UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine

Reactive oxygen species (ROS) are involved in the oxidative damage of the cyanobacterium Anabaena sp. caused by UV-B (280-315 nm) radiation. UV-B-induced overproduction of ROS as well as the oxidative stress was detected in vivo by using the ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Thiobarbituric acid reactive substances (TBARS) and fluorometric analysis of DNA unwinding (FADU) methods were adapted to measure lipid peroxidation and DNA strand breaks in Anabaena sp. Moderate UV-B radiation causes an increase of ROS production, enhanced lipid peroxidation and DNA strand breaks, yielding a significantly decreased survival. In contrast, the supplementation of UV-A in our work only showed a significant increase in total ROS levels and DNA strand breaks while no significant effect on lipid peroxidation, chlorophyll bleaching or survival was observed. The presence of ascorbic acid and N-acetyl-L-cysteine (NAC) reversed the oxidative stress and protected the organisms from chlorophyll bleaching and the damage of photosynthetic apparatus induced by UV-B significantly, resulting in a considerably higher survival rate. Ascorbic acid also exhibited a significant protective effect on lipid peroxidation and DNA strand breaks while NAC did not show a substantial effect. These results suggest that ascorbic acid exhibited significantly higher protective efficiency with respect to DNA strand breaks and survival than NAC while NAC appears to be especially effective in defending the photosynthetic apparatus from oxidative damage.     

Adaptation of cyanobacteria to UV-B stress correlated with oxidative stress and oxidative damage

Cyanobacteria must cope with the negative effects of ultraviolet B (280-315 nm) (UV-B) stress caused by their obligatory light requirement for photosynthesis. The adaptation of the cyanobacterium Anabaena sp. to moderate UV-B radiation has been observed after 2 weeks of irradiation, as indicated by decreased oxidative stress, decreased damage, recovered photosynthetic efficiency and increased survival. Oxidative stress in the form of UV-B-induced production of reactive oxygen species was measured in vivo with the oxidative stress-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate. Photooxidative damage by UV-B radiation, including lipid peroxidation and DNA strand breakage, was determined by a modified method using thiobarbituric acid reactive substances and fluorometric analysis of DNA unwinding. Photosynthetic quantum yield was determined by pulse amplitude-modulated fluorometry. The results suggest that moderate UV-B radiation results in an evident oxidative stress, enhanced lipid peroxidation, increased DNA strand breaks, elevated chlorophyll bleaching as well as decreased photosynthetic efficiency and survival during the initial exposure. However, DNA strand breaks, photosynthetic parameters and chlorophyll bleaching returned to their unirradiated levels after 4-7 days of irradiation. Oxidative stress and lipid peroxidation appeared to respond later because decreases were observed after 7 days of radiation. The survival curve against irradiation time exhibited a close relationship with the changes in photosynthetic quantum yield and DNA damage, with little mortality after 4 days. Growth inhibition by UV-B radiation was observed during the first 7 days of radiation, whereas normal growth resumed even under UV-B stress thereafter. An efficient defense system was assumed to come into play to repair photosynthetic and DNA damage and induce the de novo synthesis of UV-sensitive proteins and lipids, allowing the organisms to adapt to UV-B stress successfully and survive as well as grow. No induction of mycosporine-like amino acids (MAA) was observed during the adaptation of Anabaena sp. to UV-B stress in our work. The adaptation of the cyanobacterium correlated with and could be caused by the oxidative stress and oxidative damage.     

Involvement of reactive oxygen species in the UV-B damage to the cyanobacterium Anabaena sp

Reactive oxygen species (ROS) are involved the damage of living organisms under environmental stress including UV radiation. Cyanobacteria, photoautotrophic prokaryotic organisms, also suffer from increasing UV-B due to the depletion of the stratospheric ozone layer. The increased UV-B induces the production of ROS in vivo detected by using the ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Ascorbic acid and N-acetyl-L-cysteine (NAC) scavenged ROS effectively, while alpha-tocopherol acetate or pyrrolidine dithiocarbamate (PDTC) did not. The presence of rose bengal and hypocrellin A increased the ROS level by photodynamic action in the visible light. The presence of the herbicide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), increased ROS production slightly, and ROS formation was greatly enhanced by the addition of methyl viologen due to the fact that this redox system diverts electrons from PSI to oxygen and thus forms ROS. UV-B induces ROS generation by photodynamic action and inhibition of the electron transport by damaging the electron receptors or enzymes associated with the electron transport chain during photosynthesis.     

