Inactivation of tyrosinase photoinduced by pterin.

Tyrosinase catalyzes in mammals the first and rate-limiting step in the biosynthesis of the melanin, the main pigment of the skin. Pterins, heterocyclic compounds able to photoinduce oxidation of DNA and its components, accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder in which the protection against UV radiation fails due to the lack of melanin. Aqueous solutions of tyrosinase were exposed to UV-A irradiation (350 nm) in the presence of pterin, the parent compound of oxidized pterins, under different experimental conditions. The enzyme activity in the irradiated solutions was determined by spectrophotometry and HPLC. In this work, we present data that demonstrate unequivocally that the enzyme is photoinactivated by pterin. The mechanism of the photosensitized process involves an electron transfer from tyrosinase to the triplet excited state of pterin, formed after UV-A excitation of pterin. The biological implications of the results are discussed.

Inhibitory effect of the water-soluble polymer-wrapped derivative of fullerene on UVA-induced melanogenesis via downregulation of tyrosinase expression in human melanocytes and skin tissues.

The C60-fullerene derivatives are expected, as novel and potent anti-oxidants, to more effectively protect skin cells against oxidative stress. UVA-induced oxidative stress is considered to promote melanogenesis and serious skin damage. The effect of any fullerene derivatives on UVA-induced melanogenesis is still unknown. Here, we evaluated effects of a water-soluble polyvinylpyrrolidone (PVP)-wrapped fullerene derivative (named "Radical Radical Sponge" because of its anti-oxidant ability) on melanogenesis, which was promoted by UVA-irradiation to human melanocytes and skin tissues. Radical Sponge markedly scavenged UVA-induced reactive oxygen species (ROS) inside human melanocytes as shown by fluorometry using the redox indicator CDCFH-DA. After treatment with Radical Sponge or other agents, human melanocytes and skin tissues were irradiated by UVA. Then, cellular melanin content, tyrosinase activity and the ultrastructural change of skin melanosomes were examined. Radical Sponge showed to significantly inhibit UVA-promoted melanogenesis in normal human epidermis melanocytes (NHEM) and human melanoma HMV-II cells within a non-cytotoxicity dose range. As compared with two whitening agents, arbutin and L-ascorbic acid, Radical Sponge demonstrated the stronger anti-melanogenic potential according to spectrophotometric quantification for extracted melanin. In human skin cultures also, UVA-promoted melanin contents were repressed by Radical Sponge according to Fontana-Masson stain, suggesting its ability to repress UVA-induced tanning. Transmission electron microscopic ultrastructural images also proved that UVA-increased melanosomes in human skin tissue were obviously reduced by Radical Sponge. The UVA-enhanced tyrosinase enzymatic activity in NHEM melanocytes was inhibited by Radical Sponge more markedly than by arbutin and L-ascorbic acid. The UVA-enhanced tyrosinase protein expression, together with cell-size fatness and dendrite-formation, was also inhibited more markedly by Radical Sponge according to immunostain and flow cytometry using anti-tyrosinase antibody. Thus the depigmentating action of Radical Sponge might be due to its down-regulating effect on the tyrosinase expression, which is initiated by UVA-caused ROS generation.

Activation of the cyclic AMP pathway by alpha-melanotropin mediates the response of human melanocytes to ultraviolet B radiation.

A hallmark of sun exposure is increased melanin synthesis by cutaneous melanocytes which protects against photodamage and photocarcinogenesis. Irradiation of human keratinocytes or melanocytes with ultraviolet (UV) rays stimulates the synthesis and release of alpha-melanotropin (alpha-MSH) and adrenocorticotropic hormone (ACTH), which induce cyclic AMP (cAMP) formation and increase the proliferation and melanogenesis of human melanocytes. We report that stimulation of cAMP formation is obligatory for the melanogenic response of cultured normal human melanocytes to UVB radiation. In the absence of cAMP inducers, UVB radiation inhibited, rather than stimulated, melanogenesis. UVB radiation (28 mJ/cm2) arrested melanocytes in the G1 phase of the cell cycle, and concomitant treatment with 0.1 microM alpha-MSH enhanced their proliferation but did not increase the surviving fraction. Irradiation with UVB, with or without alpha-MSH, caused prolonged expression of p53 and p21(waf-1, cip-1), maintained pRB in a hypophosphorylated state, and reduced the expression of Bcl2. However, alpha-MSH allowed UVB-irradiated melanocytes to enter S phase, suggesting that alpha-MSH acts as a mitogen rather than a survival factor, and that overexpression of p53 is mainly a signal for cell death. Our results underscore the importance of the cAMP pathway and its physiological inducers in mediating the response of human melanocytes to UV radiation.

