J. Pineal Res. 2013; 54:303–312
© 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12018
Journal of Pineal Research
Melatonin enhances antioxidative enzyme gene expression (CAT, GPx, SOD), prevents their UVR-induced depletion, and protects against the formation of DNA damage (8-hydroxy-2’-deoxyguanosine) in ex vivo human skin Abstract: UV radiation (UVR) induces serious structural and functional alterations in human skin leading to skin aging and carcinogenesis. Reactive oxygen species are key players in UVR-mediated photodamage and induce the DNA-base-oxidized, intermediate 8-hydroxy-2’-deoxyguanosine (8-OHdG). Herein, we report the protective action of melatonin against UVR-induced 8-OHdG formation and depletion of antioxidative enzymes using ex vivo human full-thickness skin exposed to UVR in a dose (0, 100, 300 mJ/cm2)and time-dependent manner (0, 24, 48 hr post-UVR). Dynamics of depletion of antioxidative enzymes including catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), or 8-OHdG formation were studied by real-time PCR and immunofluorescence/immunohistochemical staining. UVR-treated skin revealed significant and immediate (0 hr 300 mJ/cm2) reduction of gene expression, and this effect intensified within 24 hr postUVR. Simultaneous increase in 8-OHdG-positive keratinocytes occurred already after 0 hr post-UVR reaching 71% and 99% up-regulation at 100 and 300 mJ/cm2, respectively (P < 0.001). Preincubation with melatonin (10 3 M) led to 32% and 29% significant reductions in 8-OHdG-positive cells and the prevention of antioxidative enzyme gene and protein suppression. Thus, melatonin was shown to play a crucial role as a potent antioxidant and DNA protectant against UVR-induced oxidative damage in human skin.
Introduction Ultraviolet radiation (UVR) is known as one of the major environmental factors that affects structure and function of mammalian skin. It induces numerous important changes such as acute solar dermatitis, chronic photoaging, and skin carcinogenesis [1–4]. In these UVR-mediated damaging events, oxidative stress is a known key factor represented by the generation of reactive oxygen species (ROS) or induction of oxidative DNA damage [5–7], which are also involved in skin aging and carcinogenesis [8]. UVR-mediated skin damage is also based on the evidence that singlet oxygen (1O2), superoxide anion (O2• ), hydrogen peroxide (H2O2), and the devastatingly damaging hydroxyl radical (•OH) induce the formation of 8-hydroxy-2’-deoxyguanosine (8-OHdG) representing oxidatively damaged DNA [6, 9]. Additionally, antioxidative enzymes are used in an attempt to protect cells and tissues from free radicals [10]. Therefore, an appropriate strategy for the protection of skin against these UVR-induced alterations is the use of agents with direct and indirect antioxidative properties such as the strong radical scavenger and antioxidant melatonin [11–14]. Melatonin is a phylogenetically ancient methoxyindole, present in all organisms from unicells to vertebrates [15]. Besides, the
Tobias W. Fischer*, Konrad Kleszczyn´ski*, Lena H. Hardkop, Nathalie Kruse and Detlef Zillikens Department of Dermatology, Allergology and Venerology, University of Lu¨beck, Lu¨beck, Germany
Key words: catalase, glutathione peroxidase, human full-thickness skin, 8-hydroxy-2’deoxyguanosine, melatonin, superoxide dismutase, ultraviolet radiation Address reprint requests to Tobias W. Fischer, Department of Dermatology, Allergology and Venerology, University of Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany. E-mail:
[email protected] *These authors equally contributed to the published work. Received July 27, 2012; Accepted September 21, 2012.
