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Blue Light Differentially Modulates Cell Survival and Growth

¹ Department of Oral Rehabilitation, Medical College of Georgia School of Dentistry, Augusta, GA 30912-1260; and
² University of Geneva School of Dental Medicine, Geneva, Switzerland;

^* corresponding author, watahaj{at}mail.mcg.edu

	ABSTRACT

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Previous studies have reported that blue light (400–500 nm) inhibits cell mitochondrial activity. We investigated the hypothesis that cells with high energy consumption are most susceptible to blue-light-induced mitochondrial inhibition. We estimated cell energy consumption by population doubling time, and cell survival and growth by succinate dehydrogenase (SDH) activity. Six cell types were exposed to 5 or 60 J/cm² of blue light from quartz-tungsten-halogen (QTH), plasma-arc (PAC), or argon laser sources in monolayer culture. Post-light SDH activity correlated positively with population doubling time (R² = 0.91 for PAC, 0.76 for QTH, 0.68 for laser); SDH activity increased for cell types with the longest doubling times and was suppressed for cell types with shorter doubling times. Thus, light-induced exposure differentially affects SDH activity, cell survival, and growth, depending on cell energy consumption. Blue light may be useful as a therapeutic modulator of cell growth and survival.

KEY WORDS: visible light • in vitro • MTT • fibroblasts • keratinocytes

	INTRODUCTION

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Ultraviolet light (100–400 nm wavelengths) is known to cause aging and neoplasms of the skin (Black et al., 1997; Slominski and Pawelek, 1998), and cataract formation in the lens of the eye (Zigman, 2000). Similarly, infrared light (700–1050 nm) induces cell stress responses such as induction of heat-shock protein synthesis (Noda et al., 2002) and, ultimately, cell death by denaturation of proteins. However, the biological effects of visible light (400–700 nm) are not well-characterized and often are viewed as relatively innocuous. It is for this reason that high-intensity blue light (600–2500 mW/cm², 400–500 nm) is accepted as an activator of polymerization of dental composites (Caughman et al., 1995).

However, several studies suggest that blue light is not innocuous. Blue light causes oxidative stress in the retina, ultimately leading to age-related macular degeneration (Beatty et al., 2000). Blue light also inhibits mitosis (Gorgidze et al., 1998) and mitochondrial activity (Aggarwal et al., 1978), and damages DNA (Pflaum et al., 1998). Finally, blue light modulates stress-responsive transcription factors such as NFB (Krishnamoorthy et al., 1999).

Mitochondria are one logical mediator of the effects of blue light. Cytochromes absorb light between 400 and 500 nm (CRC, 1970; Hull and Foster, 2001), depending on the porphyrin ring and protein involved. Flavins, such as those present in flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN), also absorb blue light (Hockberger et al., 1999; Massey, 2000). Although flavins and cytochromes occur to some degree throughout the cell, mitochondria contain them in high numbers. Our previous work has shown that, in Balb/c mouse fibroblasts, irradiation with as little as 6 J/cm² of blue light (500 mW/cm² for 12 sec) from a dental composite curing unit significantly suppressed (> 90%) succinate dehydrogenase (SDH) activity, a mitochondrial enzyme (Wataha et al., 2003a). This suppression was dose- and wavelength-dependent, suggesting interactions with specific cellular molecules.

Cell division is an energy-intensive process, and because mitochondria provide the majority of energy for cell functions such as division, cells that divide more frequently depend most on mitochondrial function. Our previous observations, that blue light inhibits mitochondrial SDH activity (Wataha et al., 2002), suggest that cells that divide rapidly will be more sensitive to blue light exposure. This hypothesis is supported by reports that blue light inhibits mitosis and division in some types of cells (Callaghan et al., 1996; Gorgidze et al., 1998). If true, then blue light may have therapeutic applications for diseases such as cancer, where cells are dividing inappropriately. Light from dental curing units is well-characterized, and these curing lights would be a convenient source of light for such applications.

In the current study, we used cell population doubling time to estimate cell energy consumption and SDH activity to estimate cell survival and growth. We first correlated the inhibition of SDH activity by blue light (60 J/cm²) to the population doubling times of six types of cells of normal and tumor origin. We then showed differential cellular effects of lower doses of blue light (5 J/cm²) at discrete wavelengths. The results of the current study support the hypothesis that blue light can be used as a differential modulator of cell survival and growth, and that the mitochondria at least partly mediate these effects.

