Udden M M, Patton C S
Hematology/Oncology Section, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Toxicol Lett. 2005 Mar 28;156(1):81-93. doi: 10.1016/j.toxlet.2003.09.020.
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.
溶血是大鼠急性接触乙二醇单丁醚（EGBE）时的主要毒性作用。EGBE本身并非活性溶血剂，但其代谢产物——由脱氢酶活性形成的丁氧基乙酸（BAA）却是一种强效溶血素。在此，我们探讨了悬浮缓冲液的渗透压和阳离子组成在体外BAA诱导大鼠红细胞溶血机制中的作用。通过向悬浮介质中添加蔗糖，可保护大鼠红细胞免受BAA诱导的细胞肿胀和溶血。用钾替代细胞外钠也可减少溶血和细胞肿胀。当悬浮介质中不存在钙或被乙二醇双乙胺四乙酸（EGTA）螯合时，与1 mM或2 mM BAA孵育2小时后溶血增加。加入低至0.05 mM的氯化钙可显著降低溶血，而加入氯化镁则无作用。在有或无钙存在的情况下测定BAA浓度与溶血之间的剂量反应关系，结果显示在无钙时BAA的作用增强。在无钙情况下，BAA诱导的球形红细胞增多症和细胞破碎更为明显。在有或无钙存在的情况下，BAA诱导溶血的时间进程表明，钙的作用是延迟溶血的发生。通过原子吸收光谱法证实，接触BAA会导致细胞内钙增加。钙激活钾通道抑制剂蝎毒素可阻断钙的保护作用，这表明在有钙存在的情况下溶血起始延迟是由于该通道导致的钾流失。我们得出结论，BAA的作用方式是导致大鼠红细胞发生胶体渗透溶解。溶血需要细胞外钠，且与钙摄取有关。钙似乎会延迟溶血的发生。我们推测：（1）BAA导致钠和钙进入细胞；（2）钙最初通过钙激活钾通道发挥保护作用，该通道促进钾流失，从而补偿细胞内钠增加的渗透效应；（3）随后钙可能通过激活蛋白酶和使膜外小叶中的磷脂酰丝氨酸外化而产生其他有害作用。
