In vitro and in vivo transfer of bcl-2 gene into keratinocytes suppresses UVB-induced apoptosis.

Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2-expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 x 10(6)) were transfected at 1 x 10(8) plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 x 10(9) PFU in 200 microL. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.     

Photoprotective Effect of Black Tea Extracts Against UVB-induced Phototoxicity in Skin

In previous studies, we showed that green tea and black tea extracts and their major polyphenolic constituents protect against UVB light-induced carcinogenesis in murine skin. All of these studies required chronic administration of tea extracts or specific constituents either topically or orally. However, it is not known whether acute or subchronic administration of black tea extracts or constituents can ameliorate UVB-induced early effects in skin. In the present study, cultured keratinocytes and mouse and human skin were employed to assess the effect of both oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic subfractions namely BTF1 and BTF2 against UVB-induced photodamage. In SKH-1 hairless mice, topical application of SBTE (0.2 mg/cm2) prior to UVB exposure (180 mJ/cm2) resulted in 40% reduced incidence and 64% reduced severity of erythema and 50% reduction in skinfold thickness by day 6 when compared to nontreated UVB-exposed animals. The SBTE was also effective in protecting against UVB-induced erythema in human volunteers. Administration of SBTE 5 min after UVB irradiation was similarly effective in reducing UVB-induced inflammation in both murine and human skin. The major polyphenolic subfractions, BTF1 and BTF2, were also effective in protecting in mouse skin. The SBTE subfractions inhibited UVB-induced tyrosine phosphorylation of epidermal growth factor receptor (EGFR). The UVB irradiation of human epidermoid carcinoma cells resulted in 3.3-fold induction of tyrosine phosphorylation of EGFR. Pretreatment with BTF1 and BTF2 reduced tyrosine phosphorylation of EGFR by 53% and 31%, respectively. The UVB-mediated enhanced expression of the early response genes, c-fos and c-jun in human epidermal keratinocytes was reduced in a dose-dependent manner by SBTE. Topical application of SBTE was also effective in reducing accumulation of c-fos and p53 proteins by 82% and 78%, respectively, in UVB-exposed mouse skin. These data provide evidence that constituents of black tea can abrogate UVB-induced erythema and associated early events in murine and human skin.     

DNA damage, apoptosis and langerhans cells--Activators of UV-induced immune tolerance

Solar UVR is highly mutagenic but is only partially absorbed by the outer stratum corneum of the epidermis. UVR can penetrate into the deeper layers of the epidermis, depending on melanin content, where it induces DNA damage and apoptosis in epidermal cells, including those in the germinative basal layer. The cellular decision to initiate either cellular repair or undergo apoptosis has evolved to balance the acute need to maintain skin barrier function with the long-term risk of retaining precancerous cells. Langerhans cells (LCs) are positioned suprabasally, where they may sense UV damage directly, or indirectly through recognition of apoptotic vesicles and soluble mediators derived from surrounding keratinocytes. Apoptotic vesicles will contain UV-induced altered proteins that may be presented to the immune system as foreign. The observation that UVR induces immune tolerance to skin-associated antigens suggests that this photodamage response has evolved to preserve the skin barrier by protecting it from autoimmune attack. LC involvement in this process is not clear and controversial. We will highlight some basic concepts of photobiology and review recent advances pertaining to UV-induced DNA damage, apoptosis regulation, novel immunomodulatory mechanisms and the role of LCs in generating antigen-specific regulatory T cells.     

The Protective Effect of N-Acetylcysteine on UVB-Induced Immunosuppression by Inhibition of the Action of Cis-Urocanic Acid

Abstract— A recent study has shown that N-acetylcysteine (NAC) not only has sun-protective properties but also inhibits the UVB-induced suppression of contact hypersensitivity (CHS) in mice. Because NAC does not absorb any UVA (320-400 nm radiation) or UVB (290-320 nm radiation) we have studied the underlying mechanism of protection. Irradiation of solutions of plasmid DNA with UVC (200-290 nm radiation) (10 J m-2) resulted in the formation of cyclobutane pyrimidine dimers, but the extent to which this occurred was not affected by the presence of NAC as was determined by an in vitro T4 endonuclease assay. N-acetylcysteine proved not to have any effect on the photoisomerization of trans-urocanic acid (UCA) to its cis-form in vitro; at equilibrium, approximately 55% cis-UCA was formed. The same percentage was also found in vivo on exposure of mice to UVB (15 kj m-2). Topical application of NAC 30 min prior to irradiation did not have any influence as well on the photoisomerization of trans- to cis-UCA. These in vivo experiments were performed under the same conditions used previously to show the protective effect of NAC against UVB-induced suppression of CHS.
We conclude that this protection of NAC is at least partly based on interference in the role of cis -UCA in UVB-induced suppression of CHS. This conclusion is supported by the observation that NAC completely inhibits the suppression of CHS by cis -UCA administered to mice that were always kept in the dark. In the same range of doses as used in the present study, it was shown in our previous study that NAC alone does not affect the CHS response.     

