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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

Compact fluorescent light (CFL) bulbs can provide the same amount of lumens as incandescent light bulbs, using one quarter of the energy. Recently, CFL exposure was found to exacerbate existing skin conditions; however, the effects of CFL exposure on healthy skin tissue have not been thoroughly investigated. In this study, we studied the effects of exposure to CFL illumination on healthy human skin tissue cells (fibroblasts and keratinocytes). Cells exposed to CFLs exhibited a decrease in the proliferation rate, a significant increase in the production of reactive oxygen species, and a decrease in their ability to contract collagen. Measurements of UV emissions from these bulbs found significant levels of UVC and UVA (mercury [Hg] emission lines), which appeared to originate from cracks in the phosphor coatings, present in all bulbs studied. The response of the cells to the CFLs was consistent with damage from UV radiation, which was further enhanced when low dosages of TiO2 nanoparticles (NPs), normally used for UV absorption, were added prior to exposure. No effect on cells, with or without TiO2 NPs, was observed when they were exposed to incandescent light of the same intensity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

Compact fluorescent light (CFL) bulbs work on the principal of excitation of Hg vapors and production of the Hg emission lines that are used to excite the phosphor leading to light emission in the visible range. In addition to emission of visible light, the phosphor also serves as an absorber of the UV radiation from the Hg vapor. Information provided by different manufacturers shows the emissions spectra of “typical” bulbs, which are adjusted for different colors in the visible light, without any emission in the UV range. However, a recent study (1) performed a general survey of the emissions from commercially available bulbs and found significant amounts of UV A and C light. The major source of the UV light appears to be the physical defects in the bulbs where the phosphorus coating is not present (has chipped or cracked) on the glass surface. In contrast to the linear fluorescent bulbs, CFL bulbs contain narrow glass tubes where large stresses on the phosphor are introduced in the curved geometry. Optical examination of the bulbs reveals “bald” areas, in nearly all tubes we examined, regardless of manufacturer, indicating the absence of phosphorus at those regions. In the past two years, some disturbing reports have surfaced mostly in the European Union literature, which indicate that exposure to CFL bulbs might be responsible for exacerbating certain skin conditions, such as photodermatoses and skin cancer in humans (2,3). On the basis of this information, we wanted to probe further into this problem by directly measuring the UV emissions from CFL exposure and quantitating their effects on normal skin cells. As it is also well known that TiO2 nanoparticles are activated by UV light, we also studied the effect of the CFL on cells exposed to TiO2 to test whether damage due to the CFL emissions is enhanced.

Our results indicate that commercial CFL bulbs (chosen at random) emit UV radiation, which can induce damage to various types of skin cells. Two types of primary human skin cells, keratinocytes and dermal fibroblasts, were exposed to the CFL and damage was assayed using proliferation measurements, generation of reactive oxygen species (ROS) and visual examination via confocal microscopy. In each case, the damage was greater for fibroblasts, where we also observed a decrease in their ability to contract collagen or migrate; in living tissue, this would ultimately also affect the keratinocytes and may cause premature aging and impaired wound healing. In contrast to cells exposed to incandescent lighting, where no damage was observed with the or without addition of low doses of TiO2 nanoparticles, the damage due to exposure to the CFL was enhanced by the presence of TiO2 confirming the emissions of UV radiation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

Cell culture.  Primary human dermal fibroblasts (CF-29, National Institute on Aging [NIA] Bank) and keratinocytes DO33 (Living Skin Bank, Stony Brook University) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% of penicillinstreptomycin (PS) and 10% of fetal bovine serum (FBS; all purchased from Sigma). Medium containing TiO2 (15 mL, 0.1 mg mL−1 rutile or anatase) was added to each tissue culture dish 24 h after plating (initial cell concentration was 150 000 per 75 cm2 tissue culture dish). The samples were incubated with TiO2 for 24 h and exposed to CFL bulbs. Cells were placed at a distance of 2.5 cm away from the center of the bulb, in a cooled enclosure, which ensured that the temperature was maintained in the 25–37°C range. Samples were collected at specific time points (up to 4 days) and were counted or fixed, stained and imaged. All incubations were performed at 37°C and 5% CO2.

