Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in vivo chemiluminescent detection and tape stripping methods

Although the formation of reactive oxygen species (ROS) in the skin induced by the ultraviolet (UV) light has been shown to lead to many cutaneous disorders, skin cancer and photoageing, the mechanism and distribution of ROS generation has not yet been definitively determined. In the present study, we examined the distribution of UVA-induced ROS in the skin of live hairless mice, using our proposed in vivo imaging chemiluminescent (CL) method to detect ROS combined with a CL probe (cypridina hilgendorfii luciferin analogue; CLA) and tape stripping (TS) technique. The CL intensities in the skin of live hairless mice were confirmed to significantly increase by UVA exposure. When TS was conducted five times in a maximum level after CL measurement following UVA exposure and subsequent CLA application, CL intensities due to UVA-induced ROS generation in the residual skin decreased to 10% of the original levels; and those in the stripped skin on each tape decreased in the stripped order such as 52%, 16%, 11%, 6% and 5%. Next, CLA was applied and then CL intensities were measured in the residual skin after advance 1, 3 and 5 tape strippings, and CL intensities due to ROS were detected primarily in the outer layer of the skin. On the basis of these results, we concluded that ROS induced by UVA exposure occurs and distributes in the outermost layer of the stratum corneum.     

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, beta-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2 .- in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.     

Direct monitoring of UV-induced free radical generation in HaCaT keratinocytes

BACKGROUND: Ultraviolet radiation (UVR) is one of the most important aetiological factors in the development of skin cancer, with an estimated 100,000 new cases of nonmelanoma skin cancer (NMSC) diagnosed each year in the UK. To date, little work has been carried out to investigate the role of UVR in the increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) following exposure of skin cells to simulated solar UVR. AIM: To monitor directly the effects of simulated solar UVR on ROS and RNS generation in HaCaT keratinocytes. METHODS: This study reports the use of electrochemical monitoring techniques for the direct, real-time detection of two highly reactive free radical species, superoxide (O2-) and nitric oxide (NO), from HaCaT keratinocyte cells that had been exposed to a source of UVR designed to simulate the doses of UVA and UVB found in solar light. RESULTS: An increase in both O2- and NO generation was observed in HaCaT cells that had been exposed to UVR. No detectable increase in either species was observed in cells that had not been exposed to UVR. The specificity of the electrochemical methods for O2- or NO was confirmed through the scavenging or inhibition of these species. CONCLUSION: The findings of this study demonstrated that exposure of HaCaT cells to relatively low doses of UVR resulted in the immediate generation of both O2- and NO, therefore potentially leading to the downstream generation of highly damaging metabolites and the development of a number of pathologies, including cancer.     

Irradiation of skin with visible light induces reactive oxygen species and matrix-degrading enzymes

Daily skin exposure to solar radiation causes cells to produce reactive oxygen species (ROS), which are a primary factor in skin damage. Although the contribution of the UV component to skin damage has been established, few studies have examined the effects of non-UV solar radiation on skin physiology. Solar radiation comprises <10% of UV, and thus the purpose of this study was to examine the physiological response of skin to visible light (400-700 nm). Irradiation of human skin equivalents with visible light induced production of ROS, proinflammatory cytokines, and matrix metalloproteinase (MMP)-1 expression. Commercially available sunscreens were found to have minimal effects on reducing visible light-induced ROS, suggesting that UVA/UVB sunscreens do not protect the skin from visible light-induced responses. Using clinical models to assess the generation of free radicals from oxidative stress, higher levels of free radical activity were found after visible light exposure. Pretreatment with a photostable UVA/UVB sunscreen containing an antioxidant combination significantly reduced the production of ROS, cytokines, and MMP expression in vitro, and decreased oxidative stress in human subjects after visible light irradiation. Taken together, these findings suggest that other portions of the solar spectrum aside from UV, particularly visible light, may also contribute to signs of premature photoaging in skin.     

