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The inhibitory effect of glycolic and lactic acid on melanin synthesis in melanoma cells
Abstract
Alpha-hydroxy acids (AHAs) such as glycolic acid (GA) and lactic acid (LA) have been reported to be effective in treating pigmentary lesions such as melasma, solar lentigines, and postinflammatory hyperpigmentation. The mechanism of this effect might be due to epidermal remodeling and accelerated desquamation, which would result in quick pigment dispersion. However, the direct effect of AHAs on melanin synthesis has not yet been well studied. To elucidate such a direct effect of AHAs on melanogenesis, we performed melanin assays, growth curve determinations, Northern and Western blotting for melanogenic proteins [tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2], and tyrosinase and, 4-dihydroxyphenylalaninechrome tautomerase enzyme activity assays using mouse B16 and human melanoma cells. GA or LA (at doses of 300 or 500 microg/ml) inhibited melanin formation in similar dose-dependent manner, without affecting cell growth. Although the mRNA and protein expression or molecular size of tyrosinase, TRP-1 and TRP-2 were not affected, tyrosinase activity was inhibited. To see whether GA and/or LA directly inhibit tyrosinase catalytic function, the effect of GA and LA on human tyrosinase purified from the melanosome-rich large granule fraction of human melanoma cells was performed. GA or LA were shown to inhibit tyrosinase enzyme activity directly, but this effect was not due to the acidity of GA or LA, because adjusting the pH to 5.6 (the pH of GA and LA at concentrations of 2500 microg/ml), did not affect tyrosinase activity. Taken together, these results show that GA and LA suppress melanin formation by directly inhibiting tyrosinase activity, an effect independent of their acidic nature. GA and LA might work on pigmentary lesions not only by accelerating the turnover of the epidermis but also by directly inhibiting melanin formation in melanocytes.
... Rozjaśniający mechanizm jego działania wiąże się, m.in. ze wzmożonym obrotem naskórkowym, a także modulacją syntezy melaniny poprzez hamowanie aktywności tyrozynazy [43]. Kwas glikolowy może być stosowany pojedynczo lub w połączeniu z innymi związkami rozjaśniającymi (np. ...
... Kwas glikolowy może być stosowany pojedynczo lub w połączeniu z innymi związkami rozjaśniającymi (np. hydrochinonem, kwasem mlekowym) [43,44]. Kwas glikolowy jest substancją naturalną dla naszego organizmu. ...
... Właściwości nawilżające wynikają z możliwości wbudowywania się kwasu w struktury lipidowej bariery naskórkowej. Dłuższe stosowanie ma bardzo korzystny wpływ na skórę właściwą, poprawia się jej grubość do 25% [43,44]. -kwas mlekowy, który występuje w kwaśnym mleku jest to hydroksykwas o mechanizmie działania podobnym do kwasu glikolowego. ...
W artykule wyjaśniono zagadnienia związane z występowaniem i zastosowaniem hydroksykwasów organicznych w fitokosmetykach rewitalizujących i zabiegach kosmetycznych. Dokonano charakterystyki budowy skóry oraz właściwości fizycznych i chemicznych alfa-(AHA) i beta-hydroksykwasów (BHA) (kwas glikolowy, kwas mlekowy, kwas cytrynowy, kwas jabłkowy, kwas winowy, kwas salicylowy). Scharakteryzowano sposoby oznaczania hydroksykwasów, a także przedstawiono regulacje prawne dotyczące stosowania hydroksykwasów w kosmetykach. Przedstawiono wskazania i przeciwwskazania do zastosowania zabiegów z kwasami owocowymi. Opisano czynniki warunkujące skuteczność działania biologicznego alfa-hydroksykwasów (AHA).
... Study done by Usuki et al. to identify the direct effect of GLY on melanogenesis using B16 and human melanoma cells found that glycolic acid can act as a competitive inhibitor to TYR; inhibits melanin formation without affecting the cell growth [20]. In most of the studies, combination with other active ingredient such as hydroquinone, AZ and KO give a more promising to reduce pigmentation and melasma [21,22]. ...
... Lim and Tham reported that GLY can be used to treat hyperpigmentation [22]. Our results speculate GLY can be TYR inhibitor which is in line with previous studies [20,30,31]. Determination of melanin synthesis in MNT-1 and B16-F10 showed that melanin content was significantly decreased (P ≤ 0.001) with GLY treatment. ...
... This inhibition effect of GLY could be attributed to the inhibition of TYR activity which was significantly decreased (P ≤ 0.001) with GLY treatment. Usuki and colleague reported that GLY inhibit TYR enzyme activity directly on human melanoma cells and the effect is independent of their acidic nature [20]. In our study, it is likely that GLY could be suppressing melanin formation by directly inhibiting TYR activity too as our results are in line with Usuki and colleague findings [20]. ...
Treatment for abnormal melanin accumulation is available with controversial results, thus aim of the present work was to determine azelaic acid (AZ) and glycolic acid (GLY) cytotoxicity and inhibiting melanogenesis efficacy. Safety concentration and efficacy in reducing the melanin content were access through MTT assay, Fontana-Masson staining, melanin content assay and tyrosinase (TYR) assay. Kojic acid (KO) was used as the positive control throughout the assays. AZ and GLY exhibited low cytotoxicity at 100 µg/mL and do not show suppression towards cell viability as cells were 100% viable. 100 µg/mL and 50 µg/mL AZ and GLY show comparable depigment effect to the positive control in Fontana-Masson staining and the depigmentation effect was dose dependent. 100 µg/mL AZ and 100 µg/mL GLY shows significant (P ≤ 0.05) reduction of melanin content in both melanoma cells compared to untreated group. TYR activity in both cells also shows significant lower (P ≤ 0.001) after the treatment with 100 µg/mL AZ and 100 µg/mL GLY compared to untreated group. AZ and GLY were suggested to suppress melanin formation by directly inhibiting TYR activity and act as TYR inhibitor. The depigmenting effect was further confirmed with down-regulating of TYR, TYRP1 and TYRP2. The possible depigmentation mechanisms of AZ and GLY could be due to inhibition of melanin synthesis and suppression of tyrosinase-related genes expression including which affecting TYR activity. The depigmenting potential of AZ and GLY can be evaluate further in-vivo model.
... Glucose is converted into the cellular metabolite lactate, which is lactic acid in solution and which has been reported to be effective in treating pigmentary lesions [14,15]. Therefore, we hypothesized that glucose is converted into lactic acid in melanocytes and that increased levels of lactic acid inhibit melanogenesis through tyrosinase inactivation. ...
... As expected, glucose significantly up-regulated the lactic acid content in B16 cells-cultured media ( Figure 5A). In addition, because lactic acid is known to directly inhibit tyrosinase activity [15], we evaluated if lactic acid suppressed mushroom tyrosinase activity. As shown in Figure 5B, lactic acid dramatically inhibited mushroom tyrosinase activity, unlike glucose. ...
... Lactic acid is an alpha hydroxy acid (AHA); these acids are used extensively in cosmetic formulations as superficial peeling agents [25]. In addition, lactic acid suppresses melanin formation by directly inhibiting tyrosinase activity, an effect independent of its acidic nature, which means that lactic acid's effects on pigmentary lesions are due not only to acceleration of epidermal cell turnover, but also direct inhibition of melanin formation in melanocytes [15]. Thus, we reasoned that glucose may exert its anti-melanogenic effect via lactic acid production, because glucose can be converted to lactic acid. ...
Sugars are ubiquitous in organisms and well-known cosmetic ingredients for moisturizing skin with minimal side-effects. Glucose, a simple sugar used as an energy source by living cells, is often used in skin care products. Several reports have demonstrated that sugar and sugar-related compounds have anti-melanogenic effects on melanocytes. However, the underlying molecular mechanism by which glucose inhibits melanin synthesis is unknown, even though glucose is used as a whitening as well as moisturizing ingredient in cosmetics. Herein, we found that glucose significantly reduced the melanin content of α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells and darkly pigmented normal human melanocytes with no signs of cytotoxicity. Furthermore, topical treatment of glucose clearly demonstrated its whitening efficacy through photography, Fontana-Masson (F&M) staining, and multi-photon microscopy in a pigmented 3D human skin model, MelanoDerm. However, glucose did not alter the gene expression or protein levels of major melanogenic proteins in melanocytes. While glucose potently decreased intracellular tyrosinase activity in melanocytes, it did not reduce mushroom tyrosinase activity in a cell-free experimental system. However, glucose was metabolized into lactic acid, which can powerfully suppress tyrosinase activity. Thus, we concluded that glucose indirectly inhibits tyrosinase activity through conversion into lactic acid, explaining its anti-melanogenic effects in melanocytes.
... PLGA NPs provide several advantageous properties for the delivery of therapeutic agents, with the most important being the induction of controlled release over several days, protection against pre-mature degradation by hydrolytic enzymes and proteases, reduction in the cytotoxicity to mammalian cells, the possibility for surface functionalization to target the site of infection, or co-delivery with a secondary therapeutic biomolecule (e.g., antibiofilm, antibiotics, or anti-inflammatory) [7]. PLGA degrades to alpha-hydroxyacids (AHAs), namely lactic acid and glycolic acid, which activate several skin rejuvenating functions by epidermal remodeling and desquamation, which stimulate wound healing [8,9]. Of note, AHAs are a major part of cosmetics because they promote the production of hyaluronic acid, collagen, and elastin to reverse the signs of ageing on the skin [10]. ...
... In addition, SAAP-148 NPs induced a dose-dependent inhibition of planktonic AMR S. aureus and A. baumannii after 4 h, and they maintained this level of inhibition after 24 h (Supplementary Material Figures S1 and S2). Together, this data 9 concentrations for SAAP-148, in solution, was 25.6 µM regarding both bacterial biofilms for up to 24 h, with no improvement in killing activity at longer exposure times. As before, blank NPs did not exhibit any antibiofilm activity at any of the time-points tested. ...
Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of the peptide. The aim of this study was to investigate the physical and functional characteristics of SAAP-148 NPs and to compare the selectivity index of the formulated peptide with that of the peptide in solution. SAAP-148 NPs displayed favorable physiochemical properties [size = 94.1 ± 23 nm, polydispersity index (PDI) = 0.08 ± 0.1, surface charge = 1.65 ± 0.1 mV, and encapsulation efficiency (EE) = 86.7 ± 0.3%] and sustained release of peptide for up to 21 days in PBS at 37 °C. The antibacterial and cytotoxicity studies showed that the selectivity index for SAAP-148 NPs was drastically increased, by 10-fold, regarding AMR Staphylococcus aureus and 20-fold regarding AMR Acinetobacter baumannii after 4 h. Interestingly, the antibiofilm activity of SAAP-148 NPs against AMR S. aureus and A. baumannii gradually increased overtime, suggesting a dose–effect relationship based on the peptide’s in vitro release profile. Using 3D human skin equivalents (HSEs), dual drug SAAP-148 NPs and the novel antibiotic halicin NPs provided a stronger antibacterial response against planktonic and cell-associated bacteria than SAAP-148 NPs but not halicin NPs after 24 h. Confocal laser scanning microscopy revealed the presence of SAAP-148 NPs on the top layers of the skin models in close proximity to AMR S. aureus at 24 h. Overall, SAAP-148 NPs present a promising yet challenging approach for further development as treatment against bacterial infections.
... The underlying mechanism of the anti-melanogenesis activity of malic acid is still not clear. Some AHAs, such as glycolic acid and lactic acid, have been reported to suppress melanin formation by directly inhibiting tyrosinase activity, which might be possible in malic acid [21,22]. Although Ch1M1 is not suitable for the extraction of M. alba stilbenoids, it could have potential to extract other non-stilbenoid ...
... The underlying mechanism of the anti-melanogenesis activity of malic acid is still not clear. Some AHAs, such as glycolic acid and lactic acid, have been reported to suppress melanin formation by directly inhibiting tyrosinase activity, which might be possible in malic acid [21,22]. Although Ch1M1 is not suitable for the extraction of M. alba stilbenoids, it could have potential to extract other non-stilbenoid compounds with anti-melanogenesis properties. ...
