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Keywords:

  • antioxidant;
  • DNA;
  • melatonin;
  • NQ02;
  • skin;
  • UV

Abstract

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Abstract:  Melatonin, one of the evolutionarily most ancient, highly conserved and most pleiotropic hormones still operative in man, couples complex tissue functions to defined changes in the environment. Showing photoperiod-associated changes in its activity levels in mammals, melatonin regulates, chronobiological and reproductive systems, coat phenotype and mammary gland functions. However, this chief secretory product of the pineal gland is now recognized to also exert numerous additional functions which range from free radical scavenging and DNA repair via immunomodulation, body weight control and the promotion of wound healing to the coupling of environmental cues to circadian clock gene expression and the modulation of secondary endocrine signalling (e.g. prolactin release, oestrogen receptor-mediated signalling). Some of these activities are mediated by high-affinity membrane (MT1, MT2) or specific cytosolic (MT3/NQO2) and nuclear hormone receptors (RORα), while others reflect receptor-independent antioxidant activities of melatonin. Recently, it was shown that mammalian (including human) skin and hair follicles are not only melatonin targets, but also sites of extrapineal melatonin synthesis. Therefore, we provide here an update of the relevant cutaneous effects and mechanisms of melatonin, portray melatonin as a major skin protectant and sketch how its multi-facetted functions may impact on skin biology and pathology. This is illustrated by focussing on recent findings on the role of melatonin in photodermatology and hair follicle biology. After listing a number of key open questions, we conclude by defining particularly important, clinically relevant perspectives for how melatonin may become therapeutically exploitable in cutaneous medicine.


Introduction: melatonin as a pleitropic bioregulator and direct antioxidant

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Melatonin is a phylogenetically ancient methoxyindole that was first identified as the chief secretory product of the pineal gland five decades ago (1,2). It has since then aptly been labelled ‘Nature’s most versatile biological signal’ (3), since melatonin exerts surprisingly pleiotropic bioregulatory functions in numerous and extremely diverse biological systems, ranging from single cells to very complex organisms, including man (2,4–7).

In mammals, the pineal gland secretes melatonin into the blood circulation to exert a range of well-documented physiological functions (8). Classical chronobiology considers melatonin exclusively a hormone that regulates the circadian day–night rhythm and seasonal biorhythms (8–10). At least in part, these effects of melatonin are indirectly mediated by coupling to other endocrine systems [e.g. prolactin secretion and oestrus activity (11–14)], whose output/signalling activity is modulated by the photoperiod-dependent pineal secretion of melatonin (15–18). Additionally, currently recognized physiological melatonin activities in the mammalian system include the modulation of immune defense responses (19,20), body weight and reproduction (10), tumor growth inhibitory and anti-jet-lag effects (21–29). Independent of these effects, melatonin exerts many direct, receptor-independent activities, acting for example as a potent direct antioxidant (7,30–32), as a chemotoxicity reducing agent (7,33–36) and a putative anti-aging substance (37–39).

Melatonin is a highly lipophilic substance that easily penetrates organic membranes and therefore is able to protect important intracellular structures including mitochondria and DNA from oxidative damage directly at the sites where such damage occurs (40–46). Intriguingly, melatonin also up-regulates gene expression and activity of several antioxidative enzymes such as Cu/Zn-superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase and glutathione peroxidase (GPx) (7). Thus, melatonin not only acts as a potent antioxidant itself, but also is capable of activating an entire endogenous enzymatic protective system against oxidative stress (7,47).

During the past decade, evidence for multiple extrapineal sites of melatonin synthesis has accumulated, with significant melatonin levels recorded for example in bile fluid, bone marrow, cerebrospinal fluid, ovary, eye, lymphocytes, gastral mucosa and – last but not least – skin (19,47–57). It is now evident that the physiological level of melatonin has to be defined individually for each tissue, since the body liquids, tissues or organs mentioned above reveal melatonin levels which are 10- to 1000-fold higher than plasma melatonin concentrations which formerly might have been considered as ‘pharmacological’ (47–49,58,59). However, this observation throughout several completely different body compartments is highly suggestive for local tissue-specific melatonin synthesis since plasma levels would be too low to build this high tissue levels. Therefore, the presence of tissue-specific, local melatoninergic systems have been suggested that would have the biological role of counteracting specific, tissue-related regional stressors exactly at the place where they occur (47–49,60,61). In fact, such a melatoninergic antioxidative system (MAS) has been discovered recently in a highly differentiated manner in the skin (47).

Intracutaneous synthesis of melatonin

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

The discovery that the skin of the Syrian gold hamster displays activity for arylalkylamine-N-acetyltransferase (AANAT) (62,63), the key enzyme of melatonin synthesis, and can transform serotonin to melatonin ex vivo (Fig. 1) (57) has encouraged a series of subsequent studies that have explored whether skin from other mammalian species and its constituent cells are capable of synthesizing melatonin. Indeed, mammalian skin shows abundant concentrations of precursor molecules that are required for melatonin synthesis (e.g. by the massive stores of serotonin present in mast cell granules of murine skin) (64). These studies revealed that not only several mammalian skin cell populations in vitro, but also intact rodent and human skin in situ possess a fully functioning melatoninergic system. This includes gene expression, protein synthesis and enzymatic activity for AANAT and hydroxyindole-O-methyltransferase (HIOMT) as well as for tryptophan hydroxylase (54–56,62,65–69) with its isoforms TPH1 and TPH2 (Zmijewski and Slominski, unpublished data). The essential amino acid tryptophan (Trp) is necessary for melatonin synthesis. Hydroxylation of Trp leads to 5-OH-Trp and further to serotonin by decarboxylases that are available in almost every tissue (55). Serotonin has not necessarily to enter into the pathway for melatonin synthesis, has independent biological actions by itself and enters degradation pathways independent of melatonin (55). The rate-limiting enzyme for melatonin is AANAT (which produces N-acetyl serotonin) or HIOMT (which finally synthesizes melatonin) (55). This concept is supported by the fact that these enzymes follow circadian rhythms that parallel melatonin production and that they are suppressed by light, just as melatonin itself (6). Also, an essential cofactor, (6R) 5,6,7,8-tetrahydrobiopterin (6-BH4) which is required for hydroxylation of tryptophan, the essential amino acid for synthesis of melatonin, is abundantly present in skin (70,71). Of note, tryptophan itself can be metabolized by 2,3-dioxygenase to N-formylkynurenine using superoxide anion radical as a substrate. This, interestingly, represents a pre-melatoninergic tryptophan- and 2,3-dioxygenase-related antioxidant mechanism in the skin (72–74). Tryptophan has recently also been shown to be an important chromophore of UVB-induced cell surface receptor activation, forming a dimer which is the ligand for the arylhydrocarbon receptor AhR leading to activation of p450 (75).

image

Figure 1.  Mechanisms of the protective MAS of the skin. Melatonin is synthesized from tryptophan in a multi-step process to melatonin which can be metabolized by a variety of enzymatic and non-enzymatic pathways. Melatonin acts indirectly through membrane and nuclear receptors on stimulation of gene expression and activity of antioxidant enzymes, influences NQO2 activity and directly and indirectly scavenges reactive oxygen and nitric species which lead to reduced cell damage induced by oxidative stress.

