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褐牙鲆早期生活史阶段的抗氧化和免疫系统对甲基汞毒性的响应
Alternative TitleThe responses of antioxidant and immune systems of early life stage of flounder (Paralichthys olivaceus) to the toxicity of methylmercury
任中华
Subtype博士
Thesis Advisor窦硕增
2019-05-18
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Degree Discipline海洋生态学
Keyword海水鱼,早期生活史阶段,抗氧化能力,免疫功能,蓄积,生长
Abstract

    甲基汞(Methylmercury, MeHg)是海洋中普遍存在的持久性有机污染物,对海洋生物的生命过程和人类的水产食品安全构成潜在威胁,这也引发国际社会的广泛关注。本研究以褐牙鲆(Paralichthys olivaceus)早期生活史阶段(Early life stage, ELS)为研究对象,通过急性毒性实验(胚胎: 0-24 µg/L, 48 h; 仔鱼: 0-30 µg/L, 96 h)分别计算褐牙鲆胚胎和仔鱼的MeHg半致死浓度(Lethal concentration of 50%, LC50);通过亚急性毒性实验(0-15 µg/L; 胚胎-仔鱼卵黄囊基本消失, 128 h)分析MeHg对褐牙鲆胚胎-仔鱼阶段发育、存活和生长过程的毒性效应;通过MeHg对仔鱼的慢性毒性实验(0-10.0 µg/L, 胚胎-仔鱼着底, 35 d),分析MeHg对褐牙鲆仔鱼抗氧化能力、脂质过氧化(Lipid peroxidation, LPO)水平、免疫功能和生长的影响;通过MeHg对幼鱼的慢性毒性实验(0-20.0 µg/L, 30 d),分析MeHg对褐牙鲆幼鱼不同组织抗氧化能力和免疫功能的影响,分析MeHg在幼鱼体内蓄积的组织特异性,以及MeHg对幼鱼生长的影响。以上研究结果有助于我们深入了解MeHg对褐牙鲆ELS生长、发育和存活过程和重要生理功能的毒性效应,探讨MeHg对鱼类的致毒机制,为研究近海污染条件下鱼类资源关键补充过程的衰退机理提供科学依据,也为海洋环境污染检测技术体系的构建提供理论依据。

    甲基汞急性毒性实验结果表明:MeHg对褐牙鲆胚胎的24 h-48 h-LC50分别为17.415.3 µg/L,对仔鱼的24 h-48 h-72 h-96 h-LC50分别为22.320.719.016.3 µg/L,胚胎对MeHg的毒性更敏感。甲基汞亚急性毒性实验结果表明:当暴露浓度13 µg/L时,胚胎和仔鱼的累积死亡率和仔鱼的累积畸形率均显著升高,而仔鱼的最终体长和卵黄囊吸收率均显著降低,4个生物学指标均表现出显著的浓度制约性,可作为有效的生物学指标应用于评估MeHg对褐牙鲆ELS的毒性效应。然而,MeHg对胚胎的累积孵化率无显著影响,但胚胎在15 µg/L浓度组的孵化比例相比对照组显著升高。亚急性毒性实验中设置的MeHg暴露浓度均低于胚胎和仔鱼的LC50,可能尚未达到抑制胚胎孵化率的阈值,但高浓度的MeHg暴露(15 µg/L)能延长胚胎的孵化时间,对其孵化过程产生不利影响。

    甲基汞对褐牙鲆仔鱼的慢性实验结束后,仔鱼体内的MeHg蓄积量与暴露浓度呈正相关,浓度制约性显著;仔鱼的生长在1.0 µg/L浓度组受抑制。相比对照组,超氧化物歧化酶(Superoxide dismutase, SOD)活性在各个MeHg浓度组差异均不显著,过氧化氢酶(Catalase, CAT)和谷胱甘肽还原酶(Glutathione peroxidase, GR)活性均在10.0 µg/L浓度组显著升高,而谷胱甘肽过氧化物酶(Glutathione peroxidase, GPx)活性和脂质过氧化(Lipid peroxidation, LPO)水平均在1.0 µg/L浓度组显著升高。除了sod之外,抗氧化相关基因(catgpx, 10.0 µg/L; gr, 1.0 µg/L)表达量均显著升高。此外,溶菌酶(Lysozyme, LZM)含量在10.0 µg/L浓度组相比对照组显著升高,而免疫球蛋白MImmunoglobulin, IgM)含量却在10.0 µg/L浓度组显著降低。随着暴露浓度升高,免疫相关基因(hsp70, 0.110.0 µg/L; lzmil-1β, 1.0 µg/L; il-6tnf-α, 10.0 µg/L)表达量显著升高,而igm表达量却在0.1 µg/L浓度组显著降低。总体而言,当暴露浓度1.0 µg/L时,MeHg对仔鱼造成氧化压力,而当暴露浓度达到10.0 µg/L时,MeHg对仔鱼产生免疫毒性。此外,抗氧化和免疫生物标志物的活性(含量)变化与抗氧化和免疫相关基因的表达量变化基本保持同步,共同抵御MeHg暴露对仔鱼产生的氧化压力和免疫毒性,而这些指标对MeHg的毒性响应敏感,可作为有效的指标用于MeHg对鱼类ELS的毒性效应研究。

