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Redox Activities of Melanins Investigated by Electrochemical Reverse Engineering: Implications for their Roles in Oxidative Stress

Open ArchivePublished:December 04, 2019DOI:https://doi.org/10.1016/j.jid.2019.09.010
      Melanins, the main epidermal pigments of man, have been viewed traditionally as performing photoprotective and antioxidant functions, yet increasing evidence indicates they also possess detrimental pro-oxidant activities. Understanding this duality in functional activity (anti- vs. pro-oxidant) is important because oxidative stress is believed to play a central role in melanoma pathophysiology. Here, we review current knowledge of melanin’s structure and functional activities and their relevance to redox biology and oxidative stress. We especially focus on recent efforts to develop an in vitro experimental methodology to characterize melanin’s redox activities and suggest the implications of these in vitro observations.

      Abbreviations:

      E0 (thermodynamic value of redox potential), MEP (mediated electrochemical probing), ROS (reactive oxygen species)

      Melanin Biosynthesis and Functional Role

      Melanin is a polymeric pigment whose structural complexity derives from the diversity of the precursors, their mode of polymerization, and their aggregation state. In addition, dynamic structural changes (e.g., upon exposure to oxygen and UVR) and interactions with other chemical species within the physiological environment further contribute to melanin’s structural complexity. Further, melanin’s unfavorable solubility properties and the low levels found in natural sources have greatly hampered efforts to apply standard chemical characterization methods to understand the structure-property-function relations that would allow a full understanding of melanin’s biological role (
      • D’Alba L.
      • Shawkey M.D.
      Melanosomes: biogenesis, properties, and evolution of an ancient organelle.
      ,
      • Denat L.
      • Kadekaro A.L.
      • Marrot L.
      • Leachman S.A.
      • Abdel-Malek Z.A.
      Melanocytes as instigators and victims of oxidative stress.
      ,
      • Obrador E.
      • Liu-Smith F.
      • Dellinger R.W.
      • Salvador R.
      • Meyskens F.L.
      • Estrela J.M.
      Oxidative stress and antioxidants in the pathophysiology of malignant melanoma.
      ).

      The melanogenic pathway

      The variety of human pigmentation is determined by two main classes of melanin pigments produced in epidermal melanocytes; eumelanins are responsible for the dark pigmentations, whereas pheomelanins are light in color, contain sulfur, and occur in the red-haired phenotype. Melanin pigmentation is regulated by the melanocortin 1 receptor gene MC1R that encodes a 317–amino acid G-coupled receptor, MC1R, activated by the peptide α-melanocyte-stimulating hormone. In wild-type eumelanic subjects, MC1R activation induces eumelanin synthesis via tyrosinase activation. MC1R sequence variants are associated with red hair and fair skin in the Caucasian population that show varying degrees of diminished function. The main consequence of MC1R sequence variants is a decrease in the amount of eumelanin pigments with prevalence of the pheomelanin variant. At the biochemical level, this change results from a diminished tyrosinase activity that favors the concomitant intervention of cysteine in the melanogenic pathway, as illustrated in Figure 1. Non-enzymatic addition of the SH group of cysteine to the oxidation product of tyrosine, dopaquinone, leads to the formation of isomeric cysteinyldopas. Specifically, the intramolecular cyclization pathway of 5,6-dihydroxyindole formation leading to eumelanin polymers is inhibited, and an alternate 1,4-benzothiazine route to pheomelanins becomes dominant (
      • Napolitano A.
      • Panzella L.
      • Leone L.
      • D’Ischia M.
      Red hair benzothiazines and benzothiazoles: mutation-inspired chemistry in the quest for functionality.
      ). Chemical analysis of hair melanins showed that this switching mechanism may operate to varying degrees, leading to the generation of mixed melanins responsible for phenotypes with variable shades of color (
      • Ito S.
      • Miyake S.
      • Maruyama S.
      • Suzuki I.
      • Commo S.
      • Nakanishi Y.
      • et al.
      Acid hydrolysis reveals a low but constant level of pheomelanin in human black to brown hair.
      ). Thus, human skin color is mainly determined by the ratio between the dark eumelanin and the lighter pheomelanin.
      Figure thumbnail gr1
      Figure 1Biosynthetic pathways of eumelanin and pheomelanin.
      Within melanocytes, melanin production, storage, and transport take place in specialized membrane-bound organelles termed melanosomes. Melanosomes are large (500 nm) lysosome-related organelles (
      • D’Alba L.
      • Shawkey M.D.
      Melanosomes: biogenesis, properties, and evolution of an ancient organelle.
      ,
      • Marks M.S.
      • Heijnen H.F.
      • Raposo G.
      Lysosome-related organelles: unusual compartments become mainstream.
      ,
      • Wasmeier C.
      • Hume A.N.
      • Bolasco G.
      • Seabra M.C.
      Melanosomes at a glance.
      ) that undergo maturation in a four-stage process. In Stage I, premelanosomes are non-pigmented early endosomes (
      • Wasmeier C.
      • Hume A.N.
      • Bolasco G.
      • Seabra M.C.
      Melanosomes at a glance.
      ). In Stage II, melanosomes acquire internal striations associated with the pigment cell–specific structural protein PMEL17 forming intralumenal fibrils that confer an ellipsoidal shape to the melanosome and also serve as the template for melanin deposition (
      • Marks M.S.
      • Heijnen H.F.
      • Raposo G.
      Lysosome-related organelles: unusual compartments become mainstream.
      ,
      • Wasmeier C.
      • Hume A.N.
      • Bolasco G.
      • Seabra M.C.
      Melanosomes at a glance.
      ). During Stage III, melanin is deposited on the PMEL17 scaffolds to generate fully melanized Stage IV melanosomes that no longer have tyrosinase activity. In melanocytes, the late melanosomes (Stages III and IV) bind to microtubules and undergo actin-dependent transport toward the cell periphery, where they are transferred to keratinocytes. Skin color does not depend on the number of melanocytes, which is similar among individuals with different phenotypes, but relies on the type, number, and size of melanosomes, as well as on their correct transfer, distribution, and organization through the epidermis (
      • Hurbain I.
      • Romao M.
      • Sextius P.
      • Bourreau E.
      • Marchal C.
      • Bernerd F.
      • et al.
      Melanosome distribution in keratinocytes in different skin types: melanosome clusters are not degradative organelles.
      ,
      • Thong H.Y.
      • Jee S.H.
      • Sun C.C.
      • Boissy R.E.
      The patterns of melanosome distribution in keratinocytes of human skin as one determining factor of skin colour.
      ).

