Search

Vitiligo is an acquired skin depigmentation disease involving multiple pathogenetic mechanisms, which ultimately direct cytotoxic CD8 + cells to destroy melanocytes. Abnormalities have been described in several cells even in pigmented skin as an expression of a functional inherited defect. Keratinocytes regulate skin homeostasis by the assembly of a proper skin barrier and releasing and responding to cytokines and growth factors. Alterations in epidermal proliferation, differentiation, and lipid composition as triggers for immune response activation in vitiligo have not yet been investigated. By applying cellular and lipidomic approaches, we revealed a deregulated keratinocyte differentiation with altered lipid composition, associated with impaired energy metabolism and increased glycolytic enzyme expression. Vitiligo keratinocytes secreted inflammatory mediators, which further increased following mild mechanical stress, thus evidencing immune activation. These findings identify intrinsic alterations of the nonlesional epidermis, which can be the prime instigator of the local inflammatory milieu that stimulates immune responses targeting melanocytes.

Acne is the most common skin disease in adolescent Westernized populations. Several data support the involvement of the mammalian target of rapamycin complex 1 (mTORC1) signalling in the interplay between androgens, insulin, insulin-like growth factor (IGF1) and high-glycaemic index diet in acne. The peroxisome proliferator-activated receptor γ (PPARγ) is involved in both differentiation and anti-inflammatory response. Low differentiated sebocytes showed decreased expression of PPARγ and increased level of insulin and IGF-1 receptors, resulting in the production of acne-like sebum and inflammatory mediators. In this viewpoint, we discuss how in acne the dysregulation of proliferation and differentiation processes in sebocytes and keratinocytes may be associated with altered response to androgens and other hormones, such as insulin or IGF-1. Moreover, we propose PPARγ modulation as an innovative therapeutic approach to normalize sebocyte differentiation process, interfering with the different mechanisms involved in altered pilosebaceous unit.

Apoptotic cells stimulate compensatory proliferation through the caspase-3-cPLA-2-COX-2-PGE-2-STAT3 Phoenix Rising pathway as a healing process in normal tissues. Phoenix Rising is however usurped in cancer, potentially nullifying pro-apoptotic therapies. Cytotoxic therapies also promote cancer cell plasticity through epigenetic reprogramming, leading to epithelial-to-mesenchymal-transition (EMT), chemo-resistance and tumor progression. We explored the relationship between such scenarios, setting-up an innovative, straightforward one-pot in vitro model of therapy-induced prostate cancer repopulation. Cancer (castration-resistant PC3 and androgen-sensitive LNCaP), or normal (RWPE-1) prostate cells, are treated with etoposide and left recovering for 18 days. After a robust apoptotic phase, PC3 setup a coordinate tissue-like response, repopulating and acquiring EMT and chemo-resistance; repopulation occurs via Phoenix Rising, being dependent on high PGE-2 levels achieved through caspase-3-promoted signaling; epigenetic inhibitors interrupt Phoenix Rising after PGE-2, preventing repopulation. Instead, RWPE-1 repopulate via Phoenix Rising without reprogramming, EMT or chemo-resistance, indicating that only cancer cells require reprogramming to complete Phoenix Rising. Intriguingly, LNCaP stop Phoenix-Rising after PGE-2, failing repopulating, suggesting that the propensity to engage/complete Phoenix Rising may influence the outcome of pro-apoptotic therapies. Concluding, we established a reliable system where to study prostate cancer repopulation, showing that epigenetic reprogramming assists Phoenix Rising to promote post-therapy cancer repopulation and acquired cell-resistance (CRAC).

Lipidomics is strategic in the discovery of biomarkers of neurodegenerative diseases (NDDs). The skin surface lipidome bears the potential to provide biomarker candidates in the detection of pathological processes occurring in distal organs. We investigated the sebum composition to search diagnostic and, possibly, prognostic, biomarkers of Alzheimer’s disease (AD) and Parkinson’s disease (PD). The observational study included 64 subjects: 20 characterized as “probable AD with documented decline”, 20 as “clinically established PD”, and 24 healthy subjects (HS) of comparable age. The analysis of sebum by GCMS and TLC retrieved the amounts (µg) of 41 free fatty acids (FFAs), 7 fatty alcohols (FOHs), vitamin E, cholesterol, squalene, and total triglycerides (TGs) and wax esters (WEs). Distributions of sebum lipids in NDDs and healthy conditions were investigated with multivariate ANOVA-simultaneous component analysis (ASCA). The deranged sebum composition associated with the PD group showed incretion of most composing lipids compared to HS, whereas only two lipid species (vitamin E and FOH14:0) were discriminant of AD samples and presented lower levels than HS sebum. Thus, sebum lipid biosynthetic pathways are differently affected in PD and AD. The characteristic sebum bio-signatures detected support the value of sebum lipidomics in the biomarkers search in NDDs.