Enzymatic-induced upconversion photoinduced electron transfer for sensing tyrosine in human serum.

This paper reports a novel nanosensor for tyrosine based on photoinduced electron-transfer (PET) between NaYF4:Yb, Tm upconversion nanoparticles (UCNPs) and melanin-like polymers. Melanin-like films were obtained from catalytic oxidation of tyrosine by tyrosinase, and deposited on the surface of UCNPs, and then quenched the fluorescence of UCNPs. Under the optimized conditions, the fluorescence quenching of UCNPs showed a good linear response to tyrosine concentration in the range of 0.8-100 µΜ with a detection limit of 1.1 µΜ. Meanwhile, it showed good sensitivity, stability and has been successfully applied to the detection of tyrosine in human serum.

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.

The effect of ultraviolet radiation and melanocyte-stimulating hormone on tyrosinase activity in epidermal melanocytes of the mouse.

The role of melanocyte stimulating hormone (MSH) as a mediator of the melanogenic response to ultraviolet radiation (UVR) was examined in C57 BL/6 mice. While exposure to UVR (250-300 nm) for 7, 14 and 27 days increased tyrosinase activity in epidermal melanocytes of the ear MSH had no effect and failed to alter the response to UVR. Plasma alpha-MSH concentrations were unchanged following UVR. Theophylline, a phosphodiesterase inhibitor, also had no effect on epidermal tyrosinase activity in non-irradiated and UV irradiated mice. Prostaglandin E2 and arachidonic acid were also ineffective in non-irradiated and UV irradiated mice and indomethacin, an inhibitor of prostaglandin synthesis, failed to increase epidermal tyrosinase activity after UVR. On the other hand, 12-0-tetradecanoyl phorbol 13 acetate, an activator of protein kinase C, increased epidermal tyrosinase activity in non-irradiated mice and also enhanced the effect of UVR.

Antigenicity, catalytic activity and conformation of Agaricus bisporus tyrosinase: interaction of conformation-directed antibodies with the native and irradiated enzyme.

The antiferromagnetically spin-coupled Cu2+ pair present in the active center of tyrosinase was found to be indispensable for its catalytic function. However, the metal ion did not contribute to the conformational integrity or antigenicity of the enzyme molecule. Irradiation of tyrosinase with 254 nm light resulted in dose dependent, essentially irreversible losses of its catalytic and antigenic functions. The apparent first order rate constants for the two processes were 17.6 X 10(-2) min-1 and 28.1 X 10(-2) min-1, respectively. The approximately 1.6-fold difference between the two rate constants suggests that the sites of antigenic determinants in tyrosinase are distinguishable from the enzymic active site by their higher photosensitivity. Kinetic analysis of the data as to photoinactivation, and the UV induced losses of antigenicity and structural integrity revealed that UV radiation disrupts the short-range noncovalent interactions occurring within the enzyme molecule. The disruption of the noncovalent interactions results in partial unfolding of the tyrosinase structure which in turn leads to the progressive loss of its catalytic activity and antigenicity. The anti-tyrosinase antibodies raised in rabbits were found to be directed against the native conformation of the enzyme. It is speculated that these antibodies might be useful in exploring the tyrosinase conformation and in studying the effects of various factors on the enzyme surface and molecular structure.

Activity regulation of tyrosinase by using photoisomerizable inhibitors.

