Production of ROS by photosensitized anthracene under sunlight and UV-R at ambient environmental intensities

The aim of this study was to analyze the photostability and phototoxicity mechanism of anthracene (ANT) in a human skin epidermal cell line (HaCaT) at ambient environmental intensities of sunlight/UV‐R (UV‐A and UV‐B). Photomodification of ANT under sunlight/UV‐R exposure produced two photoproducts, anthrone and 9,10 anthracenedione. Generation of ¹O₂, O₂^?? and ^?OH was measured under UV‐R/sunlight exposure. Involvement of reactive oxygen species (ROS) was further substantiated by their quenching with free radical quenchers. Photodegradation of 2‐deoxyguanosine and linoleic acid peroxidation showed that ROS were mainly responsible for ANT phototoxicity. ANT generates significant amount of intracellular ROS in cell line. Maximum cell viability (85%) was reduced under sunlight exposure (30 min). Results of MTT assay accord NRU assay. ANT (0.01 μg mL^?1) induced cell‐cycle arrest at G1 phase. RT‐PCR demonstrated constitutive inducible mRNA expression of CYP 1A1 and 1B1 genes. Photosensitive ANT upregulates CYP 1A1 (2.2‐folds) and 1B1 (4.1‐folds) genes. Thus, the study suggests that ROS and DNA damage were mainly responsible for ANT phototoxicity. ANT exposure may be deleterious to human health at ambient environmental intensities reaching the earth’s surface through sunlight.     

In Vitro Evaluation of the Photoreactivity and Phototoxicity of Natural Polyphenol Antioxidants

Polyphenols are a large family of natural compounds widely used in cosmetic products due to their antioxidant and anti-inflammatory beneficial properties and their ability to prevent UV radiation-induced oxidative stress. Since these compounds present chromophores and are applied directly to the skin, they can react with sunlight and exert phototoxic effects. The available scientific information on the phototoxic potential of these natural compounds is scarce, and thus the aim of this study was to evaluate the photoreactivity and phototoxicity of five phenolic antioxidants with documented use in cosmetic products. A standard ROS assay was validated and applied to screen the photoreactivity of the natural phenolic antioxidants caffeic acid, ferulic acid, p-coumaric acid, 3,4-dihydroxyphenylacetic acid (DOPAC), and rutin. The phototoxicity potential was determined by using a human keratinocyte cell line (HaCaT), based on the 3T3 Neutral Red Uptake phototoxicity test. Although all studied phenolic antioxidants absorbed UV/Vis radiation in the range of 290 to 700 nm, only DOPAC was able to generate singlet oxygen. The generation of reactive oxygen species is an early-stage chemical reaction as part of the phototoxicity mechanism. Yet, none of the studied compounds decreased the viability of keratinocytes after irradiation, leading to the conclusion that they do not have phototoxic potential. The data obtained with this work suggests that these compounds are safe when incorporated in cosmetic products.     

Phloxine B phototoxicity: a mechanistic study using HaCaT keratinocytes

Phloxine B (PhB) (2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein; D&C Red No. 28) is a red dye found in drugs, cosmetics and foods; it is also currently being evaluated as a phototoxin for the potential control of fruit flies. Previous studies have shown that PhB is an efficient photosensitizer of damage to cellular membranes; thus, exposure of the skin to the dye and sunlight or artificial light may result in phototoxicity. Therefore, we have studied the phototoxicity of PhB and its structural analogue 2',7'-dichlorofluorescein (DCF) to HaCaT keratinocytes. Anaerobic visible irradiation (>400 nm) of PhB generated a semiquinone type radical, as detected by direct electron paramagnetic resonance. Aerobic visible irradiation of a reaction mixture containing PhB, the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and nicotinamide adenine dinucleotide (reduced) generated a superoxide dismutase-sensitive DMPO/O(2)(.-) adduct. Irradiation of PhB and DCF in D(2)O generated singlet oxygen with quantum yields of 0.59 and 0.06, respectively. PhB was much more phototoxic than DCF when cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Visible irradiation of HaCaT keratinocytes in the presence of PhB (5 micro M) resulted in a 90% decrease in cell viability. 3beta-Hydroxy-5alpha-cholest-6-ene-5-hydroperoxide, a singlet oxygen photoproduct of cholesterol, was isolated from HaCaT keratinocytes irradiated in the presence of PhB. Furthermore, PhB phototoxicity was inhibited by histidine and cysteine, quenchers of singlet oxygen. PhB (0.5 microM) and light irradiation also resulted in DNA damage, as measured by the Comet assay. The phototoxicity mechanism of PhB most probably initially involves a Type-II reaction with free radicals playing a minor role. However, secondary oxidative species such as radicals generated as a result of lipid peroxidation may serve to further promote oxidative damage. Our findings suggest that concern is warranted about the use of this dye in cosmetic products, as a food additive and in insecticidal sprays.     

