镉对华溪蟹糖和蛋白质分解代谢的影响机制研究
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摘要
研究目的及意义：山西省在煤炭工业发展、有色金属冶炼和电子工业兴起中普遍存在着镉污染。大量研究表明,当动物受到镉胁迫时,其体内的能量代谢首先会发生变化。本学位论文在课题组前期研究的基础上,综合分析了急性和亚慢性镉处理后,河南华溪蟹(Sinopotamon henanense)糖和蛋白质分解代谢和能量产生的变化,并从细胞形态学、动物生理学和分子生物学等层面分析了可能的中毒机制,旨在明确溪蟹对镉的代谢反应,筛选敏感分子作为污染指示物,揭示S. henanense对急性和亚慢性镉处理的差异性应激反应,为进一步利用该指示生物鉴定水体的突发性污染和长期污染提供科学依据。
研究内容与方法：首先采用亚慢性毒性实验方法,研究了镉对S. henanense血淋巴、肝胰腺和肌肉等组织中糖和蛋白分解代谢基本参数的影响,主要包括与糖分解代谢相关的糖原、葡萄糖、乳酸、乳酸脱氢酶等参数；与蛋白质分解代谢相关的总蛋白、蛋白酶、游离氨基酸、转氨酶、氨、尿素、谷氨酰胺等参数,初步掌握了镉对S. henanense主要能量物质分解代谢的影响。在进一步分析胁迫条件下糖分解代谢的主要途径,即有氧氧化(能量产生的主要方式)和磷酸戊糖途径(还原力产生的主要途径)的变化之前,先分别在急性和亚慢性毒性实验条件下,采用氧电极法对有氧氧化活性的重要衡量指标“氧消耗”进行了测定。在此基础上,针对不同组织的功能特点,采用酶学检测、亚显微技术、基因克隆、实时定量PCR对主要组织糖分解代谢活性进行了评价,主要包括肝胰腺组织磷酸戊糖途径(检测指标为葡萄糖-6磷酸脱氢酶(G-6-PDH)活性和NADPH含量,还原型谷胱甘肽(GSH),氧化型谷胱甘肽(GSSG))含量和GSH/GSSG比值),心脏组织呼吸代谢活性以及肌肉组织呼吸代谢活性(检测指标包括ATP含量、三羧酸循环关键酶NAD-异柠檬酸脱氢酶(NAD-IDH)、呼吸链终端酶细胞色素C氧化酶(CCO)、糖酵解关键酶乳酸脱氢酶(LDH)、CCO活性亚基Ⅰ(cco-1)和ldh mRNA表达水平)。除此之外,为了阐明不同实验条件下氧消耗变化的原因,应用比色法、显微和亚显微技术检测了血淋巴中氧合血蓝蛋白的含量和比例,观察了鳃组织和细胞形态的变化；并采用火焰原子吸收法、镉-血红蛋白饱和法和比色法检测了两种处理对鳃组织中Cd2+蓄积、金属硫蛋白(MT)含量、总超氧化物歧化酶(SOD)活性和膜脂质过氧化产物丙二醛(MDA)含量的影响,旨在揭示急性和亚慢性镉处理导致鳃结构功能发生变化的原因。最后,基于本研究所发现的血糖水平的变化,以及甲壳动物高血糖激素(CHH)对血糖水平的调节作用和镉对CHH潜在的干扰和抑制作用,对CHH合成与分泌的器官“X器-窦腺复合体”的显微和亚显微结构进行了观察,从形态学上初步证明了镉对CHH合成和分泌的影响,为进一步深入研究镉代谢的干扰机制奠定了基础。
研究结果：
(1)Cd2+暴露7d后,肝糖原和肌糖原含量下降,蛋白质含量降低,伴随着蛋白酶活性的升高,组织中游离氨基酸及谷丙、谷草两种转氨酶活性的增强(P<0.05)；血淋巴中的乳酸脱氢酶活性升高,而乳酸的含量并没有发生显著变化(P>0.05)。血淋巴中游离氨的含量在该时间段几乎没有发生变化,而血淋巴中检测到的升高的尿素和谷氨酰胺含量则说明了游离氨的去向。Cd2+暴露21d导致了血淋巴中葡萄糖含量的下降；谷丙、谷草两种转氨酶活性在该时间段降低,这将削弱机体对游离氨基酸的利用；同时,血淋巴中游离氨的含量升高,尿素的含量没有显著变化,仅可见谷氨酰胺含量的上升。
(2)氧消耗在急性处理组增加,而在亚慢性处理组则相反；氧合血蓝蛋白在血淋巴中的含量和比例经急性和亚慢性镉处理后的变化趋势同氧消耗基本一致；鳃形态学观察结果显示,急性镉处理后鳃叶肥大增厚,部分上皮细胞中出现空泡；亚慢性处理后,鳃叶边缘通道紧缩,上皮细胞高度肿胀,鳃血腔缩小变窄,上皮细胞顶膜微绒毛减少,线粒体嵴溶解消失,细胞核膜断裂变形,核染色质浓缩,边集化；两个处理组中的Cd2+蓄积、MT含量、SOD活性和MDA含量皆高于对照组,其中Cd2+蓄积量和SOD活性在两个处理组之间没有显著差异；急性处理组MT含量显著高于亚慢性组；而亚慢性组MDA含量显著高于急性组。
(3)急性镉处理后,G-6-PDH活性显著下降,伴随着NADPH、GSH含量和GSH/GSSG比值的下降；亚慢性镉处理后,在2.86mg/L组,G-6-PDH活性和NADPH含量显著上升,GSH含量先(0.71mg/L)上升后(2.86mg/L)下降,GSSG含量随处理浓度增加显著上升,伴随着GSH/GSSG比值的下降。
(4)急性和亚慢性镉处理皆导致溪蟹心脏组织CCO活性的降低和cco-1mRNA表达水平的下调；急性镉处理后,心脏组织ATP含量升高,伴随着IDH活性的上升和LDH活性的下降,而亚慢性镉处理后,IDH活性降低,同时ldh mRNA表达上调,LDH活性升高,组织ATP含量降低,线粒体嵴断裂甚至空泡化。
(5)急性处理组肌肉组织ATP含量没有发生明显的变化,而亚慢性处理在2.86mg/L组ATP含量显著下降；IDH、CCC、LDH酶活性在急性处理组都没有发生显著变化,而在亚慢性处理组显著下降；cco-1mRNA表达在急性处理的14.28mg/L浓度组显著上调,随着浓度的升高下调至对照组水平,ldh mRNA表达则在急性处理的各浓度组都显著上调。cco-1和ldh mRNA表达在亚慢性处理组的最高浓度皆显著下调。