The melanocortin-1 receptor (MC1R) belongs to the family of the G protein-coupled receptor (GPCR). Activated GPCRs can promote the phosphoinositide 3-kinase (PI3K) pathway. Few studies deal with the role of the PI3K pathway activation in response to αMSH. On B16-F10 cell line, we investigated the αMSH-dependent modulation of pAKT/AKT, as a key element of the PI3K pathway after rapid and prolonged stimulation. We demonstrated that αMSH triggers a rapid modulation of AKT which culminates in an increase in its phosphorylation. We highlighted a comparable upregulation of pAKT after exposure to αMSH on primary cultures of normal human melanocytes (NHMs) expressing a wild-type MC1R. On B16-F10 cells, NHMs, and an ex vivo model of human skin biopsies, we explored the influence of PI3K/AKT signaling triggered by αMSH, focusing on the control of melanogenesis and pigment release. We showed that the αMSH-dependent PI3K/AKT pathway exerts a negative feedback on melanogenesis and promotes the extracellular release of pigment. We strengthened the role of the PI3K/AKT pathway triggered by αMSH in preserving redox equilibrium and genome integrity, highlighting its role in affecting cell survival.

Background Acne, a disease of the sebaceous gland with multifactorial pathogenesis, affects more than 85% of adolescents. A better deepening of the mechanisms underlying the disease is needed to define effective and mechanism-targeted treatments. Objective To understand whether the sebocyte differentiation process could be involved in the pathogenesis of the disease. Methods Protein expressions were evaluated by Western blot analysis and ELISA; mRNA levels by real-time RT-PCR, lipid analysis and lipid peroxidation were performed by gas chromatography, mass spectrometry and spectrophotometric assay. Results In vitro, low differentiated SZ95 sebocyte expressed an up-modulation of genes involved in sebogenesis and a higher level of insulin receptor respect to differentiated cells, resulting in an increased response to insulin and in the production of acne-like sebum. The induction of SZ95 sebocyte differentiation by the peroxisome proliferator-activated receptor γ (PPARγ) modulator NAC-GED0507 reduced the response to insulin normalizing the sebum production and decreasing the release of proinflammatory mediators. In vivo treatment of acne patients with NAC-GED0507 1% gel ameliorated clinical manifestations and induced in sebum the expression of PPARγ, associated with the decrease in mammalian target of rapamycin activation and levels of inflammatory molecules, confirming the results obtained in vitro. Conclusions The study provides relevant insight into acne pathogenesis, identifying an alteration of sebocyte differentiation as pathogenetic basis of the disease and the induction of the differentiation process as a therapeutic target in acne therapy interfering with all pathogenic mechanisms.

Cerium oxide nanoparticles (CNPs) are potent radical scavengers protecting cells from oxidative insults, including ionizing radiation. Here we show that CNPs prevent X-ray-induced oxidative imbalance reducing DNA breaks on HaCat keratinocytes, nearly abating mutagenesis. At the same time, and in spite of the reduced damage, CNPs strengthen radiation-induced cell cycle arrest and apoptosis outcome, dropping colony formation; notably, CNPs do not possess any intrinsic toxicity toward non-irradiated HaCat, indicating that they act on damaged cells. Thus CNPs, while exerting their antioxidant action, also reinforce the stringency of damage-induced cell integrity checkpoints, promoting elimination of the “tolerant” cells, being in fact radio-sensitizers. These two contrasting pathways are mediated by different activities of CNPs: indeed Sm-doped CNPs, which lack the Ce3+/Ce4+ redox switch and the correlated antioxidant action, fail to decrease radiation-induced superoxide formation, as expected, but surprisingly maintain the radio-sensitizing ability and the dramatic decrease of mutagenesis. The latter is thus attributable to elimination of damaged cells rather than decreased oxidative damage. This highlights a novel redox-independent activity of CNPs, allowing selectively eliminating heavily damaged cells through non-toxic mechanisms, rather reactivating endogenous anticancer pathways in transformed cells. Copyright © 2018 Caputo, Giovanetti, Corsi, Maresca, Briganti, Licoccia, Traversa and Ghibelli.

Increasing attention is addressed to identify products able to enhance skin photoprotection and to prevent skin carcinogenesis. Several studies have demonstrated that the α-melanocyte stimulating hormone (αMSH), acting on a functional MC1R, provides a photoprotective effect by inducing pigmentation, antioxidants and DNA repair. We discovered a link between αMSH and the nuclear receptor Peroxisome Proliferator-Activated Receptor-γ (PPARγ), suggesting that some of the αMSH protective effects may be dependent on PPARγ transcriptional activity. Moreover, we demonstrated that the activation of PPARγ by the parrodiene 2,4,6-octatrienoic acid (Octa) induces melanogenesis and antioxidant defence in human melanocytes and counteracts senescence-like phenotype in human fibroblasts. In this study, we demonstrate that the activation of PPARγ by Octa exerts a protective effect against UVA- and UVB-induced damage on normal human keratinocytes (NHKs), the major target cells of UV radiation. Octa promotes the antioxidant defence, augments DNA repair and reduces the induction of proteins involved in UV-induced DNA damage response. Our results contribute to deepen the analysis of the αMSH/PPARγ connection and suggest perspectives for the development of new molecules and formulations able to prevent cutaneous UV damage by acting on the different skin cell populations through PPARγ activation.

