Wavelength-dependent threshold fluences for melanosome disruption to evaluate the treatment of pigmented lesions with 532-, 730-, 755-, 785-, and 1064-nm picosecond lasers.

BACKGROUND AND OBJECTIVES: A threshold fluence for melanosome disruption has the potential to provide a robust numerical indicator for establishing clinical endpoints for pigmented lesion treatment using a picosecond laser. Although the thresholds for a 755-nm picosecond laser were previously reported, the wavelength dependence has not been investigated. In this study, wavelength-dependent threshold fluences for melanosome disruption were determined. Using a mathematical model based on the thresholds, irradiation parameters for 532-, 730-, 755-, 785-, and 1064-nm picosecond laser treatments were evaluated quantitatively. STUDY DESIGN/MATERIALS AND METHODS: A suspension of melanosomes extracted from porcine eyes was irradiated using picosecond lasers with varying fluence. The mean particle size of the irradiated melanosomes was measured by dynamic light scattering, and their disruption was observed by scanning electron microscopy to determine the disruption thresholds. A mathematical model was developed, combined with the threshold obtained and Monte Carlo light transport to calculate irradiation parameters required to disrupt melanosomes within the skin tissue. RESULTS: The threshold fluences were determined to be 0.95, 2.25, 2.75, and 6.50 J/cm² for 532-, 730-, 785-, and 1064-nm picosecond lasers, respectively. The numerical results quantitatively revealed the relationship between irradiation wavelength, incident fluence, and spot size required to disrupt melanosomes distributed at different depths in the skin tissue. The calculated irradiation parameters were consistent with clinical parameters that showed high efficacy with a low incidence of complications. CONCLUSION: The wavelength-dependent thresholds for melanosome disruption were determined. The results of the evaluation of irradiation parameters from the threshold-based analysis provided numerical indicators for setting the clinical endpoints for 532-, 730-, 755-, 785-, and 1064-nm picosecond lasers.

Photoreactivity and phototoxicity of experimentally photodegraded hair melanosomes from individuals of different skin phototypes.

Even though melanin is commonly viewed as natural photoprotectant, the pigment demonstrates residual photoreactivity, which under certain conditions could contribute to UVA-dependent melanomagenesis. Skin melanin is constantly exposed to external stressors, including solar radiation, which could induce photodegradation of the pigment. Although photodegradation of melanin pigments was studied in synthetic models and RPE melanosomes, photochemical and photobiological effects of experimental photodegradation of human skin melanin of different chemical composition remain unknown. In this work, melanosomes isolated from hair of individuals of different skin phototypes (I-III, V) were exposed to high-intensity violet light and its impact on physical and chemical properties of the pigments were analyzed using electron paramagnetic resonance (EPR), spectrophotometry and dynamic light scattering (DLS). Photoreactivity of photodegraded melanins was examined by EPR oximetry, EPR spin-trapping and time-resolved singlet oxygen phosphorescence. Antioxidant potential of the pigments was measured using the EPR DPPH assay. Cellular effect of the exposure of melanosome-loaded HaCaT cells to UV-Vis light was determined by MTT assay, JC-10 assay, and iodometric assay. The data revealed that experimental photodegradation increased photoreactivity of natural melanins, while decreasing their antioxidant capacity. Photodegraded melanin was responsible for higher cell death, a decrease in mitochondrial membrane potential and elevated levels of lipid hydroperoxides.

Membrane translocation of vinculin after UVA exposure facilitates melanosome trafficking.

Skin pigmentation is among the defenses against ultraviolet (UV) radiation. During formation of skin pigmentation, melanosomes that are transported to the cell membrane and released are internalized by keratinocytes. We here examined whether vinculin, the origin of actin fibers, is involved in this intracellular transport of melanosomes by using melanocytes with suppressed vinculin expression. Using fluorescence immunostaining, the migration of vinculin to the cell membrane due to exposure to 365-nm LED light was examined. The intracellular distribution of melanosomes after irradiation was weighted toward the pericellular region compared with non-irradiated cells. With the suppression of vinculin expression, the amount of extracellularly released melanin decreased. We conclude that the membrane migration of vinculin after UVA exposure is involved in the intracellular transport of melanosomes.

