Cellular and mitochondrial toxicity of zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC) on cultured human muscle cells

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Abstract

Zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC) are the reference antiretroviral therapy in patients with AIDS. A toxic mitochondrial myopathy can be observed in patients treated with AZT, but not with ddI and ddC. All 3 compounds can inhibit mitochondrial (mt)DNA polymerase and cause termination of synthesis of growing mtDNA strands and mtDNA depletion. The propensity to injure particular target tissues is unexplained. In our work, cultured muscle cells prepared from human muscle biopsies, were exposed to various concentrations of AZT (4–5000 μmol/l), ddI (5–1000 μmol/l) and ddC (1–1000 μmol/l) for 10 days. We evaluated cell proliferation and differentiation and measured lipid droplet accumulation, lactate production and respiratory chain enzyme activities. All 3 compounds induced a dose-related decrease of cell proliferation and differentiation. AZT seemed to be the most potent inhibitor of cell proliferation. AZT, ddI and ddC induced cytoplasmic lipid droplet accumulations, increased lactate production and decreased activities of COX (complex IV) and SDH (part of complex II). NADHR (complex I) and citrate sinthase activities were unchanged. Zalcitabine (ddC) and, to a lesser extent, ddI, were the most potent inhibitors of mitochondrial function. In conclusion, AZT, ddI and ddC all exert cytotoxic effects on human muscle cells and induce functional alterations of mitochondria possibly due to mechanisms other than the sole mtDNA depletion. Our results provide only a partial explanation of the fact that AZT, but not ddI and ddC, can induce a myopathy in HIV-infected patients. AZT myopathy might not simply result from a direct mitochondrial toxic effect of crude AZT.

Introduction

Nucleoside analogs, such as zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC), are the reference antiretroviral drugs in patients with AIDS. These analogs cannot form 3′ phosphodiester linkages, and thus terminate DNA elongation when incorporated into a growing DNA strand (Lewis et al., 1992). They inhibit the reverse transcriptase and the mitochondrial DNA polymerase (gamma polymerase) responsible for mitochondrial (mt)DNA replication (Yarchoan et al., 1989).

Long-term administration of these drugs can induce reversible neuromuscular disorders. AZT can induce a mitochondrial myopathy with ragged-red fibers (Dalakas et al., 1990, Mhiri et al., 1991, Grau et al., 1993), partial cytochrome c oxidase (COX) deficiency (9Chariot and Gherardi, 1991, Chariot et al., 1993) and depletion of mtDNA (Arnaudo et al., 1991) in muscle and increased blood lactate/pyruvate ratios (Chariot et al., 1994a). Both ddI and ddC induce painful distal axonal polyneuropathies (Yarchoan et al., 1990, Berger et al., 1993), also possibly related to mitochondrial dysfunction (Chen et al., 1991, Feldman and Anderson, 1994, Martin et al., 1994).

Several points remain unclear about mitochondrial toxicity of nucleoside analogs. (1) Animals receiving AZT showed mild structural and functional mitochondrial abnormalities (Lamperth et al., 1991, Lewis et al., 1991) but ragged-red fibers were not documented, raising questions about the ability of AZT to induce mitochondrial myopathy in the absence of concurrent HIV infection (Reyes et al., 1992, Engbretson, 1996). (2) Studies of mitochondrial function of human muscle cells exposed to AZT have yielded mixed results: mitochondrial dysfunction was reported to be detectable when cells were exposed to concentrations similar to those observed in serum of AZT receivers (Lamperth et al., 1991). Another group failed to demonstrate any dysfunction until exposure to a concentration 1000 times higher (Herzberg et al., 1992). (3) The propensity to injure particular target tissues, namely muscle in the case of AZT and peripheral nerve in the case of ddI and ddC, is unexplained.

In the present study, we intended to compare the in vitro myotoxicity of the three main nucleoside analogs. Would in vitro myotoxicity parallel clinical myotoxicity? Our general objective was to determine if assessment of toxicity on cultured human muscle cells might be used as a predictive test for clinical myotoxicity in early phases of research and development in antiretroviral therapy. We compared the effects of AZT, ddI and ddC on proliferation, differentiation, lipid accumulation, lactate production and mitochondrial enzyme activities in cultured human muscle cells.

Section snippets

Muscle cultures

Human muscle biopsies were obtained from healthy patients undergoing orthopedic surgery. Primary cultures were prepared as described (Barlovatz-Meimon et al., 1994). Culture medium was changed every fifth culture day.

Zidovudine (AZT, Burroughs Wellcome, Research Triangle, North Carolina: 4–5000 μmol/l), ddI (Bristol-Myers, Wallingford, CT: 5–1000 μmol/l) and ddC (Hoffman La Roche, Basel, Switzerland: 1–1000 μmol/l) were added to culture medium every fifth day from the fourth day after seeding.

Effect of AZT, ddI and ddC on cell proliferation and differentiation

Cultures exposed to AZT, ddI and ddC showed a remarkable decrease in cell proliferation assessed by counting viable cells using trypan blue (Fig. 1). The decrease of proliferation was observed after 10 day exposures to AZT, ddI or ddC, at concentrations ≥20, 100 and 100 μmol/l, respectively. The concentrations of AZT, ddI and ddC required to inhibit cell proliferation by 50% (IC50) were approximately 200, 1000 and 100 μmol/l after 6 days of incubation and 100, 500 and 20 μmol/l after 10 days.

Discussion

In the present study, human muscle cells exposed to AZT, ddI and ddC showed decreased proliferation and differentiation, marked lipid droplet accumulation and increased lactate production. COX (complex IV) and SDH (part of complex II) activities decreased while NADH reductase (complex I) was unchanged. Concentrations of nucleoside analogs necessary to induce a decrease of COX activity after a 10 day exposure were lower for ddI and ddC than for AZT.

Human plasma peak concentrations in patients

Acknowledgements

This work was supported in part by grants from SIDACTION (Paris, France) to Dr. Chariot and from the Agence Nationale de Recherches sur le SIDA (Paris, France) to Dr. Gherardi, and by a fellowship to E. Benbrik from the Association Française contre les Myopathies (Evry, France). We thank Professor Bombart and his group from the Department of Surgery, Centre Hospitalier Intercommunal de Villeneuve-Saint-Georges for their help in obtaining muscle samples.

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