Riddles in the dark: Decoding the relationship between neuromelanin and neurodegeneration in locus coeruleus neurons.

The noradrenergic locus coeruleus (LC) is among the first regions of the brain affected by pathology in both Alzheimer's disease (AD) and Parkinson's disease (PD), but the reasons for this selective vulnerability are not completely understood. Several features of LC neurons have been proposed as contributing factors to this dysfunction and degeneration, and this review will focus on the presence of neuromelanin (NM). NM is a dark pigment unique to catecholaminergic cells that is formed of norepinephrine (NE) and dopamine (DA) metabolites, heavy metals, protein aggregates, and oxidated lipids. We cover what is currently known about NM and the limitations of historical approaches, then discuss the new human tyrosinase (hTyr) model of NM production in rodent catecholamine cells in vivo that offers unique opportunities for studying its neurobiology, neurotoxicity, and potential of NM-based therapeutics for treating neurodegenerative disease.

Photothermal therapy-induced immunogenic cell death based on natural melanin nanoparticles against breast cancer.

A photothermal and immune co-therapy strategy based on natural melanin nanoparticles was developed for treating primary and abscopal breast cancers.

Using Sepia melanin as a PD model to describe the binding characteristics of neuromelanin - A critical review.

Parkinson's disease is characterised pathologically by a relatively selective death of dopaminergic neurons in the substantia nigra of the brain. The vulnerability of these neurons appears to be linked to the pigment neuromelanin. However, as yet there is limited understanding behind the mechanisms of this disease process. Complications arise due to the difficulty in obtaining appreciable quantities of neuromelanin. Furthermore, an appropriate model for studying neuromelanin has not been identified. To date there has been many studies looking at the binding and chemical characteristics of neuromelanin. However, a range of different synthetic and organic melanins have been used as models and leading to many varied conclusions being drawn. Therefore, the aim of this review is to present Sepia melanin as the most appropriate study model for the binding characteristics of neuromelanin. Considerations included chemical structure, surface characteristics and structural features of both synthetic and organic melanins.

Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging.

Developing multifunctional and easily prepared nanoplatforms with integrated different modalities is highly challenging for molecular imaging. Here, we report the successful transfer of an important molecular target, melanin, into a novel multimodality imaging nanoplatform. Melanin is abundantly expressed in melanotic melanomas and thus has been actively studied as a target for melanoma imaging. In our work, the multifunctional biopolymer nanoplatform based on ultrasmall (<10 nm) water-soluble melanin nanoparticle (MNP) was developed and showed unique photoacoustic property and natural binding ability with metal ions (for example, (64)Cu(2+), Fe(3+)). Therefore, MNP can serve not only as a photoacoustic contrast agent, but also as a nanoplatform for positron emission tomography (PET) and magnetic resonance imaging (MRI). Traditional passive nanoplatforms require complicated and time-consuming processes for prebuilding reporting moieties or chemical modifications using active groups to integrate different contrast properties into one entity. In comparison, utilizing functional biomarker melanin can greatly simplify the building process. We further conjugated αvß3 integrins, cyclic c(RGDfC) peptide, to MNPs to allow for U87MG tumor accumulation due to its targeting property combined with the enhanced permeability and retention (EPR) effect. The multimodal properties of MNPs demonstrate the high potential of endogenous materials with multifunctions as nanoplatforms for molecular theranostics and clinical translation.

Natural melanin: a potential pH-responsive drug release device.

This work proposes melanin as a new nanocarrier for pH-responsive drug release. Melanin is an abundant natural polymer that can be easily extracted from cuttlefish as nanoparticles with a suitable size range for drug delivery. However, despite its high potentiality, the application of this biopolymer in the pharmaceutical and biomedical fields is yet to be explored. Herein, melanin nanoparticles were impregnated with metronidazole, chosen as model antibiotic drug, using supercritical carbon dioxide. The drug release profile was investigated at acidic and physiologic pH, and the dominant mechanism was found to follow a non-Fickian transport. Drug release from melanin shows a strong pH dependency, which allied to its biocompatibility and lack of cytotoxicity envisages its potential application as nanocarrier in formulations for colon and intestine targeted drug delivery.

Potent low toxicity inhibition of human melanogenesis by novel indole-containing octapeptides.

