A melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma.

Melanocortin 1 receptor (MC1R) signaling stimulates black eumelanin production through a cAMP-dependent pathway. MC1R polymorphisms can impair this process, resulting in a predominance of red phaeomelanin. The red hair, fair skin and UV sensitive phenotype is a well-described melanoma risk factor. MC1R polymorphisms also confer melanoma risk independent of pigment. We investigated the effect of Mc1r deficiency in a mouse model of UV-induced melanoma. C57BL/6-Mc1r+/+-HGF transgenic mice have a characteristic hyperpigmented black phenotype with extra-follicular dermal melanocytes located at the dermal/epidermal junction. UVB induces melanoma, independent of melanin pigmentation, but UVA-induced and spontaneous melanomas are dependent on black eumelanin. We crossed these mice with yellow C57BL/6-Mc1re/e animals which have a non-functional Mc1r and produce predominantly yellow phaeomelanin. Yellow C57BL/6-Mc1re/e-HGF mice produced no melanoma in response to UVR or spontaneously even though the HGF transgene and its receptor Met were expressed. Total melanin was less than in C57BL/6-Mc1r+/+-HGF mice, hyperpigmentation was not observed and there were few extra-follicular melanocytes. Thus, functional Mc1r was required for expression of the transgenic HGF phenotype. Heterozygous C57BL/6-Mc1re/+-HGF mice were black and hyperpigmented and, although extra-follicular melanocytes and skin melanin content were similar to C57BL/6-Mc1r+/+-HGF animals, they developed UV-induced and spontaneous melanomas with significantly less efficiency by all criteria. Thus, heterozygosity for Mc1r was sufficient to restore the transgenic HGF phenotype but insufficient to fully restore melanoma. We conclude that a previously unsuspected melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma and postulate that this pathway is involved in human melanoma.

Melanoma induction by ultraviolet A but not ultraviolet B radiation requires melanin pigment.

Malignant melanoma of the skin (CMM) is associated with ultraviolet radiation exposure, but the mechanisms and even the wavelengths responsible are unclear. Here we use a mammalian model to investigate melanoma formed in response to precise spectrally defined ultraviolet wavelengths and biologically relevant doses. We show that melanoma induction by ultraviolet A (320-400 nm) requires the presence of melanin pigment and is associated with oxidative DNA damage within melanocytes. In contrast, ultraviolet B radiation (280-320 nm) initiates melanoma in a pigment-independent manner associated with direct ultraviolet B DNA damage. Thus, we identified two ultraviolet wavelength-dependent pathways for the induction of CMM and describe an unexpected and significant role for melanin within the melanocyte in melanomagenesis.

Retinal photodamage by endogenous and xenobiotic agents.

The human eye is constantly exposed to sunlight and artificial lighting. Light transmission through the eye is fundamental to its unique biological functions of directing vision and circadian rhythm and therefore light absorbed by the eye must be benign. However, exposure to the very intense ambient radiation can pose a hazard particularly if the recipient is over 40 years of age. There are age-related changes in the endogenous (natural) chromophores (lipofuscin, A2E and all-trans-retinal derivatives) in the human retina that makes it more susceptible to visible light damage. Intense visible light sources that do not filter short blue visible light (400-440 nm) used for phototherapy of circadian imbalance (i.e. seasonal affective disorder) increase the risk for age-related light damage to the retina. Moreover, many drugs, dietary supplements, nanoparticles and diagnostic dyes (xenobiotics) absorb ocular light and have the potential to induce photodamage to the retina, leading to transient or permanent blinding disorders. This article will review the underlying reasons why visible light in general and short blue visible light in particular dramatically raises the risk of photodamage to the human retina.

Retinal pigment epithelium response to oxidant injury in the pathogenesis of early age-related macular degeneration.

Age-related macular degeneration (AMD) represents the leading cause of vision loss in the elderly. Accumulation of lipid- and protein-rich deposits under the retinal pigment epithelium (RPE) heralds the onset of early AMD, but the pathogenesis of subretinal deposit formation is poorly understood. Numerous hypothetical models of deposit formation have been proposed, including hypotheses for a genetic basis, choroidal hypoperfusion, abnormal barrier formation, and lysosomal failure. This review explore the RPE injury hypothesis, characterized by three distinct stages (1) Initial RPE oxidant injury, caused by any number of endogenous or exogenous oxidants, results in extrusion of cell membrane "blebs," together with decreased activity of matrix metalloproteinases (MMPs), promoting bleb accumulation under the RPE as basal laminar deposits (BLD). (2) RPE cells are subsequently stimulated to increase synthesis of MMPs and other molecules responsible for extracellular matrix turnover (i.e., producing decreased collagen), affecting both RPE basement membrane and Bruchs membrane (BrM). This process leads to progression of BLD into basal linear deposits (BLinD) and drusen by admixture of blebs into BrM, followed by the formation of new basement membrane under the RPE to trap these deposits within BrM. We postulate that various hormones and other plasma-derived molecules related to systemic health cofactors are implicated in this second stage. (3) Finally, macrophages are recruited to sites of RPE injury and deposit formation. The recruitment of nonactivated or scavenging macrophages may remove deposits without further injury, while the recruitment of activated or reparative macrophages, through the release of inflammatory mediators, growth factors, or other substances, may promote complications and progression to the late forms of the disease.

