L'Ecuyer Thomas, Sanjeev Sanjeev, Thomas Ronald, Novak Raymond, Das Lauri, Campbell Wendy, Heide Richard Vander
Department of Pediatrics, Institute of Environmental Health Sciences, Detroit, MI 48201, USA.
Am J Physiol Heart Circ Physiol. 2006 Sep;291(3):H1273-80. doi: 10.1152/ajpheart.00738.2005. Epub 2006 Mar 24.
Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.
蒽环类药物是抗肿瘤药物，其主要临床局限性是心脏毒性。这种心脏毒性的机制被认为与氧化应激的产生有关，氧化应激会对心肌细胞造成致命损伤。虽然在接受蒽环类药物治疗的心肌细胞中已记录到蛋白质和脂质氧化，但DNA损伤尚未得到直接证实。本研究旨在确定蒽环类药物是否会诱导心肌细胞DNA损伤，以及这种损伤是否与导致细胞死亡的信号通路相关。用临床相关浓度的蒽环类药物阿霉素处理H9c2心肌细胞，并使用碱性彗星试验评估DNA损伤。阿霉素诱导了DNA损伤，表现为平均尾矩比对照组显著增加，加入自由基清除剂可改善这种效应。与在去除过氧化氢后观察到的H2O2处理的心肌细胞中DNA损伤完全修复不同，阿霉素处理后DNA损伤的修复并不完全。免疫印迹分析显示，p53激活在时间上发生在DNA损伤之后。通过荧光测定，阿霉素在p53激活后诱导线粒体膜电位丧失。对p53的化学抑制可防止阿霉素诱导的细胞死亡和线粒体膜电位丧失，但不能防止DNA损伤，这表明DNA损伤在从阿霉素治疗到心肌细胞死亡的事件中处于近端位置。阿霉素特异性诱导的DNA损伤包括氧化嘧啶和8-羟基鸟嘌呤。因此，DNA损伤似乎在蒽环类药物诱导的致命性心肌细胞损伤中通过涉及p53和线粒体的途径发挥重要的早期作用。
