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UV-A emission from fluorescent energy-saving light bulbs alters local retinoic acid homeostasis Julian Hellmann-Regen,* Isabella Heuser and Francesca Regen Worldwide bans on incandescent light bulbs (ILBs) drive the use of compact fluorescent light (CFL) bulbs, which emit ultraviolet (UV) radiation. Potential health issues of these light sources have already been discussed, including speculation about the putative biological effects on light exposed tissues, yet the underlying mechanisms remain unclear. We hypothesized photoisomerization of all-trans retinoic acid (at-RA), a highly light sensitive morphogen, into biologically less active isomers, as a mechanism mediating biological effects of CFLs. Local at-RA is anti-carcinogenic, entrains molecular rhythms and is crucial for skin homeostasis. Therefore, we quantified the impact of CFL irradiation on extra- and intracellular levels of RA isomers using an epidermal cell culture model. Moreover, a biologically relevant impact of CFL irradiation was assessed using highly at-RA-sensitive human neuroblastoma cells. Dose-dependent
Received 2nd July 2013, Accepted 30th September 2013 DOI: 10.1039/c3pp50206f www.rsc.org/pps
conversion of extra- and intracellular at-RA into the biologically less active 13-cis-isomer was significantly higher in CFL vs. ILB exposure and completely preventable by employing a UV-filter. Moreover, preirradiation of culture media by CFL attenuated at-RA-specific effects on cell viability in human at-RA-sensitive cells in a dose-dependent manner. These findings point towards a biological relevance of CFLinduced at-RA decomposition, providing a mechanism for CFL-mediated effects on environmental health.
Clinical Neurobiology, Department of Psychiatry, Charité, University Medicine Berlin, CBF, Eschenallee 3, 14050 Berlin, Germany. E-mail: [email protected]; Fax: +49 3 0 8445 8233; Tel: +49 30 8445 8234
physico-chemical properties with other fluorescent lamps.7 The UV output of CFLs may imply several health issues not only for photosensitive individuals,8–10 as even direct mutagenic effects have previously been discussed.11 Beyond that, health issues have been reported for light-at-night conditions, mostly discussed in the context of disrupted circadian rhythms by blue visible light at 460–500 nm.12–18 Under normal conditions, light at wavelengths below 500 nm are only present during the day, where visible blue light at 460–500 nm serves as a natural Zeitgeber for setting circadian biorhythms via retinal ganglion cells.19 In contrast, light at such wavelengths is completely absent during the night; therefore, shortwaved light below 500 nm emitted by modern CFLs may represent a condition that mankind has never been exposed to before and thus create a truly novel evolutionary environment. Rhythmic oscillations of a molecular network of so-called clock genes are traceable down to the single-cell level.20 Even the epidermal skin has repeatedly been discussed as a potential extraocular photoreceptor21 and discrete changes in its intrinsic biorhythm have been observed upon low-dose UV-B exposure.22 Photoreception via extraretinal photoreceptors (ERP) has been known for a long time23 and involves retinaldehyde-coupled receptor proteins.24,25 Retinaldehyde belongs to the family of naturally light sensitive vitamin-A derivatives which play crucial roles in many other organs, such as the brain and the skin. One of the most active metabolites of vitamin A is the all-trans isomer of retinoic acid (at-RA), which
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1.
Introduction
With the invention of the incandescent light bulb (ILB) in the late 19th century, electrical lighting at night has become a normal condition for most humans in most parts of the modern world.1,2 Due to their low efficiencies,1 legislation to phase-out ILBs has already come into effect in many countries to date.2,3 Bans on ILBs have massively stimulated the use of compact fluorescent energy-saving light bulbs (CFLs), which are distinct from ILBs when it comes to the characteristic emission spectra. While ILBs share a continuous emission spectrum with e.g. wood fires or candles, emission spectra of CFLs are often dominated by characteristic “mercury lines”, including significant invisible ultraviolet (UV) output with peaks e.g. at 254 nm, 312 nm and 365 nm, corresponding to the UV bands UV-C (200–280 nm), UV-B (280–315 nm) and UV-A (315–400 nm) per ISO-21348. These intensity peaks are thought to be the result of small “bald” areas in the internal phosphor coatings that “convert” mercury vapour fluorescence into visible light.4–6 This may even be true for CFLs with socalled warm-white spectra, which have been designed to mimic the spectra of ILBs more closely but share similar
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Paper is a highly potent small molecule with pleiotropic actions on various tissues and strong affinity to retinoic acid receptors (RAR). Retinoic acid (RA) naturally mainly occurs as 13-cis-, 9-cis- and at-RA, which differ greatly in their RAR binding affinities. While at-RA strongly binds RAR at low nanomolar concentrations, 13-cis-RA appears to be a much weaker agonist.26 In fact, biological actions of 13-cis-RA are attributed to partial isomerisation of 13-cis-RA into at-RA.27 At-RA acts in a hormonal manner with profound effects on neuronal differentiation28 and distinct anti-inflammatory effects in both, neuronal tissue29–33 and in the skin,34–37 where it is therapeutically used as one of the most potent substances in the treatment of severe acne or psoriasis. The actions of endogenous RA are thought to be determined by its local synthesis and degradation within the target tissues. Interestingly, this homeostatic balance has been shown to be regulated in a photoperiodical manner.38–40 Moreover, at-RA has been shown to be essentially involved in regulating the core feedback loops of mammalian circadian clock gene oscillations, being capable of (re)setting both central and peripheral intracellular circadian oscillatory activity, directly affecting the expression of specific regulatory proteins within the “clock gene family” via the nuclear receptors retinoic acid receptor alpha (RARa) and retinoid-X receptor alpha (RXRa).41–44 In this respect, at-RA represents one of only a few endogenously occurring small molecules identified to date to potently act as an entrainment factor on the intracellular molecular clock.45 With respect to its pronounced photosensitivity,46 it has to be noted that the absorption spectra of at-RA, in contrast to the retinaldehyde-coupled photoreceptor proteins, exhibit a maximum absorption at around 340–350 nm, which lies within the UV-A band and thus well outside the visible range of light. Interestingly, a previous study on the stability of usually topically applied retinoic acids has revealed that UV-A radiation may be the major contributor to photodegradation of retinoic acid.47 This fact may have significant implications for the use of CFLs, since at least two of their characteristic mercury peaks at short wavelengths superimpose closely with the absorption peak of at-RA at 340–350 nm, as confirmed by own experiments. Any direct, light-mediated biological effect will likely involve a direct physico-chemical interaction between light at a discrete wavelength interval and a target molecule exhibiting corresponding sensitivity, eventually resulting in light-dependent modification of exogenous (e.g. xenobiotics) or endogenous signaling molecules. Therefore, we hypothesized that light emitted by CFLs will impact local tissue levels of the potent endogenous signaling molecule at-RA, possibly via photoisomerization of the photosensitive molecule and conversion of at-RA into isomers with differential biological activity, ultimately resulting in altered biological effects on the target tissue. We furthermore hypothesized that this effect will be specifically attributable to the CFL-specific light emission at 365 nm, and thus be absent or less prominent for ILBs at a comparable gross luminal flux rating. Finally, we hypothesized that eliminating these light emission at 365 nm from the CFL’s spectrum using a UV edge filter, thus not altering the
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Photochemical & Photobiological Sciences rest of the visible spectrum, will effectively prevent the disintegration of at-RA. To test these hypotheses, we recorded and qualitatively compared the characteristic emission spectra of several commercially available single enveloped CFLs and ILBs at comparable luminous flux ratings and established a human keratinocytebased in vitro model for the standardized measurement of extraand intracellular at-RA levels pre, during, and post well-defined conditions of light exposure using various light sources. Finally, to assess the biologically relevant impact of potential CFLinduced photoisomerization of local at-RA, we used a well-established, highly at-RA-responsive human cell line and quantified the dose-dependent cellular response to at-RA from various CFL-pre-irradiated cell culture media.
