砷(III)对紫贻贝遗传毒性和免疫毒性的研究
李巧梅
学位类型硕士
导师赵建民研究员+吴惠丰研究员
2013-05-23
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业环境科学
关键词紫贻贝 亚砷酸钠 遗传毒性 免疫毒性 细胞凋亡
其他摘要
本论文以紫贻贝(Mytilus galloprovincialis)为研究对象,采用生物化学、组织病理学以及分子生物学等方法,研究了不同浓度砷(III)(1、10、100 μg/L)亚慢性暴露(30天)对紫贻贝的免疫毒性和遗传毒性。通过测定砷(III)暴露对血细胞吞噬活力、活性氧(ROS)含量和抗氧化指标的影响,评估了砷(III)对紫贻贝的免疫毒性;通过测定血细胞微核率(MN)、消化腺和鳃组织损伤以及细胞凋亡状况,结合凋亡相关基因的转录表达变化,探讨了砷(III)暴露对紫贻贝的遗传毒性。研究结果可为深入了解砷对海洋贝类的毒性作用机制提供参考,并可为我国近海砷污染的生态风险评估提供理论依据。主要研究结果如下:
(1)砷(III)暴露对紫贻贝免疫毒性的研究
采用中性红分光光度法测定了紫贻贝溶酶体膜稳定性变化,结果发现砷(III)暴露组紫贻贝的溶酶体膜稳定性随暴露浓度的增加依次降低,表明溶酶体膜的通透性增加;其中,100 μg/L砷暴露组血细胞溶酶体膜稳定性较对照组显著降低(P <0.05),提示溶酶体受到严重损伤;采用流式细胞术测定了砷(III)暴露对血细胞吞噬活力和呼吸爆发的影响,结果发现,砷(III)暴露对血细胞吞噬活力和呼吸爆发均表现为诱导作用,诱导效果随暴露剂量的增加呈现先升高后降低的趋势;呼吸爆发的变化趋势与细胞吞噬活性相一致。同时,探讨了砷(III)暴露对紫贻贝消化腺和鳃组织抗氧化指标的影响,结果发现,中剂量砷(III)暴露可导致鳃组织CAT活性和GSH含量显著增加(P <0.05),而高剂量砷(III)暴露则可导致消化腺和鳃组织MDA含量极显著增加(P <0.01)。
(2)砷(III)暴露对紫贻贝遗传毒性的研究
采用微核试验测定了砷(III)暴露对紫贻贝血细胞DNA的损伤情况,结果发现,中、高剂量(10 μg/L和100 μg/L)砷(III)暴露组的血细胞微核率显著增加(P <0.05),表明亚慢性中、高剂量砷(III)可导致紫贻贝血细胞的遗传损伤;采用组织病理切片和TUNEL技术分析了砷(III)暴露对消化腺、鳃组织结构及细胞凋亡状况的影响,结果发现,中、高剂量砷(III)暴露对消化腺和鳃组织细胞凋亡具有强烈的诱导作用,且高剂量暴露可导致消化腺和鳃组织结构严重破坏、边缘模糊,细胞坏死现象明显。为深入探讨砷(III)暴露对细胞凋亡的诱导机制,采用实时荧光定量PCR技术检测了Bcl-2、Bcl-xL、Caspase-2、Caspase-3、Ras和p63基因的表达水平。结果发现,砷(III)暴露可影响细胞凋亡信号转导通路中关键基因的转录表达;其中,低剂量(1 μg/L)砷(III)暴露对细胞的抗凋亡功能占主导作用,而中、高剂量砷(III)暴露则以促凋亡功能占主导作用。
综上所述,亚慢性低剂量砷(III)暴露可导致紫贻贝CAT活性的显著增高,但未检测到显著遗传毒性;亚慢性中、高剂量砷(III)暴露则可诱导紫贻贝血细胞活性氧的增加,导致遗传物质的明显损伤,表现为显著的遗传毒性和一定的免疫毒性。
关键词:紫贻贝,亚砷酸钠,遗传毒性,免疫毒性,细胞凋亡
;
In the present study, immunotoxicity and genotoxicity of sodium arsenite on Mytilus galloprovincialis were investigated with biochemistry, histopathology and molecular biology methods. The mussels were exposed to arsenite of 1、10 and 100 μg/L for 30 days, respectively. Immunotoxicity was evaluated by measuring activity of phagocytosis, contents of reactive oxygen species (ROS) and alteration of antioxidant enzymes. Genotoxicity was assessed by evaluating micronucleus rates of hemocytes, histopathological damage and cell apoptosis in hepatopancreas and gills. The above results can provide references for further understanding the mechanisms of arsenite on M. galloprovincialis as well as providing theoretical basis for ecological risk assessments and management decisions for coastal pollution.
