Phosphorylation of thymidine and AZT in heart mitochondria: Elucidation of a novel mechanism of AZT cardiotoxicity

Edward E. McKee, Alice T. Bentley, Matthew Hatch, Joel Gingerich, Delia Susan-Resiga

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

Antiretroviral nucleoside analogs used in highly active antiretroviral therapy (HAART) are associated with cardiovascular and other tissue toxicity associated with mitochondrial DNA depletion, suggesting a block in mitochondrial (mt)-DNA replication. Because the triphosphate forms of these analogs variably inhibit mt-DNA polymerase, this enzyme has been promoted as the major target of toxicity associated with HAART. We have used isolated mitochondria from rat heart to study the mitochondrial transport and phosphorylation of thymidine and AZT (azidothymidine, or zidovudine), a component used in HAART. We demonstrate that isolated mitochondria readily transport thymidine and phosphorylate it to thymidine 5′-triphosphate (TTP) within the matrix. Under identical conditions, AZT is phosphorylated only to AZT-5′-monophosphate (AZT-MP). The kinetics of thymidine and AZT suggest negative cooperativity of substrate interaction with the enzyme, consistent with work by others on mitochondrial thymidine kinase 2. Results show that TMP and AZT-MP are not transported across the inner membrane, suggesting that AZT-MP may accumulate with time in the matrix. Given the lack of AZT-5′-triphosphate (AZT-TP), it seems unlikely that the toxicity of AZT in the heart is mediated by AZT-TP inhibition of DNA polymerase γ. Rather, our work shows that AZT is a potent inhibitor of thymidine phosphorylation in heart mitochondria, having an inhibitory concentration (IC)50 of 7.0 0.9 μM. Thus, the toxicity of AZT in some tissues may be mediated by disrupting the substrate supply of TTP for mt-DNA replication.

Original languageEnglish (US)
Pages (from-to)155-167
Number of pages13
JournalCardiovascular Toxicology
Volume4
Issue number2
DOIs
StatePublished - Oct 14 2004

Keywords

  • AZT cardiotoxicity
  • Heart mitochondria
  • Mitochondrial DNA depletion
  • Thymidine kinase
  • TMP kinase

ASJC Scopus subject areas

  • Toxicology
  • Cardiology and Cardiovascular Medicine

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