CORRELATION BETWEEN ENDOGENOUS GLUTATHIONE CONTENT AND SENSITIVITY OF CULTURED HUMAN SKIN CELLS TO RADIATION AT DEFINED WAVELENGTHS IN THE SOLAR ULTRAVIOLET RANGE

Abstract— Glutathione depletion of cultured human skin fibroblasts by treatment with buthionine-S,R-sulfoximine (BSO) sensitises them to radiation at a series of defined wavelengths throughout the solar UV range. We now show that there is a close quantitative correlation between cellular glutathione content (as depleted by BSO) and sensitivity to radiation at 365 nm. A weaker correlation is observed when cells are depleted of glutathione using diethylmaleimide. Both fibroblasts and epidermal keratinocytes derived from the same foreskin biopsy are sensitised to radiation at 313 nm by glutathione depletion. However, the keratinocytes are sensitised to a much lesser extent, an observation which agrees quantitatively with the higher residual levels of cellular glutathione remaining after maximum depletion by BSO (approximately 25% for the keratinocytes vs less than 5% for the fibroblasts). At low to intermediate fluence levels, 10 mM cysteamine present during irradiation at 302 nm is able to almost completely reverse the sensitising effects of glutathione depletion suggesting that the endogenous thiol protects against radiation at this wavelength by a free radical scavenging mechanism. At 313 nm, the sensitisation is not reversed by cysteamine suggesting that glutathione plays a more specific role in protection against radiation at longer wavelengths. Xeroderma pigmentosum group A fibroblasts (excision deficient) are also sensitised to radiation at 313 and 365 nm by depletion of glutathione but since the sensitization is less than that observed for the normal strain, we cannot conclude that glutathione protects against a sector of DNA damage susceptible to excision repair. The results provide further evidence that endogenous glutathione is involved in protecting human skin cells against a wide range of solar radiation damage and suggest that while free radical scavenging is involved at the shortest wavelength (302 nm) tested, a more specific role of glutathione is involved in protection against radiation at longer wavelengths.     

TWO GENES CONTRIBUTE TO DIFFERENT EXTENTS TO THE HEME OXYGENASE ENZYME ACTIVITY MEASURED IN CULTURED HUMAN SKIN FIBROBLASTS AND KERATINOCYTES: IMPLICATIONS FOR PROTECTION AGAINST OXIDANT STRESS

Abstract— Activation of expression of the heme oxygenase (HO) gene appears to be involved in a cellular defense system in mammalian cells. We now demonstrate that while HO-1 mRNA levels are strongly inducible in dermal fibroblasts they are barely inducible in human epidermal keratinocytes following oxidative stress (UVA radiation and hydrogen peroxide). Paralleling this result was the observation that HO-2 mRNA levels were low in dermal fibroblasts but were high in epidermal keratinocytes. In neither case was the HO-2 gene inducible. The expression of the two HO genes led to enzymatic activity in both types of skin cells with an approximately 2.5-fold higher level of enzymatic activity present in keratinocytes compared with fibroblasts derived from the same biopsy. In addition, ferritin levels, which have been found to be augmented via the HO-dependent release of iron from endogenous heme sources, were two- to three-fold higher in keratinocytes compared with matching fibroblasts. This higher ferritin pool would result in an enhancement of cellular iron sequestering capacity that may confer increased resistance to oxidative stress. Indeed, keratinocytes showed less UVA radiation-dependent cell membrane damage than fibroblasts. These results are consistent with the hypothesis that HO expression in human epidermis and dermis is related to cellular defense mechanisms that operate in human skin.     

LONG-WAVE ULTRAVIOLET RADIATION INDUCES PROTEIN KINASE C IN NORMAL HUMAN KERATINOCYTES

Abstract Skin tumor promotion by phorbol ester is believed to be mediated by the phospholipid-dependent ser/ thr kinase, protein kinase C (PKC). Long-wave ultraviolet radiation (320-400 nm, UVA), which has also been shown to promote skin tumors, induces elevated levels of PKC in murine fibroblasts, suggesting that UVA may promote the development of basal and squamous cell skin cancers by a mechanism involving PKC. To examine UVA effects on PKC in a model relevant to skin, we maintained normal human epidermal keratinocytes (NHEK) in serum-free medium and exposed the cultured cells to various doses of UVA or to the phorbol ester, 12- O -tetradecanoylphorbol-13-acetate (TPA). Fifty minutes after exposure to UVA (5-20 J/cm²), PKC activity was elevated up to three-fold in NHEK cytosolic fractions, and membrane-associated PKC activity was elevated up to two-fold by UVA. The TPA treatment induced a 10-fold increase in membrane-associate PKC activity only. Immunoblot analysis suggested that a UVA-induced increase in PKC protein occurred. Both UVA and TPA reduced the cell number by 50-75% in the first 24-48 h; however, irradiated cultures began to recover at 72 h post-UVA due to an increased proliferative rate beginning after 48 h. Treatment with TPA induced a high level of differentiation as measured by cornified envelope formation. Ultraviolet A irradiation exposure was not followed by increased differentiation. These findings suggest that acute UVA exposure elevates PKC activity in human keratinocytes and may act through PKC to promote actinic skin cancer. The molecular mechanism is like to differ from that of the phorbol esters, however.     