BACKGROUND: Abnormal production and accumulation of melanin are characteristics of a number of skin disorders, including postinflammatory hyperpigmentation and melasma. Our objective was to develop and validate novel oligopeptides with potent inhibitory activity against mushroom and human tyrosinase with minimal toxicity toward melanocytes, keratinocytes, and fibroblasts. METHODS: A library of short sequence oligopeptides was docked against the crystal structure of mushroom tyrosinase to screen for favorable binding free energies and direct interaction with the catalytic pocket. The inhibitory activity of the octapeptides and hydroquinone (HQ) was assessed using mushroom and human tyrosinase and melanin content via human primary melanocytes. Effects on cell viability and proliferation were determined using the MTT assay and cytotoxicity via trypan blue exclusion. RESULTS: Octapeptides P16-18 outperformed HQ, the benchmark of hypopigmenting agents, in all tested categories. Prolonged incubation of human keratinocytes, fibroblasts, or melanocytes with 30-3000µM HQ led to 8- to 65-fold greater cell death than with octapeptides. After 6d of incubation with 30µM HQ, we observed 70±3% and 60±2% cell death in melanocytes and fibroblasts, respectively, versus minimal toxicity up to an octapeptide concentration of 3mM. CONCLUSION: Octapeptides P16-18 are potent competitive tyrosinase inhibitors with minimal toxicity toward the major cell types of human skin. GENERAL SIGNIFICANCE: The findings in our study suggest that all three novel octapeptides may serve as safe and efficacious replacements of HQ for the treatment of pigmentary disorders.

Neuromelanin enhances the toxicity of α-synuclein in SK-N-SH cells.

The key pathological feature of Parkinson's disease (PD) is selective degeneration of the neuromelanin (NM)-pigmented dopaminergic neurons in the substantia nigra (SN). NM, like other risk factors, such as oxidative stress (OS) and α-synuclein (α-syn), is involved in the pathogenesis of PD. But whether or not NM synergizes with α-syn or OS in the pathogenesis of PD remains unexplored. In the present study, we examined the effects of NM on cellular viability, apoptosis and free radical production in α-syn over-expressing human neuroblastoma cell line (SK-N-SH) in the presence or absence of the oxidizer Fenton's Reagent (FR). We showed that NM synergized with FR in suppressing cell viability, and in inducing apoptosis and hydroxyl radical production in all SK-N-SH cell lines. α-Syn over-expressing cells exhibited more pronounced effect, especially the A53T mutation. Our findings suggest that NM synergizes with both OS and α-syn in conferring dopaminergic vulnerability, adding to our understanding of the pathogenesis of PD.

Carnosine protects against the neurotoxic effects of a serotonin-derived melanoid.

Anesthesia-related postoperative cognitive dysfunction (POCD) leads to morbidity in the elderly. Lipid peroxidative byproducts (i.e. acrolein) accumulate in aging and may play a role. Sevoflurane, an inhaled anesthetic, sequesters acrolein and enhances the formation of a serotonin-derived melanoid (SDM). SDM may be a biologically relevant polymeric melanoid that we previously showed exhibits redox activity and disrupts lipid bilayers. In this study, we examined the toxicity of SDM in cell culture and looked at protection using L-carnosine. SDM's toxic effects were tested on neuronal-like SH-SY5Y cells, causing an exponential decrease in viability, while human dermal fibroblasts were completely resistant to the toxic effects. SDM brought about morphological changes to differentiated SH-SY5Y cells, particularly to neuronal processes. Co- but not pre-treatment with L-carnosine protected differentiated SH-SY5Y cells exposed to SDM. Our mechanism suggests focal sevoflurane-induced sequestration of age-related acrolein leading to SDM synthesis and neuronal impairment, which is prevented by L-carnosine.

A comparative evaluation of photo-toxic effect of fractionated melanin and chlorpromazine hydrochloride on human (dermal fibroblasts and epidermal keratinocytes) and mouse cell line/s (fibroblast Balb/c 3T3).

