Maria Anna Rosei

CATECHOLAMINES OXIDATION BY XANTHINE OXIDASE

Dopamine and structurally related catecholamines in the presence of hydrogen peroxide are oxidized in vitro by xanthine oxidase producing the corresponding melanin pigments. The kinetic parameters of the reaction, measured as aminochrome formation, have been calculated. The rate of peroxidation depends on enzyme and hydrogen peroxide concentration. The optimum pH for the peroxidative activity of the enzyme is around 8.5. Activation of the peroxidative reaction is also elicited by catechol compounds through a redox cycle mechanism. Implications about the possible biochemical relevance of xanthine oxidase activity on catecholamines oxidation are discussed.

LIPOXYGENASE/H2O2-CATALYZED OXIDATION OF DIHYDROXYINDOLES : SYNTHESIS OF MELANIN PIGMENTS AND STUDY OF THEIR ANTIOXIDANT PROPERTIES

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which are important intermediates in melanogenesis, can be converted into the corresponding melanin pigments by the action of the lipoxygenase/H2O2 system. Kinetic and HPLC analyses indicate that both DHI and DHICA are good substrates for this enzymatic system. Enzyme activity on both substrates was measured in comparison with peroxidase and tyrosinase; the oxidizing behaviour of lipoxygenase is more similar to that of peroxidase rather than that of tyrosinase. The antioxidant properties of DHI- and DHICA-melanins have been investigated in comparison with other kinds of melanins. DHICA-melanin shows a more pronounced antioxidant effect than that of DHI-melanin and this behaviour can be ascribed to the different structure and solubility of the two pigments. The mixed polymer synthesized from DHI and DHICA is the most effective one. Some implications about the possible explanation of the above mentioned behaviour are discussed.

Melanins From Tetrahydroisoquinolines: Spectroscopic Characteristics, Scavenging Activity and Redox Transfer Properties

Tetrahydroisoquinolines (TIQs) are endogenous compounds deriving from the nonenzymatic Pictet-Spengler condensation of catecholamines (CA) with aldehydes. TIQs have been extensively studied in the last years not only because they have been found in the brain of postmortem specimens of Parkinsons patients, but also because they are able to induce parkinsonian symptoms if injected in animals. In the present article we demonstrate that TIQs bearing a catecholic moiety (tetrahydropapaveroline, salsolinol, laudanosoline, and apomorphine) are easily oxidized in the presence of hydrogen peroxide by various enzymes--i.e., peroxidase (POD), lipoxygenase (LOX), and xanthine oxidase (XO)--into the corresponding TIQ-melanins. The kinetic parameters of the above-mentioned reactions and some spectroscopic characteristics of the synthetized pigments are reported. In particular, UV-VIS and EPR spectra emerge as very similar to those exhibited by dopa-melanin. Furthermore, TIQ-melanins appear to be similar to dopa-melanin regarding some specific physico-chemical properties: NADH-oxidizing properties, oxy-radicals scavenging activity, and ability to form soluble mixed polymers with melanins from opioid peptides.

Cytotoxicity of dopamine-derived tetrahydroisoquinolines on melanoma cells.

Tetrahydroisoquinolines (TIQs) are endogenous alkaloid compounds detected in urine, central nervous system and some peripheral tissues in Mammalia. No data are at present available on TIQ levels in skin, although in vitro biochemical evidences indicate that they may undergo auto-oxidation with production of reactive oxygen species or may be enzymatically converted into melanin pigments. The effect of two catechol-bearing TIQs, salsolinol (SAL) and tetrahydropapaveroline (THP), on the viability of melanotic or amelanotic melanoma cell lines was investigated. Both SAL and THP were well tolerated up to roughly 30 microM and became overtly toxic at higher concentrations, with SAL being better tolerated than THP. Intracellular activity of some antioxidant enzymes, tyrosinase and alpha-ketoglutarate dehydrogenase was also evaluated to assess the cell response to oxidative and metabolic challenge of TIQs treatment. Catalase and superoxide dismutase pre-treatment only partially prevented TIQs toxicity while a complete protection was obtained with N-acetylcysteine and GSH. TIQs were able to provoke upregulation of the scavenging enzymes catalase and DT-diaphorase and to determine a decrease of the alpha-ketoglutarate dehydrogenase activity. SAL and THP enhanced tyrosinase activity and melanin production, suggesting that they were indeed tyrosinase substrates leading to melanin formation. The results support the evidence that TIQs were toxic toward melanoma cells, leading to their death by necrosis. TIQs toxicity was likely due to increased oxidative stress by generation of reactive oxygen species and oxidative metabolites. Our study represents an intent to furnish an additional contribution for the comprehension of catechol cytotoxicity.

Melanins production from enkephalins by tyrosinase.

