Increased generation of reactive oxygen species (ROS), depletion of antioxidant factors (such as glutathione; GSH) and accumulation of oxidative modifications of vital molecules (exemplified by 8-hydroxyguanine; 8-OH-dG) have all been implicated in the development of senescent phenotype. We have therefore examined whether replicative senescence of human peritoneal mesothelial cells (HPMC) in vitro is associated with increased oxidative stress.
HPMC were passaged at 3-day intervals at a seeding density of 30000/cm2 until their proliferating capacity exhausted. Cell were maintained in M199 culture medium supplemented with 10% fetal calf serum. ROS generation was monitored using DCFDA probe, the intracellular GSH concentration was measured according to Akerboom et al., and the presence of 8-OH-dG was detected by immunocytochemistry. Cells were compared after their first ("early") and last ("late") passages. The data were analyzed using the Wilcoxon test for paired non-parametric data and expressed as means ± SD.
During replicative senescence of HPMC the DCFDA-fluorescence intensity (per 100 000 cells) increased from 1550±886 for early-passage cells to 8143±5160 for late-passage cells (n=6, p
These results suggest that oxidative stress may significantly contribute to replicative senescence of HPMC.