2.
Methods
2.1.
Cell culture and sample preparation
Keratinocyte cell culture was performed using human HaCaT keratinocytes. This cell culture represents a spontaneously immortalized human keratinocyte cell line and an excellent alternative to normal, primary human keratinocytes. Cells stop proliferating upon reaching confluency, but exhibit high proliferation rates earlier. Therefore, HaCaT cell culture represents an excellent tool for the rapid generation of closed keratinocyte-based epithelial layers with similarities to human skin surface.48 Moreover, the same model has repeatedly been used in UV irradiation experiments.49–51 Cells were grown in 75 cm2 cell culture flasks in a humidified atmosphere at 37 °C with 5% CO2 using Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum (FCS), 100 U ml−1 penicillin and 50 µg ml−1 streptomycin (Biomol, Berlin, Germany). Cells were detached and plated at 5 × 105 cells per dish in 30 mm Petri dishes and allowed to reach confluency prior to exposure experiments. For assessing radiation effects on extracellular RA levels, light exposure experiments were initiated by replacing the culture medium with 2 ml of fresh medium containing at-RA (1 µM). Following an adaptation period of 2 h, all dishes were placed into a sealed styrofoam box and randomly assigned to either a sham exposure condition or to the respective light exposure conditions. For assessing radiation effects on intracellular at-RA levels, a different set of dishes containing dense keratinocyte layers was washed twice with phosphate-buffered saline (PBS) and placed for 5 or 10 min directly into the radiation with only a thin layer of PBS (2 ml) covering the cells. Sham treated cells were shielded from light using aluminium foil. After removal of PBS, ice cold methanol (2 ml) was added directly to the cells and the suspension was treated in analogy to the cell culture supernatant samples. Following protein precipitation, samples were cleared by centrifugation at 15 000g, 4 °C for 20 min. The supernatants of all samples were subjected to immediate high performance liquid chromatography (HPLC) analysis. Direct UV exposure experiments were performed using a photodetector-controlled Stratalinker UV irradiator equipped
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Photochemical & Photobiological Sciences with G15T8 bulbs, which emit mainly UV-C, together with UV-B, UV-A and visible blue radiation (Stratagene, La Jolla, CA, USA). Defined amounts of radiation (0, 1, 10, 100, 500, 1000 mJ cm−2; measured by photodetector cell) were applied to the preparations of cell culture medium containing 10% FCS and at-RA at 1 µM. Exposure to regular light sources was conducted using various 60 W-ILBs and CFLs at equal luminous flux ratings. Light sources were placed at exactly 300 mm distance from cell culture plates. During light exposure, samples of 60 µl cell culture supernatant were drawn from all conditions including a sham condition and in duplicates after 0, 30 and 60 minutes, thoroughly mixed with 240 µl of ice cold methanol and placed at −20 °C for 30 min. All following steps were conducted either in complete darkness or under indirect dim yellow light. For assessing whether a biologically relevant impact of CFL irradiation is mediated by at-RA, serum-depleted cell culture media containing at-RA (1 µM) or vehicle were irradiated by defined amounts of UV radiation (100/500 mJ cm−2) and by a “warm-white” CFL (2 h at ∼100 mm distance). The distance of 100 mm was chosen to maximize potential CFL-mediated effects by narrowing the exposure distance. Treated media were subsequently diluted to final concentrations of 10% and 75% (v/v) with fresh medium, yielding final concentrations of all three RA isomers (total RA) of 0 (VEH), 100 and 750 nM respectively and subjected to the highly at-RA-responsive human SH-SY5Y cell line in a 96-multiwell plate at 5 × 104 cells per well. Human SH-SY5Y cells are a subclone of the human SK-N-SH neuroblastoma with a monoaminergic phenotype and exhibit pronounced responsiveness to at-RA, which represents one of the best studied differentiation-inducing stimuli.52,53 At the same time, at-RA strikingly enhances cell survival in serum deprivation, a condition known to induce apoptosis in this cell line.52–55 SH-SY5Y cells were cultured essentially as described in detail before.31,56,57 Following 48 h incubation, at-RA-mediated effects on cell viability were quantified using a standard colorimetric tetrazolium-based assay, which offers a sensitive method to evaluate a cell viabilitydependent response of a cell population to various external stimuli. It is based on the cells’ mitochondrial capacity to reduce the yellow soluble salt 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium Bromide (MTT) into a formazan product that can be measured photometrically at 550 nm using a multiwell photometer (Model 500, Biorad, Munich, Germany). Since the MTT assay can only estimate cell viability on the basis of mitochondrial activity, we routinely confirmed a close correlation between the MTT assay, total protein content (BCAMethod) and cell counts (trypan-blue exclusion method).
Paper composed of 95% buffer B (acetonitrile (69) : n-butanol (2) : MeOH (10) : 2% ammonium acetate (16) : glacial acetic acid (3)) and 5% buffer A (40 mM ammonium acetate, pH 3.5) at a flow rate of 0.65 ml min−1 and a detection at a 350 nm. As an important limitation to this methodology, the generation of small amounts of the 9,13-dicis isomer, which for retinal has been demonstrated to occur at approximately 4% both in dark equilibrium mixture as well as upon illumination, cannot be ruled out completely.59 Data were evaluated using the Agilent ChemStation software. All chemicals, if not otherwise stated, were purchased from Sigma Aldrich, Taufkirchen, Germany. 2.3.
Emission spectra recording
Emission spectra were recorded for different branded sets of ILBs, CFLs with “cool white” or “warm white” spectra using the wavelength-calibrated high-sensitivity photodiode array from an Agilent 1290 infinity series detector system. For recording emission spectra, a mirror was placed into the light path of the detector. Subsequently, a minimum of 6 emission spectra were recorded for each light source at a distance of 300 mm from the detector and corrected for background noise at complete darkness. 2.4.
Statistical analyses
All numerical analyses were performed using the statistical software GraphPad Prism (Ver. 5; GraphPad Software Inc., La Jolla, CA, USA). Values are presented as means ± standard deviations. Differences between the groups were analyzed by oneway ANOVA followed by Tukey’s post-test or by two-way ANOVA followed by Bonferroni post tests where appropriate. Results were considered significant for p < 0.05.
3.
Results
3.1.
