 nm) on a radiation-impaired wound-healing model in murine skin. Lasers Surg. Med., 26:41–47.

Whelan

H.T.

, Smits

R.L.

Jr. , Buchman

E.V.

et al. 2001. Effect of NASA light-emitting diode irradiation on wound healing. J. Clin. Laser Med. Surg., 19:305–314.

10.

Lim

, Lee

, Kim

et al. 2007. The anti-inflammatory mechanism of 635 nm light-emitting-diode irradiation compared with existing COX inhibitors. Lasers Surg. Med., 39:614–621.

11.

Garavello–Freitas

, Baranauskas

, Joazeiro

P.P.

, Padovani

C.R.

, Dal Pai–Silva

, da Cruz–Hofling

M.A.

2003. Low-power laser irradiation improves histomorphometrical parameters and bone matrix organization during tibia wound healing in rats. J. Photochem. Photobiol. B, 70:81–89.

12.

Al-Watban

F.A.

, Andres

B.L.

2001. The effect of He-Ne laser (632.8 nm) and Solcoseryl in vitro. Lasers Med. Sci., 16:267–275.

13.

Karu

T.I.

, Pyatibrat

L.V.

, Kalendo

G.S.

2001. Thiol reactive agents eliminate stimulation of cell attachment to extracellular matrices induced by irradiation at l ¼ 820 nm: Possible involvement of cellular redox status into low power laser effects. Laser Therapy, 11:177–187.

14.

Stelian

, Gil

, Habot

et al. 1992. Improvement of pain and disability in elderly patients with degenerative osteoarthritis of the knee treated with narrow-band light therapy. J. Am. Geriatr. Soc., 40:23–26.

15.

Gupta

AK.

, Filonenko

, Salansky

, Sauder

D. N.

1998. The use of low energy photon therapy (LEPT) in venous leg ulcers: a double-blind, placebo-controlled study. Dermatol. Surg., 24:1383–1386.

16.

Minatel

D.G.

, Frade

M.A.

, França

S.C.

, Enwemeka

C.S.

2009. Phototherapy promotes healing of chronic diabetic leg ulcers that failed to respond to other therapies. Lasers Surg. Med., 41:433–441.

17.

Schubert

2001. Effects of phototherapy on pressure ulcer healing in elderly patients after a falling trauma. A prospective, randomized, controlled study. Photodermatol. Photoimmunol. Photomed., 17:32–38.

18.

Corti

, Chiarion–Sileni

, Aversa

et al. 2006. Treatment of chemotherapy-induced oral mucositis with light-emitting diode. Photomed. Laser Surg., 24:207–213.

19.

DeLand

M.M.

, Weiss

R.A.

, McDaniel

D.H.

, Geronemus

R.G.

2007. Treatment of radiation-induced dermatitis with light-emitting diode (LED) photomodulation. Lasers Surg. Med., 39:164–168.

20.

Lim

W.B.

, Kim

J.S.

, Ko

Y.J.

et al. 2011. Effects of 635nm light-emitting diode irradiation on angiogenesis in CoCl(2) -exposed HUVECs. Lasers Surg. Med., 43:344–352.

21.

Lubart

, Lavi

, Friedmann

, Rochkind

2006. Photochemistry and photobiology of light absorption by living cells. Photomed. Laser Surg., 24:179–185.

22.

Karu

1999. Primary and secondary mechanisms of action of visible to near-IR radiation on cells, J. Photochem. Photobiol. B, 49:1–17.

23.

Wong–Riley

M.T.

, Liang

H.L.

, Eells

J.T.

et al. 2005. Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase. J. Biol. Chem., 280:4761–4771.

24.

Lim

, Kim

J.H.

, Gook

et al. 2009. Inhibition of mitochondria-dependent apoptosis by 635-nm irradiation in sodium nitroprusside-treated SH-SY5Y cells. Free Radic. Biol. Med., 47:850–857.

25.

Bai

X. C.

, Lu

, Liu

A. L.

et al. 2005. Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast. J. Biol. Chem., 280:17,497–17,506.

26.

Laurent

, Nicco

, Chereau

et al. 2005. Controlling tumor growth by modulating endogenous production of reactive oxygen species. Cancer Res., 65:948–956.

27.

Han

Y.H.

, Kim

S.Z.

, Kim

S.H.

