Post-Inflammatory Hyperpigmentation in Dark Skin: Molecular Mechanism and Skincare Implications.

Human skin is characterized by significant diversity in color and tone, which are determined by the quantity and distribution of melanin pigment in the epidermis. Melanin absorbs and reflects ultraviolet radiation (UVR), preventing the damage to genomic DNA in the epidermis and degradation of collagen in the dermis; therefore, darker skin types are thought to be well protected from the photodamage because of the high melanin content. However, increased content of melanin in combination with the extrinsic stress factors causing inflammation such as excess UVR, allergic reactions, or injury can also frequently lead to cosmetic problems resulting in discoloration and scarring. This review summarizes current knowledge on histopathology and likely molecular signatures of one of the most common problems, post-inflammatory hyperpigmentation (PIH). The mechanisms proposed so far are subsequently discussed in the context of other factors characterizing darker skin types. This includes the common cellular features, organization of upper skin layers, and major biomarkers, with particular emphasis on increased propensities to systemic and localized inflammation. Enhanced or prolonged inflammatory responses can not only affect the process of melanogenesis but also have been implicated in injury-related skin pathologies and aging. Finally, we summarize the major cosmetic treatments for PIH and their known anti-inflammatory targets, which can be beneficial for darker skin tones and combined with broad-spectrum filters against UVR.

Anti-Glycation and Anti-Aging Properties of Resveratrol Derivatives in the in-vitro 3D Models of Human Skin.

Purpose: Human skin undergoes modifications affecting its structural properties and barrier functions involved in protection against age-related damage. Glycation is a non-enzymatic reaction between macromolecules and sugars causing alterations to the elastic fibers and premature aging of the skin. Glycation can be prevented by a range of bioactive molecules; however, at present only a few of them are validated for inclusion in cosmetic products. There is also a demand for reproducible in-vitro assays demonstrating the anti-aging effect of compounds on the skin. This study aimed to define the potential targets for screening and validation of anti-glycation activity of novel cosmetic candidates from natural products and to provide a plausible mechanism for their anti-aging potential based on 3D skin models. Methods: Dermal fibroblasts and 3D skin models were treated with glycation agent and topical applications of Resveratrol derivatives. The samples were analyzed for advanced glycation end products (AGEs) alongside an organization of elastic fibers and expression of proliferative, senescence, and oxidative stress markers by autofluorescence, immunocytochemistry and quantitative assays. Results: Accumulation of AGEs in the 3D skin model is associated with reduced stratification of the epidermis and re-organization of the collagen in the upper, cell-dense layer of the dermis. Treatment of dermal fibroblasts with Resveratrol, OxyResveratrol, Piceatannol, and Triacetyl Resveratrol ameliorates the effects of glycation consistent with cellular aging. Subsequent topical application of the compounds in skin models results in a reduction in glycation-induced AGEs, an increase in collagen expression and a stratification of the epidermis. Conclusion: Glycation could result in age-related alterations in the structural and cellular organizations of the superficial layers of the skin, which can be restored by Resveratrol derivatives, pointing to their promising capacities as bioactive ingredients in cosmetic products. Insight into the potential parameters affected by skin glycation could also serve as a reference for screening the bioactive molecules for cosmetic purposes.

Effect of seasonal and geographical differences on skin and effect of treatment with an osmoprotectant: Sorbitol.

Human skin maintains an optimal permeability barrier function in a terrestrial environment that varies considerably in humidity. Cells cultured under hyperosmotic stress accumulate osmolytes including sorbitol. Epidermal keratinocytes experience similar high osmolality under dry environmental conditions because of increased transepidermal water loss (TEWL) and concomitant drying of the skin. This study was designed to determine if epidermal keratinocytes, in vitro, could be protected from high osmotic stress, with the exogenous addition of sorbitol. In addition, we evaluated the effect of a formulation containing topical sorbitol on skin barrier and moisturization of subjects living in arid and humid regions in summer as well as in winter. Results from in vitro experiments showed that 50 mM sorbitol protected epidermal keratinocytes from osmotic toxicity induced by sodium chloride. Clinical studies indicated that skin chronically exposed to hot, dry environment appeared to exhibit stronger skin barrier and a lower baseline TEWL. In addition, skin barrier was stronger in summer than in winter. Sorbitol exhibited significant improvement in both barrier repair and moisturization, especially in individuals subjected to arid environmental conditions.