The Phototoxic Potential of the Flavonoids,Taxifolin and Quercetin

Quercetin, one of the most abundant polyphenols in the plant kingdom has been shown to be photodegraded on exposure to UV light. Despite the fact, it is a component of several dermatological preparations. Its phototoxic potential has not been evaluated to date. The aim of this study was to assess whether photo‐induced degradation of quercetin is linked to phototoxic effects on living cells. Its dihydro derivative, taxifolin, was included in the study. For evaluation, the 3T3 Neutral Red Uptake Phototoxicity Test according to OECD TG 432 was used. To better approximate human skin, HaCaT keratinocytes, normal human epidermal keratinocytes and dermal fibroblasts were used, apart from the Balb/c 3T3 cell line. Quercetin showed a dose‐dependent photodegradation in aqueous and organic environments and a phototoxic effect on all used cells. Quercetin pretreatment and following UVA exposure resulted in increased reactive oxygen species production and intracellular glutathione level depletion in human dermal fibroblasts. Taxifolin was found completely nonphototoxic and photostable. As only in vitro methodology was used, further studies using 3D skin models and/or human volunteers are needed to confirm whether exposure to sunlight, tanning sunbeds and/or phototherapy in people using cosmetics containing quercetin is a health risk.     

ENDOGENOUS GLUTATHIONE PROTECTS HUMAN SKIN FIBROBLASTS AGAINST THE CYTOTOXIC ACTION OF UVB, UVA AND NEAR-VISIBLE RADIATIONS

Both the UVB (290-320 nm) and UVA (320-380 nm) regions of sunlight damage human skin cells but, particularly at the longer wavelengths, information is scant concerning the mechanism(s) of damage induction and the roles of cellular defense mechanisms. Following extensive glutathione depletion of cultured human skin fibroblasts, the cells become strongly sensitized to the cytotoxic action of near-visible (405 nm), UVA (334 nm, 365 nm) and UVB (313 nm) but not UVC (254 nm) radiations. In the critical UVB region, the magnitude of the protection afforded by endogenous glutathione approaches that of the protection provided by excision repair. The results suggest that a significant fraction of even UVB damage can be mediated by free radical attack and that a major role of glutathione in human skin cells is to protect them from the cytotoxic action of sunlight.     

Depletion of cutaneous glutathione by ultraviolet radiation

Supplemental antioxidants may have a role in ameliorating or preventing the actinic damage that can lead to cutaneous disorders such as skin cancer, hyperpigmentation, and premature aging. Glutathione is an important endogenous antioxidant and fulfills various protective functions in the skin. Irradiation of hairless mice with short (UVB) or long (UVA) wavelength ultraviolet radiation or with UVA combined with a photosensitizing psoralen (PUVA) can deplete skin glutathione levels. Ultraviolet B irradiation causes rapid transient fluctuations in the epidermal glutathione level and the relative amount present as the oxidized form. Ultraviolet A irradiation can deplete epidermal and dermal glutathione for several hours but requires much higher doses than UVB. PUVA treatments may lead to extensive and prolonged depletions of epidermal and dermal glutathione, the severity of which is dependent on the psoralen dose and may last for several days. These transient depletions, oxidations, and sometimes rapid recoveries of cutaneous glutathione levels are compatible with a role for glutathione as an endogenous photoprotective agent in the skin. Experimental evidence supports such a role: for example severe skin edema develops in mice only after about 50% of the glutathione has been depleted by PUVA treatment. Although different mechanisms are involved in each case, glutathione depletion may contribute to the production of phototoxicity by UVB, UVA, and by PUVA. Understanding the depletion mechanisms may allow the development of strategies aimed at preventing loss of cutaneous glutathione, and at reinforcing the natural protective functions of this critical cell component.     

Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.     