Tem.  Transmission electron microscopy (TEM) analysis was used to assess the size distribution of the TiO2 particles. To image them directly, we dispersed particles in ethanol and then spread a droplet of the solution on 300 mesh copper grip, which was coated with formvar film. The sample was then dried out at room temperature. Gaussian distributions of diameters were calculated from the samples with more than 170 nanoparticles. The samples were imaged using a FEI Tecnai12 BioTwinG2 transmission electron microscope (FEI Company, Hillsboro, OR, USA). Digital images were acquired with an AMT XR-60 CCD Digital Camera System (AMT Corp., Wobum, MA, USA).

Cell counting.  To determine the cell number during the growth curve experiments, cells were plated at an initial density of 150 000 cells per 75 cm2 tissue culture dish and manually counted using hemocytometer at the specific time points (at 0, 2 and 4 days after NPs were added) following exposure to the light. Each condition was performed in triplicates and all experiments were conducted three times. Cell suspensions were mixed for uniform distribution and were diluted enough so that the cells did not aggregate.

Proliferation/MTS assay.  Mitochondrial activity was evaluated with CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the standard procedure. In the typical experiment, cells were grown in the 35 mm2 Petri dish, treated with TiO2 and exposed to CFL as described elsewhere, after that cells were trypsinized, the enzyme reaction was stopped with media or soybean protein (with DO33 cells) and the 100 mL aliquot was placed in each well of the 96-wells dish. A 20 mL of the MTS solution was added, samples were incubated for 3 h at 37°C. The absorbance was read at 490 nm (BioTek EL800).

Migration.  Cell migration was evaluated using the agarose droplet assay. The 0.2% (wt/vol) agarose gel in DMEM was added to the cell pellets and the cells were resuspended to a concentration of 1.5 × 107 cells mL−1. A 1.25 μL were then loaded on the Petri dish. After that, the whole dish was placed at 4°C for 20 min to allow the agarose droplet to gel before the addition of 400 μL of DMEM into each well. Following 24 h incubation at 37°C, the cells were visualized using an Olympus optical microscope (Olympus Company, Center Valley, PA, USA). The cell migration distance was defined as the area of the outward cell migration minus the area of the agarose droplet.

Collagen gel contraction.  Cells were grown and exposed to 0.1 mg mL−1 TiO2 and CFL as described elsewhere, followed by trypsinization, counting and suspension in a prepared collagen solution. (1.8 mg mL−1 purified collagen, 2% BSA, 100 ng mL−1 PDGF in DMEM with PS/G) at a concentration of 3.5 × 105 cells mL−1. Cell/collagen gels were loaded into a BSA coated 24-well dish at 0.7 mL per well. After preincubation for 2 h to allow the mixture to gel, collagen gels were gently detached by slight tapping on the wells and 500 μL DMEM with 2% BSA and 100 ng mL−1 PDGF were added. Following 5 h incubation, images of gels were acquired and analyzed by measuring the gel size.

Cell staining for confocal microscopy.  Cell area and overall morphology were monitored using a Leica confocal microscope. After 48 h of exposure to CFL bulbs, cell were analyzed after an additional 2 and 4 days by being fixed with 3.7% formaldehyde for 15 min and followed by the addition of Alexa Fluor 488–Phalloidin (used for actin fiber staining) and Propidium Iodide (used for nuclei staining).

Cell aspect ratio and area calculation.  Using five typical confocal microscopy photographs, the cell aspect ratio and area of ca 100 cells were measured. The cell aspect ratio was determined as a ratio of the cell width to the cell length. Cell area was measured by the LEICA LITE software (Leica Microsystems, Buffalo Grove, IL, USA) after the cell edge was outlined manually.

Characterization of the TiO2 nanoparticles.  The TiO2 nanoparticles were purchased from US cosmetics. The crystalline structure of the particles was determined from X-ray diffraction, which confirmed that they were in the anatase and rutile form (Fig. S1, Supporting information). The diameter for each type of particle was determined from TEM microscopy and shown to be 134 ± 73 and 16 ± 3 nm for anatase and rutile, respectively (Fig. S2). The charge of the particles was determined from zeta-potential measurements (Table S1) and found to be −51.83 ± 1.40 and −35.01 ± 1.95 for anatase and rutile in DI water. When measured in cell culture medium it was −13.27 ± 2.07 and 13.13 ± 1.30 for anatase and rutile, respectively. Due to its larger band gap, anatase is known to be a stronger photosensitizer.

Cell exposure to CFL.  Cells were exposed in the CO2 independent media (Sigma) for 2 h at the 2.5 cm distance to the CFL bulb, in a cooled enclosure, which ensured that the temperature was maintained in the 25–37°C range, and 24 h after exposure to TiO2 nanoparticles. After the CFL exposure, media was replaced with fresh tissue culture medium. For double exposure cells were illuminated by CFL bulb second time 48 h after the first exposure.

