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Biomedical subjects

G Prota

Publications and source records attributed to G Prota.

At least 37 records · Page 2Linked to original sources
PubMed1998

Phaeomelanin versus eumelanin as a chemical indicator of ultraviolet sensitivity in fair-skinned subjects at high risk for melanoma: a pilot study.

It is now generally agreed that solar exposure is a major external factor in the causation of cutaneous melanoma in light skinned populations with red hair and a marked susceptibility to the acute effects of ultraviolet (UV) radiation. In the present study, we investigated the existence of a possible relationship between hair melanin composition and minimal erythema dose (MED), as an indicator of UV sensitivity, in a group of 15 healthy red-haired subjects aged 20-46 years. In spite of comparable skin and hair colour, marked variations were observed in the MED values as well as in the hair melanin composition. Phaeomelanin levels varied in the range 0.026-0.53% w/w and were generally comparable to or higher than eumelanin levels (0.042-0.17% w/w). No significant relationship was found between MED values and phaeomelanin, eumelanin or total melanin (eumelanin plus phaeomelanin) content. Notably, however, a gross positive correlation was found between the eumelanin/phaeomelanin ratio and the MED values. These results would suggest that a high UV sensitivity is associated with high phaeomelanin and low eumelanin levels, and point to the eumelanin/phaeomelanin ratio as a novel chemical parameter that could be used for predicting individuals at high risk for skin cancer and melanoma.

Adult↗
PubMed1997

Iron-mediated generation of the neurotoxin 6-hydroxydopamine quinone by reaction of fatty acid hydroperoxides with dopamine: a possible contributory mechanism for neuronal degeneration in Parkinson's disease.

Exposure of dopamine to an excess of linoleic acid 13-hydroperoxide (13-hydroperoxyoctadecadienoic acid) in the presence of ferrous ions in Tris buffer, pH 7.4, resulted in a relatively fast, oxygen-independent reaction exhibiting first-order kinetics with respect to both catecholamine and metal concentrations. Product analysis in the early stages revealed the presence of significant amounts of the quinone of the neurotoxin 6-hydroxydopamine, together with some aminochrome and ill-defined melanin-like material. Quinone formation required the presence of iron, either in the ferrous or ferric form, and was unaffected by peroxidase, catalase, and hydroxyl radical scavengers, e.g. mannitol, as well as biologically relevant antioxidants, like ascorbate and glutathione. Hydrogen peroxide proved as effective as linoleic acid hydroperoxide in inducing dopamine oxidation and conversion to 6-hydroxydopamine quinone. Metal chelators, including EDTA and bipyridyl, markedly suppressed quinone formation without, however, inhibiting dopamine oxidation. These and other results are consistent with a hydroxyl radical independent hydroxylation/oxidation mechanism basically different from the Fenton reaction, which involves direct interaction of the peroxide with a dopamine-Fe(III) chelate generated during the process.

Dopamine↗
PubMed1997

Diffusible melanin-related metabolites are potent inhibitors of lipid peroxidation.

Although it has long been known that epidermal melanocytes produce and excrete a number of melanin-related metabolites, including 5.6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and 5-S-cysteinyldopa (CD), the possible functional significance of these compounds has been so far largely overlooked. We report now evidence that DHI, DHICA and CD exert potent inhibitory effects in different in vitro models of lipid peroxidation. The compounds, at 100 microM concentration, substantially decreased malondialdehyde (MDA) formation by lipid peroxidation in rat brain cortex homogenates. At 1.2 microM concentration, DHI proved as effective as alpha-tocopherol (alpha-T), one of the most potent endogenous antioxidants, in suppressing azo-induced peroxidation of linoleic acid in phosphate buffer (pH 7.4), containing 0.10 M SDS, whereas CD and DHICA at the same concentration were less active. DHI, CD and DHICA (all in the range 25 microM-0.5 mM) were also found to inhibit Fe (II)/EDTA-induced oxidation of 0.5 mM arachidonic acid at pH 7.4, as well as MDA formation by iron-promoted degradation of 0.5 mM 15-hydroperoxy-5,8,11, 13-eicosatetraenoic acid (15-HPETE). In both cases the inhibitory effects were much greater than those of ascorbic acid and glutathione. These results point to melanin precursors as a novel class of biological antioxidants which may contribute to defense mechanisms against oxidative injury in human skin.