Pleural mesothelioma is a deadly form of cancer. The prognosis is extremely poor due to the limited treatment modalities. Uptake of asbestos fibres, the leading cause of mesothelioma, lead to the accumulation of reactive-oxygen-species (ROS). Interestingly, increasing ROS production by using ROS-generating drugs may offer a strategy to selectively trigger cell death. Exemestane, an aromatase inhibitor, has previously shown anti-tumor properties in mesothelioma preclinical models suggesting a role of G protein-coupled receptor 30 (GPR30) in the drug response. As exemestane, in addition to blocking estrogen biosynthesis, generates ROS that are able to arrest the growth of breast cancer, we explored the role of ROS, antioxidant defense system, and ROS-induced signalling pathways in mesothelioma cells during exemestane response. Here we report that exemestane treatment reduced cell proliferation with an increase in ROS production and reduction of cyclic adenosine monophosphate (cAMP) levels in MSTO-H211, Ist-Mes1, Ist-Mes2 and MPP89 exemestane-sensitive mesothelioma cell lines, but not in NCI-H2452 exemestane-insensitive mesothelioma cells. Exemestane induced a significant antioxidant response in NCI-H2452 cells, as highlighted by an increase in γ-glutamylcysteine levels, catalase (Cat), superoxide-dismutase and (SOD) and glutathione-peroxidase (GSH-Px) activity and nuclear factor E2-related factor 2 (Nrf2) activation, responsible for drug insensitivity. Conversely, exemestane elevated ROS levels along with increased ERK phosphorylation and a reduction of p-STA3 in exemestane-sensitive mesothelioma cells. ROS generation was the crucial event of exemestane action because ROS inhibitor N-acetyl-L-cysteine (NAC) abrogated p-ERK and p-STAT3 modulation and cellular death. Exemestane also modulates ERK and STAT3 signalling via GPR30. Results indicate an essential role of ROS in the antiproliferative action of exemestane in mesothelioma cells. It is likely that the additional oxidative insults induced by exemestane results in the lethal effects of mesothelioma cells by increasing ROS production. As such, manipulating ROS levels with exemestane seems to be a feasible strategy to selectively kill mesothelioma cells with less toxicity to normal cells by regulating ERK and STAT3 activity.

Background Vitiligo skin shows different burning capacity in people with different phototype. In normal skin antioxidant status is correlated to skin phototype, but unexpectedly it appears that there is a gradual decrease in burning susceptibility of depigmented skin of individuals with increasing phototype (IIVI). Objective To assess if the antioxidant response in the lesional vitiligo skin is involved in those protection mechanisms. Moreover, a possible correlation between cutaneous and systemic endogenous antioxidants in vitiligo patients has been investigated. Methods We enrolled in the study 29 patients with active vitiligo, divided into five groups according to skin type (II to VI). We analysed reduced and oxidized glutathione (GSH and GSSG, respectively), ubiquinone (CoQ10), catalase (Cat), superoxide dismutases (Cu/Zn-SOD and Mn-SOD), GSH peroxidase (GSH-Px), as indexes of chemical and enzymatic antioxidants, in suction blister roofs as well as in peripheral blood mononuclear cells (PBMNCs). Results The vitiligo patients showed an imbalance of antioxidant network, both in depigmented skin and PBMNCs. Interestingly, in vitiligo skin a phototype-related increase of antioxidant enzyme activities (Cat, Mn-SOD and GPx) and GSH amount have been observed. Similarly in PMBNCs Cat and total SOD activities, as well as GSH content progressively increased from skin type II to skin type VI. Endogenous antioxidants in vitiligo skin are correlated to those in PBMNCs, suggesting that systemic and epidermal antioxidant network functionalities are connected. Conclusions The correlation between antioxidant levels and clinical phototype confirmed the hypothesis that other factors than melanin determine largely the minimal erythema dose values in vitiligo lesional skin.

Given the importance of the tanning response in protecting human skin from the harmful effects of UV radiation, one important research priority is to identify novel molecules that are capable of promoting pigmentation and/or antioxidant defence. Parrodienes share some structural features with carotenoids and retinoids, stimulate cell antioxidant defence and counteract senescence-like phenotype in fibroblasts. We selected the parrodiene-derivative 2,4,6-octatrienoic acid (Octa) to study its impact on key parameters of melanogenesis and antioxidant defence in organ-cultured human skin and in normal human melanocytes. Octa promoted melanogenesis by up-regulating tyrosinase and microphthalmia-associated transcription factor expression. This correlated with an increase of melanin content in both human epidermis in situ and cultured human epidermal melanocytes. Moreover, Octa increased the biological antioxidant potential content and the expression and activity of catalase. Activation of peroxisome proliferator-activated receptor (PPAR)-? was necessary to evoke these effects. These data strongly encourage the systematic study of Octa as a novel candidate promoter of human skin photoprotection.