pineal [16] melatonin has been reported at extra pineal sites in various body compartments including human bile [17], cerebrospinal fluid [18], ovary [19], eye [20], bone marrow [21], and gastric mucosa [22], etc. It is well-documented for its functional interactions with both the neuroendocrine axis and with circadian systems [23, 24]. Melatonin has also been shown to be synthesized in the skin, and its metabolites are fully expressed in human skin where they are present in a melatoninergic system regulated by melatonin receptors [25–31]. Considerable evidence has been generated to show that melatonin is a direct scavenger of the highly toxic •OH radical [11, 14]. Additionally, because of its highly lipophilic character, melatonin penetrates easily through cellular membranes reaching intracellular compartments including mitochondria and nuclei where it seems to be accumulated [32]. Moreover, our most recent investigations show that melatonin effectively acts against UVR-induced alterations in plasma membrane potential and acidification of cytosol, which are important causes of oxidative stress in human keratinocytes [33]. Recently, a melatoninergic antioxidative system (MAS) has been described for human cutaneous biology [29]. Therefore, some of the antioxidant actions of melatonin involve gene regulation of major antioxidant enzymes including catalase (CAT), glutathione 303
Fischer et al. peroxidase (GPx), and superoxide dismutase (SOD) [34– 36], and it also increases the expression of c-glutamylcysteine synthetase (cGCS), the limiting enzyme in glutathione (GSH) synthesis [37, 38]. Finally, melatonin maintains mitochondrial homeostasis by reducing oxidative stressmediated mitochondria damage [30, 39–42]. In the present study, we investigated in an ex vivo human full-thickness skin organ culture model the negative effects of UVR with regard to oxidative stress on one hand and, on the other hand, evaluated the protective action of melatonin as a sun damage-preventing agent with special regard to its antioxidative properties. We analyzed dynamics of UVR-induced oxidative DNA damage, that is, 8-OHdG formation, and UVR-induced effects on antioxidative enzymes such as CAT, GPx, or SOD on a gene and protein expression level.
Materials and methods Reagents Reagents were purchased as follows: Williams’ E medium containing NaHCO3 (2200 mg/L), glucose (2000 mg/L), CaCl2•2H2O (265 mg/L), essential amino acids (Biochrom AG, Berlin, Germany); L-glutamine (100 9 ), penicillin– streptomycin solution (10,000 units of penicillin and 10 mg of streptomycin in 1 mL 0.9% NaCl) (Invitrogen, Carlsbad, CA, USA); insulin from bovine pancreas, hydrocortisone, acetone, ethanol, 4’,6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO, USA); normal goat serum (NGS), normal rabbit serum (NRS) (Dako, Inc., Carpinteria, CA, USA). Skin preparation and culturing Explants were full-thickness skin samples taken from independent Caucasian individuals (age range = 22–56 yr) undergoing abdominoplastic surgery after informed consent. All subjects were in good health with no evidence for systemic disease. Skin samples were delivered to the laboratory within 1–1.5 hr postsurgery in Williams’ E medium on ice. The skin was defatted, cut by scalpel into sample pieces of 0.5 9 1.0 cm, rinsed abundantly with 1 9 PBS (pH 7.2), and placed epidermis up/dermis down into 6-well plates. Skin samples were cultured in a humidified atmosphere (5% CO2, 37°C) in 2 mL serum-free Williams’ E medium according to earlier validated conditions regarding human skin or human hair follicle culture [43–45].
transferred to new 6-well plates, and 2 mL of 1 9 PBS was added again prior to UVR exposure. UV irradiation experiments were performed with a Bio-Rad UV transilluminator 2000 (Bio-Rad, Laboratories, Hercules, CA, USA) calibrated and validated by Piazena Biolabs (Berlin, Germany). The UVR source emission consisted primarily of UVB (280–320 nm; ~60%), with minor output in the UVA (320–400 nm) and UVC (120–280 nm) range (~30% and 10%, respectively) [29, 30]. UV-irradiated human skin was examined in a UVR-dose (0, 100, 300 mJ/cm2)- and time-dependent (0, 24, 48 hr post-UVR) manner where 0 hr equaled approximately 8 min post-UVR due to freezing procedure. UVR doses were achieved after 30 and 91 s of irradiation for 100 and 300 mJ/cm2, respectively, at a distance of 10.5 cm. Simultaneously, sham-irradiated samples (0 mJ/cm2) were generated by fully covering 6-well plates with aluminum foil. PBS was removed from skin samples, which were then cultured in presence of 2 mL culture medium for the next 24 and 48 hr with fresh medium changed every 24 hr. After specific times, the skin specimen was divided into halves of which one was placed in 80°C until further procedures for real-time PCR, and the other half was embedded in freezing Cryomatrix formula (Thermo Scientific Ltd., Runcore, UK), snap-frozen in liquid nitrogen, and subjected to cryosections (d = 6 lm) using Leica CM 3050S research cryostat (Leica Mikrosysteme GmbH, Wetzlar, Germany) for further immunofluorescence/immunohistochemical staining. RNA isolation Tissues were treated with TRIzol Reagent and homogenized on ice using pellet pestle. Chloroform was added, and the sample was shaken vigorously by hand for 15 s and incubated for 2–3 min at room temperature (RT). The samples were centrifuged for 15 min at 8000 9 g in 4°C resulting in separation of the mixture into a lower organic phase (proteins, lipids), an interphase (DNA), and a colorless upper aqueous phase containing exclusively RNA. The aqueous phases were transferred into the new tube, and these samples were precipitated using 2-propanol, shaken vigorously, kept for 10 min, and centrifuged for 20 min at 8000 9 g in 4°C. Supernatants were discarded, and the resultant pellets were washed using ice-cold 70% EtOH, and vortexed to release the pellet. The samples were centrifuged at 8000 9 g in 4°C, supernatants were discarded, and RNA pellets were suspended in 0.1% DEPCtreated water and stored in 80°C before cDNA synthesis and real-time PCR analysis.