(6)X器由许多神经内分泌细胞构成,正常组窦腺呈囊状,中央为血窦腔,周围是轴突、膨大的末梢和胶质细胞组成的壁,三者在腺体内紧紧缠绕,形成坚实的结构,固定了腺体的形状,轴突神经末梢的排列沿中央血腔呈放射状排列,血窦周围分布着致密的神经内分泌颗粒,包裹在不同的神经内分泌细胞的轴突膨大的末梢中,轴突末梢中含有少量的线粒体。镉处理组中的窦腺结构明显比对照组疏松,形成窦腺壁的栅栏结构消失,原本由轴突、膨大的末梢和胶质细胞紧密排列的结构变得疏松多孔,窦腺结构临近崩溃；血窦周围分布的神经分泌颗粒减少,轴突末梢中含有大量空泡和部分受损的线粒体,分泌颗粒极少。
研究结论：
(1)低浓度镉在短期胁迫下,河南华溪蟹可以依靠自身的代谢补偿机制,例如分解体内储存的能量物质,增强糖异生作用等来应对镉的能量胁迫；而相对高浓度的镉长时间暴露则可能导致机体内分泌失调,糖异生障碍,有毒代谢产物积累等。
(2)河南华溪蟹氧消耗的变化同呼吸器官鳃的摄氧能力、氧合血蓝蛋白结合和运输氧的能力有关,亚慢性镉处理对鳃组织结构的损伤程度比急性组的严重,可能与亚慢性组鳃的金属硫蛋白含量低及脂质过氧化程度高等有关。
(3)急性镉处理对磷酸戊糖途径有抑制作用,降低了细胞内还原力的生成,还原型谷胱甘肽含量减少,细胞内的抗氧化能力下降；亚慢性镉处理导致肝胰腺组织应激性得增加了还原力的生成,但是仍然难以维持谷胱甘肽的还原状态和细胞内的抗氧化水平,可能与细胞抗氧化过程对GSH的大量消耗和GSH合成受阻有关。
(4)急性和亚慢性镉处理皆导致了河南华溪蟹心脏线粒体电子传递链受阻。急性镉处理后,心脏的有氧代谢活性增强,会导致镉的吸收,转移和积累增加,将会在短期内导致严重的组织损伤；亚慢性镉处理后,线粒体功能受损,有氧代谢速率降低,减少了溪蟹在低氧摄入情况下的氧气需求。但是,同时会导致组织ATP供应不足,造成动物体的能量衰竭。
(5)亚慢性镉处理对肌肉组织的有氧呼吸和无氧呼吸活性皆有抑制作用,阻碍了其能量的产生；呼吸酶基因mRNA表达对急性镉处理较为敏感。反映了肌肉组织呼吸方式随镉浓度升高的动态转变过程。
(6)镉处理可导致窦腺结构的改变,影响了神经激素的储存和释放,会进一步影响河南华溪蟹的生理功能。
Objective:Development of the coal industry, non-ferrous metal smelting and rise of the electronics industry made cadmium pollution wide-spread in Shanxi Province. Freshwater crab, a biological indicator for heavy metal, whose sensitive responses to cadmium could help us detect the pollution earlier, and it is of interest to the monitoring, assessment and control to cadmium pollution. Metabolism is one of the most fundamental characteristics for organisms. Alteration or failure of metabolism has been considered as the initial symptoms of sublethal poisoning. To explore the metabolic responses of aquatic organisms and select the sensitive parameters as bio-indicator of cadmium pollution, the present thesis detected the alterations on carbohydrate and protein catabolism and energy production in freshwater crab Sinopotamon henanense caused by acute and subchronic cadmium exposures. The possible mechanisms on morphology, physiology and molecular biology were analyzed to reveal the differential stress responses between acute and subchronic cadmium exposure, which is in favor of the identifying of sudden and long-term cadmium pollution.
Contents and Methods:Firstly, to preliminarily investigate the catabolism responses of S. henanense to cadmium, the crab were exposed to sublethal Cd2+concentrations of0.725,1.45,2.9mg·L-1for7d,14d and21d. Glycogen, protein, free amino acid (FAA), hemolymph glucose, lactate, ammonia, urea and glutamine levels, as well as in activities of lactate dehydrogenase (LDH), protease, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in the hemolymph, muscle and hepatopancreas. Before further analyzing the aerobic oxidation, oxygen consumption, as the indicator of aerobic metabolic activity, was measured with the method of oxygen electrode respectively under the conditions of acute exposure (7.14,14.28,28.55mg/L Cd2+for96h) and subchronic exposure (0.71,1.43,2.86mg/L for three weeks), simultaneously, the oxygen uptake capacity of gills, oxygen-binding and transport capacity of hemocyanin in hemolymph, cadmium accumulation, metallothionein content, and lipid oxidation in gills were assayed to shed light on the reasons for differential changes on oxygen consumption between acute and subchronic cadmium exposure. On the basis of oxygen consumption changes, focused on the different functions of S. henanense tissues, the metabolic activities in different tissues were assessed, mainly including the pentose phosphate pathway in hepatopancreas, respiratory metabolism in heart and muscle. The parameters measured included glucose-6-phosphate dehydrogenase (G-6-PDH) activity, NADPH content, reduced glutathione (GSH), oxidized glutathione (GSSG) in hepatopancreas and GSH/GSSG ratio was calculated. For respiratory metabolism, the activities of respiratory enzymes, i.e. lactate dehydrogenase (LDH), NAD-isocitrate dehydrogenase (IDH), cytochrome c oxidase (CCO), as well as cco-1(CCO active subunit1) and ldh mRNA expression level and adenosine triphosphate (ATP) content were assessed. Lastly, based on the changes of glucose level in S. henanense hemolymph and the regulation of crustacean hyperglycemic hormone on glucose concentration, and the possible interference/inhibition of Cd2+with CHH, the present study observed the effect of Cd2+on microstructure and ultrastructure of X organ-sinus gland, which is the site for CHH synthesis and release, in eyestalk of S. henanense using light microscope and transmission electron microscope, to demonstrate the disturbance of Cd2+with CHH synthesis and release from the view of morphology, laying a foundation for further study on interference mechanism.