Animals↗
PubMed1997

Selective incorporation of the prototype melanoma seeker thiourea into nascent melanin: a chemical insight.

The mechanism of selective incorporation of thiourea into melanotic melanoma was investigated by model experiments in which the effect of the compound was examined at various stages of melanogenesis in vitro. Up to 50% inhibition of dopachrome formation was observed in the tyrosinase-dopa reaction in the presence of thiourea at a 2:1 molar ratio with respect to the substrate. Under these conditions, a major product was formed which was isolated and identified as a 1:1 dopa-thiourea adduct (adduct I). Subsequent stages of the oxidation were characterized by the development of a yellow chromophore (lambdamax 440-460nm), virtually identical to that obtained by separate oxidation of the adduct I. A less remarkable effect of thiourea was observed on the oxidative polymerization of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) which was apparent on spectrophotometric and high pressure liquid chromatography (HPLC) analysis. Radiolabelling experiments with 14C-thiourea showed that the label was initially incorporated into the adduct I, while in the subsequent stages of the oxidation it was associated with pigmented materials which escaped direct analysis. Incorporation of labelled thiourea into dopa-melanins was found to be significantly higher than incorporation into synthetic pigments from indole precursors. These results provide a chemical basis for the interpretation of the selective accumulation of thiourea in those melanoma areas with high rates of melanin synthesis seen in autoradiographic experiments.

Antineoplastic Agents↗
PubMed1997

Biosynthesis, structure, and function of neuromelanin and its relation to Parkinson's disease: a critical update.

No longer dismissed as just a mere curiosity in the family of melanin pigments, neuromelanin is attracting increasing interest as a central constituent of certain populations of dopaminergic neurons in the human substantia nigra, which may hold the key for the understanding of neuron functioning and degeneration in aging and in Parkinson's disease. It is the purpose of this article to provide a concise review of the most significant data on the origin, structure, and functional significance of neuromelanin that accrued over the past few years. It also aims at critically surveying the currently debated views regarding the role of such intriguing pigment in the etiology and biochemical pathology of Parkinson's disease.

Animals↗
PubMed1996

Mechanism of selective incorporation of the melanoma seeker 2-thiouracil into growing melanin.

The mechanism of selective incorporation of 2-thiouracil (TU), a highly specific melanoma seeker, into growing melanins was investigated both in vitro and in vivo. Methods used included direct analysis of the melanins, by evaluation of the absorption at 350 nm (A350) and chemical degradation coupled with HPLC quantitation of pigment makers, i.e., pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), as well as biosynthetic experiments involving tyrosinase-catalyzed oxidation of DOPA, 5,6-dihydroxyindole (DHI), and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Injection of radiolabeled TU into melanoma-bearing mice resulted in a rapid incorporation of the drug into the tumor pigment, with a substantial decrease in A350 and in PTCA yields. Similar changes in the absorption properties were observed in biosynthetic melanins prepared in the presence of TU, whereas the yields of PTCA and PDCA varied depending on the pigment precursor used. When incubated with DOPA in the presence of tyrosinase, TU profoundly modified the normal course of melanogenesis, favoring formation of a complex mixture of addition products consisting mainly of 6-S-thiouracil-DOPA as well as DHI-TU adducts. The latter were obtained in larger amounts by enzymatic oxidation of DHI in the presence of TU and were identified as the 3- and 2-substituted adducts 1 and 2, the dimer 3, and the trimer 4. Similar reactions carried out on DHICA yielded the 4-substituted adduct 5, the dimer 6, and the trimer 7. A new mechanistic scheme for the incorporation of TU into growing melanin is proposed, which envisages nucleophilic attack of the thioureylene moiety of TU to transient quinonoid intermediates in the melanin pathway, chiefly dopaquinone and 5,6-indolequinones, followed by entrainment of the resulting adducts into the growing pigment via oxidative copolymerization with DHICA and/or DHI.

Animals↗
PubMed1996

5-S-cysteinyldopa, a diffusible product of melanocyte activity, is an efficient inhibitor of hydroxylation/oxidation reactions induced by the Fenton system.