Atmospheric skin aging—Contributors and inhibitors

Cutaneous aging is a complex biological process consisting of 2 elements: intrinsic aging, which is primarily determined by genetics, and extrinsic aging, which is largely caused by atmospheric factors, such as exposure to sunlight and air pollution, and lifestyle choices, such as diet and smoking. The role of the solar spectrum, comprised of ultraviolet light, specifically UVB (290‐320 nm) and UVA (320‐400) in causing skin damage, including skin cancers, has been well documented. In recent years, the contribution of visible light (400‐700 nm) and infrared radiation (above 800 nm) in causing skin damage, similar to the photodamage caused by UV light, is also being elucidated. In addition, other atmospheric factors such as air pollution (smog, ozone, particulate matter, etc.) have been implicated in premature skin aging. The skin damage caused by environmental exposure is largely attributable to a complex cascade of reactions inside the skin initiated by the generation of reactive oxygen species (ROS), which causes oxidative damage to cellular components such as proteins, lipids, and nucleic acids. These damaged skin cells initiate inflammatory responses leading to the eventual damage manifested in chronically exposed skin. Novel therapeutic strategies to combat ROS species generation are being developed to prevent the skin damage caused by atmospheric factors. In addition to protecting skin from solar radiation using sunscreens, other approaches using topically applied ingredients, particularly antioxidants that penetrate the skin and protect the skin from within, have also been well documented. This review summarizes current knowledge of atmospheric aggressors, including UVA, UVB, visible light, infrared radiation (IR), and ozone on skin damage, and proposes new avenues for future research in the prevention and treatment of premature skin aging caused by such atmospheric factors. New therapeutic modalities currently being developed are also discussed.     

Effect of ultraviolet A on ornithine decarboxylase and metallothionein gene expression in mouse skin

BACKGROUND: Ultraviolet A (UVA) is known to induce the expression of many stress responsive genes due to the generation of reactive oxygen species (ROS). However, UVA's role in inducing metallothionein (MT) gene expression has not been studied. Furthermore, our group demonstrated that UVA enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of ornithine decarboxylase (ODC) activity in mouse skin (1). METHODS: We examined the interaction of UVA, TPA and antioxidants on the induction of MT and ODC mRNA in mouse skin. Female CD-1 mice were exposed to UVA (19 J/cm2) and total RNA was isolated from the skin. Northern blot analysis for MT and ODC mRNAs was performed. ODC activity in mouse epidermis was also determined in some experiments. RESULTS: UVA induced MT mRNA in mouse skin; however, it did not increase ODC mRNA. 1,4-Diazabicylo-[2,2,2]-octane (DABCO), a singlet oxygen scavenger, reduced UVA-mediated induction of MT mRNA by 40%. The data suggest that ROS produced by UVA exposure may contribute to its ability to induce MT mRNA. UVA slightly enhanced TPA-mediated ODC mRNA induction, while it enhanced ODC enzyme activity 70%. UVA additively intensified TPA-mediated MT mRNA induction. alpha-Tocopherol pretreatment inhibited the induction of ODC enzyme activity by TPA treatment combined with UVA exposure (TPA + UVA); however, alpha-tocopherol had less of an inhibitory effect on ODC mRNA induction by TPA + UVA. Curcumin, a plant pigment, dramatically inhibited both TPA- and TPA + UVA-induced expression of ODC and MT genes. CONCLUSIONS: These results demonstrate that UVA can induce MT gene expression and enhance TPA-induced ODC and MT gene expression. The data further suggest that these effects are partially mediated by ROS.     

Suppressive effect of orally administered copper(II)-aspirinate (Cu2(asp)4) complex on the generation of reactive oxygen species in the skin of animals subjected to UVA exposure

As reactive oxygen species (ROS) are involved in the pathogenesis of various diseases, superoxide dismutase (SOD) and its mimetic complexes have been intensively studied. Recently, we found that Cu(2)(aspirinate)(4) (Cu(2)(asp)(4)) has both in vitro and in vivo antioxidative activities. We investigated the suppressive effect of Cu(2)(asp)(4) on ROS generation in the skin of hairless mice that were orally administered Cu(2)(asp)(4) and followed by UVA exposure. The results were compared with those obtained from mice that were orally administered Cu(salicylate)(2) (Cu(sal)(2)) or Cu(acetate)(2), (Cu(ace)(2)) and followed by UVA exposure. After confirming that Cu(2)(asp)(4) suppressed ROS generation in the skin, we measured both SOD activity and metallothionein (MT) and SOD protein levels in the whole proteins extracted from the skin of ICR mice that were orally administered Cu(II) compounds. The Cu(2)Zn(2)-SOD activity was enhanced by the administration of Cu(II) compounds; however, no alterations in the protein levels of MT and SOD were observed. Metallokinetics of the paramagnetic Cu(II) species in the circulating blood of rats, as estimated by electron spin resonance (ESR), revealed that among the Cu(II) compounds, the residence time of the Cu(II) species from Cu(2)(asp)(4) was the longest. On the basis of these results, we conclude that Cu(2)(asp)(4) is an orally potent antioxidative compound that suppresses ROS generation in the skin. The residence time of Cu(II) in the blood and the enhanced SOD activity in the skin following the oral administration of Cu(2)(asp)(4) support this conclusion. Here, we propose that Cu(2)(asp)(4) may be used to protect the skin against ROS generation.     