Enzymatic activity of tyrosinase was controlled on the basis of cis-trans photoisomerization of inhibitors, 4-azobenzene carboxylic acid (ACA) and 4,4'-azobenzene dicarboxylic acid (ADCA). In the case of ACA, the cis form inhibited tyrosinase-catalyzed oxidation of L-tyrosine more strongly than the trans form. On the contrary, in the case of ADCA, the cis form was less inhibitory. The oxidation rate was controlled reversibly by light irradiation in the course of the reaction. In the presence of ACA, UV light irradiation, which isomerized trans to cis form, decelerated the tyrosine oxidation, while visible light irradiation, which isomerized backward, accelerated the reaction. In contrast, in the presence of ADCA, UV light accelerated and visible light decelerated the reaction.

Comparative studies on the correlation between pyrimidine dimer formation and tyrosinase activity in cloudman S91 melanoma cells after ultraviolet-irradiation.

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.

Fluoroquinolones as chemical tools to define a strategy for photogenotoxicity in vitro assessment.

Today's lifestyle is often associated with frequent exposure to sunlight, but some xenobiotics used in drugs, cosmetics or food chemicals can produce adverse biological effects when irradiated. In particular, they can increase the risk of photogenotoxicity already due to UV radiation itself. There is thus a need to design appropriate approaches in order to obtain relevant data at the molecular and cellular level in this field. For ethical and practical reasons, in vitro models can be very convenient at least for first evaluation tests. Here, we propose a strategy based on complementary experiments to study the photogenotoxic potential of a compound. The fluoroquinolones BAYy3118 and lomefloxacin were used as standards to demonstrate the performance of each test: photoinduced interaction with supercoiled circular DNA, photomutagenicity in the yeast Saccharomyces cerevisae, induction of DNA photodamage in cultured human skin cells as revealed by comet assay, and finally induction of specific phototoxic stress responses such as p53 activation or melanogenesis stimulation. Such a strategy should help to ensure the safety of products likely to undergo environmental sunlight exposure.

Biosynthesis of L-DOPA by Aspergillus oryzae.

The present investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae. There was a marked difference between the mycelial morphology and pellet type of parental and UV-irradiated mutant culture. The mutant strain of A. oryzae UV-6 exhibited pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinase is an intracellular enzyme. The mutant was found to yield 3.72 fold higher production of L-DOPA than the parental strain. The mutant strain is stable and D-glc-resistant. The comparison of kinetic parameters was also done which showed the greater ability of the mutant to yield L-DOPA (i.e., Yp/x 40.00+/-0.01 d mg/mg with parent and 182.86+/-0.02a mg/mg in case of mutant). When cultures grown for various incubation periods, were monitored for Qp, Qs and q(p), there was significant enhancement (p < 0.0025-0.005) in these variables by the mutant strain of A. oryzae UV-7 over GCB-6 on all the rates. L-DOPA (3,4-dihydroxy phenyl L-alanine) is a drug of choice in the treatment of Parkinson's disease and myocardium following neurogenic injury.

The expression of melanogenic proteins in Korean skin after ultraviolet irradiation.

For proper melanin production, several specific enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase are required. Their expressions are increased after exposure to UVB. However, it is not known how long tyrosinase and TRP-1 activities continue after UV irradiation in vivo. The purpose of this study is to measure the changes in expressions of tyrosinase, TRP1, and MITF after exposure to UV on skin in a Korean population. We established an immunohistochemical staining protocol for specimens which were obtained from UV-irradiated skin in five healthy Korean males on the 2nd, 5th, 7th, 28th, and 56th days after UV irradiation. Tyrosinase, TRP-1, and MITF expressions increased until 7 days after UV irradiation and then dropped to the basal constitutive level 4 and 8 weeks later. Interestingly, tyrosinase increased prior to TRP-1. This study reveals the time-sequence of melanin-synthesized enzymes and provides important information for the clinical evaluation of the effectiveness of whitening agents.

Tryptophan as an endogenous photosensitizer to elicit harmful effects of ultraviolet B.

