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Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3'-azido-2',3'-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK. In organello studies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 microM; 2.7 and 13.4 microg/ml), didanosine (ddI) (10 and 50 microM; 2.4 and 11.8 microg/ml), and zalcitabine (ddC) (1 and 5 microM; 0.21 and 1.1 microg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 microg/ml; 1.3 microM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 microM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard.

Nucleobase and nucleoside transporters play central roles in the biochemistry of parasitic protozoa, as they lack the ability to synthesize purines de novo and are absolutely reliant upon purine salvage from their hosts. Furthermore, such transporters are potentially critical to the pharmacology of these important human pathogens, because they mediate the uptake of purine analogues, as well as some nonpurine drugs, that can be selectively cytotoxic to the parasites. We here report the first identification and characterization of a purine nucleobase transporter in Leishmania amastigotes. Uptake of [3H]hypoxanthine by Leishmania mexicana amastigotes was mediated by a single high-affinity transporter, LmexNBT1, with a Km of 1.6 +/- 0.4 microM and high affinity for adenine, guanine, and xanthine but low affinity for nucleosides and pyrimidine nucleobases. Allopurinol, an antileishmanial hypoxanthine analogue, was apparently taken up by the same transporter. Using [3H]allopurinol, a Km value of 33.6 +/- 6.0 microM was obtained. All evidence was compatible with a model of a single purine nucleobase transporter being expressed in amastigotes. Using various purine nucleobase analogues, a model for the interactions between hypoxanthine and the transporter's permeant binding site was constructed. The binding interactions were compared with those of the LmajNBT1 transporter in Leishmania major promastigotes and found to be very similar.

The toxicity of 6-mercaptopurine was potentiated by 2 mg of either Escherichia coli 026:B6 B endotoxin or Salmonella typhosa 0901 W endotoxin per kg. Nonlethal doses of heat-killed, gram-negative bacteria were also capable of potentiating the lethality of 6-mercaptopurine (6-MP). Salmonella minnesota S, the wild-type strain, and S. minnesota Re 595, a mutant containing only the lipid A and 2-keto-3-deoxyoctonate moiety of the endotoxin molecule, exhibited the same capability to enhance the toxic action of 6-MP. Endotoxin (lipopolysaccharide [LPS]) did not affect the clearance of 6-MP from the circulation, but did alter its apparent metabolism as indicated by blood levels of a metabolite, 6-thiouric acid. The concentration of blood urea nitrogen (BUN) in mice 18 h after injection of 100 mg of 6-MP per kg simultaneously with 2 mg of LPS per kg was significantly elevated over normal values. However, these BUN values were significantly less than those resulting from the administration of one mean lethal dose of either agent. The clearance from the circulation of the gram-negative organism E. coli HB, or the gram-positive organism Staphylococcus epidermidis S, was not affected by 6-MP. Endotoxin had no effect on the clearance of S. epidermidis S, but inhibited that of E. coli HB. When 6-MP and LPS were administered simultaneously with either bacterial species, only the clearance of E. coli HB was inhibited. Mice were protected from the lethality associated with combinations of 6-MP and LPS by (i) prior treatment with phenobarbital, (ii) caffeine, (iii) methylprednisolone, and (iv) polymyxin B sulfate. With the exception of caffeine, each regimen protected mice against the lethal effects of 400 mg of 6-MP per kg, and methylprednisolone or polymyxin B protected mice against 8 mg of LPS per kg.