Summary Background Cutaneous pigmentation is regulated by a complex melanogenic network in which both keratinocytes and fibroblasts synthesize growth factors and cytokines. Solar lentigo (SL) is characterized by hyperpigmented lesions occurring on photodamaged skin areas. Despite the association of SL to ultraviolet (UV) exposure, the mechanisms underlying the development of these spots are not completely defined. Objectives To analyse the involvement of the fibroblast-derived growth factors, hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) in SL hyperpigmentation; to evaluate whether the photoageing process occurring in fibroblasts could be responsible for the altered expression of these cytokines; and to investigate a new possible role of KGF in regulating pigmentation through the specific induction of melanogenic cytokines by keratinocytes. Methods We performed immunohistochemical analysis of HGF, KGF and SCF on SL biopsies. We analysed the mRNA expression of these cytokines using an in vitro model of photoageing induced on fibroblasts. Finally, we evaluated the effects of KGF on the expression of melanogenic cytokines at the mRNA and protein levels on keratinocytes. Results We found positive staining for HGF, KGF and SCF in the upper dermis of SL lesions and a significant induction of the three cytokines in photoaged fibroblasts. We also demonstrated the contribution of KGF to pigmentation, showing its ability specifically to modulate the expression of SCF in keratinocytes. Conclusions Fibroblasts may be persistently activated by UV exposure to release melanogenic growth factors; this inducible cytokine network acts both directly and indirectly through keratinocytes and may contribute to the hyperpigmentation of SL.

Exposure of human fibroblasts to 8-methoxypsoralen plus ultraviolet-A irradiation (PUVA) results in stress-induced cellular senescence in fibroblasts. We here studied the role of the antioxidant defense system in the accumulation of reactive oxygen species (ROS) and the effect of the antioxidants alpha-tocopherol, N-acetylcysteine, and alpha-lipoic acid on PUVA-induced cellular senescence. PUVA treatment induced an immediate and increasing generation of intracellular ROS. Supplementation of PUVA-treated fibroblasts with alpha-tocopherol (alpha-Toc), N-acetylcysteine (NAC), or alpha-lipoic acid (alpha-LA) abrogated the increased ROS generation and rescued fibroblasts from the ROS-dependent changes into the cellular senescence phenotype, such as cytoplasmic enlargement, enhanced expression of senescence-associated-beta-galactosidase and matrix-metalloproteinase-1, hallmarks of photoaging and intrinsic aging. PUVA treatment disrupted the integrity of cellular membranes and impaired homeostasis and function of the cellular antioxidant system with a significant decrease in glutathione and hydrogen peroxide-detoxifying enzymes activities. Supplementation with NAC, alpha-LA, and alpha-Toc counteracted these changes. Our data provide causal evidence that (i) oxidative stress due to an imbalance in the overall cellular antioxidant capacity contributes to the induction and maintenance of the PUVA-induced fibroblast senescence and that (ii) low molecular antioxidants protect effectively against these deleterious alterations.

UV-induced DNA damage can lead to melanoma, the most dangerous form of skin cancer. Understanding the mechanisms employed by melanocytes to protect against UV is therefore a key issue. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide and we have previously shown that that low basal levels of catalase activity are associated with the light phototype in in vitro and ex vivo models. Here we investigate the possible correlation between its activity and melanogenesis in primary cultures of human melanocytes. We show that while the total melanin concentration is directly correlated to the level of pigmentation, the more the degree of pigmentation increased, the lower the proportion of pheomelanin present. Moreover, in human melanocytes in vitro, catalase-specific mRNA, protein and enzymatic activity were all directly correlated with total cellular melanin content. We also observed that immediately after a peroxidative treatment, the increase in reactive oxygen species was inversely associated with pigmentation level. Darkly pigmented melanocytes therefore possess two protective strategies represented by melanins and catalase activity that are likely to act synergistically to counteract the deleterious effects of UV radiation. By contrast, lightly pigmented melanocytes possess lower levels of melanogenic and catalase activity and are therefore more susceptible to accumulate damage after UV exposition.

An overview of agents causing hypopigmentation in human skin is presented. The review is organized to put forward groups of biological and chemical agents. Their mechanisms of action cover (i) tyrosinase inhibition, maturation and enhancement of its degradation; (ii) Mitf inhibition; (iii) downregulation of MC1R activity; (iv) interference with melanosome maturation and transfer; (v) melanocyte loss, desquamation and chemical peeling. Tyrosinase inhibition is the most common approach to achieve skin hypopigmentation as this enzyme catalyses the rate-limiting step of pigmentation. Despite the large number of tyrosinase inhibitors in vitro, only a few are able to induce effects in clinical trials. The gap between in-vitro and in-vivo studies suggests that innovative strategies are needed for validating their efficacy and safety. Successful treatments need the combination of two or more agents acting on different mechanisms to achieve a synergistic effect. In addition to tyrosinase inhibition, other parameters related to cytotoxicity, solubility, cutaneous absorption, penetration and stability of the agents should be considered. The screening test system is also very important as keratinocytes play an active role in modulating melanogenesis within melanocytes. Mammalian skin or at least keratinocytes/melanocytes co-cultures should be preferred rather than pure melanocyte cultures or soluble tyrosinase.

The harmful effects of UVA radiation (320-400 nm) on the skin have been related to the generation of reactive oxygen species. Pheomelanin, the pigment characteristic of fair-skinned individuals, amplifies these effects. In vitro, in the presence of photosensitizing agents, UVA light produces singlet oxygen, which reacts with several targets. We have investigated a possible correlation between melanin-type and the antioxidant defense system after UV, focusing on the activities of superoxide dismutase and catalase, which correlated with the phototype of epidermal reconstructs. UVA was more effective than UVB in damaging these enzymatic activities, especially catalase. Furthermore, UVA irradiation induced a free-radical-mediated damage in the cells, leading to an oxidation of cell proteins. On catalase, synthetic pheomelanin amplified this effect on specific targets, such as residues of tryptophan and methionine. UVA irradiation of low phototype reconstructed epidermis and of U937 through synthetic pheomelanin induced a modification in the electrophoretic properties of native catalase, which was counteracted by histidine, a quencher of singlet oxygen. These results demonstrate that pheomelanin could act as a photosensitizing agent, following UVA irradiation, inducing charge modifications of native catalase, by a mechanism involving singlet oxygen or its downstream products.