Photosensitizing potential of ciprofloxacin at ambient level of UV radiation

Ciprofloxacin is a widely used fluoroquinolone drug with broad spectrum antibacterial activities. Clinical experience has shown incidences of adverse effects related to skin, hepatic, central nervous system, gastrointestinal and phototoxicity. India is a tropical country and sunlight is abundant throughout the day. In this scenario exposure to ambient levels of ultraviolet radiation (UV-R) in sunlight may lead to harmful effects in ciprofloxacin users. Phototoxicity assessment of ciprofloxacin was studied by two mouse fibroblast cell lines L-929 and NIH-3T3. Generation of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2*-) and hydroxyl radical (*OH) was studied under the exposure of ambient intensities of UV-A (1.14, 1.6 and 2.2 mW cm(-2)), UV-B (0.6, 0.9 and 1.2 mW cm(-2)) and sunlight (60 min). The drug was generating 1O2, O2*- and *OH in a concentration and dose-dependent manner. Sodium azide (NaN3) and 1,4-diazabicyclo 2-2-2-octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 90-95% O2*- generation. The drug (5-40 microg mL(-1)) was responsible for linoleic acid peroxidation. Quenching study of linoleic acid peroxidation with SOD (25 and 50 U mL(-1)) confirms the involvement of ROS in drug-induced lipid peroxidation. The generation of *OH radical was further confirmed by using specific quenchers of *OH such as mannitol (0.5 M) and sodium benzoate (0.5 M). 2'-deoxyguanosine (2'-dGuO) assay and linoleic acid peroxidation showed that ROS were mainly responsible for ciprofloxacin-sensitized photo-degradation of guanine base. L-929 cell line showed 29%, 34% and 54% reduced cell viability at higher drug concentration (300 microg mL(-1)) under UV-A, UV-B and sunlight, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in NIH-3T3 cell line at higher drug concentration (300 microg mL(-1)) showed a decrease in cell viability by 54%, 56% and 59% under UV-A, UV-B and sunlight, respectively. Results of neutral red uptake assay (NRU) in L-929 cell line were in accordance with MTT assay. The NIH-3T3 cell line showed a higher photosensitizing potential than L-929. The phototoxicity end point shows a time- and concentration-dependent statistically significant (P<0.001) damage. Ciprofloxacin produced ROS by Type I and Type II photodynamic reactions, interacted with nucleic acid moiety and inhibited cell viability. Further, UV-induced photo-peroxidation of linoleic acid accorded the involvement of ROS in the manifestation of drug phototoxicity. Appearance of ciprofloxacin-induced phototoxicity at the ambient level of sunlight is a real risk for the people of India and for those of other tropical countries. We suggest that sunlight exposure should be avoided (especially peak hours) during ciprofloxacin treatment.     

