Jean-Claude Beani

Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblasts

The present study analyzes the expression of the thioredoxin/thioredoxin reductase (Trx/TR) system in UVA-irradiated human skin fibroblasts. Irradiation increases the intracellular level of Trx and a time-dependent increase of Trx mRNA is observed. Our data indicate that Trx translocates from the cytoplasm to the nucleus. In addition, UV exposure results in an increase in TR synthesis. In order to evaluate the function of Trx/TR system, we investigated the antioxidant role of Trx in transient transfected cells. The ROS accumulation in UVA irradiated cells was assessed using flow cytometry. A 3-fold decrease in ROS production was observed in transiently transfected fibroblasts. These results indicate that Trx acts as an antioxidant protein in UVA irradiated fibroblasts. As ROS are inducers of cell death, this raises the question as to whether Trx is able to protect cells from apoptosis and/or necrosis induced by UVA. Six hours after UVA-irradiation, 29.92% of cells were annexin-V positive. This population was significantly reduced in Trx-transfected cells (8.58%). Moreover, this work demonstrates that Trx prevents the loss of the membrane potential of the mitochondria, the depletion of cellular ATP content, and the loss of cell viability induced by irradiation.

INVOLVEMENT OF ZINC IN INTRACELLULAR OXIDANT/ANTIOXIDANT BALANCE

The effect of zinc (Zn) on cellular oxidative metabolism is complex and could be explained by multiple complementary interactions. In this study, we evaluated the impact of Zn on the pro-oxidant/ antioxidant balance of HaCaT keratinocytes.Cells were submitted to a diffusible metal chelator able to induce intracellular Zn deprivation, TPEN, in combination or not with Zn chloride (ZnCl2), in the culture medium. The intracellular amount of Zn, copper (Cu), and iron (Fe) was determined, as well as CuZnSOD and MnSOD activities and glutathione reserves. The consequence of the modulation of Zn concentration on lipid peroxidation was also evaluated.TPEN induced a significant dose-dependent decrease in intracellular Zn and Cu (from 394–181 and 43–21 Μg/g protein, respectively, after 6 h of TPEN 50 ΜM). No significant change in intracellular Fe concentration was found following TPEN exposure. The SOD activities were unchanged after 6 h of TPEN 50 ΜM application, either CuZnSOD or MnSOD. Cells exposure to TPEN induced a deep time- and dosedependent decrease in their glutathione content (from 65–8 ΜM/g protein after 6 h of TPEN 50 ΜM), and a concomittant increase in glutathione in the cell-culture supernatants. No significant change in lipid peroxidation products was detected.

Effect of zinc supplementation on resistance of cultured human skin fibroblasts toward oxidant stress.

In purified system zinc has been shown to have an antioxidant role. Its effects on the resistance of cultured cells towards oxidative stress in vitro were examined. Diploid human skin fibroblasts were grown for 21 d in culture media (RPMI 1640 containing 15% fetal calf serum) added with different zinc (Zn) concentrations (100, 125, and 150μM as Zinc chlorur ZnCl2). In comparison, cell controls were grown in standard culture media (6.5μM Zn). The intracellular zinc levels of treated fibroblasts increased from 3- to 7-fold (2330±120 ng/mg protein in 150-μM Zn-treated cells versus 331±21 ng/mg protein in control cells). The intracellular copper increased 3- fold whereas the iron content slightly but not significantly decreased. The index of basal lipid peroxidation measured as thiobarbituric acid reactants (TBARs) of zinc-supplemented cells was lower than that of non zinc supplemented controls (0.89 μmol/g protein in 150μM Zn-treated cells versus 1.59 μmol/g protein in controls). At these high doses of zinc, fibroblasts expressed lower antioxidant metalloenzymes activities.Diminished TBARs in Zn treated cells tends to support that Zn acts protectively against free radical mediated damage. However when the cells were challenged with extracellular oxidant stresses mediated by hypoxanthine/xanthine oxidase or hydrogen peroxide (H2O2), an increased toxicity in Zn-supplemented cells was observed. When we applied an intracellular oxidative stress as UV-B or UV-A radiation, Zn-treated fibroblasts were more resistant than cells grown in normal medium. If Zn has shown antioxidant effect in some in vitro or in vivo systems our observations clearly demonstrate that this role is not mediated by antioxidant metalloenzymes.

