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Pyridoxine (VB

Skin transparency is a cosmetic asset highly considered by Asian women. Resulting from complex light interactions within the skin, but still not fully understood, there is no simple method to measure it objectively. In this study, skin parameters from digital images were analysed to build a model predicting transparency.Initially, 71 Japanese women (between ages 50 and 60 years) were recruited. This group was then extended to 262 women (between ages 21 and 60 years). Pictures of their faces were taken with the ColorfaceIn the initial group of 71 subjects, 109 parameters correlated with transparency. Half of them are from the cheek and relate to colour or colour homogeneity. If the cheek presented the largest proportion of correlated parameters, best correlations were usually found in other facial regions. Multiple regressions from some cheek parameters can predict up to 80% of transparency. Stepwise regression on parameters from 262 subjects led to a six-parameter model, which is highly correlated (R = 84.1%) with transparency. It combines skin texture, colour, colour homogeneity and gloss parameters. If half of them are from the cheek, the others are from the tear trough, the full face and the cheekbone.Using parameters from digital pictures exclusively, we propose a model that accurately reflects transparency. Including parameters previously shown to relate to transparency, this model should be useful for future dermatology and cosmetic research.

The purpose of this study was to clarify the impact of protein carbonylation on the chemical characteristics of the hair surface focusing on hydrophobicity.First, we examined the validity of methods to evaluate hydrophobicity, one that utilizes the fluorescence of 1-anilinonaphtalene-8-sulfonic acid (1,8-ANS) compared with the contact angles against HThe fluorescence intensity of 1,8-ANS of the hair surface modified chemically correlated well with the contact angles against HThe results suggest that the carbonylation of proteins at the hair surface with aldehydes decreases hydrophobicity and promotes further damage as does bleaching.OBJECTIF: l’objectif de cette étude était de clarifier l’impact de la carbonylation des protéines sur les caractéristiques chimiques de la surface des cheveux en se concentrant sur l’hydrophobicité. MÉTHODES: nous avons d’abord examiné la validité de la méthode d’évaluation de l’hydrophobicité, une méthode qui utilise la fluorescence de l’acide 1-anilinonaphtalène-8-sulfonique (1,8-ANS) par rapport aux angles de contact avec l’H2O, de la surface des cheveux chimiquement modifiés par hydrolyse alcaline ou traités par chlorure d’ammonium stéarylique. Nous avons mesuré la fluorescence provenant du 1,8-ANS, l’angle de contact et les modifications des groupes fonctionnels, aldéhydes (le degré de carbonylation), NH2, COOH et SH des cheveux décolorés à l’H2O2 ou traités par acroléine, à l’aide de méthodes de marquage par fluorescence. RÉSULTATS: l’intensité de la fluorescence du 1,8-ANS de la surface des cheveux modifiés chimiquement était bien corrélée aux angles de contact avec l’H2O. Les résultats ont indiqué que le 1,8-ANS était adapté à l’évaluation de l’hydrophobicité de la surface des cheveux. L’hydrophobicité des cheveux décolorés à l’H2O2 ou carbonylés à l’acroléine a diminué. De plus, les modifications des groupes fonctionnels des cheveux carbonylés par l’acroléine ont augmenté, tout comme celles des cheveux décolorés à l’H2O2. CONCLUSION: les résultats suggèrent que la carbonylation des protéines à la surface des cheveux par des aldéhydes diminue l’hydrophobicité et favorise d’autres dommages, tout comme la décoloration.

Residues of olive fruit (ROF) after the extraction of oils are an increasing source of industrial waste, because olive oil is becoming more popular as a healthy food. It has been reported that olives have some polyphenols that have an antioxidation capability. On the other hand, excess oxidative stress disrupts epidermal barrier function. This study was conducted to determine whether ROF could be utilized as an antioxidant source to reduce industrial wastes and to identify possible active materials to maintain healthy skin. Olive fruits are categorized into two groups depending on the time of harvest, young fruit (YF) and mature fruit (MF). Thus, we examined the antioxidant potentials of extracts from YF and from MF to remove reactive oxygen species (ROS) from biological and chemical aspects. HaCaT keratinocytes cultured with extracts of YF or MF had reduced levels of intracellular ROS in spite of the relatively low chemical capability against ROS scavenging. The biological effects of the YF extract were superior to those of the MF extract. The YF extract showed effective reductions of intracellular ROS and carbonylated proteins that were elevated by the stress-related hormone cortisol. In addition, the YF extract reinforced the intracellular antioxidation capability through the activation of Nrf2 signaling. Taken together, the YF extract was an effective source to reinforce the intracellular antioxidation capability. We conclude from these results that utilizing ROF would lead to the reduction of industrial wastes and would supply active materials to maintain healthy skin.

Carbonylated proteins (CPs) are synthesized by reactions between amino groups in proteins and reactive aldehyde compounds (RAC) yielded from lipid peroxidation initiated by reactive oxygen species (ROS). In the skin, CPs are detected in a higher frequency at sun-exposed sites of the skin in elderly subjects. Since CPs in the stratum corneum (SC) have been reported to correlate with skin water content and transepidermal water loss, it is considered that the accumulation of CPs in the SC involves the loss of skin moisture functions. However, the roles of CPs in the dermis on skin physiology are still unclear. The purpose of this study was to investigate the roles of CPs in the dermis during the progression of photoaged skin and to propose a method to prevent or reduce the synthesis of CPs. The exposure of human normal dermal fibroblasts to CPs increased intracellular ROS levels and the synthesis of intracellular CPs. In addition, CPs caused morphological changes of fibroblasts. Furthermore, CPs caused alterations of mRNA expression levels of dermal matrix-related proteins, such as upregulating MMP-1 and IL-8. These results indicated that CPs disrupt construction of the dermal matrix. On the other hand, α-tocopherol and β-carotene suppressed the synthesis of RAC during lipid peroxidation which resulted in the reduction of UVA-induced CPs in the SC. From these results, we propose that extracellular CPs increase intracellular ROS levels and contribute to alterations of the dermal matrix. To prevent the synthesis of CPs, the application of α-tocopherol or β-carotene could be effective.

Although extracellular carbonylated proteins (CPs) are found at higher levels in sun-exposed skin, their impact on the cellular functions of fibroblasts and their involvement in the progression of photoaging skin are not fully clarified. In our previous study, we reported that extracellular CPs increase levels of intracellular oxidative stress and result in the accumulation of newly synthesized CPs in normal human dermal fibroblasts (NHDF). Furthermore, fibroblasts exposed to CP-BSA, which is a model of extracellular CPs, had upregulated expression levels of mRNAs encoding matrix metalloproteinase-1 (MMP-1) and interleukin-8/CXCL8 (IL-8/CXCL8). These facts suggested the possibility that extracellular CPs induce a fragile structure in the dermis through the degradation of collagen and elastin. The purpose of this study was to characterize the efficacy of natural carotenoids, such as astaxanthin analogs, produced by Hematococus pluvialis (CHPs) to improve the impaired functions of fibroblasts exposed to CPs. CHPs suppressed the intracellular CP levels elevated by CP-BSA, restored mRNA expression levels of factors involved in the formation and assembly of collagen and elastin fibers and improved the formation of those fibers impaired by CP-BSA. We conclude that CHPs function as antiaging substances due to their restoration of the impaired formation of collagen and elastin fibers caused by extracellular soluble CPs.

Background Phagocytosis is an essential process that maintains cellular homeostasis. In the epidermis, the phagocytosis of melanosomes into keratinocytes is important to protect their DNA against damage from ultraviolet B (UVB) radiation. Furthermore, it is considered that UVB activates the phagocytosis by keratinocytes but the detailed mechanism involved is not fully understood. Objective To clarify the mechanism of UVB-enhanced phagocytosis in keratinocytes, we investigated the relationship between the phagocytic ability of keratinocytes and the cell cycle stage of keratinocytes. Methods The phagocytic ability of keratinocytes was evaluated using the incorporation of fluorescent beads after exposure to UVB or oxidative stress. S-phase was evaluated by BrdU incorporation and immunostaining of cyclin D1. Intracellular calcium levels of keratinocytes were measured using the probe Fluo-4AM. Results The phagocytosis of fluorescent beads into keratinocytes was enhanced by UVB and also by oxidative stress. We found that keratinocytes exposed to UVB or oxidative stress were at S-phase of the cell cycle. Furthermore, keratinocytes synchronized to S-phase showed a higher phagocytic ability according to the increased intracellular ROS level. The UVB-enhanced phagocytosis and entrance into S-phase of keratinocytes was abolished by ascorbic acid, a typical antioxidant. Keratinocytes synchronized to S-phase and exposed to UVB or oxidative stress had increased levels of intracellular calcium and their enhanced phagocytic abilities were diminished by the calcium ion chelator BAPTA-AM. Conclusion Taken together, intracellular oxidative stress induced by intracellular calcium influx mediates the UVB-enhanced phagocytic ability of keratinocytes accumulating at S-phase of the cell cycle.

Photoaged skin is characterized by the appearance of pigmented spots such as solar lentigos, deep wrinkles and sags, and progresses due to chronic sun exposure. Among the wavelengths of sunlight, UVA is responsible for the appearance of wrinkles and sags that originate from structural alterations in the dermis of photoaged skin such as the depletion of collagen fibers. Thus, improving and restoring collagen fibers is an effective approach to reduce skin photoaging and maintain a youthful appearance. This study was conducted to evaluate the potential of an extract of Ocimum basilicum (OC), which contains rosmarinic acid (RA), as an anti-photoaging material focusing on the capacity to restore collagen fibers that are disrupted due to intracellular oxidative stress. In spite of their relatively low capacities for chemical scavenging of reactive oxygen species (ROS), both OC and RA showed efficient removal of biological oxidative stress by reducing levels of intracellular ROS and carbonylated proteins (CPs) in fibroblasts following exposure to single or repetitive UVA irradiations. Fibroblasts irradiated with repetitive UVA as a model for chronic sun-exposed cells showed significant increases in matrix metalloproteinase-1 and decreases in type I collagen synthesis and formed reduced numbers of collagen fibers. Since both OC and RA restored the adverse phenomena caused by repetitive UVA irradiation, we conclude that OC containing RA is an effective anti-photoaging material.

Sacran, a polysaccharide isolated from the alga Aphanothece sacrum (Suizenji-nori), has unique physical and physiological characteristics. In a previous study, we reported that sacran improves skin conditions in individuals who suffer from atopic dermatitis (AD), focusing on its trapping function against extrinsic stimuli compared with hyaluronic acid (HA). First, we examined the penetration of sacran through stratum corneum (SC) with an impaired barrier function using immature reconstructed human epidermal equivalents. Sacran penetrates the SC to living cell layers of the epidermis, which suggested that sacran would attenuate adverse influences in keratinocytes caused by extracellular factors such as irritants or proinflammatory cytokines such as interleukin 1α (IL-1α). Sacran markedly reduced the cell damage induced by a nonionic detergent, sodium lauryl sulfate (SLS). Moreover, sacran restored the elevation of intracellular reactive oxygen species (ROS) levels stimulated by SLS and by IL-1α. These effects of sacran were superior to those of HA. In order to investigate the restoration effects of sacran, the influence of sacran on the physical properties of lipid bilayers was evaluated by measuring the order parameter using the ESR spin-labeling method. Because sacran failed to cause changes in the order parameters of liposomes and HaCaT keratinocytes, these results indicate that sacran does not interact with lipid bilayers although it restored changes in the order parameter caused by SLS. The sum of these results demonstrates that sacran reduces the influence of extracellular stimuli by its trapping effects. We conclude that the improving action of sacran is based on its trapping effect.

Photoaging of the skin is marked by obvious wrinkles and mainly depends on degradation of the extracellular matrix (ECM) in the dermis. Matrix metalloproteinase (MMP)-1 is one of the most important factors involved in degradation of the ECM; however, its mechanism of activation is not fully understood. It has been thought that MMP-1 is expressed by dermal fibroblasts as an inactive precursor protein that is activated by proteinases produced by keratinocytes in the epidermis. In this study, we constructed a 3D model of the dermis using collagen-embedded fibroblasts with or without ultraviolet (UV)-A exposure to mimic photoaging in the dermis. Collagen lattices embedded with UV-A-irradiated fibroblasts miniaturized and collagen was degraded to a greater extent than collagen lattices embedded with non-irradiated fibroblasts. The results demonstrate that fibroblasts in this 3D model express activated MMP-1 in the absence of keratinocytes. Moreover, the results confirm that activation of MMP-1 depends on increased plasmin activity in this model and lattice miniaturization was inhibited by the plasmin inhibitor tranexamic acid. Our results suggest that plasmin acts as an activator of MMP-1 and the inhibition of plasmin prevents collagen degradation.

Background Autophagy is known as an intracellular cleanup system necessary to maintain homeostasis of the skin. Many studies have pointed out the relationship between aging and the inactivation of autophagy function, which suggests that the inactivation of autophagy occurs in aged skin. However, the aging process of the skin is complicated compared with other organs, because the skin is localized at the border between the inside of the body and the environment. Thus, skin aging is strongly affected by environmental factors, and it is well recognized that ultraviolet (UV) radiation is an important environmental factor that promotes skin aging. Therefore, characterizing the autophagic phenotypes induced by environmental factors is important to understand the process of skin aging. Methods In order to demonstrate the status of autophagy during environment-induced aging of the skin, we investigated the autophagy profiles of normal human dermal fibroblasts (NHDFs) treated with repetitive UVA irradiation as model fibroblasts in photoaged skin. Results Repetitively UVA-irradiated NHDFs showed increased numbers of autophagosomes, which coincided with the accumulation of p62 and increased levels of LAMP-1 and lysosomes. The behavior of repetitively UVA-irradiated NHDFs on autophagy was similar to that of NHDFs treated with hydroxychloroquine (HCQ), which is an inhibitor of lysosomal proteinase. Conclusion In summary, these results demonstrate that repetitively UVA-irradiated fibroblasts have reduced autophagy function due to the dysfunction of lysosomes.

Recent increases in air pollution have raised concerns about its adverse effects on human health. Sacran is a natural polysaccharide isolated from a cyanobacterium. We previously reported that sacran improves skin conditions because of its effects as an artificial barrier against external stimuli, which suggested that sacran might protect the skin against air pollutants. The goal of this study was to characterize the potential of sacran to protect human skin against damage from air pollutants and to compare sacran with hyaluronic acid (HA). Sacran that was topically applied on the skin stayed on the surface or in the stratum corneum. Sacran-treated filters had a shielding effect against benzo[a]pyrene (BaP) and aldehyde compounds contained in tobacco smoke. Sacran suppressed the upregulation of cytochrome P4501A1 messenger ribonucleic acid (mRNA), which is a xenobiotic-metabolizing enzyme induced by BaP, and other responses against tobacco smoke in HaCaT keratinocytes. Furthermore, topical application of a serum containing 0.04% sacran on the skin reduced levels of carbonylated proteins in corneocytes of tobacco smokers. Sacran showed superior effects in every characteristic measured, compared with HA. We conclude that sacran ameliorates the oxidative stress initiated by tobacco smoke by shielding the skin surface and protects human skin.

Sodium lauryl sulfate (SLS), a representative anionic surfactant, is well-known to induce rough skin following single or multiple topical applications. The mechanism by which SLS induces rough skin is thought to result from the disruption of skin moisture function consisting of NMF and epidermal lipids. However, a recent study demonstrated that topically applied SLS easily penetrates into the living cell layers of the epidermis, which suggests that physiological alterations of keratinocytes might cause the SLS-induced rough skin. This study was conducted to clarify the effects of SLS on keratinocytes to demonstrate the contribution of SLS to the induction of rough skin. In addition, the potentials of other widely used anionic surfactants to induce rough skin were evaluated. HaCaT keratinocytes treated with SLS had increased levels of intracellular ROS and IL-1α secretion. Application of SLS on the surface of a reconstructed epidermal equivalent also showed the increased generation of ROS. Further, SLS-treated cells showed an increase of intracellular calpain activity associated with the increase of intracellular Ca2+ concentration. The increase of intracellular ROS was abolished by the addition of BAPTA-AM, a specific chelator of Ca2+. In addition, IL-1α also stimulated ROS generation by HaCaT keratinocytes. An ESR spin-labeling study demonstrated that SLS increased the fluidity of membranes of liposomes and cells. Together, those results indicate that SLS initially interacts with cell membranes, which results in the elevation of intracellular Ca2+ influx. Ca2+ stimulates the secretion of IL-1α due to the activation of calpain, and also increases ROS generation. IL-1α also stimulates ROS generation by HaCaT keratinocytes. We conclude from these results that the elevation of intracellular ROS levels is one of the causes of SLS-induced rough skin. Finally, among the other anionic surfactants tested, sodium lauryl phosphate has less potential to induce rough skin because of its lower generation of ROS.

Skin sensitivity is a serious problem for many people, and it can be induced by various factors such as UV irradiation, physical and mental stresses, air pollution, dry air and so on. Skin dryness triggered by UV and dry air is one of the most important causes inducing the development of sensitive skin, and it has been reported that oxidative stress contributes to skin dryness. In this study, we investigated whether treatment with 3-O-laurylglyceryl ascorbate (VC-3LG), which is an amphipathic ascorbic acid derivative, can suppress the development of sensitive skin. The results demonstrate that VC-3LG restores the expression levels of interleukin-1α, nerve growth factor and matrix metalloprotease-9 in the dry skin models of reconstructed human epidermal equivalents (RHEEs) and in H2 O2 -treated keratinocytes. In addition, VC-3LG suppresses the dendrite elongation of nerve cells induced in RHEEs by dry skin conditions and by H2 O2 treatment of keratinocytes. Therefore, we consider that treatment of the skin with VC-3LG is an effective approach to improve the development of sensitive skin.

Pyridoxine (VBThis study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VBIntracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2',7'-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VBTreatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VBThese results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB

Background The induction of skin pigmentation by ultraviolet (UV) radiation has been shown to result from factors secreted from UV-exposed keratinocytes that enhance melanogenesis in melanosomes (MSs) and stimulates their transfer to keratinocytes. Among those factors, it has been reported that α-melanocyte stimulating hormone, which is converted from the precursor proopiomelanocortin (POMC) following UV exposure, stimulates the transfer of MSs from melanocytes to surrounding keratinocytes. Objective The purpose of this study was to evaluate the effects of a red pumpkin seed (RPS) extract on the transfer of MSs to keratinocytes and to clarify the involvement of reactive oxygen species (ROS) on the UVB-induced transfer of MSs. Methods The transfer of MSs into keratinocytes was examined by measuring the incorporation of fluorescent beads, which were used as pseudo-MSs. mRNA expression levels of POMC, Nrf2, and Nrf2-related genes were determined using real-time PCR. Intracellular ROS levels were estimated with H2 DCFDA. Results The incorporation of fluorescent beads into keratinocytes was enhanced by treatment with the conditioned medium (CM) from keratinocytes exposed to UVB or H2 O2 . UVB or H2 O2 exposed keratinocytes had an up-regulated mRNA expression level of POMC. Treatment of keratinocytes with the RPS extract enhanced their intracellular antioxidant system via the activation of Nrf2 signaling and suppressed their incorporation of fluorescent beads that had been stimulated by the CM from UVB or H2 O2 exposed keratinocytes. Conclusion These results indicate that the RPS extract suppresses MS transfer stimulated by ROS generated following UVB exposure through the activation of Nrf2 signaling.

Background Skin hydration is generally assessed using the parameters of skin surface water content (SWC) and trans-epidermal water loss (TEWL). To date, few studies have characterized skin conditions using correlations between skin hydration parameters and corneocyte parameters. Aims The parameters SWC and TEWL allow the classification of skin conditions into four distinct Groups. The purpose of this study was to assess the characteristics of skin conditions classified by SWC and TEWL for correlations with parameters from corneocytes. Methods A human volunteer test was conducted that measured SWC and TEWL. As corneocyte-derived parameters, the size and thick abrasion ratios, the ratio of sulfhydryl groups and disulfide bonds (SH/SS) and CP levels were analyzed. Results Volunteers were classified by their median SWC and TEWL values into 4 Groups: Group I (high SWC/low TEWL), Group II (high SWC/high TEWL), Group III (low SWC/low TEWL), and Group IV (low SWC/high TEWL). Group IV showed a significantly smaller size of corneocytes. Groups III and IV had significantly higher thick abrasion ratios and CP levels. Group I had a significantly lower SH/SS value. The SWC/TEWL value showed a decline in order from Group I to Group IV. Conclusion Groups classified by their SWC and TEWL values showed characteristic skin conditions. We propose that the SWC and TEWL ratio is a comprehensive parameter to assess skin conditions.

Background Carbonylated proteins (CPs) are generated by the reaction of basic amino acid residues in proteins with aldehyde compounds produced during lipid peroxidation. CPs in the stratum corneum (SC) impact skin conditions such as skin moisture functions including water content and transepidermal water loss (TEWL). In addition, CPs can be frequently seen in the SC from sun-exposed sites compared with sun-protected sites. Objective The aim of this study was to reveal whether CPs could be a generation source of reactive oxygen species (ROS) in the SC following exposure to ultraviolet (UV) radiation and to identify the type of ROS and its generation mechanism. Methods ROS generation was detected using a methyl cypridina luciferin analog (MCLA) chemiluminescence system and an ESR spin-trapping method. CPs in porcine SC, in a keratin film and in bovine serum albumin (BSA) were prepared by reaction with acrolein. Levels of protein carbonylation were quantified by detecting aldehyde residues. Results CP levels in the SC were increased in a UVA energy-dependent manner. That result suggested that a source of ROS generation existed in the SC initiated and produced the carbonylation of SC proteins. Carbonylated BSA and carbonylated porcine SC sheets exhibited fluorescence spectra at an excitation wavelength of 430 nm and an emission wavelength of 520 nm. Irradiation of the SC with UVA increased protein carbonylation and the amount of autofluorescence in the SC. ROS generation in the SC caused by UVA and by short-wavelength visible light (blue light, 400–470 nm) was detected by the MCLA chemiluminescence system. Artificially carbonylated porcine SCs and keratin films had increases of chemiluminescence intensity after exposure to both light sources as well. The addition of superoxide dismutase to the MCLA system completely abolished the incremental chemiluminescence intensity after both UVA and blue light exposure of the SC. In addition, acrolein-treated BSA gave ESR signals like hydroxyl radicals ( OH) converted from superoxide anion radicals ( O 2 − ) during irradiation with a xenon arc lamp containing UVA and visible light. From the sum of these results, we consider that CPs are produced from O 2 − initially generated from exposure to UVA and blue light. Conclusion CPs are excited by absorbing sunlight, particularly UVA and blue light, and result in the generation of O 2 − through a CPs progress new protein carbonylation in stratum corneum through ROS generation. photosensitizing reaction. Further, the results suggest that the O 2 − produces CPs in the SC through lipid peroxidation in the sebum, and finally affects skin conditions including color and moisture functions.

alpha-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of alpha-tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with alpha-tocopherol for 24 h, the intracellular GSH was increased at every concentration of alpha-tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with alpha-tocopherol at 50 microM for 24 h exhibited resistance against H2O2. However, a short exposure of HaCaT keratinocytes to alpha-tocopherol for 1 h did not influence either the GSH level or the resistance to H2O2. These findings suggest that GSH, which is inductively synthesized by alpha-tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of alpha-tocopherol on the GSH level, BSO, which is a typical inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of alpha-tocopherol on the GSH level was observed. On the other hand, alpha-tocopherol resulted in the up-regulation of gamma-GCS-HS (heavy subunit) mRNA. In addition, water soluble alpha-tocopherol derivatives (alpha-tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that alpha-tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of gamma-GCS-HS mRNA.

Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavonoid, was found to prevent human dermal fibroblast cell damage induced by reactive oxygen species such as hydrogen peroxide (H2O2), tert-butyl hydroperoxide (BuOOH) and superoxide anions (.O2-) in a concentration-dependent manner, and was more effective than the iron chelator, deferoxamine, hydroxyl radical (.OH) scavengers such as dimethyl sulfoxide (DMSO) and ethanol (EtOH), the lipid peroxidation chain blocker, alpha-tocopherol (Vit. E) and the xanthine oxidase inhibitor, allopurinol. To probe the mechanism of cell defense, the reaction of baicalein with oxygen free radicals was investigated using electron spin resonance (ESR) spectrometry. Baicalein decreased the signal intensities due to the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adducts of .OH, .O2- and tert-butyl peroxyl (BuOO.) radicals in a concentration-dependent manner. The IC50 values, which are the 50% inhibition concentrations of baicalein for the free radicals, were 10, 45 and 310 microM, respectively. These results suggested that baicalein possesses free radical scavenging ability which prevents the fibroblast damage induced by these free radical species.

It is recognized that reactive oxygen species (ROS) are responsible for skin damage due to UVB-radiation (UVB-R). However, the triggering substance(s) for ROS generation after UVB-R is uncertain with respect to the activation of NADPH oxidase (Nox), xanthine oxidase (XOD), and respiratory chain-chain reactions in mitochondria. As a first step in identifying the trigger(s) for UVB-induced ROS generation, we examined the relationship between Ca(2+) levels and ROS generation in HaCaT keratinocytes. UVB-R exposure of HaCaT keratinocytes resulted in an immediate elevation of ROS that recurred 7 hours later. This was accompanied by immediately elevated intracellular Ca(2+) . A Ca(2+) chelating agent, BAPTA, abolished the elevation of ROS after UVB-R completely. In addition, exogenous H(2)O(2) did not increase intracellular Ca(2+) levels. This suggests that intracellular Ca(2+) is the first trigger for UVB-induced ROS generation.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 50-52; doi:10.1038/jidsymp.2009.12.

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.

Oxidative stress caused by ultraviolet (UV) radiation generates reactive oxygen species (ROS) in the skin, induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyper-pigmentation. Thus, increasing the anti-oxidative ability of skin cells is expected to be a good strategy for skin-lightening cosmetics. Metallothionein (MT) is one of the stress-induced proteins and is known to exhibit a strong anti-oxidative property. We previously reported that a zinc(II) complex with glycine (Zn(II)(Gly)(2)) effectively induces MT expression in cultured human keratinocytes. To determine its potential as a new skin lightening active, we examined whether Zn(II)(Gly)(2) regulates the release of melanocyte-activating factors from UVB-irradiated keratinocytes and affects melanin production in a reconstructed human epidermal equivalent. Conditioned medium from UVB-irradiated keratinocytes accelerated melanocyte proliferation to 110%, and that increase could be prevented by pre-treatment with Zn(II)(Gly)(2). In addition, Zn(II)(Gly)(2) significantly reduced both the production of prostaglandin E(2) and proopiomelanocortin expression in UVB-irradiated keratinocytes. Zn(II)(Gly)(2) also decreased melanin production in a reconstructed human epidermal equivalent. These results indicate that MT-induction in the epidermis effectively up-regulates tolerance against oxidative stress and inhibits the secretion of melanocyte growth and activating factors from keratinocytes. Thus, Zn(II)(Gly)(2) is a good candidate as a new skin-lightening active.