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Definitive proof that even defined compartments of normal human skin can synthesize melatonin came from organ-cultured human scalp hair follicles. These not only showed abundant melatonin-like immunoreactivity in situ (59,66), but also can be stimulated to up-regulate their melatonin synthesis by incubation with noradrenaline, the key stimulus for the promotion of pineal melatonin synthesis (Fig. 2) (59). This corresponds well to earlier in vitro observations that another cAMP-raising (exogenous) molecule, forskolin, can raise the melatonin content, for example, of hamster skin fragments (57). Since catecholamines can actually be produced by human keratinocytes in vitro (76–78), it is reasonable to expect that intracutaneous melatonin synthesis can be stimulated not only by catecholamines released by sympathetic nerve endings in the skin, but also by catecholamines that are locally generated by the skin epithelium (77).

image

Figure 2.  Melatonin levels in murine skin organ culture, murine vibrissae follicles and human hair follicles under normal conditions and after stimulation with noradrenalin (NE) measured by double-antibody radioimmunoassay using anti-melatonin antibody, Kennaway G280 (Buehlmann, Schoenenbuch, Switzerland) [adapted from Ref. (59)].

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Interestingly, one of the most extensively studied mouse strains in investigative dermatology and skin biology exploits an important intracutaneous alternative to the classical (intrapineal) pathway of melatonin: C57BL/6J mice show an AANAT mutation that generates an inactive enzyme (79). Instead, serotonin is here acetylated to N-acetyl serotonin (NAS, obligatory precursor to melatonin) by alternative enzyme(s) (68). Thus, it is misleading to characterize C57BL/6J mice as a ‘natural melatonin knockdown’ species, as it is sometimes still claimed (79) because intracutaneously generated NAS may be methylated to melatonin at tissue sites that express HIOMT activity (54,68).

Thus, the skin, the largest organ of the mammalian body, which engages in very complex, major endocrine activities (80–91) has joined the growing number of mammalian tissues that operate as extrapineal sites of melatonin synthesis (54,56,65). Here, melatonin may serve to protect this crucial barrier organ against multiple environmental and endogenous stressors that threaten skin homeostasis.

Melatonin receptors

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Before further discussing the functions of the cutaneous melatoninergic system (55,57), the evidence that the skin also expresses membrane and nuclear melatonin receptors must be briefly reviewed. While it had long been known that both normal mouse skin and melanoma cells express melatonin-binding sites (92,93), it has only recently been demonstrated that human and rodent cells and tissues of cutaneous origin as well as murine and human hair follicles reveal specific, functionally active membrane receptors MT1 and/or MT2 (59,94,95). Furthermore, additional cytosolic (MT3/NQO2) and nuclear melatonin receptors (RORα) have been identified in rodent and human skin constituents (Table 1) (24,56,59).

Table 1.   Expression of membrane receptors MT1, MT2, cytosolic melatonin-binding site MT3/NQO2 and nuclear receptor RORα (including selected RORα isoforms RORα1 and RORα4/RZRα that are relevant in normal human skin cells and malignant melanoma cells as well as in mouse whole skin and murine epidermis and hair follicles) Thumbnail image of

The enzyme quinone reductase type 2 (NQO2) has been identified as the cytosolic melatonin receptor, MT3 (96). Recently, MT3 transcripts were demonstrated by RT-PCR in human keratinocytes, melanocytes and fibroblasts (56) as well as in several melanoma cell lines (Table 1) (24). Though the functions of NQO2 in skin biology are poorly understood, some evidence suggests its involvement in skin carcinogenesis: NQO2 knockout mice, for example, have been observed to be far more sensitive to the development of skin tumors after topical carcinogen application than their wild-type litter mates (97). Recently, inhibition of melanoma cell growth and clonogeneicity was correlated with up-regulation of NQO2 (98). This corresponds well to another recent observation of ours, namely that the growth of melanoma cell lines that express NQO2 is more suppressed by melatonin, the natural ligand of NQO2, compared to cell lines that do not express NQO2 (24). However, the detailed characterization of functions of this cytosolic melatonin receptor in cutaneous biology still remains to be clarified.

More light has been shed onto the binding characteristics of melatonin to NQO2, which support the initial hypothesis that NQO2, originally classified as MT3, is indeed a binding site for melatonin (99). Two melatonin molecules are involved in the reduction of flavin adenine dinucleotide (FAD) to FADH2 reaction: one binds as co-substrates to the catalytic site of NQO2-FAD, donating an electron to the enzyme co-factor, FAD, transforming it to FADH, while the resulting melatonin cation radical interacts with a superoxide anion radical to form N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) (6). The second electron required to build FADH2 is donated by another melatonin molecule, also forming AFMK or being converted to the melatonin neutral radical. The melatonin neutral radical scavenges a hydroxyl radical to form cyclic 3-hydroxymelatonin. Both melatonin reactions scavenge strong reactive oxygen species (ROS), the superoxide anion and the hydroxyl radical, supporting the role of melatonin not only as a direct, but also as an indirect radical scavenger, with NQO2 as a co-substrate. Furthermore, it has recently been reported that NQO2 directly reduces hydrogen peroxide and dangerous quinones protecting therefore DNA from oxidant damage (Fig. 1) (99,100).

Carlberg and colleagues have identified RZR/RORα as a candidate nuclear melatonin receptor (101–105). Regarding cutaneous cell populations and skin tissue, RORα has now been identified in adult epidermal keratinocytes and fibroblasts, in immortalized epidermal keratinocytes (HaCaT), in neonatal and immortalized melanocytes (PIG-1) (56) and in several melanoma cell lines (24). RORα was also detected in normal murine whole skin (RT-PCR) as well as in murine epidermis and hair follicles in situ (Table 1) (59). Apparently, the RORα-receptor isoform RORα1 is expressed only in adult dermal fibroblasts, whereas the isoform RORα2 is expressed in immortalized melanocytes, and RORα4 (RZRα) is expressed in adult epidermal keratinocytes, HaCaT keratinocytes, neonatal melanocytes and adult dermal fibroblasts (56).

Yet another signal transduction mechanism exploited by melatonin involves the membrane receptor-induced stimulation of MAP kinase cascades (106). Alternatively, calcium/calmodulin pathways regulated by transcriptional activity of RZR/ROR and by calmodulin-dependent protein kinases may be involved by either increasing intracellular calcium concentrations or by binding to MT1/MT2 receptor (107).

Notwithstanding the fact that the diverse biological functions of these melatonin receptors are far from understood and that their relative biological importance is controversially discussed, it is already evident that mammalian skin displays an entire repertoire of distinct melatonin receptors. This should allow fine-tuned, dose-dependent and cell-type-selective responses of this – apparently important – non-classical target tissue to melatonin stimulation.

General effects of melatonin in skin biology

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Since changes in skin and coat phenotype/function represent a major form of mammalian adaptation to changing environmental challenges, it is not surprising that melatonin – the major neuroendocrine regulator that couples photoperiod changes to complex endocrine responses (10,108,109) – impacts on mammalian skin physiology. In fact, indications that melatonin is involved in the regulation of seasonal hair growth and pigmentation can already be traced back several decades [reviewed in (110,111)]. For example, in several mammalian species, melatonin can alter wool and cashmere production, the development and frequency of pelage cycling and seasonal moulting as well as coat colour (112–117).