Fluorescence Evaluation of Scavenging Efficiency of Antioxidants Against Reactive Oxygen Species (ROS) in Cigarette Smoke

Cigarette smoke can cause cellular oxidative stress that contributes to various adverse health effects associated with smoke exposure, partially due to reactive oxygen species (ROS) present in cigarette smoke. Reduction of abundant ROS in the cigarette mainstream smoke (MSS) is of importance for human health. In this work, a simple, rapid, and reliable fluorescence evaluation of scavenging efficiency of antioxidants as potential filter additives against ROS in cigarette smoke is reported. This method was based on the combination of model glass reactor and a fluorescence assay of ROS in cigarette smoke using dihydrorhodamine 6 G (DHR-6 G). The antioxidant was added into a glass reactor attached to cigarette filter, which simplified the preparation of combined filter containing additives. The ROS scavenging efficiency of antioxidants was then determined using spectrofluorimetry by the change in fluorescence intensity of whole smoke-bubbled solutions before and after addition of antioxidants into the glass reactor. The proposed method was successfully applied to the determination of ROS scavenging efficiency of several potential additives, such as tert-butylhydroquinone (TBHQ), vitamin C, β-carotene, grape seed extract, and Ginkgo biloba extract. Moreover, the relationship between MSS ROS scavenging efficiency and antioxidant activities (DPPH radicals scavenging efficiency and Fe²⁺ reducing power) of these compounds was also investigated.     

Presence of peroxyradicals in cigarette smoke and the scavenging effect of shikonin, a naphthoquinone pigment

Using a new method having been developed for the purpose of quantitative determination for peroxyradicals, the presence of peroxyradicals was proved in cigarette smoke. In brief, peroxyradicals in cigarette smoke were measured by ESR spectrometry coupled to non-reductive scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH). As a result, peroxyradicals were found to be major reactive oxygen species (ROS) since the concentration of peroxyradicals recovered from cigarette smoke was much higher than that of any of other ROS (superoxide and hydroxyl radical) and nitric oxide. Furthermore, several antioxidants (ascorbic acid, reduced glutathione, epigallocatechin gallate, shikonin) were examined for scavenging activity against peroxyradicals in the cigarette smoke. Among them shikonin alone exerted the scavenging activity, suggesting that shikonin is promising antioxidant for cigarette filters because of its effectiveness against broad range of ROS including peroxyradicals, heat resistance, nonvolatility and high affinity to the filter.     

In Vitro and In Vivo Photoprotective Effects of (-)-Loliode Isolated from the Brown Seaweed,Sargassum horneri

Skin is the largest organ of humans. Overexposure to ultraviolet (UV) is the primary environmental factor that causes skin damage. The compound, (-)-loliode, isolated from the brown seaweed Sargassum horneri, showed strong antioxidant and anti-inflammatory activities in in vitro and in vivo models. To further explore the potential of (-)-loliode in cosmetics, in the present study, we investigated the photoprotective effect of (-)-loliode in vitro in skin cells and in vivo in zebrafish. The results indicated that (-)-loliode significantly reduced intracellular reactive oxygen species (ROS) level, improved cell viability, and suppressed apoptosis of UVB-irradiated human keratinocytes. In addition, (-)-loliode remarkably attenuated oxidative damage, improved collagen synthesis, and inhibited matrix metalloproteinases expression in UVB-irradiated human dermal fibroblasts. Furthermore, the in vivo test demonstrated that (-)-loliode effectively and dose-dependently suppressed UVB-induced zebrafish damage displayed in decreasing the levels of ROS, nitric oxide, lipid peroxidation, and cell death in UVB-irradiated zebrafish. These results indicate that (-)-loliode possesses strong photoprotective activities and suggest (-)-loliode may an ideal ingredient in the pharmaceutical and cosmeceutical industries.     

Photosensitizing mechanism and identification of levofloxacin photoproducts at ambient UV radiation

