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.