Damage In Human Keratinocytes Research Articles

Fucoidan isolated from the edible seaweed Sargassum fusiforme suppresses skin damage stimulated by airborne particulate matter

Particulate matter (PM) pollution is a major environmental factor to global health problems. It exposes to the skin of human body, induces skin diseases and aging. Sargassum fusiforme is rich in a variety of bioactive substances and possesses the potential to protect skin against PM-stimulated damage. The purpose of this study is to investigate the effect of the fucoidan (SF-F4) isolated from S. fusiforme on PM-stimulated skin damage in human keratinocytes (HaCaT cells) and human dermal fibroblasts (HDF cells). The results show that PM significantly increased ROS level and promoted the cell death in both HaCaT cells and HDF cells. However, SF-F4 effectively increased the viability of PM-treated HaCaT cells by inhibiting apoptosis via scavenging intracellular ROS. In addition, SF-F4 regulated the expressions of MMPs and pro-inflammatory molecules in PM-stimulated HDF cells in a concentration-dependent manner, thus, it increasing pro-collagen content of PM-stimulated HDF cells. In conclusion, the current findings indicate that SF-F4 can effective inhibit PM-induced oxidative stress and reduce the secretion of pro-inflammatory factors and MMPs in cells, thereby slowing down the triggering of skin damage, suggesting that SF-F4 may be a potential pharmaceutical or cosmetic ingredient.

Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the development of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.

1,2,4-trihydroxybenzene induces stress granule formation and causes DNA damage in human keratinocytes

Household chemical products are typically evaluated for toxicity through ingestion and inhalation, with limited information on skin absorption. Furthermore, current research focuses on the long-term toxic effects of harmful substances contained in these household chemical products, however not much is known about their acute toxic effects. In this study, the effects of 1,2,4-trihydroxybenzene (THB) in human keratinocytes by examining its effects on stress granule (SG) formation, a marker of acute stress response, and DNA double strand breaks caused by repeated exposure. THB effectively induced SG formation via endoplasmic reticulum stress-mediated eIF2α phosphorylation in keratinocytes. Furthermore, repeated exposure to THB causes apoptotic cell death due to DNA double strand breaks. Collectively, THB exposure leads to skin toxicity, suggesting precautions for the use of THB-containing household chemical products.

Skin Cancer-Associated S. aureus Strains Can Induce DNA Damage in Human Keratinocytes by Downregulating DNA Repair and Promoting Oxidative Stress.

Simple SummarySquamous cell carcinoma of the skin and precancerous skin lesions, called actinic keratoses, on severely photodamaged skin are often colonized with unusually high amounts of the bacterium Staphylococcus aureus. It is not yet known whether this bacterium directly contributes to cancer formation in the skin. To test this, we exposed healthy human skin cells in culture to secreted compounds from S. aureus taken from precancerous and cancerous skin lesions. We then determined if the S. aureus-treated skin cells start showing typical signs of cancer transformation by looking at the changes in RNA transcripts and proteins. We found that the secreted compounds from some S. aureus strains were able to induce cancer-promoting changes in skin cells. Our findings suggest that S. aureus colonization on precancerous skin may contribute to cancer development and that there might be benefits of eradicating S. aureus on heavily photodamaged skin.Actinic keratosis (AK) is a premalignant lesion, common on severely photodamaged skin, that can progress over time to cutaneous squamous cell carcinoma (SCC). A high bacterial load of Staphylococcus aureus is associated with AK and SCC, but it is unknown whether this has a direct impact on skin cancer development. To determine whether S. aureus can have cancer-promoting effects on skin cells, we performed RNA sequencing and shotgun proteomics on primary human keratinocytes after challenge with sterile culture supernatant (‘secretome’) from four S. aureus clinical strains isolated from AK and SCC. Secretomes of two of the S. aureus strains induced keratinocytes to overexpress biomarkers associated with skin carcinogenesis and upregulated the expression of enzymes linked to reduced skin barrier function. Further, these strains induced oxidative stress markers and all secretomes downregulated DNA repair mechanisms. Subsequent experiments on an expanded set of lesion-associated S. aureus strains confirmed that exposure to their secretomes led to increased oxidative stress and DNA damage in primary human keratinocytes. A significant correlation between the concentration of S. aureus phenol soluble modulin toxins in secretome and the secretome-induced level of oxidative stress and genotoxicity in keratinocytes was observed. Taken together, these data demonstrate that secreted compounds from lesion-associated clinical isolates of S. aureus can have cancer-promoting effects in keratinocytes that may be relevant to skin oncogenesis.