Emission spectra of ILBs and CFLs
Fig. 1 shows the emission spectra recordings of traditional 60 W ILBs and of CFLs at equal luminous flux ratings from the same light bulbs that were also used in subsequent exposure experiments. With regard to CFLs, both the so-called cool white CFLs (A; straight line), meant to mimic a typical daylight spectrum, and “warm-white” CFLs (B; straight line), designed to mimic warm, ambient light were studied. Traditional ILBs (dashed lines in A and B) exhibit continuous spectra characteristic of “black-body radiation”. The spectra emitted by both CFLs, however, are discontinuous, containing strong emission peaks at wavelengths that are characteristic of the emission spectrum of mercury (Hg), regardless of the spectral design (Fig. 1).
2.2. Retinoid quantification: reversed-phase high performance liquid chromatography
3.2. Qualitative and quantitative impact of UV radiation on RA isomers
Retinoic acids were essentially quantified as described in detail previously.31,58 In brief, 20 µl of the sample was subjected to an Agilent 1100 system equipped with a 1290 Infinity diode array detector (Agilent Technologies, Böblingen, Germany), a Suplex pkb-100 column, a mobile phase
Defined amounts of UV-radiation significantly affect RA isomers in cell culture solution. While the total amount of all the three RA isomers (total RA) is significantly affected only at 1000 mJ cm−2 (Fig. 2A), the relative amounts of the respective isomers at-(B), 9-cis (C) and 13-cis-RA (D) exhibit a pronounced
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Fig. 1 Superimposed emission spectra from 60 W traditional incandescent light bulbs (ILB; dotted lines in A and B) and single-enveloped, modern compact fluorescent light bulbs (CFL; straight lines) with luminous flux ratings comparable to a 60 W ILB. Data are plotted as relative intensity as a function of wavelength. Both CFLs with “cool white” (A) and “warm white” (B) spectral ratings exhibit a discontinuous spectrum with prominent mercury lines and significant UV emission. Data represent averages of 6 independent spectral scans.
Fig. 2 Qualitative and quantitative impact of UV irradiation on total RA and relative amounts of RA isomers in cell culture media. The total amount of retinoic acid is significantly affected by UV irradiation only at 1000 mJ cm−2 but remains unaffected at lower dosages of 1–100 mJ cm−2 (A). Conversely, the relative amounts of the three major isomers at-RA (B), 9-cis- (C), and 13-cis-RA (D) are significantly affected at lower dosages of 1–100 mJ cm−2 in a dose dependent manner and even significantly altered at the lowest dosage of 1 mJ cm−2. While relative amounts of at-RA decrease with increasing dosage (B), biologically less active 13-cis-RA increases substantially (D). While 9-cis-RA also increases significantly with UV exposure, the total amount of 9-cis-RA is rather small compared to 13-cis-RA (1, 8% vs. 22% of total RA at 100 mJ cm−2). The severe drop in total RAs at 1000 mJ cm−2 (A) was associated with the appearance of several early-eluting products in the HPLC analysis, likely representing degradation products of RAs at this irradiation dose. * significantly different from sham-treated controls (0 mJ cm−2), one-way ANOVA followed by Tukey’s post test.
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Photochemical & Photobiological Sciences and dose-dependent change compared to sham-treated controls (0 mJ cm−2). While at-RA levels significantly decreased, the two isomers 13-cis and 9-cis RA significantly increased with increasing UV dosage, indicating a photoisomerization process, at least for the lower range of UV dosages. These effects were statistically significant even at the lowest amount of UV irradiation applied (1 mJ cm−2; Fig. 2). The severe drop in total RAs at 1000 mJ cm−2 (Fig. 2A) was associated with the appearance of several early-eluting products in the HPLC analysis, likely representing degradation products of RAs at this irradiation dose. 3.3.
Impact of ILB and CFL emitted radiation on local at-RA
The qualitative and quantitative impacts of various light sources on extra- and intracellular at-RA were studied using a human keratinocyte cell line. Fig. 3 demonstrates a dose- and light source-dependent impact of light exposure on the levels of extracellular (A–C) and intracellular (D–F) RA isomers. Exposure to radiation emitted from “warm-white” (black bars) or “cool white” (grey bars) CFLs results in a significant, dosedependent decomposition of extracellular at-RA (A), mainly leading to the formation of biologically less active 13-cis-RA (C). Conversely, exposure to the light emitted from a traditional 60 W ILB results in significantly less degradation of at-RA into 13-cis-RA (A–C). Similar effects are observed for
Paper intracellular retinoids, where both “warm-white” and “cool white” CFLs result in significant at-RA degradation, while ILB exposure remains without effect on intracellular at-RA levels (Fig. 3D–F). 3.4. Prevention of CFL-induced at-RA decomposition using a UV edge filter At-RA exhibits a maximum absorption at about 345 nm (Fig. 4A). When superimposed with a characteristic emission spectrum of a CFL (“warm-white”), there are at least two strong peaks at 367 nm and 405 nm in the close vicinity of the absorption maximum of at-RA. Therefore, a polycarbonatebased UV edge filter (derived from eye protection device no. 303, UVP, Upland, CA, USA) effectively eliminating these two peaks from the spectrum of a “warm-white” CFL was selected (Fig. 4B) and deployed in a cell culture exposure paradigm similar to experiments depicted in Fig. 3. As expected when the major absorption peaks of at-RA were almost completely filtered the intra- and extracellular at-RA degradation was prevented (Fig. 5). 3.5.
Impact of irradiation on at-RA-mediated viability
Exposing the highly at-RA-responsive human SH-SY5Y cells to at-RA in serum depleted medium resulted in a dose-dependent enhancement of cell viability after 48 h of incubation, as
Fig. 3 Qualitative and quantitative impact of different light sources on at-RA (1 µM) in human keratinocyte cell cultures. A traditional 60 W ILB, a “warm-white” and a “cool white” CFL, all at comparable luminous flux ratings, were used to irradiate human keratinocyte cell cultures. Relative concentrations (fold of total RA) of extracellular RA isomers were determined after 0, 30 and 60 min exposure (A–C), while intracellular concentrations were assessed after 5 min exposure of dense, PBS-covered keratinocyte layers (D–F). Values are means of at least three independent experiments ±SD, * significantly different ( p < 0.05).
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Fig. 4 (A) Depicts the absorption spectra of at-RA (black line; right abscissa) and 13-cis-RA (dashed grey line; right abscissa) dissolved in sodium acetate (100 mM) and methanol (20% v/v), superimposed with the emission spectrum of a single-enveloped CFL with “warm-white” spectrum (straight line; left abscissa) indicating a significant UV emission peak at 365 nm, close to the absorbance maximum of at-RA. (B) Depicts the effects of a standard UV-filter (straight line), completely eliminating the strong peak at 365 nm and largely reducing a peak at 405 nm from the unfiltered spectrum (dashed line) without significantly affecting the rest of the spectrum in the visible range.