      Melanin’s role in photoprotection and oxidative stress

      The photoprotective role of eumelanin against damage induced by UVA (320–400 nm) and UVB (280–320 nm) is well documented. Eumelanin displays a characteristic broad-band optical absorption throughout the UV-visible spectrum, decreasing monotonically with increasing wavelength. Light absorption by eumelanin is followed by thermal relaxation that diverts energy from potentially harmful photochemical reactions. Epidermal eumelanin shields nuclei, forming supranuclear melanin caps in melanocytes and keratinocytes. An in vivo study comparing the levels of UVR-induced DNA damage and its clearance in the skin of human individuals with different pigmentation and ethnic origin showed an inverse correlation between melanin content and the levels of cyclobutane pyrimidine dimers, the typical UVR-induced lesions in cellular DNA (
      • Tadokoro T.
      • Kobayashi N.
      • Zmudzka B.Z.
      • Ito S.
      • Wakamatsu K.
      • Yamaguchi Y.
      • et al.
      UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin.
      ). Support for this finding was also provided by in vitro experiments with human melanocyte cultures derived from donors with different skin phototypes and different total melanin and eumelanin contents exposed to the same doses of UV (
      • Hauser J.E.
      • Kadekaro A.L.
      • Kavanagh R.J.
      • Wakamatsu K.
      • Terzieva S.
      • Schwemberger S.
      • et al.
      Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes.
      ).
      The higher susceptibility to skin cancer of red-haired individuals seems to be due not only to a reduced eumelanin content but also to the toxic properties of pheomelanins, especially upon exposure to UVR. Indeed, pheomelanin acts as a photosensitizer, exacerbating UVR-induced reactive oxygen species (ROS) production. It has been proposed that the UVA-excited pheomelanin chromophore generates ROS including the O2•− anion via electron transfer to molecular oxygen, which then generates H2O2 and hydroxyl radicals as well as singlet oxygen (1O2) (
      • Panzella L.
      • Szewczyk G.
      • D’Ischia M.
      • Napolitano A.
      • Sarna T.
      Zinc-induced structural effects enhance oxygen consumption and superoxide generation in synthetic pheomelanins on UVA/visible light irradiation.
      ,
      • Takeuchi S.
      • Zhang W.
      • Wakamatsu K.
      • Ito S.
      • Hearing V.J.
      • Kraemer K.H.
      • et al.
      Melanin acts as a potent UVB photosensitizer to cause an atypical mode of cell death in murine skin.
      ). Moreover, pheomelanin content correlates inversely with the levels and activity of the antioxidant enzyme catalase in human melanocytes, making them even more susceptible to accumulate oxidative damage after UV exposure (
      • Maresca V.
      • Flori E.
      • Briganti S.
      • Mastrofrancesco A.
      • Fabbri C.
      • Mileo A.M.
      • et al.
      Correlation between melanogenic and catalase activity in in vitro human melanocytes: a synergic strategy against oxidative stress.
      ).
      Indeed, several studies suggest that the phototoxic properties of pheomelanin contribute to UVR-induced DNA damage and melanomagenesis. Pheomelanin increased DNA strand break induction in UVA-irradiated human melanocytes derived from phenotype I skin and participated actively in cyclobutane pyrimidine dimer formation in melanocytes long after UV exposure (
      • Abdel-Malek Z.A.
      • Cassidy P.
      Dark CPDs and photocarcinogenesis: the party continues after the lights go out.
      ). A recent study has suggested that not only pheomelanin but also eumelanin can mediate delayed cyclobutane pyrimidine dimer formation in human skin brought about by ROS and reactive nitrogen species following UVA radiation by a process involving the generation of a quantum triplet state in fragments of the melanin pigments that have the energy of a UV photon and are capable of transferring it to DNA in radiation-independent mode (
      • Premi S.
      • Wallisch S.
      • Mano C.M.
      • Weiner A.B.
      • Bacchiocchi A.
      • Wakamatsu K.
      • et al.
      Photochemistry. Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure.
      ). Another study revealed that UVA-irradiated mice required melanin to develop melanoma (
      • Noonan F.P.
      • Zaidi M.R.
