Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid

Abstract

Exposure of human red blood cells to low doses of hypochlorous acid (HOCl) resulted in the loss of intracellular GSH. Oxidation occurred less than 2 min after the addition of HOCl, and required approx. 2.5 mol of HOCl per mol of GSH lost. Loss of GSH preceded oxidation of membrane thiols, the formation of chloramines and haemoglobin oxidation. The susceptibility of intracellular GSH to oxidation by HOCl was two-thirds that of GSH in cell lysates. These results indicate that HOCl can penetrate the red cell membrane, which provides little barrier protection for cytoplasmic components, and that GSH oxidation by HOCl may be a highly selective process. Virtually all of the GSH lost was converted into GSSG. If glucose was added to the medium, most of the GSH oxidized by low doses of HOCl was rapidly regenerated. At higher doses, recovery was less efficient. However, when HOCl was added as a slow infusion rather than in a single bolus, there was increased recovery at higher doses. This indicates that in metabolically active cells regeneration is rapid and GSH may protect cell components from damage by HOCl. HOCl-induced lysis was only slightly delayed by adding glucose to the medium, indicating that lytic injury is not ameliorated by GSH.

Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis

“…However, because HOCl reacts readily with a range of biological targets, it has been difficult to identify which reactions are critical for the cytotoxic effect. Although thiol groups are particularly reactive [13,14], many cells can repair oxidized thiols [14,15]. We have shown that regeneration of thiols did not prevent HOCl-mediated red cell lysis [14], and it is not clear whether thiol oxidation results in irreversible cell injury.…”

“…Although thiol groups are particularly reactive [13,14], many cells can repair oxidized thiols [14,15]. We have shown that regeneration of thiols did not prevent HOCl-mediated red cell lysis [14], and it is not clear whether thiol oxidation results in irreversible cell injury. Other reactive groups are Fe-S centres [7], thiolate bonds in zinc proteins [16] adenine nucleotides [17,18], alkenes [19,20] and amino groups, producing chloramines that are also strong oxidants [1,13].…”

“…GSH was measured by reaction with monobromobimane as described previously [14]. Red cell ghosts were prepared [31] after exposure to HOCl or HOBr, and membrane thiols were measured by reaction with 2,2-dithiobis-(5-nitrobenzoic acid) in the presence of 6 mg\ml SDS [27].…”

“…Red blood cells have been used extensively as a model system for investigating mechanisms of neutrophil-mediated cell injury [11,12,14,17,26]. Haemolysis is dependent on HOCl and is not readily mediated by chloramines [11,17].…”

“…However, the mechanism by which HOCl causes lysis is not known. We have shown previously that haemolysis occurs by a colloid osmotic mechanism that involves disruption of the membrane to cause K + leakage and swelling [14,27]. Although reduced glutathione (GSH) and membrane thiols were easily oxidized, this was not associated with lysis and the critical target remains unidentified.…”