(1) Study on arsenite-induced immunotoxicity in M. galloprovincialis
Neutral red spectrophotometry was used to measuring lysosomal membrane stability. Results showed that lysosomal membrane stability decreased with increasing exposure concentration, which suggested that the permeability of lysosomal membrane increased with the increase of arsenite concentration. In addition, lysosomal membrane stability in high-dose group (100 μg/L) decreased significantly than that in the control group (P <0.05), which indicated that the damage of lysosomal membrane increased after arsenite exposure. Phagocytosis and respiratory burst in hemocytes of M. galloprovincialis were measured by flow cytometer. It was showed that arsenite exposure could induce phagocytosis and respiratory burst, and the induction level declined with the increase of exposure concentration. Moreover, the effect of arsenite exposure on ROS production was in accordance with phagocytic activity. Meanwhile, the effects of arsenite on antioxidant indexes of hepatopancreas and gills were also studied. We found that the enzymatic activities of catalase (CAT) and the content of glutathione (GSH) of gills in medium-dose group increased significantly (P <0.05). Furthermore, the content of malondialdehyde (MDA) in both hepatopancreas and gills in high-dose group increased extremely significantly than that in control group(P <0.01).
(2) Study on arsenite-induced genotoxicity in M. galloprovincialis
The micronucleus test was used to investigate the micronucleus rates of hemocytes in M. galloprovincialis after arsenite exposure. It was found that the micronucleus rates of hemocytes in both medium- (10 μg/L) and high (100 μg/L)-dose group were significantly higher than that in the control group (P <0.05), which suggested that medium- and high-doses of arsenite could induce DNA damage seriously after sub-chronic exposure. The TUNEL assay and histopathology were used to investigate morphological alternation and cell apoptosis in hepatopancreas and gills. The results obtained from the histopathology showed disorganization and damage of gills and hepatopancreas in high-dose group. The TUNEL assay showed that cell apoptosis in medium- and high-dose group was more serious than that in the control group. To elucidate the mechanism of arsenite-induced apoptosis, effects of arsenite exposure on the expression of apoptosis related genes (Bcl-2、Bcl-xL、Caspase-2、Caspase-3、Ras and p63 genes) in hepatopancreas and gills of M.galloprovincialis were measured by RealTime-PCR (real-time polymerase chain reaction). Results showed that the expressions of key genes in apoptosis pathway altered obviously after arsenite exposure. Anti-apoptosis effect was induced strongly in low dose group, while promoting apoptosis effect was observed in high dose group compared with that in control group.
In conclusion, low level of arsenite exposure had significant influence on enzymatic activity of CAT but no obvious genotoxicity was detected on M. galloprovincialis. However, high level of arsenite could induce ROS production and DNA damage in mussels, which indicated that high level of arsenite exposure had serious genotoxicity and slight immunotoxicity on M. galloprovincialis after sub-chronic exposure.
KEY WORDS: Mytilus galloprovincialis, sodium arsenite, genotoxicity, immunotoxicity, apoptosis
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/6750
专题中国科学院烟台海岸带研究所知识产出_学位论文
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李巧梅. 砷(III)对紫贻贝遗传毒性和免疫毒性的研究[D]. 北京. 中国科学院研究生院,2013.
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