Fig. 5 Prevention of CFL-induced at-RA decomposition in human keratinocyte cell cultures using a standard UV-filter. A standard, single-enveloped “warm-white” CFL was used either with or without a UV-filter to irradiate human keratinocyte cell cultures for the indicated time periods (A–C) and cell layers for 10 min (D–F). Relative concentrations (fold of total RA) of extracellular RA isomers were determined after 0, 30 and 60 min exposure (A–C) for both conditions. Similarly, intracellular concentrations were assessed after 5 min exposure of dense, PBS-covered keratinocyte layers (D–F). Values are means of at least three independent experiments ±SD, * significantly different ( p < 0.05).
evidenced by a standard tetrazolium-based viability assay (Fig. 6). At-RA mediated effects were significantly attenuated by pretreating cell-free culture media (notably not the cells) either with UV irradiation (500 mJ cm−2) or a standard “warm-white”
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CFL for 120 min at a distance of 100 mm (Fig. 6). Importantly, irradiation of media lacking at-RA (VEH) did not have significant effects on cell viability, indicating that irradiation effects depend on – and are mediated by – the presence of at-RA.
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Fig. 6 Impact of irradiation on at-RA-mediated viability. At-RA-containing cell culture media were exposed to UV irradiation and to a CFL for 120 min. Exposed media were diluted 1 : 10 and 3 : 4 (100 nM and 750 nM total RA isomers) and subjected to the highly at-RA responsive human SH-SY5Y cells in serum depleted medium. Following 48 h incubation, at-RA-mediated effects on cell viability were quantified using a standard colorimetric tetrazolium-based assay, indicating a strong, concentration-dependent effect of at-RA, which is significantly attenuated by prior irradiation of the cell culture media. Values are presented as means ± SD of n = 6–8 samples. * p < 0.05, two-way ANOVA with Bonferroni’s post test.
4.
Discussion
For reasons of low efficiency, traditional ILBs are increasingly being replaced by various types of CFLs. These often singleenveloped fluorescent lamps emit significantly more UV radiation than ILBs. Own emission recordings and spectral analyses by others6 clearly demonstrate this fact for both, CFLs meant to mimic daylight (“cool white”) and those for ambient light applications (“warm-white”; Fig. 1A and B). Biological implications for these spectral differences, including biologically relevant effects on light-exposed tissues, are largely unclear to date. From a chronobiological perspective, exposure to blue visible light represents an important natural zeitgeber for entraining circadian rhythms in most light-exposed organisms. Under normal conditions, blue visible and UV light is absent at night, and minimal in traditional light sources such as fires, candles and ILBs. This may change with the use of CFLs, yet “clinical relevance” of the emitted amount of blue visible light and UV radiation remains subject to discussion.60 Recently, the striking differences between ILBs and CFLs have again been discussed10,61 and “clinical relevance” has gained considerable support by a study demonstrating significant effects of CFL-emitted radiation on human fibroblast and keratinocyte cell culture models, which was consistent with damage from UV irradiation.4 So far, none of these studies aimed at providing a direct molecular correlate for the divergent, putatively UV-associated cellular reactions to ILB and CFL irradiation, nor did they employ filters in a “rescue approach”. Since biologically relevant effects of light (e.g. phototransduction or photodisease) may be caused either by light-
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Paper mediated formation of toxic compounds or by decomposition of e.g. essential endogenous molecules (e.g. photoisomerization), we chose to characterize the latter by studying the direct effects of CFL- and ILB-emitted light as well as of UV irradiation on intra- and extracellular retinoids in a human keratinocyte cell culture model.48 Besides their famous roles in phototransduction or neuronal differentiation, retinoids exert profound effects on various homeostatic processes, including effects on local inflammation, lipid metabolism, photoaging, entrainment of chronobiological rhythms and, most importantly, controlling the balance between proliferation and differentiation.45,62,63 Since all of these processes are known to be affected by exposure to UV radiation, retinoids may provide a common denominator for linking some of the diverse light-mediated biological effects. In fact, many at-RA mediated effects on the human skin are antagonistic to the effects observed for UV radiation, such as at-RA being chemopreventive for keratinocyte carcionoma and protective of photoaging, conditions which are both known to be caused by UV exposure.37 Most retinoids exhibit peak absorptions between 320 and 360 nm and a dose-dependent sensitivity to UV radiation, which we were able to characterize for at-RA in cell culture media (Fig. 2). Interestingly, light-mediated effects on at-RA were found to be mainly based on isomerisation rather than oxidative modification with a clear preference for the biologically less active 13-cis-RA to be generated under UV exposure (Fig. 2). The levels observed for 13-cis-RA were surprisingly high if compared to e.g. published values for retinal.59 To preclude the possibility of an overestimation of the observed disproportionate decrease in at-RA due to differences in absorption coefficients at 350 nm between 13-cis- and at-RA, absorption spectra were recorded for both isomers at 1 µM, revealing almost identical absorption spectra. Therefore, the disproportionate decrease in at-RA and increase in 13-cis-RA may not have been overestimated but might rather be due to catalytic/ enzymatic processes. CFL, but not ILB irradiation mimicked the effects of direct UV irradiation, resulting in dose-dependent degradation of at-RA (Fig. 3). These findings highlight significant differences between CFL and ILB-emitted light on the one hand, and clearly demonstrate the strong effects of light on local levels of different isomers of RA. Interestingly, these effects were equally observed for serumand cell culture media free conditions (Fig. 3D–E) as well as in conditions containing complete DMEM cell culture media + 10% FCS (Fig. 3A–C), suggesting that potentially interfering photosensitizers may, at least in this particular case, not have significantly affected the experimental outcome. In the skin, it is foremost the all-trans isomer of RA (at-RA) that exerts potent anti-acne, anti-inflammatory effects and is effectively used as a therapeutic agent for conditions such as acne vulgaris, photoaging, psoriasis or ichthyosis.64 These findings highlight the importance of local at-RA homeostasis in extraretinal locations, where even compounds that solely
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Paper inhibit the degradation of at-RA can mimic its therapeutic actions.65 The all-trans isomer of RA is a ligand for a number of specific retinoid receptors (RAR and RXR), which act as heterodimeric, ligand-activated transcription factors. They belong to the superfamily of ‘class I nuclear receptors’ which control transcription via binding to specific ‘retinoic acid response elements’ in the promoter regions of e.g. differentiation- or plasticity-associated genes.28 While at-RA preferentially binds RAR and RXR, 13-cis RA represents a much weaker agonist at both RXR and RAR.26 In fact, biological actions of 13-cis-RA are thought to be mediated through intracellular isomerisation into at-RA.27 This highlights the functional relevance of the isomerization state of RA, ultimately determining the biological activity of the molecule. Therefore, any process directly affecting the isomerization behaviour of local at-RA may in consequence be of relevance for its biological function. Additionally supporting this line of evidence, at-RA has recently been shown to be one of only a few naturally occurring small molecules capable of entraining or even resetting intracellular transcriptional oscillations of “clock genes” in vitro.66 Thus, our findings of a pronounced impact of CFL irradiation on extra- and intracellular at-RA levels may represent a potential mechanism for CFL-induced effects on light-exposed tissue (Fig. 3). The complete prevention of CFL-mediated at-RA degradation by employing a simple edge filter proves the majority of the observed effects to be solely dependent on the short-waved portion
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UV-A emission from fluorescent energy-saving light bulbs alters local retinoic acid homeostasis Julian Hellmann-Regen,* Isabella Heuser and Francesca Regen Worldwide bans on incandescent light bulbs (ILBs) drive the use of compact fluorescent light (CFL) bulbs, which emit ultraviolet (UV) radiation. Potential health issues of these light sources have already been discussed, including speculation about the putative biological effects on light exposed tissues, yet the underlying mechanisms remain unclear. We hypothesized photoisomerization of all-trans retinoic acid (at-RA), a highly light sensitive morphogen, into biologically less active isomers, as a mechanism mediating biological effects of CFLs. Local at-RA is anti-carcinogenic, entrains molecular rhythms and is crucial for skin homeostasis. Therefore, we quantified the impact of CFL irradiation on extra- and intracellular levels of RA isomers using an epidermal cell culture model. Moreover, a biologically relevant impact of CFL irradiation was assessed using highly at-RA-sensitive human neuroblastoma cells. Dose-dependent
Received 2nd July 2013, Accepted 30th September 2013 DOI: 10.1039/c3pp50206f www.rsc.org/pps
conversion of extra- and intracellular at-RA into the biologically less active 13-cis-isomer was significantly higher in CFL vs. ILB exposure and completely preventable by employing a UV-filter. Moreover, preirradiation of culture media by CFL attenuated at-RA-specific effects on cell viability in human at-RA-sensitive cells in a dose-dependent manner. These findings point towards a biological relevance of CFLinduced at-RA decomposition, providing a mechanism for CFL-mediated effects on environmental health.
