Abstract3′-Azido-3′-deoxythymidine (AZT) is a staple of highly active antiretroviral therapy (HAART). Prior to HAART, long-term use of high-dosage AZT caused myopathy, cardiomyopathy, and hepatotoxicity, associated with mitochondrial DNA depletion. As a component of HARRT, AZT causes cytopenias and lipodystrophy. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart mitochondria, AZT phosphorylation beyond AZT-5′-monophosphate (AZTMP) was not detected. Rather, AZT was shown to be a more potent inhibitor of thymidine phosphorylation (50% inhibitory concentration (IC 50 ) of 7.0 ± 1.0 μM) than AZTTP is of polymerase γ (IC 50 of >100 μM), suggesting that depletion of mitochondrial stores of TTP may limit replication. This work has investigated whether an identical mechanism might account for the hepatotoxicity seen with long-term use of AZT. Isolated rat liver mitochondria were incubated with labeled thymidine or AZT, and the rate and extent of phosphorylation were determined by HPLC analysis of acid-soluble extracts of the incubated mitochondria. The results showed that in the phosphorylation of thymidine to TMP, liver mitochondria exhibit a higher V max and K m than heart mitochondria, but otherwise heart and liver mitochondria display similar kinetics. AZT is phosphorylated to AZTMP, but no further phosphorylated forms were detected. In addition, AZT inhibited the production of TTP, with an IC 50 of 14.4 ± 2.6 μM AZT. This is higher, but comparable to, the results seen in isolated rat heart mitochondria. KeywordsAZT; Liver mitochondria; Thymidine; Mitochondrial toxicity; Thymidine kinase 2; Reverse transcriptase inhibitors Abbreviations AIDS, acquired immunodeficiency syndrome; AZT, 3′-azido-3′-deoxythymidine; AZTDP, 3′-azido-3′-deoxythymidine-5′-diphosphate; AZTMP, 3′-azido-3′-deoxythymidine-5′-monophosphate; AZTTP, 3′-azido-3′-deoxythymidine-5′-triphosphate; HAART, highly active antiretroviral therapy; HIV, human immunodeficiency virus; IC 50 , 50% inhibitory concentration; S.E.M., standard error of mean

Long-term use of 3'-azido-3'-deoxythymidine (AZT) is associated with various tissue toxicities, including hepatotoxicity and cardiomyopathy, and with mitochondrial DNA depletion. AZT-5'-triphosphate (AZTTP) is a known inhibitor of the mitochondrial DNA polymerase gamma and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart and liver mitochondria, AZT itself was shown to be a more potent inhibitor of thymidine phosphorylation (IC50 of 7.0+/-1.0 microM AZT in heart mitochondria and of 14.4+/-2.6 microM AZT in liver mitochondria) than AZTTP is of polymerase gamma (IC50 of >100 microM AZTTP), suggesting that depletion of mitochondrial stores of TTP may limit replication and could be the cause of the mitochondrial DNA depletion observed in tissues affected by AZT toxicity. The purpose of this work is to characterize the nature of AZT inhibition of thymidine phosphorylation in isolated rat heart and rat liver mitochondria. In both of these tissues, AZT was found to be a competitive inhibitor of the phosphorylation of thymidine to TMP, catalyzed by thymidine kinase 2. The inhibition constant (Ki) for heart mitochondria is 10.6+/-4.5 microM AZT, and for liver mitochondria Ki is 14.0+/-2.5 microM AZT. Since AZT is functioning as a competitive inhibitor, increasing thymidine concentrations may be one mechanism to overcome the inhibition and decrease AZT-related toxicity in these tissues.

Zidovudine (AZT; 3-azido-3-deoxythymidine), a thymidine analog, has been a staple of highly active antiretroviral therapy. It is phosphorylated in the host to the triphosphate and functions by inhibiting the viral reverse transcriptase. However, long-term use of AZT is linked to various tissue toxicities, including cardiomyopathy. These toxicities are associated with mitochondrial DNA depletion, which is hypothesized to be caused by AZT triphosphate inhibition of mitochondrial DNA polymerase ␥. In previous work with isolated heart mitochondria, we demonstrated that AZT phosphorylation beyond the monophosphate was not detected and that AZT itself was a potent inhibitor of thymidine phosphorylation. This suggests an alternative hypothesis in which depletion of the TTP pool may limit mitochondrial DNA replication. The present work extends these studies to the whole cell by investigating the metabolism of thymidine and AZT in the intact isolated perfused rat heart. Zidovudine (AZT; 3Ј-azido-3Ј-deoxythymidine), a thymidine analog, is a member of the class of compounds called nucleoside analog reverse transcriptase inhibitors (NRTIs). Members of this class of drugs are phosphorylated by host cell enzymes to their triphosphates, which inhibit the human immunodeficiency virus (HIV) reverse transcriptase and/or act as chain terminators when they are incorporated into viral DNA, since these analogs cannot form 3Ј-phosphodiester bonds. AZT was the first drug approved by the Food and Drug Administration for the treatment of AIDS. In early monotherapy regimens, AZT was given alone at a dose of 1,200 mg per day (3). After some months of therapy, AZT was associated with a variety of common tissue toxicities that included myopathy, dilated cardiomyopathy, hepatotoxicity, various cytopenias, and lactic acidosis that were serious enough to result in the discontinuation of therapy in about 25 to 30% of patients (1, 5, 7, 9-11, 27, 29, 34, 37). In affected tissues, the mitochondria were abnormal, with reduced cristae and mitochondrial DNA (mtDNA) depletion (18,19,22). In present AIDS therapy, referred to as highly active antiretroviral therapy (HAART), AZT remains in common use but is given at a lower dose (600 mg per day) and in combination with a second NRTI and a viral protease inhibitor. As a result, the cardiovascular toxicity of the AZT used in HAART is now a rare event, and the main toxicities include lipodystrophy and cytopenias (2,12,28). AZT monotherapy is currently used to prevent the transmission of HIV from mothers to their babies prior to delivery and during the baby's first postnatal month (33). AZT is also a mainstay of HIV treatment in many resource-limited settings around the world (36). Understanding the mechanism of toxicity of AZT is of critical importance both in designing therapy to limit toxicity and in the rational design of less toxic therapeutic compounds.The phosphorylation of AZT is mediated by cellular enzymes of the thymidine salvage pathway, which convert thymidine to TTP. This pathway has two route...