Results:
(1) For short-term exposure, decrease of glycogen, breakdown of protein, lowering of FAA and enhancement of ALT and AST activities are most likely the physiological responses to the high energy requirement induced by the Cd2+treatment; an increase in LDH activity and the non-change of lactate indicate the enhanced anaerobiosis and increased utilization of lactate for energy during the Cd2+exposure; ammonia level barely changed, which was mostly due to increased urea and glutamine production. The long-term Cd2+ exposure led to a decrease in hemolymph glucose level; the lowering of ALT and AST activities may weaken the mobilization of FAA; increased ammonia was observed followed by the increased glutamine.
(2) Acute Cd2+exposure increased the oxygen consumption, which is contrary with that during subchronic Cd2+exposure. The results showed concentration of oxyhemocyanin and oxyhemocyanin/blood protein proportion were increased during acute exposure and decreased during sub-chronic exposure. The morphological results showed that subchronic exposure to cadmium induced more profound damages than acute exposure, i.e. the branchial epithelial cells were more disorganized and vacuolized, and the apical microvilli decreased in number. In addition were vacuolized mitochondria and condensed chromatin in gill epithelial cells observed in the subchronic exposure group. There were no significant differences in Cd2+accumulation, CBP and total SOD activity between acute and subchronic cadmium exposures. However, MT level in acute-exposed group was significantly increased compared with that in subchronic-exposed group, and MDA concentration was exactly the opposite.
(3) Acute cadmium exposure decreased the G-6-PDH activity, accompanied by the decreased NADPH, GSH content and GSH/GSSG. After the subchronic cadmium exposure, G-6-PDH activity and NADPH content were increased at2.86mg/L. GSH content was increased at0.71mg/L and decreased at2.86mg/L. GSSG content showed an increasing change with the increasing Cd2+concentration, and the ratio of GSH/GSSG was decreased.
(4) Both exposure schemes induced downregulation of cco-1gene expression and lowered CCO activity. For acute exposure, tissue ATP level was increased, in association with increased IDH activity and decreased LDH activity, whereas subchronic exposure caused decreased IDH activity accompanied with increased ldh gene expression and LDH activity, resulting in lowered ATP level, and the mitochondrial cristae were ruptured even vacuolized.
(5) ATP content was found not changed during acute exposure, accompanied by the unchanged IDH, CCO and LDH activity, however, cco-1and ldh mRNA expression were upregulated. ATP content was decreased by subchronic exposure, in accordance with the decreased activities of IDH, CCO and LDH, and down-regulations of cco-1and ldh mRNA expression.
(6) X organ is made up of many nerve endocrine cells and no difference on microstructure was observed between control and treated group. In normal group, sinus gland has the central hemolymph sinus, surrounded by the wall tightly arranged by the axon, whose enlarged terminal and glial cell, making sure structure of the sinus gland is fixed and solid. Large numbers of neurosecretory granules wrapped in the enlarged anox terminal from different neuroendocrine cell were distributed around the small hemocoel. A small amount of mitochondria was contained in the terminal of axon. After the exposure of cadmium, the tightly arranged structure became porous and sinus gland structure near collapsed. Nerve secretory granules around the hemolymph sinus were decreased, and a large number of vacuoles and damaged mitochondrial were found in axon terminal. A few of secretory granules were observed in the axon.