	MATERIALS & METHODS

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Cell Culture
We selected 6 cell types to provide a range of population doubling times, types of cells likely to be accessible to blue light in the oral cavity (fibroblasts and epithelial cells), and several known tumorigenic cells of oral and non-oral origin (OSC-2 and MCF-7, respectively). WI-38 (ATCC CCL75, diploid human lung fibroblasts) and MCF-7 (ATCC HTB, human epithelial breast carcinoma) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 100 units/mL of penicillin, and 100 µg/mL of streptomycin (all from Gibco BRL, Grand Island, NY, USA). Balb/c cells (ATCC CCL163, clone A31, Rockville, MD, USA; aneuploid mouse lung fibroblasts) were cultured with 3% NuSerum (Collaborative Biomedical, Bedford, MA, USA) instead of FBS. OSC-2 cells (a gift from Dr. Tokio Osaki, Nankoku, Japan; human oral squamous cell carcinoma keratinocytes) were cultured in a 50/50 (vol%) mixture of DMEM and F12 with 10% FBS and the antibiotics previously described. NHEK cells (Cambrex, East Rutherford, NJ, USA; human diploid foreskin keratinocytes) were cultured in commercial KGM-2 medium (Cambrex) between passages 2 and 5. HGF (human gingival fibroblasts) were cultured in DMEM with 5% FBS supplemented with glutamine (2 mmol/L) and 2.2 g/L of sodium bicarbonate, and were used between passages 2 and 10 after harvest from gingival tissues of patients during periodontal surgery (with patient informed consent, IRB approval, expedited procedure, from the Medical College of Georgia). We determined population doubling times of the cells by counting with a hemocytometer (n = 5) during subculture.

Light Sources
Cells were exposed to three light sources (Table), all used clinically as curing lights for dental composite restorations. Wavelength distributions and total radiant flux were measured by means of a laboratory-grade spectral radiometer (DAS 2001, Labsphere, Inc., Sutton, NH, USA). Two continuous-wavelength source lights (quartz-tungsten-halogen = QTH; plasma arc = PAC) were used. The QTH source emitted 94% of its spectral output between 400 and 500 nm, with total output defined in the 350- to 1050-nm range. The PAC source emitted 81% of its output between 400 and 500 nm, with 10–15% of its output between 360 and 400 nm. These spectral emissions are consistent with lights designed to activate camphorquinone, a common activator of composite polymerization with an absorption maximum at 460 nm, and to avoid output in the infrared wavelengths > 700 nm (to avoid heating the tooth or adjacent tissues).

Exposure of Cells to Light and Measurement of Cell Effects
Cells were exposed to the light sources as previously described (Wataha et al., 2003a). For the QTH and PAC sources, light was applied to cells continuously with a total dose of 60 J/cm² for 2 min or 30 sec, respectively. This dose of light is commonly used clinically. For the laser source, a continuous 10-second dose of 5 J/cm² was applied.

Cells were plated (n = 3 per condition) in 96-well format in 200 µL of medium at 12,500 cells/cm² and incubated for 24 hrs at 37°C and 5% CO₂. We isolated the wells to ensure that light exposure from other wells had no influence. Light was applied at the tops of the wells, 24 hrs after plating, with no tray cover, approximately 7.5 mm from the cell monolayer through 4 mm of medium. Pilot experiments showed that the attenuation of light through this distance and through the medium was approximately 15% of the light output. Control wells received no light but were in the same cell culture tray to ensure equivalent environmental changes as cell cultures were manipulated. All experiments were repeated at least once.

Mitochondrial function was estimated by the MTT assay (Pearse, 1972) customized for the assessment of mitochondrial succinate dehydrogenase (SDH) activity (Alley et al., 1988; Wataha et al., 1991). SDH activity was measured 2, 24, 48, and 72 hrs after light exposure, with the use of parallel cultures. We used transmission electron microscopy (TEM) of Balb/c fibroblasts to assess mitochondrial structure. Cells were exposed to 60 J/cm² of blue light, then processed for TEM with standard glutaraldehyde fixation, osmium staining, and embedding in epoxy resin.

