Inhibition of phosphate transport in rat heart mitochondria by 3′-azido-3′-deoxythymidine due to stimulation of superoxide anion mitochondrial production
Introduction
AZT is one of the mostly used drugs, either alone or in combination with other nucleoside or protease inhibitors, in AIDS therapy [1], [2]. Unfortunately, the clinical effectiveness of AZT is limited by its toxic side effects [3].
Mitochondria are cell targets of AZT: long-term as well as short-term treatment with AZT caused many dramatic structural and functional changes of mitochondria [4]. We have shown that adenylate kinase [5], ADP/ATP carrier [6], and nucleoside diphosphate kinase [7] are AZT targets in isolated RLM. On the other hand in rat liver mitochondria, the AZT capability to uncouple mitochondria was ruled out [8]. Finally, the existence of tissue-specific AZT effects have been also reported, with mitochondria isolated by heart and skeletal muscle more impaired than those from other tissues by AZT [9]; moreover, alterations of membrane potential caused by AZT in rat myotubes related to changes on the structural organization of the inner mitochondrial membrane has been recently shown [10].
Specific treatments with certain drugs used in different therapies were found to increase the cellular production of ROS, (see for instance [11]), particularly, ROS have been indicated as important factors in the development of myopathy and cardiomyopathy in AZT-treated rats [12]; consistently, AZT toxicity was prevented by supranutritional doses of antioxidant vitamins [13].
Mitochondria are the main cell sources of ROS [14], [15]; as well as they can be targets of ROS generated in several cellular processes as the biochemical events leading to cell death [16], [17].
In the light of this, we investigated whether externally added AZT can cause ROS production in isolated RHM. Moreover, since ROS can cause mitochondrial carrier impairment [18], a first investigation was also carried out to determine whether AZT-dependent ROS production can impair the mitochondrial permeability.
We show that the exposure of RHM to AZT causes extra-O2− mitochondrial formation that can impair the Pi transport in RHM.
Section snippets
Chemicals
All chemicals were from Sigma Chemicals Co. Mitochondrial substrates were used as Tris salts at pH 7.0–7.3.
Isolation of mitochondria
RHM were isolated essentially as described in [19] from male Wistar rats, (200–250 g) fed ad libitum and suspended in the standard medium containing 70 mM sucrose, 220 mM mannitol, 5 mM Hepes–Tris pH 7.25 and 0.1 mM EDTA in the presence of BSA (0.5 mg/mL). Mitochondrial protein was determined according to [20].
Detection of O2− produced by RHM
In order to measure O2− production in RHM, caused by AZT addition, the Fe3+-cytc
Externally added AZT can stimulate O2− production by RHM
In order to ascertain whether AZT can affect ROS production when added to isolated RHM, an experimental procedure was used which allows early O2− detection by following the Fe3+-cytc reduction [21], (see Section 2). In the absence of AZT, O2− amount in RHM was 0.3±0.02 nmol/mg protein, as measured in three different experiments. As a result of AZT addition (100 μM), that per se does not produce ROS when added to the medium in the absence of mitochondria, an increase in O2− production was found up
Discussion
We show that O2− production in RHM increases as a result of AZT addition. Since AZT cannot produce ROS, both per se and when added to respiring mitochondria (Table 1), we conclude that ROS are generated by RHM and that AZT-dependent increase of ROS generation is due to AZT interaction with certain mitochondria component/s, perhaps the highly reactive electron carriers of the respiratory chain, including flavins, non-heme iron proteins, quinols and semiquinones, that can produce ROS [14], [15].
Acknowledgements
The skilful technical assistance of Mr. Vito Giannoccaro and the linguistic consultancy by Dr. Mina Cezza are gratefully acknowledged. This work was partially financed by PRIN Bioenergetics and Transport of Membrane and by POP Molise “Studio del meccanismo di azione degli antiossidanti” to S.P.
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