Hemolysis of human erythrocytes by hypochlorous acid is modulated by amino acids, antioxidants, oxidants, membrane-perforating agents and by divalent metals.
The optimal conditions under which hypochlorous acid (NaOCl) either hemolyzes human RBC or kills monkey kidney epithelial cells (BGM) in culture had been investigated. While in Hank's balanced salt solution (HBSS), micromolar amounts of NaOCl caused full hemolysis and also killed BGM cells, in D-MEM or RPMI media rich in amino acids, 25-40 mM of hypochlorite were needed to induce cell injury. Cells exposed to high amounts of NaOCl became highly refractory to strong detergents. Hemolysis by NaOCl was strongly inhibited by a large variety of antioxidants. RBC treated by subtoxic concentrations either of peroxide, peroxyl radical, NO, cholesterol, PLA2, PLC as well as by N2, argon or by mixture of CO2 (10%) and O2 (90%) became much more susceptible to lysis by NaOCl. On the other hand, while RBC treated by Fe2+, Co2+, and V2+ and to a lesser extent with Cu2+ became highly resistant to NaOCl hemolysis presumably due to NaOCl decomposition, no such effect was found either with Co2+ or by Mn2+. RBC treated by azide to destroy catalase and then incubated with peroxide and with NaOCl failed to undergo hemolysis due to the ability of peroxide to decompose NaOCl. The inhibitory effects of the divalent metals on NaOCl-induced hemolysis were also substantiated by measuring the decrease in pH and by cyclic voltammetry. The findings that like peroxide, NaOCl also synergizes with membrane-perforating agents and with a protease to kill epithelial cells further implicate such "cocktails" in cell injury in inflammatory conditions. Taken together, because of the capacity of many agents to scavenge NaOCl, tissue damage by NaOCl-generated neutrophils can take place primarily if activated neutrophils closely adhere to target cells to avoid the scavenging effects of amino acids and of antioxidants. Therefore, the significance of the data which had tested the cytotoxic effects of NaOCl using cells suspended only in salt solutions, should be reconsidered.
Ginsburg
Isaac
I
Department of Oral Biology, Hadassha Faculty of Dental Medicine, Hebrew University, Jerusalem, Israel. ginsburg@cc.huji.ac.il
Sadovnic
Milu
M
Yedgar
Shaul
S
Kohen
Ron
R
Hrbac
Jan
J
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000633:toxicity*; D001386:Azides / Q000633:toxicity; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D010450:Endopeptidases / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000633:toxicity; D004847:Epithelial Cells / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects*; D016166:Free Radical Scavengers / Q000378:metabolism; D005740:Gases; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D006997:Hypochlorous Acid / Q000633:toxicity*; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D008670:Metals / Q000494:pharmacology; D016877:Oxidants / Q000633:toxicity*; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D011506:Proteins / Q000378:metabolism
D000596:Amino Acids; D000975:Antioxidants; D001386:Azides; D003470:Culture Media; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D005740:Gases; D008670:Metals; D016877:Oxidants; D011506:Proteins; D006997:Hypochlorous Acid; D002784:Cholesterol; D006861:Hydrogen Peroxide; D010741:Phospholipases A; D054467:Phospholipases A2; D010450:Endopeptidases