Fractionated melanin (Mel-HEV), a bleached version of natural melanin, offers protection against the high energy visible (HEV/UVA) and ultraviolet (specifically UVA) irradiation making it a potential compound to be added to skin care and sunscreen formulations and other cosmetic and personal care products. Chlorpromazine (CPZ) has been shown to exhibit photosensitivity and phototoxicity reaction in vitro and in vivo. Comparative evaluation of chemotoxicity and phototoxicity using Mel-HEV and CPZ (as positive control) was performed on mouse fibroblast cell line 'Balb/c 3T3'. This is the recommended method for evaluating the phototoxic potential of compounds under the European Center of Validation of Alternative Methods (ECVAM) guidelines (OECD, 2004). This study was expanded from a mouse cell line - Balb 3T3/c to two human cell lines - HDF and HEKn for two reasons: to compare the difference between the sensitivity and behavior of two fibroblast cell lines (Balb/c 3T3 vs. HDF) and to compare the differences between two fibroblast cell lines with the keratinocyte cell line (HDF & Balb/c 3T3 vs. HEKn). It was found that Balb/c 3T3 and HEKn were both sensitive to the phototoxic potential of CPZ. However, HDF showed insensitivity to phototoxic evaluation. The test compound, Mel-HEV, was found to be non-phototoxic. The mean toxic concentration (MTC) for CPZ during HEV and UVA exposure conditions was found to be similar using Balb/c 3T3 (36.25 µg/ml) and HEKn (39.99 µg/ml) showing that cells exhibit similar responses at HEV/UVA- conditions. However, Balb/c 3T3 showed more sensitivity to CPZ at HEV/UVA+ condition (MTC=0.87 µg/ml; mean PIF=55.33; MPE=0.395) than HEKn (MTC=5.35 µg/ml; PIF=7.61; MPE=0.276) making it the preferred cell line for phototoxicity evaluations.

Toxicity of a serotonin-derived neuromelanin.

Postoperative Cognitive Dysfunction (POCD) is associated with increased mortality in the elderly and may occur from lipid peroxidation in aging. We previously showed that sevoflurane sequesters acrolein, which promotes the formation of a novel species of a putative neuromelanin. The current study examined the properties of this serotonin-derived melanoid (SDM). The interaction of SDM with unilamellar vesicles (ULVs) was examined using lipid membrane probes. Vesicle disruption was investigated by leakage of dye from calcein-loaded ULVs. We observed that SDM decreased diphenyl-hexatriene fluorescence anisotropy and increased the temperature-dependent change in anisotropy. SDM changed the absorbance of merocyanin-bound ULVs. SDM increased detergent-mediated calcein leakage. SDM structure was dramatically altered upon interaction with ULVs. We also observed that SDM enhanced detergent-mediated leakage of loaded ULVs, suggesting that SDM may be neurotoxic. We propose that inhalational agents, which sequester acrolein, may promote the production of certain species of neuromelanin that depletes local serotonin and enhances neuronal vulnerability.

Analysis of plasmid DNA damage induced by melanin with capillary electrophoresis.

Dilute linear poly(N-isopropylacrylamide) (PNIPAM) in Tris-Mes-EDTA (TME) buffer was used as sieving matrix for capillary electrophoresis (CE) of plasmid DNA and plasmid topological isomers induced by melanin in uncoated capillary. At the optimized condition of 0.1% (w/v) PNIPAM in TME buffer, base line separation of the plasmid DNA ladder (2-12 kbp) was achieved within 15 min. Three positive clones with inserts of 468, 1147 and 1566 bp can be distinguished from the plasmid pUC 18 vector within 13 min. The migration order of the plasmid topological isomers in the dynamic coating matrix was confirmed by the enzymatically prepared and UV-induced plasmids. The covalently closed circular form appeared firstly, followed by the linear plasmid form and then the open circular form. The effect of bacterial melanin obtained from Pseudomonas maltophilia AT18 on plasmid pUC 18 was investigated by CE in uncoated capillary in vitro. Plasmid pUC 18 incubated with either melanin or copper ions alone sustained little DNA damage. The combination of melanin with Cu(II) can cause the plasmid pUC 18 conformational changes from covalently closed circular form to open form. Understanding the damage effect of melanin with copper ions on DNA would be important for the melanin-related application, such as photoprotective antioxidant in protecting the skin from cancer, pathophysiology research in clinic. The investigation of melanin induced plasmid conformational changes by CE in uncoated capillary also revealed that the application of the dynamic coating matrix could be extended to the study of plasmid conformational changes in other plasmid-based biological technologies.

Effects of melanin and manganese on DNA damage and repair in PC12-derived neurons.