The consumer and cosmetic industries have recently placed a greater emphasis on ecofriendly solvents for botanical extraction, including natural deep eutectic solvents (NADES). In this study, NADES were prepared for Morus alba callus extraction. The efficiency of extraction from the NADES and methanol was investigated by comparison of the stilbenoids yield and anti-melanogenesis activity. Prior to testing the irritability of a suitable NADES on the reconstructed human epidermis (RhE), the effect of the selected NADES on stilbenoids stability was determined. The results showed that the highest yields of stilbenoids were obtained from choline chloride-glycerol mixtures (Ch1G2) and methanol extracts, with no significant difference in yields (5.06 ± 0.05 and 6.32 ± 0.40 mg/g callus dry weight, respectively). The NADES extracts of M. alba callus showed comparable anti-melanogenesis activity compared to methanol. In term of stability, stilbenoids in Ch1G2 remained stable after six months of storage at 4 °C except resveratrol. Furthermore, Ch1G2 had no irritation effect on RhE. Thus, based on the findings of this study, Ch1G2 is an intriguing green solvent alternative for the extraction of M. alba callus and may be advantageous for the preparation of skin-lightening cosmetics.
... Here the same authors compared this index with a 0-4 categorical severity scale, before and following glycolic acid (GA) peeling. GA acts as a keratolytic [9] and a depigmenting agent [10]. Patients with pseudo-AN were included in this study rather than patients with AN, to eliminate a possible underlying condition that could influence the clinical course of the skin lesions following GA peel. ...
... The increased number of melanosomes cause hyperpigmentation [16], which also improved following GA peeling. GA inhibits the catalytic activity of tyrosinase, thus decreasing melanin synthesis [10] as well as stimulating interleukin-1α production from keratinocytes [17], which in turn suppresses the action of tyrosinase [18]. ...
Background Pseudo-acanthosis nigricans (pseudo-AN) is a common clinical scenario, which can be challenging to treat. Evaluating the efficacy of different therapeutic conditions requires a clinical outcome measure.
Objective To compare the novel score acanthosis nigricans area and severity index (ANASI) with a conventional severity scale.
Patients and methods A total of 20 patients with neck pseudo-AN were enrolled in the study. Each side of the neck was randomly subjected to receiving biweekly three sessions of either glycolic acid peel 70% or saline (serving as a control). Evaluation was done at baseline and 1 week after the last session by ANASI score versus a simple categorical 0–4 severity scale. For evaluating interrater and intrarater reliability and validity, six raters were asked to evaluate each side of the neck before and after treatment by both the ANASI and severity scale.
Results Glycolic acid caused a 43% and a 13% improvement according to the ANASI scoring system and the severity scale, respectively, with a highly statistical significance between both. ANASI score showed good reliability across and between raters and was considered valid when compared with the severity scale.
Conclusion ANASI is an easy-to-apply and reproducible scoring system that lessens the bias in subjective evaluation; thus, it offers accurate quantification of pseudo-AN. ANASI score is a reliable and valid measure for AN skin lesions.
... 5 Glycolic acid (GA), an alpha-hydroxy acid peel, is commonly used in various concentrations (20%-70%) either as a sole agent or in combination with other agents in the treatment of melasma. 2 It exerts whitening effect through loss of cohesion, desquamation, alteration in the epidermis and inhibition of melanogenesis. 6 Microdermabrasion (MDA) is a common procedure of superficial resurfacing used to treat various skin problems including post acne scars, photodamaged skin, striae distensae and melisma. 7,8 Also, it could be beneficial in enhancing transdermal drug delivery through selectively removing the stratum corneum that is considered the main barrier for percutaneous delivery. ...
... obtained a significant decline in the MASI score following six sessions of GA35% peels. 20 The authors reasoned these obtained significant results due to the whitening effect of GA which is achieved though enhancing desquamation, remodeling the epidermis, dispersing basal melanin and directly inhibiting melanogenesis, 6,20 additionally, most of the included melasma was epidermal which is superficial and responds better than deeper types. 1 In the current study, we obtained significantly superior results on the MDA/GA70% side than the GA70% side. To our knowledge, this is the first study to use such innovative combination of MDA with GA for treating melasma. ...
Background
Melasma is a disfiguring dermatologic condition and its treatment is still considered a challenge.
Objectives
To evaluate the efficacy and safety of microdermabrasion (MDA) combined with glycolic acid 70% (GA70%) peel versus GA70% alone in treating melasma in dark-skinned patients.
Patients and methods
This study included 30 female patients (skin type IV and V) with melasma. After cleansing the face, 3 passes of MDA were done on one side of the face. Then, GA70% was applied to the whole face in 1-2 uniform passes. Melasma area and severity index (MASI), modified MASI and hemi-MASI scores were used to assess the outcome.
Results
A significant decline of the mean MASI, mMASI and both hemi-MASI scores following treatment (p value= 0.000 for each). Furthermore, the hemi-MASI score on the MDA/GA70% treated side showed significantly greater decrease than the hemi-MASI score on GA70% treated side (p value=0.041).
Conclusion
MDA enhanced the improvement of GA70% peel effectively and safely.
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... After the addition of 10 mM L-DOPA, the mixture was further incubated at 37 °C for 20-30 min and read at 490 nm (SpectraMax, Molecular Devices, San Jose, CA, USA). A known tyrosinase inhibitor, glycolic acid 0.04%, served as the positive control [20]. The negative control did not receive inhibitors. ...
Skin exfoliators, specifically alpha and beta hydroxy acids, have been shown to improve overall skin health and the clinical signs of aging. A micropeeling cream was developed with hydroxy acids and a brown algae extract and the efficacy and tolerability were evaluated in two parts. In the first part of the pre-clinical investigation, the micropeeling cream and the placebo control were evaluated by ELISA, immunostaining, qPCR and an activity assay using ex vivo and in vitro models. In the second part of the clinical efficacy study, 36 female subjects were enrolled for bioinstrumental measurements, visual imaging and clinical evaluation for 28 days. Fifty percent of subjects had sensitive skin. The ex-vivo study showed an increase in loricrin, superoxide dismutase 2, and extracellular matrix expression, without stimulating inflammatory biomarkers. The dermatologist observed a significant enhancement in all the parameters evaluated at day 28, and radiance, homogeneity, and roughness were significantly better after the first cream application. The homogeneity, desquamation and pore diameter showed significant improvement at day 7. The cream improved markers associated with skin aging and protection ex vivo. It was well tolerated, even on sensitive skin, and provided a significant improvement of fine lines, skin texture, and overall skin characteristics.
... The mouse melanoma cell line B16F10 has the advantages of multiple passages, rapid development, relatively simple culture conditions, high malignancy, and good tumorigenicity. As such, B16F10 cells are widely used as the effective cells for cell evaluation of whitening activity substances [16][17][18]. In this study, we aim to further analyze the anti-melanin effect and potential mechanism of FTGML based on the previous stage in the mouse melanoma cell line B16F10. ...
Peptide Phe-Thr-Gly-Met-Leu (FTGML) is a bioactive oligopeptide with tyrosinase inhibitory activity derived from gelatin hydrolysate of grass carp scales. Previous studies have shown that FTGML addition can effectively inhibit mushroom tyrosinase activity in vitro, and also has some effect on the inhibition of melanogenesis in zebrafish in vivo, but the underlying mechanism is not fully understood. In this study, we used FTGML to treat B16F10 melanoma cells, and found a significant inhibition of tyrosinase activity and melanin synthesis. Interestingly, the treatment showed a strong correlation between antioxidant activity and anti-melanin, which was associated with FTGML reducing the involvement of reactive oxygen species in melanin synthesis. Furthermore, FTGML reduced melanogenesis in B16F10 cells by downregulating the cAMP-PI3K/Akt and MAPK pathways (p38 and JNK). These results suggested that FTGML can reduce melanin production in mouse B16F10 melanoma cells through multiple pathways.
... Cinnamic acid doesn't influence the fungal growth but decreases the yield of the pigment from the mycelium. 45,46 Sophorcarpidine Tyrosinase activity are often greatly inhibited by cinnamic acid, aloin, and sophorcarpidine, of which sophorcarpidine functions as an uncompetitive inhibitor, compared to aloin and cinnamic acid, which are mixed-type inhibitors. Tan et al. ...
Skin whitening is a term used for lightening the complexion of the skin through artificial means like creams, lotions, soaps and injections. Unfortunately, the appeal of these skin bleaching products is based on the obsession of people across the world with skin color. Melanins are produced by specialized cells, termed melanocytes, which are located primarily in the skin, hair bulbs, and eyes. The melanins can be of two basic types: eumelanin’s, which are brown or black, and phaeomelanin’s, which are red or yellow, in mammals typically there are mixtures of both types Increased production and accumulation of melanins characterize number of skin diseases, which include hyperpigmentation such as melanoma, post-inflammatory melanoderma, solar lentigo, etc. Several modalities of treatment for these problems are available including chemical agents or physical therapies. The aim of this review article is to show that some of the skin whitening creams, often sold illegally without a prescription may contain dangerous ingredients that could put people health at risk
... Nair et al. (2001) evaluated repigmentation, as determined by cell pellet color and melanin assays. The colors of cell pellets are evaluated visually for melanin content assay (Usuki et al. 2003). These observations suggest that the L. bicolor extract increased the melanogenic activity of B16 melanoma cells. ...
Lespedeza bicolor (L. bicolor) is used for medicinal purposes because of its various biological and pharmacological activities. In this study, the effects of L. bicolor ethanol extract on the treatment of vitiligo were investigated. The determination of melanin content in melanocytes was measured using B16 melanoma cells and C57BL/6J Ler-vit/vit mice. Finally, the quercetin content in L. bicolor were qualitatively analyzed using HPLC. The results obviously indicated that the L. bicolor extract enhanced melanogenesis and increased tyrosinase activity in cultured melanoma cells and C57BL/6J Ler-vit/vit mice. Treatment with L. bicolor extract led to a higher content of melanin and eumelanin in C57BL/6J Ler-vit/vit mice hair than in control (untreated) mice, which demonstrated the therapeutic effect of hair-graying associated with vitiligo. There was a notable increase in melanocytes in the skin of C57BL/6J Ler-vit/vit mice treated with L. bicolor extract compared with the control. L. bicolor extract was a potent tyrosinase and melanin synthesis activator in B16 melanoma cells. C57BL/6J Ler-vit/vit mice treated with L. bicolor extract had significantly higher melanin content in hair than the untreated control. The results suggest that L. bicolor extract is a potential alternative treatment for improvement of vitiligo.
... In addition, it has been accounted for to hinder tyrosinase compound; along these lines it was utilized in the treatment of melasma [9]. ...
... Results showed reduced overall severity of photodamage, mottled hyperpigmentation, and sallowness, and improvements in skin smoothness in response to lactic acid (Smith, 1996). These effects were related to an inhibition of melatonin formation in melanocytes through the inhibition of their tyrosinase activity (Usuki et al., 2003) and the promotion of cell apoptosis in keratinocytes (Hsiao et al., 2009). Lactic acid has further bioactive properties in the body related to the stimulation of endothelial cell migration (Beckert et al., 2006), inhibition of germ cell apoptosis in the human testis (Erkkilä et al., 2002), amelioration of cognitive impairment (Holloway et al., 2007), and improvement of cerebral energy metabolism in humans with traumatic brain injury (Bouzat et al., 2014). ...
