Expression of hydrogen peroxide and glutathione metabolizing enzymes in human skin fibroblasts derived from donors of different ages

We have examined the activities and mRNA abundance of two hydrogen peroxide metabolizing enzymes (glutathione peroxidase and catalase), glutathione concen tration, and the activities of several enzymes that influence glutathione concentration, including glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PD), and γ-glutamylcysteine synthetase (γ-GCS), in 29 skin fibroblast lines derived from donors ranging in age from 14 gestational weeks to 94 years of age. H₂O₂ metabolizing enzyme activities and mRNA abundances were greater in skin fibroblast cultures established from postnatal donors than in fetally derived cultures. There were no significant differences in either of these parameters in cell lines established from postnatal donors of different ages. Total glutathione concentration decreased with age, but GR activity appeared to be unaffected by age. In order to estimate the ability of the cultures to produce NADPH (an important component of cellular redox status and a cofactor for GR), we determined glucose-6-phosphate dehydrogenase activity and mRNA abundance. We were unable to directly measure γ-GCS activity or mRNA abundance in any of the skin lines or in fetal lung fibroblasts; however, we were able to indirectly demonstrate the presence of th is enzyme by stimulating fetal lung fibroblasts with H₂O₂ following treatment with L-buthionine-S,R-sulfoximine (BSO), an inhibiter of γ-GCS activity. These results show that some, but not all, age-associated differences in antioxidant defense levels are maintained in a culture environment and are consistent with the hypothesis that developmental stages of life are associated with lower antioxidant defense levels than are present in postnatal phases of life.