      • Wolnicka-Glubisz A.
      • Anver M.R.
      • Bahn J.
      • Wielgus A.
      • et al.
      Melanoma induction by ultraviolet A but not ultraviolet B radiation requires melanin pigment.
      ).
      Although all these data provide a convincing background to support a photoprotective or phototoxic activity for eumelanin and pheomelanin, respectively, there are other issues regarding melanin function still awaiting a rationalization. The presence in nonexposed organs like the inner ear and brain would suggest that melanins may play roles not related to the processes initiated by UVR but still dictated by their specific structural features and chemical reactivity. Moreover, why melanoma is not restricted to sun-exposed areas of the body and UV radiation signature mutations are infrequently oncogenic drivers remain also to be assessed.
      Clues to understanding melanin function beyond UVR response have been provided by recent literature. The seminal work by
      • Mitra D.
      • Luo X.
      • Morgan A.
      • Wang J.
      • Hoang M.P.
      • Lo J.
      • et al.
      An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background.
      showed that mice with a red/yellow phenotype bearing a conditional BRAFV600E mutant allele developed invasive melanoma at higher rates than albino mice with the same genetic background even in the absence of UVR (
      • Mitra D.
      • Luo X.
      • Morgan A.
      • Wang J.
      • Hoang M.P.
      • Lo J.
      • et al.
      An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background.
      ). In addition, the same study demonstrated that the skin of pheomelanic mice contained higher levels of oxidative DNA damage and lipid peroxidation than albino mice. The molecular mechanisms underlying these observations were addressed in a model study showing that purified red human hair pheomelanin was able to sustain autoxidation of cellular antioxidants (glutathione and nicotinamide adenine dinucleotide) with production of ROS (
      • Napolitano A.
      • Panzella L.
      • Monfrecola G.
      • D’Ischia M.
      Pheomelanin-induced oxidative stress: bright and dark chemistry bridging red hair phenotype and melanoma.
      ,
      • Panzella L.
      • Leone L.
      • Greco G.
      • Vitiello G.
      • D’Errico G.
      • Napolitano A.
      • et al.
      Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis.
      ). In another study, red human hair pheomelanin was shown to lower viability of keratinocytes and to promote expression of pro-inflammatory interleukins and oxidative damage more strongly than black hair melanin (
      • Lembo S.
      • Di Caprio R.
      • Micillo R.
      • Balato A.
      • Monfrecola G.
      • Panzella L.
      • et al.
      Light-independent pro-inflammatory and pro-oxidant effects of purified human hair melanins on keratinocyte cell cultures.
      ,
      • Płonka P.M.
      • Picardo M.
      • Slominski A.T.
      Does melanin matter in the dark?.
      ).
      Other mechanisms may also contribute to melanin’s biological activities. For instance, the 5,6-dihydroxyindole-2-carboxylic acid rather than the 5,6-dihydroxyindole component of eumelanin is reported to act as a potent free radical scavenger in the Fenton reaction (
      • Jiang S.
      • Liu X.M.
      • Dai X.
      • Zhou Q.
      • Lei T.C.
      • Beermann F.
      • et al.
      Regulation of DHICA-mediated antioxidation by dopachrome tautomerase: implication for skin photoprotection against UVA radiation.
      ). This observation was interpreted in terms of the mode of coupling of the indole units and delocalization of the pi electrons over this model eumelanin pigment (
      • Panzella L.
      • Gentile G.
      • D’Errico G.
      • Della Vecchia N.F.
      • Errico M.E.
      • Napolitano A.
      • et al.
      Atypical Structural and π-Electron Features of a Melanin Polymer that lead to Superior Free-radical-Scavenging Properties.
      ).
      In sum, there has been considerable recent progress in understanding the duality of functional activities of melanin pigments. In many cases, the protective or damaging mechanisms are redox-based, and thus we suggest that a better understanding of melanin’s intrinsic redox activities may be essential to understand how these pigments protect from, or contribute to, the oxidative stresses that are linked to melanoma.