ROS determination.  For ROS determination, 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was used (Invitrogen). In a typical experiment 50 000 cells were placed in each well of 96-well cell tissue culture dish in 50 mL media. After that, 100 μL of a working solution of CM-H2DCFDA were added to each well and incubated at 37 °C for 20 min. An additional 100 μL of 25 mm NaN3 solution was added to each well and cells were further incubated for 2 h. Fluorescence was determined with a Microplate reader at 490 nm excitation and 580 nm emission (BioTek EL800). All experimental conditions are outlined in Table 1.

Table 1.   Summary of experimental conditions.
ExposureTreatment with NPs
NoneRutile 0.1 mg mL−1Anatase 0.1 mg mL−1
  1. NPs, nanoparticles; CFL, compact fluorescent light; 0-0, no exposure, no NPs; SC-0, single CFL exposure, no NPs; DC-0, double CFL exposure, no NPs; SI, single incandescent exposure, no NPs; 0-R no exposure, rutile NPs; SC-R, single CFL exposure, rutile NPs; DC-R, double CFL exposure, rutile NPs; SI-R, single incandescent exposure, rutile NPs; 0-A, no exposure, anatase NPs; SC-A, single CFL exposure, anatase NPs; DC-A, double CFL exposure, anatase NPs; SI-A, single incandescent exposure, anatase NPs.

None0-0   0-R  0-A
SCSC-0 SC-RSC-A
DCDC-0DC-RDC-A
SISI-0  SI-R SI-A

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

In Table 2, we list the intensity of the various forms of UV (A and C) light emitted by the different commercially available, and randomly selected CFL bulbs. The light bulbs were placed in a typical desk lamp, and the emissions were measured in the same configuration at distances of 2.5, 7.5 and 35 cm. On the basis of these data, we chose Bulb no. 2 for further experiments as it had significant emission intensity in all three UV bands.

Table 2.   UV-light emission by CFL bulbs.
No.Power (W)2.5 cm7.5 cm35 cm
UVA μW cm−2UVC μW cm−2UVA μW cm−2UVC μW cm−2UVA μW cm−2UVC μW cm−2
1264661.42520.521.80.072
2265527.63102.2823.40.31
323471.4270.334.80.056
451011.117.240.0691.60.01
5113263.6455.30.175.90.03
611410.224.80.742.360.02
7261040.4830.80.0853.60.007
815640.22230.186.50.02
9131162640.410.10.05

In Table 3 we list threshold limit values (TLVs) for UVA and UVC that should not be exceeded within an 8 h period. The CFL emission in the UV spectra is due to the Hg excitation bands, which are 365, 253 and 184 nm and correlate to the UVA and UVC bands. The exposure time to CFL before the TLV is reached is a function of distance and frequency. The values for UVA and the two UVC wavelengths for the radiation emitted from Bulb no. 2 are listed in Table 3, from which we can see that even at a typical working distance of 35 cm, the TLV is reached in ca <6 h, which is at least 30% less than the recommended time for the exposure at workplace (4).

Table 3.   TVLs for the UV emission from the CFL no. 2.
 Wavelength rangeHg excitation bands (nm)TLV (mJ cm−2)Time for no. 2 26 W CFL to reach TLV
2.5 cm7.5 cm35 cm
  1. CFL, compact fluorescent light; TVLs, threshold limit values.

UVA400 nm–315 nm365100030 min54 min11.9 h
UVC280 nm–100 nm18410022 min  12 h  89 h
253  6  79 s44 min 5.4 h

To determine the influence on cell viability, we first examined the effects of exposure to CFL illumination on the mitochondrial activity of dermal fibroblasts and keratinocytes. The results are shown in Fig. 1, where exposure to CFL for 2 h results in a significant (P < 0.005) reduction of mitochondrial activity, 25% reduction for keratinocytes and 40% for dermal fibroblasts. When 0.1 mg mL−1 of photosensitizing TiO2 nanoparticles are added, no significant difference is observed in the unexposed samples (0-R, 0-A for all abbreviations see Table 1) at this concentration. Upon exposure, the decrease for cultures containing the rutile form (SC-R) is similar to that observed with cultures without NPs (SC-0), whereas that for anatase (SC-A) results in a decrease of 50% for the keratinocytes and 60% for the dermal fibroblasts.

image

Figure 1.  MTS analysis results of the keratinocytes DO33 and dermal fibroblasts CF-29 with and without TiO2 exposed to CFL and control. (a) DO33 2 days after first exposure, (b) DO33 4 days after first exposure, (c) CF-29 2 days after first, (d) CF-29 4 days after first exposure.