Leu-enkephalin and Met-enkephalin are oxidized in vitro by mushroom and sepia tyrosinase giving rise to synthetic melanins whose production is dependent on incubation time and on enzyme concentration. The Enk-melanins formed are acid-insoluble brownish or reddish pigments showing a continuous absorbance in the visible region when dissolved in basic solution. The presence of the amino acid chain makes them fully soluble in pH 7.4 0.05 M phosphate buffer and methanol.

Production of melanin pigments by cytochrome c/H2O2 system

In the presence of hydrogen peroxide cytochrome c can perform the oxidation of catecholamines and their S-cysteinyl-derivatives yielding melanins as final products. The initial reaction rate is linearly dependent on cytochrome c and H2O2 concentration; the reaction follows the Michaelis and Menten kinetics both for H2O2 and hydrogen donors. Sulfhydryl compounds inhibit the formation of the pigment. The reported data indicate that a heme-containing protein belonging to the mitochondrial chain can accelerate the oxidation of catecholamines to eumelanins.

The peroxidase-catalyzed oxidation of enkephalins.

In vitro experiments are reported showing that Leu-enkephalin and Metenkephalin, in the presence of hydrogen peroxide, can be oxidized by horseradish peroxidase. The products formed are strongly fluorescent and characterized by absorption peaks with maxima at 290 nm and 315 nm. The effects of substrate and enzyme concentrations on the oxidation rate of enkephalins are described. Amino acid analysis of the hydrolysates from peroxidase-treated enkephalins provides evidence for the presence of dityrosine. The data suggest that the oxidation leads to the production of enkephalin dimers with a linkage between the N-terminal tyrosine residues. Data are also obtained indicating that enkephalins function as hydrogen donors for mammalian peroxidases.

Spectroscopic features of native and bleached opio-melanins

Opioid peptides can be converted by tyrosinase into melanin-like compounds, in which the peptide moiety is retained. Such pigments, named opio-melanins, exhibit a characteristic absorption spectrum with a maximum at about 330 nm and a different solubility behaviour with respect to dopa-melanin, being completely soluble in hydrophylic solvents at neutral and basic pH. Opio-melanins precipitate in aqueous solutions below pH 5.0, and show apparent pKa values of 3.1, 3.6 and 4.4 for Tyr-Gly-melanin, Tyr-Gly-Gly-melanin and leuenk-melanin, respectively. The concomitant oxidation of dopa and opioid peptides by tyrosinase produces mixed polymers, showing the distinctive absorption peak at 330 nm. In the dark, in the pH range 5.5-7.0 the pigments are completely stable, whereas H2O2 addition provokes a slight degradation. At higher pH values or under simulated solar illumination with or without hydrogen peroxide, bleaching occurs more rapidly than in dopa-melanin. Upon photoirradiation the absorption spectrum of opio-melanins undergoes a marked variation, the peak at 330 nm being replaced by a broad shoulder in the range 280-350 nm. The absorption spectra of native and bleached pigments and the extent of opio-melanins degradation by bleaching agents, confirm the hypothesis that the different initial structure of the precursors accounts for a final diverse polymeric architecture of these pigments with respect to dopa-melanin.

Some biochemical properties of melanins from opioid peptides

Opioid peptides are converted by mushroom tyrosinase into melanin-like compounds retaining the peptide moiety (opio-melanins). Opio-melanins, owing to the presence of the linked aminoacids and in contrast with DOPA-melanin, are soluble compounds. The enkephalin-generated melanins are cleaved by carboxypeptidase A and pronase whereas aminopeptidase M cannot remove aminoacids from the pigment. Enkephalins, as well as other opioid peptides, (alpha-endorphin, kyotorphin, esorphins) if oxidized in presence of DOPA and tyrosinase are readily incorporated into DOPA-melanin. The resulting mixed-melanins (opio-melanin + DOPA-melanin) can be solubilized in hydrophilic solvents. Melanin from leu-enkephalin exhibits paramagnetism as evidenced by an EPR spectrum identical to that of DOPA-melanin, but unlike the latter pigment, it does not appear to oxidize NADH, probably for the presence of the peptide moiety that exerts a hampering effect on the oxidizing capacity.

Interaction of enkephalin derivatives with reactive oxygen species.

The oxidation of opioid peptides by tyrosinase in the presence of an excess of a thiol gives rise to cysteinyldopa derivatives. The major products arising from the reaction between Leu-enkephalin and cysteine are represented by 5-S-cysteinyldopaenkephalin (5-CDenk) and 2-S-cysteinyldopaenkephalin (2-CDenk). The interaction of 5-CDenk and 2-CDenk with reactive oxygen species (ROS) has been studied. These compounds are able to scavenge superoxide anion, hydroxyl and peroxyl radicals as well as to reduce the lipid peroxidation rate induced by ABAP. The scavenging activities in all instances are dose-dependent. In some cases CDenks are more active than compounds recognized as strong radical scavengers, such as Trolox and mannitol. As a result of the action of the Fenton system, the CDenks (as well as the Enks) are oxidized into pigmented derivatives. The possible implications of the interaction of CDenks and Enks with ROS on melanization process in Parkinsons disease are discussed.