Toxic Interaction Between Solar Radiation and Cigarette Smoke on Primary Human Keratinocytes

Solar radiation and cigarette smoke are two environmental risk factors known to affect skin integrity. Although the toxic effects of these factors on skin have been widely studied separately, few studies have focused on their interaction. The objective of this study was to evaluate and understand the synergistic harmful effects of cigarette smoke and solar rays on human primary keratinocytes. The keratinocytes were exposed to cigarette smoke extract (CSE) and then irradiated with a solar simulator light (SSL). The viability, as determined by measuring metabolic activity of skin cells, and the levels of global reactive oxygen species (ROS) were evaluated after exposure to CSE and SSL. The combination of 3% CSE with 29 kJ m⁻² UVA caused a decrease of 81% in cell viability, while with 10% to 20% CSE, the cell viability was null. This phototoxicity was accompanied by an increase in singlet oxygen but a decrease in type I ROS when CSE and SSL were combined in vitro. Surprisingly, an increase in the CSE's total antioxidant capacity was also observed. These results suggest a synergy between the two environmental factors in their effect on skin cells, and more precisely a phototoxicity causing a drastic decrease in cell viability.     

Red Light Interferes in UVA‐Induced Photoaging of Human Skin Fibroblast Cells

The possible regulation mechanism of red light was determined to discover how to retard UVA‐induced skin photoaging. Human skin fibroblasts were cultured and irradiated with different doses of UVA, thus creating a photoaging model. Fibroblasts were also exposed to a subtoxic dose of UVA combined with a red light‐emitting diode (LED) for five continuous days. Three groups were examined: control, UVA and UVA plus red light. Cumulative exposure doses of UVA were 25 J cm^?2, and the total doses of red light were 0.18 J cm^?2. Various indicators were measured before and after irradiation, including cell morphology, viability, β‐galactosidase staining, apoptosis, cycle phase, the length of telomeres and the protein levels of photoaging‐related genes. Red light irradiation retarded the cumulative low‐dose UVA irradiation‐induced skin photoaging, decreased the expression of senescence‐associated β‐galactosidase, upregulated SIRT1 expression, decreased matrix metalloproteinase MMP‐1 and the acetylation of p53 expression, reduced the horizon of cell apoptosis and enhanced cell viability. Furthermore, the telomeres in UVA‐treated cells were shortened compared to those of cells in the red light groups. These results suggest that red light plays a key role in the antiphotoaging of human skin fibroblasts by acting on different signaling transduction pathways.     

ULTRAVIOLET RADIATION-INDUCED PHOSPHOLIPASE A2 ACTIVATION OCCURS IN MAMMALIAN CELL MEMBRANE PREPARATIONS

Ultraviolet erythema in human skin is mediated in part by membrane derivatives of arachidonic acid (AA). UVA (320–400nm) and UVB (290–320nm) have been shown to induce release of AA from intact mammalian cells in culture. In order to investigate the mechanism of this release we examined the effect of UVA and UVB on release of [³H] AA from membrane preparations of murine fibroblasts. C3H 10T1/2 cells were prelabelled for 24 h with [³H] AA. The membrane fractions of the cells were separated after lysis by differential centrifugation. The membranes were irradiated in suspension and the [³H] AA released from the membranes was determined by scintillation spectroscopy of supernatants3–4 h after irradiation. Both UVA and UVB induced release of AA from the membrane preparations. The response to UVB was small but significant, reaching levels approximately 150% of control release at doses of 1,200-4,000 J/m². The response to UVA was larger; doses of 2.5-5.0 J/cm² induced release equal to twice control (200%) levels, while doses of10–20 J/cm² induced maximal release at levels approximately 400% of control. Time course studies with UVB and UVA showed maximal release at 4 h after irradiation. When the membrane preparations were incubated with a polyclonal anti-phospholipase A₂ antibody the UV induced release of [³H] AA was completely inhibited in both UVB (1200 J/m²) and UVA (10 J/cm²) treated cells. These data suggest that activation of phospholipase A₂ is responsible for the UV induced release of AA in mammalian cells and that the mechanism of this activation is due, in part at least, to direct photon-membrane interaction.     

The B6‐vitamer Pyridoxal is a Sensitizer of UVA‐induced Genotoxic Stress in Human Primary Keratinocytes and Reconstructed Epidermis

UVA‐driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3‐hydroxypyridine‐derived chromophores including B₆‐vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA‐induced photooxidative stress in human skin cells. Here, we report that the B₆‐vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA‐induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ‐H2AX (Ser139)], comet analysis indicated the formation of Fpg‐sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA‐driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B₆‐vitamers.     

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

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

Effects of Ultraviolet A-1 Radiation on Calcineurin Activity and Cytokine Production in (Skin) Cell Cultures†

Calcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has been shown to positively influence various inflammatory skin diseases. Recently, Cn activity has been demonstrated in skin and skin cell cultures. In the present study we have investigated the effects of UV(A-1) irradiation on Cn activity in skin. In total skin we found a significant reduction in Cn activity after exposure to 450 kJ m⁻² of UVA-1 (340–400 nm). In repeated experiments cultures of fibroblasts and keratinocytes also showed dose-dependent and selective reduction in Cn activity after UVA-1 irradiation. UVB irradiation caused a decrease in the Cn activity of one of two fibroblast cultures and was ineffective in keratinocytes. In Jurkat cells and PBMC UVA-1 reduced Cn activity and also the production of cytokines such as interleukin (IL)-2, γ-interferon, IL-4 and IL-10 that are controlled by the Ca²⁺–Cn pathway. These results indicate that UV(A-1) irradiation may lead to inactivation of Cn in the skin and thus suppress the skin immune system in a similar fashion to the Cn inhibitors.     

