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This paper is an attempt to summarize the current state of information on melanin and epidermal melanin pigmentation (EMP) as photoprotective agents. The chemistry and biochemistry of melanin (the particle) and its interaction, in its various forms, with UV radiation are considered. Methods of attenuation of UV radiation are discussed in terms of structure and chemical constituents. Photoprotection by constitutive and facultative pigmentation is reviewed with minimum erythema dose (MED) as the end point. The issue of acclimatization to UV radiation is discussed in terms of UVB phototherapy for psoriasis. Finally, skin cancer is considered as an end point and the reduction of its incidence with pigment level is discussed. It is concluded that whilst EMP provides protection, its extent depends on the end point chosen for evaluation. MED is a convenient photobiological end point but is rather insensitive, whereas skin cancer is sensitive but impractical for laboratory studies. Our current state of knowledge of melanin lacks information on its absorption and scattering coefficients and its refractive index. Methods for the quantitative measurement of EMP are also urgently required.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48-53, 1992).

Natural melanins are composed of two distinct portions; a protein fraction and a chromophoric backbone. There is no unequivocal evidence for covalent bonding between these two fractions, and standard protocols used in protein purification have failed to separate the protein fraction from the chromophoric fraction. In order to study the chromophoric backbone, many workers have resorted to harsh isolation and purification protocols that are now known to degrade and damage the chromophoric portion. These artifactual melanin preparations are poor models for valid chemical, physical, and biological studies. We have developed a mild isolation and purification protocol for melanins that takes into consideration both the particulate nature of natural melanins and the stability characteristics of the chromophoric fraction. Mathematical factoring of the quantitative amino acid data into the elemental analysis was used to obtain the empirical formula of the chromophoric backbone of melanins. The analyses have shown that melanins from various sources have significantly different amino acid compositions and contents, molar C/N ratios, and empirical formulae. This method successfully differentiates melanins from a variety of sources, namely, human hair, Sepia officinalis, Sigma Chemical Company (cat. no. M8631), autoxidation of dopa, and from the feathers of Rhode Island Red chickens. Analytical results from these studies are presented and discussed.

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.

An antiserum against alpha-amino-(4-hydroxy-6-benzothiazolyl)propionic acid (AHBP), a major product obtained after hydriodic acid hydrolysis of pheomelanin, was raised in rabbits immunized with AHBP coupled to bovine serum albumin (BSA). The antiserum was used to develop a radioimmunoassay (RIA) to AHBP. The limit of detection of the RIA is 0.1 ng of AHBP. The antiserum does not crossreact with other major hydriodic acid degradation products, and the assay has been used to estimate the amounts of AHBP in synthetic and natural melanins.

Cysteinyldopas, metabolic by-products of activated melanocytes, are photochemically unstable in the presence of biologically relevant ultraviolet radiation (i.e. wavelengths > 300 nm). Initial photochemical processes involve free radical production; continued photolysis yields polymeric photoproducts. Radicals produced during 5SCD photolysis were trapped by 5,5-dimethyl-l-pyrrolidine-l-oxide (DMPO) and identified by their ESR spectra. Further characterization by use of nitroso spin trap (2-methvl-2-nitrosopropane-MNP) demonstrated that homolytic cleavage of the -S-CH2 bond of the 5SCD cysteinyl side chain is a significant photochemical pathway. The potential photobiological significance of these reactive intermediates was investigated in vitro using isolated nucleic acids. Radiolabeled 5-[35S]-cysteinyldopa was found to photobind to calf thymus DNA with 300 nm light activation. Under similar conditions, 5-S-cysteinyldopa also induced single strand breakage of 3H-radiolabeled superhelical, circular pBR322 plasmid DNA. The implications of the 5SCD photoinitiated DNA damage and the production of highly reactive free radicals in this process are discussed with respect to the etiology of various skin cancers, particularly malignant melanoma.

A mixture of (−)-nicotine-[2′-3H] and (±)-nicotine-[2′-14C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (−)-nornicotine-[2′-14C,3H] and 52% (+)-nornicotine-[2′-14C]. Thus if (+)-nornicotine is formed from (−)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-14C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-14C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.

We have recently demonstrated that cysteinyldopas, pheomelanogenic precursors and excreted eumelanogenic metabolites, are photolabile and initiate DNA damage in vitro. In this study we have extended our photochemical investigations to eumelanogenic indole intermediates and metabolites. Continuous-wave photolysis of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), or its 5-methoxylated metabolite (HMICA) with biologically relevant ultraviolet radiation (i.e., wavelengths greater than 300 nm) resulted in rapid destruction of starting material. Using ESR spin-trapping techniques we observed the initial production of free radical species; prolonged photolysis resulted in the formation of polymeric photoproducts. Radicals were trapped by the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and characterized by their ESR spectra as hydrated electrons and hydrogen atoms. Experiments further demonstrated that while DHI photoionizes, the two indole-2-carboxylic acid derivatives do not ionize appreciably upon irradiation, rather homolysis of X-H bonds appears to be a significant photochemical pathway. The potential photobiological significance of melanogenic indole intermediates is discussed.

