Differential effects of antiretroviral nucleoside analogs on mitochondrial function in HepG2 cells

Abstract

Numerous studies have reported effects of antiviral nucleoside analogs on mitochondrial function, but they have not correlated well with the observed toxic side effects. By comparing the effects of the five Food and Drug Administration-approved anti-human immunodeficiency virus nucleoside analogs, zidovudine (3'-azido-3'-deoxythymidine) (AZT), 2',3'-dideoxycytidine (ddC), 2', 3'-dideoxyinosine (ddI), 2',3'-didehydro-2',3'-deoxythymidine (d4T), and beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), as well as the metabolite of AZT, 3'-amino-3'-deoxythymidine (AMT), on mitochondrial function in a human hepatoma cell line, this issue has been reexamined. Evidence for a number of mitochondrial defects with AZT, ddC, and ddI was found, but only AZT induced a marked rise in lactic acid levels. Only in mitochondria isolated from AZT (50 microM)-treated cells was significant inhibition of cytochrome c oxidase and citrate synthase found. Our investigations also demonstrated that AZT, d4T, and 3TC did not affect the synthesis of the 11 polypeptides encoded by mitochondrial DNA, while ddC caused 70% reduction of total polypeptide content and ddI reduced by 43% the total content of 8 polypeptides (including NADH dehydrogenase subunits 1, 2, 4, and 5, cytochrome c oxidase subunits I to III, and cytochrome b). We hypothesize that in hepatocytes the reserve capacity for mitochondrial respiration is such that inhibition of respiratory enzymes is unlikely to become critical. In contrast, the combined inhibition of the citric acid cycle and electron transport greatly enhances the dependence of the cell on glycolysis and may explain why apparent mitochondrial dysfunction is more prevalent with AZT treatment.

FIG. 1

Electron micrograph of HepG2 cells treated with nucleosides. After 14 days of incubation of HepG2 cells with the control (A), 10 μM AZT (B), 50 μM ddI (C), and 10 μM ddC (D), electron microscopy was undertaken (magnification, ×30,000). Disrupted mitochondrial cristae were observed in ddC- and ddI-treated cells.

FIG. 2

Effects of nucleoside analogs on mtDNA content of HepG2 cells. HepG2 cells (3 × 10⁴/ml) were treated with various concentrations of nucleoside analogs for 14 days or were not given drug treatment (control). The mtDNA of HepG2 cells on the nitrocellulose paper was detected by using ³²P-labeled human mtDNA fragment as described in Materials and Methods. The data represent the means of two experiments.

FIG. 3

Levels of lactic acid in the extracellular medium of HepG2 cells after treatment with various concentrations of nucleoside analogs for 4 days. Lactic acid levels are expressed as milligrams of lactic acid per 10⁶ cells. The lactic acid levels at each drug concentration were compared with those in cells incubated in drug-free medium, and the percentage of the control levels was determined for each concentration. The standard deviations (error bars) of the data were determined from the means of three experiments.

FIG. 4

Autoradiography of radiolabeled mitochondrially coded proteins. After 6 days of treatment with AZT, ddC, ddI, d4T, or 3TC, the medium of HepG2 cells (2.5 × 10⁶/ml) was replaced by the same medium deficient in methionine. Cytoplasmic protein synthesis was inhibited by emetine (200 μg/ml), and mitochondrially coded proteins were labeled by adding 60 μCi of [³⁵S]methionine/ml. To one set of the control cells was added chloramphenicol (200 μg/ml) to inhibit mitochondrial protein synthesis. After 3 h, the cells were washed with PBS and dissolved in 5% SDS–Tris buffer prior to electrophoresis. Electrophoresis and autoradiography were then performed as described previously (17).