Effects of 4-tertiary butylphenol on the tyrosinase activity in human melanocytes.

Vitiligo is a common dermatological disorder characterized by the development of complete pigment loss from focal lesions that tends to increase in size over time. The etiology of vitiligo, resulting in the disappearance of functional melanocytes from involved skin, is not clearly understood. As a consequence, no satisfactory therapy has been developed. A subtype of vitiligo, termed 'occupational' or 'contact' vitiligo, is increased in individuals who are exposed to materials containing phenolic derivatives, such as 4-tertiary butylphenol (4-TBP). Phenolic derivatives are structurally similar to tyrosine, the initial substrate of tyrosinase in the biochemical synthesis of melanin. Therefore, it has been proposed that phenolic derivatives compete with tyrosine for hydroxylation by tyrosinase and interfere with the completion of melanin synthesis and/or generate cytotoxic intermediates. Our results demonstrated that 4-TBP competitively inhibited both tyrosine hydroxylase and dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase, i.e., the first two catalytic steps in the biochemical conversion of tyrosine to melanin in cultured human melanocytes. This inhibition occurred at concentrations that did not influence the viability of melanocytes. The tyrosinase activity inhibited by 4-TBP was recovered after removing the treatment. 4-TBP did not affect the function of other enzymes, such as succinate-tetrazolium reductase, acid phosphatase and sulfatase. Since depigmentation occurred without a cytotoxic response after exposure of melanocytes to low concentration of 4-TBP, it is unclear whether the interaction between 4-TBP and tyrosinase leads to the destruction of the melanocytes in 'contact/occupational' vitiligo.     

Effects of 4-Tertiary Butylphenol on the Tyrosinase Activity in Human Melanocytes

Vitiligo is a common dermatological disorder characterized by the development of complete pigment loss from focal lesions that tends to increase in size over time. The etiology of vitiligo, resulting in the disappearance of functional melanocytes from involved skin, is not clearly understood. As a consequence, no satisfactory therapy has been developed. A subtype of vitiligo, termed‘occupational’or‘contact’vitiligo, is increased in individuals who are exposed to materials containing phenolic derivatives, such as 4-tertiary butylphenol (4-TBP). Phenolic derivatives are structurally similar to tyrosine, the initial substrate of tyrosinase in the biochemical synthesis of melanin. Therefore, it has been proposed that phenolic derivatives compete with tyrosine for hydroxyla-tion by tyrosinase and interfere with the completion of melanin synthesis and/or generate cytotoxic intermediates. Our results demonstrated that 4-TBP competitively inhibited both tyrosine hydroxylase and dihydrox-yphenylalanine (DOPA) oxidase activities of tyrosinase, i.e., the first two catalytic steps in the biochemical conversion of tyrosine to melanin in cultured human melanocytes. This inhibition occurred at concentrations that did not influence the viability of melanocytes. The tyrosinase activity inhibited by 4-TBP was recovered after removing the treatment. 4-TBP did not affect the function of other enzymes, such as succinate-tetra-zolium reductase, acid phosphatase and sulfatase. Since depigmentation occurred without a cytotoxic response after exposure of melanocytes to low concentration of 4-TBP, it is unclear whether the interaction between 4-TBP and tyrosinase leads to the destruction of the melanocytes in‘contact/occupational’vitiligo.     

Generation of albino Xenopus tropicalis using zinc‐finger nucleases

To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc‐finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN‐induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.     