Mangostanin, a Xanthone Derived from Garcinia mangostana Fruit, Exerts Protective and Reparative Effects on Oxidative Damage in Human Keratinocytes.

The fruit of Garcinia mangostana (mangosteen) is known in ancient traditional Asian medicine for its antioxidant, anti-inflammatory, immunomodulatory and anticancer activities. These effects are mainly due to the action of polyphenols known as xanthones, which are contained in the pericarp of the fruit. In recent years, there has been a growing interest from pharmaceutical companies in formulating new topicals based on mangosteen full extracts to prevent skin aging. However, the molecules responsible for these effects and the mechanisms involved have not been investigated so far. Here, the arils and shells of Garcinia mangostana were extracted with chloroform and methanol, and the extracts were further purified to yield 12 xanthone derivatives. Their effects were evaluated using in vitro cultures of human epidermal keratinocytes. After confirming the absence of cytotoxicity, we evaluated the antioxidant potential of these compounds, identifying mangostanin as capable of both protecting and restoring oxidative damage induced by H2O2. We showed how mangostanin, by reducing the generation of intracellular reactive oxygen species (ROS), prevents the activation of AKT (protein kinase B), ERK (extracellular signal-regulated kinase), p53, and other cellular pathways underlying cell damage and apoptosis activation. In conclusion, our study is the first to demonstrate that mangostanin is effective in protecting the skin from the action of free radicals, thus preventing skin aging, confirming a potential toward its development in the nutraceutical and cosmeceutical fields.

Alkaloids and flavonoids exert protective effects against UVB-induced damage in a 3D skin model using human keratinocytes

This study isolated six carboline alkaloids, (3S,4R)-1-(β-carbolin-1-yl)-3,4,5-trihydroxy-1-pentanone (1), β-carboline (2), 1-hydroxymethyl-β-carboline (3), perlolyrine (4), (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (5) and (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (6) from the leaves of Dendrobium officinale Kimura et Migo (Orchidaceae). These alkaloids, along with 14 flavonoids, vicenin-2 (7), vicenin-1 (8), isoschaftoside (9), schaftoside (10), neoschaftoside (11), violanthin (12), isoviolanthin (13), vicenin-3 (14), apigenin 6-C-α-l-arabinopyranosyl-8-C-β-d-xylopyranoside (15), vitexin 2″-O-glucoside (16), vitexin 2″-O-rhamnoside (17), rutin (18) and quercetin (19), which were previously found in D. officinale leaves by other researchers, and apigenin (20), which is the aglycone of flavone C-glycosides 7–17, were bioassayed to determine their protective effects on UVB-induced damage in human keratinocytes. Carboline alkaloids 1 and 3 and flavonoids 8 and 20 significantly increased cell viability in human keratinocytes irradiated by UVB. In a 3D skin model, compounds 1 and 8 showed promising protective effects, maintained reasonable skin structures and increased the expression of filaggrin and claudin-1, which was inhibited by UVB irradiation. The anti-UVB activities of compounds 1, 3 and 8 were reported for the first time, and these three compounds exhibit potential for application in sunscreen cosmetics.

Pomegranate (Punica granatum) extract and its polyphenols reduce the formation of methylglyoxal-DNA adducts and protect human keratinocytes against methylglyoxal-induced oxidative stress

Pomegranate extract (PE) and its polyphenols have been reported to show skin protective effects but their cytoprotective effects against methylglyoxal (MGO)-induced DNA damage and cell dysfunctions are unclear. Herein, we evaluated whether PE, punicalagin (PA), ellagic acid (EA), and urolithin A (UA), can alleviate MGO-induced DNA damage in human keratinocytes. PE (50 µg/mL) and PA (50 µM) protected DNA integrity and reduced the formation of MGO-DNA adducts and tailed DNA by 60.2 and 49.7%, respectively, in HaCaT cells. PE and PA reduced MGO-induced cytotoxicity by increasing the cell viability (by 17.5 and 15.0%) and decreasing reactive oxygen species (by 28.3 and 30.0%), respectively. PE and PA also ameliorated MGO-induced cell dysfunction by restoring cell adhesion, migration, and wound healing capacity. Findings from this study provide insights into the skin protective effects of PE and its polyphenols supporting their applications as potential bioactive ingredients for cosmeceuticals.