      Oxidative Stress and Redox Biology

      Oxidative stress is important in melanoma pathophysiology (
      • Denat L.
      • Kadekaro A.L.
      • Marrot L.
      • Leachman S.A.
      • Abdel-Malek Z.A.
      Melanocytes as instigators and victims of oxidative stress.
      ,
      • Obrador E.
      • Liu-Smith F.
      • Dellinger R.W.
      • Salvador R.
      • Meyskens F.L.
      • Estrela J.M.
      Oxidative stress and antioxidants in the pathophysiology of malignant melanoma.
      ), and melanin may contribute to these stresses through the pro-oxidant nature of its synthesis (
      • Denat L.
      • Kadekaro A.L.
      • Marrot L.
      • Leachman S.A.
      • Abdel-Malek Z.A.
      Melanocytes as instigators and victims of oxidative stress.
      ,
      • Gidanian S.
      • Mentelle M.
      • Meyskens F.L.
      • Farmer P.J.
      Melanosomal damage in normal human melanocytes induced by UVB and metal uptake—A basis for the pro-oxidant state of melanoma.
      ,
      • Natarajan V.T.
      • Ganju P.
      • Ramkumar A.
      • Grover R.
      • Gokhale R.S.
      Multifaceted pathways protect human skin from UV radiation.
      ), the redox activities of its diffusible intermediates (e.g., 5,6-dihydroxyindoles and cysteinyldopa) (
      • Edge R.
      • D’Ischia M.
      • Land E.J.
      • Napolitano A.
      • Navaratnam S.
      • Panzella L.
      • et al.
      Dopaquinone redox exchange with dihydroxyindole and dihydroxyindole carboxylic acid.
      ,
      • Napolitano A.
      • Memoli S.
      • Nappi A.J.
      • D’Ischia M.
      • Prota G.
      5-S-cysteinyldopa, a diffusible product of melanocyte activity, is an efficient inhibitor of hydroxylation/oxidation reactions induced by the Fenton system.
      ,
      • Panzella L.
      • Napolitano A.
      • D’Ischia M.
      Is DHICA the key to dopachrome tautomerase and melanocyte functions?.
      ), and its intrinsic properties (e.g., photosensitizing activities) (
      • Wood S.R.
      • Berwick M.
      • Ley R.D.
      • Walter R.B.
      • Setlow R.B.
      • Timmins G.S.
      UV causation of melanoma in Xiphophorus is dominated by melanin photosensitized oxidant production.
      ) that enable the generation of ROS and/or depletion of protective antioxidants (
      • Morgan A.M.
      • Lo J.
      • Fisher D.E.
      How does pheomelanin synthesis contribute to melanomagenesis?.
      ,
      • Panzella L.
      • Leone L.
      • Greco G.
      • Vitiello G.
      • D’Errico G.
      • Napolitano A.
      • et al.
      Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis.
      ,
      • Tanaka H.
      • Yamashita Y.
      • Umezawa K.
      • Hirobe T.
      • Ito S.
      • Wakamatsu K.
      The pro-oxidant activity of pheomelanin is significantly enhanced by UVA irradiation: benzothiazole moieties are more reactive than benzothiazine moieties.
      ). Additionally, early events in the etiology and progression of melanoma appear to involve morphological changes (
      • Obrador E.
      • Liu-Smith F.
      • Dellinger R.W.
      • Salvador R.
      • Meyskens F.L.
      • Estrela J.M.
      Oxidative stress and antioxidants in the pathophysiology of malignant melanoma.
      ) such as the disruption of melanosomal membranes and degradation of melanin (
      • Gidanian S.
      • Mentelle M.
      • Meyskens F.L.
      • Farmer P.J.
      Melanosomal damage in normal human melanocytes induced by UVB and metal uptake—A basis for the pro-oxidant state of melanoma.
      ). It has been suggested that such changes in melanosomal compartmentation may facilitate oxidative stress by enabling the exposure of melanins to O2 to facilitate ROS generation (
      • Farmer P.J.
      • Gidanian S.
      • Shahandeh B.
      • Di Bilio A.J.
      • Tohidian N.
      • Meyskens F.L.
      Melanin as a target for melanoma chemotherapy: pro-oxidant effect of oxygen and metals on melanoma viability.
      ,
      • Gidanian S.
      • Mentelle M.
      • Meyskens F.L.
      • Farmer P.J.
      Melanosomal damage in normal human melanocytes induced by UVB and metal uptake—A basis for the pro-oxidant state of melanoma.
      ).
      More recently, there have been efforts to place oxidative stress within a broader context of redox biology and redox signaling. Redox is believed to be a modality for cell-cell communication that is separate from the better known molecularly specific modalities (e.g., associated with kinase signaling) or the electrical modality involving ion flow across membranes (e.g., the action potentials of neuronal communication). A redox code has been proposed, (
      • Jones D.P.
      • Sies H.
      The redox code.
      ) and the transmitted redox signals are the same ROS and reactive nitrogen species implicated in oxidative stress (
      • García-Santamarina S.
      • Boronat S.
      • Hidalgo E.
