Cardiac mitochondrial DNA polymerase-gamma is inhibited competitively and noncompetitively by phosphorylated zidovudine.

Abstract

and related this to altered mitochondrial DNA replication found in vivo. 5 We explored enzymologic events in the interaction of purified DNA pol-y with AZTTP. Toxic mechanisms in AZT mitochondrial myopathy may be analogous in some ways to pharmacologic mechanisms in the antiretroviral action of AZT.2 The present study showed that AZTTP inhibited DNA pol-y with mixed inhibition kinetics. AZTTP acted both as an alternate substrate for dTTP with DNA pol-y and competed with dTTP for DNA pol-y nucleotide binding (as seen with AZTTP and some cellular polymerases). Materials and Methods MaterialsReagents were analytic grade I. AZTTP was from Moravek Biochemicals, Brea, Calif. [3H]dTTP was from Amersham. All enzyme inhibitor assays were replicated from five to eight times, and each assay point was performed in triplicate within each run. Calculated arithmetic means from the triplicate assays were used to plot kinetic data. Mitochondrial Isolation ProceduresFor mitochondrial isolation, all procedures took place either on ice or at 4°C and resembled those used by us and by others in the past.78 Fresh bovine hearts were obtained from the slaughterhouse (courtesy of Kluener Packing Co, Cincinnati, Ohio) approximately 20 minutes before beginning the preparation. Tissue was minced into 1-cm cubes and homogenized in buffer M that consisted of (mmol/L) sucrose, 250; Tris-HCI, 50 (pH 8.0); 2-mercaptoethanol (2-ME), 5; EDTA, 1; MgCl2, 5; and KCI, 25. Buffer volume was increased 10-fold, and the mixture was homogenized with a hand-held Potter-Elvejhem homogenizer (Fisher Scientific, Pittsburgh, Pa). Debris was pelleted at 500g. The mitochondria were pelleted from the supernatant solution by centrifugation at 8500g for 10 minutes.

Transgenic expression of the deoxynucleotide carrier causes mitochondrial damage that is enhanced by NRTIs for AIDS

“…17 The DNC TG here offered an approach to evaluate NRTI toxicity in vivo and linked NRTI toxicity to mitochondrial import and nucleotide homeostasis. 4 For some time, our group [26][27][28] and others [29][30][31][32][33][34][35] have studied mechanisms of mitochondrial toxicity of NRTIs focusing on inhibition of DNA pol g (reviewed in Kaguni 36 ). NRTIs are firmly linked to altered mtDNA replication 10,37,38 through the DNA pol g hypothesis.…”

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals

“…Furthermore, after prolonged assumption of these drugs, histological findings commonly associated with depletion of mtDNA, such as red-ragged fibers, are observed (27). It should be noted that the antiviral nucleotide analogs strongly interfere with the action of the viral reverse transcriptases and the mtDNA polymerase-␥ but have a very low affinity for the nuclear DNA polymerases (17,28,29). When the diphosphate and triphosphate derivatives of these drugs and other related potential antivirals become available, it should be possible to assess their potential toxicity by studying their import into proteoliposomes reconstituted with the human DNC.…”

Mitochondrial DNA Depletion, Oxidative Stress, and Mutation: Mechanisms 0f Dysfunction from Nucleoside Reverse Transcriptase Inhibitors

“…Although energy depletion from altered mtDNA replication in NRTI toxicity is a logical consequence (Lewis et al, 1991(Lewis et al, , 1994a(Lewis et al, , 1994b(Lewis et al, , 1996(Lewis et al, , 1997Lewis and Dalakas, 1995), related events of oxidative stress also impact on energetics and mtDNA replication. Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ie, superoxide, hydrogen peroxide, lipid peroxides, hydroxyl radical, and peroxynitrite) and the cellular antioxidant defenses that prevent damage from those moieties (Betteridge, 2000).…”

“…We demonstrated that "adenosine tracts" were analogous to template-related "hot spots" that were particularly sensitive to mtDNA replication inhibition and resembled those found in some heritable mitochondrial diseases (Wallace, 1992a(Wallace, , 1992b because they altered mtDNA synthesis. Adenosine tracts in DNA templates inhibit the incorporation of fialuridine monophosphate into DNA in vitro (Lewis et al, 1994b). Table 1 highlights some of the studies in the literature.…”