Photoprotective properties of Prunella vulgaris and rosmarinic acid on human keratinocytes

UVA radiation provokes the generation of reactive oxygen species (ROS), which induce oxidative stress in the exposed cells leading to extensive cellular damage and cell death either by apoptosis or necrosis. One approach to protecting human skin against the harmful effects of UV radiation is by using herbal compounds as photoprotectants. This study evaluated the protective effects of Prunella vulgaris L. (Labiatae) and its main phenolic acid component, rosmarinic acid (RA), against UVA-induced changes in a human keratinocyte cell line (HaCaT). Human keratinocytes exposed to UVA (10-30 J/cm(2)) were treated with an extract of P. vulgaris (1-75 mg/l) or RA (0.9-18 mg/l) for 4h. P. vulgaris and RA exhibited ability to reduce the UVA-caused decrease in a cell viability monitored by neutral red retention and by LDH release into medium. The P. vulgaris extract and RA significantly suppressed UVA-induced ROS production, which manifests as a decrease in intracellular lipid peroxidation, elevation of ATP and reduced glutathione. Post-treatment with P. vulgaris extract and RA also significantly reduced DNA damage. In addition, UVA-induced activation of caspase-3 was inhibited by treatment with P. vulgaris and RA. The P. vulgaris extract and RA demonstrated a concentration-dependent photoprotection (maximum at 25-50 mg/l and 9 mg/l, respectively). These results suggest that P. vulgaris and RA, used in skin care cosmetics, may offer protection against UVA-induced oxidative stress and may be beneficial as a supplement in photoprotective dermatological preparations.     

Protective effects of cyanidin-3-O-beta-glucopyranoside against UVA-induced oxidative stress in human keratinocytes

Ultraviolet-A (UVA) radiation causes significant oxidative stress because it leads to the generation of reactive oxygen species (ROS), leading to extensive cellular damage and eventual cell death either by apoptosis or necrosis. We evaluated the protective effects of cyanidin-3-O-beta-glucopyranoside (C-3-G) against UVA-induced apoptosis and DNA fragmentation in a human keratinocyte cell line (HaCaT). Treatment of HaCaT cells with C-3-G before UVA irradiation inhibited the formation of apoptotic cells (61%) and DNA fragmentation (54%). We also investigated antioxidant properties of C-3-G in HaCaT cells against ROS formation at apoptotic doses of UVA; C-3-G inhibited hydrogen peroxide (H2O2) release (an indicator of cellular ROS formation) after UVA irradiation. Further confirmation of the potential of C-3-G to counteract UVA-induced ROS formation comes from our demonstration of its ability to enhance the resistance of HaCaT cells to the apoptotic effects of both H2O2 and the superoxide anion (O2*-), two ROS involved in UVA-oxidative stress. Furthermore, in terms of Trolox Equivalent Antioxidant Activity, C-3-G treatment led to a greater increase in antioxidant activity in the membrane-enriched fraction than in the cytosol (55% vs 19%). The protective effects against UVA-induced ROS formation can be attributed to the higher membrane levels of C-3-G incorporation. These encouraging in vitro results support further research into C-3-G (and other anthocyanins) as novel agents for skin photoprotection.     

Skin photosensitizing agents and the role of reactive oxygen species in photoaging.

