UVB-induced epidermal pigmentation in mice eyes with no contact lens wear and non-UVB blocking and UVB blocking contact lens wear.

PURPOSE: Irradiation by ultraviolet (UV) B is known to increase the number of Dopa-positive melanocytes in the skin. This study examines the effectiveness of a contact lens for the defense of UVB eye irradiation-induced pigmentation. METHODS: A 2.5 kJ/m(2) dose of UVB radiation was delivered by a sunlamp to the eye of C57BL/6j male mice, and changes in the expression of Dopa-positive melanocytes in the epidermis and the plasma level of alpha-melanocyte-stimulating hormone (α-MSH) was analyzed. RESULTS: The degree of change in the Dopa-positive melanocytes expression was reduced by UVB blocking contact lens using mice given UVB irradiation to the eye. The plasma level of α-MSH increased in the C57BL/6j mice after irradiation to the eye, but there was no increase in the UVB blocking contact lens mice given UVB irradiation to the eye. Both the increase of the expression of Dopa-positive melanocytes and the plasma level of α-MSH were strongly suppressed by an alignment fitting UVB blocking contact lens and only a slightly suspended UVB blocking contact lens. In addition, these changes were successfully inhibited by a UVB blocking contact lens but not by a non-UVB blocking contact lens with a similar absorbance. CONCLUSION: These observations suggest that the UVB blocking contact lens inhibits the pigmentation of the epidermis in mice by suppressing of the α-MSH.

Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation.

The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that gamma-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604-612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions.

Photoconductivity of synthetic dopa-melanin polymer.

The photoconductivity effect in synthetic dopa-melanin polymer with relation to the charge hopping conduction has been investigated. Measurements of the rise and decay of photocurrents upon visible radiation (400-800 nm) and at temperatures of 293-326 K allowed the determination of the major trapping levels as follows: 56, 35 and 26 kJ/mol. Spectral response of the steady-state photocurrent in the range 367-1100 nm showed significant departures from the absorption spectrum of melanin. The high concentration of traps or recombination centers can explain the long time-constants calculated from the photocurrent rise and decay curves. The results obtained can support the postulated earlier polarons and hopping model of conductivity in synthetic dopa-melanin.

Effects of ultraviolet irradiation on melanogenesis from tyrosine, Dopa and dopamine: a matrix-assisted laser desorption/ionization mass spectrometric study.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry experiments were applied to study the influence of ultraviolet (UV) irradiation in melanogenesis. Samples were prepared starting from three different precursors, tyrosine, Dopa and dopamine, in the presence or absence of tyrosinase, the enzyme responsible for the synthesis of melanin. Enzymatic reactions were carried out for 10, 30, 60 and 120 min under UV irradiation at 365 nm, and aliquots were then immediately ultrafiltered and lyophilized. Samples obtained by irradiation of tyrosine solution revealed the formation of 5,6-dihydroxyindole (DHI) oligomers up to pentamers at 120 min; the reaction kinetics were markedly enhanced in the presence of tyrosinase. In the case of Dopa, UV irradiation favored melanogenesis only in the presence of the enzyme; in this case, many reaction pathways were activated, originating various oligomeric species of Dopa, DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Conversely, when dopamine was used as tyrosinase substrate under UV light, mechanisms of melanogenesis different from those generated by simple enzymatic reaction without irradiation were not activated, as the same oligomeric species were present.

The fluorescent oxidation products of dihydroxyphenylalanine and its esters.

Dihydroxyphenylalanine (DOPA), its methyl ester (DOPAM) and the N-acetylated derivative of the ester (DOPAMNA) are found to undergo rapid oxidation in air-saturated alkaline solution. Some of the products of oxidation exhibit fluorescent emission in the 300-500 nm spectral range and their excitation-emission spectra have been determined in acidic and alkaline aqueous solutions. The spectral distributions and positions of the maxima depend on the pH of the solution. Excitation-emission maxima associated with the protonated phenolic form of the compounds occur at shorter wavelengths than those of the conjugate base. At some pH values the phenolic forms of these molecules are excited and undergo rapid deprotonation in the excited state; as a consequence, emission is observed from the phenolate anion. The fluorescence excitation-emission spectrum of an authentic sample of 3,4-dihydroxycinnamic (caffeic) acid has also been determined and features of the fluorescence spectra of the principal oxidation products are consistent with the presence of 3,4-hydroxycinnamoyl compounds in solutions of oxidized DOPAM and DOPAMNA.