The mechanism of neurotoxicity produced by the interaction of melanin with manganese was investigated in PC12-derived neuronal cell cultures. The cells were incubated with melanin (25-500 microg/ml), MnCl2 (10 ng/ml-100 microg/ml) and a combination of both substances for 24 and 72 h. Incubation with either toxicant alone resulted in a minimal decrease in cell viability. The combination of melanin and manganese caused significant (up to 60%) decreases in viability of PC12 cells in a dose-dependent manner. Increases in oxidative DNA damage, indicated by levels of 8-hydroxy-2'deoxyguanosine (8-oxodG), was associated with decreased cell viability. Melanin alone, but not manganese alone, resulted in increased oxidative DNA damage. The maximal increase in 8-oxodG caused by melanin was about seven times higher than control after 24 h of exposure. The activity of the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), was increased in cells incubated with single toxicants and their combinations for 24 h. On the third day of incubation with the toxicants, activity of OGG1 declined below control levels and cell viability significantly decreased. Melanin was observed to have an inhibitory effect on OGG1 activity. Study of the regulation of OGG1 activity in response to melanin and manganese may provide insights into the vulnerability of nigral neurons to oxidative stress in Parkinson's disease.

Inhibitory effects on phospholipase A2 and antivenin activity of melanin extracted from Thea sinensis Linn.

Antivenin activity of melanin extracted from black tea (MEBT) was reported for the first time. The antagonistic effect of MEBT was evaluated for Agkistrodon contortrix laticinctus (broadbanded copperhead), Agkistrodon halys blomhoffii (Japanese mamushi), and Crotalus atrox (western diamondback rattlesnake) snake venoms administered i.p. to ICR mice. MEBT was injected i.p. immediately after the venom administration in dose of 3 mg per mouse in the same place of venom injection. MEBT demonstrated neutralization effect against all venoms tested. The greatest antivenin effect of MEBT was found against Japanese mamushi snake venom. In this case, half the mice died within 2.5 +/- 0.7 h after injection of 0.9 mg/kg of venom. An immediate injection of MEBT substantially reduced the toxic effect of venom and extended time at the 50% level of survival up to 52.3 +/- 2.3 h. The antivenin activity of MEBT is due to chelating of Ca++ and non-specific binding of phospholipase A2. The inhibitory effect of MEBT on phospholipase A2 assessed for different venoms was similar to that obtained with pure enzyme. Low toxicity of MEBT in combination with its antagonistic activity against different venoms may allow effective life-saving treatment against snakebites. Such application of MEBT is important when identification of the snake is impossible or if specific treatment is unavailable.

The dopamine receptor agonist lisuride attenuates iron-mediated dopaminergic neurodegeneration.

Many dopamine agonists used in the treatment of Parkinson's disease are suggested to be potentially neuroprotective. On the basis of its structure, the dopamine agonist lisuride may share this characteristic. In the current study discrete asymptomatic lesions were produced by the injection of iron-laden neuromelanin into the rat substantia nigra and the animals treated with lisuride to determine the protective potential of this substance. Two treatment regimes were utilised. In the neuroprotective protocol, animals were treated with 0.1 mg.kg(-1) lisuride twice daily 3 days prior to, and 7 days following, the iron lesion. In the neurorescue protocol, the animals received 0.1 mg.kg(-1) lisuride twice daily for 1 week beginning on the fourth day post surgery. Eight weeks post surgery, tyrosine hydroxylase-positive neurons surrounding the injection site (33% of total nigral volume) were counted. Dopamine neuron number in iron-lesioned animals was reduced to 50% of that in vehicle-injected animals. The absence of motoric disturbances or a striatal dopamine deficit in these animals suggests a subclinical dopaminergic lesion. Dopamine neuron number in the quantified area in sham-injected animals receiving lisuride or iron-lesioned animals receiving lisuride in both the neuroprotection and neurorescue groups were not significantly reduced. These results suggest that lisuride can protect neurons against iron-induced cell death and might thus be neuroprotective in Parkinson's disease.

Gastrointestinal enhancement of MRI with melanin derived from tea leaves (Thea sinensis Linn.).