In this paper, the role of reactive oxygen species in photoaging is presented. Many photosensitizing agents are known to generate reactive oxygen species (singlet oxygen (1O2), superoxide anion (O2.-) and .OH radicals). Although photoaging (dermatoheliosis) of human skin is caused by UVB and UVA radiation, the hypothesis tested here in the pathogenesis of photoaging of human skin is the free radical theory involving the generation of reactive oxygen species by UVA (320-400 nm) radiation and their damaging oxidative effects on cutaneous collagen and other model proteins. The UVA-generated reactive oxygen species cause cross-linking of proteins (e.g. collagen), oxidation of sulfydryl groups causing disulfide cross-links, oxidative inactivation of certain enzymes causing functional impairment of cells (fibroblasts, keratinocytes, melanocytes, Langerhans cells) and liberation of proteases, collagenase and elastase. The skin-damaging effects of UVA appear to result from type II, oxygen-mediated photodynamic reactions in which UVA or near-UV radiation in the presence of certain photosensitizing chromophores (e.g., riboflavin, porphyrins, nicotinamide adenine dinucleotide phosphate (NADPH), etc.) leads to the formation of reactive oxygen species (1O2, O2.-, .OH). Four specific observations are presented to illustrate the concept: (1) the production of 1O2 and O2.- by UVB, UVA and UVA plus photosensitizing agents (such as riboflavin, porphyrin and 3-carbethoxypsoralens) as a function of UV exposure dose, the sensitizer concentration and the pH of the irradiated solution; (2) the formation of protein cross-links in collagen, catalase and superoxide dismutase by 1O2 and O2.- (.OH) and the resulting denaturation of proteins and enzyme activities as a function of UVA exposure dose; (3) the protective role of selective quenchers of 1O2 and O2.- (e.g. alpha-tocopherol acetate, beta-carotene, sodium azide, ascorbic acid, etc.) against the photoinactivation of enzymes and the prevention of the protein cross-linking reaction; (4) the possible usefulness of certain antioxidants or quenchers that interact with the UVA-induced generation of reactive oxygen species in the amelioration of the process of photoaging.     

Silibinin is a potent sensitizer of UVA radiation-induced oxidative stress and apoptosis in human keratinocyte HaCaT cells

UVA radiation (315-400 nm), which constitutes ca 95% of the UV irradiation in natural sunlight reaching earth surface, is a major environmental risk factor associated with human skin cancer pathogenesis. UVA is an oxidizing agent that causes significant damage to cellular components through the release of reactive oxygen species (ROS) and leads to photoaging and photocarcinogenesis. Here we investigate the effect of silibinin, the flavonolignan from Silybum marianum, on UVA-induced ROS and cell death in human keratinocyte cell line HaCaT. In addition, the effect of silibinin on UVA-induced intracellular ROS-mediated endoplasmic reticulum (ER) stress was also analyzed. UVA irradiation resulted in ROS production and apoptosis in HaCaT cells in a dose-dependent manner, and the ROS levels and apoptotic index were found to be elevated significantly when the cells were treated with 75 μmsilibinin for 2 h before UVA exposure. When the cells were pretreated with 10 mmN-acetyl cysteine, the enhancement of UVA-induced apoptosis by silibinin was compromised. Furthermore, we found that silibinin enhances ER stress-mediated apoptosis in HaCaT cells by increasing the expression of CHOP protein. These results suggest that silibinin may be beneficial in the removal of UVA-damaged cells and the prevention of skin cancer.     

Caffeic acid and ferulic acid inhibit UVA-induced matrix metalloproteinase-1 through regulation of antioxidant defense system in keratinocyte HaCaT cells

Ultraviolet A (UVA) plays a vital role in the pathogenesis of premature skin aging through keratinocyte cytotoxicity and degradation of collagen, a main component of the extracellular matrix providing structural support. Oxidative stress caused by UVA irradiation can mediate induction of matrix metalloprotease-1 (MMP-1), a major enzyme responsible for collagen damage. Protection against UV-mediated disturbance of antioxidant defense system has been proposed as a possible mechanism by which botanical compounds slow down skin aging process. This study therefore aimed to assess inhibitory effects of caffeic acid (CA) and ferulic acid (FA), powerful plant-based phenolic antioxidants, on UVA-induced cytotoxicity and MMP-1 activity and mRNA level through modulation of antioxidant defense mechanism in immortalized human keratinocyte (HaCaT) cells. Pretreatment of the cells with CA or FA prior to UVA irradiation inhibited cytotoxicity, induction of MMP-1 activity and mRNA and oxidant formation. Moreover, CA and FA were able to up-regulate glutathione (GSH) content, γ-glutamate cysteine ligase (γ-GCL) mRNA as well as activities and mRNA expression of catalase and glutathione peroxidase (GPx) in irradiated cells. In conclusion, CA and FA provided protective effects on UVA-mediated MMP-1 induction in HaCaT cells possibly through restoration of antioxidant defense system at the cellular and molecular level.     