Neuromelanin activates microglia and induces degeneration of dopaminergic neurons: implications for progression of Parkinson's disease.

In Parkinson's disease (PD), there is a progressive loss of neuromelanin (NM)-containing dopamine neurons in substantia nigra (SN) which is associated with microgliosis and presence of extracellular NM. Herein, we have investigated the interplay between microglia and human NM on the degeneration of SN dopaminergic neurons. Although NM particles are phagocytized and degraded by microglia within minutes in vitro, extracellular NM particles induce microglial activation and ensuing production of superoxide, nitric oxide, hydrogen peroxide (H2O2), and pro-inflammatory factors. Furthermore, NM produces, in a microglia-depended manner, neurodegeneration in primary ventral midbrain cultures. Neurodegeneration was effectively attenuated with microglia derived from mice deficient in macrophage antigen complex-1, a microglial integrin receptor involved in the initiation of phagocytosis. Neuronal loss was also attenuated with microglia derived from mice deficient in phagocytic oxidase, a subunit of NADPH oxidase, that is responsible for superoxide and H2O2 production, or apocynin, an NADPH oxidase inhibitor. In vivo, NM injected into rat SN produces microgliosis and a loss of tyrosine hydroxylase neurons. Thus, these results show that extracellular NM can activate microglia, which in turn may induce dopaminergic neurodegeneration in PD. Our study may have far-reaching implications, both pathogenic and therapeutic.

Comparison of A2E cytotoxicity and phototoxicity with all-trans-retinal in human retinal pigment epithelial cells.

All-trans-retinal is the precursor of A2E, a fluorophore within lipofuscin, which accumulates in human retinal pigment epithelial (hRPE) cells and contributes to age-related macular degeneration. Here we have compared the in vitro dark cytotoxicity and visible-light-mediated photoreactivity of all-trans-retinal and A2E in hRPE cells. All-trans-retinal caused distinct cytotoxicity in hRPE cells measured with cell metabolic activity (MTS) and lactate dehydrogenase release assays. Significant increases in intracellular oxidized glutathione (GSSG), extracellular GSH and GSSG levels and lipid hydroperoxide production were observed in cells incubated in the dark with 25 and 50 microM all-trans-retinal. Light modified all-trans-retinal's harmful action and decreased extracellular glutathione and hydroperoxide levels. A2E (<25 microM) did not affect cell metabolism or cytoplasmic membrane integrity in the dark or when irradiated. 25 microM A2E raised the intracellular GSSG level in hRPE cells to a much smaller extent than 25 microM all-trans-retinal. A2E did not induce glutathione efflux or hydroperoxide generation in the dark or after irradiation. These studies support our previous conclusions that although A2E may be harmful at high concentrations or when oxidized, its phototoxic properties are insignificant compared to those of all-trans-retinal. The endogenous production of A2E may serve as a protective mechanism to prevent damage to the retina by free all-trans-retinal.

PPAR nuclear receptors and altered RPE lipid metabolism in age-related macular degeneration.

The pathophysiology of 'early' dry age-related macular degeneration (ARMD), characterized by the accumulation of lipid and protein-rich sub-retinal deposits remains largely unknown. Accumulation and dysregulated turnover of lipids as well as extracellular matrix (ECM) molecules in sub-retinal pigment epithelial (RPE) deposits and Bruch's membrane, itself an ECM, play a role in ARMD. Epidemiological studies have shown an increased risk for the disease associated with higher dietary intake of long chain poly-unsaturated fatty acids (LCPUFA) and specifically more so for n-6 versus n-3 fatty acids. PUFAs are membrane targets of lipid peroxidation and natural ligands for the nuclear receptors, peroxisome proliferator activated receptors (PPAR). Here we investigated the expression of genes involved in lipid metabolism and expression of the three isoforms of PPARs in an immortalized cell line of human RPE cells (ARPE19) in the presence or absence of fatty acids.

Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells.