A study of protein synthesis in cells cultured from involved psoriatic skin

Using histochemical techniques an abnormal programme of epidermal differentiation has been well documented in psoriasis. In order to characterise further the biochemistry of this process we have cultured dermal fibroblasts and epidermal keratinocytes from involved psoriatic skin. This has facilitated metabolic radiolabelling of skin cells and analysis of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The expression of keratin and differentiation markers was identical to that of normal keratinocytes, suggesting that psoriatic epidermal differentiation is not truncated in vitro as has been postulated to be the case in vivo. Low molecular mass components (5-8.5 kDa), previously shown to be upregulated in suprabasal keratinocytes, were detected in epidermal fractions from psoriatic skin enriched for basal cells. Of special interest was a component of 26 kDa, pI 5.9, which was highly upregulated in psoriatic as compared to normal cultured keratinocytes and was not detected in fibroblasts. These findings are in accord with a qualitatively abnormal pattern of differentiation for keratinocytes in the involved psoriatic epidermis.     

Ultraviolet A Eye Irradiation Ameliorates Atopic Dermatitis via p53 and Clock Gene Proteins in NC/Nga Mice

Atopic dermatitis (AD ) is a widespread chronic skin condition that severely affects quality of life and can lead to more serious complications. Although ultraviolet (UV )A eye irradiation can exert various effects on the skin, it is unknown whether UVA can affect AD . To investigate potential associations, we used an NC /Nga mouse model of AD to study the effects of UVA eye irradiation. The eyes of mice were irradiated with a UVA dose of 100 kJ m⁻² using a FL 20SBLB ‐A lamp. Our histological data demonstrated that AD symptoms could be ameliorated by UVA eye irradiation. We also observed an increase in the levels of adrenocorticotropic hormone (ACTH ), p53 and retinoid X receptor α (RXR α ) in mice with UVA ‐irradiated eyes. In contrast, the levels of thymic stromal lymphopoietin (TSLP ), period 2 (PER 2) and differentiated embryo chondrocytes 1 (DEC 1) protein were decreased in mice treated with UVA irradiation. Furthermore, UVA eye‐irradiated mice exhibited reduced DEC 1 and RXR α colocalization compared with nonirradiated mice. These results suggested that p53 and various clock gene proteins played important roles in the amelioration of AD symptoms observed after UVA eye irradiation; this technique may have therapeutic applications in AD .     

Thioredoxin Reductase Activity may be More Important than GSH Level in Protecting Human Lens Epithelial Cells against UVA Light

This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L‐buthionine‐(S,R)‐sulfoximine (BSO) or 1‐chloro‐2,4‐dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O₂, 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm^?2 of UVA radiation (338–400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O₂ compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA‐induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA‐induced cell damage.     

N-acetyl-L-cysteine prevents DNA damage induced by UVA, UVB and visible radiation in human fibroblasts

The thiol N-acetyl-L-cysteine (NAC) is a source of cysteine for the synthesis of the endogenous antioxidant glutathione (GSH) which is depleted by ultraviolet radiation. It is also associated with the scavenging of reactive oxygen species (ROS). In this study the effects of NAC were examined in cultured human fibroblasts during prolonged exposure to ultraviolet B (UVB), ultraviolet A (UVA) and visible irradiation (280-700 nm), delivered by a 150 W xenon-arc lamp. The alkaline comet assay was used to assess the DNA damage in individual cells. It was found that incubating skin and lung fibroblasts at 37 degrees C for 1 h with an optimal 6 mM NAC supplement prior to light exposure, significantly reduced the level of DNA damage in both cell types, however, the skin fibroblasts were less sensitive to xenon-arc lamp irradiation than lung fibroblasts. NAC incubation resulted in an initial delay in DNA damage when the cells were irradiated. There was also a significant reduction in the overall levels of DNA damage observed with continued irradiation. NAC significantly reduced the DNA damage produced in lung fibroblasts depleted of normal GSH protection by the glutamylcysteinyl synthetase inhibitor, L-buthionine-[S,R]-sulfoximine. Although the specific mechanism of NAC protection has not yet been elucidated, these results support the hypothesis that NAC may protect the cells directly, by scavenging ROS induced by UVA and visible radiation, and indirectly by donating cysteine for GSH synthesis.