Interest in 5-S-cysteinyldopa (5-S-CD), a major excretion product of normal and malignant melanocytes, has traditionally concentrated on its significance as a biosynthetic precursor of pheomelanins, the characteristic pigments of red hair, and as a specific biochemical marker for monitoring melanoma progression. The present study shows that 5-S-CD is a potent inhibitor of hydroxylation/oxidation reactions mediated by hydrogen peroxide and the Fe2+/EDTA complex under both aerobic and anaerobic conditions. The inhibitory effect of 5-S-CD, as determined by the deoxyribose and salicylic acid assays in phosphate buffer (pH 7.4), is much stronger than that of dopa, acetylsalicylic acid and mannitol, increases with increasing ligand-to-metal ratio, and is inversely proportional to the concentration of EDTA present in the Fenton system. Spectrophotometric evidence and competition experiments indicate that 5-S-CD forms a chelate complex with ferric ions (lambda max = 500 nm at pH 7.4), which may account for both an altered production of hydroxyl radicals by the Fenton reagent and a site-specific localization of oxidative damage on the chelate complex itself.

Aerobiosis↗
PubMed1996

Lipofuscin-like pigments by autoxidation of polyunsaturated fatty acids in the presence of amine neurotransmitters: the role of malondialdehyde.

Iron-promoted autoxidation of arachidonic acid in acetate buffer (pH 6.0) at 37 degrees C in the presence of glycine led to the formation after 24 h of a complex pattern of fluorescent products, one of which was identified as 1-carboxymethyl-4-methyl-1,4-dihydropyridine-3,5-dicarboxaldehyde (1), arising by reaction of glycine with malondialdehyde. When glutamic acid was used as the amine compound, the corresponding dihydropyridine 2 was detected in the oxidation mixture. Formation of adducts 1 and 2 was markedly decreased at pH 7.4, or in the presence of a large excess of the amino acid, and was suppressed by glutathione or ascorbic acid. In the presence of dopamine, norepinephrine or serotonin, autoxidation of arachidonic acid led likewise to complex patterns of fluorescent products, but no evidence of the dihydropyridine adducts 3-5, nor of their oxidation products 6-8 was obtained. No malondialdehyde-derived product could be detected when linoleic acid was used as the fatty acid. These and other results that are presented suggest that malondialdehyde is a possible contributor to, but not the primary determinant of fluorescent pigment formation by peroxidative degradation of polyunsaturated fatty acids in the presence of amine compounds.

Arachidonic Acid↗
PubMed1995

Iron- and peroxide-dependent conjugation of dopamine with cysteine: oxidative routes to the novel brain metabolite 5-S-cysteinyldopamine.

The mechanism of formation of 5-S-cysteinyldopamine (5-S-CDA), a putative index of oxidative stress in dopaminergic regions of the brain, was investigated by comparing the ability of a number of neurochemically relevant oxidising systems to promote the conjugation of dopamine with cysteine in vitro. Autoxidation of the catecholamine proceeds at relatively slow rate in the physiological pH range, and is little affected by 1 mM Fe(2+)-EDTA complex. In the presence of cysteine, however, the Fe(2+)-induced autoxidation is hastened, affording little amounts of 5-S-CDA. Formation of the adduct is completely suppressed by ascorbic acid. Hydrogen peroxide, in the presence of Fe(2+)-EDTA (Fenton-type oxidation) or peroxidase, promotes a relatively efficient conversion of dopamine to 5-S-CDA and the minor isomer 2-S-CDA. Noteworthy, 15-hydroperoxyeicosatetraenoic acid (arachidonic acid hydroperoxide, HPETE), in the presence of Fe(2+)-EDTA complex, can also mediate 5-S-CDA formation, whilst superoxide radicals are little effective. Overall, these results suggest that ferrous ions, hydrogen peroxide and lipoperoxides may play an important role in 5-S-CDA generation.

Brain↗
PubMed1995

Generation of the neurotoxin 6-hydroxydopamine by peroxidase/H2O2 oxidation of dopamine.

