Vanadium pentoxide induced oxidative stress and cellular senescence in human lung fibroblasts.

Both environmental exposure to vanadium pentoxide (V2 O5 , V+5 for its ionic counterparts) and fibroblast senescence are associated with pulmonary fibrosis, but whether V+5 causes fibroblast senescence remains unknown. We found in a dose-response study that 2-40 μM V+5 caused human lung fibroblasts (HLF) senescence with increased senescence-associated β-galactosidase activity and p16 expression, while cell death occurred at higher concentration (LC50 , 82 μM V+5 ). Notably, measures of reactive oxygen species (ROS) production with fluorescence probes showed no association of ROS with V+5 -dependent senescence. Preloading catalase (polyethylene-conjugated), a H2 O2 scavenger, did not alleviate the cellular senescence induced by V+5 . Analyses of the cellular glutathione (GSH) system showed that V+5 oxidized GSH, increased GSH biosynthesis, stimulated cellular GSH efflux and increased protein S-glutathionylation, and addition of N-acetyl cysteine inhibited V+5 -elevated p16 expression, suggesting that thiol oxidation mediates V+5 -caused senescence. Moreover, strong correlations between GSSG/GSH redox potential (Eh ), protein S-glutathionylation, and cellular senescence (R2  > 0.99, p < 0.05) were present in V+5 -treated cells. Studies with cell-free and enzyme-free solutions showed that V+5 directly oxidized GSH with formation of V+4 and GSSG in the absence of O2 . Analyses of V+5 and V+4 in HLF and culture media showed that V+5 was reduced to V+4 in cells and that a stable V+4 /V+5 ratio was rapidly achieved in extracellular media, indicating ongoing release of V+4 and reoxidation to V+5 . Together, the results show that V+5 -dependent fibroblast senescence is associated with a cellular/extracellular redox cycling mechanism involving the GSH system and occurring under conditions that do not cause cell death. These results establish a mechanism by which environmental vanadium from food, dietary supplements or drinking water, can cause or contribute to lung fibrosis in the absence of high-level occupational exposures and cytotoxic cell death.