Changes in blood platelets exposed to UV-B radiation.

The effects of UV-B radiation (312 nm) on the pig-blood platelet secretory process (platelet activation) and platelet lipid peroxidation have been studied. The responses of platelets to UV-B radiation are compared with the response of these cells to thrombin, which is a strong platelet agonist. The obtained results show that exposure of blood platelets to UV-B radiation (1.2 mW/cm2, 0.072-8.64 J/cm2) causes dose-dependent platelet lipid peroxidation (measured as thiobarbituric acid reactive substances, TBARS) and release of adenine nucleotides and proteins from irradiated platelets. The dose-dependent release of platelet compounds from irradiated platelet does not correlate with the activity of platelet lactic dehydrogenase (marker of cell lysis) in the extracellular medium. It seems that UV-B radiation can partly activate platelets by stimulating the platelet secretory process and metabolism of arachidonate.     

Increased UV-B Radiation Affects the Viability, Reactive Oxygen Species Accumulation and Antioxidant Enzyme Activities in Maize (Zea mays L.) Pollen

The increase in UV-B radiation reaching the earth's surface has prompted extensive studies on the effects of UV-B on plants. However, most of these studies have not addressed the close characteristics related to future survival of plant populations. The purpose of this study was to investigate the effects of UV-B radiation on reactive oxygen species (ROS) accumulation and antioxidant defense system in relation to germination, tube length and viability of maize pollen. Our results indicate that increased UV-B radiation decreased the pollen germination rate and tube length in vitro and also its fertilization ability in the field. Production of O₂^•− and H₂O₂ increased by UV-B radiation treatment, and their continuous accumulation resulted in lipid peroxidization. The activities of superoxide dismutase, catalase, peroxidase and DPPH-radical scavenging were decreased by increased UV-B radiation. The increased ROS and lipid peroxidization, and decreased activities of the antioxidants may be attributed to the effects of UV-B radiation on pollen germination, tube growth and fertilization ability.     

Spectral properties of topical retinoids prevent DNA damage and apoptosis after acute UV-B exposure in hairless mice

We showed in a recent study that topical retinyl palmitate prevented UV-B-induced DNA damage and erythema in humans. Given that retinyl palmitate is a precursor of retinoic acid, the biological form of vitamin A that acts through nuclear receptors, we wondered whether these protective effects toward UV-B exposure were either receptor dependent or linked to other properties of the retinoid molecule such as its spectral properties. We determined the epidermal retinoid profile induced by topical retinoic acid in hairless mice and analyzed its effect on markers of DNA photodamage (thymine dimers) and apoptosis following acute UV-B exposure; we compared these effects to those induced by other natural topical retinoids (retinaldehyde, retinol and retinyl palmitate) which do not directly activate the retinoid receptors. We then analyzed the direct action of these retinoids on UV-B-induced DNA damage and apoptosis in cultured A431 keratinocytes. Topical retinoic acid significantly decreased (approximately 50%) the number of apoptotic cells, as well as the formation of thymine dimers in the epidermis of mice exposed to acute UV-B. Interestingly, the other topical retinoids decreased apoptosis and DNA damage in a similar way. On the other hand, neither retinoic acid nor the other retinoids interfered with the apoptotic process in A431 keratinocytes exposed to UV-B, whereas DNA photodamage was slightly decreased. We conclude that the decrease of apoptotic cells in hairless mouse epidermis following topical retinoids and UV-B irradiation reflects a protection of the primary targets of UV-B (DNA) by a mechanism independent of the activation of retinoid nuclear receptors, rather than a direct inhibition of apoptosis.     

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.     

Contrasting effects of excess ferritin expression on the iron-mediated oxidative stress induced by tert-butyl hydroperoxide or ultraviolet-A in human fibroblasts and keratinocytes

