Assessment of mitochondrial toxicity in human cells treated with tenofovir: comparison with other nucleoside reverse transcriptase inhibitors

Abstract

Drug-associated dysfunction of mitochondria is believed to play a role in the etiology of the various adverse symptoms that occur in human immunodeficiency virus (HIV)-infected patients treated with the nucleoside reverse transcriptase inhibitors (NRTIs). Tenofovir, a nucleotide analog recently approved for use in the treatment of HIV infection, was evaluated in vitro for its potential to cause mitochondrial toxicity and was compared to currently used NRTIs. Treatment with tenofovir (3 to 300 microM) for up to 3 weeks produced no significant changes in mitochondrial DNA (mtDNA) levels in human hepatoblastoma (HepG2) cells, skeletal muscle cells (SkMCs), or renal proximal tubule epithelial cells. The potencies of inhibition of mtDNA synthesis by the NRTIs tested were zalcitabine (ddC) > didanosine (ddI) > stavudine > zidovudine (ZDV) > lamivudine = abacavir = tenofovir, with comparable relative effects in the three cell types. Unlike ddC and ddI, tenofovir did not affect cellular expression of COX II and COX IV, two components of the mitochondrial cytochrome c oxidase complex. Lactate production was elevated by less than 20% in HepG2 cells or SkMCs following treatment with 300 microM tenofovir. In contrast, lactate synthesis increased by >200% in the presence of 300 microM ZDV. Thus, treatment of various human cell types with tenofovir at concentrations that greatly exceed those required for it both to have in vitro anti-HIV type 1 activity in peripheral blood mononuclear cells (50% effective concentration, 0.2 microM) and to achieve therapeutically relevant levels in plasma (maximum concentrations in plasma, 0.8 to 1.3 microM) is not associated with mitochondrial toxicity.

FIG. 1.

Structures of tenofovir and its oral prodrug tenofovir disoproxil.

FIG. 2.

Effects of tenofovir and other NRTIs on mtDNA content in HepG2 cells. The cells were incubated with various drug concentrations for 9 days, and the relative mtDNA content was determined as the ratio of hybridization signals from mtDNA- and chromosomal DNA-specific probes. Data represent the percentages of the values for the untreated control, given as the means ± standard deviations of two independent experiments performed in duplicate.

FIG. 3.

Effects of tenofovir and other NRTIs on mtDNA content in skeletal muscle cells. The dividing cells were incubated in the presence of drugs for 9 days (A) or 18 days (B). The relative mtDNA content was determined as described in the legend to Fig. 2, with data representing the percentages of the values for the untreated control (mean ± standard deviations) from two independent experiments performed in duplicate.

FIG. 4.

Effects of ddC, ddI, and tenofovir on the expression of COX II and COX IV in HepG2 cells. Following 9 days of treatment, cells were lysed and the level of protein expression was analyzed by using specific monoclonal antibodies in combination with [³⁵S]immunoglobulin G. (A) Detection of proteins following SDS-PAGE and immunoblot analysis (10 μg of cellular proteins per each sample). (B) Quantification of the signal from immunoblot analysis. The relative expression levels were determined as a ratio between the specific cytochrome c oxidase signal and the glyceraldehyde-3-phosphate dehydrogenase signal, with the relative expression in control untreated cells being 100%.