Enzymatically generated hydrogen peroxide reduces the number of acne lesions in acne vulgaris.

A major component to the etiology of acne is the growth and invasion by Propionibacterium acnes. Hydrogen peroxide is an excellent antimicrobial agent but is unstable in most formulations. We have developed a hydrogen peroxide generation system using the enzyme glucose oxidase and glucose. This system is stable in a simple formulation and nonirritating. In a short-term clinical study (4 days), this formulation was effective in reducing the individual lesion size and total number of inflammatory acne lesions. There was a 68% reduction in acne-induced inflammation and 61% reduction in acne size within 4 days of treatment. A long-term clinical study (6 weeks in use) displayed 56% reduction in total number of inflamed lesions and a 45% reduction in noninflamed lesions after 6 weeks. This suggests that topical enzymatically generated hydrogen peroxide may help alleviate acne.

Physiological effect of a probiotic on skin.

INTRODUCTION AND OBJECTIVE: Normal human skin can produce a range of antimicrobial chemicals that play an important part in eliminating potential cutaneous pathogens. Lactobacillus plantarum is a gram-positive bacteria that produces antimicrobial peptides, which when applied to the skin can act like an anti-inflammatory as well as enhance the antimicrobial properties of the skin. Clinical studies were conducted to determine the effect of lactobacillus extract on improvement of skin barrier and reduction of erythema from chemical irritant, skin microflora, and acne. Results show that lactobacillus extract was effective in reducing skin erythema, repairing skin barrier, and reducing skin microflora, thereby exhibiting an effective reduction in acne lesion size and erythema at 5%, but not at 1%. CONCLUSION: Based on the results of these studies, lactobacillus extract (5%) can be used to treat mild acne lesions.

Modification of skin discoloration by a topical treatment containing an extract of Dianella ensifolia: a potent antioxidant.

Skin hyperpigmentation, and the reactions that precipitate it, have been linked to free radicals by the fact that free radical scavengers or antioxidants can slow that hyperpigmentation. We have screened several hundred plant extracts for antioxidants and discovered one that is both a strong antioxidant and can reduce skin hyperpigmentation. Extracts of Dianella ensifolia contain 1-(2,4-dihydrophenyl)-3-(2,4-dimethoxy-3-methylphenyl) propane (DP), which was found to inhibit the free radical 1-1-diphenyl-2-picryl-hydrazyl (DPPH) with an EC(50) value of 78 mum. DP was also found to inhibit Ultraviolet (UV)C-induced lipid oxidation with an EC(50) of about 30 mum. We next investigated the effects of this antioxidant on skin hyperpigmentation. The reduction of discoloration by different topical treatments has been assessed in human volunteers using an in vivo assay for the rate of fading of UVB-induced tan. Two pharmaceutical formulas containing 4% hydroquinone (HQ) were used as positive controls, and we tested the ability of DP, a plant-derived amphoteric antioxidant, to increase performance of non-HQ cosmetic formulations. We found that the cosmetic formula containing DP produced an increase in the rate of fading compared to the two pharmaceutical treatments containing HQ.

A novel method to study the skin-lightening effect of topical materials.

Skin without significant dyschromia is an aesthetic requirement for people worldwide. There are several in vitro methods to determine the whitening potential of actives; however, the in vivo testing of skin whiteners is a long and expensive process. We have designed a rapid clinical method to screen potential skin whiteners using a UV-induced skin tan as a model. Small areas of identical suntan are repeatably induced on the skin, and treatment of these sites allows rapid screening of several skin whiteners within the course of a month. The method provides reproducible results and valuable information about the potential skin-lightening activity of topical preparations.

Gender-linked differences in human skin.

BACKGROUND: The physiology of body organs can be affected by gender. Skin and skin appendages are influenced by sex hormones. OBJECTIVE: This review work has been undertaken to point out the most conspicuous physiological differences observed between men's and women's skin. METHODS: The literature has been searched and relevant results have been gathered. RESULTS: Men's and women's skins differ in hormone metabolism, hair growth, sweat rate, sebum production, surface pH, fat accumulation, serum leptins, etc. Examples of differences in the proneness to cutaneous diseases and skin cancer are quoted. CONCLUSION: The knowledge of gender-linked cutaneous differences might help in preparing male-specific products for more appropriate dermatological treatments or cosmetic interventions.

