UVA-induced Lipid Peroxidation Research Articles

Modulation of Redox and Inflammatory Signaling in Human Skin Cells Using Phytocannabinoids Applied after UVA Irradiation: In Vitro Studies.

UVA exposure disturbs the metabolism of skin cells, often inducing oxidative stress and inflammation. Therefore, there is a need for bioactive compounds that limit such consequences without causing undesirable side effects. The aim of this study was to analyse in vitro the effects of the phytocannabinoids cannabigerol (CBG) and cannabidiol (CBD), which differ in terms of biological effects. Furthermore, the combined use of both compounds (CBG+CBD) has been analysed in order to increase their effectiveness in human skin fibroblasts and keratinocytes protection against UVA-induced alternation. The results obtained indicate that the effects of CBG and CBD on the redox balance might indeed be enhanced when both phytocannabinoids are applied concurrently. Those effects include a reduction in NOX activity, ROS levels, and a modification of thioredoxin-dependent antioxidant systems. The reduction in the UVA-induced lipid peroxidation and protein modification has been confirmed through lower levels of 4-HNE-protein adducts and protein carbonyl groups as well as through the recovery of collagen expression. Modification of antioxidant signalling (Nrf2/HO-1) through the administration of CBG+CBD has been proven to be associated with reduced proinflammatory signalling (NFκB/TNFα). Differential metabolic responses of keratinocytes and fibroblasts to the effects of the UVA and phytocannabinoids have indicated possible beneficial protective and regenerative effects of the phytocannabinoids, suggesting their possible application for the purpose of limiting the harmful impact of the UVA on skin cells.

Photoprotective and Antiaging Effects of a Standardized Red Orange (Citrus sinensis (L.) Osbeck) Extract in Asian and Caucasian Subjects: A Randomized, Double-Blind, Controlled Study.

The increase in solar ultraviolet radiation (UVR) that reaches the Earth’s surface should make us reflect on the need to develop new approaches in protecting the skin from UVR exposure. The present study aims to evaluate the photoprotective and antiaging efficacy of a red orange extract (100 mg/day) in both Asian and Caucasian subjects. A randomized, double-blind, controlled study was carried out in 110 Asian and Caucasian subjects. Product efficacy was measured as follows: (1) the photoprotective effect was measured by the minimal erythema dose (MED) assessment; (2) the efficacy in decreasing the UVA+B-induced skin redness was measured by colorimetry; (3) the antioxidant efficacy was measured by the ferric-reducing antioxidant power (FRAP) and the malondialdehyde (MDA) assay; and (4) skin moisturization, skin elasticity, skin radiance, the intensity of melanin staining, transepidermal water loss (TEWL), and wrinkles were measured to assess the antiaging efficacy. The intake of the product for 56 days was effective in improving the skin reaction to UV exposure; in increasing the skin antioxidant capacity as well as in decreasing UVA-induced lipid peroxidation; in increasing the skin moisturization, skin elasticity, and skin radiance; and in decreasing TEWL, the intensity of melanin staining inside dark spots, and wrinkle depth. Our results suggest that the test product is effective in counteracting both the harmful effects of UVR exposure and aging signs.

Alpha-pinene attenuates UVA-induced photoaging through inhibition of matrix metalloproteinases expression in mouse skin

