Mechanism Rcsearch On Mitochondrial DNA Damage Induced By Arsenic Trioxide(As2O3)in Mouse Oocytes

ObjectiveArsenic belongs to metalloid which is widely distributed in ground water and soil around areas with extractive industries in many countries, mostly with higher levels than the currently approved maximum contaminant level. Chronic exposure to arsenic would lead to kinds of carcinogenic or non-carcinogenic diseases in mammals, like skin cancer, lung cancer, cardiovascular disease, reproductive disease and so on. During which, a burst of reactive oxygen species (ROS) is involved. The excessive amount of ROS, if not cleared timely, would induce accumulated DNA damage, both in nuclear and mitochondrial DNA (mtDNA).Mitochondria provide most of the adenosine triphosphates (ATP) through the oxidative phosphorylation pathway and the citric acid cycle necessary for cell growth and metabolism. Meanwhile, mitochondria are a major source of ROS in eukaryotic cells, which would in turn increase the oxidative damage in mtDNA. Mammal mitochondria are normally inherited exclusively from the oocytes, injury of the mitochondria or mtDNA in oocytes would definitely affect oocytes maturation or even the development of the embryo. Thus, the study on factors influencing mitochondria and mtDNA damage in oocytes induced by arsenic is essential for the further study on the mechanism and the prevention of reproductive disorders induced by arsenic. Could arsenic exposure lead to oxidative stress in oocytes? Is it possible that the mtDNA mutation in oocytes exposed to arsenic is caused by oxidative stress? If so, what are the mechanisms? All these answers remain pending further investigation.In the present study, oocytes in germinal vesicle (GV) stage were collected from healthy female Kunming mice and were treated with arsenic trioxide (As2O3) to investigate the factors and mechanisms that would influence mtDNA quality in oocytes both in vivo and in vitro. Antioxidants N-Acetyl-L-Cysteine (NAC) and Tempo were used to prevent oxidative stress caused by As2O3. Techniques like cell culture, animal model studies, semi-quantitative PCR, real-time quantitative PCR, western blotting and fluorescence electron microscopy were used during the experiment.Methods1. Oxidative damage of the blood, liver and kidney in mouse caused by AS2O3 In this part of the research, mouse body weight, organ coefficient of liver and kidney were recorded. The bioconcentration of arsenic in blood, liver and kidney as well as the oxidative stress (MDA content, SOD and GSH-Px activity) in these tissues were tested correspondingly.2. Mitochondria and mtDNA damage in mouse oocytes caused by As2O3Both in vitro and in vivo assays were set in the experiment. Oxidative stress, ATP levels, as well as mtDNA damage of oocytes treated with As2O3were investigated in this part.3. Mechanism of mtDNA damage in mouse oocytes caused by As2O3Both in vitro and in vivo assays were set in this part as well. To further verify the mechanisms leading to mtDNA damage in mouse oocytes induced by AS2O3, lysosomal activity and mitochondrial transcription factor A (mtTFA)/DNA polymerase y (pol y) levels were detected in oocytes treated with AS2O3.4. Statistical analysisData were analyzed by one way analysis of variance (ANOVA) and t-test using SPSS16.0. No significant difference was taken as p>0.05, while the criterion of statistical difference was taken as P<0.05. Origin8.0was used for graphics processing.Results1. Arsenic could accumulate in tissues of mouse, especially in liver and kidney (p<0.05). The accumulation amount of arsenic were liver> kidney> blood. Co-treatment with NAC could efficiently decrease arsenic concentration in tissues (p<0.05).2. AS2O3could increase the lipid peroxidation level and decrease the antioxidation index (GSH-Px/MDA) in mouse (p<0.05), thus lead to oxidative dammage in tissues. While intake of proper amount of NAC could eliminate excessive ROS induced by AS2O3(p<0.05) to maintain the steady level of redox state, hence to protect organism from oxidative dammage induced by arsenic.3. Both in vitro and in vivo assays demonstrated that As2O3could obviously decrease the copy number of mtDNA and cause severe3867bp deletion in mitochondria together with elevated ROS level, while ATP content was decreased (p<0.05), indicating that As2O3would cause oxidative stress and lead to mitochondrial dysfunction in mouse oocytes.4. Co-treatment with NAC efficiently eliminated ROS induced by As2O3, lessened the mtDNA damage and enhanced ATP content in mouse oocytes both in vivo and in vitro (p<0.05), revealing that As2O3could cause severe mtDNA damage and decrease ATP content by inducing excessive ROS, and this damage would then probably restrain the further development of mouse oocytes.5. Both in vitro and in vivo assays demonstrated that AS2O3induced serious mtDNA oxdative damage by the judge of8-OHdG levels in mouse oocyte, and this damage was associated with down-regulated mtTFA and pol y protein levels (p<0.05). While co-treatment with ROS scavenger lessened the injury to mtDNA along with up-regulated mtTFA and pol y expression levels (p<0.05).6. In in vitro assay, P-gal activity in oocytes treated with As2O3elevated sharply (p<0.05), and a similar change trend was shown by fluorescent intensity in oocytes stained with LysoTracker Red. Indicating that AS2O3increased lysosome activity in mouse oocytes. While co-treatment with ROS scanvenger efficiently decreased lysosome activity (p<0.05).In all, the present study suggests the roles of mtTFA, pol y and lysosome in regulating mtDNA genome integrity and quality in mouse oocytes development in a manner dependent on ROS under AS2O3exposure, whereby clearance of ROS accumulation would effectively mitigate As2O3induced mtDNA damage.