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* Department of Pathology and Laboratory Medicine,
Department of Pharmacology, and
Division of Dermatology, University of Tennessee Health Science Center, Memphis, Tennessee USA;
Department of Dermatology, University Hospital Schleswig-Holstein, University of Lübeck, Lübeck, Germany;
|| Department of Biochemistry, Belarus State University, Minsk, Belarus; and
# Department of Internal Medicine, Southern Illinois University, Springfield, Illinois, USA
1Correspondence: Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, 930 Madison Ave., Memphis, TN 38163, USA. E-mail: aslominski{at}utmem.edu
SPECIFIC AIMS
Melatonin is a strong radical scavenger, able to protect the skin against oxidative damage and cell death induced by UV radiation (UVR). So far, the influence of UVB, the most damaging wavelength of UVR, on the molecule melatonin itself has not been investigated. We studied the formation of melatonin metabolites after exposure of melatonin to UVB, determined metabolite identity, and evaluated metabolism determinants in a pure cell-free system and in HaCaT keratinocytes.
PRINCIPAL FINDINGS
1. Identification of photoproducts of melatonin in a cell-free system: 6-hydroxymelatonin, 2-hydroxymelatonin, 4-hydroxymelatonin, and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK)
Irradiation of melatonin solution in a cell-free system with UV-doses at 25, 50, and 100 mJ/cm2 generated four compounds detected by HPLC with retention times of 30 min (product 1), 34 min (product 2), 35 min (product 3), and 43 min (product 4). The relative amount of each product increased directly proportional with UV-doses (Fig. 1
). Product yield was also dependent on the preincubation concentration of melatonin, which was higher with melatonin at 10–3 M than at 10–6 M. Analysis of UV absorption spectra, HPLC retention time, and mass spectrometry of melatonin metabolites identified product 1 as 6-hydroxymelatonin, product 2 as 2-hydroxymelatonin, product 3 as 4-hydroxymelatonin, and product 4 as N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK).
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2. Determinants of post irradiation melatonin metabolism in keratinocyte supernatants
After irradiation with UVR (25, 50, or 75 mJ/cm2), supernatants of keratinocytes containing melatonin (10–3 M) produced identical compounds compared with the cell-free system. The metabolites identified in supernatants were products 2 (2-hydroxymelatonin), 3 (4-hydroxymelatonin), and 4 (AFMK), whereas product 1 (6-hydroxymelatonin) was not detected. A linear dose response to increasing UV-doses was observed, as well as a time-dependent increase in melatonin photoproducts. High levels of 2-hydroxymelatonin were detected as early as 40 min after UV exposure and remained high at 190 and 370 min post UV-exposure. Product 3, although generally lower than 2-hydroxymelatonin, increased steadily from 40 min to 370 min post-UVR exposure. AFMK also showed a time-dependent increase with highest levels reached at 40 min, remaining almost unchanged at the collection time point of 190 min to decrease at 370 min. As in the cell-free system, the overall levels of AFMK were higher than those of 2-hydroxymelatonin and 4-hydroxymelatonin.
3. Extra- and intracellular partition of melatonin metabolites
The metabolites 2-hydroxymelatonin, 4-hydroxymelatonin, and AFMK were identified at different proportions in supernatants and lysates of keratinocytes. 2-Hydroxymelatonin was present at higher levels in the extracellular compartment (supernatants) of keratinocytes, where it displayed strong increase after irradiation (50 mJ/cm2). Intracellularly, 2-hydroxymelatonin was detectable at very low levels, while still showing an increase after UVR exposure. The metabolite 4-hydroxymelatonin was not detected intracellularly, neither under basal condition (without UV irradiation) nor after UV exposure, but in supernatants preincubated with melatonin and exposed to UVR. AFMK was clearly detected in supernatants of irradiated samples (50 mJ/cm2) where it was 100-fold higher than in nonirradiated samples (Fig. 2
A, upper left inset). Lysates of cells preincubated with melatonin showed that, under nonirradiated conditions, AFMK levels were low but increased after UVR (3.5-fold) (Fig. 2A
, left). AFMK was also detected in supernatants of samples not preincubated with melatonin (Fig. 2A
, upper right inset) and UV-dependend increase was observed (13-fold). AFMK was also detected intracellularly in samples not preincubated with melatonin and showed again a distinct UV-induced 1.9-fold increase (Fig. 2A
, right).
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Melatonin, the main substrate for AFMK production, was expectedly detected at high levels in supernatants of samples preincubated with melatonin (10–3 M) with slight reduction after UV exposure (to 99.7% of levels in the nonirradiated melatonin solution) (Fig. 2B
, inset). Melatonin was not found in cell supernatants without melatonin preincubation, while cell lysates showed detectable levels of melatonin (Fig. 2B
, right). Cell lysates from cells preincubated with melatonin showed expectedly higher melatonin levels than lysates from cells not preincubated with melatonin (Fig. 2B
, left). Nevertheless, with or without melatonin preincubation, cell lysates showed a decrease in melatonin levels after UV exposure (51.4 and 18.9% of unirradiated control, respectively), reciprocal to the increase of AFMK. Under basal conditions (without UV exposure), the ratio of intra- to extracellular melatonin levels in the samples preincubated with melatonin was 
1:800 (0.125%).
