Lucia Panzella

Role of Solvent, pH, and Molecular Size in Excited-State Deactivation of Key Eumelanin Building Blocks : Implications for Melanin Pigment Photostability

Ultrafast time-resolved fluorescence spectroscopy has been used to investigate the excited-state dynamics of the basic eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA), its acetylated, methylated, and carboxylic ester derivatives, and two oligomers, a dimer and a trimer in the O-acetylated forms. The results show that (1) excited-state decays are faster for the trimer relative to the monomer; (2) for parent DHICA, excited-state lifetimes are much shorter in aqueous acidic medium (380 ps) as compared to organic solvent (acetonitrile, 2.6 ns); and (3) variation of fluorescence spectra and excited-state dynamics can be understood as a result of excited-state intramolecular proton transfer (ESIPT). The dependence on the DHICA oligomer size of the excited-state deactivation and its ESIPT mechanism provides important insight into the photostability and the photoprotective function of eumelanin. Mechanistic analogies with the corresponding processes in DNA and other biomolecules are recognized.

Pheomelanin‐induced oxidative stress: bright and dark chemistry bridging red hair phenotype and melanoma

The complex interplay of genetic and epigenetic factors linking sun exposure to melanoma in the red hair phenotype hinges on the peculiar physical and chemical properties of pheomelanins and the underlying biosynthetic pathway, which is switched on by the effects of inactivating polymorphisms in the melanocortin 1 receptor gene. In addition to the long recognized UV‐dependent pathways of toxicity and cell damage, a UV‐independent pro‐oxidant state induced by pheomelanin within the genetically determined background of the red hair phenotype has recently been disclosed. This review provides a detailed discussion of the possible UV‐dependent and UV‐independent chemical mechanisms underlying pheomelanin‐mediated oxidative stress, with special reference to the oxygen‐dependent depletion of glutathione and other cell antioxidants. The new concept of pheomelanin as a ‘living’ polymer and biocatalyst that may grow by exposure to monomer building blocks and may trigger autooxidative processes is also discussed. As a corollary, treatment of inflammatory skin diseases in RHP patients is briefly commented. Finally, possible concerted strategies for melanoma prevention in the red hair phenotype are proposed.

Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis

The highest incidence of melanoma in red haired individuals is attributed to the synthesis and phototoxic properties of pheomelanin pigments. Recently, pheomelanin has also been implicated in UV‐independent pathways of oxidative stress; however, the underlying mechanisms have remained uncharted. Herein, we disclose the unprecedented property of purified red human hair pheomelanin (RHP) to promote (i) the oxygen‐dependent depletion of major cell antioxidants, for example glutathione and NADH; (ii) the autoxidative formation of melanin pigments from their precursors. RHP would thus behave as a unique ‘living’ polymer and biocatalyst that may grow by simple exposure to monomer building blocks and may trigger autoxidative processes. These results yield new clues as to the origin of the pro‐oxidant state in the red hair phenotype, uncover non‐enzymatic pathways of melanogenesis, and pave the way to innovative strategies for melanoma prevention.

Secondary targets of nitrite-derived reactive nitrogen species: nitrosation/nitration pathways, antioxidant defense mechanisms and toxicological implications.

Nitrite, the primary metabolite of nitric oxide (NO) and a widely diffused component of human diet, plays distinct and increasingly appreciated roles in human physiology. However, when exposed to acidic environments, typically in the stomach, or under oxidative stress conditions, it may be converted to a range of reactive nitrogen species (RNS) which in turn can target a variety of biomolecules. Typical consequences of toxicological relevance include protein modification, DNA base deamination and the formation of N-nitrosamines, among the most potent mutagenic and carcinogenic compounds for humans. Besides primary biomolecules, nitrite can cause structural modifications to a variety of endogenous and exogenous organic compounds, ranging from polyunsaturated fatty acids to estrogens, tocopherol, catecholamines, furans, retinoids, dietary phenols, and a range of xenobiotics. The study of the interactions between nitrite and key food components, including phenolic antioxidants, has therefore emerged as an area of great promise for delineating innovative strategies in cancer chemoprevention. Depending on substrates and conditions, diverse reaction pathways may compete to determine product features and distribution patterns. These include nitrosation and nitration but also oxidation, via electron transfer to nitrosonium ion or nitrogen dioxide. This contribution aims to provide an overview of the main classes of compounds that can be targeted by nitrite and to discuss at chemical levels the possible reaction mechanisms under conditions that model those occurring in the stomach. The toxicological implications of the nitrite-modified molecules are finally addressed, and a rational chemical approach to the design of potent antinitrosing agents is illustrated.

