Mitochondrial DNA base excision repair and mitochondrial DNA mutation in human hepatic HuH-7 cells exposed to stavudine

Abstract

The mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is due to the inhibition of mitochondrial DNA (mtDNA) polymerase γ (Pol γ), resulting in a blockade of mtDNA replication and subsequent disruption of cellular energetics. Because mtDNA Pol γ is not only involved in mtDNA replication but also responsible for mtDNA repair, we hypothesize that mitochondrial oxidative stress leads to changes in the balance between mtDNA repair and mutation following stavudine (d4T) treatment. However, the mechanisms underlying how changes in mtDNA base excision repair (mtBER) lead to mtDNA mutation remain unclear. To test this hypothesis, total mitochondrial repair capability, different steps of mtBER, mtDNA mutations in D-loop, and oxidative stress were all assessed in cultured HuH-7 human hepatoblast cells treated with d4T for 2 weeks. Assessment by denaturing Southern blotting and quantitative PCR revealed that d4T significantly reduced in vivo repair of H 2O 2 damaged mtDNA in HuH-7 cells. d4T reduced total in vitro mtBER and DNA Pol γ capability, but did not affect mtDNA oxoguanine glycosylase and apurinic/apyrimidinic endonuclease activity in HuH-7 cells. In addition, d4T treatment is associated with a significant increase in the frequency of mtDNA mutations in HuH-7 cells. In conclusion, d4T treatment reduces mtBER and contributes mechanistically to NRTI-induced mtDNA mutation. These events may potentially be associated with some diseases linked to mtDNA mutation.