【作者】 柏世军；

【导师】 许梓荣；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2006， 博士

【摘要】 重金属镉已成为世界各地水域主要污染物之一，严重影响水生生物的生长、发育、繁殖，并通过食物链损害人类健康。鱼类与哺乳动物（包括人）生理构造相似，大多数污染物对鱼类和人类机体产生类似影响。因此，深入研究Cd²⁺对鱼类的毒害作用以及机理对实现水产养殖业可持续发展和人类健康具有重要意义。本试验以世界广泛养殖的罗非鱼（Oreochromis niloticus）为试验对象，通过对水环境Cd²⁺胁迫作用下罗非鱼内分泌、血浆渗透压和离子组成调节能力、能量代谢、GSH代谢等方面的研究，对Cd²⁺内分泌干扰作用以及ROS诱导机制进行了探讨。具体内容主要包括以下几方面：1罗非鱼对水环境Cd²⁺的敏感性采用静水水生生物急性毒性试验方法，观察罗非鱼中毒症状并计算半数致死浓度(LC₅₀)。结果发现高浓度组鱼首先出现异常行为：游动急促，上下窜动；随后身体失去平衡，游动缓慢，反应迟钝，最后呈螺旋式运动，逐渐丧失运动能力而静止在底部，直至死亡。低浓度组出现中毒症状需要时间较长，但一旦中毒症状相同。解剖显示：血液暗红色；肝脏颜色加深，质地松软；腹腔内充满黄色胶胨状物质；鱼鳃水肿，呈紫红色。计算结果显示，水环境Cd²⁺对罗非鱼鱼种24h、48h、72h和96h LC₅₀分别为27.73mg/L，16.87mg/L，12.43mg/L和9.11mg/L，安全试验浓度为0.91mg/L。1罗非鱼对水环境Cd²⁺的蓄积和排放采用室内模拟方法，将罗非鱼在不同浓度Cd²⁺（0.5mg/L和1.0mg/L）溶液中暴露60d后再转移到清洁水体排放30d。结果表明，罗非鱼对水环境Cd²⁺蓄积具有时间和剂量效应关系；蓄积速度因组织而异：鳃、肝、肾Cd²⁺浓度迅速增加，肌肉中增加缓慢。60d后Cd²⁺蓄积量为肾>肝>鳃>肌肉。排放期间，鳃和肌肉Cd²⁺迅速降低，肝Cd²⁺含量几乎维持不变，肾Cd²⁺含量略有增加。排放30d后，肌肉Cd²⁺排出率59.37％（0.5mg/L）和80.44％（1.0mg/L）。表明受Cd²⁺污染罗非鱼经清洁水排放后有望达到食用标准。3水环境Cd²⁺对罗非鱼渗透压和血浆离子组成的影响将罗非鱼分别暴露在0.5和1.5mg/L Cd²⁺溶液中，分别在5，10和20d后对血浆渗透压、离子组成及调节系统进行了检测。结果显示，0.5mg/LCd²⁺暴露5d后鳃扁平上皮细胞（PC）微脊数量增加，泌氯细胞（CC）和ATPase无明显变化，促肾上腺皮质激素（ACTH）、皮质醇（Cor）和3，5-环化腺苷酸（cAMP）含量以及头肾腺苷酸化酶（AC）活性显著升高（p<0.05）；10d后，PC微脊增多并呈不规则排列，CC表面积增加，但数量无明显变化，ATPase活性显著升高（p<0.05），其余指标恢复正常；20d后，CC数量增加，颗粒物排列密度降低，部分CC表面积增加，呈丘状突出；ATPase、ACTH、Cor和cAMP含量以及AC活性显著高于对照（p<0.05）；20d暴露期内渗透压和离子组成无显著变化（p>0.05）。1.5mg/LCd²⁺20d暴露期内PC细胞个体清晰可辨，微脊明显增多；CC数量和ATPase活性在暴露5d后呈增加趋势（p>0.05），ACTH、Cor和cAMP含量以及AC酶被显著诱导（p<0.05），血浆K⁺浓度显著升高，Na⁺和Cl^-浓度显著下降（p<0.05），Ca²⁺浓度和渗透压正常；10d后CC数量明显增加，颗粒物排列密度明显降低，ATPase显著升高（p<0.05），其余检测指标正常；20d后CC数量、Ca²⁺-ATPase、渗透压、Ca²⁺、Na⁺和Cl^-浓度显著降低（p<0.05），K⁺、ACTH浓度显著高于对照，Cor水平正常。20d暴露期内，所有处理组MDA与对照无显著差异（p>0.05）。由此可见，高浓度Cd²⁺长时间胁迫干扰ACTH信号传递，影响Cor合成和释放，损害了鱼体修复能力，从而诱发血浆渗透压和离子组成不可逆紊乱。因此，内分泌信号传递干扰作用可能是Cd²⁺毒性机理之一。4．水环境Cd²⁺对罗非鱼鳃能量代谢的影响将罗非鱼分别暴露在0mg/L、0.5mg/L和2.5mg/LCd²⁺水中7d，观察鳃线粒体超微结构并对线粒体琥珀酸脱氢酶（SDH）、细胞色素氧化酶（CCO）、超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量和鳃磷酸果糖激酶（PFK）、乳酸（LD）、游离氨基酸（FAA）、总蛋白、谷丙转氨酶（GPT）、谷草转氨酶（GOT）、磷酸烯醇式丙酮酸羧激酶（PEPCK）、血糖、血清FAA和能量负荷（EC）进行了检测。结果显示，0.5mg/LCd²⁺胁迫作用下，SOD、SDH和CCO活性在前5d暴露时间内被持续诱导，7d后恢复正常；血糖仅仅在暴露后的第3d和第5d显著高于对照（p<0.05）；其余检测指标无显著变化。2.5mg/L处理组7d后可见部分线粒体肿胀、空泡化、膜结构破损，线粒体计分显著升高（p<0.05）。SOD活性被逐渐诱导，3d后达最高，但从第5d起开始下降，7d后显著降低（p<0.05）。PFK、SDH和CCO活性逐渐增加，5d后活性最大，比对照分别高58.33％、37.00％和59.62％，同时FAA、GOT、GPT和PEPCK也显著高于对照（p<0.05）；7d后，除PEPCK恢复正常外，其余酶活指标均显著降低而LD显著增加（p<0.05）；血糖持续升高，7d后浓度达到0.97mg/L，比对照提高67.24％（p<0.005）；FAA在暴露后的第5d和第7d比对照显著高30.91％和60.31％，浓度分别为0.72和0.75mg/mL。EC仅仅在暴露7d后显著降低（p<0.05）。结果提示：低浓度Cd²⁺暴露导致罗非鱼蛋白质周转代谢和糖氧化分解加强；高浓度Cd²⁺短时间胁迫作用下罗非鱼不但加强糖氧化分解满足能量需求和加快蛋白质周转代谢合成各种应激分子，还分解部分蛋白氧化供能。但高浓度Cd²⁺胁迫长时间胁迫，将抑制线粒体SOD酶活性，导致线粒体氧化损伤，同时抑制PFK，引发鱼鳃能量供应障碍。因此，Cd²⁺引发鱼鳃能量代谢障碍可能是Cd²⁺毒性机理之一。5．水环境Cd²⁺胁迫作用下罗非鱼肝脏GSH代谢的变化和活性氧（ROS）的诱导机理将罗非鱼鱼种在3.0mg/LCd²⁺暴露1、5、10、20和40d后，对还原型谷胱甘肽（GSH）、氧化型谷胱甘肽（GSSG）和谷胱甘肽循环代谢相关酶活性进行测定，并对GSSG-GSH比率进行了计算。结果表明，GSH含量逐渐降低，GSSG-GSH比率逐渐升高；硒依赖谷胱甘肽过氧化酶（Se-GPX）、谷胱甘肽硫转移酶（GST）、葡萄糖—6—磷酸脱氢酶（G6PDH）的活性和GSSG含量在前20d暴露期内逐渐升高，40d后显著降低，但仍显著高于对照（p<0.05）。谷胱甘肽还原酶（GR）和γ—谷胺酰二酰基半胱胺酸合成酶（γ-GCS）活性在试验期间变化不明显，GR仅仅在40d后显著降低，暴露20d后γ-GCS短暂升高。40d后线粒体计分显著增加。结果表明Cd²⁺亚慢性胁迫作用初期肝脏通过加强GSH合成和周转代谢抵御Cd²⁺的毒害作用，但随着暴露时间延长，这种反应逐渐丧失。结果提示：Cd²⁺在初期诱导ROS生成增加并非通过损伤线粒体完成，但GSH耗竭将导致线粒体损伤而将加速ROS形成。更多还原

