Strong protein adduct trapping accompanies abolition of acrolein-mediated hepatotoxicity by hydralazine in mice
Strong protein adduct trapping accompanies abolition of acrolein-mediated hepatotoxicity by hydralazine in mice
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DOI：
10.1124/jpet.104.067330
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发表时间：
2004-09-01
影响因子：
3.5
通讯作者：
Burcham, PC
中科院分区：
文献类型：
--
作者：
Kaminskas, LM;Pyke, SM;Burcham, PC
丙烯醛是一种高反应性的 α,β-不饱和醛，很容易烷基化细胞大分子中的亲核中心。通常，此类反应通过迈克尔加成化学进行，形成保留亲电羰基的加合物。由于这些物质参与二次有害反应，我们假设羰基加合物的失活可能会减弱丙烯醛的毒性。事实上，我们最近确定亲核抗高血压药物肼屈嗪很容易“捕获”细胞蛋白中的丙烯醛加合物，并强烈抑制分离的肝细胞中丙烯醛介导的毒性。这项工作试图确定肼屈嗪是否可以防止丙烯醛前体烯丙醇在整个小鼠体内的肝毒性，以及加合物捕获是否伴随任何此类肝保护作用。小鼠单独接受烯丙醇或与数剂量的肼苯达嗪联合服用。四小时后，处死小鼠以测定血浆中的肝酶作为肝损伤的标志物，同时评估肝脏中的谷胱甘肽和肼苯达嗪稳定的蛋白质加合物。肼屈嗪对血浆标记酶的增加提供了强大的、剂量依赖性的保护，但对烯丙醇产生的肝脏谷胱甘肽的消耗没有作用。蛋白质印迹显示肼苯哒嗪在多种肝脏蛋白质中在 26 至 200 kDA 质量范围内产生强烈的剂量依赖性加合物捕获。与这些发现一致，肝切片的免疫组织化学分析表明，肼苯哒嗪捕获了弥漫性核外加合物，均匀分布在肝小叶上，部分定位于实质细胞膜中。这些发现与我们的假设一致，即肼屈嗪很容易使丙烯醛形成的反应性羰基保留蛋白加合物失活，从而防止引发细胞死亡的次级反应。
Acrolein is a highly reactive alpha,beta-unsaturated aldehyde that readily alkylates nucleophilic centers in cell macromolecules. Typically, such reactions proceed via Michael addition chemistry, forming adducts that retain an electrophilic carbonyl group. Since these species participate in secondary deleterious reactions, we hypothesize that inactivation of carbonyl adducts may attenuate acrolein toxicity. Indeed, we recently established that the nucleophilic antihypertensive drug hydralazine readily "traps" acrolein adducts in cell proteins and strongly suppresses acrolein-mediated toxicity in isolated hepatocytes. This work sought to determine whether hydralazine prevents the in vivo hepatotoxicity of the acrolein precursor allyl alcohol in whole mice and whether adduct trapping accompanies any such hepatoprotection. Mice received allyl alcohol alone or in conjunction with several doses of hydralazine. Four hours later, mice were sacrificed to allow for the determination of liver enzymes in plasma as markers of hepatic injury, whereas livers were assessed for glutathione and hydralazine-stabilized protein adducts. Hydralazine afforded strong, dose-dependent protection against the increases in plasma marker enzymes but not the hepatic glutathione depletion produced by allyl alcohol. Western blotting revealed intense, dose-dependent adduct trapping by hydralazine in numerous liver proteins over a broad 26- to 200-kDA mass range. In keeping with these findings, immunohistochemical analysis of liver slices indicated diffuse, extranuclear adduct trapping by hydralazine that was uniformly distributed across the liver lobule, with partial localization in parenchymal cell membranes. These findings concur with our hypothesis that hydralazine readily inactivates reactive carbonyl-retaining protein adducts formed by acrolein, thereby preventing secondary reactions that trigger cellular death.