Protective effects of gallocatechin gallate against ultraviolet B induced skin damages in hairless mice.

Epigallocatechin gallate (EGCG) has the effect to protect skin from ultraviolet B (UVB) induced damages, but it is unstable under ambient conditions, being susceptible to become brown in color. Gallocatechin gallate (GCG), an epimer counterpart of EGCG, is more stable chemically than EGCG. The potential effects of GCG against UVB-induced skin damages has not been available. The objective of this study was to investigate the protective effects of GCG against UVB-induced skin photodamages. GCG was topically applied on the skin of hairless mice at three dosage levels (LL, 12.5 mg/mL; ML 25 mg/mL; HL, 50 mg/mL), with EGCG and a commercially available baby sunscreen lotion SPF50 PA+++ as control. The mice were then irradiated by UVB (fluence rate 1.7 µmol/m2 s) for 45 min. The treatments were carried out once a day for 6 consecutive days. Skin measurements and histological studies were performed at the end of experiment. The results show that GCG treatments at ML and HL levels inhibited the increase in levels of skin oil and pigmentation induced by UVB irradiation, and improved the skin elasticity and collagen fibers. GCG at ML and HL levels inhibited the formation of melanosomes and aberrations in mitochondria of UVB-irradiated skin in hairless mice. It is concluded that GCG protected skin from UVB-induced photodamages by improving skin elasticity and collagen fibers, and inhibiting aberrations in mitochondria and formation of melanosomes.

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

The effect of aging and antioxidants on photoreactivity and phototoxicity of human melanosomes: An in vitro study.

Aging may significantly modify antioxidant and photoprotective properties of melanin in retinal pigment epithelium (RPE). Here, photoreactivity of melanosomes (MS), isolated from younger and older human donors with and without added zeaxanthin and α-tocopherol, was analyzed by electron paramagnetic resonance oximetry, time-resolved singlet oxygen phosphorescence, and protein oxidation assay. The phototoxic potential of ingested melanosomes was examined in ARPE-19 cells exposed to blue light. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was determined by flow cytometry. Irradiation of cells fed MS induced significant inhibition of the specific phagocytosis with the effect being stronger for melanosomes from older than from younger human cohorts, and enrichment of the melanosomes with antioxidants reduced the inhibitory effect. Cellular protein photooxidation was more pronounced in samples containing older melanosomes, and it was diminished by antioxidants. This study suggests that blue light irradiated RPE melanosomes could induce substantial inhibition of the key function of the cells-their specific phagocytosis. The data indicate that while photoreactivity of MS and their phototoxic potential increase with age, they could be reduced by selected natural antioxidants.

Darkening with UVRAG.

Ultraviolet radiation (UVR)-induced skin pigmentation, afforded by the dark organelles termed melanosomes, accounts for the first-line protection against environmental UVR that increases the risk of developing skin cancers including melanoma. We have recently discovered that UVRAG, originally identified as a BECN1-binding macroautophagy/autophagy protein, appears to have a specialized function in melanosome biogenesis beyond autophagy through its interaction with the biogenesis of lysosome-related organelles complex 1 (BLOC-1). This melanogenic function of UVRAG is controlled by the melanocyte-specific transcription factor MITF as a downstream effector of the α-melanocyte-stimulating hormone (α-MSH)-cAMP signaling in the suntan response, which is compromised in BRAF mutant melanoma. Thus we propose a new mode of UVRAG activity and regulation in melanocyte biology that may affect melanoma predisposition.

Melanin-concentrating hormone is a major substance mediating light wavelength-dependent skin color change in larval zebrafish.