Blue light induces DNA damage in normal human skin keratinocytes.

The generation of DNA damage by ultra-violet radiations (UV) is well established, and both the nature of the DNA lesions and their respective DNA repair pathways have largely been described. Besides UV rays, visible light constitutes a very important part of the sun spectrum where blue light is considered a significant contributor to premature aging. However, blue light-induced DNA damage has not been deeply explored yet. In the present study, we assessed in human skin keratinocytes the DNA and chromosome damaging activities of blue light rays (415nm) as well as their associated DNA repair mechanisms. Our results demonstrated that blue light induced dose-dependent DNA damage in human keratinocytes. Both oxidative and cyclobutane-pyrimidine-dimer (CPD) DNA lesions were generated. They were repaired through base excision repair (BER) and nucleotide excision repair (NER) pathways, respectively. Moreover, by using the micronucleus assay we demonstrated, for the first time, that a blue wavelength exerted a clastogenic/aneugenic effect in human keratinocytes, leading to chromosome aberration. We concluded that, in normal human keratinocytes, blue light creates genotoxic lesions which might accelerate or at least contribute to premature skin aging.

UVB protective effects of Sargassum horneri through the regulation of Nrf2 mediated antioxidant mechanism

The present study aimed to evaluate the protective effect of a methanol extract of Sargassum horneri (SHM), which contains 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (HTT) and apo-9′-fucoxanthinone, against ultraviolet B (UVB)-induced cellular damage in human keratinocytes and its underlying mechanism. SHM significantly improved cell viability of UVB-exposed human keratinocytes by reducing the generation of intracellular reactive oxygen species (ROS). Moreover, SHM inhibited UVB exposure-induced apoptosis by reducing the formation of apoptotic bodies and the populations of the sub-G1 hypodiploid cells and the early apoptotic cells by modulating the expression of the anti- and pro-apoptotic molecules, Bcl-2 and Bax, respectively. Furthermore, SHM inhibited NF-κB p65 activation by inducing the activation of Nrf2/HO-1 signaling. The cytoprotective and antiapoptotic activities of SHM are abolished by the inhibition of HO-1 signaling. In further study, SHM restored the skin dryness and skin barrier disruption in UVB-exposed human keratinocytes. Based to these results, our study suggests that SHM protects the cells against UVB-induced cellular damages through the Nrf2/HO-1/NF-κB p65 signaling pathway and may be potentially useful for the prevention of UVB-induced skin damage.

Protective effects of halophyte complex extract against UVB-induced damage in human keratinocytes and the skin of hairless mice.

Limonium tetragonum, Triglochin maritimum, Artemisia scoparia and red ginseng have been used as folk remedies for treating a variety of diseases. In the current study, the protective effects of halophyte and red ginseng against ultraviolet (UV)-induced skin damage were investigated. Halophyte red ginseng complex extract (HRCE) was prepared and its effects on UV-B irradiated human keratinocytes and mouse skin were studied through ELISA, Western blotting immunofluorescence and histological staining. HRCE inhibited peroxide-induced damage in human keratinocytes. HRCE also inhibited UVB-induced collagen and elastin degradation in human keratinocytes and mouse skin. In addition, HRCE inhibited mast cell infiltration in the skin of mice irradiated with UVB light. This effect was likely due to HRCE inhibiting the activation of MAPK and NF-κB. By protecting the skin from UVB-induced skin damage, HRCE has the potential to be used in the treatment and prevention of UV-induced skin damage and photoaging.

Cytotoxic and genotoxic effects on human keratinocytes triggered by sphingomyelinase D from Loxosceles venom.