Inflammasome Activation of IL-1 Family Mediators in Response to Cutaneous Photodamage(†)

Although keratinocytes are relatively resistant to ultraviolet radiation (UVR) induced damage, repeated UVR exposure result in accumulated DNA mutations that can lead to epidermal malignancies. Keratinocytes play a central role in elaborating innate responses that lead to inflammation and influence the generation of adaptive immune responses in skin. Apart from the minor cellular constituents of the epidermis, specifically Langerhans cells and melanocytes, keratinocytes are the major source of cytokines. UVR exposure stimulates keratinocytes to secrete abundant pro-inflammatory IL-1-family proteins, IL-1α, IL-1β, IL-18, and IL-33. Normal skin contains only low levels of inactive precursor forms of IL-1β and IL-18, which require caspase 1-mediated proteolysis for their maturation and secretion. However, caspase-1 activation is not constitutive, but dependents on the UV-induced formation of an active inflammasome complex. IL-1 family cytokines can induce a secondary cascade of mediators and cytokines from keratinocytes and other cells resulting in wide range of innate processes including infiltration of inflammatory leukocytes, induction of immunosuppression, DNA repair or apoptosis. Thus, the ability of keratinocytes to produce a wide repertoire of proinflammatory cytokines can influence the immune response locally as well as systematically, and alter the host response to photodamaged cells. We will highlight differential roles played by each IL-1 family molecule generated by UV-damaged keratinocytes, and reveal their complementary influences in modulating acute inflammatory and immunological events that follow cutaneous UV exposure.     

Nondenatured soy extracts reduce UVB-induced skin damage via multiple mechanisms

UV irradiation results in DNA damage, inflammation and immunosuppression, leading to the development of basal and squamous cell carcinomas. Earlier data show that topical treatment with nondenatured soy extracts reduced the incidence and delayed the development/progression of already-initiated skin tumors in high-risk hairless mice. Here we show that pretreatment with nondenatured soy extracts reduced UVB-induced Thymine-Thymine (TT) dimer formation. In vitro, nondenatured soy extracts enhanced UVB-induced checkpoint kinase-1 (Chk1) activation, suggesting a delay in cell cycle progression that enables longer time for DNA repair. Soy also reduced UVB-induced cyclo-oxygenase-2 (COX-2) expression and prostaglandin E2 secretion, and inhibited p38 MAP kinase activation, suggesting its anti-inflammatory activity. Mice pretreated topically with nondenatured soy extracts had reduced levels of UVB-induced TT dimers and COX-2 expression in their skins compared to UVB alone. The nondenatured soy extracts also inhibited vascular endothelial growth factor-induced endothelial tube formation in Matrigel, suggesting a possible inhibitory effect on angiogenesis and tumor progression. Taken together, nondenatured soy extracts could prevent or reduce UVB-induced skin damage via multiple mechanisms, affecting both the initiation and the progression of skin cancer. These data suggest that topical application of nondenatured soy extracts could potentially reduce the incidence of skin cancer.     

Effect of Immunosuppressants Tacrolimus and Mycophenolate Mofetil on the Keratinocyte UVB Response

Nonmelanoma skin cancer, derived from epidermal keratinocytes, is the most common malignancy in organ transplant recipients, causes serious morbidity and mortality, and is strongly associated with solar ultraviolet (UV) exposure. Preventing and treating skin cancer in these individuals has been extraordinarily challenging. Following organ transplantation, the immunosuppressants are used to prevent graft rejection. Until now, immunosuppression has been assumed to be the major factor leading to skin cancer in this setting. However, the mechanism of skin carcinogenesis in organ transplant recipients has not been understood to date; specifically, it remains unknown whether these cancers are immunosuppression‐dependent or ‐independent. In particular, it remains poorly understood what is the mechanistic carcinogenic action of the newer generation of immunosuppressants including tacrolimus and mycophenolate mofetil (MMF). Here, we show that tacrolimus and MMF impairs UVB‐induced DNA damage repair and apoptosis in human epidermal keratinocytes. In addition, tacrolimus inhibits UVB‐induced checkpoint signaling. However, MMF had no effect. Our findings have demonstrated that tacrolimus and MMF compromises proper UVB response in keratinocytes, suggesting an immunosuppression‐independent mechanism in the tumor‐promoting action of these immunosuppressants.     