On the etiology of contact/occupational vitiligo

Vitiligo is an acquired depigmentary disorder of the skin that results from the selective destruction of melanocytes, generally during the second decade of life and affecting approximately 1% of the population worldwide. Loss of cutaneous pigment appears to render the skin susceptible to premature aging and cancer. In addition this disease can be socially devastating for afflicted individuals. The etiology of vitiligo is poorly understood. The present dogma suggests that genetic factors render the melanocyte fragile thus predisposing individuals to developing vitiligo. When subjected to instigating factors, these susceptible, fragile melanocytes undergo apoptosis. Autoimmune factors then perpetuate the removal of the melanocyte component from the skin. In the majority of cases the instigating factors are not known (idiopathic vitiligo), however a small sub-set of individuals develop contact/occupational vitiligo following exposure to particular chemicals. Many of these chemicals have been implicated in both contact/occupational vitiligo and chemical leukoderma. Both conditions present with well-defined, depigmented skin lesions that develop following exposure. Only in the case of vitiligo does the depigmentation spread beyond the areas of contact, probably via an immune-mediated mechanism. The largest class of chemicals known to trigger contact/occupational vitiligo is the phenolic/catecholic derivatives. Many have been demonstrated to be preferentially cytotoxic to melanocytes, with high-dose exposure resulting in the initiation of apoptosis. Phenolic/catecholic derivatives are structurally similar to the melanin precursor tyrosine, and therefore tyrosinase was originally implicated as a mediator of cytotoxicity. However, our data suggests that tyrosinase-related protein-1, rather than tyrosinase, facilitates toxicity, possibly by catalytic conversion of the compounds, which results in the generation of radical oxygen species. The ensuing oxidative stress then triggers activation of cellular free radical scavenging pathways to prevent cell death. Genetic inability of melanocytes to tolerate and/or respond to the oxidative stress may underlie the etiology of contact/occupational vitiligo.     

Control of ketogenesis from amino acids: III. In vitro and in vivo studies on ketone body formation, lipogenesis and oxidation of tyrosine by rats

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferase (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rate limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate.For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1-¹⁴ C]tyrosine. Then their respiratory ¹⁴CO₂ and the incorporation of ¹⁴C into total lipids of liver were measured. The amounts of radioactivity in CO₂ and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U-¹⁴C] acetate the radioactivities in CO₂ and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studies gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

Association of the Acetylcholine-phosphatidyl Inositol Effect with a “Receptor” Proteolipid from Cerebral Cortex

IN spite of continuing research on the treatment of Parkinson's disease^1–3, no drug with clear advantages over L-dopa (the L-isomer of 3,4-dihydroxyphenylalanine) has yet been found. The problems of supply of L-dopa and reduction of its side effects⁴ are therefore still of interest. L-Dopa can be obtained from L-tyrosine by a hydroxylation reaction catalysed by the enzyme tyrosinase (EC 1.10.3.1). Such a reaction using immobilized tyrosinase could form the basis of an industrial method because L-tyrosine is cheap. Alternatively, in view of the fact that L-tyrosine is present in human serum, immobilized tyrosinase suitably implanted in the blood stream might be used to synthesize L-dopa in situ. We have been studying tyrosinase immobilized by covalent attachment to a cellulosic support. In the absence of a readily available mammalian tyrosinase or tyrosine hydroxylase which would be more suitable for clinical purposes we have used a polyphenol oxidase with tyrosinase activity, obtainable from mushrooms.     

Truffle Melanogenesis: Correlation With Reproductive Differentiation and Ascocarp Ripening

The present work uses histochemical techniques to investigate the correlation between reproductive differentiation and age of truffles (Tuber aestivum and Tuber melanosporum) with melanin synthesis. The dopa oxidase and tyrosine hydroxylase activities of tyrosinase have been localized within the ascocarp and melanin localization was performed by the Schmorl's reaction. A true tyrosinase is present in truffles, able to oxidize both 1-tyrosine and 1-dopa. The tyrosinase activity is on in the young ascocarps (in the peridium, hypothecium, and fertile veins) and off in the ripe ones, thus it appears correlated with the age and differentiation of the sporogenic hyphae that arise from the hypothecium; a similar correlation has been previously described in Neurospora crassa which is an ascomycete as well as the truffles.     

Potential of Cassia alata leaf extract in inducing differentiation and migration of mouse melanoblasts

In vitiligo, active melanocytes in the epidermis are not present, whereas melanoblast cells in the outer root sheath of hair follicles are not affected. The existence of these inactive melanocytes provides a source for repigmentation of vitiligo. To evaluate the potential of herbal candidates in treatment of vitiligo, we studied the effect of Cassia alata leaf extract on the differentiation, proliferation and migration of melanoblast cells. Melanin content increased in melb-a melanoblast cells in response to this herb extract in a dose-dependent manner compared to control cells. In addition, it induced tyrosinase activity and altered melb-a cell morphology. A transwell migration assay showed the potential of this herbal candidate to induce direct migration of treated cells. To our knowledge, this is the first report on activity of this kind in Cassia alata. The findings of the present study are significant in the direction of developing safer strategies for vitiligo treatment.     