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The cultures were then exposed for an additional 2 h and the MTS activity measured after another 2 days (Fig. 1). The additional exposure did not result in further reduction of the MTS activity for keratinocytes or dermal fibroblasts (DC-0). On the other hand, the second exposure resulted in a drastic decrease in MTS activity for the culture exposed to rutile (DC-R). The MTS activity of all cultures, 4 days after the initial exposure is also shown for comparison (Fig. 1).

No significant decrease in MTS activity was observed for the unexposed keratinocytes DO33 and fibroblasts CF-29 on Day 2 (Fig. 1a,c) and 4 following exposure (0-0; Fig. 1b,d). After exposure to CFL at Day 2, there was a reduction of ca 20% for both the DO33 control (SC-0) and the sample exposed to rutile (SC-R) and ca 45% in sample exposed to anatase (SC-A; Fig. 1a). For the dermal fibroblasts CF-29 exposed once to the CFL, a reduction of ca 36%, 40% and 63% at Day 2 for the control (SC-0) and the exposed sample to rutile (SC-R) and anatase (SC-A), respectively, was obtained (Fig. 1c).

In Fig. 1b,d, we show the results taken 4 days after the initial exposure for keratinocytes and dermal fibroblasts, respectively. No additional damage occurred relative to the control samples (0-0) for either cell type, regardless of NP exposure. The situation was very different at Day 4 when the cells were subjected to a second exposure to CFL, 2 days after the initial one. The second illumination (DC-0) did not significantly increase the damage compared to the control unirradiated sample (0-0). A different response is observed for both CF-29 and DO-33 cultures treated with both rutile (DC-R) and anatase (DC-A) where more than 70% of the cells were killed after the second illumination (Fig. 1d).

To directly observe the impact of exposure of the cells to the CFL, we also measured cell proliferation and compared the results to the MTS data, as was previously reported that mitochondrial activity alone may not be an accurate measure as it can be enhanced by UV light-induced DNA damage (5,6). The data obtained from counting the cells is shown in Fig. 2.

image

Figure 2.  Proliferation of keratinocytes DO33 and dermal fibroblasts CF-29 with and without TiO2 exposed to CFL. (a) DO33 samples at Day 0 (24 h after exposing samples to the TiO2 rutile and anatase) and 2 days after the first exposure to CFL, (b) DO33 4 days after the first exposure, (c) CF-29 Day 0 is 24 h after exposing samples to the TiO2 rutile and anatase, 2 days after first exposure to CFL, (d) CF-29 4 days after first exposure to CFL.

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At Day 0 and 2, the control cells (0-0) and those that were exposed to rutile (0-R) and anatase (0-A) for 24 h, but not exposed to CFL, had the same number of cells, indicating that the TiO2 particles had no effect on cell proliferation (Fig. 2a,c). In contrast, at Day 2, the DO33 cells that were exposed to CFL showed a significant decrease of ca 20%, 17% and 45%, relative to the unilluminated control (0-0; Fig. 2a) for the samples without particles (SC-0), and those containing rutile (SC-R) and anatase (SC-A), respectively. A much larger decrease of ca 45%, 49% and 88%, was observed for CF-29 samples without NPs (SC-0), and those with rutile (SC-R) and anatase (SC-A), respectively (Fig. 2c). Furthermore, after a single exposure to CFL, the cell number at Day 4 in the cultures without NPs (SC-0) had almost recovered to ca 95% of the value relative to the unilluminated control (0-0). In samples containing rutile (SC-R), the cell number remained the same, and those with anatase had a decrease relative to the unexposed control (0-0), by ca 65% in DO33 and 93% in CF-29 samples (Fig. 2b,d). The difference between the cultures, which contained NPs and those without was even larger after the second exposure to CFL. DO33 cells that were exposed twice to CFL without TiO2 (DC-0) decreased by 18% relative to the unilluminated control (0-0), whereas the cultures containing rutile (DC-R) and anatase (DC-A) NPs decreased drastically by 79% and 86%, respectively. Similarly, a decrease of 46% and 81% was observed for the CF-29 samples without NPs (DC-0) and with rutile (DC-R), respectively. No cells were observed in cultures containing anatase (DC-A; Fig. 2b,d).