Plant responses to biotic and abiotic stress can be mediated by oxidised products and in this study we analysed the relation among some of them and the growth factor indole-3-acetic acid (IAA). The plant material used was potato tuber sliced below bud and incubated for different lengths of time before analysis. Wounding in potato tuber leads, in a very short time (0–30 min), to the generation of lipid hydroperoxides (LOOH) from polyunsaturated fatty acids (PUFA). These reactive species could cause a subsequent increase of 9 and 13-lipoxygenase (LOX, E.C.1.13.12.12.), analysed by RT-PCR and spectrophotometric assay, LOOH, Jasmonates and IAA all quantified by GC–MS analysis. The activation of 9 and 13-LOX, using different timing, leads to the formation of LOOH with a subsequent generation of jasmonates and IAA as highlighted by the addition on the potato tuber slices of salicylhydroxamic acid (SHAM), an inhibitor of LOX activity. A correlation between jasmonates and IAA resulted by testing their reciprocal influence during wounding in potato tuber. The relationship occurring among each hormone analysed during wounding underlines the fact that the jasmonates level can be regulated in situ and this can suggest a role for these compounds in potato tuber which has been underestimated up to now.

The incidence rate of melanoma is higher in fair-skinned than in dark-skinned individuals. In negroid skin there is more eumelanin which is present in all skin layers and fewer polyunsaturated fatty acids (PUFA) than in caucasoid skin. The western diet, which is rich in omega-6 polyunsaturated fatty acids, is associated with more proneness to cancer including cutaneous melanoma. To study the respective influence of omega-6 PUFA and low phototype melanocytes on redox status -basal and following UV irradiation-, we used epidermal reconstructs. The addition of polyunsaturated fatty acids as well as the presence of low phototype melanocytes affected basal status similarly except for catalase activity, which decreased significantly in polyunsaturated fatty acid-supplemented reconstructs. Following UV, polyunsaturated fatty acids and low phototype melanocytes increased lipid and protein oxidative damage without affecting direct DNA damage. However, polyunsaturated fatty acids increased epidermal apoptosis whereas low phototype melanocytes decreased it. Since our data suggest that an omega-6 PUFA rich-diet may increase oxidative damage in melanocytes without inducing apoptosis, the long-term net outcome could be cumulated mutations and an increased risk of skin cancer, especially melanoma.

Due to its interface function between the body and the environment, the skin is chronically exposed to both endogenous and environmental pro-oxidant agents, leading to the harmful generation of reactive oxygen species (ROS). There is compelling evidence that oxidative stress is involved in the damage of cellular constituents, such as DNA, cell membrane lipids or proteins. To protect the skin against the over-load of oxidant species, it contains a well-organised system of both chemical and enzymatic antioxidant which are able to work in a synergistic manner. Skin antioxidant network protects cells against oxidative injury and prevent the production of oxidation products, such as 4-hydroxy-2-nonenal or malonaldehyde, which are able to induce protein damage, apoptosis or release of pro-inflammatory mediators, such as cytokines. When oxidative stress overwhelms the skin antioxidant capacity the subsequent modification of cellular redox apparatus leads to an alteration of cell homeostasis and a generation of degenerative processes. Topical application or oral administration of antioxidants has been recently suggested as preventive therapy for skin photoaging and UV-induced cancer. The recognition that ROS can act as second messengers in the induction of several biological responses, such as the activation of NF-kB or AP-1, the generation of cytokines, the modulation of signalling pathways, etc., has led many researchers to focus on the possible effects of antioxidants in many pathological processes. The recent demonstration that the peroxisome proliferators-activated receptors, whose natural ligands are polyunsaturated fatty acids and theirs oxidation products, have a central role in the induction of some skin diseases, such as psoriasis or acne, has indicated new links between free radicals and skin inflammation. Based on these findings, the review summarises the possible correlations between antioxidant imbalance, lipid oxidative breakage and skin diseases, from both a pathological and therapeutic points of view.

In this study, we compared the effects of sunscreens and antioxidants on reconstructed epidermis made with or without melanocytes 24 h after UVB, UVA or UVA+B irradiation. For this purpose, we studied sunburn cells and cyclobutane pyrimidine dimer formation, protein and lipid oxidation, catalase and superoxide dismutase activities and vitamin E levels. Topical sunscreens protected against direct cell death and thymine dimer formation whereas their protective effect against protein and lipid oxidation and antioxidant depletion was less marked partly due to the difficulty of spreading the cream. Antioxidant molecules protected against direct cell death and protein oxidation but not against thymine dimer formation. Since topical sunscreens and systemic antioxidant protected the skin differently, we speculate that their association might protect more efficiently against UV-induced damage. This model is relevant to study systemic molecules but is less practical, due to the technical limitations of studying topical molecules.

