Menadione causes endothelial barrier failure by a direct effect on intracellular thiols, independent of reactive oxidant production

Menadione (MQ), a quinone used with cancer chemotherapeutic agents, causes cytotoxicity to endothelial cells (EC). Previous studies have suggested that MQ induces an oxidative stress and dysfunction in EC by either increasing hydrogen peroxide (H2O2) production or depleting intracellular glutathione (GSH), the main intracellular antioxidant. Since a primary function of EC is to form a barrier to fluid movement into tissues, protecting organs from edema formation and dysfunction, our aim was to see if MQ would cause a barrier dysfunction and to ascertain the mechanism. Using diffusional permeability to fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) as a measure of barrier function, we found that 15 muM MQ incubated with a bovine pulmonary artery EC (BPAEC) monolayer for 4 h produced a profound barrier failure (similar to7-fold increase in permeability) with a parallel fall in glutathione, almost to depletion. These two events were highly correlated. Immunofluorescent imaging showed formation of paracellular holes consistent with a loss or rearrangement of cell-cell and cell-matrix adhesion molecules. H2O2 (100 muM), a concentration which gave about the same increase in permeability as MQ, only slightly decreased GSH concentration. Antioxidants, such as catalase (CAT) and dimethylthiourea (DMTU), which were able to block the H2O2-induced changes, had no effect on the MQ-induced permeability and GSH changes, suggesting that H2O2 was not involved in MQ-induced. effects. MQ caused a severe EC cytotoxicity as judged by lactate dehydrogenase (LDH) leakage from the EC, whereas H2O2 caused only a minor increase. Also, MQ profoundly inhibited the activities of glucose-6-phosphate dehydrogenase (G6PDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), key thiol enzymes involved in glutathione and ATP metabolism, whereas H2O2 produced only a slight decrease in these activities. We conclude that the cytotoxicity of MQ and resulting barrier dysfunction correlate with GSH depletion and inactivation of key metabolic enzymes, compromising antioxidant defenses, rather than being consistent with H2O2-mediated oxidative stress. (C) 2003 Elsevier Science B.V. All rights reserved.