Giovanna Misuraca

Mechanism of inhibition of melanogenesis by hydroquinone

Hydroquinone (HQ) is one of the most effective inhibitors of melanogenesis in vitro and in vivo, and is widely used for the treatment of melanosis and other hyperpigmentary disorders. In an attempt to get some insight into the molecular mechanism of the depigmenting action, which is still very poorly understood, we have investigated the effect of HQ on the tyrosinase catalysed conversion of tyrosine to melanin. Incubation of 0.5 mM tyrosine with 0.07 U/ml tyrosinase in phosphate buffer at pH 6.8 in the presence of 0.5 mM HQ led to no detectable melanin formation, due to the preferential oxidation of HQ with respect to tyrosine (HPLC evidence). Kinetic investigations showed that HQ is a poorer substrate of tyrosinase than tyrosine; yet, it may be effectively oxidised in the presence of tyrosine owing to the generation of catalytic amounts of dopa acting as cofactor of tyrosinase. Product analysis of HQ oxidation with tyrosinase in the presence of dopa showed the predominant formation in the early stages of hydroxybenzoquinone (HBQ), arising from enzymic hydroxylation and subsequent oxidation of HQ, along with lower amounts of benzoquinone (BQ). These results suggest that the depigmenting activity of HQ may partly be related to the ability of the compound to act as an alternate substrate of tyrosinase, thereby competing for tyrosine oxidation in active melanocytes.

Effect of metal ions on the rearrangement of dopachrome

In vitro experiments are reported showing that a number of transition metal ions exert a profound influence on both the kinetics and chemical course of the rearrangement of dopachrome, a key step in the biosynthesis of melanins. HPLC analysis shows that Cu2+, Ni2+ and Co2+ are particularly effective in inducing the non-decarboxylative rearrangement of dopachrome at physiological pH values, leading mainly to 5,6-dihydroxyindole-2-carboxylic acid, whereas in the absence of metal ions the reaction proceeds with concomitant loss of carbon dioxide to give almost exclusively 5,6-dihydroxyindole. Kinetic experiments provide evidence that the rate of the metal-promoted rearrangement is first order with respect to both aminochrome and metal concentration and decreases in the presence of increasing concentrations of EDTA, consistent with a mechanism involving a direct 1:1 dopachrome-metal ion interaction in the transition state. When considered in the light of the known metal accumulation in pigmented tissues, the results of this study provide a new entry into the regulatory mechanisms involved in the biosynthesis of melanins.

Structural modifications in biosynthetic melanins induced by metal ions

A number of transition metal ions with a wide distribution in biological systems, e.g., Cu2+, Co2+ and Zn2+, are shown to affect markedly the chemical properties of melanins formed by the tyrosinase-catalysed oxidation of dopa. Acid decarboxylation and permanganate degradation provide evidence that melanins prepared in the presence of metal ions contain a high content of carboxyl groups arising from the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DICA) into the pigment polymer. Naturally occurring melanins from cephalopod ink and B16 mouse melanoma were found to be much more similar to melanins prepared in the presence of metal ions than to standard melanins prepared in the absence of metal ions. These results suggest that the presence of carboxylated indole units in natural melanins is probably due to the intervention in the biochemical pathway of metal ions which, as recently shown, catalyse the formation of DICA versus 5,6-dihydroxyindole in the rearrangement of dopachrome.

Comparative action of dopachrome tautomerase and metal ions on the rearrangement of dopachrome

A vis-a-vis comparison between the effects of dopachrome tautomerase (DCT) and metal ions, e.g., cupric ions, on the kinetics and mode of rearrangement of dopachrome has been carried out under appropriate analytical conditions. The enzyme-promoted reaction is highly stereospecific for L-dopachrome, is unaffected by metal chelators and has an optimal pH around 6.8. By contrast, the kinetics of dopachrome rearrangement catalysed by cupric ions are not dependent on the stereochemistry of the substrate, are affected by EDTA and are not influenced by the pH of the medium in the range between 5-7.5. Both cupric ions and DCT catalyse the rearrangement of dopachrome to give 5,6-dihydroxyindole-2-carboxylic acid (DICA) rather than 5,6-dihydroxyindole (DI). However, at comparable activity, the ratio of formation DICA/DI is significantly higher in the enzyme-catalysed than in the metal-catalysed reaction. These results provide an improved background to look into the mode of action of DCT and metal ions, enabling a clear cut differentiation between the effects of the two factors when both are present in biological extracts.

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.

Isolation and structure of a new sulphur-containing aminoacid from sea urchin eggs

Abstract Unfertilized sea urchin eggs (Paracentrotus lividus) contain, in addition to glutathione, a new low molecular weight thiol, 1-methyl-5-thiol- l -histidine ( 1 ) which was isolated and characterized as the disulphide 2 by spectral and chemical evidence.

Inhibitory effect of melanin precursors on arachidonic acid peroxidation

A possible role of melanin precursors in lipid peroxidation was investigated using the lipoxygenase catalysed oxygenation of arachidonic acid (AA) as a model system. Polarographical monitoring of oxygen consumption showed that, among the metabolites examined, 5,6-dihydroxyindole (DHI) was the most active in inhibiting AA oxygenation catalysed by 15-lipoxygenase. The inhibition was found to be concentration-dependent with an IC50 value of 15 microM. Similar effects were observed in the case of the 5-lipoxygenase promoted reaction. Periodical HPLC analysis of the oxidation mixture showed that, in the presence of DHI, the rate of substrate consumption is markedly reduced. The inhibitory potency was significantly increased either by preincubation of DHI with the enzyme or by increasing the time of residence of the indole in aerated buffer solutions prior to contact with the enzyme. Addition of catalase to the incubation mixture resulted in a partial removal of DHI inhibition. From these and other experiments, an inhibition mechanism is proposed which involves inactivation of the enzyme by reactive species, especially hydrogen peroxide, arising from DHI autoxidation.

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.

Activation of mammalian tyrosinase by ferrous ions

Kinetic experiments are reported showing that mammalian tyrosinase from B16 mouse melanoma is significantly activated by catalytic amounts of ferrous ions. Monitoring of tyrosine oxidation by both dopachrome formation and oxygen consumption showed that ferrous ions at micromolar concentrations induce a marked enzymatic activity with 0.01 U/ml of highly purified tyrosinase, whereas no detectable reaction occurs in the absence of metal over a sufficiently prolonged period of time. The extent of the activating effect, which is specific for the reduced form of iron, is proportional to the concentration of the added metal with a typical saturation profile, no further effect being observed beyond a threshold value. Changing the buffer system from phosphate to hepes or tris results in a marked decrease of the Fe2(+)-induced activation. Scavengers of active oxygen species, such as superoxide dismutase, catalase, formate and mannitol have no detectable effect on the tyrosinase activity. These results are accounted for in terms of an activation mechanism involving reduction of the cupric ions at the active site of the resting enzyme.

5,6-Dihydroxyindole-2-carboxylic acid, a diffusible melanin precursor, is a potent stimulator of lipopolysaccharide-induced production of nitric oxide by J774 macrophages

Pre-incubation of J774 murine macrophages with 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible intermediate in the biosynthesis of eumelanins, leads to a marked increase in the levels of nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced NO-synthase (iNOS). The effect varies with DHICA concentration being maximum at a concentration of 1 x 10(-6)M, and is suppressed by the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). No stimulation is observed when macrophages are exposed to DHICA after activation with LPS, indicating that the indole does not affect the catalytic activity of iNOS. These results point to a hitherto unrecognized role of DHICA as a chemical messenger mediating interaction between active melanocytes and macrophages in epidermal inflammatory and immune responses.