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.     

Action of the phosphonic analogue of tyrosine and of other tyrosine derivatives on rat liver tyrosine aminotransferase

Summary Tyrosine transamination has been investigatedin vitro with a preparation of rat liver tyrosine aminotransferase in the presence of several structural derivatives of the substrate, including the phosphonic analogue. The transamination by tyrosine aminotransferase (TAT) needs the presence in the substrate molecule of free amino and carboxylic groups, a three-carbon aliphatic chain, a para-phenolic hydroxylic function and al-configuration. Some tyrosine analogues can markedly disturb the Tyr-TAT association: the chief structural modifications are (i) the removal of the free amine function in a compound still possessing a para-hydroxylic and a carboxylic group, (ii) the change of the carboxylic function by another acidic group, especially a phosphonic one, (iii) a disubstitution in positions 3 and 5. In every situation, the presence of a parahydroxylic group is compulsory to observe an inhibitory effect.     

Cycloheximide causes increased accumulation of translatable mRNA for tyrosine aminotransferase and tryptophan oxygenase in livers of cortisol-treated rats.

Messenger RNA activities for two cortisol-inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated. Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five-fold and two-fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still several fold higher than in control animals. Application of alpha-amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs. The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.     

A review of recent advances on the regulation of pigmentation in the human epidermis.

It has been recognised that the active transport of L-phenylalanine and its autocrine turnover to L-tyrosine via phenylalanine hydroxylase in the cytosol of epidermal melanocytes provides the majority of the L-tyrosine pool for melanogenesis. In this context, it has been shown that the cofactor 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) is produced de novo, recycled and regulated in both epidermal melanocytes and keratinocytes to control tyrosine hydroxylase, phenylalanine hydroxylase and tyrosinase activity. Inhibition of the enzymes by excessive 6BH4 levels is reversible with alpha-MSH by specific complex formation between 6BH4 and the hormone. This direct mechanism of alpha-MSH is supported by the presence of the entire POMC processing system in the melanosome indicating a receptor independent control of eumelanogenesis. Finally, the role of tyrosinase, TRP-1 and TRP2 is discussed in association with oxidative stress specifically related to hydrogen peroxide. These recent findings are based on detailed investigations of the depigmentation disorder vitiligo and Hermansky-Pudlák syndrome.     

The antibody response against MART‐1 differs in patients with melanoma‐associated leucoderma and vitiligo

Patients with melanoma may develop skin depigmentation spontaneously or following therapy, referred to as melanoma‐associated leucoderma (MAL). As clinical presentation of MAL may precede primary/metastatic melanoma detection, recognition of MAL is important to prevent its misdiagnosis as vitiligo and the subsequent application of immunosuppressive treatment. To reveal the immunity involved in MAL development, we investigated the presence of antibody and T‐cell immune responses directed against the melanocyte‐differentiation‐antigens MART‐1 (Melan‐A), tyrosinase and gp100 in patients with MAL, as compared to patients with vitiligo. Autoantibodies to gp100 and tyrosinase were commonly found in both diseases. Interestingly, MART‐1 antibodies were only present in patients with MAL. Melanocyte antigen‐specific T cells were found in all patients, with relatively more specific T cells in patients with active vitiligo. Although MAL and vitiligo may appear clinically similar, our results indicate that the humoral immune responses against MART‐1 differ between these diseases, which can help to differentiate MAL from vitiligo.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Vitiligo puzzle: the pieces fall in place

