Salminen W F, Roberts S M, Pumford N R, Hinson J A
Department of Pharmacology & Therapeutics, J. Hillis Miller Health Science Center, University of Florida, Gainesville, FL 32611, USA.
Drug Metab Dispos. 1998 Mar;26(3):267-71.
The hepatotoxicity of the analgesic acetaminophen is believed to be mediated by covalent binding to critical proteins. Radiolabeled 3'-hydroxyacetanilide, a regioisomer of acetaminophen, covalently binds to proteins at levels similar to those of acetaminophen, but without toxicity. Covalent binding has recently been detected by Western blot to a 50-kDa microsomal protein that comigrated with CYP2E1 and was accompanied by a loss of the CYP2E1 activity. However, radiolabel studies previously indicated that a significant amount of the radiolabel is lost during electrophoresis. In the present study, 3'-hydroxyacetanilide covalent binding was detected immunohistochemically in liver using an anti-acetaminophen antiserum. 3'-Hydroxyacetanilide (1000 mg/kg, ip) administration to mice resulted in panlobular immunostaining in liver, with the single layer of hepatocytes surrounding the central veins having the greatest intensity of staining. Staining was most intense at 1 hr and somewhat decreased at 3 and 6 hr. In contrast, immunochemical staining indicated that covalent binding of acetaminophen (250 mg/kg, ip) was confined to the centrilobular hepatocytes, the area of the ensuing necrosis. Cobaltous chloride pretreatment decreased the total intensity of the panlobular immunostaining following 3'-hydroxyacetanilide. The CYP2E1 inhibitor diallyl sulfide decreased the intensity of immunostaining in the central vein area only. Western blot analysis indicated diallyl sulfide also eliminated binding to the microsomal 50-kDa protein. These data are consistent with centrilobular binding of 3'-hydroxyacetanilide, mediated in part by CYP2E1, and panlobular binding, mediated by other P450 enzymes.
人们认为，镇痛剂对乙酰氨基酚的肝毒性是通过与关键蛋白质的共价结合介导的。放射性标记的3'-羟基乙酰苯胺是对乙酰氨基酚的区域异构体，它与蛋白质的共价结合水平与对乙酰氨基酚相似，但没有毒性。最近通过蛋白质印迹法检测到与一种50 kDa微粒体蛋白的共价结合，该蛋白与CYP2E1迁移位置相同，同时伴有CYP2E1活性丧失。然而，放射性标记研究先前表明，在电泳过程中有大量放射性标记丢失。在本研究中，使用抗对乙酰氨基酚抗血清通过免疫组织化学方法在肝脏中检测到3'-羟基乙酰苯胺的共价结合。给小鼠腹腔注射3'-羟基乙酰苯胺（1000 mg/kg）后，肝脏出现全小叶免疫染色，围绕中央静脉的单层肝细胞染色强度最大。染色在1小时时最强，在3小时和6小时时有所减弱。相比之下，免疫化学染色表明，对乙酰氨基酚（250 mg/kg，腹腔注射）的共价结合局限于小叶中央肝细胞，即随后发生坏死的区域。氯化钴预处理降低了3'-羟基乙酰苯胺注射后全小叶免疫染色的总强度。CYP2E1抑制剂二烯丙基硫醚仅降低了中央静脉区域的免疫染色强度。蛋白质印迹分析表明，二烯丙基硫醚也消除了与微粒体50 kDa蛋白的结合。这些数据与3'-羟基乙酰苯胺的小叶中央结合（部分由CYP2E1介导）和全小叶结合（由其他P450酶介导）一致。