The “Benzothiazine” Chromophore of Pheomelanins: A Reassessment†

The characteristic absorption and photochemical properties of pheomelanins are generally attributed to “benzothiazine” structural units derived biogenetically from 5‐S‐cysteinyldopa. This notion, however, conveys little or no information about the structural chromophores responsible for the photoreactivity of pheomelanins. At pH 7.4, natural and synthetic pheomelanins show a defined maximum around 305 nm, which is not affected by reductive treatment with sodium borohydride, and a monotonic decrease in the absorption in the range 350–550 nm. These features are not compatible with a significant proportion of structural units related to 2H‐1,4‐benzothiazine and 2H‐1,4‐benzothiazine‐3‐carboxylic acid, the early borohydride‐reducible pheomelanin precursors featuring absorption maxima above 340 nm. Rather, these features would better accommodate a contribution by the nonreducible 3‐oxo‐3,4‐dihydrobenzothiazine (λmax 299 nm) and benzothiazole (λmax 303 nm) structural motifs, which are generated in the later stages of pheomelanogenesis in vitro. This conclusion is supported by a detailed liquid chromatography/UV and mass spectrometry monitoring of the species formed in the oxidative conversion of 5‐S‐cysteinyldopa to pheomelanin, and would point to a critical reassessment of the commonly reported “benzothiazine” chromophore in terms of more specific and substantiated structural units, like those formed during the later stages of pheomelanin synthesis in vitro.

Oxidative conjugation of chlorogenic acid with glutathione: Structural characterization of addition products and a new nitrite-promoted pathway

Chlorogenic acid (1), a cancer chemopreventive agent widely found in fruits, tea and coffee, undergoes efficient conjugation with glutathione (GSH), in the presence of horseradish peroxidase/H(2)O(2) or tyrosinase at pH 7.4, to yield three main adducts that have been isolated and identified as 2-S-glutathionylchlorogenic acid (3), 2,5-di-S-glutathionylchlorogenic acid (4) and 2,5,6-tri-S-glutathionylchlorogenic acid (5) by extensive NMR analysis. The same pattern of products could be obtained by reaction of 1 with GSH in the presence of nitrite ions in acetate buffer at pH 4. Mechanistic experiments suggested that oxidative conjugation reactions proceed by sequential nucleophilic attack of GSH on ortho-quinone intermediates. Overall, these results provide the first complete spectral characterization of the adducts generated by biomimetic oxidation of 1 in the presence of GSH, and disclose a new possible nitrite-mediated conjugation pathway of 1 with GSH at acidic pH of physiological relevance.

Structural Effects on the Electronic Absorption Properties of 5,6‐Dihydroxyindole Oligomers: The Potential of an Integrated Experimental and DFT Approach to Model Eumelanin Optical Properties†

Elucidation of the relationships between structural features and UV–visible absorption properties of 5,6‐dihydroxyindole oligomers is an essential step towards an understanding of the unique optical properties of eumelanins. Herein, we report the first combined experimental and density functional theory (DFT) investigation of the 5,6‐dihydroxyindole oligomers so far isolated. 2,2′‐Biindolyl 2 and the 2,4′‐biindolyl 3 absorb at longer wavelengths relative to 2,7′‐biindolyl 4 and their spectra were well predicted by DFT analysis. The absorption bands of 2,4′:2′,4′′‐ and 2,4′:2′,7′′‐triindolyls 5 and 6 also fall at different wavelengths and can be interpreted by DFT simulations as being due to a combination of two main separate transitions. Tetramer 7, in which two 2,4′‐biindolyl units are linked through a 2,3′‐connection, exhibits a broad chromophore extending over the entire UV range without well defined absorption maxima. Within the dimer–tetramer range examined, three key points emerge: (1) an increase in oligomer chain length does not result in any regular and predictable bathochromic shift; (2) a marked broadening of the absorption bands occurs when going from the monomer to the tetramer structure; and (3) the mode of coupling of the indole units is a crucial, hitherto unrecognized, structural parameter affecting the electronic absorption properties of 5,6‐dihydroxyindole oligomers. It is concluded that use of experimentally characterized oligomeric scaffolds as a basis for DFT calculations is a most promising approach to building reliable structural models for studies of eumelanins optical properties.