The water-soluble nanoparticle hydroxylated fullerene [fullerol, nano-C60(OH)(22-26)] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have previously found that fullerol is both cytotoxic and phototoxic to human lens epithelial cells (HLE B-3) and that the endogenous antioxidant lutein blocked some of this phototoxicity. In the present study we have found that fullerol induces cytotoxic and phototoxic damage to human retinal pigment epithelial cells. Accumulation of nano-C60(OH)(22-26) in the cells was confirmed spectrophotometrically at 405 nm, and cell viability, cell metabolism and membrane permeability were estimated using trypan blue, MTS and LDH assays, respectively. Fullerol was cytotoxic toward hRPE cells maintained in the dark at concentrations higher than 10 microM. Exposure to an 8.5 J x cm(-2) dose of visible light in the presence of >5 microM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 microM lutein offered some protection against fullerol photodamage. Using time resolved photophysical techniques, we have now confirmed that fullerol produces singlet oxygen with a quantum yield of Phi=0.05 in D2O and with a range of 0.002-0.139 in various solvents. As our previous studies have shown that fullerol also produces superoxide in the presence of light, retinal phototoxic damage may occur through both type I (free radical) and type II (singlet oxygen) mechanisms. In conclusion, ocular exposure to fullerol, particularly in the presence of sunlight, may lead to retinal damage.

Peroxidation of lipids in liposomal membranes of different composition photosensitized by chlorpromazine.

Chlorpromazine (CPZ), a phenothiazine derivative, is a neuroleptic drug widely used in medicine because of its tranquilizing and antipsychotic properties. CPZ often causes side effects including cutaneous photosensitization and ocular damage. It was suggested that reactive oxygen species, including singlet oxygen, might play an important role in its phototoxicity. In this work, we studied effects of cholesterol and saturation of fatty acid phospholipids on peroxidation of liposomal lipids induced by UVA irradiation in the presence of chlorpromazine by measuring lipid hydroperoxides using HPLC-EC(Hg) and the iodometric assay. Our study shows that the CPZ-photosensitized peroxidation of lipids is modified by cholesterol and the type of phospholipids present in the liposomes. Thus in liposomes made of the unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), chlorpromazine photosensitized strong peroxidation of lipids, which was mainly due to generation of singlet oxygen. In such liposomes, CPZ-photosensitized peroxidation of lipids was decreased, in a dose-dependent manner, by addition of cholesterol. On the other hand, in liposomes made of dimyristoyl-phosphatidylcholine (DMPC) and cholesterol peroxidation of lipids was 5 fold slower, while generation of singlet oxygen was reduced over 20 fold. Importantly, cholesterol had no effect on lipid peroxidation in POPC and DMPC liposomes induced by other photosensitizers such as Rose Bengal and merocyanine 540. We postulate that the effect of cholesterol on peroxidation of lipids in liposomal membranes, photosensitized by chlorpromazine, is mostly due to the cholesterol-dependent modulation of CPZ partition in such membranes, which dramatically lowers the efficiency of the photoexcited CPZ to generate singlet oxygen.

Ascorbate enhances photogeneration of hydrogen peroxide mediated by the iris melanin.

The iris in the human eye is exposed to UV and visible light transmitted by the cornea. This pigmented tissue is bathed with the aqueous humor (AqH), which contains high concentration of ascorbate. It has been postulated that the presence of ascorbate in AqH can contribute to increased photoproduction of H2O2 mediated by the iris melanin. In this study, we monitored generation of H2O2 induced by UV-VIS irradiation of bovine irides, bovine and human iris homogenates and iris melanin. Our data show that exogenous ascorbate significantly amplified the rate of H2O2 photoformation in all melanotic samples. Deactivation of endogenous catalase with NaN3 in bovine irides increased the level of the accumulated H2O2 in the bathing solution following sample irradiation. Photoformation of H2O2 in samples with exogenous ascorbate was accompanied by its photo-oxidation. Both photoprocesses exhibited significant wavelength dependence. EPR spectroscopy measurements showed that ascorbyl radical is an intermediate product of the ascorbate photo-oxidation. The photoproduction of H2O2 and photo-oxidation of ascorbate appear to be stoichiometric processes. No significant differences in the photoreactivity of iridial melanin from donors of different age and iris color was found. We postulate that also in vivo ascorbate increases the rate of iris melanin-mediated photoformation of H2O2 and its steady state concentration in AqH.

Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells.

The water-soluble, hydroxylated fullerene [fullerol, nano-C60(OH)22-26] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C60(OH)22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 microM. Exposure to either UVA or visible light in the presence of >5 microM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 microM lutein, 1 mM N-acetyl cysteine, or 1 mM l-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA>visible light>dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein alpha-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C60(OH)22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo.

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells.