While the effects of melatonin on hair follicle biology have long been most obvious, yet are still insufficiently understood [for a comprehensive review of this specialized aspect of melatonin biology, see (118)]. This should not detract from the accumulating body of evidence that melatonin’s functions in skin biology and skin pathology extend far beyond the modulation of hair growth and/or pigmentation (Table 2). A few examples may suffice to illustrate this wide range of – at times, seemingly contradictory – functions.

Table 2.   Examples for the relevance of melatonin in skin biology and clinical dermatology
Site/function of effectObserved effectReferences
  1. Melatonin shows antioxidative and anti-apoptotic effects against UVR-induced damage, protection against different mechanico-physical destruction of skin, hair growth and pigmentation modulatory effects as well as oncostatic potency.

Cutaneous synthesisExtrapineal melatonin production by human keratinocytes and mouse and human hair follicles(47,55,59)
Keratinocyte and fibroblast growthStimulation of keratinocyte proliferation; inhibition of apoptosis in cell cycle-synchronized keratinocytes under serum-free condition, but inhibition of proliferation in serum-supplemented keratinocytes; increased viability in serum-free-cultured fibroblasts(95,119)
PigmentationInhibition of melanogenesis and melanocyte proliferation (hamster, rodent melanoma cells, whole skin organ culture)(92,93,234,235)
Endocrine signallingDown-regulates cutaneous expression of oestrogen receptors(59)
Hair follicle biologyExpression of melatonin receptors (MT1, RORα) in murine skin in situ is hair cycle-dependent(59,94)
Stimulation of hair shaft elongation in human hair follicles in vitro at low concentrations and inhibition at high concentrations(233)
Increased anagen rate in women with diffuse or androgenetic alopecia(232)
Mechanico-physical skin damageProtection against pressure-induced ulcer formation(122)
Restoration of skin thickness in pinealectomized rats(120)
Reduction of thermal skin injury(124)
Thermoregulation of skin blood flow(134,135)
Protection against necrosis, lipid peroxidation and reduction of antioxidative enzymes in skin flaps(125)
UVR-induced damageSuppression of UV-induced erythema by topical pretreatment with melatonin with/without combination of vitamins C and E(126,177)
Melatonin is a stronger radical scavenger than vitamins C and E(160)
Increased cell survival of HaCaT keratinocytes and ensuring keratinocyte colony growth under UV-induced stress; decrease of UV-induced DNA fragmentation(44)
Maintenance of mitochondrial membrane potential in UVR-exposed keratinocytes; reduction of casp 9, 3 and 7 activation; inhibition of PARP activation (= marker of apoptosis and DNA damage)(46)
Oxidative damageReduction of lipid peroxidation and increase of antioxidant enzyme levels (GPx, CAT); increase of GSH level(120,122,124)
Anti-tumor propertiesGrowth suppression in squamous cell carcinoma cell lines(56)
Suppression of proliferation in melanoma cells of different dignity and growth characteristics (radial growth, vertical growth, metastatic stage); MT1- and RORα receptor-dependent enhancement of suppressive effects(23,24,27,28,93,151)
Growth inhibition in breast cancer, colon carcinoma and squamous cell carcinoma cell lines(37,56,149,156)
Inhibition of benz(a)pyrene-induced skin carcinogenesis(25,133)
Potential clinical efficacy against malignant melanoma clinical stage IV(250,254)

Melatonin suppresses apoptosis and stimulates growth in both serum-starving HaCaT keratinocytes and serum-free-cultured fibroblasts (95). In contrast, the growth of serum-supplemented HaCaT keratinocytes is inhibited by melatonin at low concentrations (95), whereas very high concentrations of melatonin (4–20 × 10−6 mol) were found to stimulate cell growth under the same serum-supplemented culture condition (Table 2) (119). Strikingly, pinealectomized (i.e. melatonin-deficient) rats have been reported to show markedly reduced back, abdominal and thoracic skin thickness, along with an increase of lipid peroxidation and a decrease in the number of dermal papillae and hair follicles as well as of antioxidative enzymes (CAT, GPx). Melatonin substitution to these rats reportedly restored skin thickness, reduced lipid peroxidation and enhanced antioxidative enzyme activity (120). These results were later supplemented by the same group by ultrastructural evidence: compared to unsubstituted animals, melatonin-treated, pinealectomized rats showed reduced cytological atypia, decrease of nuclear irregularity, normalization of tonofilament distribution and mitochondrial integrity as well as of dermal collagen fibre structure (Table 2) (121). Collagen synthesis is controlled by proline hydroxylase which uses superoxide anion radical as the specific substrate together with l-proline yielding hydroxyproline on the precollagens. The removal of the ROS superoxide anion radical by melatonin would therefore prevent collagen synthesis. This corresponds well to the finding that melatonin also protects against pressure-induced ulcer formation in rat skin, as reflected by reduced lipid peroxidation, tissue neutrophil infiltration, along with increased glutathione (GSH) levels and reduced degenerative skin changes (122).

One of many arguments that advocate the administration of melatonin as a therapeutic adjuvant in burns patients (123) is that skin damage induced by thermal injury is reduced by melatonin, likely by limiting oxidative damage (124). Oxidative damage is also a key pathogeneic element in skin flap necrosis after plastic surgery: in pinealectomized rats, skin flaps of melatonin-treated animals exhibited reduced lipid peroxidation, nitric oxide formation and ratio of skin flap necrosis, along with increasing levels of GSH, GPx and superoxide dismutase (SOD) compared to non-melatonin-treated rats (Table 2) (125).

Clinically, topically applied 0.5% melatonin reduces UV-erythema when administered before, but not when applied after UV-irradiation (126,127). This was confirmed by another group showing that not only melatonin but also other antioxidants (vitamin E and vitamin C) have no effect on UV-erythema when administered after UV-irradiation, irrespective of the time course of application (128). Associated immunological skin responses, as exemplified by UV-induced suppression of the Mantoux response, are also not inhibited by melatonin when applied after UV-exposure (129). This indicates that the UV-induced free radical formation in skin is an immediate event which can only be antagonized by antioxidants that are already present at the target sites and at the time point of UV-exposure (Table 2) (44,126,128).

The antioxidant and DNA repair properties of melatonin raise the theoretical possibility that it may also prevent or reduce cutaneous photo-aging (32,130,131). In healthy skin, melatonin reduces the collagen accumulation, an indicator of skin aging (132). Melatonin also inhibits chemically induced carcinogenesis in rat skin, represented by reduction of the number of benzo(a)pyrene-induced papillomas; this is paralleled by attenuated lipid peroxidation and prevention of the binding of benzo(a)pyrene or its metabolites to DNA (25). Indeed, melatonin treatment reportedly reduced benzo(a)pyrene-induced tumor frequency by 30% in mice (133). Melatonin may also play a role in the thermoregulatory control of human skin blood flow, at least in healthy males (Table 2) (134,135).