AimThe present study was designed to examine the role of alpha-pinene (AP) against skin photoaging in UVA-irradiated mice. Materials and methodsSwiss albino mice were subjected to UVA-irradiation at the rate of 10 J/cm2 per day for ten days, totally mouse received 100 J/cm2. One hour prior to each UVA-exposure, the mouse skin was topically treated with AP (100 mg kg/b·wt). Biochemical methods were employed to study the status of antioxidant enzymes and lipid peroxidation. Histopathological observations were performed using hematoxylin and eosin (H & E) and Verhoeff van Gieson (VVG) staining in the mouse skin. The inflammatory and apoptotic protein expression was studied by immunohistochemical and Western blot methods. The mRNA expression of matrix metalloproteinases was determined by qRT-PCR and Western blot analysis. Key findingsWe found that AP pretreatment substantially ameliorated UVA-induced depletion of antioxidant enzymes and prevented UVA-induced lipid peroxidation in the mouse skin. Further, AP effectively inhibited UVA-induced activation of pro-angiogenic (iNOS and VEGF), inflammatory proteins (TNF-α, IL-6, and COX-2) expression and prevented the activation of NF-κB p65 in the mouse skin. Additionally, AP inhibited UVA-mediated apoptotic mediators (Bax, Bcl-2, caspase-3 and caspase 9) expression in the mouse skin. Moreover, AP inhibited mRNA expression of matrix metalloproteinases (MMP-13 and MMP-9) and tissue type IV collagenase (MMP-2) expression in the mouse skin. Histological studies showed that AP remarkably prevented the dermal tissue damage in UVA-irradiated mice. ConclusionThus, AP treatment effectively prevented UVA-induced photoaging probably through its antioxidant property.

Photoimmune protective effect of the phytoestrogenic isoflavonoid equol is partially due to its antioxidant activities

Topical application of lotions containing the phytoestrogenic isoflavonoid equol have been reported to protect mice against UV radiation-induced inflammation, immune suppression and photocarcinogenesis. The photoimmune protective property was shown to depend on equol's activation of oestrogen receptor signalling in the skin. However, isoflavones are also recognised for their antioxidant properties in biological systems. As endogenous cutaneous antioxidant enzymes including the inducible stress protein haem oxygenase (HO)-1, have photoprotective efficacy, this study in the Skh:hr-1 hairless mouse seeks evidence for an antioxidant role for equol in contributing to its photoimmune protection. Oxidative stress has been measured as UVA-induced lipid peroxidation in the mouse skin, and was dose-dependently inhibited by topical equol. Inhibition of the UVA (320-400 nm)-inducible HO activity significantly reduced the level of equol protection against lipid peroxidation, thereby attributing a component of equol's lipid protection capacity to this stress enzyme. It was consistent that topical equol enhanced the level of HO induction by UVA irradiation in both skin and liver. Subsequently, the dose-dependent protection by topical equol lotions against solar simulated UV radiation induced immunosuppression, measured by the contact hypersensitivity reaction, was found also to be partially reduced by the inhibition of HO activity. Therefore, in addition to the activation by equol of oestrogenic signalling pathways for photoprotection, this isoflavonoid also provides UV-protective antioxidant effects that depend partially on HO-1 induction.

Protection against ultraviolet A‐induced oxidative damage in normal human epidermal keratinocytes under post‐menopausal conditions by an ultraviolet A‐activated caged‐iron chelator: a pilot study

Human skin is constantly exposed to ultraviolet A (UVA), which can generate reactive oxygen species and cause iron release from ferritin, leading to oxidative damage in biomolecules. This is particularly true in post-menopausal skin due to an increase in iron as a result of menopause. As iron is generally released through desquamation, the skin becomes a main portal for the release of excess iron in this age group. In the present study, we examined a strategy for controlling UVA- and iron-induced oxidative stress in skin using a keratinocyte post-menopausal cellular model system. Keratinocytes that had been cultured under normal or high-iron, low-estrogen conditions were treated with (2-nitrophenyl) ethyl pyridoxal isonicotinoyl hydrazone (2-PNE-PIH). 2-PNE-PIH is a caged-iron chelator that does not normally bind iron but can be activated by UVA radiation to bind iron. Following incubation with 2-PNE-PIH, the cells were exposed to 5 J/cm² UVA and then measured for changes in lipid peroxidation and ferritin levels. 2-PNE-PIH protected keratinocytes against UVA-induced lipid peroxidation and ferritin depletion. Further, 2-PNE-PIH was neither cytotoxic nor did it alter iron metabolism. 2-PNE-PIH may be a useful deterrent against UVA-induced oxidative stress in post-menopausal women.

Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts

Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 μM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-plasmodium toxicity

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria.

Incorporation of phytosterols in human keratinocytes: Consequences on UVA-induced lipid peroxidation and calcium ionophore-induced prostaglandin release

We have designed experimental conditions allowing the replacement of 50% of cholesterol of human keratinocytes (SVK14 line) with sitosterol or stigmasterol without affecting cellular viability. We have investigated the influence of incorporating phytosterol on the ultraviolet-A-induced formation of lipid-peroxidation products (thiobarbituric reactive substances (TBARS)) in these cells. Our results show that ultraviolet-A-induced lipid peroxidation depends on the nature of the phytosterol. Sitosterol induces a significant decrease (−30%) of TBARS relative to the control whereas stigmasterol markedly increases lipid peroxidation (+70%). We have also studied the effect of plant sterols on prostaglandin release by using the Ca 2+ ionophore A23187 as an in vitro model of the inflammation induced by UVA radiation. We show that in the presence of 50% of phytosterol (particularly stigmasterol), the release of prostaglandin (6-ketoPG 1α, PGE 2) is increased compared to untreated cells. This pro-inflammatory effect of phytosterols is correlated with a loss of the regulation of the intracellular Ca 2+ concentration.

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.

Role of Phagocyte Oxidase in UVA-Induced Oxidative Stress and Apoptosis in Keratinocytes

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

Effects of periodic, low UVA radiation on cell characteristics and oxidative stress in the marine planktonic diatom Ditylum brightwellii

A continuous culture experiment was conducted with the marine diatom Ditylum brighiwellii to assess the photo-oxidative effects of transition from dim light to high Light at three successive levels. The focus was on temporary stress by ambient WA at the water surface, via a simulation of the periodic ascent of diatoms caused by vertical mixing. Within a 14 h dim- light period, cells were exposed daily to 4 h of: (A) moderate PAR (100 mu mol photon m(-2) s(-1)), (B) high PAR (400 mu mol photon m(-2) s(-1)) and (C) high PAR (400-700 nm) plus 67 pmol photon m(-2) s(-1) (2.2 W m(-2)) UVA. Cell shape, life cycle and sinking rates were not affected by high PAR per se (mode B). The daily 4 h high-irradiance mode C, with additional UVA (WA:PAR = 0.016), caused an increase in cell diameter, a decrease in length axes, and a production of vegetative resting stages. Superoxide dismutase (SOD) activities, oxidized glutathione (GSSG) pools and malondialdehyde (MDA) contents increased as well, which indicates that this biologically effective WA dose (0.78 W m(-2)) promoted active oxygen production, oxidative stress and lipid peroxidation. Sinking rates in WA-exposed D. brightwellii were higher than in those grown without UVA. A close correlation between sinking rates and MDA contents suggests that UVA-induced lipid peroxidation has altered the membrane functions which regulate buoyancy in D. brightwellii. Although UVA-induced formation of resting cells will contribute to accelerated sinking, it is not evident that this can be considered as a stress avoidance mechanism under adverse light (WA) conditions. D. brightwellii proved to be sensitive to transitions from shade to natural surface irradiance conditions. In coastal waters, such large diatom species may experience oxidative stress after a few hours' residence near the water surface at ambient UVA levels. [KEYWORDS: cell size; Ditylum brightwellii; glutathione redox;lipid peroxidation; oxidative stress; planktonic diatom; resting stage; sinking rate; superoxide; ultraviolet-A Ultraviolet-b radiation; lipid-peroxidation; chlorella-vulgaris; photosynthetic pigment; superoxide-dismutase; thylakoid membranes; antioxidant systems; maize seedlings; sinking rates; phytoplankton]

Effects of periodic, low UVA radiation on cell characteristics and oxidative stress in the marine planktonic diatom Ditylum brightwellii