Regulatory effects of heat on normal human melanocyte growth and melanogenesis: comparative study with UVB.

Although energy-rich ultraviolet B (UVB) is considered to be primarily responsible for most of the effects associated with solar radiation, small energy recorded as heat appears to contribute to the biologic effects of solar radiation on the skin. We compared the effects of heat and UVB on normal human melanocyte functions. In monolayer culture the following was found. (i) Heat-treated melanocytes showed an increased dendricity and exhibited a larger cell body compared with nontreated melanocytes. (ii) After multiple treatments with UVB (20 mJ per cm2, 312 nm) or heat (42 degrees C for 1 h) for 3 d, melanocytes had a lower survival than nontreated melanocytes, but they resumed proliferation within 6 d in the same manner as seen in control. (iii) The expression levels of cell cycle regulators, p53 and p21 proteins, were increased after multiple treatments with UVB or heat. (iv) The tyrosinase (dopa-oxidase) activity per cell was increased after the multiple treatments with UVB or heat. (v) The number of dopa-positive melanocytes in coculture with keratinocytes in epithelial sheets was greatly increased by UVB or heat treatments. (vi) Similarly, the increased number of tyrosinase-related protein 1 positive melanocytes was seen in skin equivalents after UVB (100 mJ per cm2) or heat (42 degrees C for 1 h) treatments for 7 d. These results suggest that heat shares significant biologic activities with UVB in melanocyte functions. These results could be considered as one of the protective or adaptive responses of the skin pigmentary system to the environment.

Radiation-induced formation of 3,4-dihydroxyphenylalanine in tyrosine-containing peptides and proteins as a function of X-irradiation dose.

Radiation-induced formation of 3,4-dihydroxyphenylalanine (DOPA) in Tyr and Tyr-containing peptides and proteins was investigated as a function of X-irradiation dose. Irradiated Tyr (0-30 Gy) and the acid hydrolysates of irradiated peptide and protein (0-240 Gy) were conjugated with dansyl chloride. The dansylated amino acids were analyzed by reversed-phase HPLC using fluorescence detection. Formation of DOPA, determined by integrated peak area, increased with dose. Analysis of the major product from irradiated tripeptide Tyr-Gly-Gly detected Gly and DOPA (2:1). Extension of the model study to irradiated BSA and RNase A showed correlation of DOPA formation with Tyr modification up to 120 Gy. Higher dose induced further transformation of DOPA. The fluorescence signal of dansylated DOPA was linear from 1.5 nmol to 0.5 pmol (correlation coefficient of 0.999, n = 3). The detection limit allows the detection of 1 molecule of DOPA/300 molecules of BSA in 5 micrograms of dansylated hydrolysate. Most standard amino acid analysis techniques are limited to detect normal residues of protein. Protein-bound DOPA has been suggested to have a role in the replenishment of reduced transition metal ion involved free-radical-generating system in vivo. Sensitive analysis of protein-bound DOPA will be useful to study amplification of the radical-damaging event.

Formation of protein-bound 3,4-dihydroxyphenylalanine in collagen types I and IV exposed to ultraviolet light.

Collagen was exposed to an ultraviolet (UV) lamp that emitted predominantly in the UVB range. The cross-linking of collagen type I and type IV by UV irradiation was observed. Amino acid analyses revealed that Tyr residues in both collagen types I and IV were decreased by irradiation. In collagen type IV, losses of His and Met residues were also observed. These losses of collagen type IV may be due to the degradation of Trp, which exists in collagen type IV and decreased drastically during UV irradiation. To clarify the mechanism of Tyr modification in both types of collagen, the degradation products of Tyr were analyzed. Dityrosine, which is a dimer of the Tyr residue, could not be detected in the acid hydrolysates of UV-irradiated collagen. However, 3,4-dihydroxyphenylalanine, DOPA, was detected in the hydrolysates using HPLC with an electrochemical detector. The amounts of DOPA in the acid hydrolysates of collagen exposed to UV light for 24 h were approximately 350 pmol/mg protein (collagen type IV) and 80 pmol/mg protein (collagen type I). The DOPA formed may partially contribute to photoaging of the skin.

Free radicals in melanin-cationic porphyrin complexes in the dark and under light irradiation.