A few selected aspects of melatonin’s proposed role as a major skin protectant deserve to be discussed in more detail, since they are of particular clinical and/or pharmaceutical interest.

Enhancement of antioxidative enzyme gene expression and activity

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

First reports that melatonin not only operates as a potent, direct free radical scavenging molecule, but also enhances the activity of antioxidative enzyme systems in various organs and organ systems were published in the mid-1990s (136–140). The activity of GPx, which reduces H2O2 to water, is up-regulated by melatonin in rat brain (136) and in several chicken tissues (139). Intriguingly, not only enzyme activity but also gene transcription of antioxidant enzymes, namely of Mn-SOD, CuZn-SOD and GPx, is up-regulated by melatonin during porphyrin-induced cell damage in rat brain cortex and in neuronal cell lines (137).

The effective melatonin concentration to up-regulate enzyme gene expression was 10−9 mol, a concentration that corresponds to the physiological night time peak of plasma melatonin (8), while higher concentrations had no effect on gene expression (141). Culturing cells for 1 h in the presence of a melatonin-containing solution was sufficient to generate elevated levels of mRNA for both SOD and GPx and maintain their levels for at least 24 h; this suggests a possible involvement of membrane and/or nuclear melatonin receptor activation with signal transduction on transcriptional mRNA level to modify the regulation of antioxidant enzymes by melatonin following outer signals to stress (142). Interestingly, melatonin is an even stronger antioxidant than GSH (143). At high melatonin concentrations (1 mmol), these effects were attributed to the indole’s direct radical scavenging actions, whereas at lower concentrations (100 nmol), the antioxidative effects might have been indirectly mediated via up-regulation of GPx gene expression and antioxidant activity (143).

SOD and catalase are stimulated by melatonin not only under conditions of oxidative stress, but also under basal (non-stress) conditions – presumably as a tissue’s baseline protection against subsequent stressful conditions that generate free radicals (144,145). In the context of the age-related brain deterioration, mediated by toxic free radicals as harmful products of natural aerobic metabolism, melatonin acts more efficiently than vitamin E or vitamin C in detoxifying the highly reactive hydroxyl and peroxyl radical, and stimulates GPx, the major antioxidant enzyme in the brain (146). Given the common neuro-ectodermal origin of brain and epidermis, it is at least conceivable that these findings are relevant to skin, and helps to understand why mammalian skin engages in constitutive and inducible melatonin synthesis (47,55,57,59,67).

Melatonin up-regulates mRNA levels for several antioxidative enzymes (Zn-SOD, CuZn-SOD, GPx and γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme of GSH synthesis (137,138,147). Currently, it is known that melatonin exerts quite a few of its effects via signal transduction ways mediated by membrane, cytosolic and nuclear receptors (24,56,106,148–156). It has been proposed, but not yet definitively demonstrated that melatonin’s ability to up-regulate antioxidative enzymes involves at least both membrane and nuclear melatonin receptors (157). Evidence for the latter rises from the observation that melatonin itself and a synthetic nuclear melatonin receptor agonist (CGP 52608) stimulate GPx and glutathione reductase activities in mouse brain (158).

A central question with all of the numerous, sometimes seemingly contradictory, and not uncommonly biphasic effects that have been reported for melatonin (dependent on cell type and experimental condition) is whether these effects reflect direct (receptor-dependent or -independent) melatonin activities (24,56,94,106,148–156) or indirect melatonin-mediated secondary processes with subsequent modulation of unrelated genes, for example up-regulation of antioxidant enzyme transcription or the modulation of other endocrine signalling systems (e.g. suppression of prolactin release, down-regulation of oestrogen receptor expression) (59,159).

Direct protective effects against skin photodamage

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

It is on this background that recent photobiological in vitro studies are of special interest, since they clearly demonstrate direct protective effects of melatonin on isolated cell populations of cutaneous origin. There is clear evidence that these protective effects are considerably mediated by the strong antioxidative capacities of melatonin (30). In fact, melatonin even has a higher reduction potential (0.73 V) than vitamin C (0.23 V) (160,161). The lower reduction potential of vitamin C makes the latter even a pro-oxidant, due to its ability to reduce Fe3+ to Fe2+, thus resulting in the formation of the highly toxic hydroxyl radical, while to date no such pro-oxidative properties of melatonin have been unequivocally demonstrated (160). Regarding UV-induced oxidative damage, it has been shown that melatonin is a strong scavenger against UV-induced formation of ROS, and even biologically more potent in this capacity than vitamin C or vitamin E analogue, trolox (Figs 3 and 4) (42,160,162). In UV-exposed fibroblasts, only 56% of the cells survived the exposure to UV-irradiation (140 mJ/cm2), whereas preincubation with 1 nmol melatonin lead to a cell survival rate of 92.5% which was paralleled by a significant inhibition of lipid peroxidation and apoptosis (163). A similar study showed cell protection against UV-induced suppression of cell viability by anti-apoptotic effects in fibroblasts when preincubated with melatonin at the concentration of 100 nmol (164).

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Figure 3.  Melatonin significantly suppresses ROS under UV-exposure at the concentrations of 10 nmol and 1 mmol compared to non-melatonin-treated control; **P < 0.01; ***P < 0.001; CPM: counts per minute as measured by chemiluminescence [modified after Fischer et al., Ref. (162)].

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Figure 4.  Melatonin leads to stronger reduction of ROS formation under UV-irradiation of human leukocytes than vitamin C or trolox. ***P < 0.001 [modified after Fischer et al., Ref. (160)].

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Human keratinocytes, the main target cells in epidermal photodamage, irradiated with UVR at increasing doses (25, 50 mJ/cm2) and at a wavelength combination that mimicks the skin’s natural UVR exposure (combined UVB/UVA irradiation) were investigated for proliferation, colony formation and apoptotis by [3H]-thymidine-labelled DNA incorporation, clonogeneicity assay and TUNEL assay, respectively. Melatonin significantly increased the cell survival rate and colony formation ability, mainly by preventing UV-induced apoptosis (44). Also, transcription of several classical target genes that are up-regulated after UV-exposure and play an important role in the execution of skin photodamage (165–169) were down-regulated in HaCaT keratinocytes by melatonin pretreatment: interstitial collagenase (MMP-1), stromelysin 1 (MMP-3), stromelysin 2 (MMP-10) and aldehyde dehydrogenase 3 type A1 (56). Most recently, Cho et al. (170) performed cDNA microarray analysis from HaCaT keratinocytes, exposed to 100 mJ/cm2 and pretreated with melatonin at the concentration of 100 nmol for 30 min. A great variety of genes related to apoptosis (apoptosis-related protein-3), cancer induction, cell cycle (cyclin-dependent kinase 2 interacting protein), enzymes (GPx, ubiquitin-conjugating enzyme E2M) and signal transducer genes (fibroblast growth factor, TGFβ-stimulated protein TSC-22) were down-regulated by melatonin compared to UV-exposed keratinocytes not pretreated with melatonin (170).

Analysis of mitochondrial (intrinsic) and death receptor-mediated (extrinsic) apoptosis pathways suggests that melatonin inhibits the intrinsic, but not the extrinsic apoptotic pathway, and down-regulates its downstream effector caspases (46). This pathway is induced by reduction of the mitochondrial membrane potential which is caused by UV-related formation of mitochondrial ROS (mROS). The reduced membrane potential is normalized by melatonin (at the concentration of 10−6, 10−4, 10−3 mol).