【Abstract】 Contamination of fresh water systems by heavy metal cadmium（Cd） has been recognized as a global environmental problem, exposure of fish to even low Cd²⁺ concentrations can have severe toxic effects on growth, development and reproduction. The transfer of Cd²⁺ through food chains also endangers the human health. The effects of pollutants on fish are similar to that of mammalian（including human being） due to the semblable physiological structure. Thus, the deep investigation on the toxic effects of Cd²⁺ on fish is quite important for the realization of sustained development of aquaculture, environmental protection and human health. In current study, the variations of tilapia Oreochromis niloticus cultured worldwide in endocrine, osmo-and ion-regulation and energy metabolism as well as the GSH metabolism have been investigated, the possible mechanisms for the endocrine disruption and the induction of reactive oxygen species（ROS） have been intensively discussed. The main contents are as following:1 The sensitivity of tilapia O. niloticus to waterborne Cd²⁺The acute toxicity of Cd²⁺ to tilapia O. niloticus was studied with the static test method. The intoxication symptom and the LC₅₀（median lethal concentration） value through linear regression based on the recording mortality rate were investigated. The results demonstrated that the fish exposed to the highest Cd²⁺ concentration showed abnormal behavior initially. The main symptoms showed that the swimming ability of fish was affected heavily, the fish hastily swim up and down at the beginning of intoxication, subsequently, fish loss body balance and swam slowly and responses obtuse, with the prolonged exposure period the fish swam spirally and gradually lose the swimming ability and died finally. The intoxication symptom appeared after a longer exposure period in the fish exposed to lower Cd²⁺ concentrations, however, the pathological changes were similar in spite of the different exposure concentrations. The results of dissection showed that the fish were characteristic of dull-redness blood, purple gill covered with much mucous and swollen abdominal cavity filled with yellow jelly materials. The LC₅₀ of 24, 48, 72 and 96h for Cd²⁺ to juvenile tilapia are 27.73mg/L, 16.87mg/L, 12.43mg/L and 9.11mg/L, respectively. The safe concentration of Cd²⁺ to juvenile tilapia is 0.91 mg/L.2 Kinetics of cadmium accumulation, distribution and elimination in tilapiaThe Cd²⁺ accumulation and elimination in the tilapia after exposure to sublethal concentrations(0.05, 0.5 mg l^-1 Cd²⁺) for 60 days followed by a 30-day depuration under a simulated pollution had been investigated. The results showed that Cd²⁺ exposure resulted in an increased Cd uptake in tissues with the exposure periods and concentrations, and the patterns of accumulation varied with the organs. For gill, liver, and kidney Cd²⁺concentrations increased sharply. However, Cd²⁺ level in muscle was increased slowly. After 60 days Cd²⁺ exposure, the order of Cd²⁺ accumulation was in organs kidney>liver>gill>muscle. During the elimination days, the loss of accumulated Cd²⁺ was rapid and immediate in gill whereas the Cd²⁺ concentration increased slightly in kidney and remained constant in liver and muscle. At the end of the cleaning period, the elimination rate in muscle was about 59.37 % at 0.5mg/L Cd²⁺ and 80.44 % at 1.0mg/LCd²⁺. Hence, it is expected that the tilapia contaminated by waterborne Cd can reach the edible standard after elimination in the cleaning water.3 The effects of waterborne Cd²⁺ on the plasma osmolality and ion compositionJuvenile tilapia were exposed to different Cd²⁺ concentrations（0.5, 1.5mg/L） for 20 days. The plasma osmolality and ion composition as well as the regulation system were investigated after the exposure of 5, 10 and 20 days. The results showed that the exposure ot 0.5mg/L Cd²⁺ for 5 days caused to an increase in the microdige of pavement cells（PC） and cortisl（Cor）, adrenocorticotrophic hormone（ACTH） concentrations in plasma as well as the adenylate cyclase（AC） activity and cAMP