Pheomelanin, the red-brown polymeric pigment in the skin and hair of red-headed humans, is composed of a protein fraction covalently bound to a colored chromophore. Photolysis of aerated aqueous phemelanin solutions, isolated from human red hair, results in destruction of the chromophore and liberation of the peptide fraction. The rate of photolysis depends on the pH and the concentration of both pigment and oxygen and is slightly inhibited by the enzyme superoxide dismutase (superoxide:superoxide oxidoreductase EC 1.15.1.1). Pheomelanin photolyzed in the presence of nitroblue tetrazolium results in the formation of a blue diformazan, whether or not oxygen is present. Superoxide dismutase inhibits the aerobic photoreduction of nitroblue tetrazolium whereas, in the absence of oxygen, no inhibition is observed. These experiments strongly suggest the involvement of superoxide in the aerobic photolysis of pheomelanin and point out a possible mechanism for ultraviolet-induced cell damage in redheads.

Etude de l'isomerisation cis-trans du cyclobutane dans les produits issus de la reaction du titre: les acides cyclobuta [c] furo [3,2-g] benzopyranne-1 caprylique-1 et -2

— A new in vitro model system for studying the photochemistry and photobiology of melanins in epidermal tissue has been developed. The photochemistry of phaeomelanin in this model system follows the same course as in pH 8.0 aqueous buffered solutions, i.e. photodegradation accompanied by production of superoxide radicals. These photoreactions may be effectively quenched by application of a broad spectrum sunscreen prior to irradiation.

Two pheomelanin model compounds were prepared and their photochemistries examined. Upon irradiation, an oxygen-mediated photopolymerization reaction was observed. The photopolymers obtained were characterized by their apparent molecular weights, UV and fluorescence spectra. The implications of these findings with respect to the structure and photobiology of pheomelanin are discussed.

13C-NMR spectroscopy of pheomelanin biopolymers, prepared from isotopically enriched precursors, has been developed as a tool for structure elucidation of melanins. By employing large pulse-widths and short cycle time, only the signals originating from labeled carbons are observed in the high-resolution spectra of these polymers.

— The photochemical interaction between 8-methoxypsoralen (8-MOP) and the melanin precursorL–3,4-dihydroxyphenylalanine(dopaH2) has been studied using laser flash photolysis. Triplet excited 8-MOP was thus found to abstract electrons from dopaH2 (k∼ 2 × 109 dm3 mol-1 s-1) to form semireduced 8-MOP and semioxidised dopaH2.The technique of pulse radiolysis was used to establish separately the spectra of (a) the semi-reduced form of 8-MOP at pH 6.5 and (b) the semioxidised forms of dopaH2 at pH 6.5, 5.8, 4.6 and 3.3. The corresponding λmax and extinction coefficients found were: for 8-MOP at pH 6.5, λmax= 350 nm (= 9050 dm3 mol-1 cm-1); for dopa at pH 6.5, λmax= 305 nm (e= 12000 dm3 mol-1 cm-1) and for dopaH at pH 3.3, λ= 305 nm (e= 5900 dm3 mol-1 cm-1).

The rate constants associated with the series of successive transient absorptions initiated by one-electron oxidation of 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-car☐ylic acid (DHICA), precursors of melanin, and N -methyl-5,6-dihydroxyindole (NMDHI), a model compound, have been studied by pulse radiolysis. The initial transient species resulting from N 3 oxidation reaction at pH 7.3–7.4 are assigned as the corresponding semiquinones. In each case, these radicals decayed, probably by disproportionation, into products most readily monitored in the 400–430 nm region. For DHI, the decay in this region could be fitted by two parent concentration independent first-order processes. These may correspond to transformations between 5,6-indolequinone, and its quinone-imine and quinone-methide tautomers. With NMDHI, on the other hand, a single longer-lived product with a peak around 430 nm predominated after decay of the corresponding radical, due almost certainly to N -methyl-5,6-indolequinone. The data appear to exclude significant melanin polymerisation by condensation of semiquinones, reaction of semiquinones with dihydroxyindoles, self-addition of indolequinones or tautomers, or reaction of indolequinones or tautomers with the parent dihydroxyindoles. It is suggested that polymerisation of melanin may rather occur by stepwise addition of indolequinone methide/imine to reduced oligomeric species.