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, β-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2′-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with β-carotene and SOD effectively suppressed CL intensity, indicating the generation of ¹O₂ and O₂ ^{· ?} in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.     

Nox1-based NADPH oxidase is the major source of UVA-induced reactive oxygen species in human keratinocytes

UVA radiation is a major environmental stress on skin, causing acute and chronic photodamage. These responses are mediated by reactive oxygen species (ROS), although the cellular source of these ROS is unknown. We tested the hypotheses that UVA-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is required for ROS generation in human keratinocytes (HK) and that these ROS initiate rapid prostaglandin E2 (PGE2) synthesis. Treatment of HK with a non-toxic dose of UVA rapidly increased NADPH oxidase activity and intracellular ROS, which were partially blocked by an inhibitor of NADPH oxidase and by a mitochondria-selective antioxidant. Depleting the Nox1 isoform of the catalytic subunit of NADPH oxidase using small interfering RNA (siRNA) blocked the UVA-induced ROS increase, indicating that ROS produced by mitochondria or other sources are downstream from Nox1. Nox1 siRNA also blocked UVA-initiated PGE2 synthesis. The mechanism for activation of Nox1 is mediated by an increase in intracellular calcium. Ceramide, which has been proposed to mediate responses to UVA in HK, also activated NADPH oxidase. These results indicate that UVA activates Nox1-based NADPH oxidase to produce ROS that stimulate PGE2 synthesis, and that Nox1 may be an appropriate target for agents designed to block UVA-induced skin injury.     

Photoprotection of bacterial-derived melanin against ultraviolet A–induced cell death and its potential application as an active sunscreen

Background  The increase in the incidence of non-melanoma skin tumours, photoaging, and immunosuppression demand for more effective sunscreen on ultraviolet A (UVA) irradiation.
Objectives  The aim of the study is to evaluate the photoprotective effects of a bacterial-derived melanin against UVA-induced damages in vitro and in vivo .
Methods  Human fibroblasts were used to assess the role of the bacterial-derived melanin on cell viability against UVA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and nuclear morphology were employed to evaluate the photoprotection at the cellular level. Fluorometric assays were performed to detect the formation of reactive oxygen species (ROS) in the cells. Evaluations of the bacterial-derived melanin as a sunscreen were measured by transmission test and persistent pigment darkening on human skin.
Results  Bacterial-derived melanin efficiently scavenged ROS in the fibroblasts after UVA irradiation. The cell viability of xeroderma pigmentosum (XP) fibroblast treated with varied doses of melanin increased dramatically in comparison with untreated control and the treated XP fibroblasts became more resistant to UVA-induced apoptosis than normal fibroblasts. Although the relative transmission didn't change too much with different concentration of bacterial-derived melanin, this melanin could keep UVA-irradiated skin from pigment darkening and act as an active sunscreen on skin.
Conclusions  The bacterial-derived melanin provided significant protection to fibroblast cell and human skin against the UVA radiation. It has the potential to be developed as an active sunscreen for the patients with photosensitivity skin to sun exposure.     

Effects of repeated sunbed exposures on the human skin. In vivo measurements with confocal microscopy