Peroxisome proliferator-activated receptor gamma (PPARγ) may be involved in a key mechanism of the skin aging process, influencing several aspects related to the age-related degeneration of skin cells, including antioxidant unbalance. Therefore, we investigated whether the up-modulation of this nuclear receptor exerts a protective effect in a stress-induced premature senescence (SIPS) model based on a single exposure of human dermal fibroblasts to 8-methoxypsoralen plus + ultraviolet-A-irradiation (PUVA). Among possible PPARγ modulators, we selected 2,4,6-octatrienoic acid (Octa), a member of the parrodiene family, previously reported to promote melanogenesis and antioxidant defense in normal human melanocytes through a mechanism involving PPARγ activation. Exposure to PUVA induced an early and significant decrease in PPARγ expression and activity. PPARγ up-modulation counteracted the antioxidant imbalance induced by PUVA and reduced the expression of stress response genes with a synergistic increase of different components of the cell antioxidant network, such as catalase and reduced glutathione. PUVA-treated fibroblasts grown in the presence of Octa are partially but significantly rescued from the features of the cellular senescence-like phenotype, such as cytoplasmic enlargement, the expression of senescence-associated-β-galactosidase, matrix-metalloproteinase-1, and cell cycle proteins. Moreover, the alterations in the cell membrane lipids, such as the decrease in the polyunsaturated fatty acid content of phospholipids and the increase in cholesterol levels, which are typical features of cell aging, were prevented. Our data suggest that PPARγ is one of the targets of PUVA-SIPS and that its pharmacological up-modulation may represent a novel therapeutic approach for the photooxidative skin damage.

Several hypotheses have been made about the pathogenesis of vitiligo, and some of them have considered a systemic involvement in the course of the disease. Evidence has been presented on the role of oxidative stress as the initial event in melanocyte degeneration. In accordance with this view, we determined the levels of some antioxidants, i.e., superoxide dismutase, catalase, reduced glutathione, and vitamin E, in erythrocytes and/or peripheral blood mononuclear cells from patients with active or stable vitiligo and from a control group of healthy subjects. In erythrocytes the parameters evaluated were not significantly different. On the contrary, in peripheral blood mononuclear cells, superoxide dismutase activity was increased in both groups of patients, whereas catalase activity, reduced glutathione and vitamin E levels were decreased exclusively in subjects with active disease. The imbalance of antioxidants was associated with hyperproduction of reactive oxygen species due to a mitochondrial impairment as cyclosporin A, an inhibitor of the permeability transition pores opening, significantly reduced the reactive oxygen species production. Moreover an alteration of the mitochondrial transmembrane potential and a higher percentage of apoptotic cells were observed in active vitiligo patients. Based on these results, we suggest that, in vitiligo, mitochondria might be the target of different stimuli, such as reactive oxygen species generation, cytokines production, catecholamine release, alteration of Ca2+ metabolism, all of which capable of inducing melanocyte degeneration.

We developed a sensitive and specific liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous determination of reduced and oxidized glutathione (GSH and GSSG) in peripheral blood mononuclear cells (PBMC). Following derivatization with N -ethylmaleimide to prevent GSH auto-oxidation, addition of thiosalicylic acid as internal standard, and protein precipitation with cold acetonitrile, the samples were injected into a diol column, eluted with acetonitrile–1% aqueous acetic acid (25:75) and detected by the ESI-MS system. The optimized method exhibited a good detection limit for both analytes (0.01 and 0.05 μ M for GSH and GSSG, respectively). Good linearity was reached in the 0.01–20 μ M range for GSH and 0.05–20 μ M for GSSG. The mean recoveries of GSH and GSSG were 98.5–100.6% and 105.8–111.5%, respectively. The run-to-run repeatability for retention time and peak area was RSD% 0.06 and 1.75 for GSH and 0.18 and 2.50 for GSSG. The optimized method was applied to GSH and GSSG assay in PBMC analyzing 20 healthy individuals.

Despite considerable advances in the past decade, melanin research still suffers from the lack of universally accepted and shared nomenclature, methodologies, and structural models. This paper stems from the joint efforts of chemists, biochemists, physicists, biologists, and physicians with recognized and consolidated expertise in the field of melanins and melanogenesis, who critically reviewed and experimentally revisited methods, standards, and protocols to provide for the first time a consensus set of recommended procedures to be adopted and shared by researchers involved in pigment cell research. The aim of the paper was to define an unprecedented frame of reference built on cutting-edge knowledge and state-of-the-art methodology, to enable reliable comparison of results among laboratories and new progress in the field based on standardized methods and shared information.

Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long-time treatment with AzA. We previously unrevealed that anti-inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo-aggravated disease, we investigated the ability of AzA to counteract stress-induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8-methoxypsoralen (PUVA), previously reported to activate a senescence-like phenotype, including long-term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence-associated β-galactosidase (SA-β-gal). We found that PUVA-treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP-1 release and SA-β-galactosidase-positive cells. Moreover, AzA induced a reduction in ROS generation, an up-modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA-treated HDFs. Further evidences of AzA anti-senescence effect were repression of p53 and p21, increase in type I pro-collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA-SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA-induced senescence-like phenotype and its ability to activate PPAR-γ provides relevant insights into the anti-senescence mechanism.