— The synthesis of 4-(N-methylpyridinium) t-butyl nitrone (4-MePyBN) and its use as a spin trap for superoxide radicals produced in aqueous solutions is reported (aN= 13.78 G, aHs= 1.65 G, and g = 2.0091). The half-life of the spin adduct as a function of pH (83 s at pH 5.5, 78 s at pH 7.0, and 65 s at pH 8.0) the effects of iron salts, diethylenetriaminepentacetic acid (DETAPAC), and superoxide dismutase were examined, and comparisons made between 4-MePyBN and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin trapping agents for the superoxide radical.

Convallaria majalis plants were fed dl -methionine-[1- 14 C]. [1- 14 C, 4- 3 H], and [1- 14 C, 2- 3 H], S -adenosyl- l -methionine-[1- 14 C], and dl -homoserine-[1- 14 C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1- 14 C, 4- 3 H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1- 14 C, 2- 3 H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S -adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.

Normal melanocytes produce specialized subcellular organelles called melanosomes within which the biochemical processes of melanogenesis occurs. During sunlight-induced melanogenesis, the melanocyte-specific enzyme tyrosinase catalyzes the oxidative polymerization of 3,4-dihydroxyphenyl-alanine (DOPA) to melanin. Nucleophilic addition of cysteine to tyrosinase-generated dopaquinone leads to the formation of cysteinyldopas, precursors of pheomelanin and excreted by-products of eumelanogenesis. Under conditions of low sulfhydryl content, dopaquinone undergoes a 1,4 intramolecular cycloaddition to yield, after further oxidation, 5,6-dihydroxyindoles and/or 5,6-dihydroxy-2-carboxyindoles. These indolic melanogenic intermediates and their O-methylated metabolites, like cysteinyldopas, are excreted by actively pigmenting as well as dormant melanocytes. Indeed, it has been determined that in humans, the serum and urine concentrations of these melanogenic metabolites increase dramatically following exposure to sunlight, UVA (315-400 nm), UVB (290-315 nm) exposure, as well as during PUVA therapy and in melanoma patients, and thus have proved to be excellent biochemical markers of normal and pathological melanocyte function. While controlled light exposure or PUVA therapy generally lead to 100-300% increases in 5-S-cysteinyldopa (5SCD) and 5-methoxy-6-hydroxyindole-2-carboxylic acid (6HMICA) serum levels (normal concentration about 4–16 nmol l-1), the local concentrations in the skin and especially in the actively pigmenting melanocyte may be as high as 200 μM. Evidence is presented to document that a number of catecholic melanin precursors, including cysteinyldopas and dihydroxyindoles, are photochemically unstable in the presence of biologically relevant ultraviolet radiation (i.e. wavelengths ± 300 nm). Initial photochemical processes involve free radical production; continued photolysis yields polymeric photoproducts. Radicals produced during melanogenic metabolite photolysis have been identified by ESR spin trapping, laser flash photolysis and pulse radiolysis techniques and include hydrated electrons (eaq), hydrogen atoms (H'), hydroxyl radicals (OH), semiquinones, aryl thiyl (ArS), and alanyl carbon-based radicals. In vitro investigations of the potential photobiological significance of these reactions have demonstrated photolysis of cysteinyldopas may lead to photoinitiated DNA binding and single strand break induction. The above mentioned radical species may also damage proteins and initiate lipid peroxidation. Definitive evidence for the occurrence of these phototoxic reactions in vivo is currently unavailable, however our in vitro studies suggest a possible role for melanogenic metabolite photolysis in acute and chronic solar responses of human skin.

o-Aminothiophenols are an important class of starting material for synthesis of a number of heterocycles, e.g., phenothiazines1, benzothiazoles2 and benzothiazines3. However, most of the methods for their preparation either lack generality or are inefficient, giving low yields or hard to purify mixtures of products. Further, preparation and purification of these compounds is complicated by their inherent lack of stability.

— The action spectrum for superoxide production from aerated aqueous solutions of pheomela-nin was determined by utilizing the nitroblue tetrazolium-superoxide dismutase assay for superoxide. Superoxide production was greatest in the UVC regions, but continued well into the visible wavelengths. The marked increase in superoxide production noted in the UVC-UVB regions of the spectrum suggests that superoxide production may be involved in a number of actinic disorders in fair-skinned humans.

Phaeomelanin readily decomposes by the action of UV light and oxygen. This finding may explain many of the abnormal reactions of the skin of redheads and blondes to sunlight, including their high susceptibility to skin cancer. In aqueous solution, phaeomelanin displays an EPR signal rich in hyperfine splitting, consistent with structural information. SUMMARY Phaeomelanin readily, decomposes by the action of UV light and oxygen. This finding may explain many of the abnormal reactions of the skin of redheads and blondes to sunlight, including their high susceptibility to skin cancer. We have yet to identify any of the fragmentation products. Chromatographic analysis of the photolysate indicates at least 17 different compounds. Work on the reactions of phaeomelanin with superoxide, and the identification, synthesis, and physiological evaluation of these photoproducts as etiological factors in sunburn and carcinogenesis is in progress. In aqueous solutions, phaeomelanin displays an air stable EPR signal rich in hyperfine splitting. Complete analysis of this signal will provide a better understanding of the structure of the pigment.