Levofloxacin (LVFX) is a broad spectrum third generation fluoroquinolone antibiotic, used in the treatment of severe or life-threatening bacterial infections. Photosensitizing mechanism of LVFX was investigated under the ambient environmental intensities of UV-A, UV-B and sunlight exposure. Phototoxic effects of LVFX were assessed on NIH-3T3 and HaCaT cell lines. Results identified first time three photoproducts of LVFX at ambient levels of UV-R by LC-MS/MS. The generation of reactive oxygen species (ROS) was investigated photochemically as well as intracellularly in HaCaT cell line. ROS were significantly quenched by specific quenchers like DABCO, NaN(3), D-mannitol and NAC. Photosensitized LVFX caused lipid peroxidation at different concentrations. Quenching study with superoxide dismutase confirms the LVFX-induced lipid photoperoxidation. Further, photocytotoxicity of LVFX showed significant reduction in cell viability by MTT and neutral red uptake assays. LVFX caused cell arrest in G2/M phases as well as induced apoptosis through ROS-dependent pathway. In addition, photosensitized LVFX also induced upregulation of p21 and Bax/Bcl-2 genes ratio. India is a tropical country and most of the human activities such as agriculture, commerce, sports, etc. take place in bright sunlight; therefore, photosensitive LVFX may lead to skin/ocular disorders and immune suppression. Information is needed regarding the phototoxicity of LVFX for human safety.     

Defensive effects of fullerene-C60/liposome complex against UVA-induced intracellular reactive oxygen species generation and cell death in human skin keratinocytes HaCaT,associated with intracellular uptake and extracellular excretion of fullerene-C60

The UVA-irradiation of 10 J/cm² on HaCaT keratinocytes increased 59.1% of the intracellular reactive oxygen species (ROS) by NBT assay and the cell viability decreased to 31.5% by WST-1 assay, comparing to the non-irradiated control. In the presence of fullerene-C60 (C60) incorporated in phospholipid membrane vehicle (LiposomeFullerene: Lpsm-Flln) of 250–500 ppm, they were restored to ?9.1% to  + 2.3% of the ROS and 83.0–84.8% of the cell viability, but scarcely restored by the liposome without C60 (Lpsm). In HaCaT cells administered with Lpsm-Flln (150 ppm), C60 was ingested at the intracellular concentrations of 1.4–21.9 ppm for 4–24 h, and, intracellular C60 was excreted by 80% at 4 h after rinsing-out, and decreased to 2–10% after 24–48 h. C60 was predominantly distributed around the outside of nuclear membrane without deterioration of intact cell morphology according to fluorescent immunostain. Thus Lpsm-Flln is found to be an effective antioxidant that could preserve HaCaT keratinocytes against UVA-induced cellular injury. Lpsm-Flln has a potential to serve as a cosmetic material for skin protection against UVA.     

Photo‐co‐carcinogenesis of Topically Applied Retinyl Palmitate in SKH‐1 Hairless Mice

Cosmetic products that contain retinyl palmitate are popular as antiaging skin treatments; however, recent studies suggest a risk for enhanced skin tumor development with topical retinyl palmitate applications and exposure to solar ultraviolet radiation (UVR). In this study, we investigated the potential of retinyl palmitate to enhance UVR‐induced photo‐co‐carcinogenesis. Groups of 36 male and 36 female SKH‐1 hairless mice were exposed to simulated solar light (SSL) and treated with the control cream or creams containing retinyl palmitate, 5 days per week for 40 weeks. Other groups of mice were exposed to SSL and received no cream treatment or received cream treatments and were exposed to ultraviolet‐A or ultraviolet‐B. Mice were monitored for the development of skin tumors, and the incidences and multiplicities of squamous cell neoplasia were determined by histopathology. In both the absence and presence of SSL, mice administered the control cream developed skin tumors earlier and had higher incidences and multiplicities of skin squamous cell neoplasms than mice that received no cream treatment. Compared to the control cream groups, mice exposed to SSL and administered the retinyl palmitate creams demonstrated earlier onsets of skin tumors and had increased incidences and multiplicities of squamous cell skin neoplasms.     

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.     

Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin

Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 x 10(-3) mol of ROS that react with DHR are generated in the stratum corneum of an average adult-size face (258 cm(-2)). Approximately 10(-4) mol are potentially generated in the lower epidermal strata. The data show that two-photon fluorescence imaging can be used to detect ROS in UV-irradiated skin.     

Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells

Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.     