, Park

W.H.

2008. Enhancement of arsenic trioxide-induced apoptosis in HeLa cells by diethyldithiocarbamate or buthionine sulfoximine. Int. J. Oncol., 33:205–213.

28.

Jenkins

P.A.

, Carrol

J.D.

2011. How to report low-level laser therapy (LLLT)/photomedicine dose and beam parameters in clinical and laboratory studies. Photomed. Laser Surg., 29:785–787.

29.

Sato

, Mokudai

, Niwano

, Mokudai

2011. Knetic anaylsis of reactive oxygen species generated by the in vitro reconstityted NADPH oxidase and xanthine oxidase systems. J. Biochem., 150:173–181.

30.

Zhang

, Kang

K.A.

, Piao

M.J.

, Maeng

et al. 2009. Cellular protection of morin against the oxidative stress induced by hydrogen peroxide. Chem. Biol. Interact., 177:21–27.

31.

Srour

M.A.

, Bilto

Y.Y.

, Juma

, Irhimeh

M.R.

2000. Exposure of human erythrocytes to oxygen radicals causes loss of deformability, increased osmotic fragility, lipid peroxidation and protein degradation. Clin. Hemorheol. Microcirc., 23:13–21.

32.

Kohen

1999. Skin antioxidants: their role in aging and in oxidative stress––new approaches for their evaluation. Biomed. Pharmacother., 53:181–192.

33.

Trouba

K.J.

, Hamadeh

H.K.

, Amin

R.P.

, Germolec

D.R.

2002. Oxidative stress and its role in skin disease. Antioxid. Redox. Signal., 4:665–673.

34.

Dinu

, Groenenboom

G.C.

, van der Zande

W.J.

2003. Competition between photodetachment and photodissociation in O. J. Chem. Phys., 119:8864.

35.

Masuhara

, Mataga

1981. Ionic photodissociation of electron donor–acceptor systems in solution. Acc. Chem. Res., 14:312–318.

36.

Creighton

J.A.

, Lippincott

E.R.

1964. Vibrational frequency and dissociation energy of the superoxide ion. J. Chem. Phys., 40:1779.

37.

Avni

, Levkovitz

, Maltz

, Oron

2005. Protection of skeletal muscles from ischemic injury: low-level laser therapy increases antioxidant activity. Photomed. Laser Surg., 23:273–277.

38.

Szundi

, Liao

G.L.

, Einarsdottir

2001. Near-infrared time-resolved optical absorption studies of the reaction of fully reduced cytochrome c oxidase with dioxygen. Biochemistry, 40:2332–2339.

39.

Karu

T.I.

, Pyatibrat

L.V.

, Kolyakov

S.F.

, Afanasyeva

N.I.

2005. Absorption measurements of a cell monolayer relevant to phototherapy: reduction of cytochrome c oxidase under near IR radiation. J. Photochem. Photobiol. B, 81:98–106.

40.

Hayworth

C.R.

, Rojas

J.C.

, Padilla

, Holmes

G.M.

, Sheridan

E.C.

, Gonzalez–Lima

2010. In vivo low-level light therapy increases cytochrome oxidase in skeletal muscle. Photochem. Photobiol., 86:673–680.

41.

Brown

G.C.

2001. Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase. Biochim. Biophys. Acta., 1504:46–57.

Effect of 635 nm Light-Emitting Diode Irradiation on Intracellular Superoxide Anion Scavenging Independent of the Cellular Enzymatic Antioxidant System

Abstract

Introduction

Materials and Methods

Cell culture

Chemical treatments

Light source and irradiation

Cell viability

Electron spin resonance (ESR) spin trapping determination of superoxide anion (•O2 -)

Flow cytometric analysis for detection of H2O2 formation

Lipid peroxidation

Statistical analysis

Results

The effect of antioxidants, inhibitors of intracellular antioxidants, and ROS generation on cell viability

Level of superoxide anion/H2O2 and inhibitory effect of lipid peroxidation

Discussion

Conclusions

Footnotes

Acknowledgments

Author Disclosure Statement

References

Electron spin resonance (ESR) spin trapping determination of superoxide anion (•O₂ ^-)

Flow cytometric analysis for detection of H₂O₂ formation

Level of superoxide anion/H₂O₂ and inhibitory effect of lipid peroxidation