In recent years, acid whey production has increased due to a growing demand for Greek yogurt and acid-coagulated cheeses. Acid whey is a dairy by-product for which the industry has long struggled to find a sustainable application. Bulk amounts of acid whey associated with the dairy industry have led to increasing research on ways to valorize it. Industry players are finding ways to use acid whey on-site with ultrafiltration techniques and biodigesters, to reduce transportation costs and provide energy for the facility. Academia has sought to further investigate practical uses and benefits of this by-product. Although modern research has shown many other possible applications for acid whey, no comprehensive review yet exists about its composition, utilization, and health benefits. In this review, the industrial trends, the applications and uses, and the potential health benefits associated with the consumption of acid whey are discussed. The proximal composition of acid whey is discussed in depth. In addition, the potential applications of acid whey, such as its use as a starting material in the production of fermented beverages, as growth medium for cultivation of lactic acid bacteria in replacement of commercial media, and as a substrate for the isolation of lactose and minerals, are reviewed. Finally, the potential health benefits of the major protein constituents of acid whey, bioactive phospholipids, and organic acids such as lactic acid are described. Acid whey has promising applications related to potential health benefits, ranging from antibacterial effects to cognitive development for babies to human gut health.
... Overall, the role of each microorganism in the De-E11 starter could be inferred as follows: during fermentation of UBR by the defined microbial starter, both R. oryzae and S. cerevisiae were the main enzyme producers to catalyze degradation of UBR ( Fig 4A) and carbohydrate metabolic processes (Fig 5A), respectively. S. fibuligera may play a role in UBR degradation (Fig 4) while enzymes from P. pentosaceus regulate the carbohydrate metabolic process, especially in the biosynthesis of lactic acid (Fig 5), another known melanogenesis inhibitor [59]. This overview is summarized as a schematic diagram related to rice degradation and carbohydrate metabolic processes in Fig 9. First, various components in the rice grain, such as xylan, cellulose, starch, protein, and lipid, were degraded into monomers. ...
Fermentation of rice grains requires diverse metabolic enzymes to be synchronously synthesized by the microbial community. Although many studies have used a metaproteomic approach to investigate the roles of microorganisms in improving the flavor of fermented foods, their roles in producing compounds with biological activity have not yet been reported. In a previous study the ferment obtained from unpolished black rice (UBR) fermented with a defined microbial starter (De-E11), comprised of Rhizopus oryzae, Saccharomycopsis fibuligera, Saccharomyces cerevisiae, and Pediococcus pentosaceus, (fermented UBR; FUBR) showed a strong melanogenesis inhibition activity in B16F10 melanoma cells. Hence, in this study, the roles of these microorganisms in producing the melanogenesis inhibitor(s) in FUBR was investigated using a metaproteomic approach. The melanogenesis inhibition activity of the FUBR liquid (FR-Liq) was found to increase with longer fermentation times. R. oryzae and S. cerevisiae were the major hosts of proteins related to the biosynthesis of melanogenesis inhibitor(s) in the FUBR. During fermentation, the enzymes involved in the degradation of UBR and in the carbohydrate metabolic process were identified. These enzymes were associated with the process of releasing of bioactive compound(s) from UBR and the synthesis of organic acids from the microorganisms, respectively. In addition, enzymes involved in the synthesis of some known melanogenesis inhibitor(s) and in the degradation of the melanogenesis stimulator (arsenate) were detected. Varying the combination of microorganisms in the De-E11 starter to produce the FR-Liq revealed that all four microorganisms were required to produce the most potent melanogenesis inhibition activity. Taken together with the metaproteomics results, this suggested that the microorganisms in De-E11 synchronously synthesize the FR-Liq with melanogenesis inhibition activity. In conclusion, this information on the metaproteome in FUBR will increase our understanding of the microbial metabolic modes and could lead to knowledge-based improvements in the fermented rice process to produce melanogenesis inhibitor(s).
... 73 AHA may also decrease melanin deposition in the skin in vivo. 69,74 AHA at lower concentrations has none of these effects and is highly useful in dermatologic practice. ...
Infection preventive practice of using disinfectants against SARS-CoV-2 has become the new normal due to the COVID-19 pandemic. Although disinfectants may not be applied directly to the human body, it remains at high risk of exposure including close skin contact on disinfected surfaces or during handling. This dermal contact, on a regular basis, can induce hazardous skin reactions like irritation, inflammation, and burning in severe conditions. Disinfectants are germicide chemicals that can penetrate the skin and create skin reactions that are usually regarded as irritant and allergic contact dermatitis. More importantly, disinfectants can react with skin components (proteins and lipids) to facilitate their skin penetration and disrupt the skin barrier function. Whereas the antimicrobial actions of disinfectants are well understood, much less is known regarding their dermatologic reactions, including but not limited to irritation and hypersensitivity. We reviewed the skin reactions created by those disinfectants against SARS-CoV-2 approved by the European Chemical Agency and the United States Environmental Protection Agency.
... 8 GA has also been demonstrated to increase epidermal and dermal hyaluronic acid levels, 9 increase keratinocyte and fibroblast proliferation rates, 10,11 stimulate collagen production, 2,[9][10][11][12] and improve the quality of elastic fibers. 2 An inhibitory effect on melanin synthesis has also been reported. 13 The ability of GA to invoke these biological responses is determined by its capacity to penetrate into skin. 1 This itself depends upon the amount of GA in its most biologically active free acid form, its pH and concentration, its contact time with the skin, and the vehicle used to deliver it. 1 Like all acids, however, GA can cause skin irritation and erythema, and these effects are greatest when the concentration of GA is high, and its pH is low. ...
Background
Glycolic acid (GA) is an effective way of reversing the signs of age and photodamage. GA enhances desquamation of the stratum corneum and induces biological responses that can help restore skin’s integrity. GA can, however, cause irritation, especially when its concentration is high, and its pH is low. Thus, most commercially available products for home use contain relatively low GA concentrations and are partially neutralized to a pH around 4.
Aims
The aim of this study was to determine the biological effects and relative efficacy of cosmetic formulations containing GA at concentrations ranging from 8% to 25% at pH 4 in human ex vivo skin explants.
Methods
Human skin explants were topically treated with gel formulations and oil‐in‐water creams containing 8, 10, 15, or 25% GA, adjusted to pH 4, daily for 5 days. The degree of desquamation, their effect on cell proliferation, and their impact upon total collagen levels were determined 24 hours later. Levels of tumor necrosis factor‐alpha (TNF‐α) were measured after days 3 and 6.
Results
All formulations effectively induced desquamation in a concentration‐dependent manner. Total collagen levels were increased at all concentrations, with greatest effects at higher GA concentrations. No effect on TNF‐α expression was observed.
Conclusions
These data suggest that partially neutralized GA formulations retain skin rejuvenating properties without causing irritation and inflammation, and that their use can be tailored to individual needs based on the concentration of GA in the formulation.
... Salicylic acid had been well demonstrated to have a whitening effect among the Asian population.22 Alpha hydroxy acid (e.g., lactic acid) had been demonstrated to inhibit tyrosinase activity, which further led to the suppression of melanin formation.23 ...
Introduction and Objective
Antibiotics and retinoids have been used for acne vulgaris for decades. Though effective, each has its own drawbacks. Chemical peels have been used for treatment of acne vulgaris with inadequate clinical evidence. We sought to determine the efficacy and safety of Jessner’s solution (JS) in comparison with salicylic acid (SA) 30% in the management of acne vulgaris and postacne hyperpigmentation in patients with colored skin.
Methods
A total of 36 subjects (94.5% Fitzpatick Type IV‐V) were recruited in this randomized double‐blinded, split‐face, controlled trial. Each side of the face was randomly assigned for treatment with either JS or SA. Subjects were treated once fortnightly for a total of three sessions. Lesion counting, Michaelsson acne score (MAS), photographs, and postacne hyperpigmentation index (PAHPI) were used to objectively assess the improvement. Complications were assessed during each visit. Statistical analysis was conducted using SPSS v22.0. Significance was set at P = 0.05.
Results
At the end of therapy, significant reduction in inflammatory, noninflammatory lesions, MAS, and PAHPI scores (P < 0.001, respectively) were noted in comparison to baseline. Mixed model analysis revealed no significant outcome difference between the two groups. Patients who reported good and very good outcome were 76.4% (JS) and 85.3% (SA). Burning, stinging sensation, and exfoliation were the common complications reported. Postinflammatory hyperpigmentation was reported only once in the JS arm.
Conclusion
Both JS and SA were equally effective in the treatment of acne vulgaris and reducing postacne hyperpigmentation in patients with colored skin.
... The previous study reported that A. oryzae-fermented rice bran extracts contained various organic acids, including oxalic acid, citric acid, succinic acid, kojic acid and acetic acid 32 . However, some organic acids, such as glycolic acid and lactic acid, could directly inhibit tyrosinase activity during melanin synthesis in melanoma cells 38 . Accordingly, lactic acid in Lactobacillus rhamnosus spent culture supernatant suppressed tyrosinase activity in vitro in a dose-dependent manner 39 . ...
This study aimed to investigate kojic acid production from the fermentation of rice by using Amylomyces rouxii TISTR 3182 and Aspergillus oryzae TISTR 3259. Steamed rice was fermented by A. rouxii followed by A. oryzae for 15 days, and mixed and separated cultures of both strains were compared. Kojic acid was analysed using Bentley’s colorimetric method while glucose was analysed by the dinitrosalicylic acid (DNS) method. The results showed that separation of A. rouxii from rice medium before the addition of A. oryzae produced more kojic acid than the mixed culture, reaching a maximum yield of approximately 1.54 ± 0.10 g/l at day 10, pH 4 and 27 ± 2 °C. The fermented broth was evaluated for antioxidant activity by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, producing a maximum inhibition of 84 ± 11% and antityrosinase activity by dopachrome method with a maximum inhibition of 89 ± 11%. It may be concluded that microbial fermentation of rice could provide a good source of kojic acid. Separation of A. rouxii TISTR 3182 before the addition of A. oryzae TISTR 3259 in rice medium resulted in more effective kojic acid production. The fermented mixtures may have the potential to be further utilized as functional food and cosmetic ingredients, providing antioxidant and whitening activity. © 2019 Science Society of Thailand under Royal Patronage. All rights reserved.
... 17 It is used at concentrations ≤10%, and at final pH ≥ 3.5 in cosmetic and pharmaceutical products for the treatment of skin diseases including actinic keratosis, hyperkeratosis, and seborrheic eczema. [18][19][20][21][22] ...
The nutricosmetics are products and ingredients that act as nutritional supplements to care skin, nails, and hair natural beauty. They work from the inside to promote beauty from within. Nutricosmetic is the latest trend in the beauty industry. This tendency rapidly gained many followers because it fits with the modern culture: Today, consumers are very careful with the food that they introduce into their body, and there is also an increasing demand for natural products able to enhance one's health and beauty without side effects and significant traction before use. However, many nutricosmetic products are considered effective due to the historical use and word of mouth. Comprehensive analysis of the global nutricosmetics market is conducted considering form, end‐user applications, and some product components such as collagen, peptides, proteins, vitamins, carotenes, minerals, and omega‐3 fatty acid are reported. Plant extract ingredients used in nutricosmetic are also described.
... 17 It is used at concentrations ≤10%, and at final pH ≥ 3.5 in cosmetic and pharmaceutical products for the treatment of skin diseases including actinic keratosis, hyperkeratosis, and seborrheic eczema. [18][19][20][21][22] ...
The present study was carried out to provide the allantoin and glycolic acid contents in the Helix aspersa Muller mucus of common Campania land (Italy) by using chromatographic method. The study continued with the formulation of a snail mucus cosmetic cream, whose ability to hydrate the skin was evaluated comparing the skin hydration and trans-epidermal water loss (TEWL) effects of a stable cosmetic preparation. The skin TEWL and skin hydration effects were measured by TEWAMETER and corneometer probe, respectively, at the beginning, after 1 hour, and 24 hours.
... Lactic acid is also used as skin whitener (Table 1). At a dose of 500 µg/mL it inhibits melanin formation in a dose-dependent manner without affecting cell growth [151]. Recent studies have shown that species of Rhizopus could offer a valuable alternative source for lactic acid production [152]. ...