A rapid method to clinically assess the effect of an anti-acne formulation.

Historically, clinical evaluation of acne treatment has been based on direct visual assessment and the counting of lesions over a period of several weeks of treatment. However, with advancing technology there has been ever-increasing speed in the effectiveness of these treatments. To successfully assess these faster treatments, acne pathology needs to be evaluated in a shorter time frame. The object of these studies was to develop techniques to evaluate individual acne lesions in a shorter time frame and to assess speedier treatment technologies. Ten healthy volunteers with acne lesions on their upper backs were recruited for the study. Two inflamed acne lesions were selected for each treatment, along with lesions to be left untreated, on each volunteer. Each lesion was marked, photographed, and visually graded. A skin surface microscope (Scopeman) was used to visualize size and to grade the lesions by two experts every day for five days. The sites were treated once a day for the course of the study. There was a remarkable reduction in the size and erythema of acne lesions after treatment with the acne formulation as compared to the untreated and vehicle-treated lesions. Individual lesions, both treated and untreated, appeared resolved in 14 days. This resolution can be noticeably accelerated by topical treatments. We have developed a simple and faster clinical method to evaluate the effects of topical anti-acne technology.

SIRT1 promotes differentiation of normal human keratinocytes.

Sir2 regulates lifespan in model organisms, which has stimulated interest in understanding human Sir2 homolog functions. The human Sir2 gene family comprises seven members (SIRT1-SIRT7). SIRT1, the human ortholog of the yeast Sir2 by closest sequence similarity, is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase with enzymatic properties indistinguishable from the yeast enzyme. We studied the involvement of SIRT1 in normal human keratinocyte physiology by a transcriptional microarray analysis of primary keratinocytes either overexpressing or underexpressing SIRT1. Using a systems biology analytical approach, we predicted that SIRT1 induces keratinocyte differentiation through a pathway integral to or overlapping with that of calcium-induced differentiation. We experimentally assayed this prediction and found that the SIRT1 inhibitor nicotinamide inhibited expression of keratinocyte differentiation markers, whereas a SIRT1 activator, resveratrol, enhanced expression of keratinocyte differentiation markers. Similar results were obtained in keratinocytes manipulated to overexpress or underexpress SIRT1, and modulating SIRT1 significantly affected keratinocyte proliferation rates. We conclude that SIRT1 functions in normal human keratinocytes to inhibit proliferation and to promote differentiation.

Osmotic stress induces terminal differentiation in cultured normal human epidermal keratinocytes.

The signals for epidermal differentiation and barrier formation are largely unknown. One possible signal is dehydration or osmotic stress. To test this hypothesis, we investigated the effects of osmotic stress on markers of differentiation of normal human keratinocytes in culture. Hyperosmotic stress treatment of normal human keratinocyte cultures by elevated sorbitol concentrations was observed to induce markers of terminal differentiation. Sorbitol was added to keratinocyte media at 50, 100, 200, and 300 mM final concentration. These concentrations of sorbitol induce a dehydration effect or osmotic stress on the keratinocytes. These sorbitol treatments increased the levels of messenger RNA for the differentiation markers involucrin, transglutaminase, and filaggrin as measured by reverse transcription-polymerase chain reaction. Keratin K1 and K10 and involucrin protein levels were also increased in normal human keratinocyte cultures exposed to increasing osmotic stress. These observations suggest that keratinocytes in the epidermis may use dehydration as a sign to trigger the differentiation of the skin barrier.

UV protective effects of DNA repair enzymes and RNA lotion.

Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes (Micrococcus luteus) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.

Identification of histamine receptors and reduction of squalene levels by an antihistamine in sebocytes.