Melanosome dispersion is important for protecting the internal organs of fish against ultraviolet light, especially in transparent larvae with underdeveloped skin. Melanosome dispersion leads to dark skin color in dim light. Melanosome aggregation, on the other hand, leads to pale skin color in bright light. Both of these mechanisms are therefore useful for camouflage. In this study, we investigated a hormone thought to be responsible for the light wavelength-dependent response of melanophores in zebrafish larvae. We irradiated larvae using light-emitting diode (LED) lights with peak wavelengths (λmax) of 355, 400, 476, 530, and 590 nm or fluorescent light (FL) 1-4 days post fertilization (dpf). Melanosomes in skin melanophores were more dispersed under short wavelength light (λmax ≤ 400 nm) than under FL. Conversely, melanosomes were more aggregated under mid-long wavelength light (λmax ≥ 476 nm) than under FL. In addition, long-term (1-12 dpf) irradiation of 400 nm light increased melanophores in the skin, whereas that of 530 nm light decreased them. In teleosts, melanin-concentrating hormone (MCH) aggregates melanosomes within chromatophores, whereas melanocyte-stimulating hormone, derived from proopiomelanocortin (POMC), disperses melanosomes. The expression of a gene for MCH was down-regulated by short wavelength light but up-regulated by mid-long wavelength light, whereas a gene for POMC was up-regulated under short wavelength light. Melanosomes in larvae (4 dpf) exposed to a black background aggregated when immersing the larvae in MCH solution. Yohimbine, an α2-adrenergic receptor antagonist, attenuated adrenaline-dependent aggregation in larvae exposed to a black background but did not induce melanosome dispersion in larvae exposed to a white background. These results suggest that MCH plays a key role in the light wavelength-dependent response of melanophores, flexibly mediating the transmission of light wavelength information between photoreceptors and melanophores.

Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage.

Melanosomes are membrane-bound intracellular organelles that are uniquely generated by melanocytes (MCs) in the basal layer of human epidermis. Highly pigmented mature melanosomes are transferred from MCs to keratinocytes (KCs), and then positioned in the supra-nuclear region to ensure protection against ultraviolet radiation (UVR). However, the molecular mechanism underlying melanosome (or melanin pigment) transfer remains enigmatic. Emerging evidence shows that exo-/endo-cytosis of the melanosome core (termed melanocore) has been considered as the main transfer manner between MCs and KCs. As KCs in the skin migrate up from the basal layer and undergo terminal differentiation, the melanocores they have taken up from MCs are subjected to degradation. In this study, we isolated individual melanocores from human MCs in culture and then induced their destruction/disruption using a physical approach. The results demonstrate that the ultrastructural integrity of melanocores is essential for their antioxidant and photoprotective properties. In addition, we also show that cathepsin V (CTSV), a lysosomal acid protease, is involved in melanocore degradation in calcium-induced differentiated KCs and is also suppressed in KCs following exposure to UVA or UVB radiation. Thus, our study demonstrates that change in the proportion of melanocores in the intact/undegraded state by CTSV-related degradation in KCs affects photoprotection of the skin.

Photodegradation of Eumelanin and Pheomelanin and Its Pathophysiological Implications.

Eumelanin is photoprotective for pigmented tissues while pheomelanin is phototoxic. In this review, we summarize current understanding of how eumelanin and pheomelanin structures are modified by ultraviolet A (UVA) and also by visible light and how reactive oxygen species participate in those processes. Alkaline hydrogen peroxide oxidation was employed to characterize eumelanin and benzothiazole-type pheomelanin, giving pyrrole-2,3,5-tricarboxylic acid (PTCA) and thiazole-2,4,5-tricarboxylic acid (TTCA), respectively. Reductive hydrolysis with hydroiodic acid gives 4-amino-3-hydroxyphenylalanine (4-AHP) from the benzothiazine moiety of pheomelanin. The results show that the photoaging of eumelanin gives rise to free PTCA (produced by peroxidation in situ) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA, produced by cross-linking). The TTCA/4-AHP ratio increases with photoaging, indicating the conversion of benzothiazine to the benzothiazole moiety. Analysis of those markers and their ratios show that both eumelanin and pheomelanin in human retinal pigment epithelium melanosomes undergo extensive structural modifications due to their lifelong exposure to blue light. Using synthetic melanins, we also found that singlet oxygen, in addition to superoxide anions, is photogenerated and quenched upon UVA irradiation. The (patho)physiological significance of those findings is discussed in relation to the tanning process, to melanomagenesis in the skin and to age-related macular degeneration in the eyes.

Loss of Melanin by Eye Retinal Pigment Epithelium Cells Is Associated with Its Oxidative Destruction in Melanolipofuscin Granules.

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p < 0.05, 150 eyes) lower than in a melanosome, which indicates melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.

Induction of retinal-dependent calcium influx in human melanocytes by UVA or UVB radiation contributes to the stimulation of melanosome transfer.