Melanin was extracted from tea leaves (Thea sinensis Linn.) for the first time. Characterization of melanin proved similarity of the original compound to standard melanin. The Langmuir adsorption isotherms for gadolinium (Gd) binding were obtained using melanin. Melanin-Gd preparation demonstrated low acute toxicity. LD(50) for this preparation was in a range of 1250-1500 mg/kg in mice. Magnetic Resonance Imaging (MRI) properties of melanin itself and melanin-Gd complexes have been estimated. Gd free melanin fractions possess slighter relaxivity compared with its complexes. The relaxivity of lower molecular weight fraction was two times higher than relaxivity of Gd(DTPA) standard. Postcontrast images demonstrate that oral administration of melanin complexes in concentration 0.1 mM provides essential enhancement to longitudinal relaxation times (T(1))-weighted spin echo image. The required contrast and delineation of the stomach wall demonstrated uniform enhancement of MRI with proposed melanin complex.

Effects of melanin on high- and low- linear energy transfer (LET) radiation response of human epithelial cells.

The search for effective radioprotectors is of major concern in the medical, military, environmental, and space sciences. Conventional radioprotectors are generally effective only during a single irradiation and display their radioprotective properties only at high, toxic concentrations. In addition, they reduce somatic radiation effects but are poorly efficient in protecting from hereditary stochastic radiation effects. In this respect, the pigment melanin merits attention. Experiments referring to potential melanin effects on the ionising radiation response have been carried out with different biological systems, both in vivo and in vitro. In this paper, we present results on the response to high- and low-linear energy transfer (LET) radiation of a human mammary epithelial cell line, H184B5 F5-1 M/10, supplemented by melanin. The incorporation of auto-oxidative (L-dopa) melanin was linear for concentrations from 3 to 10 micrograms/ml in the growth medium. Concentrations of up to 250 micrograms/ml did not significantly impair the cells proliferative ability. No significant protective effect of melanin on the survival of cultured cells after exposure to alpha-particles (130 keV/micron) or x-rays was observed.

Dopamine-induced apoptosis is inhibited in PC12 cells expressing Bcl-2.

1. Degeneration of nigrostriatal dopaminergic neurons is the major pathogenic substrate of Parkinson's disease (PD). It is assumed that the lethal trigger is the accumulation of oxidative reactive species generated during metabolism of the natural neurotransmitter dopamine. 2. We have recently shown that dopamine is capable of inducing programmed cell death (PCD) or apoptosis in cultured postmitotic chick sympathetic neurons and rat PC12 pheochromocytoma cells. 3. The bcl-2 gene encodes a protein which blocks physiological PCD in many mammalian cells. In an attempt to elucidate further the mechanism of dopamine toxicity, we examined the potential protective effect of bcl-2 in PC12 cells which were transfected with the protooncogene. 4. In our experiments, Bcl-2 producing cells showed a marked resistance to dopamine toxicity. The percentage of nuclear condensation and DNA fragmentation visualized by the end-labeling method following dopamine treatment was significantly lower in bcl-2 expressing cells. Bcl-2 did not protect PC12 cells against toxicity induced by exposure to dopamine-melanin. Extracts of PC12 cells containing Bcl-2 inhibited dopamine autooxidation and formation of dopamine-melanin. Furthermore, the presence of Bcl-2 protected cells from thiol imbalance and prevented thiol loss following exposure to dopamine. 5. The protective effects of Bcl-2 against dopamine toxicity may be explained, in part, by its action as an antioxidant and by its interference in the production of toxic agents. The possible protection by Bcl-2 against neuronal degeneration caused by dopamine may play a role in the pathogenesis of PD and may provide a new direction for the development of neuroprotective therapies.

Melanin-containing hydrogel intraocular lenses: a histopathological study in animal eyes.

Poly(2-hydroxyethyl methacrylate) hydrogel intraocular lenses, containing adrenochrome-melanin, were manufactured and implanted in animal eyes in order to assess the effect of melanin upon (a) biocompatibility of implants with the eye tissues, and (b) fibrous proliferation of lens epithelium responsible for the opacification of the posterior capsular membrane. An equal number of control lenses were also implanted. The animals were followed up for durations up to two years, and a detailed histopathological examination of the eyes was performed subsequent to their enucleation. The postoperative complications were minor and probably caused by surgical trauma. The study failed to give any indication of the postulated antiproliferative activity of adrenochrome-melanin since minimal capsular opacification occurred in the operated eyes, regardless of the presence of melanin.