contents in head kidney but no variation in chloride cells（CC） and ATPase. After 10 days of exposure, the microdige of PC becomes irrgeluar and the surface area of CC was elevated without significant changes in the number, ATPase activities werer enhanced and other indicators return to normal level. Subsequently, the amounts were enhanced with a decreas in the denstity of granule in the CCs while the surface area of partial CCs were increased and showed hillock prominence after 20-day exposure, meanwhile, ATPase and AC activities and ACTH, Cor, cAMP concentrations were higher significantly than the control. There was no significant change in the plasma osmolality and ion composition during the whole exposure period. On the other hand, there was an obvious increase in the microridge of the squamous PCs, and the individual was discriminable during the whole exposure period at 1.5mg/LCd²⁺, while the CC amount and ATPase activities showed an increase trend after 5-day exposure without significant difference, however, ACTH, Cor, cAMP, K⁺ concentration as well as AC were elevated significantly, in addition, the osmolality and Ca²⁺ concentration reamined at normoral level whereas Na⁺ and Cl^-concentrations were decreased significantly（p<0.05）. After 10 days of exposure, CC amounts increased and the density of granule decrease obviously, ATPase were increased significantly with normal value in other indicators assayed. A similar change to that of 5-day exposure in K⁺, Na⁺ and Cl^-concentrations was detected after 20 days of exposure, meanwhile a significant decrease in CC amount, Ca²⁺-ATPase, osmolality and Ca²⁺ concentration were observed, whereas, a normal Cor and augmented ACTH concentration in plasma were detected. Furthermore, the MDA levels in kidney were kept at the control levels in two treatment groups. The results showed that the impairment of gill, especial the inhibition of ATPase would lead to the disruption of plasma ion composition and osmolality, however, the fish can cope with the stress through enhancing secretion of Cor, which can increase CC amount and surface and ATPase activity and strengthen the regulation ability. But exposure of fish to the higher concentration for a longer time, the regulation ability of plasma osmolality and ion composition. The normal ACTH concentration and AC activity suggest that impairment of ion regulation and osmolality may be ascribe to the disruption of ACTH signal transduction and/or inhibit the synthesis of Cor, which would lead to a decrease in the repair ability. Hence, the disruption of endocrine signal transduction would be one of the possible mechanisms for Cd²⁺toxicity.4 The effects of waterborne Cd²⁺ on the energy metabolism in the gills of tilapia and the possible mechanismThe activities of succinate dehydrogenase（SDH）, cytochrome C oxidase（CCO）, superoxide dismutase（SOD） and MDA in mitochondria in the gills of tilapia and phosphorfucto kinase（PFK）, glutamate oxaloacetate transaminase（GOT）, glutamate pyruvate transaminase（GPT）, phosphoenolpyruvate carboxykinase（PEPCK）, lactic acid（LD）, total protein, free amino acid（FAA）, ATP, ADP, AMP in gills and plasma glucose as well as FAA in plasma were determined after exposure to different Cd²⁺ concentrations(0mg/L、0.5mg/L and 2.5mg/L for 7 days. The results showed that 5-day exposure of fish to 0.5mg/L Cd²⁺ led to a constant elevation in SOD, SDH and CCO activites while returned to normal level after 7 days of exposure, meantime a transitional increase in plasma glucose on the third and fifth day were detected. Howver, exposure to 0.5mg/L Cd²⁺ had no significant