Melatonin-incubated skin and UV irradiation After 24 hr of normal culture to allow recovery from possible preparation-induced tissue stress, culture medium was replaced with fresh medium containing melatonin (Sigma) or with melatonin-free medium as control. Melatonin was dissolved in absolute ethanol (final ethanol concentration< 0.2%) and further diluted with PBS to yield 10 2 M stock solution, while skin samples were incubated with 10 3 M final concentration. After 1-hr incubation with melatonin, skin samples were washed twice with 1 9 PBS to remove remnants of medium and melatonin, 304
cDNA synthesis and real-time PCR Isolated RNA samples were measured for total RNA ratio using NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Every sample was taken for cDNA synthesis using SuperScriptTM III First-Strand Synthesis System for reverse transcriptasePCR (RT-PCR) according to protocol described by the supplier (Invitrogen). Briefly, an appropriate volume of RNA was taken and mixed with DEPC-treated water, 50 ng/lL random hexamers, and 10 mM dNTPs mix.
Melatonin and UVR-induced oxidative damage Denaturation and annealing reaction were carried out using C1000 Thermal Cycler (Bio-Rad) at 65°C for 5 min and subsequently placed on ice. Meanwhile, cDNA synthesis mix was prepared containing 10 9 RT buffer, 25 mM MgCl2, 0.1 M DTT, RNaseOUT (40 U/lL), and SuperScriptTM III RT (200 U/lL). Next, the sample was added to RNA/primer mixture and incubated as follows at 25°C for 10 min, 50°C for 50 min. The reaction was terminated at 85°C for 5 min and cooled on ice. RNase H was added, the reaction was carried out at 37°C for 20 min, and the resultant cDNA was stored at 20°C prior to real-time PCR. Real-time PCRs were carried out in triplicate using a LightCycler® 480 (Roche, Mannheim, Germany) in a mixture containing Maxima® SYBR Green qPCR Master Mix (Fermentas GmbH, St. Leon-Rot, Germany) and 10 ng of cDNA in a total volume of 20 lL in LightCycler® borosilicate glass capillaries (Roche). The following primers were used: CAT, 5’-CGTGCTGAATG AGGAACAGA-3’ (forward) and 5’-AGTCAGGGTGGA CCTCAGTG-3’ (reverse); GPx, 5’-ACGATGTTGCCT GGAACTTT-3’ (forward) and 5’-GATGTCAGGCTCGA TGTCAA-3’ (reverse); Cu/Zn–SOD, 5’-GGCAAAGGT GGAAATGAAGA-3’ (forward) and 5’-GGGCCTCAGA CTACATCCAA-3’ (reverse); Mn–SOD, 5’-GCTCATGC TTGAGACCCAAT-3’ (forward) and 5’-CACCCGAT CTCGACTGATTT-3’ (reverse); b-actin, 5’-TGGCACCCAGCACAATGAAG-3’ (forward) and 5’-GACTCGTC ATACTCCTGCTTGC-3’ (reverse). PCRs were performed using a 10-min initial denaturation at 95°C followed by 50 three-step cycles of 15 s at 95°C (denaturation), 30 s at 59°C (annealing), and 30 s at 72°C (extension). Relative expression of the genes was calculated with the DCT method with the elongation of b-actin gene used as a housekeeping gene. Immunofluorescence detection of CAT and SOD expression The immunofluorescence detection focused on the UVRinduced expression of antioxidative enzymes (CAT and Cu/ Zn–SOD). Cryosections were dried for 10 min at RT, fixed with ice-cold acetone for 10 min at 20°C, and washed three times for 5 min each with 1 9 PBS. The preincubation was carried out using 5% NRS for 1 hr at RT, and sections were incubated overnight at 4°C with the respective specific antibodies: polyclonal goat anti-CAT IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:50), polyclonal sheep anti-Cu/Zn–SOD IgG (Merck KGaA, Darmstadt, Germany; 