Iron and/or ferritin accumulation are known to occur under pathological conditions in many inflammatory skin diseases or in human skin chronically exposed to UV light. Under such conditions, ferritin is believed to play an effective protective role in accommodating and 'deactivating' excess 'free' iron produced by the inflammatory process or the UV illumination. The present study compares the relationship between ferritin over-expression and effects of an oxidative stress induced chemically by tert-butyl hydroperoxide or photochemically by UV-A radiation. As shown by immunoassay, cultured MRC 5 and HS 68 fibroblasts treated for at least one day with transferrin or overnight with non-toxic concentrations of the ferric nitrilotriacetate complex express up to 10 times more ferritin than untreated cells, whereas a five-fold increase is obtained with NCTC 2544 keratinocytes. In all cases a parallel increase in soluble cellular iron is measured by inductive plasma emission spectroscopy. The superoxide dismutase and catalase activities and total glutathione levels are not modified by the iron treatment, whereas a transient increase in the Se-dependent glutathione peroxidase activity of keratinocytes is observed after a short incubation with the iron complex. In keratinocytes and fibroblasts, ferritin over-expression after iron treatment markedly inhibits lipid peroxidation but, paradoxically, not the mortality induced by tert-butyl hydroperoxide. In contrast, this excess ferritin does not protect cells from both the peroxidation and mortality induced by moderate doses (30 J/cm2) of UV-A radiation. As a consequence, protection against oxidative damage by excess ferritin synthesis clearly depends on the nature of the oxidative stress on cell targets and it seems to be of lesser importance in the case of photochemically induced oxidation.     

Production of gamma induced reactive oxygen species and damage of DNA molecule in HaCaT cells under euoxic and hypoxic condition

The paper deals with the study of gamma radiation induced reactive oxygen species (ROS) generation in normal human keratinocytes (HaCaT) cells and quantification of subsequent damages induced on DNA molecules. The DNA damages induced in cells after gamma irradiation has been analyzed using Alkaline comet assay. The ROS produced in the cells were quantified by measuring fluorescence after loading the cells with 2′, 7′ dichlorofluorescin diacetate, a dye that is oxidized into a highly fluorescent form in the presence of peroxides. Studies reveal that in HaCaT cells radical generation occurs when exposed to ionizing radiation and it increases with dose. The induced DNA damages also increases with dose and ROS generation. The study clearly shows the importance of ROS in DNA damage induction and the cells possessing elevated levels of DNA damage after radiation exposure is due to the effect of increased levels of intracellular ROS.     

Oxidative stress and defence mechanisms of the freshwater cladoceran Daphnia longispina exposed to UV radiation

The aim of the present study is to evaluate the occurrence of oxidative stress in the cladoceran Daphnia longispina exposed to UV-A and UV-B radiation. The activity of antioxidant enzymes and lipid peroxidation markers is investigated and the protective action of ascorbic acid determined. Results show differences in the lethality radioinduced by UV-A and UV-B. Both UV-A and UV-B exposure cause an important increase in malonaldehyde (MDA) concentration and catalase activity. Ascorbic acid addition reduces the MDA concentration, indicating that the oxidative stress caused by either UV-A or UV-B radiation can be controlled by antioxidants. The increase of the antioxidant enzymes may be a response mechanism to oxidative stress.     

Nano-TiO2-Induced Apoptosis by Oxidative Stress-Mediated DNA Damage and Activation of p53 in Human Embryonic Kidney Cells

The aim of the present study is to explore the mechanism of cytotoxic and genotoxic effects of TiO₂ nanoparticles on human embryonic kidney (HEK-293) cells. Toxicity was evaluated using changes in various cellular parameters of HEK-293 cells like morphology, viability, metabolic activity, oxidative stress and apoptosis. Oxidative stress was measured by the level of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase, catalase and glutathione peroxidase. Apoptosis induced by nano-TiO₂ was characterized by PI staining and DNA ladder assay. Furthermore, apoptotic proteins such as p53 and Bax were analysed by western blot. Our results indicate that nano-TiO₂ induces cytotoxicity in a time- and dose-dependent manner. Oxidative stress and apoptosis were induced by exposure to nano-TiO₂. Moreover, the expression of p53, Bax and caspase-3 were increased in a dose-dependent pattern. In conclusion, ROS-mediated oxidative stress, the activation of p53, Bax, caspase-3 and oxidative DNA damage are involved in the mechanistic pathways of nano-TiO₂-induced apoptosis in HEK-293 cells.     

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.     