    甲基汞对褐牙鲆幼鱼的慢性实验结束后,幼鱼体内MeHg蓄积量具有显著的浓度制约性和组织特异性,各个组织内的MeHg蓄积量均随MeHg暴露浓度的升高而升高,各组织对MeHg的蓄积能力总体上呈肝脏>鳃>脾脏>肌肉的趋势。肝脏的LPO水平高于鳃和肌肉,其抗氧化能力生物标志物对MeHg暴露相比鳃和肌肉组织更敏感。具体而言,肝脏内4种抗氧化剂的活性(含量)均在低浓度组被诱导升高,但在高浓度组其活性(含量)被抑制;鳃内CAT活性在各个实验组均无显著差异,但其它3种抗氧化剂的活性(含量)表现出在低浓度组被诱导升高而高浓度组被抑制的趋势;肌肉内CAT活性在各个处理组无显著差异,但其它3种抗氧化剂的活性(含量)在各个浓度组均被诱导升高。此外,肝脏、鳃和脾脏的免疫功能生物标志物活性(含量)因暴露浓度而异,3种组织的酸性磷酸酶(Acid phosphatase, ACP)、碱性磷酸酶(Alkaline phosphatase, AKP)和LZMMeHg暴露敏感,能较好的反映对MeHg暴露的浓度制约关系和组织特异性。同时,当暴露浓度达到20.0 µg/L时,幼鱼的生长被抑制。由此可见,褐牙鲆幼鱼抗氧化能力(SODCATGlutathione S-transferase GST)、Glutathione GSH))和免疫功能生物标志物(ACPAKPLZM),均可作为有效的生物标志物用于MeHg对海水鱼类的毒性效应研究。

Other Abstract

    Methylmercury (MeHg) is a common persistent organic pollutant in the ocean posing potential threats to biological processes of marine organisms as well as human food safety, which has attracted widespread attention worldwide. In this study, flounder (Paralichthys olivaceus) at early life stage (ELS) was used for MeHg toxicity tests. Acute toxicity tests for flounder embryos and larvae (0-24 µg/L, 48 h for embryos; 0-30 µg/L, 96 h for larvae) were performed to calculate the lethal concentration of 50% (LC50) of MeHg for the embryos and larvae, respectively. Subacute toxicity test (0-15 µg/L; embryos-yolk-sac larvae, 128 h) was performed to analyze the toxic effects of MeHg on the development, survival and growth of embryonic-larval flounder. The effects of MeHg on the antioxidant defenses, the level of lipid peroxidation (LPO), immune responses and growth of flounder larvae were analyzed through a chronic toxicity test (0-10.0 µg/L; embryos-settling larvae, 35 d). A chronic toxicity test for flounder juveniles (0-20.0 µg/L, 30 d) was also performed to analyze the antioxidant defenses and immune responses in different tissues, characteristics of MeHg accumulation and the growth of juveniles under MeHg exposure. These results can help us to better understand the toxic effects of MeHg on the growth, development and survival as well as the main physiological functions of flounder at ELS, and to investigate the mechanisms underlying the toxicity of MeHg on fish. Additionally, this study can provide scientific basis for the study of compensatory mechanism of fish resources under marine pollution, and for the construction of detection system of marine pollution.