Clinical Neurobiology, Department of Psychiatry, Charité, University Medicine Berlin, CBF, Eschenallee 3, 14050 Berlin, Germany. E-mail: [email protected]; Fax: +49 3 0 8445 8233; Tel: +49 30 8445 8234
physico-chemical properties with other fluorescent lamps.7 The UV output of CFLs may imply several health issues not only for photosensitive individuals,8–10 as even direct mutagenic effects have previously been discussed.11 Beyond that, health issues have been reported for light-at-night conditions, mostly discussed in the context of disrupted circadian rhythms by blue visible light at 460–500 nm.12–18 Under normal conditions, light at wavelengths below 500 nm are only present during the day, where visible blue light at 460–500 nm serves as a natural Zeitgeber for setting circadian biorhythms via retinal ganglion cells.19 In contrast, light at such wavelengths is completely absent during the night; therefore, shortwaved light below 500 nm emitted by modern CFLs may represent a condition that mankind has never been exposed to before and thus create a truly novel evolutionary environment. Rhythmic oscillations of a molecular network of so-called clock genes are traceable down to the single-cell level.20 Even the epidermal skin has repeatedly been discussed as a potential extraocular photoreceptor21 and discrete changes in its intrinsic biorhythm have been observed upon low-dose UV-B exposure.22 Photoreception via extraretinal photoreceptors (ERP) has been known for a long time23 and involves retinaldehyde-coupled receptor proteins.24,25 Retinaldehyde belongs to the family of naturally light sensitive vitamin-A derivatives which play crucial roles in many other organs, such as the brain and the skin. One of the most active metabolites of vitamin A is the all-trans isomer of retinoic acid (at-RA), which
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1.
Introduction
With the invention of the incandescent light bulb (ILB) in the late 19th century, electrical lighting at night has become a normal condition for most humans in most parts of the modern world.1,2 Due to their low efficiencies,1 legislation to phase-out ILBs has already come into effect in many countries to date.2,3 Bans on ILBs have massively stimulated the use of compact fluorescent energy-saving light bulbs (CFLs), which are distinct from ILBs when it comes to the characteristic emission spectra. While ILBs share a continuous emission spectrum with e.g. wood fires or candles, emission spectra of CFLs are often dominated by characteristic “mercury lines”, including significant invisible ultraviolet (UV) output with peaks e.g. at 254 nm, 312 nm and 365 nm, corresponding to the UV bands UV-C (200–280 nm), UV-B (280–315 nm) and UV-A (315–400 nm) per ISO-21348. These intensity peaks are thought to be the result of small “bald” areas in the internal phosphor coatings that “convert” mercury vapour fluorescence into visible light.4–6 This may even be true for CFLs with socalled warm-white spectra, which have been designed to mimic the spectra of ILBs more closely but share similar
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Paper is a highly potent small molecule with pleiotropic actions on various tissues and strong affinity to retinoic acid receptors (RAR). Retinoic acid (RA) naturally mainly occurs as 13-cis-, 9-cis- and at-RA, which differ greatly in their RAR binding affinities. While at-RA strongly binds RAR at low nanomolar concentrations, 13-cis-RA appears to be a much weaker agonist.26 In fact, biological actions of 13-cis-RA are attributed to partial isomerisation of 13-cis-RA into at-RA.27 At-RA acts in a hormonal manner with profound effects on neuronal differentiation28 and distinct anti-inflammatory effects in both, neuronal tissue29–33 and in the skin,34–37 where it is therapeutically used as one of the most potent substances in the treatment of severe acne or psoriasis. The actions of endogenous RA are thought to be determined by its local synthesis and degradation within the target tissues. Interestingly, this homeostatic balance has been shown to be regulated in a photoperiodical manner.38–40 Moreover, at-RA has been shown to be essentially involved in regulating the core feedback loops of mammalian circadian clock gene oscillations, being capable of (re)setting both central and peripheral intracellular circadian oscillatory activity, directly affecting the expression of specific regulatory proteins within the “clock gene family” via the nuclear receptors retinoic acid receptor alpha (RARa) and retinoid-X receptor alpha (RXRa).41–44 In this respect, at-RA represents one of only a few endogenously occurring small molecules identified to date to potently act as an entrainment factor on the intracellular molecular clock.45 With respect to its pronounced photosensitivity,46 it has to be noted that the absorption spectra of at-RA, in contrast to the retinaldehyde-coupled photoreceptor proteins, exhibit a maximum absorption at around 340–350 nm, which lies within the UV-A band and thus well outside the visible range of light. Interestingly, a previous study on the stability of usually topically applied retinoic acids has revealed that UV-A radiation may be the major contributor to photodegradation of retinoic acid.47 This fact may have significant implications for the use of CFLs, since at least two of their characteristic mercury peaks at short wavelengths superimpose closely with the absorption peak of at-RA at 340–350 nm, as confirmed by own experiments. Any direct, light-mediated biological effect will likely involve a direct physico-chemical interaction between light at a discrete wavelength interval and a target molecule exhibiting corresponding sensitivity, eventually resulting in light-dependent modification of exogenous (e.g. xenobiotics) or endogenous signaling molecules. Therefore, we hypothesized that light emitted by CFLs will impact local tissue levels of the potent endogenous signaling molecule at-RA, possibly via photoisomerization of the photosensitive molecule and conversion of at-RA into isomers with differential biological activity, ultimately resulting in altered biological effects on the target tissue. We furthermore hypothesized that this effect will be specifically attributable to the CFL-specific light emission at 365 nm, and thus be absent or less prominent for ILBs at a comparable gross luminal flux rating. Finally, we hypothesized that eliminating these light emission at 365 nm from the CFL’s spectrum using a UV edge filter, thus not altering the
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Photochemical & Photobiological Sciences rest of the visible spectrum, will effectively prevent the disintegration of at-RA. To test these hypotheses, we recorded and qualitatively compared the characteristic emission spectra of several commercially available single enveloped CFLs and ILBs at comparable luminous flux ratings and established a human keratinocytebased in vitro model for the standardized measurement of extraand intracellular at-RA levels pre, during, and post well-defined conditions of light exposure using various light sources. Finally, to assess the biologically relevant impact of potential CFLinduced photoisomerization of local at-RA, we used a well-established, highly at-RA-responsive human cell line and quantified the dose-dependent cellular response to at-RA from various CFL-pre-irradiated cell culture media.
