Miles R. Chedekel

PHOTOCHEMISTRY OF PHEOMELANIN: ACTION SPECTRUM FOR SUPEROXIDE PRODUCTION

Abstract— 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.

A pulse radiolysis investigation of the oxidation of indolic melanin precursors: evidence for indolequinones and subsequent intermediates

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-carboxylic 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 N3. 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.

PHOTOCHEMISTRY AND PHOTOBIOLOGY OF EPIDERMAL MELANINS

There are a number of well-documented aging phenomena which occur in human skin as a result of chronic exposure to sunlight, and it is generally accepted that melanin and its distribution in human epidermal tissue is the single most important factor in protection of human skin from the biochemical devastation produced by chronic solar irradiation. A number of plausible mechanisms whereby melanins accomplish this photoprotection have been postulated; these include the filtering and attenuation of impinging radiation by scattering, absorption and subsequent dissipation (as heat), absorption accompanied by redox reactions, and absorption accompanied by electron transfer processes [36]. In addition, in the past decade several studies documenting the photosensitizing ability of melanins have appeared [24,30]. This review will focus on recent advances aimed at testing and evaluating these mechanisms on a molecular level.

PHOTOINITIATED DNA DAMAGE BY MELANOGENIC INTERMEDIATES IN VITRO

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.

Chemically induced Parkinson's disease: intermediates in the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to the 1-methyl-4-phenyl-pyridinium ion

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.

PHOTOCHEMISTRY and PHOTOBIOLOGY OF MELANOGENIC METABOLITES: FORMATION OF FREE RADICALS*

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.

The photochemical interaction between the triplet state of 8-methoxypsoralen and the melanin precursor L-3,4 dihydroxyphenylalanine.

Abstract— 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 (ε= 12000 dm3 mol‐1 cm‐1) and for dopaH at pH 3.3, λ= 305 nm (ε= 5900 dm3 mol‐1 cm‐1).

Chemically induced parkinson's disease II: Intermediates in the oxidation and reduction reactions of the 1-methyl-4-phenyl-2,3-dihydropyridinium ion and its deprotonated form†

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.

Biosynthetic and structural studies on pheomelanin

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.

Photogeneration of free radicals from eumelanogenic intermediates and metabolites

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.