      Reversible cysteine oxidation in hydrogen peroxide sensing and signal transduction.
      ). Information transfer occurs when the redox signal undergoes an electron transfer chemical reaction, and thus a signal’s lifetime is controlled by its intrinsic reactivity (ROS reactivities can vary by 11 orders of magnitude), whereas additional control can be exerted by the signal-generating and signal-attenuating enzymes (e.g., NOX, SOD, and catalase) (
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ). In addition, reactions of these ROS signals can generate reactive electrophilic species that can serve as second messengers, enabling information to be transferred over larger distances (
      • Parvez S.
      • Long M.J.C.
      • Poganik J.R.
      • Aye Y.
      Redox signaling by reactive electrophiles and oxidants.
      ). Importantly, the rapid interconversion of ROS/ reactive electrophilic species is context dependent, which has made it difficult to parse out mechanistic details (
      • Parvez S.
      • Long M.J.C.
      • Poganik J.R.
      • Aye Y.
      Redox signaling by reactive electrophiles and oxidants.
      ). The best known examples of signal reception are atomically specific, with sulfur playing a particularly prominent role (e.g., the sulfur-switching of protein cysteine thiols to disulfides). For instance, the H2O2 redox signal can be recognized through the oxidation of S-switches of transcription factors that in some cases serve as master regulators (e.g., NRF2/KEAP1 and NK-κB) (
      • Rampon C.
      • Volovitch M.
      • Joliot A.
      • Vriz S.
      Hydrogen peroxide and redox regulation of developments.
      ,
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Young D.
      • Pedre B.
      • Ezeriņa D.
      • De Smet B.
      • Lewandowska A.
      • Tossounian M.A.
      • et al.
      Protein promiscuity in H 2 O 2 signaling.
      ). Thus, from the perspective of redox signaling, oxidative stress is an imbalance in which excessive ROS signal generation (e.g., as a result of prolonged inflammation) overwhelms the system’s signal attenuating antioxidant activities, suggesting that oxidative stress reflects a dysregulation in redox homeostasis (
      • Kemp M.
      • Go Y.M.
      • Jones D.P.
      Nonequilibrium thermodynamics of thiol/disulfide redox systems: A perspective on redox systems biology.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Timme-Laragy A.R.
      • Hahn M.E.
      • Hansen J.M.
      • Rastogi A.
      • Roy M.A.
      Redox stress and signaling during vertebrate embryonic development: regulation and responses.
      ).
      To understand redox biology, it is often helpful to compare redox reactions (loss/gain of electrons) with acid/base reactions (loss/gain of protons) (
      • Kim E.
      • Li J.
      • Kang M.
      • Kelly D.L.
      • Chen S.
      • Napolitano A.
      • et al.
      Redox is a global biodevice information processing modality.
      ). There are two important differences: first, unlike protons, free electrons do not typically exist in water, and thus redox reactions must be coupled such that the electron is transferred from the molecule being oxidized directly to the molecule being reduced; second, whereas acid/base reactions are generally believed to be rapid and exist in dynamic equilibrium, redox reactions can have significant kinetic barriers. An important implication of these kinetic barriers is that they allow reductants (e.g., nicotinamide adenine dinucleotide phosphate) and oxidants (e.g., O2) to coexist, requiring a catalyst for a reaction to proceed toward equilibrium (e.g., NOX). Some have described such kinetic barriers as kinetic insulation preventing redox couples (e.g., the reduced and oxidized forms of nicotinamide adenine dinucleotide and of glutathione) from equilibrating even when they exist together in the same subcellular compartment (
      • Kemp M.
      • Go Y.M.
      • Jones D.P.
      Nonequilibrium thermodynamics of thiol/disulfide redox systems: A perspective on redox systems biology.
      ).
      Another consequence of the kinetic barriers of redox reactions is that it is not obvious how to measure redox (
      • Flohé L.
      The fairytale of the GSSG/GSH redox potential.
      ). Although pH measurements are well-accepted for providing a reliable quantitative measure of pH context, analogous efforts to develop an electrode measurement of redox potential are plagued by the problem that only the most reactive redox couple or couples will be measured, whereas couples with higher kinetic barriers may not contribute to these measurements. It can even be challenging to measure the thermodynamic value of redox potential (E0), which is commonly used to characterize the power of an oxidant (
      • Parvez S.
      • Long M.J.C.
      • Poganik J.R.
      • Aye Y.
      Redox signaling by reactive electrophiles and oxidants.
      ).