1:200). Next, the slides were washed three times for 5 min each with 1 9 PBS and treated in presence of 2% NRS with secondary rabbit anti-goat monoclonal IgG conjugated with rhodamine (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:400) for CAT, and secondary rabbit anti-sheep monoclonal IgG conjugated with rhodamine (Jackson ImmunoResearch Laboratories, Inc.; 1:800) for Cu/Zn–SOD. Sections were counterstained for cell nucleus detection with DAPI solution (1 lL/mL) for 1 min, washed three times for 5 min each with 1 9 PBS, and mounted in Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL, USA). All fluorescent images were then obtained
using the Keyence inverted fluorescence microscope, BZ8000 (Keyence GmbH, Neu-Isenburg, Germany). Immunohistochemical localization of 8-OHdG Immunohistochemical assessment of 8-OHdG was carried out by a modified procedure described earlier [46]. Sections were defrosted for 10 min at RT, fixed with ice-cold acetone for 20 min at 20°C, and washed three times for 5 min each with 1 9 PBS. Endogenous peroxidases were quenched using 3% H2O2 for 20 min at RT, washed three times for 5 min each with 1 9 PBS, and subsequently preincubated with 10% NGS for 20 min at RT. After that, sections were incubated overnight at 4°C with monoclonal mouse anti-8-OHdG (N45.1) IgG (JaICa, Shizuoka, Japan; 1:250). After incubation with the primary antibodies, the cryosection slides were washed three times for 5 min each with 1 9 PBS and incubated with secondary biotin-labeled goat anti-mouse IgG (Beckman Coulter, Marseille, France; 1:200) for 60 min at RT. Tissue sections were then again washed three times for 5 min each with 1 9 PBS and treated with Vectastain ABC Kit (Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min at RT. Followed by washing with 1 9 PBS, immunoreactivity was developed in the presence of H2O2 with 3,3’-diaminobenzidine (DAB) (Vector Laboratories, Inc.) for approximately 1 min. Skin sections were washed three times for 5 min each with 1 9 PBS and mounted in aqueous mounting medium (Dako, Inc.). All immunohistochemical images were obtained using the BH-2 light microscope (Olympus Optical Co., Tokyo, Japan). Statistical analysis Data were expressed as pooled means ± standard error of the mean (S.E.M.) of three independent experiments containing six taken images per condition by cell counting of immunoreactivity-positive cells (CAT, 8-OHdG). Quantification of Cu/Zn–SOD expression was performed using Image J 1.38d software (National Institute of Health, USA). Real-time PCR analysis was carried out in triplicates per condition using three independent experiments. Comparative data were analyzed using one-way ANOVA test with GraphPad Prism 5.02 software (La Jolla, CA, USA). Regarding immunolabeling, values were normalized and expressed as percentage of the control value, that is, UV-irradiated, non-melatonin-treated sample at 300 mJ/ cm2 after 24 hr post-UVR for 8-OHdG or sham-irradiated non-melatonin-treated sample at 0 hr post-UVR for CAT and Cu/Zn–SOD. In case of real-time PCR, values were normalized to 0 hr 0 mJ/cm2-Mel sample. Data were presented as fold change of relative gene expression (CAT, GPx, Cu/Zn– SOD, Mn–SOD) calculated with the DCT method with the elongation of b-actin gene used as a housekeeping gene. Differences were considered statistically significant and indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.