These findings raise the possibility that melatonin may induce relative protection against sunburn cell formation in situ (i.e. intraepidermal keratinocyte apoptosis induced by excessive UV-irradiation) not only by reducing the UVR-induced free radical load, but also by up-regulating the expression of apoptosis inhibitory genes. Melatonin also inhibits the UV-induced activation of poly(ADP-ribose) polymerase (PARP), a key DNA-damage-mediating enyzme within the first 24 h after activation (46). Complementary morphological analyses have revealed that melatonin also counteracts the UV-related increased detachment of apoptotic keratinocytes resulting in empty spaces between otherwise confluent cells, the formation of dysmorphic cell shape and nuclear chromatin condensation. This documents potent and direct protective effects of melatonin in vitro at all relevant molecular and cellular levels of UV-induced apoptosis (46).

Melatonin levels, autonomous and UV-enhanced melatonin metabolism

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Melatonin has been first identified in human epidermal keratinocytes and human malignant melanoma cells (55,67) which has later been confirmed for keratinocytes (47). When melatonin-containing skin cells or melatonin in cell-free systems are exposed to UVR, multiple melatonin metabolites are observed. At certain wavelengths (300–575 nm, UV–visible light) melatonin degradation products are produced after UVR exposure (171), of which 6-OH-melatonin and AFMK represent the main photoproducts of melatonin. AFMK as well as its degradation product, N1-acetyl-5-methoxykynuramine (AMK), are strong free radical scavengers themselves: for example, AFMK is an up to four times stronger antioxidant than melatonin itself (172,173). AFMK and AMK are also known to have anti-inflammatory properties [e.g. reduction of lipopolysaccharide (LPS)-induced COX-2 up-regulation, decrease of inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2)] (174). Moreover, AFMK inhibits 5-aminolevulinic acid-induced DNA damage (175) and prevents protein destruction (176).

It has to be noted that these interesting effects of melatonin photodegradation products had until recently exclusively been observed in cell-free testing systems, and with UV-wavelengths that are only partly related to the most damaging wavelengths encountered in cutaneous biology, that is the UVB range (280–320 nm). Recently, melatonin photoproducts have been identified in a cell-free system using wavelengths that are relevant to human skin. Under increasing UV-doses (25, 50, 100 mJ/cm2) of a mixed UV-source (UVB 60%; UVA 30%) that is more closely to naturally occurring solar irradiation, four metabolites were identified by HPLC and LC-MS: 2-OH-melatonin, 4-OH-melatonin, 6-OH-melatonin and AFMK (47). The levels of these metabolites were directly proportional to applied UV-doses as well as to melatonin substrate concentration.

Additionally, intracellular melatonin levels were measured and UV-enhanced metabolism with the formation of 2-OH-melatonin and AFMK observed (47). These studies provided the first evidence for UV-enhanced photolytic and/or enzymatic melatonin metabolism in cultured human keratinocytes: keratinocytes, incubated with melatonin revealed a melatonin uptake and showed an UV-induced enhancement of melatonin metabolism with formation of 2-OH-melatonin and AFMK. Interestingly, active autonomous melatonin metabolism in vitro (without UV-induced enhancement) over 24 h was also documented: ‘naïve’ (i.e. non-melatonin-treated) keratinocytes showed intracellular melatonin levels and a time-dependent 24 h metabolism of melatonin leading also to the formation of 2-OH-melatonin and AFMK (47). Thus, it can be concluded with almost certainty, that human keratinocytes, and likely the skin and its appendages in vivo themselves, are not only targets for protective exogenous melatonin treatment, but also an extrapineal site of melatonin synthesis and of fully functioning local autonomous melatonin metabolism. Intriguingly, this cutaneous local synthesis and metabolism are inducible by UV-irradiation, possibly to provide a self-regulated protective system that is switched-on by environmental stressors such UVR and ionizing radiation (IR), and also by inflammation (32,44,46,47,54–57,92,95,126,127,163,164,177–179).

The cutaneous melatoninergic antioxidative system (MAS)

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

The photo-induced melatonin metabolism leading to the generation of antioxidant melatonin metabolites in human keratinocytes represents an antioxidative cascade which has been described earlier for chemical or other tissue homogenate systems (6,172,173) and has now been identified in the skin to protect this important barrier organ against UVR-induced oxidative stress-mediated damaging events on DNA subcellular, protein and cell morphology level (Figs 1 and 5) (47). This newly identified MAS of the skin (47) likely extends to skin compartments beyond the epidermis, namely to the dermis and the hair follicle (56,59,118), and may have evolved as a defense mechanism against the multi-facetted threats of environmental stress, especially UVR, to which the skin is life-long exposed (32,54,56,180–186).

image

Figure 5.  Overview of the pleiotropic effects of melatonin as a major skin protectant. Melatonin acts as a strong radical scavenger with formation of AFMK and AMK, strong antioxidants themselves. This results in reduced lipid peroxidation, protein oxidation and DNA damage. Melatonin also reduces directly or indirectly through AFMK apoptosis/necrosis and maintains mitochondrial integrity. It further acts as a cell proliferation substance in benign cells while reducing tumor cell growth. Direct or indirect molecular/subcellular effects are presented within the dotted circle line, and functional, histological, clinical effects are building the circle around these melatonin-related effects.

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The UV-induced melatonin metabolites, especially AFMK, are themselves potent antioxidants (6,7,172,173,187). ROS – mainly the hydroxyl radical – occurring under UV-irradiation in the skin react directly with melatonin (7,180,188,189). The latter is either autonomously produced in epidermal and/or hair follicle keratinocytes where it engages in intracrine signalling/interactions or released into the extracellular space to regulate auto-, para- or endocrine signalling (47,59,190). The reaction of melatonin with hydroxyl radicals induces the formation of 2-OH-melatonin and 4-OH-melatonin which are then further metabolized to AFMK and by arylamine formamidase or catalase to AMK (173,191). During this process, hydroxyl radicals are scavenged, and resulting damaging events are either indirectly or directly reduced via decrease of lipid peroxidation, protein oxidation, mitochondrial damage and DNA damage (Fig. 1) (32,44,46,178).

For application in clinical dermatology, exogenous melatonin should rather be used topically than orally, since orally administered melatonin appears in rather low levels in the blood due to prominent first-pass degradation in the liver, thus limiting skin access (32,178).

Topical administration circumvents this problem. In addition, as we could show in our own investigations, melatonin can penetrate into the stratum corneum and build there a depot due to its distinct lipophilic chemical structure (192). Therefore, endogenous intracutaneous melatonin production, together with topically applied exogenous melatonin, can be expected to provide the most potent defense system against cutaneous photodamage (47) and multiple other pathologic conditions that produce oxidative stress (e.g. in chronic skin inflammation, such as atopic dermatitis) (193).