Conclusions:
(1) A compensatory metabolism, such as breakdown of reserved energy substance and enhanced gluconeogenesis, is used by crabs to overcome the energy stress caused by low and short-term Cd2+exposure and that the higher and long-term Cd2+exposure may disturb the endocrine, impair the energy metabolism and lead to the accumulation of toxic metabolites in S. henanense. These biological effects shed light on the early assessment of Cd2+pollution.
(2) Oxygen consumption was related to oxygen uptake capacity of gill and oxygen-binding and transport capacity of hemocyanin. Lower MT content and higher lipid peroxidation level in subchronically exposed group may be responsible for the more profound gill damage.
(3) Acute cadmium exposure inhibited the PPP, lowering the production of reducing power NADPH and GSH, as well as the cellular antioxidant capacity. Subchronic cadmium exposure induced the stress responses of increased NADPH, however, it is difficult to maintain the cellular reduced state, possibly due to the large consumption of GSH during the cellular antioxidant response or impaired GSH synthesis.
(4) Both acute and subchronic Cd2+exposures led to the inhibition of the mitochondrial electron transfer chain in S. henanense heart. Acute Cd2+exposure caused increased oxygen consumption and enhanced aerobic respiration, which could increase the Cd2+intake, transport and accumulation, further resulting in serious damages within a short period of time. Under subchronic Cd2+exposure, the oxygen consumption decreased, and the impairing mitochondria function made the aerobic metabolism rate lower, which led to insufficient energy production but could provide survival of crabs under the condition of lower oxygen uptake.
(5) Both aerobic and anaerobic respiratory metabolism in S. henanense muscle was inhibited by subchronic cadmium exposure, as well as the energy production. For acute exposure, respiratory gene mRNA expression was more sensitive, reflecting the dynamic changes of respiratory manner from aerobic to anaerobic in muscle with the increasing exposure concentration.