The spiders of the Loxosceles genus (called brown or violin spiders) are of medical relevance in several countries due to the many human envenomation cases reported. The main component of Loxosceles venom is the enzyme sphingomyelinase D (SMase D), which is responsible for the local and systemic effects induced by the whole venom. Here, we investigated the cytotoxic and genotoxic effects caused by Loxosceles laeta venom and SMase D on human keratinocytes to better understand the dermonecrosis development mechanism. Our findings indicate that whole venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage. These effects appear to be dependent on the binding of SMase D to the cell surface, although the complete pathway triggered as a result of the binding still needs to be elucidated. Moreover, after SMase D treatment, we observed the presence of histone γH2AX, suggesting that the cells are undergoing DNA repair. Moreover, when ATR kinase was inhibited, the cell viability of human keratinocytes was decreased. Together, our findings strongly suggest that L. laeta venom, as well as SMase D, increases intracellular superoxide levels, leading to DNA damage in human keratinocytes. Additionally, the induced DNA damage is repaired through the activation of an apparent ATR-mediated DNA-damage response. This knowledge may contribute to a better understanding of the behaviour of human keratinocytes during cutaneous loxoscelism, a condition that affects thousands of people around the world.

Rosemary Diterpenes and Flavanone Aglycones Provide Improved Genoprotection against UV-Induced DNA Damage in a Human Skin Cell Model.

Overexposure to solar ultraviolet (UV) radiation is the major cause of a variety of cutaneous disorders, including sunburn, photoaging, and skin cancers. UVB radiation (290–320 nm) causes multiple forms of DNA damage, p53 induction, protein and lipid oxidation, and the generation of harmful reactive oxygen species (ROS). In recent years, botanicals containing polyphenols with antioxidant and anti-inflammatory properties as skin photoprotective agents have emerged. This study evaluated the protective effects of two formulations against UVB-induced damage in a skin cell model. One of the formulations (F2) contained a combination of citrus and olive extracts and the other one (F1) also contained a rosemary extract. The antioxidant capacity of both formulations was estimated by different in vitro methods, and the cell viability, intracellular ROS generation, mitochondrial depolarization, and DNA damage were studied in UVB-irradiated human keratinocytes. Both formulations exerted photoprotective effects on skin cells and decreased mitochondrial depolarization and DNA damage. F1 which contained iridoids, rosemary diterpenes, glycosides and aglycones of citrus flavanones, and monohydroxylated flavones exhibited higher cellular photoprotective effects and mitochondrial membrane potential restoration, as well as an enhanced capacity to decrease DNA double strand breaks and the DNA damage response. In contrast, F2, which contained mostly iridoids, citrus flavanone aglycones, and mono- and dihydroxylated flavones, exhibited a higher capacity to decrease intracellular ROS generation and radical scavenging capacity related to metal ion chelation. Both formulations showed a similar capability to decrease the number of apoptotic cells upon UVB radiation. Based on our results and those of others, we postulate that the stronger capacity of F1 to protect against UVB-induced DNA damage in human keratinocytes is related to the presence of rosemary diterpenes and citrus flavanone aglycones. Nevertheless, the presence of the dihydroxylated flavones in F2 may contribute to inhibiting the generation of metal-related free radicals. To confirm the efficacy of these formulations as potential candidates for oral/topical photoprotection, human trials are required to circumvent the limitations of the cellular model.

Effects of Vitis vinifera L. leaves extract on UV radiation damage in human keratinocytes (HaCaT)

Vitis vinifera L. water extract from red grapevine leaves contains high levels of polyphenols in quantities similar to those found in red grape and grape seeds. Phenolic compounds are the largest group of natural antioxidants with also an anti-inflammatory activity, widely demonstrated both in vitro and in vivo. Interestingly, their antioxidant effect relies not only on the direct radical scavenging activity but also on their ability in modulating cellular signalling transduction pathways. UV radiation exerts multiple effects on skin cells inducing apoptosis, senescence and carcinogenesis. The aim of this study was to investigate the antioxidant and the DNA protective potentials of Vitis vinifera L. water extract against UV-A and UV-B radiation in HaCaT cells, a human keratinocytes cell line. Comet and ɣH2AX assays were used to assess DNA damage in UV irradiated cells pre-treated or not with the extract (100 μg/mL). For UV-B, DNA damage resulted significantly increased at 40 mJ/cm2 dose determining cell cycle arrest and apoptosis. For UV-A, DNA damage was significant at 10 J/cm2 while cell cycle arrest and apoptosis were evident only at 25 J/cm2. The extract (1h of pre-treatment) highlights the antioxidant and scavenger activity on the UV-A, while the maintenance of the apoptosis with both UV-A and UV-B must be interpreted as an anti-mutagenic effect.