Glutathione modulates the level of free radicals produced in UVA-irradiated cells

We have developed an assay to detect reactive oxygen species (ROS) generated by UVA radiation utilising chemical probes which become fluorescent upon oxidation. Using a human bladder carcinoma cell line (MGH-U1) and spontaneously immortalised keratinocytes (HaCaT), we have shown a UVA (narrow band 365+/-5 nm) dose-dependent increase in fluorescence by flow cytometry following loading of the cells with either dihydrorhodamine 123 (DHR) or 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The UVA response of both DHR and DCFH was enhanced by elevation of intracellular levels of the photosensitiser protoporphyrin IX by incubation for 2.5 h with 5-aminolaevulinic acid. Depletion of the antioxidant glutathione (GSH) using the inhibitor D,L-buthionine-sulphoximine (BSO), resulted in an increase in the UVA-induced fluorescence of DCF but not of rhodamine 123. Conversely, raising intracellular GSH levels with N-acetyl cysteine (NAC) had relatively little protective effect in terms of degree of induced fluorescence.     

Hydrogen peroxide is responsible for UVA-induced DNA damage measured by alkaline comet assay in HaCaT keratinocytes

We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay). Superoxide anion (O2*-) was a likely substrate for H2O2 production since diethyldithiocarbamate (DDC), a superoxide dismutase blocker, decreased the level of intracellular H2O2. Hydrogen peroxide was shown to play a central role in DNA damage. Increasing the intracellular levels of H2O2 with aminotriazole (AT) (a catalase blocker) and buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis) potentiated the UVA-induced DNA damage. Exogenous H2O2 was also able to induce DNA damage. Since H2O2 alone is not able to damage DNA directly, we investigated the significance of the H2O2-derived hydroxyl radical (*OH). Addition of FeSO4, that stimulates *OH formation from H2O2 (Fenton reaction) resulted in a twofold increase of DNA-damage. Desferrioxamine, an iron chelator that blocks the Fenton reaction, prevented UVA-induced DNA damage. We also employed a panel of less specific antioxidants and enzyme modulators. Sodium selenite (Na-Se) present in glutathione peroxidase and thioredoxin reductase and addition of glutathione (GSH) prevented DNA-damage. Tocopherol potently prevented UVA-and H2O2-induced DNA damage and reduced intracellular H2O2 -levels. Ascorbic acid reduced H2O2 production, but only partly prevented DNA damage. Singlet oxygen (1O2) did not seem to play an important role in the UVA-induced DNA-damage since the specific 1O2 scavenger sodium azide (NaN3) and the less specific 1O2 scavenger beta-carotene did not markedly prevent either DNA-damage or H2O2 production. In conclusion the conversion of H2O2 to *OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O2*- generated by UVA irradiation.     

Santamarine Shows Anti-Photoaging Properties via Inhibition of MAPK/AP-1 and Stimulation of TGF-β/Smad Signaling in UVA-Irradiated HDFs

Chronic UVA exposure results in elevated reactive oxygen species in skin which leads to photoaging characterized as upregulated matrix metalloproteinase (MMP)-1 and loss of collagen. Therefore, natural antioxidants are hailed as promising agents to be utilized against photoaging. In the current study, reynosin and santamarine, two known sesquiterpene lactones isolated from Artemisia scoparia, were analyzed for their anti-photoaging properties in UVA-irradiated human dermal fibroblasts (HDFs). Results showed that UVA irradiation (8 J/cm²) upregulated the MMP-1 secretion and expression, and suppressed collagen production, which were significantly reverted by santamarine treatment (10 µM). Although both reynosin and santamarine exhibited ROS scavenging abilities, reynosin failed to significantly diminish UVA-stimulated MMP-1 release. UVA-irradiated HDFs showed increased collagen production when treated with santamarine. As a mechanism to suppress MMP-1, santamarine significantly suppressed the UVA-induced phosphorylation of p38 and JNK and nuclear translocation of p-c-Fos and p-c-Jun. Santamarine promoted collagen I production via relieving the UVA-induced suppression on TGF-β and its downstream activator Smad2/3 complex. Antioxidant properties of santamarine were also shown to arise from stimulating Nrf2-dependent expression of antioxidant enzymes SOD-1 and HO-1 in UVA-irradiated HDFs. In conclusion, santamarine was found to be a promising natural antioxidant with anti-photoaging properties against UVA-induced damages in HDFs.     