BACKGROUND: Ultraviolet (UV) lamps used in commercial sunbeds are usually defined as UVA sources. Although it is well accepted that sunbed exposure significantly increases melanin pigmentation, its capacity to induce epidermal thickening is discussed controversially. OBJECTIVES: The aim of this study was to assess non-invasively the effects of repeated sunbed exposures on epidermal thickness, cell size, and pigmentation by means of confocal laser-scanning microscopy (CLSM) in vivo. METHODS: Eight volunteers had sunbed exposures six times in a 3-week period (cumulative dose: 126 J/cm(2) UVA). During irradiation, a small site (2 cm x 2 cm) on the lateral aspect of the inner forearm was covered with a UV-opaque sheet (non-exposed site). CLSM was performed with the Vivascope (Lucid, Henrietta, NY, USA) 24 h after the last UVA exposure on non-exposed sites and UVA-exposed sites that were on the medial aspect of the inner forearm at a distance of 2 cm to the non-exposed measurement site. The following parameters were assessed: thickness of the horny layer (DSC), minimal thickness of the epidermis (E(min)), minimal thickness of the viable epidermis (VE(min)), cell size of the granular layer (A(gran)), and the epidermal melanin content (MI). Additionally, colorimetric measurements have been carried out on non-exposed and UVA-exposed sites. RESULTS: DSC of the UVA-exposed skin was significantly higher than the one of non-exposed sites (mean+/-SD: 15+/-2.9 microm vs. 12.8+/-3 microm). Although E(min) was significantly higher in UVA-exposed sites (mean+/-SD: 40.4+/-3.6 microm vs. 39+/-2.9 microm), a slight but not statistically significant (P>0.05) decrease of VE(min) was observed (25.5+/-2.1 microm vs. 26.2+/-2.4 microm). The median of cell size of the granular layer (A(gran)) significantly (P=0.008) differed between non-exposed (752.1 microm(2)) and UVA-exposed sites (600 microm(2)). MI was significantly (P=0.014) higher for the UVA-exposed skin (1.12 vs. 1.34). Accordingly, colorimetry revealed significantly (P< 0.01) lower skin brightness for UVA-exposed sites (L*=60.2+/-4.3) as compared with non-exposed sites (L*=63.4+/-3.9). CONCLUSIONS: Sunbed exposures seem to induce photoadaptation not only by skin pigmentation but also by epidermal thickening that is predominantly due to an increase in thickness of the horny layer. Moreover, our data indicate that UVA radiation has an influence on the cell size of the granular layer. CLSM is a promising tool for photobiological studies in vivo.     

In situ antioxidant activity of a dermo‐cosmetic product: A randomized controlled clinical study

Ultraviolet light enhances the generation of reactive oxygen species that are responsible for skin photoageing. The aim of this randomized, vehicle‐ and active‐controlled double‐blind, intra‐individual monocentric study was to evaluate in situ the antioxidant activity of a dermo‐cosmetic product in photoaged skin. Twenty healthy volunteers had defined skin areas randomized to receive a topical product containing 3 antioxidants (pre‐tocopheryl^®, retinaldehyde and glycylglycine ole‐amide), its vehicle and a positive antioxidant control cream. The products were applied daily for 30‐day period. The skin areas were exposed to a controlled dose of UVA rays, and the skin oxidative status was evaluated 4 and 24 hours post‐UVA exposure at D0 (basal value) and after 15 and 30 days of product application. Skin layers were collected by stripping, and antioxidant capacity was measured using the ferric reducing ability of a plasma assay. Lipid peroxidation (LPO) was assessed using the malonyldialdehyde test. The tested product significantly improved the skin antioxidant capacity after 15 and 30 days and significantly decreased the basal level of the skin LPO. The skin LPO level significantly decreased 4 and 24 hours after UVA exposure at 15 and 30 days. These findings were comparable to positive control treated sites and were significantly different from the vehicle and untreated sites. This minimally invasive methodology enabled a quantitative evaluation of potent antioxidant activity in situ in the stratum corneum reflecting real‐life skin conditions and confirming the benefits of the topical application of a product containing 3 antioxidants in the prevention of UVA‐induced oxidative damage.     

Pentosidine in advanced glycation end-products (AGEs) during UVA irradiation generates active oxygen species and impairs human 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.     

Direct detection of singlet oxygen generated by UVA irradiation in human cells and skin

UVA light produces deleterious biological effects in which singlet oxygen plays a major role. These effects comprise a significant risk of carcinogenesis in the skin and cataract formation of the eye lens. Singlet oxygen is generated by UVA light absorption in endogenous molecules present in the cells. To elucidate the primary processes and sources of singlet oxygen in tissue, it is a major goal to uncover the hidden process of singlet oxygen generation, in particular in living tissue. When exposing keratinocytes or human skin in vivo to UVA laser light (355 nm) at 6 J/cm2, we measured the luminescence of singlet oxygen at 1,270 nm. This is a positive and direct proof of singlet oxygen generation in cells and skin by UVA light. Moreover, a clear signal of singlet oxygen luminescence was detected in phosphatidylcholine suspensions (water or ethanol) irradiated by UVA. Oxidized products of phosphatidylcholine are the likely chromophores because phosphatidylcholine itself does not absorb at 355 nm. The signal intensity was reduced by mannitol or super oxide dismutase. Additionally, the monochromatic UVA irradiation at 355 nm leads to upregulation of the key cytokine IL-12. This affects the balance of UV radiation on the immune system, which is comparable to effects of broadband UVA irradiation.     

Role of phagocyte oxidase in UVA-induced oxidative stress and apoptosis in keratinocytes

Chronic exposure to ultraviolet radiation including ultraviolet A (315-400 nm) (UVA) may cause photocarcinogenesis and photoaging. The UVA-induced production of reactive oxygen species (ROS) and the resultant oxidative stress exposure play an important role in these biological processes. Here we have investigated the role of phagocyte oxidase (PHOX, gp91phox) in the production of ROS, redox status change, and apoptosis after UVA exposure by using gp91phox-deficient (gp91phox-/-) primary keratinocytes. UVA radiation resulted in increased ROS production and oxidation of reduced glutathione (GSH) to its oxidized form (GSSG). The presence of diphenylene iodonium (DPI) inhibited ROS production by UVA. In comparison with wild-type cells, gp91phox-/- cells produced slightly less ROS and GSH oxidation. UVA radiation induced apoptosis in wild-type keratinocytes as detected by phosphatidylserine (PS) translocation, caspase activation, and DNA fragmentation. As compared with wild-type cells, UVA induced less PS translocation in gp91phox-deficient cells. No difference, however, was observed in caspase activation and DNA fragmentation after UVA exposure in wild-type and gp91phox-/- cells. These findings suggest that gp91phox plays a limited role in the UVA-induced ROS production, oxidative stress, and therefore the PS translocation, but has no effect on UVA-induced caspase activation and DNA fragmentation during apoptosis.     

In vivo assessment of pigmentary and vascular compartments changes in UVA exposed skin by reflectance-mode confocal microscopy

Exposure of the skin to ultraviolet A (UVA) results in various biological responses, skin-colour changes being among the major ones. Although intense research has been performed on UVA-induced pigmentation and vascular changes, the process of skin-colour changes after UVA irradiation remains unclear. For a better understanding of the UVA tanning mechanism, we here performed a human study in 27 healthy volunteers with skin phototype (SPT) II to VI. After a single UVA exposure to inner forearm, the skin sites were imaged using reflectance-mode confocal microscopy (RCM), for analysis of melanin and vascular changes. Punch biopsies were also taken from the UVA-exposed or non-exposed sites for histological examination. Skin sections were stained with Fontana-Masson and evaluated by a sensitive tyrosinase assay for comparison with RCM images. Furthermore, the effect of blood flow on skin-colour changes was evaluated visually after administration of an intradermal anesthesia of lidocaine with or without epinephrine. Our RCM analysis showed dendritic melanocytes and a different melanin distribution in the epidermal layer, clearly visible 1 week after the UVA exposure in subjects of SPT V which were supported by histological examination. However, no melanin distribution pattern changes were apparent immediately after the exposure, while RCM images showed accelerated capillary flow patterns. The restriction of this UVA induced-accelerated blood flow by epinephrine inhibited partially or completely the immediate pigment darkening and delayed tanning. These in vivo studies confirmed that vascular change is an important factor for the development of the immediate pigment darkening and delayed tanning.     

紫外线照射后生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用

目的：明确对UVA及UVB照射后皮肤成纤维细胞生成的微囊泡对成纤维细胞氧化损伤及凋亡的作用。方法：紫外线照射人皮肤成纤维细胞,提取细胞上清液中的微囊泡,利用光散射分析技术鉴定分析微囊泡的大小及数量。将紫外线照射后生成的微囊泡与正常成纤维细胞共孵育,荧光酶标仪定量检测活性氧含量,流式细胞仪检测细胞凋亡率。结果：UVA及UVB照射后皮肤成纤维细胞释放的微囊泡数量及大小明显高于正常成纤维细胞释放的微囊泡。正常纤维细胞、UVA和UVB照射后的成纤维细胞与微囊泡共孵育后活性氧荧光值分别为（52.76±1.4347）、（82.60±4.082）和（85.94±6.264）,凋亡率分别为（3.260±1.732）%,（28.94±2.430）%和（34.48±2.718）%,细胞的氧化损伤和凋亡可被抗氧化剂逆转。结论：急性中长波紫外线照射可诱导皮肤成纤维细胞释放微囊泡进一步介导细胞的氧化损伤和凋亡。     