Results Significant UVR-induced down-regulation of gene expression of all investigated antioxidative enzymes (CAT, GPx, 305
Fischer et al. Cu/Zn–SOD and Mn–SOD) was observed in a UVR-doseand time-dependent manner (Fig. 1; Figure S1). First distinct differences were detected directly after UVR exposure (0 hr) at 100 mJ/cm2 reaching the level of 68% (P < 0.001) and 76% (P < 0.05) for CAT and GPx, respectively, compared to the control (0 hr 0 mJ/cm2Mel). Suppression of SOD gene expression reached 91% (Cu/Zn–SOD) and 76% (Mn–SOD) compared to the control; however, the difference was not statistically significant. The higher UVR dose of 300 mJ/cm2 at 0 hr caused distinct down-regulation of antioxidative enzyme gene expression to 42% (P < 0.001), 58% (P < 0.001), 71% (P < 0.05), and 54% (P < 0.001) for CAT, GPx, Cu/Zn– SOD, and Mn–SOD, respectively. Preincubation with melatonin (10 3 M) led to a significant prevention of UVRinduced gene down-regulation, ranging for all enzymes between 1.01- to 1.37-fold and 1.49- to 1.90-fold upregulation at 0 hr 100 mJ/cm2 and 0 hr 300 mJ/cm2, respectively. Moreover, after 24 hr post-UVR 300 mJ/cm²,
gene expression was significantly further down-regulated by 32% (P < 0.01; CAT), 40% (P < 0.05; GPx), 41% (P < 0.01; Cu/Zn–SOD), and 13% (P < 0.01; Mn–SOD) compared to 300 mJ/cm2 at 0 hr. At 48 hr post-UVR, no further significant down-regulation compared to 24 hr was observed; however, gene expression levels in melatonintreated skin samples were still high at the same level as at 24 hr. It arrested the negative influence of UVR by 60% (CAT), 51% (GPx), 24% (Cu/Zn–SOD), and 47% (Mn– SOD) up-regulation in case of 100 mJ/cm2 at 48 hr post-UVR. Furthermore, melatonin counteracted the UVR-induced down-regulation of gene expression at the higher dose of 300 mJ/cm2 maintaining the gene expression level by 60% (CAT), 56% (GPx), 59% (Cu/Zn– SOD), and 45% (Mn–SOD). Parallel to suppression of gene expression, UVR led to significant reduction in in situ protein detection of antioxidative enzymes as exemplarily shown for CAT and Cu/Zn–SOD by immunofluorescence microscopy (Fig. 2A–D; Figure S2). Exposure with GPx
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Fig. 1. Gene expression of antioxidative enzymes in the epidermis in UVR-treated human skin. Evaluation was carried out by real-time PCR in dose- and time-dependent manner as described in Materials and methods. Data were expressed as pooled means ± S.E.M. of three independent experiments conducted in triplicates for each condition. Relative expression of the genes was calculated with the DCT method with the elongation of b-actin gene used as a housekeeping gene. Statistically significant differences were indicated as *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.
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Fig. 2. Protective effect of melatonin against UVR-mediated down-regulation of antioxidative enzymes in human skin in dose- and timedependent manner. UVR-induced alterations of CAT (A) or Cu/Zn–SOD (B) activity were noticed already at 0 hr 300 mJ/cm2 and deepened within culture time. Enzymes were detected using antibodies conjugated with rhodamine (red); DAPI was used for the nucleus (blue). One representative experiment of three is shown. Dashed line shows the basement membrane. Arrows show CAT- and Cu/Zn–SOD-positive cells. Bars = 50 lm. Evaluated data (C,D) were presented as pooled means ± S.E.M. of three independent experiments containing six taken images per condition. Values were expressed as percentage of the control value, that is, sham-irradiated without melatonin at 0 hr post-UVR. Statistically significant differences were indicated as *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.
