Resistance of cultured human skin fibroblasts from old and young donors to oxidative stress and their glutathione peroxidase activity

Abstract

Background: It has been suggested that oxidative stress is involved in the aging process and that the resistance of animals to oxidative stress may decrease with advancing aging. However, there are only a limited number of reports of studies on the relationship between aging and resistance to oxidative stress.

Objective: The aim of this work is to examine the relationship between the resistance of human skin fibroblasts to oxidative stress and donor age, and the relevance of antioxidant enzyme activities to this resistance.

Methods: Percent cell survival was determined by the trypan blue exclusion test and the neutral red method. Superoxide dismutase activity was assayed by the method of Oyanagi, catalase activity by the method of Aebi, and glutathione peroxidase activity by the method of Flohé and Günzler. Reduced glutathione concentration was measured by the method of Griffith. Antioxidant enzyme mRNA levels were estimated by reverse transcription polymerase chain reactions (RT-PCR).

Results: The percent survivals of cultured human skin fibroblasts, derived from young and old donors (referred to as young and old cells, respectively), under oxidative stress from hydrogen peroxide, linoleic acid hydroperoxide, or ultraviolet light B were examined. Old cells were more resistant to such oxidative stress than young cells. The activity of glutathione peroxidase was higher by 46.1% in old cells than in young cells, although there was no difference between their relative glutathione peroxidase mRNA levels. Further, there was no difference between their activities of copper/zinc superoxide dismutase, manganese superoxide dismutase, or catalase. However, the relative mRNA levels of copper/zinc superoxide dismutase and manganese superoxide dismutase were lower by 13.9 and 20.9% in old cells than in young cells, respectively, while there was no difference between the levels of catalase.

Conclusions: These results suggest that old cells are more resistant to oxidative stress than young cells, presumably because of an increase in cellular glutathione peroxidase activity.