At physiological pH values, oxidation of the neurotransmitter dopamine (DA) by the peroxidase/H2O2 system leads to, besides dopaminochrome and 5,6-dihydroxyindole resulting from oxidative cyclization of dopaminequinone (DQ), significant amounts of the neurotoxin 6-hydroxydopamine (6-OHDA) in the oxidized quinonoid form (topaminequinone, TQ). Formation of TQ was shown to depend critically on the presence of hydrogen peroxide in the reaction medium and was not observed when DA oxidation was carried out using the tyrosinase/O2 system or chemical agents such as periodate or ferricyanide. These and other data suggest that, under the conditions adopted, nucleophilic attack of the hydrogen peroxide anion on DQ leading to TQ significantly competes with the intramolecular cyclization path. In line with this mechanism, the reaction course was not affected by the presence of hydroxyl radical scavengers. Peroxidase/H2O2 oxidation of the model N-acetyldopamine (1) gave, as expected, the 2-hydroxy-1,4-benzoquinone 3 in yields up to 55%, depending on the catecholamine/H2O2 mole ratio. Likewise, reaction of 4-methyl-1,2-benzoquinone (4) with hydrogen peroxide afforded 2-hydroxy-5-methyl-1,4-benzoquinone (5) in good yields. Collectively, these results would point to the possibility that intraneuronal formation of 6-OHDA is associated with an increased production of hydrogen peroxide under oxidative stress conditions.

Anions↗
PubMed1995

Comparative analysis of melanins and melanosomes produced by various coat color mutants.

Pigmentation in the skin, hair, and eyes of animals is influenced by a number of genes that modulate the activity of melanocytes, the intervention of enzymatic controls at different stages of the melanogenic process, and the physico-chemical properties of the final pigment. The results of combined phenotypic, ultrastructural, biochemical, and chemical analyses of hairs of a variety of defined genotypes on a common genetic background performed in this study are consistent with the view that pigmentation of dark to black hairs results from the incorporation of eumelanin pigments whereas that of yellow hairs results from the incorporation of eu- and pheomelanins. It is also clear that relatively minor differences in melanin content can have dramatic effects on visible hair color. A good correlation was found for expression of (and enzyme activities associated with) TRP1 and TRP2 with eumelanin synthesis and eumelanosome production.

Animals↗
PubMed1995

The effects of hydroxyl radical attack on dopa, dopamine, 6-hydroxydopa, and 6-hydroxydopamine.

High pressure liquid chromatography with electrochemical detection (HPLC-ED) was employed in conjugation with a sensitive and specific salicylate hydroxylation assay to evaluate the immediate effects of hydroxyl radical (.OH) attack on four catechol intermediates of eumelanin, dopamine (3,4-dihydroxyphenylethylamine), its precursor dopa (3,4-dihydroxyphenylalanine), and their respective neurotoxic trihydroxyphenyl derivatives, 6-hydroxydopamine (2,4,5-trihydroxyphenylethylamine,6-OHDA) and 6-hydroxydopa(2,4,5-trihydroxyphenylalanine, TOPA). Semiquinone and quinone species were identified as the initial products of the oxidation of these four catechol substrates. The enhanced oxidations of the catechols when exposed to .OH attack was accompanied by marked decreases in the level of each semiquinone species. Quinone levels were elevated in reactions involving .OH attack on dopamine and 6-OHDA, but absent in reactions involving radical attack on dopa or TOPA, suggesting that dopaquinone (DOQ) and TOPA p-quinone (TOPA p-Q) are oxidized more rapidly by .OH than are the quinones of dopamine and 6-OHDA. The formation of 6-OHDA p-quinone (6-OHDA p-Q) in incubations involving DA and .OH suggest that the .OH-mediated hydroxylation of DA may be a mechanism for generating this potentially cytotoxic trihydroxyphenyl. The results of this study demonstrate for the first time that semiquinone and quinone intermediates of eumelanin are the initial products derived from the .OH-mediated oxidations of dopa, DA, TOPA, and 6-OHDA. These observations suggest that if .OH is generated beyond the capabilities of cytoprotective mechanisms, the radical can rapidly oxidize catechol precursors, augment melanogenesis, and generate additional cytotoxic quinoid intermediates of eumelanin.

Benzoquinones↗
PubMed1994

A new dopachrome-rearranging enzyme from the ejected ink of the cuttlefish Sepia officinalis.

