Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity.
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 gamma. 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 microM. Thus, the toxicity of AZT in some tissues may be mediated by disrupting the substrate supply of TTP for mt-DNA replication.
McKee
Edward E
EE
Indiana University School of Medicine, South Bend Center for Medical Education, Notre Dame, IN 46556, USA. McKee.6@nd.edu
Bentley
Alice T
AT
Hatch
Matthew
M
Gingerich
Joel
J
Susan-Resiga
Delia
D
D000818:Animals; D019380:Anti-HIV Agents / Q000008:administration & dosage / Q000493:pharmacokinetics* / Q000633:toxicity; D023241:Antiretroviral Therapy, Highly Active; D001692:Biological Transport; D002851:Chromatography, High Pressure Liquid; D004272:DNA, Mitochondrial / Q000378:metabolism; D054306:Dideoxynucleotides; D066298:In Vitro Techniques; D008929:Mitochondria, Heart / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013936:Thymidine / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism; D013997:Time Factors; D015215:Zidovudine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics* / Q000633:toxicity
D019380:Anti-HIV Agents; D004272:DNA, Mitochondrial; D054306:Dideoxynucleotides; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D013936:Thymidine