Butoxyacetic acid-induced hemolysis of rat red blood cells: effect of external osmolarity and cations.
Hemolysis is the principal toxicity of acute exposure to ethylene glycol monobutyl ether (EGBE) in rats. EGBE itself is not an active hemolytic agent, but its metabolite, butoxyacetic acid (BAA) formed as a result of dehydrogenase activity is a potent hemolysin. Here we address the role of osmolarity and cation composition of the suspending buffers in the mechanism of BAA-induced hemolysis of rat red blood cells in vitro. Rat erythrocytes were protected from BAA-induced cell swelling and hemolysis by the addition of sucrose to the suspending media. Hemolysis and cell swelling were also reduced by replacing external sodium with potassium. When calcium was not present in the suspending medium or when chelated by EGTA, hemolysis was increased after 2 h incubation with 1 mM or 2 mM BAA. Addition of as little as 0.05 mM CaCl(2) reduced hemolysis significantly while the addition of MgCl(2) had no effect. The dose-response relationship between BAA concentration and hemolysis determined in the presence or absence of calcium showed an increased effect of BAA in the absence of calcium. BAA-induced spherocytosis and cell fragmentation were more pronounced in the absence of calcium. The time course of BAA-induced hemolysis in the presence and absence of calcium demonstrated that the effect of calcium is to delay the onset of hemolysis. Increased intracellular calcium as a result of exposure to BAA was verified by atomic absorption spectroscopy. Charybdotoxin, an inhibitor of the calcium activated potassium channel, blocked the protective effect of calcium suggesting that the delay of onset of hemolysis in the presence of calcium is due to potassium loss caused by this channel. We conclude that the mode of action of BAA is to cause a colloid osmotic lysis of the rat red blood cell. Hemolysis requires external sodium and is associated with calcium uptake. Calcium appears to delay the onset of hemolysis. We speculate that: (1) BAA causes sodium and calcium to enter the cell; (2) calcium initially has a protective effect via the calcium activated potassium channel which facilitates the loss of potassium thereby, compensating for the osmotic effect of increased cell sodium; (3) calcium subsequently may have other deleterious effects through activation of proteases and externalization of phosphatidylserine in the exterior leaflet of the membrane.
Udden
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MM
Hematology/Oncology Section, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. udden@bcm.tmc.edu
Patton
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CS
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism* / Q000473:pathology; D005026:Ethylene Glycols / Q000633:toxicity*; D006016:Glycolates / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000633:toxicity*; D006461:Hemolysis / Q000187:drug effects*; D008297:Male; D009994:Osmolar Concentration; D026902:Potassium Channel Blockers / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344
D005026:Ethylene Glycols; D006016:Glycolates; D006460:Hemolysin Proteins; D026902:Potassium Channel Blockers; C017095:n-butoxyacetic acid; D002118:Calcium