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

Both environmental exposure to vanadium pentoxide (V₂O₅, 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 H₂O₂ 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.