A continuous culture experiment was conducted with the marine diatom Ditylum brightwellii to assess the photo-oxidative effects of transition from dim light to high light at three successive levels. The focus was on temporary stress by ambient UVA at the water surface, via a simulation of the periodic ascent of diatoms caused by vertical mixing. Within a 14 h dim-light period, cells were exposed daily to 4 h of : (A) moderate PAR (100 μmol photon m−2 s−1), (B) high PAR (400 μmol photon m−2 s−1) and (C) high PAR (400–700 nm) plus 6·7 μmol photon m−2 s−1 (2·2 W m−2) UVA. Cell shape, life cycle and sinking rates were not affected by high PAR per se (mode B). The daily 4 h high-irradiance mode C, with additional UVA (UVA:PAR = 0·016), caused an increase in cell diameter, a decrease in length axes, and a production of vegetative resting stages. Superoxide dismutase (SOD) activities, oxidized glutathione (GSSG) pools and malondialdehyde (MDA) contents increased as well, which indicates that this biologically effective UVA dose (0·78 W m−2) promoted active oxygen production, oxidative stress and lipid peroxidation. Sinking rates in UVA-exposed D. brightwellii were higher than in those grown without UVA. A close correlation between sinking rates and MDA contents suggests that UVA-induced lipid peroxidation has altered the membrane functions which regulate buoyancy in D. brightwellii. Although UVA-induced formation of resting cells will contribute to accelerated sinking, it is not evident that this can be considered as a stress avoidance mechanism under adverse light (UVA) conditions. D. brightwellii proved to be sensitive to transitions from shade to natural surface irradiance conditions. In coastal waters, such large diatom species may experience oxidative stress after a few hours' residence near the water surface at ambient UVA levels.

Relative roles of metal ions and singlet oxygen in UVA-induced liposomal lipid peroxidation

Unilamellar liposomes prepared from phosphatidylcholine were exposed to relatively low doses (36–72 kJ m −2) of UVA to study the relative role of metal ions and singlet oxygen. Electron spin resonance (ESR) spectroscopy showed that singlet oxygen was produced during UVA radiation of liposomes and that the signal was enhanced when liposomes and the buffer were prepared in D 2O. However, in liposomes and buffer prepared in deionized water (dH 2O), UVA-induced lipid peroxidation (measured as formation of thiobarbituric acid reactive substances, TBARS) was strongly dependent on adventitious metal ions because formation of TBARS was inhibited ∼80% by inclusion of 10 mM EDTA or desferrioxamine (desferal) during 1-h UVA radiation (72 kJ m −2). Dimethysulfoxide (DMSO) and mannitol had no effect and NaN 3 and histidine only slightly inhibited TBARS, suggesting that singlet oxygen is a minor factor in UVA-induced liposomal peroxidation. To further elucidate the relative roles of singlet oxygen and metal ions, Fe(III) was added to liposomes during UVA irradiation, which resulted in markedly enhanced formation of TBARS. Singlet oxygen was detected at UVA doses of 72 and 108 kJ m −2 by N,N-dimethyl- p-nitrosoaniline bleaching test carried out in D 2O but not in dH 2O. Formation of TBARS was completely inhibited by 10 mM EDTA and desferal but was unaffected by either 50 mM DMSO, 50 mM mannitol, 50 mM NaN 3, 4 mM tryptophan or 0.1 mM β-carotene. The present study demonstrates that, although singlet oxygen is produced by UVA radiation, its role in the formation of TBARS appears to be relatively minor compared with that of metal ions in unilamellar liposomes irradiated with physiological doses of UVA.

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Apoptosis, the Role of Oxidative Stress and the Example of Solar UV Radiation

Expression of the 72-kD heat shock protein is induced by ultraviolet A radiation in a human fibrosarcoma cell line.