Melatonin and vitiligo

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Vitiligo is a model disease that deserves special interest in the context of MAS functions and dysfunctions. Though the aetiology of this common progressive pigmentary disorder is still controversially debated (194), two main pathogeneic theories have gained most publicity (195–198). The autoimmune theory identifies an immune response to melanocyte-associated autoantigens as the main, if not the sole pathogenic factor (199). Others have proposed that exogenous or endogenous noxious and/or other hormonal/neurotransmitter factors trigger cytotoxic events based on the excessive accumulation of insufficiently scavenged free radicals which then lead to vitiligo, with autoimmune responses representing only secondary phenomena (200–202).

Almost two decades ago, a melatonin hypothesis on the pathogenesis of vitiligo was proposed that linked pathological activation of melatonin receptors to the deregulation of melanogenesis followed by sequential damage and ultimately destruction of both melanocytes and keratinocytes due to uncontrolled production of ROS and toxic quinone/semiquinone intermediates of melanogenesis (111,203). A secondary autoimmune response to melanocyte-associated autoantigens exposed by the above autodestruction would lead to further progression of vitiligo and render the disease process irreversible (203). Over the last decade, part of this concept of vitiligo pathogenesis that had linked oxidative stress with endogenous production of toxic metabolites through a self-amplifying process resulting in melanocyte destruction has attained some experimental support (197,200,201,203–205). However, following recent advances in melatonin biology on antioxidative actions of melatonin and its metabolites (6,32,42,105,187,206), and on the intracutaneous expression of local melatoninergic system (47,54–56,66,95), the original hypothesis has been modified (197): Considering that the synthesis of melanin (111) and melatonin (3,41,42,78,162) both serve important ROS scavenging functions, it is now postulated that a dysregulation of the MAS contributes to the development and progression of vitiligo (197). Specifically, insufficient local production of melatonin and/or of its antioxidant/cytoprotective intermediate AFMK (6,47,54) would impair the epidermal buffering capacity against oxidative and/or genotoxic stress, induced by a variety of exo- and/or endogenous noxious factors. This melatonin shortage is proposed to result in context-dependent melanocyte and keratinocyte damage, which leads to melanocyte death.

It also remains possible that overactive melatonin receptor-mediated signalling (e.g. the availability of excessive ligand or by acquired receptor mutations) additionally inhibits melanin formation (203). Relative shortage of the recognized ROS scavenger, melanin (111) together with active melanogenesis which still actively produces toxic intermediates of the pathway without generating the final ROS-scavenging product would promote the intracellular accumulation of cytotoxic ROS formation (203). Thus, not only insufficient intracutaneous melatonin synthesis and/or insufficient melatonin metabolism to generate potent ROS-scavenging melatonin photoproducts such as AFMK, but also dysregulated melatonin receptor activity that reduces melanin production could explain the pathogenesis of vitiligo. This vicious circle can be envisaged to lead to unbalanced ROS, H2O2, quinones and semiquinones accumulation, along with the associated accumulation of DNA damage, resulting in the development/progression of vitiligo (197).

Thus, the revised melatonin hypothesis of vitiligo integrates the emerging cutaneous MAS concept (47) with the historical melatonin receptor theory (203) and the oxidative damage scenario of vitiligo pathogenesis (197,200–202). Irrespective of whether or not this reasonable pathogenesis scenario withstands further experimental testing, it already illustrates convincingly, why vitiligo offers a uniquely instructive model disease for exploring the pathological consequences of MAS deregulation.

Melatonin as a chemotoxicity and ionizing radiation protectant

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Investigative dermatology is well-advised to more systematically explore that there is more to melatonin than ROS- and photoprotection (see Table 2 and Fig. 5). In the following, this will be illustrated by summarizing key insights on the activities of melatonin as a chemotoxicity and ionizing radiation (IR) protectant.

In chemotherapy-induced damage, melatonin significantly reduces cisplatin-induced testicular toxicity in rats (34,36,207). Also, amicacin- or cisplatin-induced nephrotoxicity in rats is prevented by melatonin through enhancement of the GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio, reduction of lipid peroxidation and restoration of the enzymatic antioxidant GPx (36,207). In primary rat renal tubular cisplatin-treated epithelial cells, melatonin exerts its protective effects via scavenging ROS and reducing DNA fragmentation, much stronger than its precursors or metabolites such as tryptophan, serotonin or 6-hydroxymelatonin (208). Melatonin also protects against doxorubicin-induced cardiotoxicity in rats by stimulating the activity of antioxidative enzymes (CAT, GSH), reducing lipid peroxidation and protecting against mitochondrial damage (35,209). This suggests that melatonin can potently protect against chemotherapy-induced damage through different biological mechanisms in a number of organs. Unfortunately, this has not yet been investigated in a dermatological context.

Melatonin may even protect the skin against the highly destructive effects of IR (179,210,211). The skin ranks among the chief target tissues for the well-recognized undesired effects of IR (212), with basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) representing the most common IR-induced skin malignancies. BCC specially develops when IR occurs before the age of 20 years, while SCC development is strongly correlated with IR in combination with cumulative UV-irradiation exposure (213). The molecular precondition for IR-induced skin cancer development is severe and widespread DNA damage, predominantly due to IR-mediated hydroxyl radical generation (214). Hydroxyl radicals are a result of IR-induced radiolysis of water, leading to formation of oxidized bases, DNA–DNA intrastrand adducts, DNA single- and double-strand breaks and DNA–protein cross-linking which all lead to genomic instability, a prerequisite for tumor promotion and development (215–218).

Since melatonin is a highly efficient hydroxyl radical scavenger (30), it is not unexpected that it acts highly protective against IR-induced damage (219). Melatonin markedly inhibited formation of chromosome aberrations and micronuclei in IR-exposed lymphocytes separated before IR from healthy volunteers who orally took melatonin (300 mg) at a single time point or from lymphocytes which were preincubated in vitro with melatonin at the concentration of 2 mmol (220,221). When cultured human fibroblasts were exposed to 8 Gy of IR, the cell survival rate was reduced to 37%, whereas preincubation with melatonin (10 μmol) led to an increased survival rate of 68% (179). These survival enhancing effects of melatonin correlated with reduced lipid peroxidation of the cell membranes (represented by lowered malondialdehyde levels) and decreased apoptotic pre-G1 peak (179). Of note, the pathways influenced by melatonin were not p53- nor p21-dependent. Interestingly, the use of different antioxidants (including trolox, the water-soluble analogue of α-tocopherol) has shown that the antioxidant must be applied before IR-exposure in order to effectively scavenge ROS formed during IR (222), just as it is true for the antioxidant effects of melatonin in connection with UVR (44,126).

In one in vivo experiment, lethal whole-body IR treatment of mice induced rapid death of all mice within 12 days after the exposure, while 43% of the melatonin-treated mice survived for up to 30 days (223). In a similar study, melatonin also promoted the 30-day survival of IR-exposed mice and inhibited bone marrow damage (224). In IR-exposed rats, pretreatment with melatonin almost completely prevented the sharp rise in hepatic levels of 8-hydroxy deguanosine, an indicator of DNA oxidation (215).