Melanocytes are cells of neural crest origin residing at the dermal-epidermal juncture. They produce specialized organelles called melanosomes within which the biochemical processes of melanization occurs. UV radiation is capable of inducing melanogenesis and, during the biosynthesis of melanins, several of the putative precursors "leak out" of the melanosome and can be detected in the skin, serum and urine of individuals undergoing active melanogenesis. Most notable are the cysteinyldopas (formed by nucleophilic addition of cysteine to dopaquinone) and several dihydroxyindoles (formed by intramolecular cyclization of dopaquinone). These catechols often are methylated in the melanocyte to afford a mixture of the monomethoxy derivatives and, in some cases, the dimethoxy species. Recent investigations in our laboratories have demonstrated that the cysteinyldopas, dihydroxyindoles, and their various methylated derivatives are photochemically unstable. Irradiation with biologically relevant ultraviolet radiation (i.e. wavelengths greater than 300 nm) results in the rapid destruction of the precursors/metabolites and the production of a variety of free radical species. The photochemistry and potential photobiological significance of melanogenic intermediates is discussed.

The photochemistries of the melanin precursors dopa, 5-S-cysteinyldopa (5-SCD) and 2.5-S,S'-dicysteinyldopa (2.5-SCD) were investigated by 265-nm laser flash photolysis. The quantum yield of hydrated electron following flash photolysis of dopa (9.1%) was half the yield of dopasemiquinone (19.6%), implying that dopasemiquinone is formed via two primary photochemical mechanisms: photionisation (giving e) or photohomolysis (giving H˙). Dopasemiquinone rapidly disproportionates to form dopaquinone and re-form dopa. Dopaquinone in turn decays via a base-catalysed unimolecular cyclisation eventually to form dopachrome. Assignment of the transient species was confirmed by previous pulse radiolysis studies of the one-electron oxidation of dopa. In contrast, flash photolysis of the cysteinyldopas, 5-SCD and 2,5-SCD results in lower photoionisation quantum yields and the production of initial transient species whose absorption spectra were markedly different from their semiquinone absorption spectra previously determined pulse radiolytically. These observations indicate that the primary cysteinyldopa photochemical species is not such a semiquinone, but rather results from S-CH2 bond photohomolysis. Absorption spectra and rate constants for the formation and decay of various transient species are reported.

The one-electron reduction product of 1-methyl-4-phenyl-2,3-dihydropyridinium ion has been generated by pulse radiolysis and its absorption spectrum recorded. This radical was found to decay by second-order kinetics (2k = 9.5 x 10(8) M-1 s-1) to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion. Reactions of the above radical species and that formed by one-electron reduction of 1-methyl-4-phenylpyridinium ion, which can also be generated by one-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, with a number of molecules of biochemical interest have been studied. The one-electron reduction product of oxidised nicotinamide adenine dinucleotide efficiently reduced 1-methyl-4-phenyl-2,3-dihydropyridinium ion (k = 2.2 x 10(9) M-1 s-1). The relevance of these results in relation to redox cycling, a possible mechanism for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, is discussed.

Various unstable intermediate oxidation states have been postulated in the metabolic activation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to the 1-methyl-4-phenyl pyridinium ion. We now report the first direct observation of these free radical intermediates by pulse radiolysis and flash photolysis. Studies are described of various reactions of such species, in particular with dopamine whose autoxidation to dopamine quinone is reported to be potentiated by 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine.

IN the past few years deuterated compounds have become readily available and are in wide use, especially as solvents for NMR spectroscopy. D. Arigoni has informed us (personal communication) that heavy water (D2O) can contain appreciable quantities of tritium. It has been known for some time that tritium is present in D2O obtained as a residue after the prolonged electrolysis of natural water1. But most users of deuterium compounds seem to be unaware of the presence of tritium, often in amounts which require a licence for possession. Those using tritium as a tracer should be cautioned regarding the potential contamination resulting from the use of D2O and other deuterated compounds. We have measured the tritium content of several commercial deuterated compounds which were in routine use in our laboratory, and the results are summarized in Table 1. All the compounds had a nominal deuterium content >99%.

Pulse radiolysis and flash photolysis-initiated one-electron oxidation of 3,4-dihydroxyphenylalanine (dopa) and other catecholamines have allowed the direct measurement of the kinetics of semiquinone disproportionation leading successively to the corresponding melanin precursors dopaquinones and dopachromes.