The 72-kD heat shock protein (hsp72) belongs to a family of stress inducible proteins (heat shock proteins, hsp) and its expression is associated with increased survival of cells in culture following exposure to ultraviolet radiation (UV). Hsp72 can be induced by a number of stresses, including heat, cold, and toxic chemicals. The purpose of this study was to evaluate whether UV is able to activate transcription of hsp72. The human fibrosarcoma cell line HT1080 was used for these experiments because hsp72 is not detectable in these cells under normal culture conditions. Cells were exposed to UVA and UVB using a solar simulating source and hsp72 was determined in whole cell extracts by immunoblotting. For inhibition of mRNA and protein synthesis cordycepin (20 microg/ml) and cycloheximide (10 microg/ml) were added to the cultures, respectively. UVA-induced lipid peroxidation was inhibited by alpha-tocopherol and butylated hydroxytoluene (BHT). UVA but not UVB induced hsp72 with maximal expression at 40 J/cm2, 8-12 h after exposure. Induction was blocked by cordycepin as well as by cycloheximide indicating that both, mRNA and protein synthesis, are required for UVA-induction of hsp72. Inhibition of cell lipid peroxidation with alpha-tocopherol and BHT had no effect on hsp72 expression. These results suggest that induction of hsp72 is part of an adaptive response mechanism in human cells to UV-related stress.

Modulation of UVA-induced lipid peroxidation and suppression of UVB-induced ornithine decarboxylase response by all-trans-retinoic acid in human skin fibroblasts in vitro.

Although clinical evidence of improvement of human skin photodamage by all-trans-retinoic acid (atRA) has accumulated, evidence for its preventative effects against photodamage is limited. Here we studied human skin fibroblasts in vitro. For determination of atRA uptake and metabolism, cells were treated with 3 or 10 microM atRA and retinoids analyzed by HPLC. After 16h a peak of cellular retinoid levels was reached, mainly atRA and 13-cis-RA. At this time point cells were irradiated with UVA (1-20 J/cm2) or UVB (5-500 mJ/cm2). TBARS in medium supernatant were used as an indicator of lipid peroxidation; ornithine decarboxylase (ODC) activity served as a marker of the mutagenic and carcinogenic effects of UV light. 1 h post irradiation (p.i.) with 20 J/cm2 UVA, TBARS production was enhanced by a 3 microM atRA treatment (121+/-5% of vehicle treated cells) and decreased by a 10 microM atRA treatment (75+/-2% of vehicle treated cells), and not significantly altered in UVB irradiated cells. 24 h p.i. with 50 mJ/cm2 UVB, ODC activity peaked in vehicle treated cells at a 7.4+/-0.2-fold increase compared to sham irradiated control cells, and was reduced to a 4.9+/-0.2-fold increase by 3 microM atRA. Treatment with 10 microM atRA further decreased ODC activity (3.7+/-1.0-fold increase) and this delayed activity peak occurred at 36 h p.i. ODC activity was not significantly enhanced by UVA irradiation. These results suggest that in normal human skins fibroblast atRA and/or its metabolites influence the UVA-induced lipid peroxidation by at least two distinct antagonistic mechanisms, while the ODC response to UVB-induced DNA damage possibly involves a ROS-independent, retinoid-sensitive regulatory pathway.

L-Ascorbic Acid Inhibits UVA-Induced Lipid Peroxidation and Secretion of IL-1α and IL-6 in Cultured Human Keratinocytes In Vitro

