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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.

7.Relationship between the oncosis of pancreatic acinar cells and activation of macrophage in rat model of acute pancreatitis

Guanghai LU ; Weihui ZHANG ; Yingmei ZHANG ; Dongho XUE ; Bei SUN

Chinese Journal of Digestive Surgery 2009;8(4):275-277

Objective To study the relationship between the oncosis of pancreatic acinar cells and activa-tion of macrophage in rat model of acute pancreatitis (AP). Methods The pancreatic acinar cells were isolated by two-step enzyme digestion, and then they were divided into control group, AP group and test group. Pancreatic acinar cells were cultured with caerulein in AP group, with caerulein and endothelin in test group, and with culture medium in control group. The oncesis rate of the pancreatic acinar cells was detected after acridine orange and ethidium bromide fluorescent staining. The supernatant was collected to detect the release of amylase and lactate dehydrogenase (LDH). The macrophages were cultured with 1 ml of supematant for 6 hours, and then the protein level of tumor necrosis factor-α (TNF-α) was measured by ELISA. Results Few oncotic pancreatic acinar cells were observed in the control group, and the levels of amylase and LDH secreted by pancreatic acinar cells and TNF-α secreted by macrophage were (1175±165)kU/L, (846±118)U/L and (36±5)μg/L, respectively. Oncotic pancreatic acinar cells were observed in AP group, and the levels of amylase, LDH and TNF-α were (7130±680) kU/L, (4262±626) U/L and (155±18) μg/L, respectively, which were significantly higher than those in control group (t = 5.184, 4.277, 3.665, P < 0.05). The levels of amylase, LDH and TNF-α were even higher in test group, and they were (9240±1177) kU/L, (6937±893)U/L and (268±35)μg/L, respectively, which were significantly higher than those in AP group (t = 2.251, 2.825, 2.843, P < 0.05). Conclusions The release of amylase was changed as the oncosis of pancreatic acinar cells occurred. The secretion of TNF-α was along with the degree of oncosis of pancreatic acinar cells. The results of the study indicate that a relationship exists among the inflammatory response of macrophage, the release of contents of pancreatic acinar cells and the oncosis of the pancreatic acinar cells.