Original Contribution ROLE OF REACTIVE OXYGEN SPECIES AND POLY-ADP-RIBOSE POLYMERASE IN THE DEVELOPMENT OF AZT-INDUCED CARDIOMYOPATHY IN RAT ESZTER SZABADOS,* GABOR M. FISCHER,* KALMAN TOTH, ² BELA CSETE,* BALAZS NEMETI,* KAROLY TROMBITAS, ‡ TAMAS HABON, ² DORA ENDREI, ² and BALAZS SUMEGI* *Department of Biochemistry, ² 1st Department of Medicine, Division of Cardiology, and ‡ Central Laboratory, University Medical School Pecs, 7624-Pecs, Hungary (Received 10 July 1998; Accepted 13 July 1998) Abstract—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. © 1998 Elsevier Science Inc. Keywords—Cardiomyopathy, Myopathy, Mitochondria, HIV-AIDS, AZT, mtDNA, Respiratory defect, Energy pro- duction INTRODUCTION AZT (zidovudine) is widely used for the treatment of AIDS patients as the first drug that inhibits the replica- tion of HIV virus at the point of reverse transcription [1,2]. It has also been well documented that AZT has numerous side effects [3,4]. In mammalian cells AZT is phosphorylated giving AZT-triphosphate that directly in- hibits the HIV reverse transcriptase [2]. Additionally, AZT-triphosphate inhibits the mitochondrial DNA poly- merase (polymerase gamma) [5]. The AZT metabolites Address correspondence to: B. Sumegi, University Medical School Pecs, Department of Biochemistry, Szigeti ut 12, 7624-Pecs, Hungary; Tel/Fax: 36-72-326-466; E-Mail: SUMEGIB@APACS.POTE.HU. Free Radical Biology & Medicine, Vol. 26, Nos. 3/4, pp. 309 –317, 1999 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter PII S0891-5849(98)00199-3 309