【作者】 杨丽萍；

【导师】 李莉；

【作者基本信息】 华中农业大学 ， 水生生物学， 2021， 硕士

【摘要】 随着世界各地水体富营养化的加剧,蓝藻水华已成为一个全球关注的环境污染问题。在蓝藻水华期间,除了由蓝藻细菌产生的微囊藻毒素,缺氧环境导致的水体亚硝酸盐水平的急剧上升常常被忽视。然而,环境中微囊藻毒素和亚硝酸盐可以在水中共存,对水生生物产生潜在的联合毒性作用。因此,本研究对两者联合的神经毒性作用及其可能的机制进行了探究,主要结果如下:在单独的亚硝酸盐暴露实验中,将成年雄性斑马鱼(Danio rerio)暴露在0、0.2、2、20 mg/L的亚硝酸盐养殖环境中30 d,观察记录斑马鱼的神经行为学变化、脑组织病理学变化,测定神经递质系统相关指标以及氧化-抗氧化系统参数。研究结果显示:亚硝酸盐暴露导致斑马鱼的行为出现异常,包括运动能力下降、对同类鱼群聚集的倾向性降低以及学习和记忆能力减弱,表明亚硝酸盐发挥了神经毒性作用。在测定神经递质系统相关指标时发现,多巴胺(DA)的含量下降,DA代谢相关基因(dat、drd1b和drd4a),胆碱能系统相关基因(ache、chrna4b、chrna7),GABA系统中的gabra1和gat1以及5-羟色胺受体家族基因(htr1aa、htr1ab、htr2a)的m RNA表达水平均有不同程度的下调,说明神经递质系统信息传递的功能可能会受到抑制。另外,我们发现水环境中亚硝酸盐的浓度升高导致鱼体血浆中高铁血红蛋白的累积,提示鱼体缺氧问题。脑组织病理学结果显示,随着亚硝酸盐暴露浓度的升高,中脑小球周灰质带的神经细胞数量减少,并在最高亚硝酸盐浓度组中出现血栓,进一步表明大脑缺氧情况的出现。同时丙二醛(MDA)含量的浓度依赖性增加以及总抗氧化能力(T-AOC)的下降,表明斑马鱼脑部产生了氧化应激并破坏了抗氧化防御系统与活性氧之间的平衡,而超氧化物歧化酶(T-SOD)活性的降低和Cu/Znsod、Mnsod转录水平的下调进一步证实亚硝酸盐暴露导致了斑马鱼大脑抗氧化能力的降低。综上所述,亚硝酸盐慢性暴露诱导氧化应激发生,使机体抗氧化能力下降、脑组织结构损伤,进而造成神经递质系统功能紊乱及行为异常。在联合暴露实验中,成年雄性斑马鱼(Danio rerio)被暴露在MC-LR(0、3、30μg/L)和亚硝酸盐(0、2、20 mg/L)的不同组合溶液中30 d。通过采集斑马鱼脑组织样品,观察其组织病理变化并测定神经递质系统和抗氧化系统相关参数和基因表达水平。结果显示,单独MC-LR或亚硝酸盐对大脑超微结构造成了浓度依赖性的损害,两者联合暴露时的毒性作用更加强烈。与神经纤维髓鞘有关的基因mbp和bdnf的表达下调,进一步证实了MC-LR和亚硝酸盐可以对神经元纤维的结构和功能造成损害。多巴胺含量、乙酰胆碱酯酶活性和相关基因m RNA转录水平的下降表明,MC-LR和亚硝酸盐对斑马鱼大脑中的多巴胺能和胆碱能系统的正常功能产生了不利影响。此外,丙二醛(MDA)的显著增加表明,MC-LR、亚硝酸盐及其联合暴露引起的氧化应激的发生,这与抗氧化酶Mn-SOD活性及其转录水平的显著下降相一致。总之,MC-LR+亚硝酸盐的联合暴露对鱼脑的结构和神经递质系统有协同的神经毒性作用,而这两个因素引起的抗氧化能力的破坏可能是潜在的协同机制之一。因此,在持续的蓝藻水华事件中,存在着鱼类被诱发神经中毒的风险。更多还原

【Abstract】 With the increasing eutrophication of water bodies around the world,cyanobacterial blooms have become a common environmental pollution problem.During cyanobacterial blooms,apart from the microcystins,the dramatic increase in nitrite levels in the water due to anoxic conditions is often ignored.However,environmentally relevant concentrations of microcystins and nitrite can co-exist in water and may have potential combined toxicity to aquatic organisms.In order to investigate their potential neurotoxic effects and possible mechanisms,the following experiments were designed and main results were as follows,In toxicological experiment with nitrite alone,adult male zebrafish(Danio rerio)were exposed to 0,0.2,2 and 20 mg/L nitrite for 30 d.After 30-day exposure period,zebrafish behavior changes were recorded with alterations in brain histopathology,the neurotransmitter system-related indicators and oxidative-antioxidative system parameters.We detected that nitrite induced abnormal behavior of zebrafish,including significant decreases in locomotor activity,the tendency to gather in similar fish groups and learning and memory abilities.The detection of indicators related to the neurotransmitter system found that dopamine levels were decreased along with the m RNA expression levels of genes related to DA metabolism(dat,drd1 b,drd4a).Meantime,the transcription levels of cholinergic system related-gene(ache,chrna4 b,chrna7),gabra1 and gat1 in the GABA system,and 5-hydroxytryptamine receptor family genes(htr1aa,htr1 ab,htr2a)were also significantly down-regulated.Moreover,the results found the increase of plasma methemoglobin,implied a lack of oxygen problem.Brain histopathology results displayed a decrease in the number of neuronal cells in periglomerular gray zone of mesencephalon with increasing nitrite exposure concentrations and the formation of thrombus.The concentration-dependent increase of MDA content and the decrease in total antioxidant capacity(T-AOC)indicated that zebrafish brain suffered from nitrited-induced oxidative stress.The decreases of T-SOD activity and Cu/Znsod and Mnsod m RNA expression levels further confirmed that nitrite exposure caused a decrease in the antioxidant capacity of zebrafish brain.In summary,chronic exposure to nitrite can cause oxidative stress,affect the tissue structure of zebrafish brain,which further cause behavioural abnormalities and causes dysfunction of the neurotransmitter system.In the joint experiment,the concentrations of Microcystin-LR and nitrite were set to0,3,30 μg/L and 0,2,20 mg/L,respectively,and then adult zebrafish were immersed into 9different joint solutions of MC-LR and nitrite for 30 d to explore the neurotoxic effects and underlying mechanisms.The results showed that single factor MC-LR or nitrite has caused a concentration-dependent damage in brain ultrastructure and the effects of their joint exposure much more intense.Downregulated expression of genes mbp and bdnf associated with myelination of nerve fibers further confirmed that MC-LR and nitrite could cause damage to the structure and function of neuron fibers.The decreases of dopamine content,ACh E activity and related gene m RNA levels indicated that MC-LR and nitrite adversely affected the normal function of the dopaminergic and cholinergic systems in zebrafish brain.In addition,a significant increase in MDA indicated that oxidative stress caused by MCLR,nitrite,and their joint-exposure was generated in the brain,which paralleled a significant decrease in antioxidant enzyme Mn-SOD activity and its transcription level.In conclusion,MC-LR + nitrite joint-exposure has synergistic neurotoxic effects on the structure and neurotransmitter systems of fish brain,and antioxidant capacity disruption caused by these two factors might be one of the underlying synergistic mechanisms.Therefore,there is a risk of being induced neurotoxicity in fish during sustained cyanobacterial bloom events.更多还原