A melanogenic enzyme catalysing the rearrangement of dopachrome has been identified in the ejected ink of the cuttlefish Sepia officinalis. This enzyme occurs as a heat-labile protein which co-migrates with tyrosinase under a variety of chromatographic and electrophoretic conditions. On SDS/PAGE it shows like a single band with an approx. molecular mass of 85 kDa. The enzyme possesses high substrate specificity, acting on L-dopachrome (Km = 1 mM at pH 6.8) and on L-alpha-methyl-dopachrome, but not on D-dopachrome, L-dopachrome methyl ester, dopaminochrome and adrenochrome. Significant inhibition of the catalytic activity was observed with tropolone and L-mimosine. H.p.1.c. analysis of the enzyme-catalysed rearrangement of L-dopachrome revealed the quantitative formation of the decarboxylated product, 5,6-dihydroxyindole. These results point to marked differences between melanogenesis in cephalopod pigment cells and in melanocytes, which may have important implications in relation to the use of sepiomelanin as a model for studies of mammalian melanins.

Animals↗
PubMed1994

The inherent cytotoxicity of melanin precursors: a revision.

The potential cytotoxicity of the melanogenic intermediates DOPA, (L-3,4-dihydroxyphenylalanine) and DHI (5,6-dihydroxyindole) has long been recognized and exploited as a targeting concept in experimental melanoma therapy. In recent years, however, a novel branchpoint in the melanin biosynthetic pathway has been shown to divert the metabolism of DOPAchrome to a carboxylated derivative termed DHICA (DHI-2-carboxylic acid) rather than to DHI. In order to evaluate the biological implications of this regulatory control, we have reexamined the inherent cytotoxicity of DHICA versus DHI on different cell lines. We found that under the usual conditions of the biological assay, the apparent cytotoxicity of the two indoles reflect their instability in the culture medium, the less stable DHI being generally more toxic than DHICA to melanoma cells and nonmelanocytic cells. Moreover, the observed cytotoxic effects increased with the time of incubation and were markedly reduced by the addition of catalase to the medium, suggesting that they were probably due to the generation of reactive oxygen species (particularly H2O2) during the autoxidation of the melanin precursors outside the cells. To circumvent this problem, we then tested the diacetylated derivatives of DHI and DHICA (DAI and DAICA) which are sufficiently stable until taken up into the cells whereupon they may be converted by endogenous esterases back to the parent indoles. Although DAI proved to be cytotoxic for nonmelanocytic cells, it had no detectable activity on melanoma cells, whereas DAICA showed no effect on any of the cells examined. These results, when combined with other studies, point to a reconsideration of the inherent cytotoxicity of the 5,6-dihydroxyindoles, as well as DOPA, to melanin producing cells.

3T3 Cells↗
PubMed1994

Melanogenesis as a targeting strategy against metastatic melanoma: a reassessment.

Following the early study of Hochstein and Cohen in 1963 suggesting the potential toxicity of quinonoid melanin precursors, considerable interest has been focused on the possibility of exploiting the pigment-producing pathway as a targeting strategy against metastatic melanoma. Several melanocytotoxic phenolic pro-drugs have been developed, and some of them have reached the preclinical stage. Here we review the progress made so far and supply some insight into the chemical and biochemical mechanisms underlying the melanocytotoxic response.

Animals↗
PubMed1994

Photochemistry of 5-S-cysteinyldopa.

The photochemical behavior of 5-S-cysteinyldopa (5-S-CD), a colorless product of melanocyte metabolism, was investigated in neutral phosphate buffer with biologically relevant UV radiation. Exposure of 5-S-CD to pyrex-filtered UV light (wavelengths > 320 nm) was found to induce an oxygen-dependent reaction, leading to, besides abundant polymeric materials, the benzothiazine derivatives I and II (two diastereoisomers). Superoxide dismutase exerted a small inhibitory effect on 5-S-CD consumption, whereas other active oxygen scavengers had no effect on the reaction course. Addition of glutathione as a hydrogen donor completely suppressed the reaction. With UVB light (wavelength range 280-320 nm) photolysis of 5-S-CD proceeded mainly with formation of 3,4-dihydroxyphenylalanine, arising presumably by photohomolytic cleavage of the S-CH2 bond followed by desulfuration. These results are of interest in relation to the high susceptibility of fair-complexioned individuals to actinic damage and skin cancer.

Cysteinyldopa↗