Effect of Enhanced UV-B Radiation and Low-Energy N+ Ion Beam Radiation on the Response of Photosynthesis, Antioxidant Enzymes, and Lipid Peroxidation in Rice (Oryza sativa) Seedlings

To understand the effect of enhanced UV-B radiation and low-energy N⁺ ion beam radiation on the response of photosynthesis, antioxidant enzymes, and lipid peroxidation in rice seedlings, Oryza sativa was exposed to three different doses of low-energy N⁺ ion beam and enhanced UV-B alone and in combination. Enhanced UV-B caused a marked decline in some photosynthetic parameters (net photosynthetic rate, transpiration rate, and stomatal conductance) and photosynthetic pigments, whereas it induced an increase in hydrogen peroxide (H₂O₂) accumulation, the rate of superoxide radical production, and the content of malondialdehyde (MDA). Enhanced UV-B also induced an increase in the activity of antioxidant enzymes (superoxide dismutase [SOD], peroxidase (POD), and catalase [CAT]) and some nonenzymatic antioxidants such as proline. Under the combined treatment of enhanced UV-B and low-energy N⁺ ion beam at the dose of 3.0?×?10¹⁷ N⁺ cm^?2, the activity of antioxidant compounds (SOD, POD, CAT, proline, and glutathione), photosynthetic pigments, and some photosynthetic parameters (net photosynthetic rate, transpiration rate, and stomatal conductance) increased significantly; however, the MDA content, H₂O₂ accumulation, and rate of superoxide radical production showed a remarkable decrease compared with the enhanced UV-B treatment alone. These results implied that the appropriate dose of low-energy N⁺ ion beam treatment may alleviate the damage caused by the enhanced UV-B radiation on rice.     

Topically applied eicosapentaenoic acid protects against local immunosuppression induced by UVB irradiation, cis-urocanic acid and thymidine dinucleotides

UVB-induced immunosuppression, a promoter of photocarcinogenesis, involves the formation of pyrimidine dimers and cis-urocanic acid (cis-UCA), but reactive oxygen species (ROS) also plays an important role. Eicosapentaenoic acid (EPA) can inhibit photocarcinogenesis, but due to its polyunsaturated nature it is susceptible to oxidative damage by ROS. The antioxidant defense system may therefore be challenged upon ultraviolet-B (UVB) irradiation in the presence of EPA. We investigated whether topically applied EPA in mice could protect against local immunosuppression (contact hypersensitivity response to dinitrofluorobenzene) induced by UVB radiation (1.5 J/cm2), or topically applied cis-UCA (150 nmol/cm2) or thymidine dinucleotides (pTpT) (5 nmol/cm2). The influence of EPA on epidermal lipid peroxidation and antioxidant status was also measured. UVB irradiation, cis-UCA and pTpT all caused 70% immunosuppression. Topical pretreatment of mice with EPA partially protected against immunosuppression; the EPA dose needed to accomplish this was 10 nmol/cm2 for UVB irradiation, 100 nmol/cm2 for cis-UCA and 1000 nmol/cm2 for pTpT. Higher EPA doses caused higher UVB-induced lipid peroxidation and lower vitamin C levels. Glutathione only decreased with the highest EPA dose whereas vitamin E was not decreased after UVB irradiation. In conclusion, topically applied EPA protects against UVB-, cis-UCA- and pTpT-induced immunosuppression and maintenance of an adequate antioxidant defense seems to be an important prerequisite for the protective action by EPA.     

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.     

Antioxidant protection in cultured corneal cells and whole corneas submitted to UV-B exposure

Several corneal pathologies are characterized by the presence of reactive oxygen species (ROS); therefore, we evaluated the protection afforded by pirenoxine and melatonin to corneal cell culture and whole rabbit cornea from ultraviolet exposure and other oxidant systems. Rabbit cornea cell (SIRC) plates and whole corneas were exposed to UV-B (80 or 800 mJ/cm2) or incubated with fMLP-stimulated autologous macrophages, in the presence or absence of pirenoxine or melatonin (10(-5) M). The protective activity of compounds was assessed by measuring superoxide anion formation, inhibition of oxidation and mitochondrial viability. Moreover the ex vivo protective effect of pirenoxine and melatonin was verified in the whole cornea submitted to UV-B exposure in vitro. Our experimental data demonstrate that pirenoxine and melatonin were able to inhibit the superoxide formation and oxidative effect in cell culture and whole rabbit corneas submitted to UV-B exposure or to incubation with fMLP-stimulated autologous macrophages. Mitochondrial viability was restored in epithelial cells of rabbit cornea but not in SIRCs. Moreover, both compounds are also able to increase ex vivo epithelial corneal cell defences against the in vitro UV-B induced lipid peroxidation.     

