Effects Of Chronic Exposure To Microcystin-LR On Somatic Mitochondrial DNA In Mice

Background and Objective:Microcystins(MCs)are the most common toxins produced by contaminated cyanobacteria in aquatic ecosystems,and have toxic effects through inhibition of serine/threonine protein phosphatases 1 and 2A(PP1 and PP2A).And microcystin-LR(MC-LR)is the most toxic and common among 90 MC variants.MC-LR damages DNA,either by directly through mutageneis or indrectly through reactive oxygen species(ROS)generation.Mitochondria are a sensitive target of environmental toxicants,and its dysfunction involves in the pathogenesis of many diseases.Therefore,in the thesis,the mitochonfiral DNA(mtDNA)copy number in hepatpatocyte and pneumonocyte of mice exposed to microcystin-LR were studied.Expression of mitochondria-related genes and mtDNA replication related genes in liver and lung tissues of MC-LR-exposed mice were studied.And the risk and mechanism of lung injury exposure to MC-LR were studied.Methods:1.Chronic exposrue to MC-LR on mice:The C57BL/6 mice of 6-8 weeks were randomly divided to 6 groups(5 experimental groups and a control group)(n=5).After one week's adaptation,all the mouse of different groups began to feeding microcystin-LR of 1,5,10,20 and 40?g/L,respectively and drank freely for 12 month.2.After sacrifice,the liver and lung tissues were sectioned and fixed,then were analyed by hematoxylin-eosin(H&E)and masson's trichrome(Masson)staining.3.The changes of mtDNA copy number were detected by Q-PCR,and the integrity of mtDNA and nuclear DNA(nDNA)were detected by long-range PCR.4.The enzyme linked immunosorbent assay(ELISA)were used to test the 8-OHdG levels in DNA of liver tissues and the expression of inflammatory cytokines IL-6,TNF-aand TGF-p in serum and lung tissue.5.QRT-PCR and Western blotting were used to detected the mRNA and protein expresion of mitochondrial genes and mtDNA replication genes in liver and lung tissues.Results:1.Compared with organs-to-body weigh ratio in the control,the experimental mice showed liver and kidney to body weigh ratios decreased,but the ratio of abdominal adipose increased in 20?g/L group of MC-LR.However,after the mice exposure with different concentations of MC-LR for 12 months,there was not statistically significant differences-noted in weight gain,and liver,lung,kidney,and abdominal asipose to body weight ratios,in comparison with the control.And our studies showed that the mtDNA copy number was changed in the 1,5,10,20 and 40?g/L groups of compared to the control groups.2.Long term and presistent exposure of mice to MC-LR increased the oxidative damage of liver and lung.And long term and presistent exposure to MC-LR induced the fat vacuoles and necrotic cells in liver with the concentration-dependent manner(10,20 and 40?g/L group of MC-LR).And MC-LR also result in thickenig of the alveolar septa,inflammaroty responses,and changes of inflammatory cytokines(5,10,20 and 40?g/L group of MC-LR).3.Chronic exposure to MC-LR increased the 8-hydroxy-2'-deoxyguanosine(8-OHdG)levels of DNA,damaged the integrity of mtDNA and nDNA and while notably altered the mtDNA copy number in liver cells.And chronic exposure to MC-LR increased the generation of ROS,disturbed the balance of redox system,decreased the integrity of mtDNA and nDNA,and changed the mtDNA copy number in lung cells.4.Long term exprosure to MC-LR did not only change the mtDNA copy number but also altered the mRNA and protein expression of mitochondrial genes and nuclear genes that are critical for regulating mtDNA replication and repairing oxidized DNA in liver and lung cells.Conclusion:1.Due to the organ specificity of toxic effects,the sensitivity of different tissues to MC-LR exposure was different in mice.Although the liver cells were more senstive to MC-LR,lung cells were the vulnerable targent of MC-LR.2.The repairing dysfunction of oxidative damage,and the changes of mtDNA structure and mtDNA copy number were the important toxicological effects of MC-LR.3.Effects of long term and chornic exposure to MC-LR on somatic mtDNA stability cells in mice associated with the expression of inflammatory cytokines,proliferation of mitochondria and mtDNA,and the regulatory mechanism for the aberrant expression of nuclear genes that are critical for reuglating mtDNA replication...