It is well known that UV light increases the formation of ROS in cells, and therefore induces indirect DNA damage, which can cause cell death (7–9). The relative intensity of the ROS products in DO33 keratinocytes and CF-29 fibroblasts at Day 2 for all samples exposed to the CFL (SC-0) showed a slight increase (<10%) in comparison to the control unilluminated cultures (0-0; Fig. 3a,c). In contrast, 4 days after the exposure to CFL, we observed a greater increase of ROS in the samples containing rutile (SC-R) and anatase (SC-A; ca 20% and 35%, respectively, for both DO33 and CF-29), whereas no changes were detected with CFL exposure in the control sample (SC-0; Fig. 3b,d). With cells double exposed, ROS increased by ca 7% (control DC-0), whereas for those exposed to rutile (DC-R) and anatase (DC-A) NPs, ROS increased by ca 35% and 48%, in DO33 and ca 27% and 48% in the CF-29, respectively (Fig. 3b,d).

image

Figure 3.  ROS of DO33 keratinocytes and dermal fibroblasts CF-29 with and without TiO2 exposed to CFL and control. (a) DO33 2 days after first exposure, (b) DO33 4 days after first exposure, (c) CF-29 2 days after first exposure, (d) CF-29 4 days after first exposure.

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Next, we wanted to monitor the effects of the NPs and CFL on the overall morphology of DO33 cells. Using confocal microscopy, we show that 4 days after exposure to CFL, damage to the cells as a function of time is seen in all samples (Fig. 4). The damage was worse in the samples containing anatase (SC-A), where remnants of dead cells were observed (Fig. 4f), and this effect was more pronounced with the double CFL exposure (DC-R, DC-A; Fig. 4h,i). Even without exposure, the cell number was reduced in the samples containing NPs (0-R, 0-A; absence of a uniform cell monolayer, Fig. 4b,c, compared with a). Further, exposure to CFL results in an even more disrupted cell monolayer (DC-0, DC-R and DC-A; Fig. 4d,e,f).

image

Figure 4.  Confocal microscopy pictures of DO33 keratinocytes. Samples containing rutile TiO2 (b, e and h) and anatase (c, f and i) on Day 4 after first exposure to CFL and control (a, d and g). Unexposed samples (a, b and c), samples after single exposure (b, e and h), samples after double exposure (c, f and i).

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Similarly, no morphological changes in the unilluminated populations of CF-29 cells were detected regardless of the condition (control [0-0] or those exposed with rutile [0-R] or anatase [0-A]) or in the organization of the monolayer (Fig. 5a,b,c). In contrast, whereas there were no changes in the control cells exposed to CFL (SC-0; Fig. 5d), the samples exposed to the NPs were morphologically different; cells treated with rutile (SC-R) did not form a uniform monolayer and begun to lift off (Fig. 5e), whereas those treated with anatase (SC-A) became less spindle-shaped (Fig. 5f). These morphological changes were much more pronounced in the samples, which were double exposed, including the control (DC-0, DC-R and DC-A; Fig. 5g,h,i).

image

Figure 5.  Confocal microscopy pictures of CF-29 dermal fibroblasts. Samples containing rutile TiO2 (b, e and h) and anatase (c, f and i) on Day 4 after first exposure to CFL and control (a, d and g). Unexposed samples (a, b and c), samples after single exposure (b, e and h), samples after double exposure (c, f and i).

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To quantitate the changes in cell morphology, we also measured the cell aspect ratio and cell area. The only statistically significant difference was seen at Day 4 between the double illuminated (DC-0) and unilluminated (0-0) control cultures without NPs (Fig. S3). Control cultures after double illumination (DC-0) have smaller aspect ratio (ca 233%) and larger area (ca 455%) than the unilluminated ones (0-0; Fig. S3a,b).