Protective effects of Cordyceps extract against UVB‑induced damage and prediction of application prospects in the topical administration: An experimental validation and network pharmacology study

Ethnopharmacological relevanceUVB is a high energy source that causes the major risk factor for sunburn and skin tumor. However, photochemical interactions lead to beneficial effects such as synthesis of vitamin D and corticosteroids. Therefore, a reasonable therapeutic regime is advocated to reduce UVB injuries but makes use of synthesizing sunlight metabolite. Many natural compounds improving plant cells resistant to oxidative stress by the harnessing of solar energy may be also used to protect human cells. Although many nature plants have shown photoprotective effects on skin, the mechanisms underlying of the effects are still ambiguous. Aim of the studyThis study evaluates the protective effects of cultivated Cordyceps against UVB-induced damage in human keratinocytes and identifies the photoprotective mechanisms using a transcriptomic network approach. Materials and MethodsCordyceps extract compositions were investigated by HPLC analysis. Cell survival, reactive oxygen species (ROS) generation, H2O2 content, aquaporin 3 (AQP3) level and DNA damage were determined upon UVB irradiation in the presence of Cordyceps extract. In addition, next-generation sequencing was used to profile transcriptomic alteration of 20 mJ/cm2 UVB and non-UV. Finally, a network pharmacology method was applied to study Cordyceps extract-related natural compounds and their UVB-induced differentially change targets using the Cytoscape 3.7.1 software. ResultsAdenosine and mannitol were the major contents in Cordyceps extract. Cordyceps caused a significant diminished in intracellular UVB-induced oxidative stress, including ROS production and intracellular H2O2 content. Besides, AQP3 which mediated intracellular signal transmission and transported H2O2 into cells was significantly increased in the presence of Cordyceps extract against UVB irradiation. In addition, DNA repair effect of Cordyceps extract after UV irradiation was proven to be effective by comet assay. Moreover, KEGG analysis showed steroid hormone biosynthesis, ovarian steroidogenesis, fat digestion and absorption were enriched in top 3 between 20 mJ/cm2 UVB and non-UV. Gene ontology (Go) analysis showed that steroid metabolic process, sterol metabolic process, and cholesterol metabolic process were enriched in top3 biology process. By using network analysis, 125 potential bioactive ingredients in Cordyceps and 201 targets were identified. Finally, signal pathway analyses suggested that the protective effects of Cordyceps compounds against low dose UVB‑induced changes might target PPAR signaling pathway, cholesterol metabolism, and ovarian steroidogenesis. ConclusionCordyceps extract may be an ideal product for external use of skin which could not only avoid UVB-induced adverse effects, but also could application of metabolite products by UVB such us steroid hormone and vitamin D3.

글루텐으로 자극한 피부각질형성세포에서 참모자반 열수 추출물의 염증 및산화적 손상 억제 효과

글루텐으로 자극한 피부각질형성세포에서 참모자반 열수 추출물의 염증 및산화적 손상 억제 효과

Protective effects of novel derivatives of vitamin D3 and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and p53 defense mechanisms

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50–200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.