      Reverse Engineering Approach to Measure Melanin’s Redox Activity

      To understand the redox properties of melanin, we are developing an electrochemical reverse engineering approach illustrated in Figure 2a (further details of this method are provided in a recent review [
      • Kang M.
      • Kim E.
      • Temoçin Z.
      • Li J.
      • Dadachova E.
      • Wang Z.
      • et al.
      Reverse engineering to characterize redox properties: revealing melanin’s redox activity through mediated electrochemical probing.
      ]). Unlike conventional approaches for characterizing a material, this method does not focus on chemical structure but rather focuses on redox properties. Unlike typical reverse engineering approaches that often begin with little understanding of a sample (i.e., the sample is a black box), the existing knowledge of melanin provides considerable chemical intuition to guide our reverse engineering (i.e., our melanin sample is more of a gray box). Our method probes the melanin with controlled redox inputs to yield response characteristics that provide insights on the functionally relevant redox properties of melanin.
      Figure thumbnail gr2
      Figure 2Reverse engineering approach to measure melanin’s redox activity. (a) Electrochemical reverse engineering approach to characterize melanin’s redox activities. (b) Melanins that are entrapped within a permeable hydrogel are probed using diffusible mediators that shuttle electrons between the electrode and melanin, and the output responses (electrical currents and in some cases optical absorbance) are interpreted to understand melanin’s redox properties. (c) Properties revealed through mediated electrochemical probing. (d) Thermodynamic plot illustrating that pheomelanin has a more oxidative redox potential than eumelanin. Oxidative stresses may be exerted through a redox buffering mechanism. GSH, glutathione; NADH, nicotinamide adenine dinucleotide; ROS, reactive oxygen species.
      Figure 2b shows there are three important features of our mediated electrochemical probing (MEP) approach (
      • Kang M.
      • Kim E.
      • Temoçin Z.
      • Li J.
      • Dadachova E.
      • Wang Z.
      • et al.
      Reverse engineering to characterize redox properties: revealing melanin’s redox activity through mediated electrochemical probing.
      ). First, the insoluble melanin particles are localized adjacent to an electrode surface by entrapping these particles within a nonconducting hydrogel (typically ∼0.5-mm wet film of the aminopolysaccharide chitosan). Where the hydrogel entraps the melanin, this gel is permeable, allowing the free diffusion of smaller molecules. Given the difficulty of obtaining melanins from natural sources, most studies have used model pigments prepared from the oxidation of the biosynthetic precursors like tyrosine, DOPA, or cysteinyldopa, or natural melanins from more accessible biological sources like Sepia and fungi. The results obtained with these model pigments have been confirmed substantially using human melanins obtained from black or red hairs by careful enzymatic digestion of the keratin matrix (
      • Kim E.
      • Panzella L.
      • Micillo R.
      • Bentley W.E.
      • Napolitano A.
      • Payne G.F.
      Reverse engineering applied to red human hair pheomelanin reveals redox-buffering as a pro-oxidant mechanism.
      ).
      Second, soluble mediators (i.e., electron shuttles) are purposefully added to the surrounding solution. These mediators can diffuse into and throughout the hydrogel matrix to electronically connect the electrode with the melanin (
      • Kang M.
      • Kim E.
      • Temoçin Z.
      • Li J.
      • Dadachova E.
      • Wang Z.
      • et al.
      Reverse engineering to characterize redox properties: revealing melanin’s redox activity through mediated electrochemical probing.
      ). For instance, Figure 2b illustrates that a reducing mediator can accept an electron at an electrode, diffuse into the film, and transfer it to the melanin particle. Alternatively, an oxidizing mediator can accept an electron from the melanin, diffuse through the film, and transfer it to the electrode. Each of these redox cycling electron exchange processes is constrained by thermodynamics; electron transfer generally proceeds from more reducing potentials (i.e., more negative voltages) to more oxidizing potentials (i.e., more positive voltages) as illustrated in Figure 2b. Importantly, a variety of mediators are available to span a range of E0 values (analogous to the variety of pH buffers available to span various pH ranges). Typically, the mediators selected are well behaved in that they can exchange electrons rapidly with minimal kinetic barriers. Analogous to pH buffers that primarily exchange protons when the pH is near their pKa, a well-behaved mediator primarily exchanges electrons at an electrode when the imposed electrode voltage is close to the mediator’s E0. Also analogous to pH buffers, multiple mediators can be added to the same solution but each mediator only exchanges electrons at the electrode when the electrode voltage approaches the mediator’s E0. Also important is that electrochemical instrumentation allows the voltage to be precisely set to control the direction of this mediator-based flow of electrons to either transfer electrons to melanin or extract electrons from melanin.
      The third feature of this MEP approach is the use of controlled sequences of imposed input voltages and measurement of output currents and in some cases the simultaneous measurement of optical absorbance (
      • Kang M.
      • Kim E.
      • Temoçin Z.
      • Li J.
      • Dadachova E.
      • Wang Z.
      • et al.
      Reverse engineering to characterize redox properties: revealing melanin’s redox activity through mediated electrochemical probing.
      ). As discussed, these output response characteristics can be interpreted using chemical and biological intuition to gain insights of the redox properties of melanin.