In albino rats, ultrastructural analyses of IR-induced skin damage showed that control animals (non-melatonin-treated) exhibited severe condensation of nuclei, vacuolization of cytoplasm, destruction of ribosomes and swelling of mitochondria as well as destruction of the ribosomes and intermediate filaments and fragmentation of the keratohyaline in keratinocytes. In sebaceous glands, the central cells of the alveoli showed larger irregular nuclei and few lipid droplets in their cytoplasm, and hair follicle keratinocytes revealed heterochromatic nuclei and less electron-dense cytoplasm containing only few complements of the organelles. Increased metabolic activity was observed including increased euchromatin, irregularity of the nuclear membrane and increased branching of melanocytes. Finally, an increased number of the Birbeck granules was seen in the Langerhans cells of IR-exposed skin (210). All these severe IR-induced damaging effects were mild or absent in animals which were pretreated with melatonin. This very detailed analysis of IR-induced damage in different compartments and structures of rat skin showed impressingly how broadly melatonin is able to prevent harmful destruction of the skin by such a powerful external stressor like IR. These promising observations remain to be systematically followed up in the human system, namely in skin (225,226).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

Since the discovery of the strong antioxidant properties of melatonin (30), which until then had exclusively been appreciated as a circadian and seasonal biorhythm regulator (109), a tremendously wide spectrum of targets and effects of melatonin has evolved in a great variety of tissues and organisms (3,4,6). The predominant feature of melatonin that has surfaced in consequence is that of a potent cytoprotective substance on multiple different levels of cell damage, both in physiological and pharmacological concentrations (4,6,7,19,23,33,35,41,43,48,52,58,61,124,139,144,146,163,174,190,221,227–232).

The presence of specific and functionally active membrane, cytosolic and nuclear melatonin receptors in mammalian (including human) skin and its appendages (55,56,59,95) suggests the skin to be a major melatonin target. Parallely the demonstration of AANAT activity in hamster skin (57,62,63), of transcripts for melatonin-synthesizing enzymes in human skin and hair follicle cells as well as in cutaneous tissues (55) and of inducible melatonin synthesis and metabolism in keratinocytes and hair follicles (47,59) identifies mammalian skin and its appendages as major extrapineal sites of melatonin synthesis and metabolism (54,56,59,118). A steadily growing body of evidence now supports that the functional role of melatonin and its metabolites fully extends to skin and hair biology/pathology including the effects of melatonin on heat- and pressure-induced skin injury, ulcer formation, apoptosis, necrosis, melanogenesis, hair shaft growth and hair follicle receptor modulation as well as tumor growth suppression (Fig. 5) (24,44,46,56,59,118,122,124,125,233–235). Finally, the main environmental skin stressors (UVR, IR) are effectively counteracted by melatonin in the context of a complex intracutaneous MAS (42,44,46,56,66,160,162,164,179).

In fact, in human biology, the skin may be unrivalled as a model organ for elucidating the full range of melatonin functions, targets, metabolism, receptors and regulation in health and disease. Moreover, growing evidence suggests that ligands of membrane, nuclear and cytosolic melatonin receptors (including antioxidant melatonin photoproducts) may be recruited as adjuvant therapy in a wide range of problems in clinical dermatology, ranging from wound healing via vitiligo, atopic eczema, sarcoidosis, diabetic foot syndrome and pruritus to carcinoma and melanoma (236–244) (see Table 3).

Table 3.   Selected specific skin diseases, pathologic conditions and syndromes in which melatonin may serve as a major skin protectant by topical and/or systemic administration
Potential1 applicationMode of adminstrationReferences
  1. Potential applications of melatonin in clinical dermatology. Mode of administration (T = topical, O = orally).

  2. 1Note that none of these potential clinical applications can currently claim convincingly to represent evidence-based medicine, and conclusive, corresponding clinical trials remain to be performed.

Wound healingT(122)
VitiligoT(197)
Androgenetic/diffuse alopeciaT(232)
Hair greyingT(186)
Atopic eczemaT + O(239,242)
Psoriasis vulgarisT + O(243,244)
Dermatitis solarisT(126,127,177)
Skin agingT + O(32,120,178)
CarcinomaT + O(23,250,253)
MelanomaO(24,27–29,151,251,254)
SarcoidosisT + O(240)
PruritusT(236,237)
Diabetic foot syndromeT(241)

Open questions and future perspectives

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

A curious observation that has recently joined the list of reasons, why the ‘melatonin–skin connection’ deserves full attention in experimental and clinical dermatology, nicely illustrates both, how many open questions there still are, and how complex this connection is likely to be. Reportedly, laughter increases the levels of breast milk melatonin in both mothers with atopic eczema and healthy mothers, and feeding infants with increased levels of melatonin-containing milk supposedly reduces their ‘allergic responses’ (245). Thus, even the mammary gland – an epidermal derivative – contributes a piece to the ‘melatonin–skin’ puzzle, and offers yet another reason to believe that maternal laughter benefits the management of infantile atopic eczema, whether or not this has anything to do with breast milk melatonin levels.

The clinical and pharmaceutical key challenge now is to explore the most effective, cost-efficient, and least risky strategies for boostering the skin’s endogenous MAS (including its capacity to generate the potent antioxidant melatonin product, AFMK), for example for photodamage prevention and repair and in selected clinical conditions (Tables 1 and 3, and Fig. 5). Naturally, the unfolding ‘melatonin–skin connection’ raises many open questions that will stimulate experimental and clinical skin research in the future. For example, it needs to be clarified what is the optimal melatonin concentration in/around the cutaneous cell populations that are to be targeted for any of the potential indications listed in Table 3. Melatonin reduces UV-induced ROS formation at both low (10−8 mol) and high (10−3 mol) concentrations (162). However, the stronger ROS-suppressive effect of melatonin is observed at the higher concentration which may reflect melatonin’s direct radical scavenging properties, whereas melatonin at the lower concentration might act through activation of membrane, cytosolic and/or nuclear melatonin receptors (32,54,56,178).

Likewise, the regulation of skin melatonin receptor expression and differential signalling/distinct target genes as well as signalling cross-talk between different classes of intracutaneous melatonin receptors all remain to be elucidated. For this, it is critical to continue exploring selective, and ideally well-tolerated, agonists and antagonists for the different classes of melatonin receptors, following published leads for ligand development strategies (104,246–248). In fact, we are still very far from understanding what are the key target genes of MT1- versus MT2-, MT3/NQO2- or RORα-mediated signalling in distinct human skin cell populations in situ. Also, even though the observation that both MT2 and RORα transcription in murine skin is stringently coupled to synchronized hair follicle cycling offers some leads in this respect (59), a clear insight into the key controls of intracutaneous melatonin receptor expression and into the modulators of postreceptor signalling events in mammalian skin is still lacking. Also, the role of MT3/NQO2 (96) in skin biology is almost completely unknown and deserves further scrutiny (24,56). Clearly, specific blocking of defined melatonin receptors, along with the development of highly selective melatonin receptor agonists, is needed to make progress in this area.

