Original ContributionsRole of reactive oxygen species and poly-ADP-ribose polymerase in the development of AZT-induced cardiomyopathy in rat
Introduction
AZT (zidovudine) is widely used for the treatment of AIDS patients as the first drug that inhibits the replication 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 inhibits the HIV reverse transcriptase [2]. Additionally, AZT-triphosphate inhibits the mitochondrial DNA polymerase (polymerase gamma) [5]. The AZT metabolites can incorporate into the host DNA [6] inducing premature chain termination in the cell own DNA, especially in the mitochondrial DNA (mtDNA) lacking of DNA repair processes [7]. As a result, significant reduction of mtDNA content was seen in the skeletal muscle of long-term zidovudine treated HIV patients [8] associated with mitochondrial myopathy, leading to the assumption that the depletion of mitochondrial DNA(mtDNA) can be the primary cause of the myopathy [9], [10]. The mtDNA encodes 13 polypeptide that are components of the respiratory complexes [11]. Therefore, any damage of mtDNA will cause defective mitochondrial protein synthesis that results in abnormal respiratory complexes and impaired oxidative energy production.
There are additional data suggesting that AZT (or its metabolites) has an inhibitory effect on the mitochondrial oxidative energy production that can not be associated with depletion of mtDNA [12]. The inhibitory effect of AZT or its metabolites was localized on NADH:citochrome C oxidoreductase although succinate:citochrome C oxidoreductase was not effected [13]. Furthermore, there are data suggesting that the primary intracellular metabolite of AZT (AZT-monophosphate) inhibits glycosphingolipid and ganglioside biosynthesis, and suppresses the incorporation of both sialic acid and galactose into proteins [14]. These new data indicate that the molecular mechanism of AZT induced cell damage is more complex than expected.
We observed that AZT treatment significantly increased the production of reactive oxygen intermediates (ROS) in rat hearts raising the possibility that ROS mediated processes may play an important role in the cardiotoxicity of AZT. Therefore, we investigated whether AZT treatment induce in vivo lipid peroxidation, protein oxidation and the formation of DNA breaks that are the major changes occur at elevated ROS levels. Because DNA breaks activate the nuclear poly-ADP-ribose polymerase (PARP) an enzyme that play an important role in the NAD+ catabolism and ROS induced cell death [15], [16], the effect of AZT treatment on the endogenous poly- and mono-ADP-ribosylation reaction were also investigated. These data may provide new evidence for the role of ROS mediated processes including endogenous ADP-ribosylation reactions in the development of the cardiotoxicity of AZT.
Section snippets
Materials
AZT, cytochrome c, CoA-SH, NAD, NADH, were obtained from Sigma. EcoR I was purchased from Amersham. Sst I was purchased from BRL. Anti-ADP-ribose antibody was a kind gift from Alexander Buerkle (Heidelberg, Germany) and Masanao Miwa (Tsukuba, Japan). All other chemicals were of the highest purity commercially available.
General methods
Approximately 80-100 g rats were intraperitoneally treated daily with AZT (50 mg/kg) for up to 14 days. Schiller AT-6 ECG was used to monitor cardiac function during and after
ECG studies
Developing (80–100 g) rats were treated daily with 50 mg/kg AZT (that is respected as a high human dose) up to 2 wk. The rat heart function was monitored by ECG, and after 1 wk of treatment we could already detect the development of abnormal heart function. The RR, PR and QT intervals prolonged significantly after 1 wk of treatment and the abnormalities further developed by the end of the second week (Table 1). In leads I and aVL that represent the main muscle mass of the left ventricle,
Discussion
Previous studies indicated that the long-term (several months) AZT treatment inhibits the replication of mtDNA and causes mtDNA depletion [9], [10] that was considered as the primary cause of AZT-induced myopathy. However, model studies indicated that a significant reduction of mtDNA level requires several months of AZT treatment. This work showed that a relatively short-term (2 wk) of AZT treatment that did not decrease the quantity of mtDNA (Fig. 3) caused significant functional damages as
Acknowledgements
Acknowledgements — The authors are grateful to Laszlo Giran and Bertalan Horvath for their technical assistance. Support for this research was given by OTKA T5057, T006360, T006018, T2010 from the Hungarian Science foundation and by the Ministry of Public Health, grant number: T06629/93.
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