Protection against UVB-induced damage to keratinocytes by antioxidant nutrients



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Texas Tech University


The ultraviolet component of sunlight causes increases in reactive oxygen species, and to the extent that these species exceed the ability of antioxidant defenses to remove them, oxidative damage occurs. Antioxidant systems have evolved to protect organisms from endogenous and exogenous reactive oxygen species — in humans these systems are enzymatic and non-enzymatic, and the non-enzymatic systems include certain nutrients. Evidence from biological and epidemiological studies suggests that vitamins C, E, B-carotene and selenium have useful roles in chemoprevention.

8-OHdG is an oxidative DNA lesion that can be sensitively measured as an index of oxidative stress in cells, using HPLC coupled with ultraviolet and electrochemical detection. These lesions can lead to transversion mutations and can therefore potentially contribute to skin cancer.

Using Balb/c MK-2 cells the level of 8-OHdG residues, normalized to normal dG residues, was measured following UVB exposure for keratmocytes grown in several nutrient-specific media. UVB doses from 4-500 mJ/cm increased the level of adducts for cells grown m EMEM, but with supplementation of 5 pg/mL selenite, 0.8 pg/mL ascorbate, or 20 pg/mL trolox (a vitamin E analog), the level of adducts was reduced to the level seen in unirradiated controls.

Within 24 hours of a 500 mJ/cm^2 UVB insult, the activity of antioxidant enzymes superoxide dismutase and catalase increased; the level of induction or activity increase was greatest for cells grown with the lowest levels of antioxidant nutrients. This suggests some complementarity for enzymatic and non-enzymatic defenses.

Cell culture medium contains little or no selenium, and glutathione peroxidase is consequently very low unless cells are supplemented. With supplementation there is an increase in glutathione peroxidase, but the level of this enzyme is unchanged by increasing Se to concentrations five times greater.

P53 constitutively arrests the cell cycle to allow repair of damaged DNA. Immunoblots of protem from cells pre-incubated with supplemental nutrients showed little difference across treatment. Immunoblotting for bcl-2, a protein that inhibits apoptosis, showed a greater that 5x stronger band for Se-treated cells than for the negative control.

Collectively these data show that the ultraviolet component of sunlight causes damage that is free-radical mediated, and that some of this damage can be reduced by antioxidant nutrients.