THIOLS AND SELENIUM : PROTECTIVE EFFECT ON HUMAN SKIN FIBROBLASTS EXPOSED TO UVA RADIATION

The sensitivity of human dermal fibroblasts to UVA radiation has been linked to a decrease in intracellular glutathione (GSH) levels. GSH (gamma-glutamyl-cysteinyl-glycine) is a radical scavenger and a cofactor for protective enzymes such as selenium-dependent GSH peroxidases. In this study, we examine the possibility of a cooperative interaction between three cysteine delivery systems and selenium in protecting human cultured fibroblast exposed to UVA radiation. Cells were irradiated (9, 15 and 20 J cm-2) following incubation with N-acetyl-cysteine (NAC, 5 mM), N-acetyl-homocysteine-thiolactone (citiolone (CIT), 1 mM) or L-2-oxothiazolidine-4-carboxylate (OTC, 1 mM). The modulation of the intracellular GSH levels by the addition of the different compounds was determined by enzymatic and separative methods. Cells were harvested for survival analysis by measuring the ability of the cell to adhere and proliferate. Treatments with NAC and CIT resulted in a significant rise in GSH levels compared with control cells, with protection against UVA radiation. OTC did not induce any rise in GSH level; nevertheless, the protective effect afforded by OTC is similar to that observed with NAC and CIT. Moreover, selenium (0.1 mg 1-1), as sodium selenite, significantly increased the protective efficiency of NAC and CIT, but not of OTC. Although the precise mechanism is not known, thiol molecules can inhibit the deleterious effects of UVA radiation. These results provide evidence that compounds capable of inducing GSH synthesis can act with selenium to protect cells against UVA damage.

Plasmacytoid dendritic cells and dermatological disorders: focus on their role in autoimmunity and cancer.

Dendritic cells (DC), considered as immunological sentinels of the organism since they are antigen presenting cells, create the link between innate and adaptive immunity. DC include myeloid dendritic cells (MDC) and plasmacytoid dendritic cells (PDC). The presence of PDC, cells capable of producing large quantities of interferon alpha (IFN-alpha) in response to pathogenic agents or danger signals, seems to be closely related to pathological conditions. PDC have been observed in inflammatory immunoallergic dermatological disorders, in malignant cutaneous tumours and in cutaneous lesions of infectious origin. They seem to play a crucial role in the initiation of the pathological processes of autoimmune diseases such as lupus or psoriasis. Their function within a tumour context is not as well known and is controversial. They could have a tolerogenic role towards tumour cells in the absence of an activator but they also have the capacity to become activated in response to Toll-like receptor (TLR) ligands and could therefore be useful for therapeutic purposes.

Individual Photosensitivity of Human Skin and UVA-Induced Pyrimidine Dimers in DNA

Delineation of the DNA-damaging properties of UVA radiation is a major issue in understanding solar carcinogenesis. Emphasis was placed in this study on the formation of cyclobutane pyrimidine dimers (CPDs), which are now well established as the most frequent UVA-induced DNA lesions in human skin. The yield of CPDs was determined by a chromatographic assay following ex vivo UVA and UVB irradiation of biopsies taken from either phototype II or IV volunteers. A clear correlation was found between the frequency of UVB-induced CPDs and both the phototype and the minimum erythemal dose (MED). Similar results were obtained for the induction of CPDs upon exposure to UVA. Moreover, an excellent correlation was observed for each donor between the yield of DNA damage induced by either UVB or UVA. These observations show that the key parameters driving UVA-induced formation of CPDs are attenuation of radiation in the skin and the number of photons reaching skin cells rather than the cellular content in photosensitizers. In addition, the results show that both MED and phototype are good predictors of the vulnerability of DNA toward UVB and UVA in the skin. This result is of importance for the identification of individuals to be extensively protected.

Effects of cadmium and zinc on solar-simulated light-irradiated cells: potential role of zinc-metallothionein in zinc-induced genoprotection.

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.

L-arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide.

