Zidovudine phosphorylation and mitochondrial toxicity in vitro.
Zidovudine (ZDV) is a thymidine analogue activated to its triphosphate (ZDVTP) by the host's intracellular enzymes. The initial phosphorylation step is conversion to ZDV monophosphate (ZDVMP). The poor affinity of ZDVMP for thymidylate kinase results in intracellular accumulation of ZDVMP. Clinical use of ZDV is associated with cytotoxicity, thought to be mediated through mitochondrial damage. It has been suggested that ZDV cytotoxicity correlates with intracellular ZDVMP. Here we have further studied the role of ZDVMP in cytotoxicity and some of the mechanisms involved. Intracellular metabolism of ZDV in five lymphocyte/monocyte cell lines, U937, BSM, MOLT 4, JJAHN, and RAJI (4 x 10(6) cells), was investigated following 24 h incubation with [(3)H]ZDV (1.2 microCi; 0.1 microM) and cytotoxicity was determined by the MTT assay. Cytotoxicity was closely related to intracellular concentrations of the major metabolite (ZDVMP) but not with the active metabolite ZDVTP. ZDVMP was the only metabolite detected following incubation of viable mitochondria isolated from U937 cells with ZDV (1.2 microCi; 0.1 microM; 24 h) with mitochondrial levels of 0.27 +/- 0.11 pmol/microg protein (mean +/- SD; n = 3). No MTT toxicity was seen in isolated mitochondria. Following phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells there was an increase in ZDV cytotoxicity compared to unstimulated cells. The results suggest that the mitochondrial isozyme of thymidine kinase (TK2) plays only a minor part in ZDVMP formation. Following PHA stimulation, activation of the cytosolic thymidine kinase isozyme (TK1) is associated with increased toxicity of ZDV. We conclude that ZDVMP responsible for mitochondrial toxicity is formed in the cytosol.
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Department of Pharmacology and Therapeutics, University of Liverpool, New Medical Building, Liverpool, United Kingdom L69 3GE.
Hoggard
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Sunderland
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Khoo
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Hart
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D004305:Dose-Response Relationship, Drug; D006801:Humans; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D010835:Phytohemagglutinins / Q000494:pharmacology; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism; D020298:U937 Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015215:Zidovudine / Q000378:metabolism / Q000633:toxicity*
D010835:Phytohemagglutinins; D015215:Zidovudine; D013385:Succinate Dehydrogenase; D013937:Thymidine Kinase