Cystinosis is a rare autosomal recessive disease characterized by cystine crystal accumulation leading to multiorgan dysfunctions and caused by mu- tation in CTNS. CTNS encodes cystinosin, a cystine/H symporter that exports cystine out of the lysosomes. Patients with cystinosis frequently exhibit blond hair and fair complexion, suggesting an alteration in mela- nogenesis. However, the pigmentation singularities of these patients have not been studied, and the role of cystinosin in melanogenesis has remained unknown. In our study, a clinical evaluation of 27 patients with cystinosis showed that 44% had a cutaneous pigmenta- tion dilution compared to their relatives. Analysis of the hair melanin content in these patients by HPLC dem- onstrated a 50% decrease in eumelanin (4360 vs. 9360 ng/mg), and a 2-fold increase in pheomelanin (53 vs. 20 ng/mg), the yellow/red pigments. Cystinosin-deficient mice also showed a 4-fold increase in hair pheomelanin content. In vitro studies showed that cystinosin was located at melanosomes. CTNS silencing led to a 75% reduction of melanin synthesis that was caused by a degradation of tyrosinase by lysosomal proteases. Our results objectify the pigmentation defect in patients with cystinosis. We also identify the role of CTNS in melanogenesis and add a new gene to the list of the genes involved in the control of skin and hair pigmen- tation.—Chiaverini, C., Sillard, L., Flori, E., Ito, S., Briganti, S., Wakamatsu, K., Fontas, E., Berard, E., Cailliez, M., Cochat, P., Foulard, M., Guest, G., Niau- det, P., Picardo, M., Bernard, F.-X., Antignac, C., Ortonne, J. P., Ballotti, R. Cystinosin is a melanosomal protein that regulates melanin synthesis. FASEB J. 26, 000 - 000 (2012). www.fasebj.org

Given the importance of the tanning response in protecting human skin from the harmful effects of UV radiation, one important research priority is to identify novel molecules that are capable of promoting pigmentation and/or antioxidant defence. Parrodienes share some structural features with carotenoids and retinoids, stimulate cell antioxidant defence and counteract senescence-like phenotype in fibroblasts. We selected the parrodiene-derivative 2,4,6-octatrienoic acid (Octa) to study its impact on key parameters of melanogenesis and antioxidant defence in organ-cultured human skin and in normal human melanocytes. Octa promoted melanogenesis by up-regulating tyrosinase and microphthalmia-associated transcription factor expression. This correlated with an increase of melanin content in both human epidermis in situ and cultured human epidermal melanocytes. Moreover, Octa increased the biological antioxidant potential content and the expression and activity of catalase. Activation of peroxisome proliferator-activated receptor (PPAR)-γ was necessary to evoke these effects. These data strongly encourage the systematic study of Octa as a novel candidate promoter of human skin photoprotection.

Malignant melanoma incidence is increasing rapidly in Western countries. Its prevention requires a deep knowledge of the biological basis of the neoplasm leading to the identification of new biological risk markers. In in-vitro and ex-vivo models we demonstrated that catalase was modified not only in its activity but also in its charge properties after ultraviolet A irradiation through pheomelanin. Here we focus on the electrophoretic behaviour of catalase in the human skin in vivo, in association with cutaneous phototype. Zymographic analysis of the enzyme on skin biopsies from Caucasian population (phototype I-IV), collected from the trunk in autumn-winter, to exclude possible influences of an acute photoexposure, evidenced a protein doublet, representing the coexistence of two active isoforms of catalase with different charge properties. In the skin from low-phototype subjects, the percent contribution of the more acidic component of the doublet was prevalent, inversely correlated with total melanin concentration in hair, and associated with a high number of melanocytic nevi. In summary, this study shows for the first time the existence of an acidic catalase in association with clinically defined risk characteristics in low phototype skin in vivo, contributing to the knowledge of a new biochemical marker of cutaneous photosusceptibility.

Background Exposure to ultraviolet (UV) radiation causes a complex cellular response, mostly mediated by the production of reactive oxygen species (ROS), which can be counteracted by exogenous treatments and endogenous mechanisms with anti-oxidant and scavenger properties. Keratinocyte growth factor (KGF/FGF7), a member of the fibroblast growth factor family, promotes epithelial growth and differentiation and is involved in cell survival after oxidant injuries. Objective We analyzed the role of KGF in the control of intracellular ROS production and oxidative stress after UVB exposure on KGF receptor (KGFR) transfected cells and human immortalized and primary keratinocytes. Methods We assessed the intracellular ROS production measuring the intensity of the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) by confocal microscopy, as well as the catalase activity by spectrophotometric assay. Moreover, morphological and biochemical analysis of actin cytoskeleton reorganization was evaluated as a further marker of oxidative damage. Results Our data show that KGF significantly reduces intracellular ROS generation in response to UVB, preserves the decrease of catalase activity and prevents actin cytoskeleton rearrangement. Conclusion Our results provide a further evidence that KGF may be crucial for an efficient skin photoprotection, demonstrating a direct role for KGF in the reduction of intracellular ROS content following UVB exposure.

New topical immunomodulators have been reported to cause repigmentation of vitiligo lesions. However, time-kinetics of such repigmentation in different anatomic locations is not well known. We performed a randomized double-blind placebo control study with tacrolimus versus the vehicle and a nonrandomized control study with pimecrolimus to evaluate the time to reach significant pigmentation, its duration and extent in treated areas. Antioxidant status of serum was also assessed. Twenty patients, in the tacrolimus study, had one pair of lesions on different localizations, and 20 on face and/or upper limbs for pimecrolimus. The extent of repigmentation was evaluated by slides and mapmakings at baseline and every 4 weeks during 7 months. Adverse events were recorded. The derivatives of oxygen metabolites, the ferric reducing ability of serum and vitamin E were assessed. Three groups of patients were identified with the tacrolimus study. Eight had no significant change in response characterized by a parallel increase of repigmentation or none in treated and control areas. Nine had a better repigmentation to tacrolimus at fifth month of treatment. Three had a marked repigmentation in control areas at the end of treatment. Repigmentation was significant on the face compared to upper-limbs with pimecrolimus from fourth to seventh month. A significant reduction of oxidative stress and an increase in antioxidant capacity in serum of patients treated with topical tacrolimus was observed, while those treated with pimecrolimus did not show any significant changes but an increase in vitamin E. Our work defines three periods in repigmentation, triggering during the first 4 months, increase in pigmentation with tacrolimus and a plateau or a sustained repigmentation. The continuity of the treatment seems necessary to ensure a prolonged repigmenting effect and even an enhanced one, such as the one we observed on the face with pimecrolimus. The extent of repigmentation was more significant on the face compared to other locations probably due to differences in melanocyte density. Furthermore, we did not find any relationship between repigmentation and the duration of vitiligo. Tacrolimus was able to reduce the systemic oxidative stress independently from its repigmenting capacity. Both drugs were well tolerated.

UV-induced DNA damage can lead to melanoma, the most dangerous form of skin cancer. Understanding the mechanisms employed by melanocytes to protect against UV is therefore a key issue. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide and we have previously shown that that low basal levels of catalase activity are associated with the light phototype in in vitro and ex vivo models. Here we investigate the possible correlation between its activity and melanogenesis in primary cultures of human melanocytes. We show that while the total melanin concentration is directly correlated to the level of pigmentation, the more the degree of pigmentation increased, the lower the proportion of pheomelanin present. Moreover, in human melanocytes in vitro, catalase-specific mRNA, protein and enzymatic activity were all directly correlated with total cellular melanin content. We also observed that immediately after a peroxidative treatment, the increase in reactive oxygen species was inversely associated with pigmentation level. Darkly pigmented melanocytes therefore possess two protective strategies represented by melanins and catalase activity that are likely to act synergistically to counteract the deleterious effects of UV radiation. By contrast, lightly pigmented melanocytes possess lower levels of melanogenic and catalase activity and are therefore more susceptible to accumulate damage after UV exposition.

The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent (Fe2+/EDTA/H2O2) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after 1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-H-2)-15-HETE (1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of la as a potential precursor of the cytotoxic gamma-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between la and the epoxy-alcohol 2a, previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Recently, after the identification of ferritin light chain (L-ferritin) gene and protein over-expression in human metastatic melanoma cells, we engineered, starting from the LM metastatic melanoma cell line, clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. The present investigation started from the observation that L-ferritin down-regulated LM cells displayed a less pigmented phenotype, confirmed by a major decrease of total melanin, when compared to control LM cells. This finding was accompanied by a dramatic decrease in tyrosinase activity, which was not paralleled by a concomitant reduction of the amount of tyrosinase specific mRNA. Western blot analysis of tyrosinase in control LM cells displayed a pattern, which corresponds to the progressive glycosylation of the native protein up to the 80 kDa form, considered the functional one. Tyrosinase pattern assayed in L-ferritin down-regulated LM cells showed the remarkable absence of the 80 kDa form and a prevalence of endoglycosidase H (endo H)-sensitive immature (70 kDa) tyrosinase, accumulated in the endoplasmic reticulum (ER), as confirmed by confocal microscopy analysis. These results demonstrate that, in a human metastatic melanoma cell line, the stress condition promoted by L-ferritin down-modulation, can substantially influence proper maturation of tyrosinase.

Many modalities of treatment for acquired skin hyperpigmentation are available including chemical agents or physical therapies, but none are completely satisfactory. Depigmenting compounds should act selectively on hyperactivated melanocytes, without short- or long-term side-effects, and induce a permanent removal of undesired pigment. Since 1961 hydroquinone, a tyrosinase inhibitor, has been introduced and its therapeutic efficacy demonstrated, and other whitening agents specifically acting on tyrosinase by different mechanisms have been proposed. Compounds with depigmenting activity are now numerous and the classification of molecules, based on their mechanism of action, has become difficult. Systematic studies to assess both the efficacy and the safety of such molecules are necessary. Moreover, the evidence that bleaching compounds are fairly ineffective on dermal accumulation of melanin has prompted investigations on the effectiveness of physical therapies, such as lasers. This review which describes the different approaches to obtain depigmentation, suggests a classification of whitening molecules on the basis of the mechanism by which they interfere with melanogenesis, and confirms the necessity to apply standardized protocols to evaluate depigmenting treatments.