(6) Cadmium treatment can lead to changes in the structure of sinus gland, affect the storage and release of neurohormone, may further disturb the physiological activities of the crabs.
研究内容与方法：首先采用亚慢性毒性实验方法,研究了镉对S. henanense血淋巴、肝胰腺和肌肉等组织中糖和蛋白分解代谢基本参数的影响,主要包括与糖分解代谢相关的糖原、葡萄糖、乳酸、乳酸脱氢酶等参数；与蛋白质分解代谢相关的总蛋白、蛋白酶、游离氨基酸、转氨酶、氨、尿素、谷氨酰胺等参数,初步掌握了镉对S. henanense主要能量物质分解代谢的影响。在进一步分析胁迫条件下糖分解代谢的主要途径,即有氧氧化(能量产生的主要方式)和磷酸戊糖途径(还原力产生的主要途径)的变化之前,先分别在急性和亚慢性毒性实验条件下,采用氧电极法对有氧氧化活性的重要衡量指标“氧消耗”进行了测定。在此基础上,针对不同组织的功能特点,采用酶学检测、亚显微技术、基因克隆、实时定量PCR对主要组织糖分解代谢活性进行了评价,主要包括肝胰腺组织磷酸戊糖途径(检测指标为葡萄糖-6磷酸脱氢酶(G-6-PDH)活性和NADPH含量,还原型谷胱甘肽(GSH),氧化型谷胱甘肽(GSSG))含量和GSH/GSSG比值),心脏组织呼吸代谢活性以及肌肉组织呼吸代谢活性(检测指标包括ATP含量、三羧酸循环关键酶NAD-异柠檬酸脱氢酶(NAD-IDH)、呼吸链终端酶细胞色素C氧化酶(CCO)、糖酵解关键酶乳酸脱氢酶(LDH)、CCO活性亚基Ⅰ(cco-1)和ldh mRNA表达水平)。除此之外,为了阐明不同实验条件下氧消耗变化的原因,应用比色法、显微和亚显微技术检测了血淋巴中氧合血蓝蛋白的含量和比例,观察了鳃组织和细胞形态的变化；并采用火焰原子吸收法、镉-血红蛋白饱和法和比色法检测了两种处理对鳃组织中Cd2+蓄积、金属硫蛋白(MT)含量、总超氧化物歧化酶(SOD)活性和膜脂质过氧化产物丙二醛(MDA)含量的影响,旨在揭示急性和亚慢性镉处理导致鳃结构功能发生变化的原因。最后,基于本研究所发现的血糖水平的变化,以及甲壳动物高血糖激素(CHH)对血糖水平的调节作用和镉对CHH潜在的干扰和抑制作用,对CHH合成与分泌的器官“X器-窦腺复合体”的显微和亚显微结构进行了观察,从形态学上初步证明了镉对CHH合成和分泌的影响,为进一步深入研究镉代谢的干扰机制奠定了基础。
研究结果：
(1)Cd2+暴露7d后,肝糖原和肌糖原含量下降,蛋白质含量降低,伴随着蛋白酶活性的升高,组织中游离氨基酸及谷丙、谷草两种转氨酶活性的增强(P<0.05)；血淋巴中的乳酸脱氢酶活性升高,而乳酸的含量并没有发生显著变化(P>0.05)。血淋巴中游离氨的含量在该时间段几乎没有发生变化,而血淋巴中检测到的升高的尿素和谷氨酰胺含量则说明了游离氨的去向。Cd2+暴露21d导致了血淋巴中葡萄糖含量的下降；谷丙、谷草两种转氨酶活性在该时间段降低,这将削弱机体对游离氨基酸的利用；同时,血淋巴中游离氨的含量升高,尿素的含量没有显著变化,仅可见谷氨酰胺含量的上升。
(2)氧消耗在急性处理组增加,而在亚慢性处理组则相反；氧合血蓝蛋白在血淋巴中的含量和比例经急性和亚慢性镉处理后的变化趋势同氧消耗基本一致；鳃形态学观察结果显示,急性镉处理后鳃叶肥大增厚,部分上皮细胞中出现空泡；亚慢性处理后,鳃叶边缘通道紧缩,上皮细胞高度肿胀,鳃血腔缩小变窄,上皮细胞顶膜微绒毛减少,线粒体嵴溶解消失,细胞核膜断裂变形,核染色质浓缩,边集化；两个处理组中的Cd2+蓄积、MT含量、SOD活性和MDA含量皆高于对照组,其中Cd2+蓄积量和SOD活性在两个处理组之间没有显著差异；急性处理组MT含量显著高于亚慢性组；而亚慢性组MDA含量显著高于急性组。
(3)急性镉处理后,G-6-PDH活性显著下降,伴随着NADPH、GSH含量和GSH/GSSG比值的下降；亚慢性镉处理后,在2.86mg/L组,G-6-PDH活性和NADPH含量显著上升,GSH含量先(0.71mg/L)上升后(2.86mg/L)下降,GSSG含量随处理浓度增加显著上升,伴随着GSH/GSSG比值的下降。
(4)急性和亚慢性镉处理皆导致溪蟹心脏组织CCO活性的降低和cco-1mRNA表达水平的下调；急性镉处理后,心脏组织ATP含量升高,伴随着IDH活性的上升和LDH活性的下降,而亚慢性镉处理后,IDH活性降低,同时ldh mRNA表达上调,LDH活性升高,组织ATP含量降低,线粒体嵴断裂甚至空泡化。
(5)急性处理组肌肉组织ATP含量没有发生明显的变化,而亚慢性处理在2.86mg/L组ATP含量显著下降；IDH、CCC、LDH酶活性在急性处理组都没有发生显著变化,而在亚慢性处理组显著下降；cco-1mRNA表达在急性处理的14.28mg/L浓度组显著上调,随着浓度的升高下调至对照组水平,ldh mRNA表达则在急性处理的各浓度组都显著上调。cco-1和ldh mRNA表达在亚慢性处理组的最高浓度皆显著下调。