We also investigated the effect of CFL exposure to other primary functions of dermal fibroblasts such as collagen contraction and migration. Figure 6a shows that for the cells without exposure to NPs, their migration velocity clearly increased about ca 20% following exposure to CFL (SC-0). Illuminating the cells for a second time resulted in a further increase of ca 40%, for both cells incubated with (DC-R) and without NPs (DC-0; Fig. 6a). We also observed that prior to CFL exposure, cells treated with rutile (0-R) migrated ca 35% slower than the control sample. However, after CFL exposure, the migration rate of samples with (SC-R) and without rutile particles (SC-0) showed a similar increase of ca 35–40% compared to the unilluminated cultures (0-0; Fig. 6a).

image

Figure 6.  Migration and collagen gel contraction study of dermal fibroblasts CF-29 with rutile TiO2 and control 4 days after first exposure to CFL. (a) Droplets (15 × 106 mL−1) were platted on the collagen gel and allowed to migrate for 24 h. (b) Gels (700 × 105 cells mL−1) were allowed to contract for 5 h.

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A decrease in the degree of contraction is frequently associated with skin damage and premature aging. In the unilluminated samples, cells incubated with rutile particles (0-R) showed no statistically significant difference in their ability to contract collagen gels (Fig. 6b). CFL exposure reduced the ability of dermal fibroblasts (SC-0) to contract the collagen fiber gels by ca 40% relative to the unilluminated control samples (0-0). For cells incubated with rutile particles (SC-R), the decrease was nearly 100%. Similar results were obtained when the cells were exposed to the CFL a second time; the effect was more pronounced with cells cultured with rutile (DC-R; ca 110%) as compared to those cultured without NPs (DC-0; ca 68%).

To confirm that these effects were specific to CFL exposure, we also illuminated the CF-29 dermal fibroblasts (as they appeared to be more sensitive than the DO33 cells) with an incandescent bulb and measured their mitochondrial activity. Exposure to the incandescent light bulb (SI-0) did not result in any statistically significant differences with respect to MTS or morphology as compared to the unilluminated control cells (0-0; Fig. S4). Similarly, no additional effects were observed after multiple illuminations (DI-0).

Finally, we also wanted to see if there were any morphological differences in the cells exposed to CFL or incandescent light bulbs for 4 days. Cells exposed to the incandescent bulb (DI-0) looked identical to the unilluminated control cells (0-0), with the presence of a uniform monolayer of spindle-shaped cells (Fig. S4b,c). In contrast, cells exposed to CFL (DC-0), did not produce a similar uniform monolayer and appeared to be lifting from the surface (Fig. S4d). All of the experimental results obtained at Day 4 are summarized in the Table 4.

Table 4.   Day 4 experimental results summary.
SamplesViabilityROSCell areaMigrationCollagen gel contraction
DFKDFKDFDFDF
  1. *, no change; +, small significant increase; ++, medium significant increase; +++, large significant increase; −, all cells died; #, significant decrease; N/A, not applicable; DF, dermal fibroblasts; K, keratinocytes.

0-0++++++    *    *+    +   +
SC-0  ++  ++    *    *N/A  ++  ++
DC-0  ++  ++    *    *+++++++++
SI-0+++ N/A N/A N/AN/A N/A N/A
0-R++++++    +    ++    #    +
SC-R  ++  ++  ++    +N/A  +++++
DC-R    +    ++++  +++++++++
SI-R+++ N/A N/A N/AN/A N/A N/A
0-A  ++  ++    +    +N/A N/A N/A
SC-A    +    +  ++  ++N/A N/A N/A
DC-A     −    −++++++N/A N/A N/A
SI-A  ++ N/A N/A N/AN/A N/A N/A

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

Despite claims (not having the UV emission; 10,11), our measurements of emissions spectra from CFL bulbs, indicated significant levels of UVA and UVC. The amount of emissions varied randomly between different bulbs and different manufacturers. CFL bulbs work primarily through the excitation of Hg vapor that has fluorescence with the characteristic wavelength of 184 and 253 nm (UVC) and 365 nm (UVA; 12). The enclosure of the bulbs is coated with different types of phosphors, which absorb the X-ray emissions and fluoresce within the visible range. CFLs consist of tightly coiled small diameter tubes; this introduces larger stresses in the fluorescent coating, and causes cracks or uncoated areas, whose location and number varied greatly. Closer examination of some of these commercially available bulbs showed multiple defects in their coating, thus allowing UV-light emission.

Even though there are no formal restrictions on UV exposure, the American Conference of Governmental Industrial Hygienists (ACGIH) has published recommended TLV as a function of wavelength (4). When we determined the TLV values for wavelength regions corresponding to the Hg excitations from the CFL bulbs we found that the TLV was exceeded after only 79 s at 2.5 cm and 5 h at a typical working distance of 35 cm of a desk lamp. These data are particularly disturbing as the UVC emission is even larger than ambient sunlight on a mountain (13,14).