    The results of acute toxicity tests showed that the 24 h- and 48 h-LC50 values of MeHg for the embryos were 17.4 and 15.3 µg/L, respectively. The 24 h-, 48 h-, 72 h- and 96 h-LC50 values of MeHg for the larvae were 22.3, 20.7, 19.0 and 16.3 µg/L, respectively. These results demonstrated that flounder embryos were more sensitive to MeHg toxicity than were the larvae. The results of subacute toxicity test for embryonic-larval flounder showed that MeHg exposures at 13 μg/L increased accumulative mortality of embryos and larvae as well as larval morphological deformities; reduced final total length of larvae; and inhibited yolk absorption rate of larvae. These four endpoints were sensitive to MeHg and their responses were dose-dependent, which could be used as effective bioindicators for assessing the toxic effects of MeHg to the ELS of flounder. However, the accumulative hatchability of flounder embryos did not significantly differ between MeHg concentrations and the control, but the hatching rate of the embryos at 15 μg/L was significantly increased relative to the control. These results suggested that MeHg exposure in the subacute test at concentrations LC50 values of embryos and larvae did not reach the threshold at which it starts to affect the hatchability of flounder embryos. However, MeHg exposure at 15 µg/L could prolong the hatching time and adversely affect the hatching process of embryos.

    After chronic test for flounder larvae, MeHg accumulation in the larvae was positively correlated with MeHg concentration in a dose-dependent manner. Moreover, MeHg exposure at 1.0 µg/L concentrations reduced larval growth. Compared to the controls, the superoxide dismutase (SOD) activity did not significantly differ among MeHg concentrations, while the activities of catalase (CAT) and glutathione reductase (GR) were significantly increased at 10.0 µg/L concentration, and the glutathione peroxidase (GPx) activity as well as lipid peroxidation (LPO) level were significantly increased at 1.0 µg/L concentrations. The corresponding antioxidant-related genes were upregulated under MeHg exposure (cat and gpx at 10.0 µg/L; gr at 1.0 µg/L), while the transcription of sod was not significantly affected by MeHg exposure. Furthermore, lysozyme (LZM) content was significantly increased, but immunoglobulin M (IgM) content was significantly decreased at 10.0 µg/L concentration relative to the controls. As MeHg concentration increased, the immune-related genes were significantly upregulated (hsp70 at 0.1 and 10.0 µg/L; lzm and il-1β at 1.0 µg/L; tnf-α and il-6 at 10.0 µg/L), while igm was significantly downregulated at 0.1 µg/L concentrations. Overall, MeHg exposure induced oxidative stress at 1.0 µg/L concentrations and caused immunotoxicity at 10.0 µg/L concentration, respectively. Moreover, the transcription of selected genes correlated with the corresponding antioxidant- or immune-related biochemical markers to defense against oxidative stress and immunotoxicity induced by MeHg exposure. These markers were sensitive to MeHg toxicity and could be used as effective indicators to assess the toxicity of MeHg to the ELS of fish.

    After chronic test for flounder juveniles, MeHg accumulation in vivo showed a significant concentration-dependent and tissue specificity tendency. In particular, MeHg accumulation in different tissues increased significantly as MeHg concentration increased as followed in the order of livergillsspleenmuscle. The level of LPO in the liver was higher than those in the gills and muscle, and the activities or contents of antioxidants (e.g., SOD, CAT, Glutathione S-transferase (GST) and Glutathione (GSH)) in the liver were more sensitive to MeHg exposure than those in the gills and muscle. Specifically, the activities or contents of these four antioxidants in the liver were significantly induced at low MeHg concentrations, whereas inhibited at high exposure. Furthermore, CAT activity in the gills and muscle showed no significant difference among MeHg concentrations and the controls. However, the activities or contents of the other three antioxidants in the gills also showed a trend the same to the liver. Meanwhile, the activities or contents of SOD, GST and GSH in the muscle were all significantly induced at each MeHg concentration compared to those in the controls. Additionally, the activities or contents of acid phosphptase (ACP), alkaline phosphatase (AKP), LZM and IgM responded differently to MeHg exposure at different concentrations. Generally, ACP, AKP and LZM in the gills, liver and spleen can better reflect the concentration-dependent and tissue specificity tendency of immune responses to MeHg exposure. Finally, the growth of juvenile was significantly inhibited at 20.0 µg/L. Therefore, the activities or contents of antioxidants (SOD, CAT, GST and GSH) as well as the activities or contents of ACP, AKP and LZM in different tissues of flounder juvenile can all be used as effective bioindicators for the toxicity study of MeHg on marine fish.

Subject Area生物学
MOST Discipline Catalogue理学
Pages89
Funding ProjectNational Key Basic Research Program of China (973 Program)[2015CB453302]
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156889
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
任中华. 褐牙鲆早期生活史阶段的抗氧化和免疫系统对甲基汞毒性的响应[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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