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丙烯酰胺致小鼠细胞DNA损伤及抗氧化剂的保护作用研究
【作者】 马红莲；
【导师】 杨建一；
【作者基本信息】 山西医科大学 ， 遗传学， 2008， 硕士
【摘要】 实验目的应用彗星试验检测丙烯酰胺(AA)染毒后小鼠多个脏器细胞DNA的损伤及损伤后的修复情况,以进一步研究AA遗传毒性,探讨AA体内遗传毒性可能的靶器官,比较不同细胞对AA的敏感性。并在一定剂量的维生素C和番茄红素干预时,检测二者对AA引起的小鼠淋巴细胞及肝细胞DNA损伤情况的影响,测定血浆及肝组织中脂质过氧化物含量及抗氧化酶活性的改变情况,以揭示AA遗传毒性与氧化损伤之间的联系,为AA遗传毒性机制及其预防提供一定的实验依据。实验方法1.在研究AA作用的时效关系时,以50mg/kg的AA给小鼠一次性腹腔注射,应用彗星试验检测AA染毒0、3、6、12、24h后小鼠外周血淋巴细胞、骨髓、肝、肺、脾、肾及睾丸细胞的DNA损伤情况。量效关系研究中,分别以0、10、25、50mg/kg的AA腹腔注射,检测12h后不同细胞DNA的损伤。2.为了研究抗氧化剂对AA毒性的影响,分别以50mg/kg的维生素C或10mg/kg剂量的番茄红素连续灌胃小鼠10天,并从第6天开始腹腔注射AA,给药结束后测定小鼠淋巴细胞、肝细胞DNA损伤及血浆和肝组织中MDA含量、SOD酶活力的变化。实验结果1.以50mg/kg的AA给小鼠腹腔注射,肝细胞在染毒后3小时,骨髓和淋巴细胞在6小时,彗星尾长、尾部DNA%及尾矩开始增加(P<0.05),到12小时达到最大值(P<0.01),24h时有所下降,但未恢复到对照组水平(P<0.05);睾丸细胞在12h时损伤程度与对照组比较有显著性差异(P<0.01);脾细胞在3h时出现DNA损伤,至24h时又基本恢复到对照组水平;各时间点未发现肺和肾脏细胞的明显损伤(P>0.05)。2.骨髓细胞在低剂量(10mg/kg)时,彗星各指标即对照组相开始增加(P<0.05),淋巴细胞、肝及睾丸细胞在中剂量(25mg/kg)时,彗星指标较对照组有明显增加(P<0.05),并随AA剂量的加大而增大。脾细胞只在AA高剂量(50mg/kg)时彗星指标才较对照组有明显增加(P<0.05)3.AA染毒5天后,除出现淋巴细胞和肝细胞的DNA损伤外,小鼠血浆及肝组织中MDA含量升高,SOD活力下降,与阴性对照组相比差别有显著性(P<0.05)。与AA组相比,给予维生素C或番茄红素的小鼠,其两种细胞彗星尾长、尾部DNA%及尾矩,都有显著下降(P<0.05),但仍高于阴性对照组(P<0.05),血浆及肝组织中MDA含量均有明显下降,SOD活力显著升高(P<0.05)。结论1. 50mg/kg剂量的AA能引起小鼠淋巴细胞、骨髓、肝、脾及睾丸等细胞的DNA损伤,机体对这种损伤具有一定的修复能力;2.不同细胞对AA的敏感性及DNA损伤修复能力不同;3.AA染毒后外周血淋巴细胞在彗星试验中的拖尾现象可反映体内组织器官DNA损伤的一般情况,可将其作为监测机体接触AA后组织细胞DNA损伤作用的生物标志。4.AA引起的小鼠细胞DNA损伤与脂质过氧化作用有关; 5.维生素C和番茄红素对AA染毒小鼠的脂质过氧化有拮抗作用,对AA引起的细胞DNA损伤有一定保护作用;
【Abstract】 Objective This study was to detect the DNA damage and repair of various cells of mice treated with acrylamide(AA) by using comet assay, so as to investigate the genotoxicity of AA further, compare the sensitivity of different cells to AA and determine its possible genotoxic target organs.After being administered certain dose of AA、vitamin C or lycopene+AA, The DNA damage of peripheral lymphocytes and liver cells and the degree of lipid peroxidation、the activity of antioxidase of mice were also detected. It can reveal the correlation between genetoxic of AA and lipid peroxidation, and provide some experimental evidence for the mechanisms and prevention of genetoxic of AA.Method 1.In the study of time-effect relationship, DNA damage in the cells of peripheral lymphocytes, bone marrow, liver, lung, spleen, kidney and testicle of mice were detected by using comet assay at 0、3、6、12、24h after 50mg/kg AA intraperitoneal injection. And in the study of dose-effect relationship, DNA damage were detected at 12h after 0、10、25、50mg/kg AA intraperitoneal injection. 2. In order to study the effect of vitamin C and lycopene on the toxicity of AA, mice were administered 50mg/kg vitamin C or 10mg/kg lycopene for successive 10 days, and 50mg/kg AA from 6th day. The DNA damage of the peripheral lymphocytes and liver cells and the content of malondialdehyde(MDA)、the activity of superoxide dismutase (SOD) were detected.Result 1.Significant increase in comet tail length、tail DNA% and tail moment(TM)were indu- ced at 3h in the liver and spleen cells and at 6h in bone marrow cells and peripheral lymphocyt- es after intraperitoneal treatment of AA at a dose of 50mg/kg(P<0.05).The indexes achieved maxlmum at 12h(P<0.01)and decreased at 24h,but were still significantly higher than the control (except spleen). Significant DNA damage of testicle cells was induced at 12h(P<0.01). No obvious increase in DNA damage was observed in the lung and kidney cells(P>0.05). 2. compared with the control group,the comet indexes showed significant increase in bone marrow cells in the 10mg/kg group,while in peripheral lymphocytes、liver and testicle cells,they increased in the 25mg/kg group(P<0.05).3. Treatment of AA for 5 days resulted in a significant increase in the content of MDA and decrease in the activity of SOD in serum and liver of mice(P<0.05). Compare with the AA group,The comet tail length、tail DNA% and TM of liver and peripheral lymphoc- ytes obviously decreased in the mice administered vitamin C or lycopene(P<0.05),the content of MDA decreased and the activity of SOD increased significantly(P<0.05).Conclusion 1. AA could induce DNA damage of peripheral lymphocytes, liver, bone marrow, testicle and spleen cells in mice. The DNA damage caused by AA could be repaired to a certain degree. 2. There are differences in sensitivity to acry1amide and the capability of DNA damage repaire of various cells in the mice. 3. It is suggested that the comet tail of peripheral lymphocytes is suitable as a biomarker for reflecting the DNA damage of organs after treatment of AA. 4. The DNA damage caused by AA is relevant with the lipid peroxidation in mice. 5. Vitamin C and lycopene could protect against the lipid peroxidation in mice treated with AA, and significantly decrease the levels of DNA damage in mice.