Proteomic identification of cathepsin B and nucleophosmin as novel UVA-targets in human skin fibroblasts

Solar UVA exposure plays a causative role in skin photoaging and photocarcinogenesis. Here, we describe the proteomic identification of novel UVA-targets in human dermal fibroblasts following a two-dimensional-difference-gel-electrophoresis (2D-DIGE) approach. Fibroblasts were exposed to noncytotoxic doses of UVA or left untreated, and total protein extracts underwent CyDye-labeling followed by 2D-DIGE/mass-spectrometric identification of differentially expressed proteins, confirmed independently by immunodetection. The protein displaying the most pronounced UVA-induced upregulation was identified as the nucleolar protein nucleophosmin. The protein undergoing the most pronounced UVA-induced downregulation was identified as cathepsin B, a lysosomal cysteine-protease displaying loss of enzymatic activity and altered maturation after cellular UVA exposure. Extensive lysosomal accumulation of lipofuscin-like autofluorescence and osmiophilic material occurred in UVA-exposed fibroblasts as detected by confocal fluorescence microscopy and transmission electron microscopy, respectively. Array analysis indicated UVA-induced upregulation of oxidative stress response gene expression, and UVA-induced loss of cathepsin B enzymatic activity in fibroblasts was suppressed by antioxidant intervention. Pharmacological cathepsin B inhibition using CA074Me mimicked UVA-induced accumulation of lysosomal autofluorescence and deficient cathepsin B maturation. Taken together, these data support the hypothesis that cathepsin B is a crucial target of UVA-induced photo-oxidative stress causatively involved in dermal photodamage through the impairment of lysosomal removal of lipofuscin.     

Protective effects of an antioxidant derived from serine and vitamin B6 on skin photoaging in hairless mice

The generation of reactive oxygen species (ROS) by ultraviolet radiation (UVR) accelerates skin aging, which is known as photoaging. Because cutaneous iron catalyzes ROS generation, sequestering iron by chelating agents is thought to be an effective approach toward preventing photoaging. Previously, N-(4-pyridoxylmethylene)-l-serine (PYSer) was designed as an antioxidant to suppress iron-catalyzed ROS generation by its iron-sequestering activity. In this study, PYSer showed protective effects against skin damage in hairless mice irradiated with ultraviolet B (UV-B). Topical application of PYSer to the skin significantly delayed and/or decreased the visible wrinkle formation induced by chronic UV-B irradiation. A histological study indicated that UV-B-induced epidermal hypertrophy and lymphocytic infiltration were suppressed by PYSer. Moreover, PYSer showed suppressive activity against the UV-B-induced increase in glycosaminoglycans (GAG). These results indicate that PYSer is a promising antioxidant for the prevention of chronic skin photoaging by its iron-sequestering activity.     

Protective Effects of Ginseng Leaf Extract Using Enzymatic Extraction Against Oxidative Damage of UVA-Irradiated Human Keratinocytes

UVA is responsible for numerous biological effects on the skin, including premature aging characterized by wrinkles, leathery texture, and mottled pigmentation. The objective of this study was evaluating the protective effect of ginseng leaf extract prepared by Ultraflo L on skin from photodamage. Anti-wrinkle effect of ginseng leaf extract with or without Ultraflo L treatment were tested on human keratinocyte cells (HaCaT) irradiated with ultraviolet (UV) A. Ginseng leaves inhibited ROS generation, GHS depletion, and expression of MMP-2 and MMP-9 induced by UVA irradiation. The glutathione (GSH) content of the cells was significantly increased by over 25 μg mL^?1 of Ultraflo-treated extract (UTGL) as well as by over 100 μg mL^?1 of nonenzyme-treated extract (NEGL) compared to control. UTGL and NEGL treatments significantly decreased expression of metalloproteinase (MMP)-2 and 9 compared with control, but inhibitory effects of two groups on expression of MMPs were not significantly different. Overall, ULtraflo L-treated ginseng leaves inhibited ROS generation, GHS depletion, and expression of MMP-2 and MMP-9 in UVA photodamaged HaCat cells. From these results, enzyme-treated ginseng leaf extract has advantages over untreated ginseng leaves and have potential as a skin protective ingredient against UVA-induced photodamage.     