2.
Methods
2.1.
Cell culture and sample preparation
Keratinocyte cell culture was performed using human HaCaT keratinocytes. This cell culture represents a spontaneously immortalized human keratinocyte cell line and an excellent alternative to normal, primary human keratinocytes. Cells stop proliferating upon reaching confluency, but exhibit high proliferation rates earlier. Therefore, HaCaT cell culture represents an excellent tool for the rapid generation of closed keratinocyte-based epithelial layers with similarities to human skin surface.48 Moreover, the same model has repeatedly been used in UV irradiation experiments.49–51 Cells were grown in 75 cm2 cell culture flasks in a humidified atmosphere at 37 °C with 5% CO2 using Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum (FCS), 100 U ml−1 penicillin and 50 µg ml−1 streptomycin (Biomol, Berlin, Germany). Cells were detached and plated at 5 × 105 cells per dish in 30 mm Petri dishes and allowed to reach confluency prior to exposure experiments. For assessing radiation effects on extracellular RA levels, light exposure experiments were initiated by replacing the culture medium with 2 ml of fresh medium containing at-RA (1 µM). Following an adaptation period of 2 h, all dishes were placed into a sealed styrofoam box and randomly assigned to either a sham exposure condition or to the respective light exposure conditions. For assessing radiation effects on intracellular at-RA levels, a different set of dishes containing dense keratinocyte layers was washed twice with phosphate-buffered saline (PBS) and placed for 5 or 10 min directly into the radiation with only a thin layer of PBS (2 ml) covering the cells. Sham treated cells were shielded from light using aluminium foil. After removal of PBS, ice cold methanol (2 ml) was added directly to the cells and the suspension was treated in analogy to the cell culture supernatant samples. Following protein precipitation, samples were cleared by centrifugation at 15 000g, 4 °C for 20 min. The supernatants of all samples were subjected to immediate high performance liquid chromatography (HPLC) analysis. Direct UV exposure experiments were performed using a photodetector-controlled Stratalinker UV irradiator equipped
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Photochemical & Photobiological Sciences with G15T8 bulbs, which emit mainly UV-C, together with UV-B, UV-A and visible blue radiation (Stratagene, La Jolla, CA, USA). Defined amounts of radiation (0, 1, 10, 100, 500, 1000 mJ cm−2; measured by photodetector cell) were applied to the preparations of cell culture medium containing 10% FCS and at-RA at 1 µM. Exposure to regular light sources was conducted using various 60 W-ILBs and CFLs at equal luminous flux ratings. Light sources were placed at exactly 300 mm distance from cell culture plates. During light exposure, samples of 60 µl cell culture supernatant were drawn from all conditions including a sham condition and in duplicates after 0, 30 and 60 minutes, thoroughly mixed with 240 µl of ice cold methanol and placed at −20 °C for 30 min. All following steps were conducted either in complete darkness or under indirect dim yellow light. For assessing whether a biologically relevant impact of CFL irradiation is mediated by at-RA, serum-depleted cell culture media containing at-RA (1 µM) or vehicle were irradiated by defined amounts of UV radiation (100/500 mJ cm−2) and by a “warm-white” CFL (2 h at ∼100 mm distance). The distance of 100 mm was chosen to maximize potential CFL-mediated effects by narrowing the exposure distance. Treated media were subsequently diluted to final concentrations of 10% and 75% (v/v) with fresh medium, yielding final concentrations of all three RA isomers (total RA) of 0 (VEH), 100 and 750 nM respectively and subjected to the highly at-RA-responsive human SH-SY5Y cell line in a 96-multiwell plate at 5 × 104 cells per well. Human SH-SY5Y cells are a subclone of the human SK-N-SH neuroblastoma with a monoaminergic phenotype and exhibit pronounced responsiveness to at-RA, which represents one of the best studied differentiation-inducing stimuli.52,53 At the same time, at-RA strikingly enhances cell survival in serum deprivation, a condition known to induce apoptosis in this cell line.52–55 SH-SY5Y cells were cultured essentially as described in detail before.31,56,57 Following 48 h incubation, at-RA-mediated effects on cell viability were quantified using a standard colorimetric tetrazolium-based assay, which offers a sensitive method to evaluate a cell viabilitydependent response of a cell population to various external stimuli. It is based on the cells’ mitochondrial capacity to reduce the yellow soluble salt 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium Bromide (MTT) into a formazan product that can be measured photometrically at 550 nm using a multiwell photometer (Model 500, Biorad, Munich, Germany). Since the MTT assay can only estimate cell viability on the basis of mitochondrial activity, we routinely confirmed a close correlation between the MTT assay, total protein content (BCAMethod) and cell counts (trypan-blue exclusion method).
Paper composed of 95% buffer B (acetonitrile (69) : n-butanol (2) : MeOH (10) : 2% ammonium acetate (16) : glacial acetic acid (3)) and 5% buffer A (40 mM ammonium acetate, pH 3.5) at a flow rate of 0.65 ml min−1 and a detection at a 350 nm. As an important limitation to this methodology, the generation of small amounts of the 9,13-dicis isomer, which for retinal has been demonstrated to occur at approximately 4% both in dark equilibrium mixture as well as upon illumination, cannot be ruled out completely.59 Data were evaluated using the Agilent ChemStation software. All chemicals, if not otherwise stated, were purchased from Sigma Aldrich, Taufkirchen, Germany. 2.3.
Emission spectra recording
Emission spectra were recorded for different branded sets of ILBs, CFLs with “cool white” or “warm white” spectra using the wavelength-calibrated high-sensitivity photodiode array from an Agilent 1290 infinity series detector system. For recording emission spectra, a mirror was placed into the light path of the detector. Subsequently, a minimum of 6 emission spectra were recorded for each light source at a distance of 300 mm from the detector and corrected for background noise at complete darkness. 2.4.
Statistical analyses
All numerical analyses were performed using the statistical software GraphPad Prism (Ver. 5; GraphPad Software Inc., La Jolla, CA, USA). Values are presented as means ± standard deviations. Differences between the groups were analyzed by oneway ANOVA followed by Tukey’s post-test or by two-way ANOVA followed by Bonferroni post tests where appropriate. Results were considered significant for p < 0.05.
3.
Results
3.1.
