INTRODUCTION
Previous studies indicate that human skin is a target for infrared A radiation (IRA).1 Infrared A (IRA) irradiation leads to increased expression of matrix metalloproteinase 1 (MMP1), an enzyme involved in wrinkle formation in human dermis after treatment with physiological IRA doses.1 In addition, IRA irradiation results in a decrease of the skin´s vitamin content.1Mechanistic studies have shown that IRA exerts these effects through the generation of oxidative stress in human skin fibroblasts, indicating the possibility that appropriate antioxidant may be able to prevent IRA-induced gene expression in this cell type. To this end, we studied a water-soluble extract made from blueberry plants grown along the coast of Maine (BerrimatrixTM). This antioxidant matrix contains polyphenols, vitamins, and other natural ingredients. In order to test whether this antioxidant matrix is able to prevent IRA-induced damage in human dermal fibroblasts, these cells were pre-incubated (24 hours) and post-incubated (24 hours) in the presence or absence of the extract. IRA-induced expression of MMP-1 has been assessed as read-out.
MATERIALS AND METHODS
Cell CultureHuman dermal fibroblasts (HDFs) prepared from neonatal foreskin were cultured in EMEM (PAA Laboratories GmbH, Kölbe, Germany) supplemented with 5% fetal calf serum (FCS, Invitrogen, Karlsruhe, Germany), 1% L-glutamine, 1% non-essential amino acids (Invitrogen, Karlsruhe, Germany), and 1 % streptomycin/amphotericin B (Invitrogen, Karlsruhe, Germany) in a humidified atmosphere containing 5% CO2 for 4 days until they reached confluence as described.1 For all studies, only early passage (<12) fibroblasts have been used to avoid changes in their original phenotype during subculture. Cells were kept in 6 well plates for culture and irradiation.Viability TestingIn order to test the concentrations to be applied on the human dermal fibroblasts, dose response studies have been performed. Viability of the cells during a period of 48 hours in the presence and absence of the antioxidant matrix has been assessed as read-out.Cytotoxicity of the antioxidant matrix was evaluated using the MTT colorimetric assay according to Mosmann as described earlier.2,3 MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide), the classical tetrazolium salt, turns bright blue and may form an insoluble precipitate when reduced in a cell either enzymatically or through direct reaction with NADH or NADPH. So, loss of coloring indicates diminished cell metabolism and increased toxicity.Human dermal fibroblasts were seeded in 96-well plates at 3.000 cells/200 μl in each well. The next day the cells were treated with the substances of interest for 48 hours. After 45 hours the medium was supplemented with 25 μl of MTT (2 mg/ml phosphate-buffered saline) and the mixture was incubated for another 3 hours. Finally, solutions were removed, formazan crystals were dissolved in 200 μl of Me2SO and absorption was measured using a microplate reader (Monochromator