      Figure 2c illustrates several observations from our recent MEP studies (
      • Kang M.
      • Kim E.
      • Temoçin Z.
      • Li J.
      • Dadachova E.
      • Wang Z.
      • et al.
      Reverse engineering to characterize redox properties: revealing melanin’s redox activity through mediated electrochemical probing.
      ). First, MEP studies support and extend previous observations that melanins (i) are redox active (
      • D’Ischia M.
      • Napolitano A.
      • Pezzella A.
      • Meredith P.
      • Sarna T.
      Chemical and structural diversity in eumelanins: unexplored bio-optoelectronic materials.
      ,
      • Meredith P.
      • Sarna T.
      The physical and chemical properties of eumelanin.
      ,
      • Meredith P.
      • Bettinger C.J.
      • Irimia-Vladu M.
      • Mostert A.B.
      • Schwenn P.E.
      Electronic and optoelectronic materials and devices inspired by nature.
      ,
      • Mostert A.B.
      • Powell B.J.
      • Pratt F.L.
      • Hanson G.R.
      • Sarna T.
      • Gentle I.R.
      • et al.
      Role of semiconductivity and ion transport in the electrical conduction of melanin.
      ,
      • Sheliakina M.
      • Mostert A.B.
      • Meredith P.
      An all-solid-state biocompatible ion-to-electron transducer for bioelectronics.
      ,
      • Watt A.A.R.
      • Bothma J.P.
      • Meredith P.
      The supramolecular structure of melanin.
      ); (ii) can be repeatedly oxidized and reduced; and (iii) possess redox potentials in the mid-physiological range (
      • Kim E.
      • Liu Y.
      • Leverage W.T.
      • Yin J.J.
      • White I.M.
      • Bentley W.E.
      • et al.
      Context-dependent redox properties of natural phenolic materials.
      ,
      • Temoçin Z.
      • Kim E.
      • Li J.
      • Panzella L.
      • Alfieri M.L.
      • Napolitano A.
      • et al.
      The analgesic acetaminophen and the antipsychotic clozapine can each redox-cycle with melanin.
      ). These observations suggest that melanins may not be inert bystanders in redox biology but rather may be active participants in redox-based molecular signaling, antioxidant protection, and pro-oxidant activities.
      Second, MEP (in combination with other methods) supports and extends previous suggestions that melanins have redox catalytic activities capable of transferring electrons from physiological reductants to oxidants (e.g., O2) (
      • Kim E.
      • Liu Y.
      • Leverage W.T.
      • Yin J.J.
      • White I.M.
      • Bentley W.E.
      • et al.
      Context-dependent redox properties of natural phenolic materials.
      ). Thus, melanins could contribute to oxidative stress both by depletion of physiological reductants (i.e., disrupt redox homeostasis) and the generation of damaging ROS. Importantly, this catalytic activity is context-dependent; ROS-generation requires melanins to have access to reducing equivalents and also exposure to O2. Possibly, such contexts could exist in regions where there are steep O2 gradients or potentially upon disruptions of the melanosomes that could expose melanin to O2-containing conditions. In a similar way, we observed that melanin can quench free radicals in a redox-linked context-dependent manner, either by the donation or acceptance of electrons as suggested in Figure 2c (
      • Kim E.
      • Kang M.
      • Tschirhart T.
      • Malo M.
      • Dadachova E.
      • Cao G.
      • et al.
      Spectroelectrochemical reverse engineering Demonstrates That melanin’s redox and radical scavenging activities are linked.
      ). One additional observation in Figure 2c involves redox cycling; we have tested a handful of redox-active chemicals (the agricultural chemical paraquat, the antipsychotic drug clozapine, and the analgesic acetaminophen) and observed in vitro that these chemicals can redox cycle with melanin (
      • Kim E.
      • Leverage W.T.
      • Liu Y.
      • Panzella L.
      • Alfieri M.L.
      • Napolitano A.
      • et al.
      Paraquat–melanin redox-cycling: evidence from electrochemical reverse engineering.
      ,
      • Temoçin Z.
      • Kim E.
      • Li J.
      • Panzella L.
      • Alfieri M.L.
      • Napolitano A.
      • et al.
      The analgesic acetaminophen and the antipsychotic clozapine can each redox-cycle with melanin.
      ). We believe such redox interactions are underappreciated but could be integral to understanding the activities and toxicities of exogenous agents.
      Finally, we used a series of MEP-based approaches to characterize differences between eumelanin and pheomelanin (
      • Kim E.
      • Panzella L.
      • Micillo R.
      • Bentley W.E.
      • Napolitano A.
      • Payne G.F.
      Reverse engineering applied to red human hair pheomelanin reveals redox-buffering as a pro-oxidant mechanism.
      ). Starting with synthetic models, we observed that the synthetic pheomelanin had a more oxidative redox potential than the synthetic eumelanin, as indicated in Figure 2d. Next, blends of these insoluble synthetic models showed intermediate redox properties with a systematic increase in redox-based pro-oxidant activity with increasing pheomelanin content. Finally, we performed these same MEP studies with natural melanin samples extracted from human hair. The melanin extracted from black hair showed similar redox signatures as the synthetic eumelanin model. In contrast, the melanin extracted from red hair showed redox signatures that were intermediate between the synthetic eumelanin and synthetic pheomelanin, which is consistent with the mixed polymeric nature of natural pheomelanins. Further, these measurements indicated that the melanin extracted from red hair had a greater redox-based pro-oxidant activity than the melanin from black hair. Overall, these observations support the use of synthetic melanin models to probe for redox activities and suggest that pheomelanin’s pro-oxidant activities may result from a redox buffering mechanism.