It is well known that terrestrial environments host an immense microbial biodiversity. Exposed to different types of stress, such as UV radiation, temperature fluctuations, water availability and the inter- / intra-specific competition for resources, terrestrial microorganisms have been evolved to produce a large spectrum of bioactive molecules. Bacteria, archaea, protists, fungi and algae have shown a high potential of producing biomolecules for pharmaceutical or other industrial purposes as they combine a sustainable, relatively low-cost and fast-production process. Herein, we provide an overview of the different bioactive molecules produced by terrestrial microorganisms with skin protecting applications. The high content in polyphenolic and carotenoid compounds produced by several strains, as well as the presence of exopolysaccharides, melanins, indole and pyrrole derivatives, mycosporines, carboxylic acids and other molecules, are discussed in the context of their antioxidant, photo-protective and skin-whitening activity. Relevant biotechnological tools developed for the enhanced production of high added value natural products, as well as the protecting effect of some antioxidant, hydrolytic and degrading enzymes are also discussed. Furthermore, we describe classes of microbial compounds that are used or have the potential to be used as antimicrobials, moisturizers, biosurfactants, pigments, flavorings and fragrances.
... Currently, kefir-based face mask has attracted a great attention, associated with numerous beneficial effects. This is due to presence of acid, alcohol, and other compounds capable of serving as antibacterial and antioxidant agents, moisturizer, regeneration of skin cells [2,9], and as well as promoting suppression of skin darkening [16]. ...
Antibacterial properties are one of the crucial factors that determine quality of face mask. To acquire this feature, incorporation of herb-based additives can be a promising way. “Bedak Lotong” is a traditional product known to exert antibacterial activity as shown in black glutinous rice (Oryza sativa Linn. var. glutinosa), temulawak (Curcuma xanthorrhiza Roxb) and tamarind (Tamarindus indica L.). This present work aimed to investigate the effects of “Bedak Lotong” addition on quality of kefir mask. The experimental work was performed using completely randomized design, consisting of two stages. First, the effect of incubation time (12, 18, and 24 h) was studied, while the second stage was carried out to investigate the effect of different ratios (60:40; 50:50; 40:60). The results showed that addition of “Bedak Lotong” could enhance antibacterial activity.
... It was shown to decrease melanin synthesis by directly inhibiting the tyrosinase enzyme, thereby explaining its skin whitening effects. 25 LA is a safe peel with minor side effects. ...
Acne vulgaris is the most common skin disorder in adolescents and young adults. It carries a significant psychological and economic burden to patients and society. A wide range of therapeutic options are available, including topicals and systemic therapies. Chemical peeling is a skin resurfacing procedure intended to regenerate normal skin from the application of exfoliative agents. It has been used for the treatment of acne vulgaris and other skin disorders for decades. There are several chemical agents with variable mechanisms of action, usually classified as superficial, medium, and deep peels. When selecting the patient and the appropriate peel, the dermatologist individualizes therapy, and performs an extensive interview, including past medical history and physical exam. Several host factors can affect the outcome of this procedure, including current psychological state, medications, history of surgery, and immune system, among others. The physician must also be confident that the peel is safe and effective for the target patient. The Fitzpatrick skin type scale is a useful tool to classify patients based on skin color and ability to tan, but also can be used to evaluate preoperative risk of postpeel response and complications. Dark-skinned patients (Fitzpatrick skin type IV–VI), including blacks, Asian, and Hispanic/Latino, are at higher risk of postinflammatory/postpeel hyperpigmentation. When treating these populations, deep chemical peels should be avoided, and preoperative preparation emphasized. There are many studies available in the literature supporting the use of superficial to medium depth peels as adjuvant therapy for acne vulgaris. This review article aims to present the most important factors when selecting a patient for a chemical peel, the evidence behind its safety and efficacy, and special considerations when choosing a specific agent.
... In melasma, it can be used twice a month for 4-5 sessions. [4,12] ...
Melasma is a notorious dermatosis, often resistant to treatment. Chemical peeling constitutes an acceptable option of management of melasma (of any type and duration). In this article, as a group of experts from Pigmentary Disorders Society (PDS) in collaboration with South Asian Pigmentary Forum (SPF), we have tried to elaborate the various chemical peeling agents for the treatment of melasma. Besides, we have reviewed the indications, mechanism of action, rationality and the detailed procedure of peeling. The evidence in favor of various peeling agents have been summarized as well.
... Lactic acid, which is an alpha hydroxy acid, accelerates epidermis turnover or acts as a chemical peel. It can lighten skin by lifting the pigmented cells [4] and increasing the penetration of its active ingredients, α arbutin and niacinamide, which affects melanin. Niacinamide has a depigmentation effect, via the inhibition of melanosome transfer from melanocytes to keratinocytes [5]. ...
Objective: The present study aimed to formulate and determine the physical stability of microemulsion and W/O/W multiple emulsions, containingα arbutin, lactic acid, and niacinamide, as skin-whitening cosmetics.Methods: A skin lightening effect can also be obtained from lactic acid, which accelerates the turnover of pigmented cells in the epidermis, andfrom niacinamide, which inhibits the transfer of melanosomes from melanocytes to keratinocytes. These active ingredients were combined inmicroemulsion and W/O/W multiple emulsions dosage forms in various concentrations of Tween 80 as an emulsifier. An evaluation and physicalstability test were carried out during 12 weeks of storage at 28±2°C, 4±2°C, and 40±2°C, as well as a cycling test.Results: The results showed that a microemulsion could be prepared in 25-35% of Tween 80 (surfactant) and 10% of ethanol (co-surfactant), andthat it had globule sizes of 2.397-16.8 nm, a transparent, pseudoplastic flow, and was most stable in storage at 28±2°C. Microemulsion with 35% ofTween 80 was the most stable microemulsion formula because it had the smallest globule size, the most stable distribution profiles of globule sizes,and the highest viscosity. W/O/W multiple emulsions could formulated made with 2.5-4.5% of Tween 80 (external emulsifier) and 3% of Span 80(internal emulsifier); these emulsions had a pseudoplastic-thixotropic flow and were most stable in storage at 28±2°C.Conclusions: The formulation of multiple emulsions with 2.5% of Tween 80 was the most stable formula, with a stable distribution profile of globulesizes during 8 weeks of storage at temperatures of 28±2°C, 4±2°C, and 40±2°C.
Rice contains numerous nutrients and biologically active compounds. The phytochemical composition of rice varies among cultivars that leads to diversities in biological activities. Fermentation is an efficient way for improving nutrient bioavailability and functional properties of raw materials. It enhances and/or synthesizes the compounds with health promoting or decreased antinutritive compounds during fermentation process. Rice-based fermented products have been reported for enhancing various biological activities including antioxidant, anti-cancer, anti-diabetes anti-wrinkle and anti-melanogenesis activities. Melanogenesis, melanin biosynthesis, is the cause of human skin pigmentation, however the accumulation of melanin leads to skin hyper-pigmentary disorders, such as freckles and melasma. In this review, the information on rice-based fermented products have been assembled to illustrate the fermented rice properties especially melanogenesis inhibition activity including functional roles of the microorganisms in the fermented rice products.
Introduction:
Polygonatum kingianum is a traditional medicinal plant, and processing has significantly impacts its quality.
Methods:
Therefore, untargeted gas chromatography-mass spectrometry (GC-MS) and Fourier transform-near-infrared spectroscopy (FT-NIR) were used to analyze the 14 processing methods commonly used in the Chinese market.It is dedicated to analyzing the causes of major volatile metabolite changes and identifying signature volatile components for each processing method.
Results:
The untargeted GC-MS technique identified a total of 333 metabolites. The relative content accounted for sugars (43%), acids (20%), amino acids (18%), nucleotides (6%), and esters (3%). The multiple steaming and roasting samples contained more sugars, nucleotides, esters and flavonoids but fewer amino acids. The sugars are predominantly monosaccharides or small molecular sugars, mainly due to polysaccharides depolymerization. The heat treatment reduces the amino acid content significantly, and the multiple steaming and roasting methods are not conducive to accumulating amino acids. The multiple steaming and roasting samples showed significant differences, as seen from principal component analysis (PCA) and hierarchical cluster analysis (HCA) based on GC-MS and FT-NIR. The partial least squares discriminant analysis (PLS-DA) based on FT-NIR can achieve 96.43% identification rate for the processed samples.
Discussion:
This study can provide some references and options for consumers, producers, and researchers.
Two Helichrysum italicum extracts, OPT-1 (rich in phenolic acids) and OPT-2 (rich in total phenols and flavonoids), were prepared using hydroxypropyl-β-cyclodextrin (HP-β-CD)-assisted extraction. The prepared extracts were rich in phenolic compounds, including flavonoids and phenolic acids. GC-MS analysis of the extracts identified neryl acetate, neo-intermedeol, β-selinene, γ-curcumene, italidione I, and nerol as the main volatile components of the extracts, as well as plant sterols, γ-sitosterol, campesterol, and stigmasterol. The antioxidant (DPPH radical scavenging, reducing power, and a carotene linoleic acid assay) and cosmeceutical (anti-hyaluronidase, anti-tyrosinase, anti-lipoxygenase, ovalbumin anti-coagulation, and a UV-absorption assay) activity of the extracts in most of the assays was better than the activity of the applied positive controls. Especially low were the IC50 values of the extracts in the anti-hyaluronidase (14.31 ± 0.29 μL extract/mL and 19.82 ± 1.53 μL extract/mL for OPT-1 and OPT-2, respectively) and the anti-lipoxygenase (0.96 ± 0.11 μL extract/mL and 1.07 ± 0.01 μL extract/mL for OPT-1 and OPT-2, respectively) assays. The extracts were non-toxic to HaCaT cells in concentrations of up to 62.5 µL extract/mL assuring their status as excellent candidates for cosmeceutical product development appropriate for direct use in cosmetic products without solvent evaporation.
Chemical peeling is a “lunch-time”, or office based, clinical, cutaneous resurfacing procedure which is chiefly used for the management of pigmentary conditions, photoaging and acne scars. Thorough knowledge of gender-based differences in cutaneous morphology goes a long way in planning chemical peels. Preliminary evidence suggests that commonly used chemical peels have higher therapeutic index and are safer in pregnancy and lactation, but convincing evidence is yet to present.KeywordsChemical peels in womenPeels in pregnancySpecial consideration of peels
The prevalence of metabolic syndrome is increasing day by day, owing to the sedentary lifestyle, unhealthy food habits, genetic factors, environmental influences and many other conspicuous variables. Skin, aptly considered to be a mirror of internal organs, often manifests with certain signs and symptoms, suggestive of a diagnosis of metabolic syndrome. In this chapter, we have attempted to touch upon the dermatological manifestations (both common and uncommon) of metabolic syndrome.KeywordsMetabolic syndromeDiagnosisSkin manifestations
In this work, the suppression of tyrosinase-related genes, including an improvement in UV absorption effects of bioconverted CS extracts (BCS), was investigated to improve the skin-whitening effect. Total polyphenols and total flavonoids, which are bioactive components, increased 2.6- and 5.4-times in bioconversion using Lactiplantibacillus plantarum SM4, respectively, as compared to ultrasound-assisted extracts (UCS). The effect of BCS on radical scavenging activity, UV-A absorption, and tyrosinase activity inhibition, contributing to skin-whitening, were 1.3-, 1.2-, and 1.2-times higher than those of UCS, respectively. The main component identified in high-performance liquid chromatography (HPLC) was gallic acid in both UCS and BCS, which increased by 2.9-times following bioconversion. The gene expression of tyrosinase-related proteins, including TRP-1 and TRP-2 genes, was studied to confirm the suppression of melanin synthesis by BCS in order to identify the skin-whitening mechanism, and BCS decreased both genes’ expression by 1.7- and 1.6-times, demonstrating that BCS effectively suppressed melanin synthesis. These findings imply that the chestnut inner shell can be employed as a cosmetic material by simultaneously inhibiting melanogenesis and enhancing UV-A absorption through bioconversion using L. plantarum SM4.
