|
6th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV25–28 October 2004 - Washington, DC, USA |
ZIDOVUDINE INHIBITS THYMIDINE PHOSPHORYLATION: A NOVEL SITE OF POTENTIAL TOXICITY IN NON-MITOTIC CELLS
Antiviral Therapy 2004; 9(6):L9 (abstract no. 13)
EE McKee1,2, MD Lynx1,2, D Susan-Resiga2, AT Bentley2, JP D'Haenens1,2, D Cullen2 and M Ferguson2
1 Indiana University School of Medicine South Bend Center for Medical Education, Notre Dame, Ind., USA; and 2 University of Notre Dame, Notre Dame, Ind., USA
Zidovudine (3´-azido-3´deoxythymidine, AZT) is a thymidine analogue pro-drug that can be phosphorylated by host cell enzymes to the triphosphate, which is a potent inhibitor of the viral reverse transcriptase. AZT has been a staple of highly active antiretroviral therapy (HAART) for many years. In the early days of AIDS therapy, AZT was given alone at relatively high concentrations and its long-term use was associated with various tissue toxicities, including hepatotoxicity and cardiomyopathy, associated with mitochondrial DNA depletion. In more recent therapy, AZT in combination with other nucleoside analogues is associated with a lipodystrophy that may be of mitochondrial origin. The triphosphate form of AZT (AZT-TP) is a known inhibitor of the mitochondrial polymerase γ and has been targeted as the potential source of the mitochondrial DNA depletion. This laboratory’s previous work with isolated rat heart mitochondria in shortterm experiments suggested that AZT was not phosphorylated beyond the monophosphate (AZT-MP). AZT-MP accumulated in the matrix and AZT-DP and AZT-TP were not detected. Instead, we found that AZT was a potent inhibitor of thymidine phosphorylation (7.0 ±1.0 μM) acting at the level of the mitochondrial thymidine kinase (TK2). In non-mitotic tissues such as cardiomyocytes, cytoplasmic thymidine kinase (TK1) is not expressed and the cell’s supply of TTP is generated solely through TK2. We therefore suggest the novel hypothesis that toxicity of AZT in non-mitotic tissue is caused by the direct inhibition of TK2 limiting the cellular pool of TTP. The low level of TTP limits mitochondrial DNA replication, ultimately leading to mitochondrial dysfunction associated with mitochondrial DNA depletion. This hypothesis was tested in this work using two approaches. In one, an isolated perfused heart system was used to extend our investigation of AZT and thymidine phosphorylation to the whole cell [Susan-Resiga et al. Antiviral Therapy 2004; 9(6):L22 (Abstract 32)] In the second, we sought to extend our observations in isolated heart mitochondria to mitochondria isolated from other tissues, beginning with liver mitochondria [Lynx et al Antiviral Therapy 2004; 9(6):L22 (Abstract 31)]. Thymidine was readily phosphorylated to TTP in both of these systems, with TTP the predominant product in the perfused heart and TMP the predominant product in isolated liver mitochondria. However, the phosphorylation of AZT beyond AZT-MP was not detected in either system. The production of TTP and AZT-MP in isolated liver mitochondria was an order of magnitude higher than observed in heart mitochondria. Finally, AZT inhibited thymidine phosphorylation in both systems with IC50s of 8.9 ± 1.8 μM in liver mitochondria and 24 ± 5 μM in the perfused heart.
ACKNOWLEDGEMENTS:: This work was supported by a grant from the National Institute of Health, HL 72710 to EEM.
2004-10-25
13
Copyright © 2004 - International Medical Press Ltd.. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the Medical Editor, International Medical Press, 36 St Mary-at-Hill, London EC3R 8DU, United Kingdom.