Electron Paramagnetic Resonance (EPR) spectroscopy was employed in the study of the interaction between L-3,4-dihydroxyphenylalanine (L-Dopa) melanin and the cationic porphyrins meso-tetrakis(1-methylpyridinium-4yl)-porphyrin (TMPyP), meso-tetrakis-(1-benzylpyridinium-4-yl)-porphyrin (TBzPyP), and their respectives complexes ZnTMPyP and ZnTBzPyP. By monitoring signal intensities and progressive microwave power saturation it was shown that the interaction increases the equilibrium concentration of free radicals in L-Dopa melanin in the dark. The extent of increase is dependent on the presence of molecular oxygen and on the type of porphyrin. Not all interacting sites available for complexation in L-Dopa melanin are involved in the formation of free radicals. It was also observed that the interaction with porphyrins promotes an increase in the number of photoinduced free radicals in L-Dopa melanin during illumination with visible light.

[Photo-induced destruction of DOPA-melanin]. / Fotoindutsirovannaia destruktsiia DOFA-melanina.

Studies of the effect of illumination at different wavelengths revealed a high stability of DOPA-melanin to the visible light. Contrariwise, UV-visible light of high intensity upon prolonged (many hours) illumination caused a significant bleaching of the diluted aqueous solution of the pigment. The absorption spectrum of DOPA-melanin in the UV- and IR-regions did not differ from the initial one; however, the illuminated pigment acquired an ability for fluorescence. Stepwise gel chromatography on Toyopearl-55 and Toyopearl-40 columns resulted in three fractions of photobleached DOPA-melanin which differed in their molecular masses, absorption spectra (UV, IR) and fluorescence. It was concluded that the photoinduced bleaching of DOPA-melanin was mainly due to the pigment depolymerization. A possible mechanism of melanin photodestruction is discussed.

[Effect of melanin on the free-radical states of gamma-irradiated proteins and lipids]. / Vliianie melanina na svobodno-radikal'nye sostoianiia gamma-obluchennykh belkov i lipidov.

It was shown that the concentration of paramagnetic centres of dihydroxyphenylalanine-melanin increased after gamma-irradiation (60Co) both at room temperature (an irreversible increase) and at 77 K (a reversible increase). The accumulation of paramagnetic centres in gamma-irradiated albumin at room temperature was found to slow down appreciably in the presence of melanin. This effect is thought to be associated with the antiradical activity of the pigment.

Free radicals from eumelanins: quantum yields and wavelength dependence.

Formation of light-induced free radicals from natural eumelanin (from bovine eyes) and synthetic melanin (from oxidation of 3,4-dihydroxyphenylalanine) has been studied by electron spin resonance spectroscopy. Action spectra measured for natural melanins are very similar to that found for synthetic melanin, and are unaffected by the removal of associated protein. A comparison of action spectra with optical absorbance spectra shows that the former has a more marked wavelength dependence, suggesting that the chromophore that is most active in free-radical production is not the major melanin chromophore that absorbs visible light. Measurements of quantum yields for free-radical production have been made over a wavelength range from 600 to 230 nm. The efficiency of radical production from natural eumelanin is about three times greater than from the synthetic material. Although production of the melanin radicals detected is independent of oxygen, some correlation with oxygen consumption is evident; quantum yields for radical production are approximately three times those for oxygen consumption obtained under similar conditions. Possible reasons for this are discussed.

Radical production during tyrosinase reaction, dopa-melanin formation, and photoirradiation of dopa-melanin.

It has been suggested that superoxide anion (O2-) may be produced during eumelanin formation and during the photoirradiation of eumelanin , but no direct evidence for this has yet been reported (although O2- production during photoirradiation of pheomelanin has been shown). In this report, the production of O2- was investigated during the formation and photoirradiation of dopa-melanin, a synthetic eumelanin . It was found that cytochrome c was reduced during the tyrosinase reaction and dopa-melanin formation in vitro; this reduction could not be inhibited by superoxide dismutase (SOD). When dopa-melanin was irradiated by UV radiation or by visible light, high nitroblue tetrazolium (NBT) reduction was observed; this reduction was proportional to the light energy and the amount of dopa-melanin. NBT reduction by visible light could be slightly inhibited by SOD, but a 12% decrease of NBT reduction by UV radiation could be shown with the addition of SOD. These observations indicate that some radicals were produced during the tyrosinase reaction and dopa-melanin formation. Further, when dopa-melanin was irradiated, radicals were also produced, some of which were thought to consist of O2-, but others were unknown.