Tibetan kefir grain as the starter of milk fermentation has been applied as functional food with many bioactive characteristics. In this study, the milk whey product (TKG-MW) was obtained through the milk fermentation of Tibetan kefir grain containing the dominant Lactobacillus, Acetobacter, and Bacillus after 3 and 6 days of cultivation. Antioxidant, anti-inflammatory, and melanogenesis inhibition capacities under TKG-MW treatment were analyzed. Results revealed that the antioxidation of TKG-MW at 6 days of fermentation was higher than that at 3 days of fermentation according to the DPPH and ABTS+ radical scavenging analysis. However, the anti-inflammation of TKG-MW was only observed at 6 days of fermentation by using lipopolysaccharide-stimulated RAW 264.7 macrophages. The inhibition of mushroom tyrosinase activity by TKG-MW was demonstrated. The decrease of melanin content was verified using α-melanocyte-stimulating hormone-stimulated B16-F10 cell. The real-time quantitative reverse transcription polymerase chain reaction result indicated that the mRNA levels of Tyr, Trp-1, and Trp-2 of the B16 cell involved in melanin synthesis were down-regulated over a two-fold change by the TKG-MW treatment. Additionally, the protein expressions of Tyr, Trp-1, Trp-2, and Mitf of the B16 cell were reduced with the TKG-MW treatment. Organic acids, such as lactic acid, succinic acid, 3-phenyllactic acid, l-pyroglutamic acid, and malic acid, were identified by liquid chromatography-mass spectrometry in TKG-MW and were found to significantly inhibit tyrosinase activity. To the best of our knowledge, this work is the first to report melanogenesis suppression by TKG-MW. Results suggested that the fermentation product of TKG could be applied as a depigmenting agent in food and cosmetics.
Arbutin, a natural glycoside, is well known as a commercial tyrosinase inhibitor and thus to prevent pigmentary disorders of skin. Indeed, tyrosinase is involved in the biosynthesis of melanin, the skin main pigment. However, arbutin is subject to hydrolysis, which limits its bioactivity. In general, thioglycosides are known to be very resistant to both chemical and enzymatic hydrolysis, which increases the interaction time with their biological targets. A biocatalytic approach allowed us to access to thioglycosidic analogs of arbutin in a green approach with good to excellent yields. Such compounds have then been tested as tyrosinase inhibitors and also as inhibitors of melanin transfer from melanocytes to keratinocytes. This latter mechanism takes place via lectin (or lectin‐like) receptors present on the cells surface. p‐Aminophenyl ß‐D‐thiogalactopyranoside appears to be an excellent candidate thanks to its tyrosinase inhibitory activity comparable to arbutin, while having the ability to interact with glycan receptors allowing to reduce melanin transfer.
Background: Melasma is an acquired Hypermelanosis often difficult to treat. In this study, we assessed efficacy and safety of 5% hydroquinone cream plus non-ablative fractional 1540 erbium YAG laser and 5% hydroquinone cream alone in the treatment of melasma Methods: We selected 40 patients of melasma aged between 18 and 60 years for the study. Group 1 were treated with a cream of 5% hydroquinone and group 2 treated with a solution of hydroquinone 5% plus three sessions of non-ablative fractional 1540 erbium YAG laser at monthly interval. Improvement was assessed by the melasma-area-and-severity-index (MASI) score. Results: The two groups did not differ significantly in (MASI( score in the first month of treatment (p = 0/135), while during the second to fifth months, there were significant differences between the two groups (p <0.05) and (MASI( score was more reduced in the second group. The two groups did not show significant differences in the incidence of complications (erythema, hyperpigmentation, hypopigmentation). (p= 0/746, 0/158, 0/574, 0/135, 0/135 respectively, the first month, second month, third month and fourth month) Conclusion: A combination of hydroquinone and laser therapy had better results in the treatment of melasma. But due to some disagreements about the use of lasers in the treatment of melasma we recommend more studies with larger sample size to be taken to definitely show better therapeutic effect of laser.
Introdução: O Melasma é uma condição dermatológica crônica com patogênese não completamente compreendida que impacta na qualidade de vida do paciente, sendo uma causa comum de cuidados dermatológicos. Objetivo: analisar os benefícios do tratamento do melasma por intermédio do ácido lático associado ao Light Emitting Diode, através de uma revisão integrativa de literatura. Método: foi realizada uma busca no LILACS, Medline, SciELO, PubMed e Google Acadêmico por meio dos descritores: melasma, tratamento, ácido lático e díodo emissor de luz. Resultados: 9 artigos compuseram a amostra. Verificou-se benefícios da utilização do peeling de ácido lático e do Light-emitting diode principalmente ao serem avaliados através do Melasma Area and Severety Index e do Melasma Quality of Life Scale. Conclusão: os tratamentos com o peeling de ácido lático e com o Light-emitting diode apresentaram respostas benéficas no controle do melasma, no entanto, foi faltam estudos avaliem os benefícios das duas terapias em conjunto.
Background
Melanin is synthesized in melanocytes and transferred to keratinocytes through dendrites. Endogenous pyruvate is a key metabolite for ATP production in glycolysis, and the tricarboxylic acid (TCA) cycle and exogenous pyruvate provide protection against oxidative stress and acidosis in the intercellular space. The function of pyruvate in the regulation of dendrite outgrowth remains to be elucidated.
Objective
We examined the effect of pyruvate on dendritic elongation and skin pigmentation
Methods
Murine B16F10 melanoma cells and human primary melanocytes were used for in vitro analysis. Melanin quantitation and histochemical staining were performed in a 3D pigmented human skin model.
Results
We demonstrated the participation of monocarboxylate transporters (MCTs) responsible for the membrane transport of pyruvate in B16F10 melanoma cells. The accumulation of pyruvate occurred in a pH-dependent manner, which was highly sensitive to a specific MCT inhibitor (α-cyano-4-hydroxycinnamic acid). α-MSH-induced morphological changes, including dendrite elongation and growth-cone-like structure, were diminished in B16F10 cells upon treatment with pyruvate. In addition, the number of dendrite branches was reduced in normal human epidermal melanocytes. As the Rho-subfamily of monomeric GTP-binding proteins modulates dendrite formation, we subsequently examined the suppression of Rac1 activation by pyruvate, but not RhoA and Cdc42. Furthermore, pyruvate showed anti-melanogenic effects against UV-induced pigmentation in reconstructed pigmented epidermis, established by co-seeding autologous melanocytes and keratinocytes, which act similar to in vivo skin tissue.
Conclusion
These results suggest that pyruvate treatment may be an alternative or additive therapeutic strategy to prevent hyperpigmentation.
Bedak Lotong kefir face mask is a combination of kefir and a traditional black face powder of the Buginese in South Sulawesi, Indonesia. The Bedak Lotong face powder used natural ingredients that are rich in phenol compounds, i.e., black glutinous rice ( Oryza Sativa L. glutinosa ), Curcuma ( Curcuma xanthorrhiza Roxb) and tamarind ( Tamarindus indica L.). This study was aimed to analyze the effect of different concentrations of curcuma ( Curcuma xanthorrhiza Roxb) on the chemical quality of Bedak Lotong kefir face mask. This research was conducted at the Laboratory of Biotechnology of Dairy Processing, Hasanuddin University, Makassar. A completely randomized design was used, the curcuma concentrations were 0%, 15%, 30% and 45%, and with oven drying. Each concentration was done three times. The result showed that an increase in the concentration of curcuma on Bedak Lotong increased antioxidant activity and alcohol content and also reduced the level of fat oxidation or Thiobarbituric acid (TBA) value of Bedak Lotong kefir face mask with oven drying.
Lactic acid bacteria are beneficial to human health. Lactic acid bacteria have wide applications in food, cosmetic and medicine industries due to being generally recognized as safe (GRAS) and multitude of therapeutic and functional properties. Previous studies have long reported the beneficial effects of lactic acid bacteria, their extracts or ferments on skin health, including improvements in skin conditions and the prevention of skin diseases. Lipoteichoic acid isolated from Lactobacillus plantarum was reported to inhibit melanogenesis in B16F10 melanoma cells. In particular, lipoteichoic acid also exerted anti-photoaging effects on human skin cells by regulating the expression of matrix metalloproteinase-1. The oral administration of Lactobacillus delbrueckii and other lactic acid bacteria has been reported to inhibit the development of atopic diseases. Additionally, the clinical and histologic evidence indicates that the topical application of lactic acid is effective for depigmentation and improving the surface roughness and mild wrinkling of the skin caused by environmental photo-damage. This review discusses recent findings on the effects of lactic acid bacteria on skin health and their specific applications in skin-whitening cosmetics.
Superficial skin resurfacing is a process causing controlled injury to the epidermis. Processes can be chemical or mechanical and classified as laser or nonlaser. Although our armamentarium for skin resurfacing ranges from topical medicaments to ablative lasers, superficial skin resurfacing with chemical peels and microdermabrasion (MDA) has maintained its popularity. These well-established therapies have proven safe and effective, offer minimal risk, low cost, and are well-tolerated by patients often with little to no post-procedure downtime. Potential benefits extend to all Fitzpatrick skin types and if done appropriately are highly unlikely to cause significant dyspigmentation or scarring. Indications for treatment are acne vulgaris, rosacea, post-inflammatory hyperpigmentation, melasma, and photodamage including lentigines, fine rhytides, and actinic keratoses. Superficial resurfacing with chemical peels or MDA can be used in combination with other therapies (e.g., laser) and may enhance the efficacy of other topical treatments. A superficial depth of penetration may be of significant benefit to the patient but does have limitations. It is important for patients to understand the likely need for a series of treatments, the importance of pre- and post-care regimens, and expected outcomes.
Objective
The aim of the study was in vitro and in vivo characterization of cosmetic cream with 6% of standardized wild apple fruit extract, stabilized by conventional non‐ionic emulsifier‐CEW, in order to determine the influence of emulsifiers (conventional vs biodegradable) on the characteristics of creams and their effects on the skin.
Methods
Organoleptic and physico‐chemical (pH values and electrical conductivity) analysis was performed, determination of fruit acids‐FAs content (using HPLC analysis) and estimation of its antioxidant activity‐AA (using DPPH test) during 180 days. In vivo study included following examinations: screening of safety profile (after creams application under occlusion during 24h at human skin); skin moisturizing potential, transepidermal water loss‐TEWL, skin pH after 28 days of cream application and hypopigmentation efficacy 7 days of cream application at artificially induced skin hyperpigmentation.
Results
Investigated cosmetic cream‐CEW showed satisfactory organoleptic, physico‐chemical characteristics, stability, FAs content (0.13%) and AA (19.25±0.67%RSC) after preparation, which remained unchanged over the study period. In vivo investigation revealed absence of skin irritation after CEW's application under occlusion. An increase of skin moisturization (after 14 days ΔEC was 18.52 ± 11.51 and after 28 days of applications 16.52 ± 9.36) during 28 day‐study, with unchanged TEWL and skin pH values was shown. Decrease of melanin index was revealed, too (after 7 days ΔMI was ‐31.40±16.50).
Conclusion
Cosmetic cream stabilized by conventional emulsifier showed better antioxidant potential and weaker moisturizing and hypopigmentation effects related to the cream with same composition but stabilized by biodegradable emulsifiers. Based on all mentioned above, investigated cosmetic cream might be considered for potential use as modern, stable, safe and efficient cosmetic product in the prevention and/or treatment of oxidative stress‐related skin changes and/or damages, for moisturization of dry even irritated skin as well as for lightening of hyperpigmented skin.
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Malignant melanoma may be a life-threatening disease caused by various conditions. Cutaneous bacteria could play a role in melanoma development or regression. The present work aimed to analyze the bacterial species present on the epidermis of piglets with hereditary melanoma. Bacteria isolated by swabs directly from melanomas and healthy epidermis were analysed using MALDI-TOF mass spectrometry. From the total of 290 isolates, 92 were identified, while the extraction by ethanol turned out to be more efficient compared to identification by direct transfer. Staphylococcus sciuri, Staphylococcus cohnii, and Lactococcus lactis were significantly more frequent on healthy skin, whereas Staphylococcus chromogenes , Staphylococcus hyicus, and Enterococcus faecalis have thrived significantly better on melanoma. Overall, the results indicate that the micro-biota of melanoma is different from that of healthy epidermis, so piglet skin bacteria inspections are recommended.