Crataegus laevigata Suppresses LPS-Induced Oxidative Stress during Inflammatory Response in Human Keratinocytes by Regulating the MAPKs/AP-1, NFκB,and NFAT Signaling Pathways

Crataegus laevigata belongs to the family Rosaceae, which has been widely investigated for pharmacological effects on the circulatory and digestive systems. However, there is limited understanding about its anti-oxidative stress and anti-inflammatory effects on skin. In this study, 70% ethanol C. laevigata berry extract (CLE) was investigated on lipopolysaccharide (LPS)-stimulated keratinocytes. The LPS-induced overproduction of reactive oxygen species (ROS) was suppressed by the treatment with CLE. In response to ROS induction, the overexpression of inflammatory regulating signaling molecules including mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB), and nuclear factor of activated T-cells (NFAT) were reduced in CLE-treated human keratinocytes. Consequently, CLE significantly suppressed the mRNA levels of pro-inflammatory chemokines and interleukins in LPS-stimulated cells. Our results indicated that CLE has protective effects against LPS-induced injury in an in vitro model and is a potential alternative agent for inflammatory treatment.     

Effects of Light Exposure and Use of Intraocular Lens on Retinal Pigment Epithelial Cells In Vitro

To investigate the effect of a blue light-filtering intraocular lens (IOL) and a UV-absorbing IOL on light-induced damage to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore N -retinylidene- N -retinylethanolamine (A2E), A2E-laden RPE cells were exposed to white light which was filtered by either a blue light-filtering IOL or a UV-absorbing IOL. After 30 min of illumination the cell viability and the level of reactive oxygen species (ROS), free glutathione (GSH), vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) were determined. In the absence of an IOL, the white light exposure decreased cell viability to 37.2% of the nonirradiated control. The UV-absorbing IOL tended to reduce light-induced cell death; however, the decrease was not significant. The blue light-filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 79.5% of the nonirradiated control. The presence of the blue light-filtering IOL significantly increased GSH and PEDF levels, and decreased ROS and VEGF levels. This study suggests that a blue light-filtering IOL may be more protective against A2E-induced light damage and inhibit more light-induced ROS and VEGF production than a conventional UV-absorbing IOL.     

The relationship between UVB screening and cytoprotection by microcorpuscular ZnO or ascorbate against DNA photodamage and membrane injuries in keratinocytes by oxidative stress.

Decreased cell viability and increased formation of cyclobutane-type pyrimidine dimers (CPDs) in DNA of UVB-irradiated keratinocytes were shown to be appreciably restored by the addition of w/o emulsion of microcorpuscular zinc oxide (mcZnO) with a corpuscle diameter of 0.15 microm. The cytoprotection was exerted only by 20 wt/wt% mcZnO at levels equivalent to 40- to 100-microm-thick emulsion layers, which screened 90-92% of the incident UVB. However, protection was not seen by mcZnO below 20-microm thickness, which, unexpectedly, screened 79% of the incident radiation. This suggests that thorough UVB screening is necessary for cytoprotection. This may be attributable to involvement of intracellular reactive oxygen species (ROS) secondarily generated from UVB-irradiated mcZnO. Intracellular ROS was increased in mcZnO-added cells in a time-dependent manner even after UVB irradiation, contrasting with reduction of intracellular ROS in ascorbic acid-added cells. UVB-induced disruption of cell membrane integrity was reduced by mcZnO at 100-microm thickness, equivalent to the addition of ascorbic acid of 50 microM. Thus, mcZnO was thought to be cytoprotective through reductions of intracellular ROS generation, CPD formation and cell membrane disintegration when added so abundantly so as to achieve UVB-screening more than 90%.     

