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

Both environmental exposure to vanadium pentoxide (V(2)O(5), 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 (LC(50), 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 H(2)O(2) 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 (E(h)), protein S-glutathionylation, and cellular senescence (R(2) > 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 O(2). 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.