We investigated the antioxidative effect of L-ascorbic acid on lipid peroxidation and on secretion and mRNA expression of IL-1alpha and IL-6 after UVA irradiation (20 J/cm2) in cultured human keratinocytes. Lipid peroxidation was measured by (i) high performance liquid chromatography with UV detection of malondialdehyde (MDA) at 256 nm and (ii) spectrometric measurement of thiobarbituric acid-reactive substances (TBARS). To evaluate UV-induced cytotoxicity, we assessed cell membrane damage by measuring lactate dehydrogenase (LDH) release. UVA-induced lipid peroxidation in cultured human keratinocytes was inhibited by ascorbic acid in a concentration-dependent manner: MDA protein equivalent was reduced by 47% (10(-6)), compared to keratinocytes not exposed to L-ascorbic acid (p < 0.05), and the TBARS showed a concentration-dependent decrease of 49% (10(-6) M) in L-ascorbic acid-supplemented cultures compared to controls (p < 0.05). LDH release was decreased by 45% in L-ascorbic acid-supplemented keratinocyte cultures, indicating protection against cell death (p < 0.05). L-Ascorbic acid was able to downregulate IL-1alpha mRNA expression in both UVA-irradiated and nonirradiated cells; however, IL-6 mRNA expression remained unaffected. The secretion of these cytokines was reduced nearly to normal in the presence of L-ascorbic acid. These findings indicate a major cell-protective effect of L-ascorbic acid on UVA-induced lipid peroxidation and the secretion of pro-inflammatory cytokines by UVA-irradiated human keratinocytes.

Ultraviolet-A induces activation of AP-1 in cultured human keratinocytes

UV-A irradiation induces a time-dependent activation of AP-1 in NCTC 2544 human keratinocytes. 4 h after irradiation, a 2–3-fold increase in AP-1 activity is observed in human keratinocytes and fibroblasts. Activation is still detectable 24 h later. The UV-A induced AP-1 binding complex is shown to contain c-Fos and c-Jun proteins. Lipophilic vitamin E impedes UV-A induced lipid peroxidation but does not prevent AP-1 activation which is inhibited by N-acetylcysteine, a hydrophilic antioxidant. This finding suggests that UV-A-dependent AP-1 activation is sensitive to the cellular redox state but is not related to membrane lipid peroxidation.

Effects of ultraviolet A and antioxidant defense in cultured fibroblasts and keratinocytes.

Lipid peroxidation, measured by the thiobarbituric acid-reactive substances assay, was evaluated for cultured human skin fibroblasts and keratinocytes exposed to ultraviolet A radiation (320-400 nm, UVA). Peroxidation increases with increasing UVA doses and is much lower for keratinocytes than for fibroblasts. Immediate UVA-induced cytotoxicity, monitored by the trypan blue exclusion assay, is also lower for keratinocytes. Thus, cultured human skin keratinocytes are less sensitive than fibroblasts to the immediate deleterious effects of UVA with respect to membrane damage and lipid peroxidation. As a first attempt to understand this lower sensitivity of keratinocytes, basal levels of antioxidant defenses including total glutathione, superoxide dismutase, glutathione peroxidase and catalase were evaluated in both keratinocytes and fibroblasts from the same donors. We failed to correlate this lower susceptibility of keratinocyte to UVA-induced lipid peroxidation and cytotoxicity with a higher antioxidant status.

Hydrogen peroxide and catalase in UVA-induced lipid peroxidation in cultured fibroblasts

UVA-induced lipid peroxidation in cultured human skin fibroblasts, as measured by the release in the supernatant of thiobarbituric acid-reactive substances, is found to be linear with increasing irradiation dose (up to about 250 kJ m(-2)). Concomitantly, within this dose range catalase is strongly inactivated by UVA radiation according to an exponential process (k≈0.01 kJ(-1) m(2)). This suggests that catalase is not involved in modulating the peroxidation process. Inactivation of catalase by 3-amino-1,2,4-triazole can be efficiently achieved prior to irradiation. This inactivation has no consequence on the extent of peroxidation triggered by subsequent exposure to UVA radiation. It may be therefore strongly suggested that catalase is not, via H2O2 removal, a key enzyme in the cellular defence equipment towards UV A-peroxidative stress. An alternative interpretation may be formulated which supports the view that H2O2 produced upon exposure to UVA has no or very little role in triggering the lipid peroxidation process.