Effects of hypoxia and glutathione depletion on hemoglobin- and myoglobin-mediated oxidative stress toward endothelium

We investigated the toxicity of hemoglobin/myoglobin on endothelial cells u nder oxidative stress conditions that include cellular hypoxia and reduced antioxidant capacity. Bovine aorta endothelial cells (BAECs), grown on micr ocarrier beads, were subjected to cycles of hypoxia and reoxygenation in a small volume of medium, and endothelial cell monolayers were depleted of th eir intracellular glutathione (GSH) by treatment with buthionine sulfoximin e. Incubation of diaspirin crosslinked hemoglobin (DBBF-Hb) or horse skelet al myoglobin (Mb) with BAECs subjected to 3 h of hypoxia caused transient o xidation of the hemoproteins to the ferryl form (Fe4+). Formation of the fe rryl intermediate was decreased in a concentration-dependent manner by the addition of L-arginine, a substrate of NO synthase, after 3 h of hypoxia. O ptimal inhibition of ferryl formation, possibly due to the antioxidant acti on of NO, was achieved with. 900 mu M L-arginine. Addition of hydrogen pero xide to GSH-depleted cells in the presence of DBBF-Hb or Mb significantly d ecreased cell viability. Ferryl Mb, but not ferryl DBBF-Hb, was observed in samples analyzed at the end of treatment, which may explain the greater to xicity observed with Mb as opposed to DBBF-Hb. This model may be utilized t o identify causative agent(s) associated with hemoprotein cytotoxicity and in designing strategies to suppress or control hems-mediated injury under p hysiologically relevant conditions. (C) 2000 Elsevier Science B.V. All righ ts reserved.