OBJECTIVES: The transfer of melanosomes from melanocytes to neighbouring keratinocytes is critical to protect the skin from the deleterious effects of ultraviolet A (UVA) and ultraviolet B (UVB) irradiation; however, the initial factor(s) that stimulates melanosome transfer remains unclear. In this study, we investigated the induction of retinal-dependent calcium (Ca2+ ) influx in melanocytes (MCs) by UVA or UVB irradiation and the effect of transient receptor potential cation channel subfamily M member 1 (TRPM1) (melastatin1)-related Ca2+ influx on melanosome transfer. MATERIALS AND METHODS: Primary human epidermal MCs were exposed to physiological doses of UVB or UVA light and loaded with a calcium indicator Fluo-4 dye. The change of intracellular calcium of MCs was monitored using a two-photon confocal fluorescence microscopy. MCs were co-cultured with human epidermal keratinocytes (KCs) in the absence or presence of voriconazole (a TRPM1 blocker) or calcium chelators. MCs were also transfected with TRPM1 siRNA for silencing the expression of TRPM1 gene. The melanosome transfer in the co-cultured cells was quantitatively analysed using flow cytometry and was further confirmed by immunofluorescent double-staining. The protein levels and distributions of TRPM1, OPN3 and OPN5 in MCs were measured by Western blotting or immunofluorescent staining. RESULTS: The retinal-dependent Ca2+ influx of UVA-exposed melanocytes differed greatly from that of UVB-exposed melanocytes in the timing-phase. The protein expression of TRPM1 in mono- and co-cultured MCs was dose-dependently up-regulated by UVA and UVB. TRPM1 siRNA-mediated knockdown and the blockage of TRPM1 channel using a putative antagonist (voriconazole) significantly inhibited melanosome transfer in co-cultures following UVA or UVB exposure. CONCLUSIONS: The distinct time-phases of Ca2+ influx in MCs induced by UVA or UVB contribute to the consecutive stimulation of melanosome transfer, thereby providing a potent photoprotection against harmful UV radiation.

Inhibitory effect of 2-methyl-naphtho[1,2,3-de]quinolin-8-one on melanosome transport and skin pigmentation.

Melanosomes are lysosome-related organelles with specialized capabilities of melanin synthesis and movement mediated by the Rab27a-Melanophilin-MyosinVa protein complex. In this study, we found that 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO) induced melanosome aggregation around the nucleus in melan-a melanocytes and in melan-a melanocytes/SP-1 keratinocyte co-cultures without inducing toxicity or changing the melanin content. Western blot and real-time PCR analyses showed that MNQO decreased expression of the Rab27a, Melanophilin and MyosinVa proteins and mRNAs, respectively, in melan-a melanocytes. In a reconstituted human epidermis model, treatment with 0.001% MNQO reduced skin pigmentation. Also, MNQO reduced skin pigmentation in brown guinea pigs induced by UVB irradiation. These results indicated that regulation of melanosome transport may serve as a good target for new skin depigmenting agents and MNQO itself could be a candidate.

Photoaging of retinal pigment epithelial melanosomes: The effect of photobleaching on morphology and reactivity of the pigment granules.

To elucidate the mechanism of age-related changes in antioxidant and photoprotective properties of human retinal pigment epithelium (RPE) melanosomes, the effect of in vitro photoaging of bovine RPE melanosomes was examined employing an array of complementary spectroscopic and analytical methods. Electron paramagnetic resonance (EPR) spectroscopy, saturation recovery EPR, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to determine melanin content of control and photobleached melanosomes, and to monitor changes in their morphology. Methylene blue (MB), TEMPO choline, dysprosium(III) ions and singlet oxygen were employed as molecular probes to characterize the efficiency of control and photobleached melanosomes to interact with different reagents. EPR oximetry, UV-vis absorption spectroscopy, iodometric assay of lipid hydroperoxides and time-resolved singlet oxygen phosphorescence were used to analyze the efficiency of photobleached and untreated melanosomes to inhibit MB-photosensitized oxidation of liposomal lipids. The obtained results revealed that, compared to untreated melanosomes, moderately photobleached melanosomes protected unsaturated lipids less efficiently against photosensitized peroxidiation, while weakly photobleached melanosomes were actually better antioxidant and photoprotective agents. The observed changes could be attributed to two effects - modification of the melanosome morphology and oxidative degradation of the melanin functional groups induced by different degree of photobleaching. While the former increases the accessibility of melanin nanoaggregates to reagents, the latter reduces the efficiency of melanin to interact with chemical and physical agents.

