Protective effect of selenium and zinc on UV-A damage in human skin fibroblasts.

Ultraviolet A radiation participates in cytotoxicity and carcinogenesis of the skin by a mechanism involving the generation of reactive oxygen species. Endogenous antiradical defense systems utilize metalloenzymes including Se-dependent glutathione peroxidase and Cu and Zn superoxide dismutase. The aim of the present work was to determine the protective effect of two trace elements, Se and Zn, on cultured human diploid fibroblasts exposed to UV-A radiation (broad-spectrum source with a maximum intensity at 375 nm). Selenium in the culture medium (0.1 mg/L) in the form of sodium selenite increased the synthesis and activity of glutathione peroxidase by 60.5% in the absence of exposure to UV-A radiation and by 35% after irradiation with 5 J/cm2 (P = 0.043). The presence of this element significantly increased the survival of UV-A-irradiated fibroblasts (P < 0.0001). This confirms the essential role of Se in the detoxifying activity of the enzyme. In addition, thiobarbituric acid-reacting substances (TBAR), which are lipid peroxidation markers, decreased in the presence of exogenous Se: -19% and -22% without irradiation and after irradiation with 5 J/cm2 (P = 0.056). When Zn was added at the dose of 6.5 mg/L as ZnCl2, fibroblasts subjected to oxidizing stress induced by UV-A were protected from cytotoxicity (P < 0.0001). The TBAR production decreased significantly: -33% without irradiation and -34% after irradiation with 5 J/cm2 (P = 0.008). Superoxide dismutase activity, however, decreased after supplementing with Zn: -26% without irradiation and -20% after UV-A irradiation (P = 0.017). The antioxidant properties of Zn are thus apparently independent of superoxide dismutase activity.

Cultured human skin fibroblasts absorb 65Zn. Optimization of the method and study of the mechanisms involved.

The radioactive isotope 65Zn was used to study the incorporation of zinc by cultured human skin fibroblasts. The development of the method for studying cell uptake of 65Zn in a minimal synthetic medium is presented. Kinetics carried out on control cultures up to 240 min indicated that zinc uptake occurred in three phases, the first being the most rapid. Temperature and pH affect zinc uptake, in favor of an active transport process. In addition, the rate of incorporation is considerably decreased during the first phases after adding potassium cyanide, during the last phases after adding sodium iodoacetate, and during all the phases if dithioerythritol is used. A hypothesis is therefore proposed according to which several types of mechanisms would be involved in zinc uptake by fibroblasts. At least a part of these mechanisms is energy-dependent.

Development of a simple antioxidant screening assay using human skin fibroblasts.

The purpose of this study was to develop a simple antioxidant screening assay for quantifying the protective effects of antioxidant enzymes, inhibitors and scavengers against extracellularly generated oxygen species on human skin fibroblast cytotoxicity. Different in vitro oxidative stresses have been studied: xanthine oxidase-hypoxanthine, flavin mononucleotide-NADH, and hydrogen peroxide. Cytotoxicity and protection were evaluated by two procedures: evaluation of the living cells using a colorimetric method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT), and ability of the viable cells to adherate and proliferate. Hypoxanthine-xanthine oxidase and H2O2 induced a dose dependent cytotoxicity only when we considered the delayed toxicity. The influence of the cell density was also investigated. The delayed toxicity was higher when cell density increased. One hundred percent protection against free radical cytotoxicity induced by the three systems were obtained with catalase (500 U/ml). When the oxidative stress used was H2O2 90-96% protection was obtained with deferoxamine an iron chelating agent that prevents iron catalysed radical reactions. Using the colorimetric method no significant protection was obtained when SOD was added before and during the stresses. Using the fibroblasts ability to proliferate SOD (10-150 micrograms/ml) reduced xanthine oxidase (20 U/l)-hypoxanthine (0.10-0.30 mM) or H2O2 (1-6 mM) cytotoxicity by 15-20%. SOD did not act as antioxidant when the applied stress was mediated by flavin. In this study we showed a paradoxical effect and the cytotoxicity of flavin-NADH system increased when we added SOD to the cell medium. This simple and reliable antioxidant screening assay required no costly or radioactive equipment.