4. Kinetics of melatonin metabolism in keratinocytes
Studies in keratinocytes not preincubated with melatonin showed that melatonin and its metabolites, AFMK and 2-hydroxymelatonin, were detected dynamically over 24 h, indicating intense melatonin metabolism. The absolute concentration of melatonin in keratinocytes was 146.0 pmoles/1000 cells, which decreased to 65.0 pmoles/1000 cells after 24 h. AFMK was detected at 17.4 pmoles/1000 cells and showed an increase to 33.6 pmoles/1000 cells after 24 h. The intracellular concentration of 2-hydroxymelatonin increased at 24 h, although to levels lower than AFMK or melatonin (7.8 pmoles/1000 cells that increased to 20.4 pmoles/1000 cells after 24 h). The metabolite 6-hydroxymelatonin was also detected (53.4 pmoles/1000 cells) but showed a decrease after 24 h (16.8 pmoles/1000 cells).
CONCLUSIONS AND SIGNIFICANCE
The present study provides evidence for intense metabolism of melatonin under the influence of UVR in skincells and in cell-free conditions with generation of 6-hydroxymelatonin, 2-hydroxymelatonin, 4-hydroxymelatonin and AFMK and also shows that melatonin can be produced endogenously in untreated keratinocytes. 6-hydroxymelatonin is the chief product of circulating (endogenous) melatonin in humans and the main metabolite of exogenous melatonin (oral intake). In our study, 6-hydroxymelatonin was detected only in non UV-exposed keratinocytes, which is a fact most likely less relevant in cutaneous biology. The second product, AFMK, is generated by enzymatic and nonenzymatic metabolism, both in vitro and in vivo. AFMK generation has been linked to melatonin oxidization by reactive oxygen species (ROS), which are produced at high levels after exposure to UVR. The major and most damaging ROS, the hydroxyl radical, can be scavenged by melatonin, which is consecutively transformed to an indolyl cation radical and, in the presence of O2–, to AFMK. We identified AFMK after irradiation of melatonin in a cell-free system and in supernatants and cell lysates of keratinocytes. The most likely mechanism of AFMK production following UV-exposure is photoinduced cleavage of the pyrrol ring and oxidization by ROS, namely the hydroxyl radical. The third metabolite was 2-hydroxymelatonin, previously identified only in Fenton-type OH-generating systems or in reaction with hypochloric acid. 2-Hydroxymelatonin is the product of a chemical reaction induced by UVR-generated oxygen-based radicals or by the combination of ROS with enzymes such as cytochrome c. The fourth product was identified as 4-hydroxymelatonin, a hydroxylated form of melatonin closely related to 2-hydroxymelatonin.
Kinetic studies on the UVR-induced generation of photoproducts of melatonin in cell supernatants clearly showed progressive increase over time (6 h). The increase of 2-hydroxymelatonin and 4-hydroxymelatonin may be explained by oxidation of melatonin suspended in supernatants and simultaneous metabolism of intracellular melatonin with extracellular release. AFMK showed a late decrease between 3 and 6 h after UVR exposure, which may be related to additional metabolism of AFMK to AMK by arylamine formamidase.
The UVR-induced production of 2-hydroxymelatonin and AFMK in cell lysates of keratinocytes is reported for the first time, together with reduction of melatonin, indicative of substrate consumption during UV irradiation. Over a time period of 24 h, an UVR-independent consumption of melatonin was observed and, in turn, an increase of AFMK and 2-hydroxymelatonin.
We used an UV source emitting UVB (280–320 nm; 60%) and UVA (320–400 nm; 30%), wavelengths that both occur naturally and are, therefore, relevant in cutaneous biology. UVB causes harmful effects in the epidermis, represented by lipid peroxidataion, protein oxidation, and direct DNA damage in proliferating keratinocytes, whereas UVA induces skin aging in the dermis. These processes are the major target for the protective effects of melatonin. AFMK is known to be a strong radical scavenger that protects against these processes. The increased formation of AFMK under progressively higher doses of UVR would, therefore, support the use of melatonin in topically applied sun protectants since increase in the damaging effect of UVR would reciprocably be accompanied by increase of AFMK production. As a result, the organ could remain in equilibrium between damaging effects of UVR and the protective effects of AFMK.
Both the presence of an enzymatic melatoninergic and functionally active system in the skin and the detection of melatonin levels in human and murine hair follicles support the present findings. We have also shown recently that melatonin, at the same concentration as used in the present study (10–3 M), prevents UVR-induced damage in skin cells.
To conclude, metabolism of melatonin is present in keratinocytes. This metabolism is accompanied by a parallel increase of 2-hydroxymelatonin and AFMK. Most importantly, this process can be independently activated by UVR. Our study supports a novel role of melatonin as protector for the skin against solar radiation. Moreover, the UV-induced production of melatonin metabolites, which are strong antioxidants themselves, defines a novel melatoninergic antioxidative system (MAS) of the skin (Fig. 3
). Finally, the combination of endogenous melatonin with externally applied melatonin may successfully counteract the multiple processes of skin damage induced by UVR.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5227fje
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