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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 Correspondence: * corresponding author, watahaj{at}mail.mcg.edu
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
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 NF 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.
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
A laser light source was used to test the effects of discrete wavelengths between 400 and 500 nm, but at lower energies (Table  ). The source was an argon laser with emission wavelengths of approximately 456, 465, 475, 488, 495, and 503 nm, with the most intense emission at 488 nm (approximately 40 mW/nm). The laser source emitted 85% of its radiant flux between 400 and 500 nm.
Exposure of Cells to Light and Measurement of Cell Effects 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 (
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.
The population doubling times of the cells were (mean, standard deviation): Balb/c, 13 (1); OSC-2, 19 (2) hrs, MCG-7, 38 (4) hrs, HGF, 45 (5); NHEK, 70 (7); and WI-38, 79 (8) hrs. Positive correlations between population doubling times and SDH activities (72 hrs) were evident following treatment with all light sources (Fig. 2 ).
Exposure to the laser source (5 J/cm2) suppressed Balb/c fibroblast SDH activity to the same degree as the QTH and PAC sources (Fig. 2D ), but caused significantly less 72-hour SDH suppression in the MCF-7 and OSC-2 cells than did the PAC or QTH sources. The laser increased SDH activity of NHEK, WI-38, and HGF by 30%, 42%, and 53%, respectively, vs. no-light controls. The WI-38 and HGF SDH activity increases were significantly different (p < 0.05).
Blue light from all sources significantly reduced the ability of Balb/c fibroblasts (the cells most sensitive to the blue light; Figs. 1
Although the light sources used in the current study emitted no infrared radiation, blue light increases temperature in in vitro systems (Hannig and Bott, 1999; Wataha et al., 2003a). A role for temperature in the suppression or stimulation of cell mitochondrial activity observed in the current work is not known, but cannot be ruled out on the basis of the current results. Blue light at doses used in the current study does not activate heat-shock protein (HSP-72) in THP-1 monocytes (Wataha et al., 2003b). Furthermore, suppression of SDH activity of Balb/c fibroblasts (the cells most sensitive to blue light in the current study) was observed at only 5 J/cm2, when temperature rise was only 3°C for less than 60 sec (Wataha et al., 2002). Nevertheless, temperature rise and its possible synergism with the effects of light deserve further study.
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 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
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 for publication December 16, 2002. Revision received October 3, 2003. Accepted for publication November 4, 2003.
Journal of Dental Research, Vol. 83, No. 2,
104-108 (2004)
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ABSTRACT
INTRODUCTION
B (
= 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. 