Zeolite encapsulation decreases TiO2-photosensitized ROS generation in cultured human skin fibroblasts

Sunscreens protect skin against sunburn. However, studies have demonstrated that UV-irradiated sunscreen components such as titanium dioxide (TiO2) promote the photogeneration of reactive oxygen species (ROS). Because encapsulation of TiO2 within zeolites alters its photocatalytic activity, supramolecular composites based on NaY zeolite hosts containing TiO2 guests were prepared, and the effects on ROS formation in cells under UVA-irradiation evaluated. DCFH-DA (2',7'-dichlorofluorescein diacetate) was used as a profluorescent probe to monitor intracellular ROS. The detection of intracellular 2',7'-dichlorofluorescein (DCF) fluorescence by confocal microscopy revealed that DCFH-DA was taken up, hydrolyzed and oxidized by yeast cells and cultured human skin fibroblasts within 20 and 6 min, respectively. Higher DCF fluorescence was observed in fibroblasts following UVA irradiation in the absence but not in the presence of the radical nitroxide, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl), which exhibits superoxide dismutase-mimetic and catalase-mimetic activity. UVA-induced fluorescence increased by approximately 50% in the presence of 32-nm anatase TiO2 particles and decreased by essentially an equal amount in the presence of TiO2 encapsulated within NaY zeolites (TiO2@NaY). Addition of the uncomplexed NaY host also decreased (by approximately 30%) the amount of UVA-induced fluorescence but, unexpectedly, the combination of the free guest and host (TiO2+NaY) caused a doubling of the fluorescence. Protection of cells against TiO2-induced intracellular ROS by encapsulation suggests that supramolecular species may be beneficial in photoprotection of the skin. In contrast, the potentiation of TiO2-induced ROS by uncomplexed NaY points to a critical role for formulation when free TiO2 is used as a sun screen ingredient.     

Changes in ultraviolet absorbance and hence in protective efficacy against lipid peroxidation of organic sunscreens after UVA irradiation

Owing to the spectral distribution of solar UV, the UVA component of sunlight is now believed to be the main cause of photoaging and photocarcinogenesis and is much more effective than UVB in inducing peroxidative damage. Consequently, most skin care cosmetic products now include UVA filters in their formulations along with UVB filters. These modern sunscreens should provide and maintain their initial absorbance, hence protection, throughout the entire period of exposure to sunlight. However, not all UVA and UVB filters are sufficiently photostable. In this study, we examine the correlation between the photochemical degradation of sunscreen agents under UVA irradiation, with particular reference to the UVA-absorber 4-tert-butyl-4'-methoxydibenzoylmethane, alone and in combination with other organic UV filters (2-ethylhexyl 4 methoxycinnamate and 2-ethylhexyl 2-cyano-3,3-diphenylacrylate) and their ability to prevent UVA-induced lipid peroxidation. Since antioxidants are also added to formulations to deactivate free radicals generated during UVA exposure, vitamin E and the synthetic antioxidant, bis(2,2,6,6-tetramethyl-1-oxyl-piperidine-4-yl)sebacate, a nitroxide derivative, were also included in this study. By using simple in vitro tests, the results show that a decrease in spectral absorbance of the UV filters correlates in most cases with increased UVA-induced lipid peroxidation; this depends on the specific UV absorber analysed and also on whether they are alone or in combination. Furthermore, the combined presence or absence of antioxidants has a profound effect on this oxidative event. In particular, the nitroxide appears to be a more efficient photo-antioxidant than vitamin E. Similar experiments were also performed under natural sunlight and the results obtained did not differ substantially from those performed under UVA. The results presented and discussed in this work may help in understanding the effects of UVA/UVB absorbers and antioxidants upon the level of UV-induced ROS generated under UVA exposure and in natural sunlight which could be relevant for improving the photoprotection and efficacy of skin care cosmetic formulations.     

PHOTO-INDUCED OXIDATIVE ACTIVATION OF THE ANTITUMOR DRUG 2N-METHYL-9-HYDROXYELLIPTICINIUM IN VITRO

Abstract— The phenolic antitumor drug 2N-methyl-9-hydroxyellipticine (NMHE) has been found to undergo oxidative activation upon irradiation with near-UV light at 365 nm (UVA) in aqueous medium. In the presence of leucine used as biological nucleophile, UVA-induced activation of NMHE results in covalent binding as shown by the generation of the corresponding adduct isobutyl-oxazolopyridocarbazole (IB-OPC). The reaction involves as the initial step the one-electron transfer from the drug to molecular oxygen yielding superoxide anion (O₂^-) whereas the generation of IB-OPC may proceed either through a free radical reaction or a Michael addition reaction. The UVA light-induced production of IB-OPC is markedly increased by superoxide dismutase (SOD) and by the singlet oxygen quencher diazabicyclooctane (DABCO) but not affected by β-carotene. It is concluded that UVA induces the oxidation of NMHE through an oxidasic process which may result in the covalent binding of the drug to biological nucleophiles. This finding leads to further investigation of the photodynamic action of NMHE in tumor cells.     