Overproduction of sebum, especially during adolescence, is causally related to acne and inflammation. As a way to reduce sebum and its interference with the process of follicular keratinization in the pilosebaceous unit leading to inflammatory acne lesions, antihistamines were investigated for their effect on sebocytes, the major cell of the sebaceous gland responsible for producing sebum. Reverse transcriptase-PCR analysis and immunofluorescence of an immortalized sebocyte cell line (SZ95) revealed the presence of histamine-1 receptor (H-1 receptor), and thus indicated that histamines and, conversely, antihistamines could potentially modulate sebocyte function directly. When sebocytes were incubated with an H-1 receptor antagonist, diphenhydramine (DPH), at non-cytotoxic doses, a significant decrease in squalene levels, a biomarker for sebum, was observed. As determined by high-performance liquid chromatography, untreated sebocytes contained 6.27 (+/-0.73) nmol squalene per 10(6) cells, whereas for DPH-treated cells, the levels were 2.37 (+/-0.24) and 2.03 (+/-0.97) nmol squalene per 10(6) cells at 50 and 100 microM, respectively. These data were further substantiated by the identification of histamine receptors in human sebaceous glands. In conclusion, our data show the presence of histamine receptors on sebocytes, demonstrate how an antagonist to these receptors modulated cellular function, and may indicate a new paradigm for acne therapy involving an H-1 receptor-mediated pathway.

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures.

Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

Salicylic acid protects the skin from UV damage.

Aspirin(acetyl salicylate) has long been used as an analgesic. Salicylic acid has been reported to have anti-inflammatory properties. These activities include inhibiting activity of cox-1, cox-2, and NF-kb. In addition, salicylic acid has also been shown in some systems to induce Hsp70. We have demonstrated that salicylic acid inhibits UVB-induced sunburn cell formation, as well as increase the removal of UVB induced TT dimer formation in living skin equivalents. Given these protective properties of salicylic acid, we propose the use of salicylic acid as a topical therapeutic to protect the skin from sun damage.

A water soluble extract from Uncaria tomentosa (Cat's Claw) is a potent enhancer of DNA repair in primary organ cultures of human skin.

Cat's Claw (Uncaria tomentosa) water extracts, essentially free of oxindole alkaloids, have been shown to possess a broad spectrum of biological activity including DNA repair enhancement and antiinflammatory properties. These two biological mechanisms are key molecular targets to develop treatments that protect skin exposed to ultraviolet light from the sun. Because C-Med-100, a Cat's Claw water extract, is the only documented natural source of components that can up-regulate simultaneously both DNA repair and antiinflammation, its ability to modulate DNA repair in human skin organ cultures was undertaken. For this purpose skin cultures were treated with or without 5 mg/mL C-Med-100, irradiated with 0-100 mJ/cm2 UVB, and microscopically analysed for necrosis as well as the level of pyrimidine dimers using immunofluorescent TT-dimer antibody staining. The data clearly demonstrated that co-incubation with C-Med-100 reduced skin cell death from UV exposure, and this protection was accounted for by a concomitant increase in DNA repair. Based on these results, it was concluded that C-Med-100 was a natural plant extract worthy of further consideration as a sunscreen product.

Keratinocytes act as a source of reactive oxygen species by transferring hydrogen peroxide to melanocytes.

Basal hydrogen peroxide (H(2)O(2)) levels in normal human epidermal keratinocytes (NHEK) and melanocytes (mel) were compared on a per cell basis and found to be significantly higher in keratinocytes. Since H(2)O(2) is a neutral molecule capable of permeating through cellular membranes, we then investigated the possibility that H(2)O(2) transfer might occur between these two types of cells. Because the ratio of keratinocytes to mel in skin is 36:1, keratinocytes may act as a source of reactive oxygen species (ROS) even by passive diffusion and, thus, affect melanocytic functions. In order to measure H(2)O(2) transfer, a fluorescence-based co-culture system was developed in which mel were first pre-labeled with 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate (DCFdA). When mel were co-cultured with keratinocytes, fluorescence increased as a function of keratinocyte cell number. Thus, for mel incubated with 1-, 1.5-, and 2-fold the number of keratinocytes, fluorescence increased by 22.6% (+/-2.8%), 25.6% (+/-4.8%), and 39.9% (+/-4.1%), respectively. Separating the cells with a transwell membrane did not prevent the transfer, whereas the addition of catalase to media significantly reduced the transfer of H(2)O(2) to mel. In conclusion, keratinocytes appear to be a previously unexamined source of ROS that may affect neighboring skin cells, such as mel, and, as a result, may influence the process of melanogenesis or contribute to the progression of vitiliginous lesions.