Summary Background Vitamin C, which is a strong anti-oxidant, plays an important role in maintaining physiological states. In dermatology, Vitamin C is used for treatment of various skin problems such as de-pigmentation of hyperpigmented spots. However, Vitamin C has limited stability and permeability, and development of a Vitamin C derivative with improved properties is needed. Objective We evaluated the effect of a lipophilic Vitamin C derivative, tetra-isopalmitoyl ascorbic acid (VC-IP), on ultraviolet (UV)-induced skin pigmentation, to determine its potential as a more effective form of Vitamin C. Methods The release of Vitamin C from VC-IP was examined using a reconstructed skin model following topical application of VC-IP. Anti-oxidative and anti-inflammatory activities of VC-IP were tested in cultured human keratinocytes. Subsequently, clinical test was done to clarify the effect of VC-IP on UVB-induced skin pigmentation. Results VC-IP released Vitamin C in physiological conditions and worked as pro-Vitamin C. In subsequent experiments, we found that VC-IP suppressed the elevation of intracellular peroxide after UVB irradiation, and enhanced cellular tolerance against UVB and reactive oxygen species such as hydrogen peroxide and tert-butyl hydroperoxide. Furthermore, VC-IP reduced the production of interleukin-1α and prostaglandin E2 in UVB-irradiated keratinocytes and suppressed melanocyte proliferation in conditioned culture medium prepared from UVB-irradiated keratinocytes. Finally, in a clinical study, topical application of a 3% VC-IP cream for 3 weeks suppressed pigmentation after UVB irradiation. Conclusions These results demonstrate that VC-IP is a precursor of Vitamin C, and effectively suppresses UVB-induced skin pigmentation, possibly through its anti-oxidative activity.

Summary Background It is well established that the alterations of dermal matrix contributes to skin aging characterized by wrinkles. On the other hand, physiological NO is useful to maintain skin homeostasis such as a vasodilatation. However, a role of NO on production of dermal matrix has been clarified. Objective In this study, we have attempted to analyze the role of NO on type I collagen synthesis of normal human dermal fibroblasts including expression of procollagen αI S(1) mRNA/protein and heat shock protein 47 (HSP47). Methods The effects of NO which was generated by two types of NO donors, sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP), on type I collagen and HSP47 and their related mRNA expression were examined with ELISA and RT-PCR. Results NO was significantly accelerated the production of type I collagen by fibroblasts corresponding with up-regulation of procollagen αI (1) mRNA. Furthermore, NO increased both levels of HSP47 protein and mRNA in fibroblasts in a dose-dependent manner. Conclusions These results suggest that NO has dual effects on collagen synthesis by fibroblasts as follows; one is the direct stimulation of collagen synthesis due to the up-regulation of procollagen αI(1) mRNA, and the other is an indirect effect through the increase of HSP47 mRNA expression. This is the first report that exogenous NO stimulates HSP47 production by dermal fibroblasts.

Glycolic acid (GA), one of the alpha-hydroxy acids, is widely used as an agent for chemical peeling. Although there are several reports about the clinical effects of GA in the literature, its biological mechanism remains mostly unclear, and there are only a few reports about its effects on skin rejuvenation mediated by keratinocytes. The aim of this study was to investigate the effect of GA on the dermal matrix metabolism of keratinocytes and fibroblasts using in vitro and ex vivo systems. Our study shows that GA not only directly accelerates collagen synthesis by fibroblasts, but it also modulates matrix degradation and collagen synthesis through keratinocyte-released cytokines. We confirm that IL-1alpha is one of the primary mediators for matrix degradation released from keratinocytes after GA treatment. These results suggest that GA contributes to the recovery of photodamaged skin through various actions, depending on the skin cell type.

Advanced glycation end-products (AGEs) have been reported to accumulate in the dermal skin. However, it remains unknown whether the AGEs interact with the dermal fibroblasts and influence their function. Previously, we demonstrated that AGEs hastened photoaging of the skin by means of active oxygen species such as *O(2)(-), H(2)O(2), and *OH, generated during UVA irradiation. The purpose of the present study was to clarify the influence of AGEs on the functions of dermal fibroblasts under physiological conditions. It was found that AGEs decreased both hyaluronic acid (HA) synthesis and activity of elastase-type matrix metalloproteinase (ET-MMP). Because the reactions of both HA synthesis and ET-MMP were found to take place at the cell membrane region, it appeared that AGEs modulated cellular dysfunction through an interaction with the cell membrane. To clarify the mechanisms of these dysfunction in relation to AGEs, we examined the interaction between AGEs and cell membranes, and obtained the following results: (1) AGEs associated with the cell membranes and liposomal membrane prepared with phosphatidyl choline; (2) AGEs hydrophobically modified the circumstances of the cell membrane and liposome membrane as evaluated by experiments using a fluorescence probe; (3) AGEs increased the fluidity of the cell membrane and liposomal membrane as estimated by ESR spin-labeling using 5-doxylstearic acid; and (4) AGEs accelerated lactate dehydrogenase (LDH) leakage from the cells. On the basis of these experimental results, we proposed that AGEs modulated cell function through a nonspecific interaction with the membranes of dermal fibroblasts.