L'hyperpigmentation résulte d’une production excessive de mélanine au niveau de certaines zones cutanées. Elle peut avoir différentes causes et se traduit par l'apparition de taches foncées sur la peau, d'étendue et de forme variables. Ces dernières années, les femmes sont de plus en plus promptes à agir pour tenter de lutter efficacement contre ces taches parfois préoccupantes ou jugées disgracieuses. Différents moyens peuvent être envisagés qu’ils soient préventifs, mécaniques, médicamenteux ou cosmétiques. Les laboratoires cosmétiques proposent d’ailleurs de plus en plus de gammes ayant pour objectif d’améliorer « l’éclat du teint » et de faire disparaître ces taches.Après avoir présenté les causes de l’hyperpigmentation cutanée et les approches envisageables actuellement ou en devenir pour sa prise en charge, cette thèse proposera plus particulièrement une revue des approches cosmétiques pouvant être proposées en 2017.
Alpha hydroxy acids (AHAs) are a class of chemicals that have been used in cosmeceuticals for many years. They are well known for their ability to rejuvenate the skin and their anti-ageing, pigmentation-reducing and volume-restoring properties. The PCA Skin Perfecting Peel (Church Pharmacy) contains 20% mandelic acid and 10% lactic acid. It is a superficial peel formulated to treat a variety of skin concerns, including hyperpigmentation, acne and common signs of ageing. This article looks at the evidence available to support the key ingredients in the PCA Skin Perfecting Peel, as well as its indications and contraindications. It also presents three case studies with results obtained following a course of just two peels and an at-home regimen. The author concludes this peel is an asset for any aesthetic practice, with just one treatment producing results for a variety of skin concerns, affecting a broad range of patients. The nature of chemical peels means that patients often return more frequently than with injectable therapies, which can provide ongoing contact and regular income.
Melasma is rare before puberty and most commonly occurs in women during their reproductive years. It not only has a notable effect on the appearance of a patient, but also it has a significant impact on his or her psychological wellbeing. Safe and effective treatment of melasma remains a substantial challenge, and it is difficult to find a way to manage the condition in the long term as it tends to reoccur. This article aims to give a comprehensive overview of the treating principles for melasma using tailored topical bleach peel protocols. The article will discuss relevant topical depigmeting agents that can be applied to effectively treat and manage melasma. Treatment protocols are based on more than 20 years of expertise and experience of a dermatologist specialised in treating this skin disease.
Rice, wheat, maize, and konjac extracts are rich in ceramide and used for cosmetic applications. However, the differences in efficacy between ceramides from different sources have not been elucidated. Therefore, we compared the anti‐melanogenesis effects of ceramide‐rich food extracts (yeast, rice, and maize) and purified food‐derived ceramides (from yeast, mushrooms, rice, wheat, and maize). We also investigated the mechanism of the inhibitory effect of yeast extract. The yeast extract suppressed melanogenesis to a greater extent than other food extracts; however, purified rice‐ and maize‐derived ceramides inhibited melanogenesis more than yeast‐derived ceramide. Additionally, ceramide‐free yeast extract suppressed melanogenesis in a dose‐dependent manner. In the melanin synthetic pathway, purified yeast‐derived ceramide attenuated melanogenesis by inhibition of tyrosinase. However, ceramide‐free yeast extract decreased the protein expression of tyrosinase related protein‐1 (TRP1), resulting in inhibition of melanogenesis. These results indicate that ceramides, as well as other unidentified components of yeast extract inhibit melanogenesis.
Practical applications
The yeast extract might have two synergistic effects on melanogenesis; tyrosinase inhibition and tyrosinase related protein‐1 inhibition. The safety of yeast extract for human oral administration is already proven, and it can be supplied inexpensively using yeast fermentation systems. These characteristics are advantages of ceramide‐rich yeast extract over the existing ceramide‐rich food extracts. It is also expected that yeast extract could be developed as an effective skin‐whitening agent.
Two pigmentation related genes have recently been cloned which map to the brown (b) and albino (c) loci of mice; these loci influence the quality and quantity, respectively, of melanin produced by melanocytes. Both these gene products are biochemically similar and have extensive amino acid sequence similarity to each other and to lower forms of tyrosinase (EC 1.14.18.1), a copper binding enzyme responsible for melanin production. In order to characterize the catalytic activities of these molecules, we have synthesized peptides and prepared antibodies to them which specifically recognize the gene products in question. By use of immune affinity purification protocols, we have isolated the proteins encoded by the brown and albino loci and have determined that both have the catalytic functions ascribed to tyrosinase, i.e. hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to DOPAquinone. These are the critical reactions to melanogenesis since melanin pigment can be spontaneously produced from those products. The specific activity of the albino locus encoded product is considerably higher than that of the protein encoded by the brown locus, although the latter protein is present in higher quantity in melanocytes than is the protein encoded by the albino locus. These results are surprising since it was anticipated that tyrosinase was the product of single gene locus, and suggest that regulation of melanogenesis in mammals is controlled at the enzymatic level by several different gene products.
The production of melanin pigment in mammals requires tyrosinase, an enzyme which hydroxylates the amino acid tyrosine to DOPA (3,4-dihydroxyphenylalanine), thus allowing the cascade of reactions necessary to synthesize that biopolymer. However, there are other regulatory steps that follow the action of tyrosinase and modulate the quantity and quality of the melanin produced. DOPAchrome tautomerase is one such melanogenic enzyme that isomerizes the pigmented intermediate DOPAchrome to DHICA (5,6-dihydroxyindole-2-carboxylic acid) rather than to DHI (5,6-dihydroxyindole), which would be generated spontaneously. This enzyme thus regulates a switch that controls the proportion of carboxylated subunits in the melanin biopolymer. Efforts to clone the gene for tyrosinase have resulted in the isolation of a family of tyrosinase related genes which have significant homology and encode proteins with similar predicted structural characteristics. Using specific antibodies generated against synthetic peptides encoded by unique areas of several of those proteins, we have immuno-affinity purified them and studied their melanogenic catalytic functions. We now report that TRP-2 (tyrosinase related protein-2), which maps to and is mutated at the slaty locus in mice, encodes a protein with DOPAchrome tautomerase activity.
Visible pigmentation in mammals results from the synthesis and distribution of melanin in the skin, hair bulbs, and eyes. The melanins are produced in melanocytes and can be of two basic types: eumelanins, which are brown or black, and phaseomelanins, which are red or yellow. In mammals typically there are mixtures of both types. The most essential enzyme in this melanin biosynthetic pathway is tyrosinase and it is the only enzyme absolutely required for melanin production. However, recent studies have shown that mammalian melanogenesis is not regulated solely by tyrosinase at the enzymatic level, and have identified additional melanogenic factors that can modulate pigmentation in either a positive or negative fashion. In addition, other pigment-specific genes that are related to tyrosinase have been cloned which encode proteins that apparently work together at the catalytic level to specify the quantity and quality of the melanins synthesized. Future research should provide a greater understanding of the enzymatic interactions, processing, and tissue specificity that are important to pigmentation in mammals.
Two pigmentation related genes have recently been cloned which map to the brown (b) and albino (c) loci of mice; these loci influence the quality and quantity, respectively, of melanin produced by melanocytes. Both these gene products are biochemically similar and have extensive amino acid sequence similarity to each other and to lower forms of tyrosinase (EC 1.14.18.1), a copper binding enzyme responsible for melanin production. In order to characterize the catalytic activities of these molecules, we have synthesized peptides and prepared antibodies to them which specifically recognize the gene products in question. By use of immune affinity purification protocols, we have isolated the proteins encoded by the brown and albino loci and have determined that both have the catalytic functions ascribed to tyrosinase, i.e. hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to DOPAquinone. These are the critical reactions to melanogenesis since melanin pigment can be spontaneously produced from those products. The specific activity of the albino locus encoded product is considerably higher than that of the protein encoded by the brown locus, although the latter protein is present in higher quantity in melanocytes than is the protein encoded by the albino locus. These results are surprising since it was anticipated that tyrosinase was the product of single gene locus, and suggest that regulation of melanogenesis in mammals is controlled at the enzymatic level by several different gene products.
The mouse brown locus encodes a putative membrane-bound metalloenzyme, tyrosinase-related protein-1 (TRP-1). We have examined the effect on mRNA expression of the locus of a number of mutant alleles. The common null mutant allele, brown, produces wild-type levels of TRP-1 mRNA, which is nonfunctional. Another recessive allele, cordovan-Harwell, has an intermediate, dark-brown phenotype and produces only very low levels of presumably normal TRP-1 mRNA. Two dominant alleles appear to act by killing the melanocyte in which they are expressed. One of them, Light, has normal size and amounts of TRP-1 mRNA. The other, White-based brown, produces no detectable TRP-1 mRNA. It has a gross DNA rearrangement at the 5' end, and we speculate that this results in activation of transcription of sequences not usually seen in melanocytes, and that this is toxic to the cell. The relationship between phenotype and molecular structure at the locus is discussed, and we draw some general principles applicable to other developmental genes.
The gp75 antigen is an abundant intracellular glycoprotein expressed in melanosomes of human pigmented melanocytes and melanomas. IgG antibodies in sera of a patient with metastatic melanoma have been shown to immunoprecipitate gp75, suggesting that immunological tolerance against gp75 can be broken. The mouse mAb TA99, which specifically recognizes gp75, was used to isolate and purify the antigen. Amino acid sequences of three internal peptides were determined from the purified gp75 polypeptide. cDNA clones were isolated by screening with oligonucleotides based on these peptide sequences. The gp75 peptides and cDNA had approximately 90% identity with, respectively, the derived amino acid and nucleotide sequences of a mouse gene that maps to the b (brown) locus. The brown locus determines coat color in the mouse, suggesting that gp75 regulates or influences the type of melanin synthesized.
The effect of melanocyte-stimulating hormone (MSH) on the differentiation of mammalian melanocytes has been widely studied since the early 1950s. There have been many reports about the stimulatory effect of MSH on melanin production and specifically on the activity of tyrosinase, the critical enzyme in the melanogenic pathway. However, few and variable results have been obtained concerning the effect of this hormone on the regulation of DOPAchrome tautomerase (TRP2), another melanogenic enzyme which functions later in the melanogenic pathway, or on other melanogenic activities, such as TRP1. In this study, we show that the MSH-induced stimulation of tyrosinase is accompanied by no significant change in the synthesis or catalytic activities of other melanogenic enzymes such as TRP1 or TRP2. This in turn elicits a dramatic increase in melanin production accompanied by a significant decrease in the incorporation of carboxylated precursors into that melanin biopolymer, although the biological implication of that is still unclear.
Since the characterization of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as a major melanogenic intermediate, the fate of this compound and the mechanisms of its incorporation into the melanin polymer have become major issues in the study of melanogenesis. DHICA is a stable dihydroxyindole with a low rate of spontaneous oxidation, suggesting that enzymatic mechanism(s) might contribute to its evolution. The most obvious candidates are the melanosomal tyrosinases. We have recently shown that mouse melanosomes contain two electrophoretically distinct tyrosinase isoenzymes, termed low electrophoretic mobility tyrosinase (LEMT) and high electrophoretic mobility tyrosinase (HEMT), that can be resolved and purified. In this study, we report immunological evidence indicating that LEMT corresponds to the protein encoded by the brown locus (termed tyrosinase-related protein-1, TRP1), while HEMT corresponds to the tyrosinase encoded by the albino locus. We have compared the ability of both isoenzymes to catalyze DHICA evolution as determined by high performance liquid chromatography; although LEMT is a relatively poor tyrosine hydroxylase and DOPA oxidase as compared to HEMT, it was readily able to accelerate DHICA consumption concomitant with the production of a brownish product. However, the DHICA conversion activity of HEMT was barely detectable. The ability of purified LEMT to catalyze DHICA conversion could be almost completely abolished by treatment with heat or trypsin, and was inhibited in a concentration dependent way by the tyrosinase inhibitor 2-phenylthiourea and by L-tyrosine. Moreover, in the presence of low concentration of ascorbate, the DHICA conversion activity of LEMT displayed a lag period which was progressively longer at higher ascorbate concentrations. Based on the relationship between ascorbate added, enzyme activity, and lag period, it is very likely that the DHICA converting activity is indeed a DHICA oxidase activity. This was further proven by the demonstration that the product reacts rapidly and efficiently with the quinone trapping reagent 3-methyl-2-benzothiazolinone hydrazone, yielding a colored adduct similar to the one obtained with DOPAquinone. The DHICA oxidase activity of LEMT displayed a Km for DHICA of about 0.8 mM, as compared to 1.9 mM for L-DOPA and 0.23 nM for L-tyrosine. These results suggest that TRP1, the product of the brown locus, is indeed a tyrosinase with DHICA oxidase activity. However, as opposed to the tyrosinase encoded by the albino locus, TRP1's role in melanogenesis could be more directly related to DHICA metabolism than to the first steps of the pathway.
Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.
This chapter focuses on melanoma cells. Melanocytes synthesize melanin in a reaction controlled by the enzyme monophenyl, dihydroxyphenylalanine: oxygen oxidoreductase. Melanocytes in the skin of adult mammals divide infrequently, perhaps once a year and malignant melanoma cells divide once a day. Commonly used lines are the B16, Harding–Passey, and Cloudman strains (mouse), the Fortner (RPM1) strain (hamster), and many human strains. The chapter describes the techniques for culturing Cloudman S91 in monolayers and clones, and to outline some of the problems unique to melanin-producing cells that are encountered in normal culturing procedures and in some biochemical analyses. Culture procedures are straightforward. Low amounts of cyclic adenosine monophosphate (AMP) markedly stimulate the proliferative rate of Cloudman S91 cells, while higher amounts of cyclic AMP inhibit proliferation. Melanin interferes with the isolation of RNA because it binds tightly to RNA molecules.
The gp75 antigen is an abundant intracellular glycoprotein expressed in melanosomes of human pigmented melanocytes and melanomas. IgG antibodies in sera of a patient with metastatic melanoma have been shown to immunoprecipitate gp75, suggesting that immunological tolerance against gp75 can be broken. The mouse mAb TA99, which specifically recognizes gp75, was used to isolate and purify the antigen. Amino acid sequences of three internal peptides were determined from the purified gp75 polypeptide. cDNA clones were isolated by screening with oligonucleotides based on these peptide sequences. The gp75 peptides and cDNA had approximately 90% identity with, respectively, the derived amino acid and nucleotide sequences of a mouse gene that maps to the b (brown) locus. The brown locus determines coat color in the mouse, suggesting that gp75 regulates or influences the type of melanin synthesized.
Background:
Melasma is difficult to clear. Many agents have been used, such as hydroquinone, and glycolic acid and glycolic acid peels, kojic acid, a tyrosinase inhibitor in the fungus Aspergilline oryzae.
Objective:
To see if the addition of 2% kojic acid in a gel containing 10% glycolic acid and 2% hydroquinone will improve melasma further.
Methods:
Forty Chinese women with epidermal melasma were treated with 2% kojic acid in a gel containing 10% glycolic acid and 2% hydroquinone on one half of the face. The other half was treated with the same application but without kojic acid. The side receiving the kojic acid was randomized. Determination of efficacy was based on clinical evaluation, photographs and self-assessment questionnaires at 4 weekly intervals until the end of the study at 12 weeks. The non-parametric Wilcoxon's rank sum test was used for statistical analysis.
Results:
All patients showed improvement in melasma on both sides of the face. The side receiving the kojic acid did better. More than half of the melasma cleared in 24/40 (60%) patients receiving kojic acid compared to 19/40 (47.5%) patients receiving the gel without kojic acid. In 2 patients, there was complete clearance of melasma, and this was on the side where kojic acid was used. Side effects include redness, stinging, and exfoliation. These were seen on both sides of the face, and they settled by the third week.
Conclusion:
The addition of kojic acid to a gel containing 10% glycolic acid and 2% hydroquinone further improves melasma.
Backgroound: alpha-Hydroxy acids (AHAs) have been reported to improve aging skin. The mechanisms of action of AHAs on epidermal and dermal compartments need clarification. Objective: Our purpose was to determine the effects of AHAs on photoaged human skin by clinical and microanalytic means. Methods: Patients applied a lotion containing 25% glycolic, lactic, or citric acid to one forearm and a placebo lotion to the opposite forearm for an average of 6 months. Thickness of forearm skin was measured throughout the study. Biopsy specimens from both forearms were processed for analysis at the end of the study. Results: Treatment with AHAs caused an approximate 25% increase in skin thickness. The epidermis was thicker and papillary dermal changes included increased thickness, increased acid mucopolysaccharides, improved quality of elastic fibers, and increased density of collagen. No inflammation was evident. Conclusion: Treatment with AHAs produced significant reversal of epidermal and dermal markers of photoaging.
Alpha Hydroxy Acids are a group of organic acids which have recently become popular in the treatment of several skin conditions.
This article explores some of the clinical uses of these products in the treatment ofichthyosis, acne, hyperpigmentation and photo‐damage. Specific recommendations are given for the treatment of each condition.
Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of 3H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.
Several genes critical to the regulation of melanin production in mammals have recently been cloned and characterized. They map to the albino, brown, and slaty loci in mice, and encode proteins with similar structures and features, but with distinct catalytic capacities. The albino locus encodes tyrosinase, an enzyme with three distinct catalytic activities—tyrosine hydroxylase, 3,4-dihydroxyphenylalanine (DOPA) oxidase and DHI (5,6-dihydroxyindole) oxidase. The brown locus encodes TRP-l (tyrosinase-related protein-I), which has the same, but greatly reduced, catalytic potential. The slaty locus encodes TRP-2, another tyrosinase related-protein, which has DOPAchrome tautomerase activity. In this study we have examined the enzymatic interactions of these proteins, and their regulation by a novel melanogenic inhibitor. We observed that tyrosinase activity is more stable in the presence of TRP-l and/or TRP-2, but that the catalytic function of TRP-2 is not affected by the presence of TRP-1 or tyrosinase. Other factors also may influence melanogenesis and a unique melanogenic inhibitor suppresses tyrosinase and DOPAchrome tautomerase activities, but does not affect the spontaneous rate of DOPAchrome decarboxylation to DHI. The results demonstrate the catalytic functions of these proteins and how they stably interact within a melanogenic complex in the melanosome to regulate the quantity and quality of melanin synthesized by the melanocyte.
When human skin is exposed to ultraviolet (UV) light, a highly complex cascade of events ensues that culminates, among other things, in increased skin melanin content. From analyses at the tissue and cellular level, it has been shown that following exposure to UV light there is an increase in the number of active melanocytes in the basal layer of the epidermis, and individual melanocytes are stimulated to produce more melanin. In addition, the rate of transfer of melanosomes from melanocytes to keratinocytes is apparently increased, although the role of UV light in this process remains to be demonstrated. Recent biochemical evidence is reviewed on factors that regulate these processes. A plausible explanation for the effects of UV on pigmentation is that there are mechanisms in the skin for the orderly, regulated reception of UV signals that are then transduced to initiate the cascade. The signals involve both melanocytes and keratinocytes, and available evidence supports a model in which melanotropins and their receptors play a central role in the process.
alpha-MSH was found to decrease the recently characterized dopachrome tautomerase activity in cultures of B16/F10 mouse melanoma cells. Other stimulating agents of melanogenesis, like dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, theophylline, retinol, and retinoic acid, caused the same effect. The grade of inhibition depended on the nature of the agent and the time of exposure. In all cases, both melanin production and tyrosinase activity were activated by these treatments, although the grade of tyrosine hydroxylase and dopa oxidase stimulation was different. Moreover, no correlation among the intensities of dopachrome tautomerase inhibition and tyrosinase activation by the tested agents could be obtained. The significance of these results in the regulation of mammalian melanogenesis is discussed.
Melanogenesis is regulated in large part by tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1), and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmented (Blt/Blt) coat versus the wild-type black (B/B). We show that the b locus codes for a glycoprotein with the activity of a catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, we conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. Our studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the Blt mutation renders the protein susceptible to rapid proteolytic degradation.
Alpha hydroxy acids and alpha keto acids applied topically in lower concentrations reduce the thickness of hyperkeratotic stratum corneum by reducing corneocyte cohesion at lower levels of the stratum corneum. This property permits efficient clinical control of dry skin, ichthyosis, follicular hyperkeratosis, and other conditions characterized by retention of stratum corneum. Applied topically in higher concentrations, these acids cause epidermolysis. This property provides a new alternative for treating seborrheic keratoses, keratoses commonly known as "age spots," actinic keratoses, and verrucae vulgares; all of which lesions involve distinct epidermal hyperplasia as well as retention of stratum corneum. Facial wrinkles can be modified with topical alpha hydroxy acids, applied in higher concentrations as office procedures, and concomitant daily home application of lower concentrations.
A mouse melanoma cDNA clone was isolated by virtue of its reactivity with two antisera raised against tyrosinase (EC 1.14.18.1) from two species, hamster and mouse. The cDNA (5A) cross-hybridizes with another, pMT4 [Shibahara, S., Tomita, V., Sakakura, T., Nager, C., Bhabatosh, C. & Muller, R. (1986) Nucleic Acids Res. 14, 2413-2427], previously thought to encode mouse tyrosinase. Two other cDNAs, one human and one mouse, have been reported recently [Kwon, B. S., Haq, A. K., Pomerantz, S. H. & Halaban, R. (1987) Proc. Natl. Acad. Sci. USA 84, 7473-7477; and Yamamoto, H., Takeuchi, S., Kudo, T., Makino, K., Nakata, A., Shinoda, T. & Takeuchi, T. (1987) Jpn. J. Genet. 62, 271-277] as candidates for tyrosinase, and they map at or very close to the mouse albino (c) locus. The proteins they encode are very similar to each other but are distinct from (although related to) the pMT4-encoded protein. Here I use recombinant inbred strains to localize pMT4 at or close to the mouse brown (b) locus. I suggest that the gene mapping to c is the authentic tyrosinase gene, whereas that mapping to b encodes a tyrosinase-related protein. All b mutations in laboratory strains are associated with the same diagnostic Taq I fragment, suggesting that all derive from the same original mutation. I discuss possible function(s) of the tyrosinase-related protein.
Melanin biosynthesis involves the enzymatic conversion of tyrosine, via dihydroxyphenylalanine (dopa), to melanin through a number of intermediate steps. It is generally assumed that the major rate-limiting factor in this process is the amount of active tyrosinase (monophenol monoxygenase; monophenol, dihydroxyphenyl- alanine: oxygen oxidoreductase, EC 1.14.18.1) available since when dopa has been oxidized by tyrosinase to dopa quinone the succeeding steps can occur spontaneously through auto-oxidation. We now present evidence that a later step, namely the conversion of dopachrome to 5,6- dihydroxyindole-2-carboxylic acid may also be under regulatory control. We have found a factor(s) from 3 different melanomas (Cloudman, B16, and Greene) which catalyzes this step. This activity is also present in mushrooms, which contain a potent pigment-synthesizing system, but not in a variety of cell lines of nonmelanocytic origin. We have not yet identified the factor but it is of relatively low molecular weight (less than 1000 daltons by Sephadex G10 gel filtration), negatively charged at pH 6.8, and stable to boiling. The factor contains no detectable sulfhydryl groups. In crude cell extracts the factor appears to be complexed to tyrosinase since it cochromatographs with tyrosinase through the early steps of purification. The factor binds tightly to QAE- Sephadex anion exchange resin and can be separated from tyrosinase by differential salt elution. We have not yet demonstrated that the factor has a physiological role in melanogenesis but we feel such a role is likely since the activity was found only in cells with melanin-synthesizing systems.
