Zidovudine phosphorylation and mitochondrial toxicity in vitro

Zidovudine (ZDV) is a thymidine analogue activated to its triphosphate (ZDV TP) by the host's intracellular enzymes. The initial phosphorylation step i s conversion to ZDV monophosphate (ZDVMP). The poor affinity of ZDVMP for t hymidylate kinase results in intracellular accumulation of ZDVMP. Clinical use of ZDV is associated with cytotoxicity, thought to be mediated through mitochondria) damage. It has been suggested that ZDV cytotoxicity correlate s with intracellular ZDVMP. Here we have further studied the role of ZDVMP in cytotoxicity and some of the mechanisms involved. Intracellular metaboli sm 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 wit h [H-3]ZDV (1.2 mu Ci; 0.1 muM) 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. ZDVM P was the only metabolite detected following incubation of viable mitochond ria isolated from U937 cells with ZDV (1.2 mu Ci; 0.1 muM; 24 h) with mitoc hondrial levels of 0.27 +/- 0.11 pmol/mug protein (mean +/- SD; n = 3). No MTT toxicity was seen in isolated mitochondria. Following phytohemagglutini n (PHA) stimulation of peripheral blood mononuclear cells there was an incr ease in ZDV cytotoxicity compared to unstimulated cells. The results sugges t that the mitochondrial isozyme of thymidine kinase (TK2) plays only a min or part in ZDVMP formation. Following PHA stimulation, activation of the cy tosolic thymidine kinase isozyme (TK1) is associated with increased toxicit y of ZDV. We conclude that ZDVMP responsible for mitochondrial toxicity is formed in the cytosol. (C) 2001 Academic Press.