An animal model of solar-aged skin: histological, physical, and visible changes in UV-irradiated hairless mouse skin

Albino hairless mice (Skh:HR-l) exposed to sub-erythemal doses of UVB or UVA radiation display physical, visible, and histological alterations. Skin surface replicas, transepidermal water loss, and skin fold thickness were found to change with irradiation. Visibly, the skin wrinkled with UVB and sagged with UVA exposure. These changes were graded on 3-point scales. Histological alterations included tissue thickening, loss of elastic fibers, elastosis, loss of collagen, and increases in muco-substances. The UVB alterations occur to a much lesser extent with an SPF-15 (7% PABA and 3% oxybenzone) sunscreen product. This sunscreen product had little effect on development of UVA-induced changes. However, an efficient UVA sunscreen (Parsol 1789) did reduce the UVA-induced changes. Many of the UVB-induced alterations regressed after UVB irradiation was stopped. No regression in UVA-induced alterations was observed when UVA irradiation was stopped. Qualitatively, the effects with UVA irradiation were like those observed in mouse chronological aging. These models and the convenient physical and visible grading methods described can be used to determine the effectiveness of topical treatments, such as sunscreens.     

Gp91phox‐derived Reactive Oxygen Species/Urocortin 2/Corticotropin‐releasing Hormone Receptor Type 2 Play an Important Role in Long‐term Ultraviolet A Eye Irradiation‐induced Photoaging

Photoaging is induced by long‐term ultraviolet A (UVA) eye irradiation. However, the mechanism of skin damage due to UVA eye irradiation is still not well understood. In this study, we used C57BL/6j and gp91phox knockout (gp91phox^?/?) mice for the long‐term effects of UVA irradiation. The eye or dorsal skin of the mice was locally exposed to UVA for 12 months. The reactive oxygen species (ROS), gp91phox, corticotropin‐releasing hormone (CRH), urocortin 2, and CRH receptor (CRHR) type 1 and type 2 levels in the brain and mast cell tryptase and histamine levels in the dorsal skin all increased after UVA irradiation. The levels of CRH, urocortin 2, CRHR type 1 and type 2 in the brain also increased more after UVA eye irradiation than after UVA skin irradiation. Moreover, photoaging of the UVA eye irradiation mice was not induced following the administration of a ROS inhibitor in the brain. In addition, in gp91phox^?/? mice, photoaging by UVA eye irradiation was not induced. These results indicate that long‐term UVA eye irradiation led to increased gp91phox‐derived ROS in the brain and the increased expression of urocortin 2 and CRHR type 2, resulting in photoaging; however, further studies are needed to confirm these findings.     

Near-ultraviolet photolysis of beta-phenylpyruvic acid generates free radicals and results in DNA damage

Ultraviolet A (UVA) light (315-400 nm) is ubiquitously found in our environment and constitutes about 95% of the total solar UV; all UVC and most UVB being absorbed by the stratospheric ozone layer. Compared with UVB and C, UVA does not show any direct effect on biological systems. Indirect effects of UVA, however, have been recognised overwhelmingly and this includes photosensitization of biological and non-biological compounds and production of free radicals many of which include oxygen and are hence known as reactive oxygen species or ROS. Several types of free radicals have been identified although their impacts on various macro- and micro-biomolecules are yet to be fully elucidated. beta-Phenylpyruvic acid is ubiquitously found in eukaryotic cells as a metabolite of phenylalanine, which is subsequently converted to phenyllactate and/or to 2-hydroxyphenylacetate and mandelate. In patients suffering from phenylketonuria the hydroxylation of phenylalanine to tyrosine is defective due to lack of phenylalanine hydroxylase. These result in accumulation and excretion of this compound in the urine. Here we present evidence that photolysis of beta-phenylpyruvic acid by a skin tanning lamp, emitting 99% UVA (315-400 nm) and 1% UVB (290-315 nm) generates carboxyl radicals (CO(2)(*)) and also possibly causes direct electron transfer (or type 1) reactions. Electron paramagnetic resonance was used to detect the free radicals. To determine the biological effects of this photolytic reaction, T7 was exposed to these photolytic reactive agents and found to lead to high levels of phage inactivation. Damage to DNA and/or components such as tail fibre proteins may be involved in T7 inactivation. In addition, our unpublished data suggest that certain phenylketonuria cell lines are more sensitive to PPA+NUV, lending importance to photolytic studies of this agent.     