Emission spectra of ILBs and CFLs
Fig. 1 shows the emission spectra recordings of traditional 60 W ILBs and of CFLs at equal luminous flux ratings from the same light bulbs that were also used in subsequent exposure experiments. With regard to CFLs, both the so-called cool white CFLs (A; straight line), meant to mimic a typical daylight spectrum, and “warm-white” CFLs (B; straight line), designed to mimic warm, ambient light were studied. Traditional ILBs (dashed lines in A and B) exhibit continuous spectra characteristic of “black-body radiation”. The spectra emitted by both CFLs, however, are discontinuous, containing strong emission peaks at wavelengths that are characteristic of the emission spectrum of mercury (Hg), regardless of the spectral design (Fig. 1).
2.2. Retinoid quantification: reversed-phase high performance liquid chromatography
3.2. Qualitative and quantitative impact of UV radiation on RA isomers
Retinoic acids were essentially quantified as described in detail previously.31,58 In brief, 20 µl of the sample was subjected to an Agilent 1100 system equipped with a 1290 Infinity diode array detector (Agilent Technologies, Böblingen, Germany), a Suplex pkb-100 column, a mobile phase
Defined amounts of UV-radiation significantly affect RA isomers in cell culture solution. While the total amount of all the three RA isomers (total RA) is significantly affected only at 1000 mJ cm−2 (Fig. 2A), the relative amounts of the respective isomers at-(B), 9-cis (C) and 13-cis-RA (D) exhibit a pronounced
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Fig. 1 Superimposed emission spectra from 60 W traditional incandescent light bulbs (ILB; dotted lines in A and B) and single-enveloped, modern compact fluorescent light bulbs (CFL; straight lines) with luminous flux ratings comparable to a 60 W ILB. Data are plotted as relative intensity as a function of wavelength. Both CFLs with “cool white” (A) and “warm white” (B) spectral ratings exhibit a discontinuous spectrum with prominent mercury lines and significant UV emission. Data represent averages of 6 independent spectral scans.
Fig. 2 Qualitative and quantitative impact of UV irradiation on total RA and relative amounts of RA isomers in cell culture media. The total amount of retinoic acid is significantly affected by UV irradiation only at 1000 mJ cm−2 but remains unaffected at lower dosages of 1–100 mJ cm−2 (A). Conversely, the relative amounts of the three major isomers at-RA (B), 9-cis- (C), and 13-cis-RA (D) are significantly affected at lower dosages of 1–100 mJ cm−2 in a dose dependent manner and even significantly altered at the lowest dosage of 1 mJ cm−2. While relative amounts of at-RA decrease with increasing dosage (B), biologically less active 13-cis-RA increases substantially (D). While 9-cis-RA also increases significantly with UV exposure, the total amount of 9-cis-RA is rather small compared to 13-cis-RA (1, 8% vs. 22% of total RA at 100 mJ cm−2). The severe drop in total RAs at 1000 mJ cm−2 (A) was associated with the appearance of several early-eluting products in the HPLC analysis, likely representing degradation products of RAs at this irradiation dose. * significantly different from sham-treated controls (0 mJ cm−2), one-way ANOVA followed by Tukey’s post test.
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Photochemical & Photobiological Sciences and dose-dependent change compared to sham-treated controls (0 mJ cm−2). While at-RA levels significantly decreased, the two isomers 13-cis and 9-cis RA significantly increased with increasing UV dosage, indicating a photoisomerization process, at least for the lower range of UV dosages. These effects were statistically significant even at the lowest amount of UV irradiation applied (1 mJ cm−2; Fig. 2). The severe drop in total RAs at 1000 mJ cm−2 (Fig. 2A) was associated with the appearance of several early-eluting products in the HPLC analysis, likely representing degradation products of RAs at this irradiation dose. 3.3.
Impact of ILB and CFL emitted radiation on local at-RA
The qualitative and quantitative impacts of various light sources on extra- and intracellular at-RA were studied using a human keratinocyte cell line. Fig. 3 demonstrates a dose- and light source-dependent impact of light exposure on the levels of extracellular (A–C) and intracellular (D–F) RA isomers. Exposure to radiation emitted from “warm-white” (black bars) or “cool white” (grey bars) CFLs results in a significant, dosedependent decomposition of extracellular at-RA (A), mainly leading to the formation of biologically less active 13-cis-RA (C). Conversely, exposure to the light emitted from a traditional 60 W ILB results in significantly less degradation of at-RA into 13-cis-RA (A–C). Similar effects are observed for
Paper intracellular retinoids, where both “warm-white” and “cool white” CFLs result in significant at-RA degradation, while ILB exposure remains without effect on intracellular at-RA levels (Fig. 3D–F). 3.4. Prevention of CFL-induced at-RA decomposition using a UV edge filter At-RA exhibits a maximum absorption at about 345 nm (Fig. 4A). When superimposed with a characteristic emission spectrum of a CFL (“warm-white”), there are at least two strong peaks at 367 nm and 405 nm in the close vicinity of the absorption maximum of at-RA. Therefore, a polycarbonatebased UV edge filter (derived from eye protection device no. 303, UVP, Upland, CA, USA) effectively eliminating these two peaks from the spectrum of a “warm-white” CFL was selected (Fig. 4B) and deployed in a cell culture exposure paradigm similar to experiments depicted in Fig. 3. As expected when the major absorption peaks of at-RA were almost completely filtered the intra- and extracellular at-RA degradation was prevented (Fig. 5). 3.5.
Impact of irradiation on at-RA-mediated viability
Exposing the highly at-RA-responsive human SH-SY5Y cells to at-RA in serum depleted medium resulted in a dose-dependent enhancement of cell viability after 48 h of incubation, as
Fig. 3 Qualitative and quantitative impact of different light sources on at-RA (1 µM) in human keratinocyte cell cultures. A traditional 60 W ILB, a “warm-white” and a “cool white” CFL, all at comparable luminous flux ratings, were used to irradiate human keratinocyte cell cultures. Relative concentrations (fold of total RA) of extracellular RA isomers were determined after 0, 30 and 60 min exposure (A–C), while intracellular concentrations were assessed after 5 min exposure of dense, PBS-covered keratinocyte layers (D–F). Values are means of at least three independent experiments ±SD, * significantly different ( p < 0.05).
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Fig. 4 (A) Depicts the absorption spectra of at-RA (black line; right abscissa) and 13-cis-RA (dashed grey line; right abscissa) dissolved in sodium acetate (100 mM) and methanol (20% v/v), superimposed with the emission spectrum of a single-enveloped CFL with “warm-white” spectrum (straight line; left abscissa) indicating a significant UV emission peak at 365 nm, close to the absorbance maximum of at-RA. (B) Depicts the effects of a standard UV-filter (straight line), completely eliminating the strong peak at 365 nm and largely reducing a peak at 405 nm from the unfiltered spectrum (dashed line) without significantly affecting the rest of the spectrum in the visible range.
