ABSTRACT
OBJECTIVE To investigate the arsenic trioxide(As2O3)-induced oxidative damage to mitochondrial DNA (mtDNA) in mouse oocytes and possible mechanisms. METHODS ① For in vitro assay,the mouse oocytes were denuded from ovaries of normal mice and incubated in medium for 20 h in different treatment groups:control,As2O3 1 and 2 μmol · L- 1,N-acetylcysteine (NAC) 5 mmol · L-1, 2,2,6,6-tetramethyl-1-piperidinyloxy(Tempo)1 mmol · L-1, As2O3(1 and 2 μmol · L-1)+NAC 5 mmol · L-1,As2O3 (1 and 2 μmol · L-1)+Tempo 1 mmol · L-1. ② For in vivo assay,mice were subjected to ip injection with physiological saline (normal control),As2O3 1 and 2 mg · kg-1,or As2O3 (1 and 2 mg · kg-1)+NAC 200 mg · kg-1, respectively. After 60 d,all the mice were sacrificed and their ovaries were quickly excised. Intracellular reactive oxygen species(ROS) levels were determined by 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The oxidative damage to mtDNA was induced using enzyme-linked immunosorbent assay(ELISA)for 8-hydroxy-2′-deoxyguanosine(8-OHdG). The expression of DNA polymerase γ(Polγ)and mitochondrial transcription factor A(mtTFA)was detected by Western blotting and the vitality of lysosomes was monitored by β-galactosidase(β-Gal)Assay Kit. RESULTS ①In vitro experiments,As2O3 elevated 8-OHdG levels of mtDNA in mouse oocytes accom? panied by increased levels of ROS (P<0.05),but co-treatment with NAC or Tempo significantly reduced ROS and 8- OHdG levels (P<0.05). Meanwhile, the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05),but were markedly elevated by the addition of NAC or Tempo (P<0.05). ②In vivo assay,As2O3 elevated ROS as well as 8-OHdG levels of mtDNA in mouse oocytes,while the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05). Co-treatment with NAC significantly reduced ROS and 8-OHdG levels,but markedly elevated Pol γ and mtTFA levels(P<0.05). Besides,a notable increase in β-Gal activity was shown in As2O3-treated mouse oocytes in vitro (P<0.05),while antioxidants efficiently reduced the activity (P<0.05). However,no significant changes were observed in the in vivo study. CONCLUSION The oxidative damage to mtDNA induced by As2O3 in mouse oocytes may be mediated by ROS and associated with down-regulation of protein levels of Pol γ and mtTFA as well as increment of lysosomal activity.