the UVR dose of 100 mJ/cm2 led to depletion of the enzymes; however, significant differences compared to the control value were noticed only at 48 hr post-UVR reaching a reduction of 67% (CAT) and 32% (Cu/Zn–SOD) (P < 0.001). Preincubation with melatonin caused increased number of immunoreactivity-positive cells by 40% (CAT) and 32% (Cu/Zn–SOD); however, the difference was only
significant in Cu/Zn–SOD enhancement (P < 0.01). At 300 mJ/cm², first significant decreases in CAT and Cu/Zn– SOD were observed directly after UVR exposure (i.e. 0 hr) reaching 46% (CAT) (P < 0.001) and 17% (Cu/Zn–SOD) (P < 0.001) reductions, respectively, compared to 0 hr 0 mJ/cm2-Mel. In contrast, melatonin-incubated samples prevented UVR-induced reduction of both enzymes at 0 hr 307
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Discussion This study demonstrates a melatonin-mediated enhancement of the endogenous antioxidative enzyme activities network of the skin as an important and protective mechanism against UVR-induced oxidative damage. This action of melatonin overcame the consecutive reduction in UVRinduced depletion of antioxidative enzymes and prevention of formation of 8-OHdG. It is well accepted that skin contains an antioxidant network, which is mainly represented by the antioxidative enzymes CAT, GPx, or SOD [47–49]. The enclosed results provide evidence that the antioxidant network follows a quite specific time-dependent and UVR-dose-dependent manner in human full skin, and melatonin protects it. By doing so it significantly reduces the appearance of 8-OHdG-positive cells in the epidermis. Interestingly, investigations regarding the general activity 308
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300 mJ/cm2 by 30% (CAT) (P < 0.001) and 11% (Cu/Zn– SOD) (P < 0.05). At 24 hr post-UVR, a significantly increased depletion of protein detection was observed by 60% for CAT and 33% for Cu/Zn–SOD compared to nonirradiated skin at 0 hr (P < 0.001). Also compared to 300 mJ/ cm2 irradiated skin at 0 hr, the enzyme depletion at 24 hr had significantly increased (P < 0.01). Again, skin that had been preincubated with melatonin showed distinct protective effects leading to 38% (P < 0.05) and 40% (P < 0.001) protein expression enhancement for CAT and Cu/Zn–SOD, respectively. At 48 hr post-UVR, the UVR-induced depletion of both antioxidative enzymes was slightly increased, however, without further statistically significant difference compared to 24 hr. Again, melatonin-preincubated skin analyzed at 48 hr after irradiation with 300 mJ/cm2 showed significantly higher levels of protein detection compared to non-melatonin-treated skin resulting in 45% (P < 0.01) and 36% (P < 0.001) increased number of positive cells of CAT and Cu/Zn–SOD, respectively. Distinct UVR-induced oxidative DNA damage represented by 8-OHdG formation was located in the epidermis in all living layers (stratum basale, spinosum, granulosum) as shown by immunohistochemical staining (Fig. 3A). Investigations of dose- and time-dependent oxidative DNA damages showed fast response upon UVR exposure at 100 and 300 mJ/cm2 at 0 hr reaching 71% and 99% increase, respectively, compared to sham-irradiated control samples (P < 0.001) (Fig. 3B). Skin samples cultured for the following 24 hr did not reveal significant differences in the number of 8-OHdGpositive cells at 300 mJ/cm2; however, after 48 hr, a significant decrease of 8-OHdG-positive cells was observed by 59% and 49% at 100 and 300 mJ/cm2 (P < 0.001), respectively, compared to 24 hr. Comparative analysis regarding melatonin-preincubated skin showed the reduction in 8OHdG formation, which was significant at 0 and 24 hr at both UVR doses (P < 0.01 and P < 0.001). Melatonin distinctly inhibited oxidative DNA damage by 32% and 29% at 100 and 300 mJ/cm2, respectively, after 0 hr post-UVR. At 24 hr post-UVR, the protective action of melatonin was more pronounced by 47% and 38% decrease of 8-OHdGpositive cells at 100 and 300 mJ/cm2. At 48 hr, no significant differences between melatonin-treated and nontreated samples were observed.
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Fig. 3. Dynamics of UVR-induced 8-hydroxy-2’-deoxyguanosine in human skin and protective action of melatonin. Investigations were conducted in dose-dependent manner (0, 100, 300 mJ/cm2) and cultured for 0, 24, and 48 hr post-UVR. Sections were labeled using immunohistochemical staining (A) for 8-OHdG and were detected by catalyzed signal amplification using 3,3’-diaminobenzidine (yields brown-colored precipitate). One representative experiment of three is shown. Dashed line shows the basement membrane. Bars = 50 lm. Evaluated data (B) were presented as pooled means ± S.E.M. of three independent experiments containing six taken images per condition. Values were expressed as percentage of the control value, that is, UV-irradiated and without melatonin sample at 300 mJ/cm2 after 24 hr post-UVR. Statistically significant differences were indicated as *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.