(6)X器由许多神经内分泌细胞构成,正常组窦腺呈囊状,中央为血窦腔,周围是轴突、膨大的末梢和胶质细胞组成的壁,三者在腺体内紧紧缠绕,形成坚实的结构,固定了腺体的形状,轴突神经末梢的排列沿中央血腔呈放射状排列,血窦周围分布着致密的神经内分泌颗粒,包裹在不同的神经内分泌细胞的轴突膨大的末梢中,轴突末梢中含有少量的线粒体。镉处理组中的窦腺结构明显比对照组疏松,形成窦腺壁的栅栏结构消失,原本由轴突、膨大的末梢和胶质细胞紧密排列的结构变得疏松多孔,窦腺结构临近崩溃；血窦周围分布的神经分泌颗粒减少,轴突末梢中含有大量空泡和部分受损的线粒体,分泌颗粒极少。
研究结论：
(1)低浓度镉在短期胁迫下,河南华溪蟹可以依靠自身的代谢补偿机制,例如分解体内储存的能量物质,增强糖异生作用等来应对镉的能量胁迫；而相对高浓度的镉长时间暴露则可能导致机体内分泌失调,糖异生障碍,有毒代谢产物积累等。
(2)河南华溪蟹氧消耗的变化同呼吸器官鳃的摄氧能力、氧合血蓝蛋白结合和运输氧的能力有关,亚慢性镉处理对鳃组织结构的损伤程度比急性组的严重,可能与亚慢性组鳃的金属硫蛋白含量低及脂质过氧化程度高等有关。
(3)急性镉处理对磷酸戊糖途径有抑制作用,降低了细胞内还原力的生成,还原型谷胱甘肽含量减少,细胞内的抗氧化能力下降；亚慢性镉处理导致肝胰腺组织应激性得增加了还原力的生成,但是仍然难以维持谷胱甘肽的还原状态和细胞内的抗氧化水平,可能与细胞抗氧化过程对GSH的大量消耗和GSH合成受阻有关。
(4)急性和亚慢性镉处理皆导致了河南华溪蟹心脏线粒体电子传递链受阻。急性镉处理后,心脏的有氧代谢活性增强,会导致镉的吸收,转移和积累增加,将会在短期内导致严重的组织损伤；亚慢性镉处理后,线粒体功能受损,有氧代谢速率降低,减少了溪蟹在低氧摄入情况下的氧气需求。但是,同时会导致组织ATP供应不足,造成动物体的能量衰竭。
(5)亚慢性镉处理对肌肉组织的有氧呼吸和无氧呼吸活性皆有抑制作用,阻碍了其能量的产生；呼吸酶基因mRNA表达对急性镉处理较为敏感。反映了肌肉组织呼吸方式随镉浓度升高的动态转变过程。
(6)镉处理可导致窦腺结构的改变,影响了神经激素的储存和释放,会进一步影响河南华溪蟹的生理功能。
Objective:Development of the coal industry, non-ferrous metal smelting and rise of the electronics industry made cadmium pollution wide-spread in Shanxi Province. Freshwater crab, a biological indicator for heavy metal, whose sensitive responses to cadmium could help us detect the pollution earlier, and it is of interest to the monitoring, assessment and control to cadmium pollution. Metabolism is one of the most fundamental characteristics for organisms. Alteration or failure of metabolism has been considered as the initial symptoms of sublethal poisoning. To explore the metabolic responses of aquatic organisms and select the sensitive parameters as bio-indicator of cadmium pollution, the present thesis detected the alterations on carbohydrate and protein catabolism and energy production in freshwater crab Sinopotamon henanense caused by acute and subchronic cadmium exposures. The possible mechanisms on morphology, physiology and molecular biology were analyzed to reveal the differential stress responses between acute and subchronic cadmium exposure, which is in favor of the identifying of sudden and long-term cadmium pollution.