Fig. 5 Prevention of CFL-induced at-RA decomposition in human keratinocyte cell cultures using a standard UV-filter. A standard, single-enveloped “warm-white” CFL was used either with or without a UV-filter to irradiate human keratinocyte cell cultures for the indicated time periods (A–C) and cell layers for 10 min (D–F). Relative concentrations (fold of total RA) of extracellular RA isomers were determined after 0, 30 and 60 min exposure (A–C) for both conditions. Similarly, intracellular concentrations were assessed after 5 min exposure of dense, PBS-covered keratinocyte layers (D–F). Values are means of at least three independent experiments ±SD, * significantly different ( p < 0.05).
evidenced by a standard tetrazolium-based viability assay (Fig. 6). At-RA mediated effects were significantly attenuated by pretreating cell-free culture media (notably not the cells) either with UV irradiation (500 mJ cm−2) or a standard “warm-white”
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CFL for 120 min at a distance of 100 mm (Fig. 6). Importantly, irradiation of media lacking at-RA (VEH) did not have significant effects on cell viability, indicating that irradiation effects depend on – and are mediated by – the presence of at-RA.
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Fig. 6 Impact of irradiation on at-RA-mediated viability. At-RA-containing cell culture media were exposed to UV irradiation and to a CFL for 120 min. Exposed media were diluted 1 : 10 and 3 : 4 (100 nM and 750 nM total RA isomers) and subjected to the highly at-RA responsive human SH-SY5Y cells in serum depleted medium. Following 48 h incubation, at-RA-mediated effects on cell viability were quantified using a standard colorimetric tetrazolium-based assay, indicating a strong, concentration-dependent effect of at-RA, which is significantly attenuated by prior irradiation of the cell culture media. Values are presented as means ± SD of n = 6–8 samples. * p < 0.05, two-way ANOVA with Bonferroni’s post test.
4.
Discussion
For reasons of low efficiency, traditional ILBs are increasingly being replaced by various types of CFLs. These often singleenveloped fluorescent lamps emit significantly more UV radiation than ILBs. Own emission recordings and spectral analyses by others6 clearly demonstrate this fact for both, CFLs meant to mimic daylight (“cool white”) and those for ambient light applications (“warm-white”; Fig. 1A and B). Biological implications for these spectral differences, including biologically relevant effects on light-exposed tissues, are largely unclear to date. From a chronobiological perspective, exposure to blue visible light represents an important natural zeitgeber for entraining circadian rhythms in most light-exposed organisms. Under normal conditions, blue visible and UV light is absent at night, and minimal in traditional light sources such as fires, candles and ILBs. This may change with the use of CFLs, yet “clinical relevance” of the emitted amount of blue visible light and UV radiation remains subject to discussion.60 Recently, the striking differences between ILBs and CFLs have again been discussed10,61 and “clinical relevance” has gained considerable support by a study demonstrating significant effects of CFL-emitted radiation on human fibroblast and keratinocyte cell culture models, which was consistent with damage from UV irradiation.4 So far, none of these studies aimed at providing a direct molecular correlate for the divergent, putatively UV-associated cellular reactions to ILB and CFL irradiation, nor did they employ filters in a “rescue approach”. Since biologically relevant effects of light (e.g. phototransduction or photodisease) may be caused either by light-
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Paper mediated formation of toxic compounds or by decomposition of e.g. essential endogenous molecules (e.g. photoisomerization), we chose to characterize the latter by studying the direct effects of CFL- and ILB-emitted light as well as of UV irradiation on intra- and extracellular retinoids in a human keratinocyte cell culture model.48 Besides their famous roles in phototransduction or neuronal differentiation, retinoids exert profound effects on various homeostatic processes, including effects on local inflammation, lipid metabolism, photoaging, entrainment of chronobiological rhythms and, most importantly, controlling the balance between proliferation and differentiation.45,62,63 Since all of these processes are known to be affected by exposure to UV radiation, retinoids may provide a common denominator for linking some of the diverse light-mediated biological effects. In fact, many at-RA mediated effects on the human skin are antagonistic to the effects observed for UV radiation, such as at-RA being chemopreventive for keratinocyte carcionoma and protective of photoaging, conditions which are both known to be caused by UV exposure.37 Most retinoids exhibit peak absorptions between 320 and 360 nm and a dose-dependent sensitivity to UV radiation, which we were able to characterize for at-RA in cell culture media (Fig. 2). Interestingly, light-mediated effects on at-RA were found to be mainly based on isomerisation rather than oxidative modification with a clear preference for the biologically less active 13-cis-RA to be generated under UV exposure (Fig. 2). The levels observed for 13-cis-RA were surprisingly high if compared to e.g. published values for retinal.59 To preclude the possibility of an overestimation of the observed disproportionate decrease in at-RA due to differences in absorption coefficients at 350 nm between 13-cis- and at-RA, absorption spectra were recorded for both isomers at 1 µM, revealing almost identical absorption spectra. Therefore, the disproportionate decrease in at-RA and increase in 13-cis-RA may not have been overestimated but might rather be due to catalytic/ enzymatic processes. CFL, but not ILB irradiation mimicked the effects of direct UV irradiation, resulting in dose-dependent degradation of at-RA (Fig. 3). These findings highlight significant differences between CFL and ILB-emitted light on the one hand, and clearly demonstrate the strong effects of light on local levels of different isomers of RA. Interestingly, these effects were equally observed for serumand cell culture media free conditions (Fig. 3D–E) as well as in conditions containing complete DMEM cell culture media + 10% FCS (Fig. 3A–C), suggesting that potentially interfering photosensitizers may, at least in this particular case, not have significantly affected the experimental outcome. In the skin, it is foremost the all-trans isomer of RA (at-RA) that exerts potent anti-acne, anti-inflammatory effects and is effectively used as a therapeutic agent for conditions such as acne vulgaris, photoaging, psoriasis or ichthyosis.64 These findings highlight the importance of local at-RA homeostasis in extraretinal locations, where even compounds that solely
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Paper inhibit the degradation of at-RA can mimic its therapeutic actions.65 The all-trans isomer of RA is a ligand for a number of specific retinoid receptors (RAR and RXR), which act as heterodimeric, ligand-activated transcription factors. They belong to the superfamily of ‘class I nuclear receptors’ which control transcription via binding to specific ‘retinoic acid response elements’ in the promoter regions of e.g. differentiation- or plasticity-associated genes.28 While at-RA preferentially binds RAR and RXR, 13-cis RA represents a much weaker agonist at both RXR and RAR.26 In fact, biological actions of 13-cis-RA are thought to be mediated through intracellular isomerisation into at-RA.27 This highlights the functional relevance of the isomerization state of RA, ultimately determining the biological activity of the molecule. Therefore, any process directly affecting the isomerization behaviour of local at-RA may in consequence be of relevance for its biological function. Additionally supporting this line of evidence, at-RA has recently been shown to be one of only a few naturally occurring small molecules capable of entraining or even resetting intracellular transcriptional oscillations of “clock genes” in vitro.66 Thus, our findings of a pronounced impact of CFL irradiation on extra- and intracellular at-RA levels may represent a potential mechanism for CFL-induced effects on light-exposed tissue (Fig. 3). The complete prevention of CFL-mediated at-RA degradation by employing a simple edge filter proves the majority of the observed effects to be solely dependent on the short-waved portion