Photosensitized DNA damage and its protection via a novel mechanism

UVA, which accounts for approximately 95% of solar UV radiation, can cause mutations and skin cancer. Based mainly on the results of our study, this paper summarizes the mechanisms of UVA-induced DNA damage in the presence of various photosensitizers, and also proposes a new mechanism for its chemoprevention. UVA radiation induces DNA damage at the 5'-G of 5'-GG-3' sequence in double-stranded DNA through Type I mechanism, which involves electron transfer from guanine to activated photosensitizers. Endogenous sensitizers such as riboflavin and pterin derivatives and an exogenous sensitizer nalidixic acid mediate DNA photodamage via this mechanism. The major Type II mechanism involves the generation of singlet oxygen from photoactivated sensitizers, including hematoporphyrin and a fluoroquinolone antibacterial lomefloxacin, resulting in damage to guanines without preference for consecutive guanines. UVA also produces superoxide anion radical by an electron transfer from photoexcited sensitizers to oxygen (minor Type II mechanism), and DNA damage is induced by reactive species generated through the interaction of hydrogen peroxide with metal ions. The involvement of these mechanisms in UVA carcinogenesis is discussed. In addition, we found that xanthone derivatives inhibited DNA damage caused by photoexcited riboflavin via the quenching of its excited triplet state. It is thus considered that naturally occurring quenchers including xanthone derivatives may act as novel chemopreventive agents against photocarcinogenesis.     

Suppression of an established immune response by UVA--a critical role for mast cells

Exposing experimental animals or human volunteers to UVA II (320-340 nm) radiation after immunization suppresses immunologic memory and the elicitation of delayed-in-time hypersensitivity reactions. Previous studies indicated that the mechanisms underlying UVA-induced immune suppression are similar to those described for UVB-induced immune suppression, i.e. transferred by T regulatory cells, overcome by repairing DNA damage, neutralizing interleukin (IL)-10 activity, or injecting recombinant IL-12. Here we continued our examination of the mechanisms involved in UVA II-induced suppression. Antibodies to cis-urocanic acid blocked UVA-induced immune suppression. Treating UVA-irradiated mice with histamine receptor antagonists, calcitonin gene-related peptide (CGRP) receptor antagonists or platelet activating factor receptor antagonists blocked immune suppression in UVA-irradiated mice. In light of the fact that cis-urocanic acid and CGRP target mast cells, which can then release platelet activating factor and histamine, we measured UVA-induced immune suppression in mast cell-deficient mice. No immune suppression was noted in UVA-irradiated mast cell-deficient mice. These findings indicate that exposure to UVA II activates many of the same immune regulatory factors activated by UVB to induce immune suppression. Moreover, they indicate that mast cells play a critical role in UVA-induced suppression of secondary immune reactions.     

Protective Effect of Baicalin Against TLR4‐mediated UVA‐induced Skin Inflammation

UVA irradiation is known to cause photoaging via production of reactive oxygen species (ROS) and activation of inflammatory processes. Previously, we have demonstrated that baicalin, a plant‐derived flavonoid possessing both antioxidant and anti‐inflammatory activity, protects mouse keratinocytes against damage from UVB irradiation. However, the role of baicalin in vivo has not been well studied, particularly in the setting of UVA irradiation. To explore the protective effects and mechanisms of baicalin treatment in mice after UVA irradiation, mice were exposed to acute and chronic doses of UVA irradiation with or without baicalin or vehicle. Skin samples were collected for histological staining, RNA isolation, flow cytometry and protein extraction. Our results demonstrate the protective effect of baicalin against UVA‐induced oxidative damage and inflammation in mouse skin. These effects are likely mediated via the TLR4 pathway, which may serve as a target for photochemoprevention against skin inflammation.