Elsevier

Biochemical Pharmacology

Volume 64, Issue 2, 15 July 2002, Pages 201-206
Biochemical Pharmacology

Inhibition of phosphate transport in rat heart mitochondria by 3′-azido-3′-deoxythymidine due to stimulation of superoxide anion mitochondrial production

https://doi.org/10.1016/S0006-2952(02)01065-1Get rights and content

Abstract

In order to gain some insight into the mechanism by which 3′-azido-3′-deoxythymidine (AZT) damages mitochondria, we investigated whether externally added AZT can stimulate reactive oxygen species (ROS) production by rat heart mitochondria (RHM). An increase in superoxide anion (O2radical dot) production was measured in RHM added with AZT, by using a photometrically method which allows an early O2radical dot detection by following the absorbance increase at 550 nm due to the ferricytochrome c reduction. Such an increase was found to be prevented from externally added superoxide dismutase. The stimulation of O2radical dot mitochondrial production induced by AZT was found to occur under conditions in which mitochondrial oxygen consumption was prevented by both inhibitors of electron flow and ATP synthesis. Since ROS can cause mitochondrial carrier impairment, we investigated whether AZT can affect mitochondrial permeability in virtue of its capability to stimulate ROS production. In this regard, we studied the transport of phosphate (Pi), by measuring the mitochondrial shrinkage that takes place as a result of Pi uptake by RHM previously swollen in a calcium acetate medium. As a result of the AZT-dependent O2radical dot production, uncompetitive inhibition of the rate of Pi transport in RHM was found (Ki of about 10 μM), consistently, such an inhibition was found to prevent by certain known ROS scavengers, i.e. superoxide dismutase, the antioxidant Vitamin C and reduced gluthatione.

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-O2radical dot 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 O2radical dot produced by RHM

In order to measure O2radical dot production in RHM, caused by AZT addition, the Fe3+-cytc

Externally added AZT can stimulate O2radical dot 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 O2radical dot detection by following the Fe3+-cytc reduction [21], (see Section 2). In the absence of AZT, O2radical dot 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 O2radical dot production was found up

Discussion

We show that O2radical dot 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.

References (29)

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