Owing to stratospheric ozone depletion (SOD) the natural flux of ultraviolet B (UVB) radiation (290-320 nm) is likely to increase on the earth surface. In our efforts to identify endogenous chromophores which may absorb significantly in the UVB range and subsequently induce phototoxic reactions, we have observed that tryptophan (Trp) was quite photoreactive under UVB. It enhanced considerably the oxygen-dependent photooxidation of tyrosine (Tyr) to dopachrome, a precursor of melanin. Our data suggest that UVB-sensitized Trp produces singlet oxygen (1O2) and superoxide radicals (O2-.), and these reactive forms of oxygen may contribute to membrane-, cytoplasm- and DNA-damaging effects. In the event of an increasing SOD level, other UVB chromophores may also exhibit similar phototoxic properties to lead to a definitive imbalance between cell life, injury and death.

[The effect of low doses of a synthetic analog of a quinoid radiotoxin on the viability of normal and gamma-irradiated animals]. / Vliianie malykh doz sinteticheskogo analoga khinoidnogo radiotoksina na zhiznesposobnost' normal'nykh i gamma-obluchennykh zhivotnykh.

The method for a production of synthetic quinoid radiotoxins in vitro has been developed and described. Synthetic quinoid radiotoxins like quinoid radiotoxins (qRT) which are being produced from irradiated tissues of the organisms have demonstrated high toxicity at relatively high qRT concentrations. However, when synthetic qRT is introduced into the organisms in ultra-small concentrations, one can observe the opposite action: the resistance of the organism increases and a number of essential functions are activated. Quinoid radiotoxins are assumed to take part in regulatory processes responsible for radiation hormesis.

Effect of gamma radiation on Phenoloxidase pathway, antioxidant defense mechanism in Helicoverpa armigera (Lepidoptera: Noctuidae) and its implication in inherited sterility towards pest suppression.

PURPOSE: To investigate age-correlated radiosensitivity in highly radioresistant lepidopteran pest, Helicoverpa armigera, upon exposure to ionizing radiation and to examine the irradiation impact on stress-molecular responses in F1 (first-filial) progeny of irradiated (100 Gy) male moths in relation to its reproductive behavior. MATERIALS AND METHODS: Efficacy of sub-lethal gamma radiation was evaluated on two markedly apart ontogenic stages, neonates and adult moths. Differential growth, reproductive behavior and stress-indicating molecular responses were examined upto F1 progeny of sub-sterilized moths. Free-radical scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and Phenoloxidase cascade enzymes, pro-phenoloxidase (PPO), its activating enzyme (PPAE) were studied in irradiated and irradiated plus microbial challenge regimen (dual-stress) by Real-time RT-PCR (reverse-transcription-polymerase-chain-reaction). RESULTS: An inverse correlation of radiosensitivity with developmental age of insect was observed. F1 sterility was higher than parent sterility. F1 progeny exhibited protraction in development and decreased survival upon irradiation. Sex ratio in F1 progeny was skewed towards males. PPO, PPAE, SOD and CAT transcripts were downregulated upon neonate irradiation resulting in enhanced vulnerability of larvae to incidental microbial challenge. These transcripts were upregulated in F1 progeny of sub-sterilized male moths (100 Gy) upon dual-stress. CONCLUSIONS: Irradiation impact on stress-indicating molecular responses in F1 progeny is correlated with its reproductive performance. These observations will permit defining regimen having pragmatic viability of 'F1 sterility technique' for pest suppression. Gamma dose of 100 Gy would ensure balance between induced sterility of males and their field competitiveness. These parameters would facilitate integration of biocontrol strategy with parabiological 'Sterile Insect Release Technique'.

Influence of ultrasound to the activity of tyrosinase.

The influence of ultrasound to the activity of tyrosinase was investigated. According to the analyses of ultraviolet-visible spectrometry and Fourier transform infrared spectroscopy, both monophenolase and diphenolase activities of tyrosinase treated by ultrasound were higher than that of control, with the decrease of lag time and the increase of activity. No oxytyrosinase was observed and ß-sheet conformation was predominant in the tyrosinase under ultrasound treatment. Moreover, with the observation of atomic force microscopy, the large molecular groups of tyrosinase were broken into small ones under the treatment of ultrasound. The present result suggested the activity of tyrosinase could be activated under the tested ultrasound treatment, mainly due to the increased likelihood of the combination of substrate and enzyme, or the possible change and exposure of the active site in tyrosinase.