of antioxidative enzymes in human skin show many-fold higher expression within the epidermis than in the dermis [47, 48]. Investigations related to UVR-exposed human skin conducted by Sander et al. [50] showed that the
Melatonin and UVR-induced oxidative damage dermal levels of antioxidative enzymes are not significantly reduced compared to epidermal layers. In contrast to our study, Sander et al. [50] found the highest levels in the stratum corneum, whereas in our study, antioxidative enzymes were detected in all layers with slight prominence in the basal layer, which makes perfectly sense, because the DNA of proliferating basal layer keratinocytes is the major chromophore for UVB radiation [51]. In this context, the fast reaction of the antioxidative defense system on a gene expression level suggests that this system is very sensible to UV irradiation. This hypothesis shows that single exposure of UVR directly results in an extremely fast reduction in gene expression of all antioxidative enzymes with CAT and GPx being significant already at 100 mJ/ cm² and all being significant at 300 mJ/cm² at 0 hr postUVR. Such fast response is consistent with previous investigations carried out by Punnonen et al. [52]. Additionally, the significant higher gene expression for CAT and Cu/Zn –SOD in melatonin-pretreated skin was noticed already at 0 hr 100 mJ/cm², and for all enzymes at 300 mJ/cm². This shows how melatonin may function as an immediate ‘SOS’-like response protectant. In the context of DNA damage, there is a close relationship between hypergeneration of ROS and 8-OHdG [53, 54]. Accumulation of highly reactive ROS triggers subsequent damaging events including formation of 8-OHdG [9, 55]. Depending on the conditions, 8-OHdG may constitute 20–30% of the deoxyguanosine damage in DNA, equivalent to 5–11% of the total DNA nucleoside damage [6, 9]. Moreover, 8-OHdG reveals mutagenic ability to pair with adenine, instead of cytosine, leading to G:C into T:A transversions if the damage is not repaired prior to DNA replication [56, 57]. Here, melatonin as an antioxidative agent has been shown to directly scavenge hydrogen peroxide [58], hydroxyl radicals [59], singlet oxygen (1O2) [60], superoxide anion (O2•-), nitric oxide (NO•) [61], and peroxynitrite anion (ONOO-) [37]. Regarding the chemical structure of melatonin, it possesses an electronrich aromatic indole ring and functions as an electron donor, thereby being able to reduce the amount of electrophilic radicals [62]. A melatoninergic system expressing all essential enzymes for melatonin synthesis and melatonin regulatory receptors is fully expressed in human skin [26, 27]; thus, it is defined as a key agent of the melatoninergic antioxidative system of the skin to reduce excessive ROS generation [49]. Therein, melatonin is transformed into its metabolites including 2-OH-melatonin, 4-OH-melatonin, and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) or N1-acetyl-5-methoxykynuramine (AMK) [29], which are strong antioxidants themselves, therefore building a continuously self-potentiating antioxidative cascade as a defense line against UVR-induced oxidative stress. It was also reported earlier that melatonin’s indirect antioxidative action is mediated through the stimulation of enzyme mRNA levels via membrane and/or nuclear receptors [63]. However, regarding its role of protecting antioxidative enzymes and preventing UVR-induced oxidative DNA damage, these effects have – to the authors’ knowledge – not yet been described in human skin and thus fill the very essential gap of missing information. While the dynamics of UVR-induced 8-OHdG formation have been demon-
strated using human skin [8], the protective effects of melatonin in UV-irradiated human skin shown here have been described for the first time. Regarding the distribution of antioxidative enzymes shown earlier in animal skin [64], our results using human full-thickness skin organ culture are consistent with those data. For instance, Chang and Zheng [64] presented also gradual reduction in CAT and SOD protein levels over time, which is confirmative to our analysis. Chang and Zheng [64], using the same UVR doses, reported that irradiation with 300 mJ/cm2 induced immediate (0 hr) reduction in CAT by 30% (P < 0.05), while SOD did not show significant changes compared to the control. Moreover, extended culture for 24 hr revealed prominent reduction in both enzymes reaching approximately 50% down-regulation (P < 0.001) of CAT and SOD with ongoing slight down-regulation after 48 hr; however, differences between these two time points were not statistically significant. Our study confirms these results with regard to UVR effect size and time dependency in all antioxidative enzyme gene expressions and at protein