1,25-Dihydroxyvitamin D3 and analogues protect primary human keratinocytes against UVB-induced DNA damage

Exposure to UVB irradiation is a major risk factor for the development of skin cancer. Therefore, it is important to identify agents that can offer protection against UVB-caused damage. Photocarcinogenesis is caused largely by mutations at sites of incorrectly repaired DNA photoproducts, of which the most common are the cyclobutane pyrimidine dimers (CPDs). In this study, we demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against the induction of CPDs by UVB. This protection required pharmacologic doses 1,25(OH)2D3 and an incubation period of at least 8 h before irradiation. Furthermore, we provided arguments indicating that the anti-proliferative capacity of 1,25(OH)2D3 underlies its protective effect against UVB-induced DNA damage. Finally, we showed that 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), two low-calcemic analogues of 1,25(OH)2D3, were even 100 times more potent than the parent molecule in inhibiting UVB-caused DNA damage. These molecules are therefore promising candidates for the chemoprevention of UVB-induced skin cancer.     

The Effect of Cold Stress on UVB Injury in Mouse Skin and Cultured Keratinocytes

Abstract— The effect of cold stress on skin damage caused by UVB irradiation was investigated both in vivo and in vitro. Ear skin of mice that had been exposed to cold stress at 0°C for 20 min and at 5°C for 24 h was exposed to UVB radiation. Sunburn cell production was less in mice exposed to the lower temperature. In addition, the effect of cold stress on the survival rate of UVB-irradiated rat keratinocytes was examined in a cytotoxicity test, with the results showing that keratinocytes exposed to cold stress of 0°C had a higher survival rate than control cells. To pursue a promising clue for explaining the result, we examined metallothionein (MT) production in rat keratinocytes that had been exposed to cold stress at 0°C. Microfluorometric quantification showed a positive correlation between the time course and the intensity of immunofluorescence for MT, indicating that the molecule is inducible by exposure to cold stress in our experimental system. These results suggest that epidermal cells that have been exposed to cold stress maintain a higher resistance to UV radiation than nonexposed controls in vivo and in vitro , and that MT with radical-scavenging activity might contribute, at least in part, to photoprotection against UVB-induced oxidative damage in mammalian skin.     

Noninvasive Assessment of UV-induced Skin Damage: Comparison of Optical Measurements to Histology and MMP Expression

Acute exposure to UV radiation (UVR) causes visible skin damage such as erythema and results in local and systemic immunosuppression while chronic exposure can result in photocarcinogenesis. These deleterious effects can be quantified by histology and by bioassays of key biological markers, including matrix metalloproteinases (MMPs), or tryptophan moieties. We now report our results in quantifying UV skin damage with noninvasive optical methods based on reflectance and fluorescence spectroscopy and compare these noninvasive measurements to histopathology and MMP-13 expression. A solar simulator with spectral output nearly identical to that of solar radiation was developed and used in our experiments. SKH1 hairless mice were exposed to solar-simulated UVR at a total dose of 21 MED delivered over 10 weeks. Changes in oxygenated and deoxygenated hemoglobin were measured by diffuse reflectance spectroscopy, and tryptophan changes were monitored via a fluorescence monitor. Our results show that there is an increase in erythema, skin fluorescence, sunburn cells and MMP-13 after a series of suberythemal doses of UV irradiation on a hairless mouse animal model. Increased skin fluorescence is observed with increasing UV exposure. The levels of MMP-13 increase as the cumulative UV dose increases but their increase does not correspond to noninvasively measured changes.     

Inhibitory effects of silk protein, sericin on UVB-induced acute damage and tumor promotion by reducing oxidative stress in the skin of hairless mouse

This study was conducted to assess protective effect of an antioxidant protein, sericin, on UVB-induced acute damage and tumor promotion in mouse skin. In experiment 1, HR-1 hairless mice were treated with 180 mJ/cm2 of ultraviolet B light (UVB) once daily for 1 and 7 days. The treatment for 7 days caused red sunburn lesions of the skin. The intensity of red color and area of these lesions were inhibited by the topical application of sericin at the dose of 5 mg after UVB treatment. Immunohistochemical analyses showed that the application of sericin significantly suppressed UVB-induced elevations in 4-hydroxynonenal (4-HNE), expression of cyclooxygenase-2 (COX-2) protein, and proliferating cell nuclear antigen (PCNA)-labeling index in the UVB-exposed epidermis. In experiment 2, HR-1 hairless mice were treated with 200 nmol of 7,12-dimethylbenz [alpha] anthracene (DMBA) followed 1 week later by irradiation with 180 mJ/ cm2 of UVB twice weekly for 22 weeks. The protective effect of sericin was evident in terms of significant reduction in tumor incidence and tumor multiplicity at the dose of 5 mg. The results suggest that sericin possesses photoprotective effect against UVB-induced acute damage and tumor promotion by reducing oxidative stress, COX-2 and cell proliferation in mouse skin.     