Normal human epidermal keratinocytes treated with 7-dehydrocholesterol express increased levels of heat shock protein.

Human skin, and its isolated cells, respond to insults with a variety of repair and protective mechanisms. One such mechanism is the production of heat shock proteins (HSPs). Heat shock proteins help the other cellular proteins fold correctly into their active three-dimensional structures. Therefore, they can enhance the survival of cells under harsh, denaturing conditions. In order to develop a means of promoting the heat shock response to prepare the skin to withstand insult, we are investigating materials that appear to protect the skin biologically. One such material is vitamin D3 and its precursors. We have observed that keratinocytes treated with 7-dehydrocholesterol (7-DHC), a precursor of vitamin D3, have increased levels of protein and mRNA for heat shock proteins. In addition, we observed that topically applied 7-DHC increases the minimal dose of UVB required to induce erythema. These data suggest that 7-DHC can induce heat shock proteins in skin keratinocytes and that they will be more resistant to UVB insult.

Evidence and utility of melanin degrading enzymes.

The biochemical synthesis of human melanin is understood in some detail. However, little is known about melanin degradation and catabolism of melanin. We hypothesize that human skin contains enzymes that degrade melanin and these enzymes can be used to reduce skin color. To test this hypothesis, HaCaT keratinocytes and normal human keratinocytes in culture were pulse labeled for one hour with radiolabeled synthetic melanin. This melanin was synthesized in vitro using tyrosinase enzyme from mushrooms and using radiolabeled [14C]3,4-dihydroxyphenylalanine (DOPA) as a substrate. After the initial pulse labeling, samples of both the cells and media were taken at 2, 4, 6 and 18 hours. Over these time periods the counts remaining in the media and cell fraction were significantly decreased. This data suggests the need for new protein synthesis and the lysosome organelle function for the degradation. Melanin degrading extracts isolated from Aspergillus fumigatus and Saccharomyces cerevisiae were applied to human skin. These extracts cause significant reduction in UVB induced pigmentation. These extracts may be useful in developing new whitening products to even skin color and tone.

Ultraviolet-B-induced oxidative DNA base damage in primary normal human epidermal keratinocytes and inhibition by a hydroxyl radical scavenger.

To evaluate the effects of ultraviolet-induced environmental trauma on human skin cells, primary normal human epidermal keratinocytes were exposed to ultraviolet-B radiation (290-320 nm). We found that relatively low doses of ultraviolet-B (62.5-500 mJ per cm2) caused dose-dependent increases in 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a biomarker of oxidative DNA damage. Unirradiated normal human epidermal keratinocytes contained 1.49 (+/- 0.11) 8-oxo-dG per 10(6) 2'-deoxyguanosine (dG) residues in cellular DNA, which increased linearly to as high as 6.24 (+/- 0.85) 8-oxo-dG per 10(6) dG after irradiation with 500 mJ per cm2. Further, this oxidative damage was reduced by 60.7% when the cells were pretreated with 1 mM mannitol. As hydrogen peroxide (H2O2) is known to be generated during oxidative stress, its accumulation in ultraviolet-B-irradiated normal human epidermal keratinocytes was also assessed and correlated to 8-oxo-dG formation. An ultraviolet-B-induced increase in H2O2 was observed in normal human epidermal keratinocytes and its production was inhibited by the addition of catalase. Based on the ability of a neutral molecule like H2O2 to permeate membranes, our data indicate that, after ultraviolet-B irradiation, H2O2 migrates from the cytosol to the nucleus where it participates in a Fenton-like reaction that results in the production of hydroxyl radicals (OH*), which may then cause 8-oxo-dG formation in cellular DNA. This conclusion is supported by our data showing that OH* scavengers, such as mannitol, are effective inhibitors of oxidative DNA base damage. Although increased levels of 8-oxo-dG were previously found in immortalized mouse keratinocytes exposed to ultraviolet-B radiation, we now report the induction of 8-oxo-dG in normal human skin keratinocytes at ultraviolet-B doses relevant to human skin exposure.