SDH activity at each time interval was compared with no-light controls by ANOVA and Tukey’s multiple-comparison intervals ( = 0.05). For the laser source, SDH activity was compared among cell types 72 hrs after light exposure, with no-light controls defined for each cell type as 100% and comparison with ANOVA and Tukey tests. Correlation between population doubling time of the cells and SDH activity 72 hrs after exposure to light was estimated by a least-squares regression, with R² as an estimator of the variation accounted for by the linear model.

	RESULTS

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QTH and PAC sources altered SDH activity depending on cell type (Fig. 1). These sources suppressed SDH activity in Balb/c, MCF-7, and OSC-2 cells as early as 24 hrs after light exposure, and this effect persisted through 72 hrs. The PAC source generally suppressed SDH activity more than the QTH source. Light-treated MCF-7 recovered some SDH activity between 24 and 72 hrs. Compared with the Balb/c fibroblasts, MCF-7, and OSC-2 cells, the slower-growing HGF, WI-38, and NHEK cells reacted differently to blue light exposure. Exposure of HGF to the PAC light resulted in a 30% transient suppression of SDH activity at 24 hrs; however, this effect was reduced to 20% suppression by 72 hrs. Furthermore, SDH activity of light-exposed WI-38 (PAC) and NHEK (QTH) increased 10–20% over no-light controls at 72 hrs.

View larger version (34K): [in this window] [in a new window]   Figure 1. Blue light differentially altered survival and growth of cells. Succinate dehydrogenase (SDH) activity (estimating cell survival and growth, MTT method, expressed as optical density [OD] of solubilized formazan @ 560 nm) was measured at 24-hour intervals for 72 hrs after exposure to QTH (2 min, 60 J/cm2) and PAC (30 sec, 60 J/cm2) light sources (Table). Cells were fibroblastic (Balb/c 3T3-mouse lung, HGF-human gingival, WI-38-human lung) or epithelial (MCF-7-human breast carcinoma, OSC-2-human oral squamous cell carcinoma, NHEK-normal human foreskin) in origin. Cells were plated at 12,500 cells/cm2 24 hrs prior to light exposure at time zero. Both suppression (Balb/c, OSC-2, MCF-7) and stimulation (WI-38, NHEK) of cell growth were observed. Error bars indicate standard deviations, and asterisks (*) indicate SDH activity statistically different from no-light control within each time period (ANOVA, Tukey, = 0.05, n = 3).

View larger version (44K): [in this window] [in a new window]   Figure 2. Effect of blue light on cellular succinate dehydrogenase activity correlated with population doubling time of cells. Cells were fibroblasts (Balb/c 3T3-mouse lung, HGF-human gingival, WI-38-human lung) or epithelial (MCF-7-human breast carcinoma, OSC-2-human oral squamous cell carcinoma, NHEK-normal human foreskin). Correlation (least-squares method, linear model) of population doubling time of cells vs. succinate dehydrogenase (SDH) activity (MTT method) as a percentage of no-light controls, 72 hrs after exposure to either the plasma-arc curing (A, PAC, 2 min, 60 J/cm2), quartz-tungsten halogen (B, QTH, 10 sec, 5 J/cm2), or laser (C, Laser, 30 sec, 60 J/cm2) light sources (Table). In each case (A,B, and C), the slope of the fitted line was significantly different from zero (p < 0.05). The strongest correlation between the SDH effect and doubling time was with the PAC source (C). The laser induced significant (D, 40–50%, p < 0.05, ANOVA, Tukey, n = 3) stimulation of SDH activity of WI-38 and HGF at 72 hrs and some stimulation of NHEK (30%, not significant). Error bars in D indicate standard deviations.

Blue light from all sources significantly reduced the ability of Balb/c fibroblasts (the cells most sensitive to the blue light; Figs. 1, 2) to convert MTT to its formazan salt, indicating suppression of oxidative phosphorylation and mitochondrial function (Fig. 3). TEM micrographs of Balb/c fibroblasts showed distinct structural changes in mitochondria after blue-light exposure, including a loss of inner mitochondrial membrane structure and a dark staining of the outer mitochondrial membrane (Fig. 3, panels E, F).