The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE-B3) were severely damaged when incubated with berberine (25 microM) and exposed to UVA (5 J cm(-2)). Under the same conditions, palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N-acetylcysteine (5 mM). When exposed to UVA (5 J cm(-2)) both berberine (10 microM) and palmatine (10 microM) caused mild DNA damage as determined by the alkaline comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the adult human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (lambda > 400 nm) approximately 10 times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged DNA in hRPE. Infusions of Goldenseal are estimated to contain approximately 1 mM berberine, while in tinctures the alkaloid concentration may be more than 10 times higher. Our findings show that eyewashes and lotions derived from Goldenseal or containing berberine must be used with caution when the eyes are exposed to bright sunlight but that oral preparations are not likely to cause ocular phototoxicity.

Phototoxicity in human retinal pigment epithelial cells promoted by hypericin, a component of St. John's wort.

St. John's wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges. In vivo studies have determined that hypericin is phototoxic to skin and our previous in vitro studies with lens tissues have determined that it is potentially phototoxic to the human lens. To determine if hypericin might also be phototoxic to the human retina, we exposed human retinal pigment epithelial (hRPE) cells to 10(-7) to 10(-5) M hypericin. Fluorescence emission detected from the cells (lambda(ex) = 488 nm; lambda(em) = 505 nm) confirmed hypericin uptake by human RPE. Neither hypericin exposure alone nor visible light exposure alone reduced cell viability. However when irradiated with 0.7 J cm(-2) of visible light (lambda > 400 nm) there was loss of cell viability as measured by MTS and lactate dehydrogenase assays. The presence of hypericin in irradiated hRPE cells significantly changed the redox equilibrium of glutathione and a decrease in the activity of glutathione reductase. Increased lipid peroxidation as measured by the thiobarbituric acid reactive substances assay correlated to hypericin concentration in hRPE cells and visible light radiation. Thus, ingested SJW is potentially phototoxic to the retina and could contribute to retinal or early macular degeneration.

Melanin in human irides of different color and age of donors.

Melanin is the main chromophore of the human iris. This pigment is considered to be the most important factor that determines the color of the irides. Previous studies based mainly on chemical degradation methods showed that brown irides contain more melanin than blue ones. In our study, we used electron spin resonance (ESR) spectroscopy to detect and characterize melanin free radical centers and associated iron in human irides. Based on this method, we determined the amount of melanin in the irides and the relative content of iron in iridial melanin as a function of their color, shade, and the age of their donors. Chemical degradation of iridial homogenates enabled us to characterize the structure of eumelanin and determine the content of pheomelanin present in human and bovine irides. The ESR amplitude, the normalized intensity obtained by double integration of the ESR signal of melanin, and the content of the pigment in the irides depended on color and shade of the eyes being 40% higher in the brown group of the irides compared with all other groups. On the other hand, the relative iron content normalized to the melanin content in light blue irides showed a small decrease with age of donors. Melanin in human and bovine irides was mostly composed of eumelanin, and pheomelanin content was of the order of a few percent. Although some differences in the structure of eumelanin present in the human and bovine irides are possible, the results obtained in this study suggest that human irides contain eumelanin with very similar chemical properties.

Spectroscopic properties and reactivity of free radical forms of A2E.

A pyridinium bisretinoid (A2E) is the only identified blue-absorbing chromophore of retinal lipofuscin that has been linked to its aerobic photoreactivity and phototoxicity. Pulse radiolysis has been used to study both the one-electron oxidation and the one-electron reduction of A2E in aqueous micellar solutions. The reduction to the semireduced A2E (lambda(max) broad and between 500 and 540 nm) was achieved with formate radicals and the subsequent decay of A2E* was slow (over hundreds of milliseconds) via complex kinetics. The long lifetime of the A2E* should facilitate its reactions with other biomolecules. For example, with oxygen, the A2E* produced the superoxide radical anion with a rate constant of 3 x 10(8) M(-1) s(-1). The A2E was also reduced by the NAD radical, the corresponding rate constant being 2.3 x 10(8) M(-1) s(-1). Other experiments showed that the one-electron reduction potential of A2E lies in the range -640 to -940 mV. The semioxidized form of A2E (lambda(max) 590 nm) was formed via oxidation with the Br2*- radical and had a much shorter lifetime than the semireduced form. With strongly oxidizing peroxyl radicals (CCl3O2*) our kinetic data suggest the formation of a radical adduct followed by dissociation to the semioxidized A2E. With milder oxidizing peroxyl radicals such as that from methanol, our results were inconclusive. In benzene we observed an efficient oxidation of zeaxanthin to its radical cation by the A2E radical cation; this may be relevant to a detrimental effect of A2E in vision.