Furthermore, the key stimulators and inhibitors of extrapineal melatonin synthesis in the skin need to be unravelled. While intracutaneously generated and released catecholamines (77,78) are chief candidates for synthesis promoters (59), they are unlikely to be the only regulators. Furthermore, it is of interest whether intracutaneous melatonin synthesis follows a circadian rhythm as the pineal gland does, and whether they are coupled or independently developing. Most interestingly, the activities of antioxidant enzymes exhibit circadian rhythms that correspond to melatonin rhythmicity as well as to the total antioxidant status of the plasma in chicken brain, liver and lung (231). In chicken, gene expression for these enzymes exhibited daily rhythms as well. Similar results were found in the rat where daily fluctuations in SOD gene expression corresponding to day–night changes in circulating melatonin levels were observed (249). This raises the question whether circadian changes in antioxidant enzyme activities are indeed melatonin-mediated.

Though increasing evidence supports that melatonin significantly up-regulates the free radical scavenging systems in many body compartments (136–139,146) the indication that this is indeed the case in the skin remains to be demonstrated in vivo. Also, despite fascinating indications that point to ‘oncostatic’ activities of melatonin in various in vitro and animal models (24,29,151,250), it is far from clear whether melatonin really enhances tumor immunosurveillance, counteracts carcinogenic stimuli and/or exerts tumor growth inhibitory properties in human skin in vivo [for discussion with emphasis on the role of melatonin in melanoma therapy, see (251–255)]. Another, important area for future skin research is to elucidate how chronic hyperproliferative, inflammatory dermatoses like psoriasis and atopic dermatitis affect cutaneous melatonin synthesis, and how any abnormalities of melatonin synthesis/metabolism in diseased human skin can be therapeutically manipulated, for example by topical agents (96).

While melatonin’s chief function in mammalian skin may well be that of a general skin protectant that promotes ROS scavenging and DNA repair, multiple other potential functions await systematic investigation (see Table 2). For example, do the well-recognized, central clock gene-phasing effects of melatonin (18,256,257) also extend to the skin? Interestingly, mammalian skin expresses major clock genes and apparently utilizes these to subject skin physiology to chronobiological rhythms (258–260). Given that, furthermore, UVR not only stimulates the synthesis of melatonin photoproducts (see earlier), but also modulates the transcription of circadian clock genes in human keratinocytes (261); it is reasonable to expect that locally synthesized melatonin also impacts on the intracutaneous expression and function of circadian clock genes. Thus, cutaneous melatonin may also serve regional/local synchronization purposes in mammalian skin biology and may be able to modulate this putative function in response to environmental changes such as UVR.

As a prototypic agent of neuroendocrine coupling, melatonin impacts on several other endocrine systems, most prominently on pituitary prolactin secretion (13,15,18). Since both murine and human skin, including human scalp hair follicles, are prominent extrapituitary sites of prolactin synthesis (262,263), it is therefore reasonable to ask whether intracutaneous melatonin production modulates skin prolactin expression and secretion in a manner that is comparable to the regulation of pituitary prolactin release by melatonin. Also, given the much debated role of oestrogens in the physiology and aging of mammalian skin and its appendages (85,87,264), the intracutaneous interactions of melatonin with oestrogen biology must be elucidated. It is recognized that melatonin exerts anti-oestrogenic effects, which range from the inhibition of oestrogen receptor activation/transactivation (11,12,265,266) through the inhibition of enzymatic oestrogen synthesis (267) to the down-regulation of oestrogen receptor expression on the gene and protein level in human and mouse hair follicles (59). Vice versa, in Chinese hamster ovary (CHO) cells, 17-β-estradiol differentially modulates MT1 and MT2 melatonin receptor functions (attenuation of melatonin responses through activation of MT1 receptors increase the MT2 receptors density) (268). Therefore, it is conceivable that intracutaneous melatonin synthesis renders the skin and its appendages less sensitive to oestrogen-mediated signalling (59) – a putative melatonin property that would likely be most relevant, and can best be studied, in the (highly oestrogen-sensitive) pilosebaceous unit (85,87,269).

It remains another crucial challenge to clarify conclusively in convincing clinical studies whether and how melatonin can be exploited to protect the skin and its appendages from the undesired effects of chemotherapy and/or IR. A recently developed new assay system for dissecting the damaging effects of cyclophosphamide metabolities on normal, organ-cultured human scalp hair follicles (225) and a new assay for the long-term organ culture of full thickness human skin (226) provide pragmatic and instructive preclinical research tools for this purpose. The same assays may be modified with relative ease also to study preclinically, whether melatonin is an effective protectant from IR in human skin, before corresponding clinical studies are designed.

Finally, the broadest appeal of the ‘melatonin–skin connection’ probably arises from pilot studies that have raised the question whether melatonin may be a key determinant in skin aging and whether it may slow down both intrinsic skin senescence and extrinsic skin photoaging. Apart from melatonin’s ROS scavenging and DNA-repair-promoting properties, the impaired skin architecture and function in pinealectomized rats (121), the declining melatonin plasma levels with age (270,271) and the partial normalization of age-related deterioration of defined skin functions/architecture by exogenous melatonin substitution therapy (31,37,120) have served as main arguments in support of such sweeping claims. The final verdict on whether long-term oral (272) or topical (192) melatonin administration can indeed slow, halt and/or revert human skin aging in vivo can only be supported from randomized, double-blind, well-controlled, prospective trials of large and homogeneous proband cohorts. The available evidence summarized here strongly encourages such studies. Yet, even if melatonin should fail to emerge from such rigorous testing as the exceptionally well-tolerated, biochemical ‘magic wand’ for cosmetic dermatology as which it is sometimes hailed, there can be little doubt: More than 60 years after its discovery (1), the future of melatonin in experimental and clinical dermatology has just begun.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References

This review is dedicated to the memory of Aaron B. Lerner (2007), the discoverer of melatonin, with whom one of the authors (AS) has spent a crucial, formative postdoctoral research period at Yale, while another co-author (from a directly adjacent laboratory) (RP) got himself married under the critical eyes of this giant in skin research and dermatology. The first author (TWF) is thankful for having been the recipient of a scholarship from the Friedrich-Schiller-University Jena, Germany, named after Aaron B. Lerner which enabled him to set new milestones for characterizing the role of melatonin in the skin at the University of Tennessee Health Science Center, Memphis, Tennessee, USA.

The authors gratefully acknowledge the funding agencies which have supported some of their original studies cited in this review: German Academy of Natural Scientists Leopoldina, Halle, and ‘Federal Ministry of Education and Research’ BMBF-LPD 9901/8-113 (TWF), Foundation ‘Rene Touraine’ Short-Term International Fellowship (TWF), Deutsche Forschungsgemeinschaft (Pa 345/11-2) (RP) and University of Tennessee Cancer Center Pilot Grant (AS, TWF).

References

  1. Top of page
  2. Abstract
  3. Introduction: melatonin as a pleitropic bioregulator and direct antioxidant
  4. Intracutaneous synthesis of melatonin
  5. Melatonin receptors
  6. General effects of melatonin in skin biology
  7. Enhancement of antioxidative enzyme gene expression and activity
  8. Direct protective effects against skin photodamage
  9. Melatonin levels, autonomous and UV-enhanced melatonin metabolism
  10. The cutaneous melatoninergic antioxidative system (MAS)
  11. Melatonin and vitiligo
  12. Melatonin as a chemotoxicity and ionizing radiation protectant
  13. Conclusions
  14. Open questions and future perspectives
  15. Acknowledgements
  16. References