View larger version (86K): [in this window] [in a new window]   Figure 3. Blue light suppressed mitochondrial activity in Balb/c 3T3 fibroblasts. Balb/c fibroblasts were chosen for further study because they were most sensitive to blue light (Figs. 1, 2). Photomicrographs (A, B, C, and D, 50x, bar = 500 µm) of cells (fixed with 4% formalin) 72 hrs post-exposure to blue light from QTH (B, 2 min, 60 J/cm2), PAC (C, 30 sec, 60 J/cm2), or Laser (D, 10 sec, 5 J/cm2) sources (Table). No-light controls are shown in A. Presence of the blue formazan dye is indicative of active mitochondrial succinate dehydrogenase (SDH). Cells in the no-light controls (A) show high levels of formazan, whereas light-exposed cells (B,C,D) show little or no formazan, indicating inactive SDH. TEM (E,F) of identically treated (fixed and processed as described in METHODS) cells 6 hrs post-QTH exposure show evidence of degenerating mitochondria (DM, in panel F) vs. normal mitochondria (M) in no-light controls in panel E. Light-exposed mitochondria in F show loss of inner mitochondrial membrane structure and a dark staining of the outer membrane. TEM micrographs courtesy of Dr. Franklin Tay, University of Hong Kong.

	DISCUSSION

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Variations in serum concentration and components in the culture media of the different cell types also are a possible cause of the differential cell responses observed in the current study (Figs. 1, 2). Serum concentrations ranged from less than 1% (NHEK) to 10% (OSC-2, WI-38, HGF, MCF-7), and exposure to blue light could have altered serum proteins, leading to differential cell responses. Although this possibility deserves further attention, altered serum proteins probably were not the primary mediator of the effects of blue light. For example, both WI-38 fibroblasts and OSC-2 carcinoma cells were cultured in 10% FBS, yet exhibited markedly different responses to light (Fig. 1). Furthermore, there is little theoretical basis for altered protein structure by blue light, because polypeptides, per se, do not absorb light of 400–500 nm, and temperature changes caused by the light were not sufficient to denature proteins (Wataha et al., 2003a).

Because SDH activity as measured in the current study is a composite of cell number, mitochondrial number, and mitochondrial activity, it is not possible to use the current results to relate the effects of light directly to cell division and number. However, our previous work with Balb/c fibroblasts indicates that, at least for this cell line, blue light at 5–60 J/cm² killed the cells (Wataha et al., 2003a). Furthermore, observation of the cells during the current experiments revealed rounding and loss of cells from the monolayers after exposure to the light for cell types with short population doubling times (Balb/c, OSC-2, MCF-7) and increases in cell density for slower-growing cultures (WI-38, NHEK, HGF).

The current work does not explain how blue light differentially affected cell survival and growth. One hypothesis is that blue light induces an oxidative stress via absorption by flavins and cytochromes that affects the balance of pro-survival or pro-apoptotic forces depending on the energy needs, gene expression, or other undetermined factors. Several reports have documented differential responses to oxidative stress (Aw, 1999), and blue light is known to induce reactive oxygen species when it is absorbed by flavins (Massey, 2000). Yamamoto et al.(2003) have recently reported a significant deficit in the ability of OSC-2 cells to process oxidative stress vs. NHEK, which would support this oxidative stress hypothesis. Other experiments are currently under way to evaluate the biochemical events and cell responses triggered by the absorption of blue light.

The most novel finding of the current work was the differential responses of cells to the light (Fig. 2). This effect could be exploited therapeutically in the treatment of neoplasm or to promote activity of normally slowly dividing cells during wound healing. However, any such use will require more knowledge about the basic mechanisms by which the light alters cell activity. Furthermore, studies to assess the attenuation of light through tissue or tissue fluids will be critical to the establishment of effective doses.

	ACKNOWLEDGMENTS

 
The authors acknowledge the support of the MCG Biocompatibility Program for this work, Dr. Tokio Osaki (Kochi Medical School, Japan) for his generous donation of the OSC-2 cells, and Dr. Franklin R. Tay (University of Hong Kong) for his help with the transmission electron microscopy.

Received December 16, 2002; Last revision October 3, 2003; Accepted November 4, 2003

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