274 - Protective effects of novel derivatives of vitamin D3 and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and P53 defense mechanisms

Objective Ultraviolet B (UVB) plays an important role in the photo-damage of skin through formation of cyclobutane pyrimidine dimers (CPD), the primary mutagenic DNA photoproducts, and regulation of the p53 protective responses against genotoxic damage. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor regulating antioxidant genes, also has a protective role against photo-oxidative damage of the skin. Since the protective role of the active form of vitamin D (1,25(OH)2D3) against UVB induced damage is recognized, we tested whether novel CYP11A1-derived D3-hydroxyderivatives and lumisterol including its hydroxy-derivatives can protect against UVB-induced damage in human keratinocytes. Materials and Methods 1,25(OH)2D3, biochemically generated 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, and 1,20,23(OH)3D3, synthetic lumisterol and its hydroxy-derivatives generated by the action of CYP11A1 including 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3), were used for testing. Cells were treated with compounds for 24 h pre-and post-UVB irradiation at doses of 25, 50, 75, and 200 mJ/cm2. Oxidant formation was determined by the DCFA-DA method. DNA damage was assessed using the comet assay. Nuclear expressions of CPD, phospho-p53 and Nrf2 were assayed by immunofluorescence and western blotting, respectively. Results Trea tment with compounds at concentrations 10-9, 10-8, and 10-7 M showed a dose-dependent reduction of oxidant formation in comparison to non-treated cells. Compounds (10-7 M) were also able to protect against DNA damage and/or induce DNA repair for cells irradiated with UVB (50 mJ/cm2) by reducing CPD levels and the tail moment of comet images. They stimulated p53 phosphorylation at Ser-15 and increased its concentration in the nucleus, and enhanced Nrf2 translocation to the nucleus. Conclusion 1,25(OH)2D3, CYP11A1-derived D3-hydroxyderivatives as well as lumisterol and its hydroxy-derivatives protect keratinocytes against UVB-induced damage via activation of DNA damage protective pathways that include Nrf2 activation and p53-phosphorylation. These compounds represent promising anti-photodamaging agents.

Hydroxytyrosol from olive fruits prevents blue-light-induced damage in human keratinocytes and fibroblasts.

Skin aging is a complex biological process influenced by a combination of endogenous or intrinsic and exogenous or extrinsic factors due to environmental damage. The primary environmental factor that causes human skin aging is the ultraviolet irradiation from the sun. Recently, it was established that the long-term exposure to light-emitting-diode-generated blue light (LED-BL) from electronic devices seems to have a relevant implication in the molecular mechanisms of premature photoaging. BL irradiation induces changes in the synthesis of various skin structures through DNA damage and overproduction of reactive oxygen species (ROS), matrix metalloproteinase-1 and -12, which are responsible for the loss of the main components of the extracellular matrix of skin like collagen type I and elastin. In the current study, using human keratinocytes and fibroblasts exposed to specific LED-BL radiation doses (45 and 15 J/cm 2 ), we produced an in vitro model of skin photoaging. We verified that, compared with untreated controls, the treatment with LED-BL irradiation results in the alteration of metalloprotease-1 (collagenase), metalloprotease-12 (elastase), 8-dihydroxy-2'-deoxyguanosine, proliferating cell nuclear antigen, and collagen type I. Moreover, we showed that the photoaging prevention is possible via the use of hydroxytyrosol extracted from olive fruits, well known for antioxidant properties. Our results demonstrated that hydroxytyrosol protects keratinocytes and fibroblasts from LED-BL-induced damage. Thus, hydroxytyrosol might be proposed as an encouraging candidate for the prevention of BL-induced premature photoaging.

P-383 - Circadian clock and oxidative Stress: functional cross-talk in skin homeostasis

Ozone (O3) is among the most reactive chemical to which living organisms are routinely exposed and due to its critical location, the skin is one of the most susceptible tissues to environmental stressors. It is well known a significant role of the circadian system in the regulation of protein involved in the cellular response to oxidative stress. In this work, we investigated a possible protective role of the circadian system to O3 induced damage in human keratinocytes evaluating the cross-talk between defensive and inflammatory pathway. Synchronized keratinocytes exhibited a faster cellular response, with a more efficient interplay among these two important pathways, compared to the arrhythmic after O3 exposure. Analysis of clock gene expression in rhythmic cells reveals a more rapid induction of the cardinal clock gene Bmal1. Taken together these data suggest that an adequate coordination of the circadian system and antioxidant/inflammatory pathway might be essential to maintain the skin homeostasis. Alteration of metabolic pathways as occurs in many diseases or simply irregular schedule of life activity (shift work, transcontinental journey) could negatively influence tissue gene expression programs and associated organ physiology via its effect on the circadian system.

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.