The Eumelanin Intermediate 5,6-Dihydroxyindole-2-Carboxylic Acid Is a Messenger in the Cross-Talk among Epidermal Cells

Interest in colorless intermediates of melanocyte metabolism has traditionally been related to their role as melanin precursors, though several lines of evidence scattered in the literature suggested that these compounds may exert an antioxidant and protective function per se unrelated to pigment synthesis. Herein, we disclose the remarkable protective and differentiating effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible dopachrome tautomerase (DCT)-dependent eumelanin intermediate, on primary cultures of human keratinocytes. At micromolar concentrations, DHICA induced: (a) time- and dose-dependent reduction of cell proliferation without concomitant toxicity; (b) enhanced expression of early (spinous keratins K1 and K10 and envelope protein involucrin) and late (loricrin and filaggrin) differentiation markers; (c) increased activities and expression of antioxidant enzymes; and (d) decreased cell damage and apoptosis following UVA exposure. The hitherto unrecognized role of DHICA as an antiproliferative, protective, and antiapoptotic endogenous cell messenger points to a reappraisal of the biological functions of melanocytes and DCT in skin homeostasis and photoprotection beyond the mere provision of melanin pigments, and provides, to our knowledge, a previously unreported possible explanation to the higher resistance of the dark-skinned eumelanic phenotypes to sunburn and skin cancer.

Uncovering the Structure of Human Red Hair Pheomelanin: Benzothiazolylthiazinodihydroisoquinolines As Key Building Blocks

Biomimetic oxidation of the pheomelanin precursor 5-S-cysteinyldopa in the presence of Zn(2+) ions led to the isolation of two isomeric products, one of which could be identified as the benzothiazolylthiazinodihydroisoquinoline 5, while the other proved too unstable for a complete characterization. Both these products were converted into more stable oxidized forms, which after ethylchloroformate derivatization were characterized as the ethyl ester/ethoxycarbonyl isoquinolines 8 and 9. Compound 5 exhibited absorption characteristics similar to those of red hair pheomelanin, including a main band around 400 nm in acids. Similarly to red hair pheomelanin and synthetic pigments, 5 afforded on chemical degradation a thiazolylpyridinecarboxylic acid fragment. Model chemical studies allowed the proposal of a formation mechanism for the benzothiazole and dihydroisoquinoline systems in compound 5.

Isomeric cysteinyldopas provide a (photo)degradable bulk component and a robust structural element in red human hair pheomelanin

Alkaline H2O2 degradation of red hair pheomelanin gave, besides 6‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA), a new product which was identified as 7‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA‐2) originating from 2‐S‐cysteinyldopa (2SCD) derived units. BTCA‐2 was also obtained from a variety of pheomelanic tissues and synthetic pigments. Simultaneous determination of BTCA and BTCA‐2 in segments of red hair locks taken at variable distances from the scalp in a group of 19 individuals indicated an abrupt drop of BTCA yields on passing from root to tip, whereas BTCA‐2 values remained virtually constant throughout hair length. Analysis of 4‐amino‐3‐hydroxyphenylalanine (AHP) and 3‐aminotyrosine (AT) in the same lock segments showed a closely similar trend, whereas yields of thiazole‐2,4,5‐tricarboxylic acid (TTCA) increased with increasing the distance from the scalp. Prolonged exposure of hair locks to sunlight caused a significant decrease in BTCA‐, but not BTCA‐2‐yielding elements. Finally, model studies showed a substantial degradation of 5SCD‐, but not 2SCD‐derived units, during pheomelanin synthesis in vitro. It is concluded that red hair pheomelanin consists of a degradable 5SCD‐derived bulk component associated with stable 2SCD‐derived units. Structural degradation occurs during hair growth probably as a result of oxidative processes related in part to sun exposure.