The damage repair role of He-Ne laser on plants exposed to different intensities of ultraviolet-B radiation

Light-grown broad bean (Vicia faba L.) seedlings were subjected to different intensities of UV-B radiation (0, 0.05, 0.15, 0.45, 0.90, 1.45 and 1.98 W m(-2)) for 7 h under photosynthetically active radiation (70 micromol m(-2) s(-1)) and then exposed to He-Ne laser (632.8 nm, 5.43 mW mm(-2)) radiation for 5 min or red light radiation for 4 h without ambient light radiation. When He-Ne laser radiated leaves were treated using lower intensity UV-B, the activities of superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and catalase (EC 1.11.1.6) improved significantly. Moreover, the UV-B-injured plants treated with laser light recovered faster from UV-B treatment because the concentration of malondialdehyde and the rate of electrolyte leakage from leaf disks reached control levels (no UV-B or laser treatment) early compared with those exposed only to ambient light or in dark conditions. Laser treatment, however, had no repair effect on seedling damage induced by higher UV-B radiation (1.45 and 1.98 W m(-2)), even with higher laser flux rates and longer laser treatment. In addition, the red light treatment had no repair effect on UV-B-induced damage. Meanwhile, the long-term physiological effect of He-Ne laser treatment on UV-B damaged plants was presented and evaluated. The results showed that the laser had a long-term positive physiological effect on the growth of UV-B-damaged plants. With the exception of the severe damage caused by higher UV-B radiation, a laser with the proper flux rate and treatment time can repair UV-B-induced damage and shorten the recovery time.     

(-)-Epicatechin-3-gallate, a green tea polyphenol is a potent agent against UVB-induced damage in HaCaT keratinocytes

(-)-Epicatechin-3-gallate (ECG) is a polyphenolic compound similar to (-)-epigallocatechin-3-gallate (EGCG) which is abundant in green tea. Numerous workers have proposed that EGCG protects epidermal cells against UVB-induced damage. However, little has been known about whether ECG protects keratinocytes against UVB-induced damage. We decided to investigate the protective effects and underlying mechanisms of ECG on UVB-induced damage. Cell viability was determined by the MTT assay. Activation of ERK1/2, p38 and JNK was analyzed by Western blotting. Intracellular H2O2 production and DNA content was analyzed by flow cytometry. Lipid peroxidation was assayed by colorimetry. In our study, we found that ECG dose-dependently attenuated UVB-induced keratinocyte death. Moreover, ECG markedly inhibited UVB-induced cell membrane lipid peroxidation and H2O2 generation in keratinocytes, suggesting that ECG can act as a free radical scavenger when keratinocytes were photodamaged. In parallel, H2O2-induced the activation of ERK1/2, p38 and JNK in keratinocytes could be inhibited by ECG. UVB-induced pre-G1 arrest leading to apoptotic changes of keratinocytes were blocked by ECG. Taken together, we provide here evidence that ECG protects keratinocytes from UVB-induced photodamage and H2O2-induced oxidative stress, possibly through inhibition of the activation of ERK1/2, p38 and JNK and/or scavenging of free radicals.     

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.     

An immunomodulator from Tinospora cordifolia with antioxidant activity in cell-free systems

Several plant products are known to exhibit immense medicinal value against human diseases. Our earlier studies showed that dry stem crude extract (DSCE) ofTinospora cordifolia contained a polyclonal B cell mitogen, G1-4A. DSCE as well as G1-4A also enhanced immune response in mice. In order to explore the possibility of using G1-4A/PPI (partially purified immunomodulator) to modulate radiation induced immunosuppression, the antioxidant effect of PPI from this plant was examined against reactive oxygen and nitrogen species (ROS/RNS), generated by photosensitization/peroxynitrite. Levels of lipid peroxidation products, superoxide dismutase (SOD) and catalase in liver/spleen homogenate from mouse were monitored. Photosensitization induced significant increase in thiobarbituric acid reactive substances (TBARS) in liver. The activities of SOD and catalase were reduced considerably. PPI, present during photosensitisation, prevented lipid peroxidation and restored the activities of both the enzymes. Likewise, oxidative damage induced by peroxynitrite was inhibited by PPI. The degradation of proteins due to photosensitization as assessed by SDS-PAGE was effectively reduced by simultaneous treatment with PPI during photosensitization. Selective inhibitors of ROS like mannitol, SOD, sodium azide and antioxidants, GSH and vitamin C brought about significant inhibition of formation of TBARS suggesting possible involvement of O₂ ^•,^•OH and¹O₂. Photosensitization in deuterated buffer enhanced formation of TBARS thus indicating generation of¹O₂. Thus, the action of PPI may be against oxidative damage through Type I and II photosensitization mechanisms. Therefore, the immunomodulator fromTinospora cordifolia may also be beneficial as an antioxidant.