Green tea phenol extracts reduce UVB-induced DNA damage in human cells via interleukin-12

Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation-induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV-induced DNA damage. These effects appeared to be mediated via interleukin (IL)-12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL-12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB-induced apoptosis was reduced in UVB-exposed cells treated with GTP. GTP induced the secretion of IL-12 in keratinocytes. The reduction in UV-induced cell death by GTP was almost completely reversed upon addition of an anti-IL-12-antibody, indicating that the reduction of UV-induced cell death by GTP is mediated via IL-12. The ability of IL-12 to reduce DNA damage and sunburn cells was confirmed in "human living skin equivalent" models. Hence the previously reported UV-protective effects of GTP appear to be mediated in human cells via IL-12, most likely through induction of DNA repair.     

Naringenin protects HaCaT human keratinocytes against UVB-induced apoptosis and enhances the removal of cyclobutane pyrimidine dimers from the genome

Many naturally occurring agents are believed to protect against UV-induced skin damage. In this study, we have investigated the effects of naringenin (NG), a naturally occurring citrus flavonone, on the removal of UVB-induced cyclobutane pyrimidine dimers (CPD) from the genome and apoptosis in immortalized p53-mutant human keratinocyte HaCaT cells. The colony-forming assay shows that treatment with NG significantly increases long-term cell survival after UVB irradiation. NG treatment also protects the cells from UVB-induced apoptosis, as indicated by the absence of the 180 base pair DNA ladders and the appearance of sub-G1 peak using agarose gel electrophoresis and flow cytometric analysis, respectively. The UVB-induced poly (ADP-ribose) polymerase-1 (PARP-1) cleavage, caspase activation and Bax/Bcl2 ratio were modulated following NG treatment, indicating an antiapoptotic effect of NG in UVB-damaged cells that occurs at least in part via caspase cascade pathway. Moreover, treatment of UVB-irradiated HaCaT cells with NG enhances the removal of CPD from the genome, as observed by both direct quantitation of CPD in genomic DNA and immuno-localization of the damage within the nuclei. The study provides a molecular basis for the action of NG as a promising natural flavonoid in preventing skin aging and carcinogenesis.     

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

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

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.     

DETECTION OF UVR-INDUCED DNA DAMAGE IN MOUSE EPIDERMIS in vivo USING ALKALINE ELUTION

Abstract— Alkaline elution has been used to detect ultraviolet radiation (UVR)-induced DNA damage in the epidermis of C3H/Tif hr/hr mice. This technique detects DNA damage in the form of single-strand breaks and alkali-labile sites (SSB) formed directly by UVA (320–400 nm) or indirectly by UVB (280–320 nm). The latter induces DNA damage such as cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4)-photoproducts, which are then converted into transient SSB by cellular endonucleases, during nucleotide excision repair (NER). The irradiation system used had a spectral output similar in effect to solar UVR, with the UVB component inducing 94% of the edema response observed in mice. Consequently, the majority of SSB detected were those formed via NER of UVB-induced photoadducts. The number of SSB detected immediately after 8 kj/m² (2.7 minimum erythema doses determined at 48 h post-UVR [MED]) was low, indicating the formation of only small numbers of transient SSB. When DNA repair inhibitors hydroxyurea and 1 -β-D-arabinofuranosylcytosine were administered (intraperi-toneally) to mice 30 min before UVR, they prevented sealing of the DNA SSB formed during NER. A four-fold increase in the number of SSB detected resulted, which was found to be linearly related to the UVR dose. The SSB induced by 2 kj/m² (less than an MED) were readily detected, with the ear showing lower numbers of SSB than the dorsum. When repair inhibitors were added post-UVR, the rate of formation of SSB declined rapidly with time of administration, reflecting repair of DNA lesions. After a UVR dose of 6 kj/m² (2 MED), 50% of the initial repair-dependent SSB had been removed after approximately 2 h in the ear and 4 h in the dorsum; no more SSB appeared to be incised by 24 h post-UVR. The technique described is an efficient and highly sensitive one for the quantification of SSB induced in UV-irradiated skin samples in vivo.     

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.     

Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells

The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man. Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation. However, there is little data on the effects of heat treatment on damage caused by UV irradiation. Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair. For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay. Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls. However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types. Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked. In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h. This is not necessarily caused by elevated heat-shock protein levels themselves. Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.     

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.     

(-)-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.