Mitochondrial DNA base excision repair and mitochondrial DNA mutation in human hepatic HuH-7 cells exposed to stavudine.
The mitochondrial toxicity of nucleoside reverse transcriptase inhibitors (NRTIs) is due to the inhibition of mitochondrial DNA (mtDNA) polymerase gamma (Pol gamma), resulting in a blockade of mtDNA replication and subsequent disruption of cellular energetics. Because mtDNA Pol gamma 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(2)O(2) damaged mtDNA in HuH-7 cells. d4T reduced total in vitro mtBER and DNA Pol gamma 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.
HIV/AIDS Research Center, Beijing You An Hospital, Capital Medical University, Beijing 100069, China.
Yasong
Y
Wu
Ning
N
Li
Tong
T
Zhang
Hao
H
Wu
Chun
C
Huang
Dexi
D
Chen
D019380:Anti-HIV Agents / Q000633:toxicity*; D001483:Base Sequence; D002460:Cell Line; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D000074002:DNA Polymerase gamma; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000187:drug effects*; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D008969:Molecular Sequence Data; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000633:toxicity*; D018119:Stavudine / Q000633:toxicity*
D019380:Anti-HIV Agents; D017931:DNA Primers; D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D018894:Reverse Transcriptase Inhibitors; D006861:Hydrogen Peroxide; D018119:Stavudine; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase