Marked Variation in Free Radical Injury Between Bovine and Porcine Endothelial Cells Cultured in Different Media

Abstract

Previous studies produced models of oxygen-derived free radical (OFR) injury, using H₂O₂ or xanthine/xanthine oxidase (X/XO), in cultured porcine aortic endothelium (PAE) and rat coronary endothelium. H₂O₂ at 0.1 mM resulted in 50% viability in both cell types. To determine if comparable H₂O₂ or X/XO concentrations have the same injurious effect on endothelium from other sources, models of OFR injury were developed for bovine aortic endothelium (BAE) and bovine brain microvessel endothelium (BBME). Varying concentrations of H₂O₂ (0.01 to 6 mM) or X/XO (10 μM/0.1 to 0.3 U/mL) were added to medium 24 h prior to evaluating cell damage. Injury was assessed using the Trypan blue exclusion test (% viability) and by measuring the release of lactate dehydrogenase into medium. H₂O₂ concentrations required to produce 50% viability were >6 mM in BAE and BBME versus 1 mM in PAE when cells were grown in Dulbecco's modified Eagle's medium (DMEM). Similarly, BAE and BBME were less sensitive than PAE to damage by X/XO. Cells from both species were more sensitive to H₂O₂ or X/XO injury when grown in Medium 199 (M199) versus DMEM. The most profound difference was observed with PAE where 50% viability was obtained with 0.12 versus 1.05 mM H₂O₂ in M199 versus DMEM. These results indicate that bovine endothelial cells from aorta and brain are more resistant to free radical injury than PAE. The presence or absence of key media components (iron, pyruvate, cysteine, histidine) likely influences the extent of OFR injury.