To determine directly if these emissions were in a range to be physiologically harmful to human skin, we tested their effects on human keratinocytes and dermal fibroblasts using a variety of different biological assays. We chose to expose the cells at a distance of 2.5 cm to minimize the exposure time, as the experiments were performed under ambient conditions. It should be noted that the UV intensity achieved at this distance over the exposure time corresponds to the same intensity achieved in 45 h at a distance of 35 cm, which is equivalent to the typical working distance from a desk lamp. It should also be noted that even though the damage is probably less severe if the amount of radiation is received over a longer period of time, the results show that the damage is significant and the consequents of continuous exposure even at reduced dosage over a long period of time require further study. Furthermore, when a CFL bulb is commercially sold without a limiting distance, it may be used in certain applications which require very short working distances, such that the distance studied here is not unreasonable.

In addition, we also investigated exposure of these cells to TiO2 rutile and anatase NPs, common light sensitive components (15), to see whether their ability to absorb UV radiation would mitigate the effects of the UV exposure. The particle concentrations chosen for these studies were based on our previous results where no effect on keratinocyte proliferation or other functions was observed for 2 days with NP treatment. In contrast, the effects observed at the longer time point of 4 days with NP treatment resulted in an increased NP uptake, thereby affecting proliferation and other cellular functions. Contrary to expectations, exposure to CFL resulted in a large decrease in cell proliferation, especially for the cultures containing anatase NPs. As exposure to ambient light is continuous, we also explored the effects of multiple exposures to CFL. We found that double exposure promotes additional cell damage in cultures incubated with rutile and anatase NPs as compared with cells incubated without NPs. Hence, consistent with earlier studies (16,17) these NPs behave as photosensitizers amplifying cell damage after exposure to CFL.

It is well known that photochemical reactions between, light, oxygen and photosensitizers cause formation of ROS (18,19). UVC radiation is known to cause direct DNA damage. UVA light also produces singlet molecular oxide, 1O2 and other ROS products by interacting with endogenous photosensitizers in human skin. Together, these are also known to result in membrane lipid peroxidation and eventually cell death (6,20,21). A recent study by Dodd et al. (17) showed that TiO2 exposed to UV light produces a carboxyl radical (CO2), which is involved in oxidative cell damage. Our data shows that in all samples after 2 days ROS slightly increased with CFL exposure and 4NPs, whereas at Day 4 significant increases were observed especially with samples exposed twice to CFL. These results confirm that when NPs are present, recovery from the CFL exposure is not efficient, supporting the notion that the NPs were behaving as photosensitizers (16,17). Thus, the emissions of CFL bulbs can be especially dangerous, and maybe even the primary contributor to a large extent of cell death observed in the presence of photosensitizers.

In healthy intact skin, the dermal fibroblasts, which comprise the next layer of cells beneath the keratinocytes would not normally be exposed to UVC unless the keratinocytes layer is significantly damaged. This layer would be exposed to the more penetrating UVA rays, which are the primary radiation emitted from CFL bulbs. Therefore, we also conducted similar studies with dermal fibroblasts and found that they were even more susceptible to damage by CFL than keratinocytes in all assays.

The overall fibroblast morphology also changed upon TiO2 exposure and CFL illumination. It is interesting to note that the morphology of the cells that were subjected to the double CFL exposure was different in terms of both area and aspect ratio in comparison to those with a single exposure. Banrud et al. (22) and also Girard et al. (23) have reported that UVA irradiation inhibit cell proliferation due to a temporary accumulation of cells in S-phase of the cell cycle. Ultimately, by 48 h post irradiation these cells had recovered from cell cycle arrest (22,23). Another study proposed that inhibition of S-phase progression is due to impaired replication fork progression (24). On the basis of these results, we can speculate that the abnormal cell size observed for the double exposed control samples may be due to the cell’s inability to divide as a result of being “locked” in the S-phase. Additional time (more than 48 h) may be required for DNA repair due to the UVA exposure, but additional experiments are required to verify this.

