Biological effects of menadione photochemistry: Effects of menadione on biological systems may not involve classical oxidant production

Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca²⁺](i)) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca²⁺](i) response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca²⁺](i). Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca²⁺](i) effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340 nm) stimulates menadione-dependent formation of the DMPO/OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca²⁺](i); and (c) light (maximum effect at 340 nm) augments the deleterious effects of menadione on cell viability as determined by ⁵¹Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.