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), which metabolize L‐arginine (L‐Arg) for producing nitric oxide (NO•). This report delineates the relations between NO• and UVA in the human keratinocyte cell line HaCaT. NOS activity was stimulated by exposure of cells to L‐Arg just after irradiation. L‐Arg (5 mM) supply led to an increase in UVA (25.3 J/cm2) cytotoxicity (% of viability 18 ± 3%) whereas neither L‐Arg itself nor UVA irradiation induced cell death at the doses used in this study. Cells were also treated either with L‐thiocitrulline (L‐Thio), an irreversible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and catalase. L‐Thio and SOD prevented L‐Arg‐mediated deleterious effects in irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L‐Arg stress altered catalase (66% decrease) and glutathione peroxidase (83% decrease). DNA damage was evaluated using the ‘comet assay’ and quantified using the ‘tail moment’. UVA alone was genotoxic (mean tail moment: 25.43 ± 1.23, P<0.001 compared control cells). The addition of L‐Arg potentiated DNA damage (mean tail moment: 41.05±3.9) whereas L‐Thio prevented them (mean tail moment 9.86 ± 0.98). We attempted to assess the effect of poly(ADP‐ribose) polymerase (PARP) inhibition on cell death. Using the PARP inhibitor 3‐aminobenzamide, we established that PARP determines both cell lysis and DNA damage induced by UVA and/or L‐Arg. Our findings demonstrated that L‐Arg was able to increase UVA‐mediated deleterious effects in keratinocytes (both DNA damage and cytotoxicity) and that the ratio NO•/O2•‐plays a key role in these processes.—Didier, C., Emonet‐Piccardi, N., Béani, J.‐C., Cadet, J., Richard, M.‐J. L‐arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide. FASEB J. 13, 1817–1824 (1999)

Impairment of cultured cell proliferation and metallothionein expression by metal chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylene diamine

Metallothioneins (MT) are a family of intracellular, cysteine-rich, zinc-binding proteins. Their expression is constitutive but can also be induced at the transcriptional level by various stimuli. In this study, we exposed HaCaT human keratinocytes to excess zinc (ZnCl2) or to zinc deprivation by the diffusible chelator NNN’N’-tetrakis(2-pyridylmethyl) ethylene diamine (TPEN), and to ultraviolet B (UVB) irradiation. We examined both cell proliferation and MT expression. Cell proliferation was maximally stimulated by 100 ΜM Zn2+ supply and was markedly inhibited by zinc deprivation or UVB irradiation. Zinc and UVB irradiation both increased MTI and/or MTII as detected by immunocytochemistry and enhanced the baseline level of MT-IIA mRNA, whereas TPEN treatment inhibited MT basal expression. Zinc partially prevented the concentration-dependent, UVB-induced decrease in cell proliferation. On the other hand, TPEN partially prevented the UVB-induced increase in MTIIA mRNA. These results suggest that zinc is involved in defense mechanisms of skin keratinocytes and in their stress-induced response.

Differential p53-mediated responses to solar-simulated radiation in human papillomavirus type 16-infected keratinocytes.

Abstract:  In immunocompromised patients, cooperative effects of human papillomavirus (HPV) and ultraviolet (UV) radiation have been postulated in the development of non‐melanoma skin cancers. The tumor suppressor p53 is a key component of the cellular response to genotoxic agents, such as UV radiation. We have previously demonstrated that in HPV16‐infected cells, a higher E6* level was associated with a higher resistance to UV and oxidative stress. Using the two same SKv cell lines, the aim of the present study was to investigate p53 and p21 expression and cell death in HPV‐infected keratinocytes in response to UV irradiation and to determine the role of HPV oncoprotein levels on the p53‐mediated cellular response. We demonstrated that the weakly E6*‐expressing level SKv‐e cell line presented both higher cytotoxicity and apoptosis to UV. This high sensitivity was associated with both p53 and p21 nuclear accumulation, while a high E6* level and resistance were associated with no p53 accumulation and a p21 nuclear down‐regulation after UV. Moreover, in SKv‐e cell line, p21 promoter activation was p53 dependent. Our results suggest that an alteration and/or a modulation of the p53–p21 pathway in response to UV could be determinant for HPV‐infected keratinocyte survival and HPV‐associated carcinogenic process.