effect on other index. The exposure of fish to 2.5mg/L lead to damage to the mitochondrion in the gill, and some swollen and blank regions and broken membranes in mitochondria were observed after 7 days of exposure. Meanwhile, the mitochondrial scoring was significant higher than the control. The SOD activities was induced significantly in the initial period and rose to the maximum after 3 days of exposure and showed a decreasing trend and the difference became significant after 7-day exposure. PFK, SDH and CCO activities also were enhanced continuously at the beginning and were up to the highest value and elevated by 58.33%, 37.00%and 59.62% than the control, respectively, at one time, FAA contents and PEPCK, GPT, GOT activities were increased significantly. However, all indicators assayed were decreased significantly apart from the return of PEPCK to normal levels and significant elevation in LD contents after 7 days of exposure. Plasma glucose were increased continuously during the exposure period and reached the highest with 0.97mg/L, which was increased by 67.24%（p<0.005）, at the end of the experiment. The significant increase in plasma FAA concentration was detected on the 5th day and 7th day, which was increased by 30.91 % and 60.31 % as compared to the control, respectively.The present results demonstrated that tilpia speed up the protein turnover metabolism and carbohydrate aerobics glycolytic to satisfy the energy requirement after exposure of tilapia to lower concentration of Cd²⁺ for a short time. For the short-term exposure of fish to higher concentration of Cd²⁺, fish also enhance the supply of energy through speeding the carbohydrate aerobics glycolytic and decompose partial protein to cope with the stress, but for a long-term exposure there is obstacle in the supply of energy due to the decrease in PFK activity and the impairment of mitochondrion resulting from the inhibition of SOD. Hence, it can be concluded that the obstacle of energy metabolism was one of the possible mechanism for Cd²⁺ toxicity.5 The variation of hepatic GSH metabolism and the induction of ROS after exposure to waterborne Cd²⁺To explore the effects of Cd²⁺ exposure of tilapia（Oreochromis niloticus） on hepatic glutathione（GSH） metabolism, tilapias were exposed to 3.0 mg/l Cd for 1,5,10, 20 and 40 days. The contents of reduced GSH and oxidized GSH（GSSG） and the activities of enzymes involved in GSH metabolism and the GSSG-GSH ratio were investigated. The results showed that reduced GSH were depleted progressively whereas the GSSG-GSH ratio increased. The activities of selenium-dependent glutathione peroxidase, glutathione S-transferase and glucose-6-phosphate dehydrogenase and GSSG levels increased initially and decreased subsequently but still higher than the controls. Glutathione reductase activity dropped on the 40th day. A transient increase inγ-glutamylcysteine synthetase activity was detected on the 20th day. Mitochondrion scoring was elevated significantly only after 40 days of exposure. These results indicated that sub-chronic exposure to waterborne Cd²⁺ affect the hepatic GSH metabolism. During the initial period, the synthesis and turnover were strengthened to deal with the stress while the response ability weakened for a longer exposure. In addition, the significant elevation in mitochondrion scoring after 40 days of exposure while the GSH was depleted suggested the ROS in the initial period was induced via other routines not mitochondrion impairment, but the depletion of GSH accelerate the formation of ROS due to the impairment of mitochondrion.更多还原