Hesperidin Exhibits Protective Effects against PM2.5-Mediated Mitochondrial Damage,Cell Cycle Arrest,and Cellular Senescence in Human HaCaT Keratinocytes

Particulate matter 2.5 (PM_2.5) exposure can trigger adverse health outcomes in the human skin, such as skin aging, wrinkles, pigment spots, and atopic dermatitis. PM_2.5 is associated with mitochondrial damage and the generation of reactive oxygen species (ROS). Hesperidin is a bioflavonoid that exhibits antioxidant and anti-inflammatory properties. This study aimed to determine the mechanism underlying the protective effect of hesperidin on human HaCaT keratinocytes against PM_2.5-induced mitochondrial damage, cell cycle arrest, and cellular senescence. Human HaCaT keratinocytes were pre-treated with hesperidin and then treated with PM_2.5. Hesperidin attenuated PM_2.5-induced mitochondrial and DNA damage, G₀/G₁ cell cycle arrest, and SA-βGal activity, the protein levels of cell cycle regulators, and matrix metalloproteinases (MMPs). Moreover, treatment with a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, along with hesperidin markedly restored PM_2.5-induced cell cycle arrest and cellular senescence. In addition, hesperidin significantly reduced the activation of MMPs, including MMP-1, MMP-2, and MMP-9, by inhibiting the activation of activator protein 1. In conclusion, hesperidin ameliorates PM_2.5-induced mitochondrial damage, cell cycle arrest, and cellular senescence in human HaCaT keratinocytes via the ROS/JNK pathway.     

Photochemistry and phototoxicity of aloe emodin

Photochemical pathways leading to the phototoxicity of the aloe vera constituent aloe emodin were studied. The results indicate a photochemical mechanism involving singlet oxygen to be the most likely pathway responsible for the observed phototoxicity. Aloe emodin was found to efficiently generate singlet oxygen when irradiated with UV light (phidelta = 0.56 in acetonitrile). The survival of human skin fibroblast cells in the presence of aloe emodin was found to decrease upon irradiation with UV light. A further decrease in cell survival was observed in D2O compared with H2O, suggesting the involvement of singlet oxygen as the primary pathway. Laser flash photolysis experiments were also carried out on aloe emodin alone and in the presence of various biological substrates. Aloe emodin proved to be relatively photostable (phi = 1 x 10(-4)) and a poor photo-oxidant (E*red = +1.02 V). Only absorption bands caused by the triplet state of aloe emodin (lambdamax = 480 nm) and the aloe emodin conjugate base (lambdamax = 520 nm) were observed in the transient spectra.     

Simvastatin inhibits induction of matrix metalloproteinase-9 in rat alveolar macrophages exposed to cigarette smoke extract

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IκB, and nuclear AP-1 or NF-κB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IκB-NF-κB are involved.     

INHIBITORY EFFECT OF 8-METHOXYPSORALEN PLUS ULTRAVIOLET-A ON INTERLEUKIN-1 PRODUCTION BY MURINE KERATINOCYTES

We report the effects of 8-methoxypsoralen (8-MOP) plus ultraviolet-A (UV-A) irradiation on interleukin-1 (IL-1) production by murine epidermal keratinocytes, correlating its effect on IL-1 with cell viability, DNA synthesis, and 8-MOP-DNA photoadduct formation. Freshly isolated murine keratinocytes were treated with various doses of 8-MOP (5-100 ng/mL; incubation time, 30 min) plus 1 J/cm2 UV-A and cultured for 1-3 days. The IL-1/epidermal cell-derived thymocyte-activating factor (ETAF) activity in both supernatant and cell extract was reduced proportionately with increasing doses of 8-MOP/UV-A. Interleukin-1 inhibitors induced by 8-MOP plus UV-A were not detected in either supernatant or cell extract. A clear reduction of the IL-1 production was induced by the treatment as low as 15 ng/mL 8-MOP plus 1 J/cm2 UV-A, which led to the formation of 0.52 8-MOP photoadducts per million DNa bases and affected neither cell viability nor DNA synthesis of the treated cells. Cells treated with 100 ng/mL 8-MOP and 1 J/cm2 UV-A exhibited 57% suppression of IL-1 production in both 2- and 3-day culture samples. This treatment resulted in the formation of 3.8 photoadducts per million bases as well as significant abrogation of DNA synthesis although cell viability was unchanged. These observations provide some insights into the phototoxicity mechanisms of 8-MOP and the effect of PUVA therapy on the cytokine regulation in keratinocytes.