Szabados E, Fischer G M, Toth K, Csete B, Nemeti B, Trombitas K, Habon T, Endrei D, Sumegi B
Department of Biochemistry, University Medical School Pecs, Hungary.
Free Radic Biol Med. 1999 Feb;26(3-4):309-17. doi: 10.1016/s0891-5849(98)00199-3.
The short term cardiac side-effects of AZT (3'-azido-3'-deoxythymidine, zidovudine) was studied in rats to understand the biochemical events contributing to the development of AZT-induced cardiomyopathy. Developing rats were treated with AZT (50 mg/kg/day) for 2 wk and the structural and functional changes were monitored in the cardiac muscle. AZT treatment provoked a surprisingly fast appearance of cardiac malfunctions in developing animals characterized by prolonged RR, PR and QT intervals and J point depression. Electron microscopy showed abnormal mitochondrial structure but the cardiomyocyte had normal myofibers. The AZT treatment of rats significantly increased ROS and peroxynitrite formation in heart tissues as determined by the oxidation of nonfluorescent dihydrorhodamine123 and dichlorodihydro-fluorescein diacetate (H2DCFDA) to fluorescent dyes, and induced single-strand DNA breaks. Lipid peroxidation and oxidation of cellular proteins determined from protein carbonyl content were increased as a consequence of AZT treatment. Activation of the nuclear poly-ADP-ribose polymerase and the accelerated NAD+ catabolism were also observed in AZT-treated animals. Western blot analysis showed that mono-ADP-ribosylation of glucose regulated protein (GRP78/BIP) was enhanced by AZT treatment, that process inactivates GRP78. In this way moderate decrease in the activity of respiratory complexes was detected in the heart of AZT-treated animals indicating a damaged mitochondrial energy production. There was a significant decrease in creatine phosphate concentration resulting in a decrease in creatine phosphate/creatine ratio from 2.08 to 0.58. ATP level remained close to normal but the total extractable ADP increased with 45%. The calculated free ATP/ADP ratio decreased from 340 to 94 in the heart of AZT-treated rats as a consequence of increased free ADP concentration. It was assumed that the increased free ADP in AZT-treated cardiomyocyte may help cells to compensate the defective ATP production in damaged mitochondria by activating the ATP synthesis in undamaged mitochondria. Southern blot analysis did not show decreased quantity of mtDNA deriving from AZT-treated rat hearts indicating that under our experimental conditions AZT-induced heart abnormalities are not the direct consequence of the mtDNA depletion. These data show that ROS-mediated oxidative damages, activated ADP-ribosylation reactions and accelerated NAD+ catabolism play basic roles in the development of AZT-induced cardiomyopathy in our animal model and indicated that these ROS-mediated processes can be important factors in the development of myopathy and cardiomyopathy in zidovudine-treated AIDS patients.
为了解导致齐多夫定（AZT，3'-叠氮-3'-脱氧胸苷）诱发心肌病的生化事件，研究了AZT对大鼠的短期心脏副作用。给发育中的大鼠每日注射AZT（50mg/kg），持续2周，并监测心肌的结构和功能变化。AZT处理使发育中的动物心脏功能异常迅速出现，表现为RR、PR和QT间期延长以及J点压低。电子显微镜显示线粒体结构异常，但心肌细胞肌纤维正常。通过非荧光二氢罗丹明123和二氯二氢荧光素二乙酸酯（H2DCFDA）氧化为荧光染料测定，AZT处理大鼠心脏组织中活性氧（ROS）和过氧亚硝酸盐生成显著增加，并诱导单链DNA断裂。AZT处理导致脂质过氧化和基于蛋白质羰基含量测定的细胞蛋白氧化增加。在AZT处理的动物中还观察到核多聚ADP核糖聚合酶激活和NAD+分解代谢加速。蛋白质印迹分析表明，AZT处理增强了葡萄糖调节蛋白（GRP78/BIP）的单ADP核糖基化，该过程使GRP78失活。通过这种方式，在AZT处理动物的心脏中检测到呼吸复合体活性适度降低，表明线粒体能量产生受损。磷酸肌酸浓度显著降低，导致磷酸肌酸/肌酸比值从2.08降至0.58。ATP水平保持接近正常，但总可提取ADP增加了45%。由于游离ADP浓度增加，AZT处理大鼠心脏中计算出的游离ATP/ADP比值从340降至94。推测AZT处理的心肌细胞中游离ADP增加可能通过激活未受损线粒体中的ATP合成，帮助细胞补偿受损线粒体中ATP产生的缺陷。Southern印迹分析未显示AZT处理大鼠心脏中mtDNA数量减少，表明在我们的实验条件下，AZT诱导的心脏异常不是mtDNA耗竭的直接后果。这些数据表明，ROS介导的氧化损伤、激活的ADP核糖基化反应和加速的NAD+分解代谢在我们的动物模型中AZT诱导的心肌病发展中起基本作用，并表明这些ROS介导的过程可能是齐多夫定治疗的艾滋病患者肌病和心肌病发展的重要因素。