level, which showed no significant decrease between 24 and 48 hr. Apart from this, we hypothesize that 8-OHdG formation observed here is presumably a consequence of massive production of ROS. Reduced levels of 8-OHdG are a result of reduced ROS ratio due to potent direct radical scavenging by melatonin itself as well as indirect ROS reduction by antioxidative enzymes. The decreased detection of 8-OHdG at 48 hr post-UVR compared to 0 and 24 hr and lack of melatonin effect at this time point are most likely due to degradation processes of oxidatively damaged DNA. Of note, resultant depletion of CAT, GPx, or SOD is relatively fast (8 min post-UVR) and synchronic to direct appearance of 8-OHdG-positive cells in the epidermis. Additionally, the UVR-dose-dependent manner of antioxidative enzyme gene reduction, particularly SOD, allows interesting conclusions. On the basis of obtained data presented here, it is obvious that the mitochondrial form (Mn –SOD) shows obviously more prominent gene down-regulation compared to the cytosolic one (Cu/Zn–SOD). From this observation, it might be concluded that UVR causes oxidative stress-mediated harmful actions. These are more prominent in intracellular organelles such as mitochondria than in the cytosol leading to depolarization of mitochondrial transmembrane potential and resultant mitochondria-mediated down-stream activation of apoptosis as presented earlier in keratinocytes [30]. This observation also finds its confirmation in earlier reports presenting the crucial role of Mn–SOD against cumulative oxidative stress events on one side and the superior role of mitochondria-generated ROS (mROS) being the major contributor to oxidative stress in the cell on the other side [65– 67]. Very importantly, melatonin has been reported to reduce mROS generation and calcium release as well as inhibiting the opening of the MPTP as shown in rat brain astrocytes [40], mouse striatal neurons [68], and rat cerebellar granule neurons [69]. It is in good accordance with the presently observed highly significant Mn–SOD gene up-regulation at both UVR doses (100 and 300 mJ/cm²) and at all time points (0, 24 and 48 hr) shown in human full skin for the first time to the authors knowledge. 309
Fischer et al. In conclusion, considering the capacity of melatonin not only as an effective direct radical scavenger, but also as an indirect antioxidant through enhancement of antioxidative enzyme gene expression and as a counteracting substance against oxidative stress-induced DNA damage in human skin, these triple mechanisms of action render melatonin literally a ‘super-potent’ antioxidant against UVR-induced damage on all relevant levels. In contrast to classical antioxidants such as vitamin C and E, which do not show such effects of gene up-regulation of antioxidative enzymes, melatonin has a clear advantage over these classical antioxidants to efficiently protect against skin aging and photocarcinogenesis.
Acknowledgements This investigation was carried out with funds from the Medical Faculty of the University of Lu¨beck, Germany.
Author contributions T.W.F. and K.K. contributed to design of experiments and wrote the manuscript. K.K., L.H.H., and N.K. carried out most of the experiments, presentation, and interpretation of data. T.W.F. contributed to conception and design of the study, specific design of experiments, analysis and interpretation of data as well as drafting the manuscript, and together with D.Z. revised and approved the final version of the manuscript.
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Supporting Information Additional Supporting Information may be found in the online version of this article:
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Figure S1. Gene expression of antioxidative enzymes in the epidermis in UVR-treated human skin without melatonin. Evaluation was carried out by real-time PCR in doseand time-dependent manner as described in Materials and methods. Data were expressed as pooled means ± S.E.M. of three independent experiments conducted in triplicates for each condition. Relative expression of the genes was calculated with the DCT method with the elongation of bactin gene used as a housekeeping gene. Statistically significant differences were indicated as *P < 0.05, §P < 0.01, # P < 0.001. Figure S2. UVR-induced alterations in activity of antioxidative enzymes in human skin in dose- and timedependent manner without melatonin. Evaluated data were presented as pooled means ± S.E.M. of three independent experiments containing six taken images per condition. Values were expressed as percentage of the control value i.e. sham-irradiated without melatonin at 0 hr postUVR. Statistically significant differences were indicated as *P < 0.05, §P < 0.01, #P < 0.001.