Over the years, the role of biochemical, immunological, genetic, and other biological aspects in the pathogenesis of vitiligo has been studied. So far, no convincing model describing the interplay of these contributing factors has been formulated. Based on existing research, we propose that vitiligo has a multi-factorial etiology, characterized by multiple steps, but always involving an increase of external or internal phenol/catechol concentration, serving as a preferred surrogate substrate of tyrosinase, competing with its physiological substrate tyrosine. The conversion of these substrates into reactive quinones is reinforced by a disturbed redox balance (increasing hydrogen peroxide). Such reactive quinones can be covalently bound to the catalytic centre of tyrosinase (haptenation). This could give rise to a new antigen, carried by Langerhans cells to the regional lymph node, stimulating the proliferation of cytotoxic T cells. However, the activation of such cytotoxic cells is only a first step in skin melanocyte killing, which also depends on a shift in the balance between immune defence and tolerance, e.g. resulting from a decrease in properly functioning T-regulatory cells. With this new model, based on a synthesis of several of the existing theories, in mind, the external and internal factors involved in the etiopathogenesis of vitiligo are reviewed, against the background of reported clinical data, experimental studies and existing and potential new therapies. A similar complex mechanism may also lead to some other autoimmune diseases.     

Natural,semisynthetic and synthetic tyrosinase inhibitors

Tyrosinase plays a pivotal role in the synthesis of melanin pigment synthesis on skin utilizing tyrosine as a substrate. Melanin is responsible for the protection against harmful ultraviolet irradiation, which can cause significant pathological conditions, such as skin cancers. However, it can also create esthetic problems when accumulated as hyperpigmented spots. Various skin-whitening ingredients which inhibit tyrosinase activity have been identified. Some of them, especially ones with natural product origins, possess phenolic moiety and have been employed in cosmetic products. Semi-synthetic and synthetic inhibitors have also been developed under inspiration of the natural inhibitors yet some of which have no phenolic groups. In this review, tyrosinase inhibitors with natural, semi-synthetic and synthetic origins are listed up with their structures, activities and characteristics. Further, a recent report on the adverse effect of a natural melanin synthesis inhibitor which was included in skin-whitening cosmetics is also briefly discussed.     

Transient phase calibration of tyrosinase-based carbaryl biosensor

The inhibition of tyrosinase, used as a selective compound in amperometric biosensor for the detection of carbaryl and the possibilities of calibration of carbaryl biosensor have been studied. The kinetic analysis of biosensor data was based on the application of the biosensor dynamic model, allowing a quick calculation of independent on each other kinetic and steady-state parameters. It was found that carbaryl acts as an inhibiting substrate of tyrosinase and at low concentrations accelerates the tyrosinase-catalyzed oxidation of tyrosine by dissolved oxygen. The reaction mechanism is analogous to that usually considered for uncompetitive inhibition and the plot of kinetic parameter as a function of carbaryl concentration has a flat asymmetric maximum. Consequently, the kinetic parameter alone is not sufficient for the calibration of carbaryl biosensor and simultaneous application of other carbaryl-dependent parameters, like steady-state parameter, is essential.     

Redefining the skin's pigmentary system with a novel tyrosinase assay

In mammalian skin, melanin is produced by melanocytes and transferred to epithelial cells, with the epithelial cells thought to receive pigment only and not generate it. Melanin formation requires the enzyme tyrosinase, which catalyzes multiple reactions in the melanin biosynthetic pathway. Here, we reassess cutaneous melanogenesis using tyramide-based tyrosinase assay (TTA), a simple test for tyrosinase activity in situ. In the TTA procedure, tyrosinase reacts with biotinyl tyramide, causing the substrate to deposit near the enzyme. These biotinylated deposits are then visualized with streptavidin conjugated to a fluorescent dye. In the skin and eye, TTA was highly specific for tyrosinase and served as a sensitive indicator of pigment cell distribution and status. In clinical skin samples, the assay detected pigment cell defects, such as melanocytic nevi and vitiligo, providing confirmation of medical diagnoses. In murine skin, TTA identified a new tyrosinase-positive cell type--the medullary cells of the hair--providing the first example of cutaneous epithelial cells with a melanogenic activity. Presumably, the epithelial tyrosinase originates in melanocytes and is acquired by medullary cells during pigment transfer. As tyrosinase by itself can generate pigment from tyrosine, it is likely that medullary cells produce melanin de novo. Thus, we propose that melanocytes convert medullary cells into pigment cells by transfer of the melanogenic apparatus, an unusual mechanism of differentiation that expands the skin's pigmentary system.