Our previous study reported that advanced glycation end-products (AGE)-modified BSA produced active oxygen species, *O2-, H2O2, and *OH under UVA irradiation and enhanced the cytotoxicity of UVA light. We examined whether pentosidine in AGE-modified BSA was involved in one of the mechanisms generating the active oxygen species. In biological investigations, fibroblasts exposed to UVA (20 J/cm2) in the presence of pentosidine-rich compounds (PRCs), which were prepared with L-arginine, L-lysine and glucose, showed a time-dependent leakage of the cytosolic enzyme LDH. In addition, release of LDH was suppressed by addition of DMSO and deferoxamine under UVA irradiation. From these results, it was determined that PRCs exposed to UVA damaged the plasma membrane of human dermal fibroblasts due to the conversion of *OH from H2O2 via a Fenton-like reaction. These features of PRCs exposed to UVA were consistent with those of AGE-modified BSA. In an ESR study, PRCs under UVA irradiation yielded DMPO-OH (DMPO-OH adduct) using DMPO as a spin-trapping reagent. *O2- generation from UVA-irradiated PRCs was also indicated by the combination of NBT reduction and SOD. When PRCs were exposed to UVA light controlled with a long-pass filter, WG-360, it was found that their production of *O2- was prohibited less than 50% in the NBT reduction assay. The *O2- production profile of PRCs depending on the wavelength of UVA light was similar to that of AGE-modified BSA. Furthermore, it was found that the H2O2 level was increased by PRCs exposed to UVA. These results indicated that pentosidine is an important factor of AGE-modified BSA in active oxygen generation under UVA irradiation.

Advanced glycation end-products (AGEs) have been reported to be accumulated in dermal skin. However, the role of AGEs in the photoaging of human skin remains unknown, and for this reason, we have examined the interaction between AGEs and ultraviolet A light (UVA) from both the chemical and biological aspects. Previously, we reported that exposing human dermal fibroblasts to UVA in the presence of AGEs that were prepared with bovine serum albumin (BSA) decreased the cell viability due to superoxide anion radical s (.O2(-)) and hydroxyl radicals (.OH) generated by AGEs under UVA irradiation, and active oxygen species are detected with ESR spin-trapping. To identify the active oxygen species in detail and to clarify the cell damaging mechanism, we performed several experiments and the following results were obtained. (1) In ESR spin-trapping, by addition of dimethyl sulfoxide and superoxide dismutase, ESR signals due to .O2(-) -derived DMPO-OOH and .OH-derived DMPO-OH adducts, respectively, were detectable. (2) UVA-irradiated AGEs elevated the lipid peroxide levels in both fibroblasts and liposomes. But the peroxidation in liposomes was inhibited by addition of deferoxamine. (3) Survival of fibroblasts exposed to UVA in the presence of AGEs was elevated by addition of deferoxamine. And finally, (4) survival of fibroblasts was found to be regulated by the level of H2O2. On the basis of these results, we propose a possible mechanism in which AGEs under UVA irradiation generate active oxygen species involving .O2(-), H2O2, and .OH, and the .OH species plays a harmful role in promoting cell damage.

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S,R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 microM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 microM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.

Ergothioneine (EGT) is a sulfur-containing amino acid, and is presumed to function as a natural antioxidant. The purpose of this study was to identify the nature of the antioxidant activity and investigate the effects of EGT on UV-induced cellular response. In chemical studies, EGT scavenged the superoxide anion radical (*O(2)(-)) and singlet oxygen ((1)O(2)). In cultured fibroblasts, EGT suppressed TNF-alpha up-regulation by UVB irradiation. In addition, in fibroblasts exposed to UV-A, EGT suppressed the expression of matrix metalloproteinase 1 (MMP-1) protein by nearly 50% and reduced MMP-1 mRNA expression. From these results, we conclude that EGT scavenges reactive oxygen species generated by both Type I and Type II photosensitization and suppresses both TNF-alpha expression and MMP-1 at their transcriptional level. EGT may reduce skin anti-aging effects after UV irradiation by the scavenging of *O(2)(-) and (1)O(2), and reducing signals for protease and inflammatory activity.