The dermal fibroblasts’ ability to migrate and contract collagen fibers is the most important feature of the wound healing process. First, cells migrate into the wound site and the tissue repair begins when they contract the collagen fibers allowing keratinocytes to differentiate and form the subsequent layers of new skin. Our results indicate that both migration and gel contraction are significantly affected by exposure to CFL, and the degree to which these processes are changed is greater in the cells treated with TiO2. Specifically, we found that migration is faster for all samples exposed to CFL, regardless of exposure to NPs. This is probably due to decrease in the number of focal adhesions leading to changes in the traction force that enables cells to migrate faster, as was previously demonstrated by different groups (3, 25, 26, 27). In addition, exposure of cells to NPs, prior to CFL irradiation, leads to decreases in actin production, which negatively affect the formation of actin fibers (28). Consequently, failure of actin fiber formation can affect traction forces by interfering with normal focal adhesion signaling thereby affecting migration and overall collagen contraction. This may offer a rational explanation as to why we observed such decreases in the migration in cells exposed only with Rutile but not exposed to CFL. Further, in the case of cells exposed to rutile but not CFL, we also observed a decrease in collagen gel contraction probably as a result of insufficient actin fiber formation and reduced traction forces as explained above. Finally, when cells were exposed to CFL, we also observed the same pattern in collagen contraction, as a result of decreases in both actin fiber and focal adhesions affecting traction forces. Hence, we propose that irradiation with CFL decreases the focal adhesions, whereas exposure to NPs decreases actin fiber formation” The influence of the faster migration velocity on wound healing is not clear at this point, but the inability to contract collagen is probably related to damage to the cell’s molecular machinery.

Finally, we also compared the relative mitochondrial activity of cells, which were not exposed to any form of light to those exposed to CFL and to a 60 W incandescent bulb emitting the same visible light as the CFL bulb. The UV emissions from the incandescent bulb were lower than the detection sensitivity of the UV meters used. Our results showed no statistically significant cell damage when the fibroblasts were exposed to the incandescent light bulb or any apparent morphological changes. Therefore, we conclude, that the observed cell damage is fully due to the UV irradiation emanating from the CFL bulb.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

The data presented herein confirm that higher than expected levels of UVA and UVC irradiation can be emitted by commercially available CFL bulbs as a result of the limitation of the current production process and the possible physical defects in the bulbs where the phosphorus coating was compromised. Skin cells exposed to the CFL exhibit 25% and 50% attrition for DO33 and CF-29, respectively. For the surviving cells, a significant increase in the production of ROS, and in the case of the CF-29, a decrease in their ability to contract collagen and abnormal migration behavior, are observed and consistent with previous reports of exposure to UVA and UVC radiation.

In addition, we also examined the effects of CFL in conjunction to the exposure of cells to low dosages (0.1 μg mL−1) of anatase and rutile TiO2 NPs prior to irradiation. In the absence of exposure to CFL, this dose was found to have minimal or no effects on the cells. In contrast, after even a single exposure to CFL cells containing anatase died. Cells containing rutile sustained slightly more damage than the cells without NPs after a single CFL exposure, but were completely destroyed following a second one. In contrast, cells without NPs sustained less damage after the second CFL exposure. Even though both keratinocytes and dermal fibroblasts sustained damage after exposure to the CFL, the extent was larger for fibroblasts. Illumination of all cell to incandescent light (where no UV emissions were detected), had no significant effect on proliferation, ROS production, or mitochondrial activity. Taken together, our results confirm that UV radiation emanating from CFL bulbs (randomly selected from different suppliers) as a result of defects or damage in the phosphorus coating is potentially harmful to human skin.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. References
  9. Supporting Information

Figure S1. TiO2 imaged by TEM and Gaussian particles size distribution histograms. (a) and (c) rutile 16 ± 3 nm particles, (b) and (d) anatase 134 ± 73 nm particles.

Figure S2. X-ray diffraction spectra of TiO2. (a) rutile, (b) anataise.

Figure S3. Cell aspect ratio for the CF-29 dermal fibroblasts. Samples with rutile unexposed and double exposed to CFL respectively and control; cell area (b) of dermal fibroblasts CF-29 samples with rutile unexposed and double exposed to CFL respectively and control.

Figure S4. (a) MTS results for the CF-29 cells 2 and 4 days after first exposure to CFL bulb, incandescent light bulb and control. After first exposure (2 days) incandescent bulb and CFL bulb; 4 days after first exposure incandescent bulb, CFL bulb; double exposed samples: incandescent and CFL bulb. (b–d) Confocal images of the cells exposed to different lighting. (b) Unexposed control, (c) double exposure to incandescent light bulb, (d) double exposure to CFL.

Table S1. Zeta-potential of TiO2 nanoparticles.

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php1192_sm_FiguresS1-S4-TableS1.pdf429KSupporting info item

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