Trends in nanosecond melanosome microcavitation up to 1540 nm.

Thresholds for microcavitation of bovine and porcine melanosomes were previously reported, using single nanosecond (ns) laser pulses in the visible (532 nm) and the near-infrared (NIR) from 1000 to 1319 nm. Here, we report average radiant exposure thresholds for bovine melanosome microcavitation at additional NIR wavelengths up to 1540 nm, which range from ∼0.159 J∕cm2 at 800 nm to 4.5 J∕cm2 at 1540 nm. Melanosome absorption coefficients were also estimated, and decreased with increasing wavelength. These values were compared to retinal pigment epithelium coefficients, and to water absorption, over the same wavelength range. Corneal total intraocular energy retinal damage threshold values were estimated and compared to the previous (2007) and recently changed (2014) maximum permissible exposure (MPE) safe levels. Results provide additional data that support the recent changes to the MPE levels, as well as the first microcavitation data at 1540 nm, a wavelength for which melanosome microcavitation may be an ns-pulse skin damage mechanism.

Multifaceted pathways protect human skin from UV radiation.

The recurrent interaction of skin with sunlight is an intrinsic constituent of human life, and exhibits both beneficial and detrimental effects. The apparent robust architectural framework of skin conceals remarkable mechanisms that operate at the interface between the surface and environment. In this Review, we discuss three distinct protective mechanisms and response pathways that safeguard skin from deleterious effects of ultraviolet (UV) radiation. The unique stratified epithelial architecture of human skin along with the antioxidant-response pathways constitutes the important defense mechanisms against UV radiation. The intricate pigmentary system and its intersection with the immune-system cytokine axis delicately balance tissue homeostasis. We discuss the relationship among these networks in the context of an unusual depigmenting disorder, vitiligo. The elaborate tunable mechanisms, elegant multilayered architecture and evolutionary selection pressures involved in skin and sunlight interaction makes this a compelling model to understand biological complexity.

Consideration of dynamic photothermal effect for evaluation of scanning light sources in optical devices using pulsed source criteria.

Quantitative evaluation of the potential radiation hazards of scanning light sources in medical optical devices is critical. Currently, point scanning light sources of continuous radiation are treated as pulsed sources, where the dwell time at each point is equal to the pulse duration. This study compares the photothermal effects from scanning light and pulsed sources using numerical calculation for scanning without restricting aperture and with various spot sizes. The calculation results show that the thermal damage threshold of scanning source not restricted by measurement aperture does not significantly differ from that of pulsed source. Temporal temperature response and size-dependent photothermal effect also confirm the similarity between scanning and pulsed sources.

Trends in melanosome microcavitation thresholds for nanosecond pulse exposures in the near infrared.

Thresholds for microcavitation of bovine and porcine melanosomes were determined using nanosecond laser pulses in the near-infrared (1000 to 1319 nm) wavelength regime. Isolated melanosomes were irradiated by single pulses (10 or 50 ns) using a Q-switched Spectra Physics Nd:YAG laser coupled with an optical parametric oscillator (1000 to 1200 nm) or a continuum laser at 1319 nm. Time-resolved nanosecond strobe photography after the arrival of the irradiation beam allowed imaging of microcavitation events. Average fluence thresholds for microcavitation increased nonlinearly with increasing wavelength from ∼0.5 J/cm2 at 1000 nm to 2.6 J/cm2 at 1319 nm. Fluence thresholds were also measured for 10-ns pulses at 532 nm and found to be comparable to visible nanosecond pulse values published in previous reports. Calculated melanosome absorption coefficients decreased from 925 cm-1 at 1000 nm to 176 cm-1 at 1319 nm. This trend was found to be comparable to the decrease in retinal pigmented epithelial layer absorption coefficients reported over the same wavelength region. Estimated corneal total intraocular energy retinal damage threshold values were determined in order to compare to current and proposed maximum permissible exposure (MPE) safe levels. Results from this study support recently proposed changes to the MPE levels.