      Conclusions

      Understanding the role(s) of melanin in redox biology may be a key to addressing long-standing and emerging questions of the biological activities of these pigments. Specifically, interpretation of the dramatic differences in susceptibility to skin cancer and melanoma of individuals with different phenotypes may not be restricted to consideration of the degree of photoprotection ensured by the basal pigmentation. Rather, the different redox activities (associated with the different structural features) of eumelanin and pheomelanin may yield nuanced differences in redox interactions within the biological context that could be responsible for their different contributions to the oxidative stresses that underlie pathological conditions that include melanomagenesis. Potentially, our electrochemical reverse engineering measurements may provide important new insights on melanin’s context-dependent redox-based contributions to such complex biological processes as oxidative stress and UVR-independent molecular and cellular damage.

      Data availability statement

      This is a review article and there are no datasets related to this article.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported in part by the United States National Science Foundation ( DMREF 1435957 ) and the Department of Defense, Defense Threat Reduction Agency ( HDTRA11910021 ). The content of the information does not necessarily reflect the position or the policy of the federal government, and no official endorsement should be inferred.

      Author Contributions

      Conceptualization: GFP, AN; Funding Acquisition: GFP; Investigation: EK, LP, AN, GFP; Supervision: GFP, AN; Visualization: EK, LP, AN, GFP; Writing - Original Draft Preparation: GFP, AN; Writing - Review and Editing: EK, LP, AN, GFP

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