The biological functions of pyrroloquinoline quinone (PQQ), a bacterial redox coenzyme and potent radical scavenger, have not been elucidated in mammals. In this paper, we studied the effects of PQQ on tyrosinase activity and subsequent melanogenesis in murine B16-F10 melanoma and found that alpha-Melanocyte stimulating hormone (MSH)-induced melanogenesis was inhibited by 6.3 to 25 microM PQQ in a dose-dependent manner. Moreover, PQQ inhibited MSH-induced tyrosinase activity by suppressing tyrosinase mRNA expressed by MSH. However, PQQ had no effect on MSH-stimulated cyclic adenosine 3', 5'-monophosphate (cAMP) production. These observations suggest that PQQ inhibits the expression of tyrosinase mRNA at a post receptor level and that PQQ may be useful in investigating hormone actions mediated by cAMP.
Several genes critical to the regulation of melanin production in mammals have recently been cloned and characterized. They map to the albino, brown, and slaty loci in mice, and encode proteins with similar structures and features, but with distinct catalytic capacities. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, the brown locus encodes TRP1 (tyrosinase-related protein-1), and the slaty locus encodes TRP2, another tyrosinase related-protein. TRP2 functions as DOPAchrome tautomerase, an enzyme that preserves the carboxylic acid content of melanins, which would be spontaneously lost in its absence, while TRP1 is able to oxidize the DHICA produced by TRP2. In this study we have used three different systems (immune-affinity purified melanogenic enzymes, mutant melanocytes, and transfected cells) to examine the enzymatic interactions of these proteins, and their stabilization in a complex which significantly increases their physiological half-life. When extrapolated to the melanocyte, our results demonstrate the catalytic functions of these proteins and suggest how they might stably interact within a melanogenic complex in the melanosome to regulate the quantity and quality of melanin synthesized.
Several genes critical to the enzymatic regulation of melanin production in mammals have recently been cloned and mapped to the albino, brown and slaty loci in mice. All three genes encode proteins with similar structures and features, but with distinct catalytic capacities; the functions of two of those gene products have previously been identified. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, while the slaty locus encodes tyrosinase-related protein 2 (TRP2), an enzyme with a single specific, but distinct, function as DOPAchrome tautomerase. Although the brown locus, encoding TRP1, was actually the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) has been in general dispute. In this study we have used two different techniques (expression of TRP1 in transfected fibroblasts and immunoaffinity purification of TRP1 from melanocytes) to examine the enzymatic function(s) of TRP1. The data demonstrate that the specific melanogenic function of TRP1 is the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to a carboxylated indole-quinone at a down-stream point in the melanin biosynthetic pathway. This enzyme activity appears to be essential to the further metabolism of DHICA to a high molecular weight pigmented biopolymer.
Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (< or = 0.8 microCi/ml of 3H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.
Retinoic acid (RA) is a hormone-like agent involved in the control of cell differentiation. The most characteristic feature of melanocyte differentiation, melanogenesis, is stimulated by UV radiations. Excessive chronic sun exposure results in irregular skin hypermelanosis that can be partially corrected by topical RA. The basic mechanisms underlying this effect of RA are unknown. To determine whether RA can directly modulate excessive melanin synthesis, we analyzed the in vitro effect of cis- and trans-RA on UVB-induced melanogenesis in S91 mouse melanoma cells and in normal human melanocytes (NHM). In both cells types, the two RA isoforms significantly decreased the UVB-stimulated melanogenesis in term of tyrosinase activity and melanin neosynthesis. To correlate changes in melanogenesis with the expression of melanogenic enzymes, we determined the neosynthesis rate of tyrosinase, tyrosinase-related protein-1 (TRP-1/gp 75) and tyrosinase-related protein-2 (TRP-2/DOPAchrome tautomerase). Here we show that UVB-induced melanogenesis in NHM is related to an increased synthesis of tyrosinase and TRP-1 and to a dramatic decrease of TRP-2 expression. RA inhibition of UVB-induced melanogenesis acts at the post-transcriptional level leading to a decreased tyrosinase and TRP-1 synthesis. We also show that in NHM, inhibition of TRP-2 following UVB-treatment is significantly reversed by RA. This demonstrates a negative correlation between melanogenesis and TRP-2 expression.
Using melanotic cells (SK-MEL-23 and G361) and amelanotic cells (C32 and SK-MEL-24) of human melanoma, this study examined whether UV-B irradiation has a direct stimulatory effect on the expression of genes involved in melanogenesis. Our initial screening of methylthiazol tetrazolium (MTT)-formazan formation assay indicated a low dose of ultraviolet (UV)-B irradiation, 2.5 and 5.0 mJ/cm², can metabolically stimulate these cells. Repeated exposure of UV-B at 5.0 mJ/cm² for seven consecutive days resulted in increased tyrosinase activity and melanin synthesis in SK- MEL-23 and G361 cells, but not in C32 and SK-MEL-24 cells. On reverse-transcription – polymerase chain reaction and immunoprecipitation studies, the two melanotic cell lines exhibited upregulated expression of mRNA and antigenic epitopes of tyrosinase, tyrosinase-related protein (TRP-1; gp75/HMSA-5), and lysosomal membrane associated protein (Lamp-1). The amelanotic cell line, C32, expressed the tyrosinase gene and protein constitutively but revealed no active tyrosinase or melanin synthesis even after UV-B exposure. Another amelanotic cell line, SK-MEL-24, exhibited no expression of tyrosinase gene and protein before and after UV-B exposure and, therefore, no melanin synthesis. Both C32 and SK-MEL-24 showed no gene or protein expression of TRP-1 before or after UV exposure, but upregulation of the Lamp-1 gene and protein expressions after exposure.
We conclude that tyrosinase is the key enzyme responsible for UVB-induced melanogenesis. Both TRP-1 and Lamp-1 act together in melanogenesis, TRP-1 being essential and necessary. There is no change in the expression of CD63 lysosomal membrane protein at either the mRNA or protein level.
Many genes mapping to pigmentation loci are involved in the regulation of melanin synthesis in the mouse. The brown (b) locus controls black/brown coat coloration, and its product has significant homology to the key melanogenic enzyme tyrosinase. This has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have established lines of mouse fibroblasts stably expressing the b-protein by co-transfection of a b-protein expression vector and a plasmid conferring resistance to the antibiotic G418. The b-protein synthesised by these cells has the expected molecular mass of 75 kDa and reacts with three different anti-b-protein antibodies. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid. Since this activity is not present in untransfected cells we conclude that the b-protein has dopachrome tautomerase activity. Fibroblasts do not contain melanosomes, the specialised organelles in which the b-protein is located in melanocytes. Nevertheless, indirect immunofluorescence localisation of the b-protein in transfected fibroblasts produces a distinctive pattern of intense juxtanuclear staining combined with punctate cytoplasmic staining. Double-labelling shows co-localisation of the b-protein with the late endosomal/lysosomal markers beta-glucuronidase and LAMP-1, both in transfected fibroblasts and in mouse melanoma cells. These findings are consistent with the hypothesis that melanosomes are closely related to lysosomes.
Pigmentation in mammals is under complex genetic control. Amongst the genes involved in this process, those encoding tyrosinase and the tyrosinase-related-proteins 1 and 2 have been well characterized and share a number of features. Recently, the murine tyrosinase-related-protein-2 gene was shown to encode dopachrome-tautomerase activity and was mapped to the slaty locus. Human tyrosinase and tyrosinase-related-protein-1 genes have been isolated and demonstrate a high degree of similarity with their murine counterparts. However, there has been limited data regarding the existence of a human homologue for tyrosinase-related-protein-2 and its relationship to the other tyrosinase-related proteins. In this study, we report the molecular isolation of a cDNA encoding a human homologue of the murine tyrosinase-related-protein-2/dopachrome tautomerase. We have characterized its expression in human melanocytic cells and have analyzed the relationship between dopachrome tautomerase and tyrosinase activities with the level of visible pigmentation in these cells. TYRP2 has been mapped to the chromosomal region 13q32, thus extending a region of synteny with mouse-chromosome 14.
Using antibodies that recognize either tyrosinase, tyrosinase-related protein-1 (TRP1), or tyrosinase-related protein-2 (TRP2, DOPAchrome tautomerase), the quantities of those melanogenic enzymes were analyzed in five melanoma cell lines that possess various degrees of melanin production. All cells except JB/MS-W increased melanin production four to 30 times after 4 d of melanocyte-stimulating hormone (MSH) treatment. Melanin production by JB/MS-W cells was always under background, with or without MSH treatment. There was a positive correlation between quantities and synthetic rates of those melanogenic enzymes and their melanin formation or DOPAchrome tautomerase activities. The activity of a heat-resistant melanogenic inhibitory factor was also analyzed. The results showed, surprisingly, that pigmented cells showed higher levels of melanogenic inhibitors activity. Tyrosinase activity was increased dramatically whereas the level of melanogenic inhibitor was remarkably decreased following MSH treatment. Interestingly, melanogenic inhibitor derived from JB/MS-W cells suppressed not only tyrosinase but also DOPAchrome tautomerase, another enzyme functional in melanin production. These results clearly suggest that melanin production is regulated by a subtle balance between the activities of these enzymes and other factors such as the melanogenic inhibitor.
The levels of tyrosinase mRNA and tyrosinase activity were analyzed in two amelanotic melanoma cell lines, D1(178) (hamster) and G-361 (human). Neither tyrosinase mRNA nor tyrosinase activity were detected in D1(178) cells. On the other hand, both tyrosinase mRNA and weak tyrosinase activity were detected in G-361 cells. Assuming that the different types of melanogenic inhibitors affected melanogenesis in these two amelanotic melanoma cells in different manners, we performed a screening of melanogenic inhibitors in these two cell lines. As an isolated tyrosinase suppressive melanogenic inhibitor, ascorbic acid and glutathione were identified from D1(178) cells and G-361 cells, respectively. Furthermore, lactic acid was identified from D1(178) cells as an isolated tyrosinase non-suppressive melanogenic inhibitor. B-16 mouse melanotic melanoma cells were depigmented by treatment with lactic acid. The melanogenesis suppression by lactic acid in B-16 cells was found to be due to inhibition of tyrosinase gene expression.
The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
Recent advances in melanogenesis have focused on the role of dihydroxyindole-2-carboxylic acid[(HO)2IndCOOH]. For example, it has been shown that formation of (HO)2IndCOOH from dopachrome is catalyzed by dopachrome tautomerase, that the melanogenic protein tyrosinase-related protein (TRP)-1 can oxidize (HO)2IndCOOH to its indole quinone, that (HO)2IndCOOH-melanins can be synthesized chemically, that mammalian melanins are naturally rich in (HO)2IndCOOH subunits, and that (HO)2IndCOOH is incorporated into melanins of melanomas in mice. The question thus emerges as to the mechanism(s) by which (HO)2IndCOOH and other precursors become incorporated into melanins in vivo. Accordingly, an activity was partially purified that catalyzed melanin formation with (HO)2IndCOOH as a substrate. Analyses of the (HO)2IndCOOH polymerization factor from Cloudman melanoma cells revealed the following: it was proteinaceous in that it was heat labile and destroyed by proteinase K; it was a glycoprotein in that it adhered to wheat germ agglutinin and was eluted with N-acetyl glucosamine; it was located predominantly in the melanosomal fraction of cell homogenates; the activity was reduced by exposure to the metal chelators EDTA and EGTA, but not by phenylthiourea, a tyrosinase inhibitor; the (HO)2IndCOOH polymerization reaction was inhibited by superoxide dismutase. In addition, the activity was found with the mouse pmel 17/silver locus protein immunopurified from human melanoma cells, and was significantly reduced in extracts of mouse melanocytes cultured from silver (si/si) mice compared to extracts from Si/Si melanocytes. In summary, an activity has been identified in human and mouse melanoma cells that catalyzes the superoxide-dependent polymerization of (HO)2IndCOOH to melanin in vitro, and appears to be a function of the pmel 17/silver protein of the human pmel 17 gene and the mouse silver locus.