Antiretroviral nucleosides, deoxynucleotide carrier and mitochondrial DNA: evidence supporting the DNA pol gamma hypothesis.
METHODS
Nucleoside reverse transcriptase inhibitors (NRTIs) exhibit mitochondrial toxicity. The mitochondrial deoxynucleotide carrier (DNC) transports nucleotide precursors (or phosphorylated NRTIs) into mitochondria for mitochondrial (mt)DNA replication or inhibition of mtDNA replication by NRTIs. Transgenic mice (TG) expressing human DNC targeted to murine myocardium served to define mitochondrial events from NRTIs in vivo and findings were corroborated by biochemical events in vitro.


METHODS
Zidovudine (3'-azido-2',3'-deoxythymidine; ZDV), stavudine (2', 3'-didehydro-2', 3'-deoxythymidine; d4T), or lamivudine ((-)-2'-deoxy-3'-thiacytidine; 3TC) were administered individually to TGs and wild-type (WT) littermates (35 days) at human doses with drug-free vehicle as control. Left ventricle (LV) mass was defined echocardiographically, mitochondrial ultrastructural defects were identified by electron microscopy, the abundance of cardiac mtDNA was quantified by real time polymerase chain reaction, and mtDNA-encoded polypeptides were quantified.


RESULTS
Untreated TGs exhibited normal LV mass with minor mitochondrial damage. NRTI monotherapy (either d4T or ZDV) increased LV mass in TGs and caused significant mitochondrial destruction. Cardiac mtDNA was depleted in ZDV and d4T-treated TG hearts and mtDNA-encoded polypeptides decreased. Changes were absent in 3TC-treated cohorts. In supportive structural observations from molecular modeling, ZDV demonstrated close contacts with K947 and Y951 in the DNA pol gamma active site that were absent in the HIV reverse transcriptase active site.


CONCLUSIONS
NRTIs deplete mtDNA and polypeptides, cause mitochondrial structural and functional defects in vivo, follow inhibition kinetics with DNA pol gamma in vitro, and are corroborated by molecular models. Disrupted pools of nucleotide precursors and inhibition of DNA pol gamma by specific NRTIs are mechanistically important in mitochondrial toxicity.
Lewis
William
W
Department of Pathology, Emory University, Atlanta, Georgia 30322, USA. wlewis@emory.edu
Kohler
James J
JJ
Hosseini
Seyed H
SH
Haase
Chad P
CP
Copeland
William C
WC
Bienstock
Rachelle J
RJ
Ludaway
Tomika
T
McNaught
Jamie
J
Russ
Rodney
R
Stuart
Tami
T
Santoianni
Robert
R
D000818:Animals; D004247:DNA / Q000032:analysis; D004272:DNA, Mitochondrial / Q000032:analysis / Q000187:drug effects*; D004452:Echocardiography; D015751:Genes, pol; D015658:HIV Infections / Q000188:drug therapy / Q000473:pathology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D019259:Lamivudine / Q000494:pharmacology; D026901:Membrane Transport Proteins; D051379:Mice; D008822:Mice, Transgenic; D046529:Microscopy, Electron, Transmission; D008929:Mitochondria, Heart / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins; D008958:Models, Molecular; D009206:Myocardium / Q000648:ultrastructure; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018119:Stavudine / Q000494:pharmacology; D015215:Zidovudine / Q000494:pharmacology*
D004272:DNA, Mitochondrial; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D018894:Reverse Transcriptase Inhibitors; C434255:SLC25A19 protein, human; D019259:Lamivudine; D015215:Zidovudine; D004247:DNA; D018119:Stavudine