Ultraviolet A radiation-induced biological effects in human skin: relevance for photoaging and photodermatosis

There is increasing evidence that longwave ultraviolet (UV) radiation (UVA; 320-400 nm) plays an important role in the pathogenesis of photodermatoses such as polymorphous light eruption as well as photoaging. In order to fully understand these detrimental effects it is critical to analyze the photobiological and molecular mechanisms by which UVA radiation affects the function of human skin cells. In this review, our current knowledge about the signal transduction pathway involved in UVA radiation-induced expression of proinflammatory genes relevant to the pathogenesis of polymorphous light eruption will be summarized. In addition, recent studies on the role of mitochondrial DNA mutations in UVA radiation-induced photoaging of human skin will be discussed. For both biological endpoints the UVA radiation-induced generation of singlet oxygen within human skin appears to be of critical importance. These studies are of enormous clinical relevance because they indicate that prevention of the generation of singlet oxygen or inhibition of singlet oxygen-induced signaling pathways may prove to be critical for effective protection of human skin against UVA radiation-induced damage.     

Nitroxides and a nitroxide-based UV filter have the potential to photoprotect UVA-irradiated human skin fibroblasts against oxidative damage

Background

Antioxidants are now being incorporated into sunscreens as additional topical measure for delaying the aging process and reducing photo-damage to skin induced by excessive UVA exposure. UVA radiation reaching the skin leads to the generation of ROS (reactive oxygen species) implicated in DNA damage and activation of matrix metalloproteinase-1 (MMP-1) responsible for collagen damage and photo-aging. Nitroxides are a class of compounds endowed with versatile antioxidant activity and recently, nitroxide-based UV filters in which a nitroxide moiety has been attached to the most popular UV filter present in sunscreens have been developed.

Objective

This study explores the potential photo-protective effects of these compounds on ROS production and induction of MMP-1 in cultured human dermal fibroblasts exposed to UVA. For comparison, vitamin E was also tested.

Methods

The effects were assessed by measuring intracellular ROS production using a ROS-index probe and MMP-1 mRNA expression levels using quantitative real-time PCR (qPCR).

Results

Exposure of fibroblasts to 18 J/cm² UVA lead to a two-fold increase in ROS production which was reduced to non-irradiated control levels in the presence of 50 μM nitroxide compounds and vitamin E. Under the same conditions, a ten-fold increase in MMP-1 mRNA expression levels was observed 24 h post-UVA treatment which was significantly reduced by all nitroxide compounds but not vitamin E.

Conclusion

The results of this study support the potential use of nitroxide compounds, including novel nitroxide-based UV filters, as a useful and alternative strategy for improving the efficacy of topical formulations against photo-aging and possibly photo-carcinogenesis.     

Positive photocontact responses are not elicited to sunscreen ingredients exposed to UVA prior to application onto the skin

Photocontact allergic reactions to sunscreen chemicals are investigated by photopatch testing. It has generally been assumed that for photocontact allergy to be shown, the putative pro-allergen must be in the skin at the time of ultraviolet A (UVA) exposure. However, this assumption has not, to our knowledge, been tested. The objective of this study was to determine whether positive photocontact responses can still be elicited when sunscreen chemicals are exposed to UVA prior to application onto the skin. 3 patients known to have positive photocontact reactions to a total of 6 sunscreen chemicals were studied. For conventional photopatch testing, patch test strips were applied onto the back and removed 1 D later, and the area was irradiated with UVA (5 J/cm(2)). For pre-irradiated testing, patches were exposed to the same dose of UVA immediately before application onto the back and then removed 1 D later. Skin responses were visually assessed by a blinded investigator 1 and 2 D after patch test removal. The same photocontact responses of the same magnitude, as previously documented for each patient, were seen at each of the conventional UVA-exposed patch test sites. However, in no patient was a positive response elicited at any of the sites where pre-irradiated patches had been applied. This study shows that positive photocontact responses to sunscreen chemicals do not occur when the putative pro-allergen is irradiated prior to application onto the skin. This suggests that for a photoallergic reaction to occur, the sunscreen chemical needs to be within the skin when activated by UVA.