Contents and Methods:Firstly, to preliminarily investigate the catabolism responses of S. henanense to cadmium, the crab were exposed to sublethal Cd2+concentrations of0.725,1.45,2.9mg·L-1for7d,14d and21d. Glycogen, protein, free amino acid (FAA), hemolymph glucose, lactate, ammonia, urea and glutamine levels, as well as in activities of lactate dehydrogenase (LDH), protease, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in the hemolymph, muscle and hepatopancreas. Before further analyzing the aerobic oxidation, oxygen consumption, as the indicator of aerobic metabolic activity, was measured with the method of oxygen electrode respectively under the conditions of acute exposure (7.14,14.28,28.55mg/L Cd2+for96h) and subchronic exposure (0.71,1.43,2.86mg/L for three weeks), simultaneously, the oxygen uptake capacity of gills, oxygen-binding and transport capacity of hemocyanin in hemolymph, cadmium accumulation, metallothionein content, and lipid oxidation in gills were assayed to shed light on the reasons for differential changes on oxygen consumption between acute and subchronic cadmium exposure. On the basis of oxygen consumption changes, focused on the different functions of S. henanense tissues, the metabolic activities in different tissues were assessed, mainly including the pentose phosphate pathway in hepatopancreas, respiratory metabolism in heart and muscle. The parameters measured included glucose-6-phosphate dehydrogenase (G-6-PDH) activity, NADPH content, reduced glutathione (GSH), oxidized glutathione (GSSG) in hepatopancreas and GSH/GSSG ratio was calculated. For respiratory metabolism, the activities of respiratory enzymes, i.e. lactate dehydrogenase (LDH), NAD-isocitrate dehydrogenase (IDH), cytochrome c oxidase (CCO), as well as cco-1(CCO active subunit1) and ldh mRNA expression level and adenosine triphosphate (ATP) content were assessed. Lastly, based on the changes of glucose level in S. henanense hemolymph and the regulation of crustacean hyperglycemic hormone on glucose concentration, and the possible interference/inhibition of Cd2+with CHH, the present study observed the effect of Cd2+on microstructure and ultrastructure of X organ-sinus gland, which is the site for CHH synthesis and release, in eyestalk of S. henanense using light microscope and transmission electron microscope, to demonstrate the disturbance of Cd2+with CHH synthesis and release from the view of morphology, laying a foundation for further study on interference mechanism.
Results:
(1) For short-term exposure, decrease of glycogen, breakdown of protein, lowering of FAA and enhancement of ALT and AST activities are most likely the physiological responses to the high energy requirement induced by the Cd2+treatment; an increase in LDH activity and the non-change of lactate indicate the enhanced anaerobiosis and increased utilization of lactate for energy during the Cd2+exposure; ammonia level barely changed, which was mostly due to increased urea and glutamine production. The long-term Cd2+ exposure led to a decrease in hemolymph glucose level; the lowering of ALT and AST activities may weaken the mobilization of FAA; increased ammonia was observed followed by the increased glutamine.
(2) Acute Cd2+exposure increased the oxygen consumption, which is contrary with that during subchronic Cd2+exposure. The results showed concentration of oxyhemocyanin and oxyhemocyanin/blood protein proportion were increased during acute exposure and decreased during sub-chronic exposure. The morphological results showed that subchronic exposure to cadmium induced more profound damages than acute exposure, i.e. the branchial epithelial cells were more disorganized and vacuolized, and the apical microvilli decreased in number. In addition were vacuolized mitochondria and condensed chromatin in gill epithelial cells observed in the subchronic exposure group. There were no significant differences in Cd2+accumulation, CBP and total SOD activity between acute and subchronic cadmium exposures. However, MT level in acute-exposed group was significantly increased compared with that in subchronic-exposed group, and MDA concentration was exactly the opposite.
(3) Acute cadmium exposure decreased the G-6-PDH activity, accompanied by the decreased NADPH, GSH content and GSH/GSSG. After the subchronic cadmium exposure, G-6-PDH activity and NADPH content were increased at2.86mg/L. GSH content was increased at0.71mg/L and decreased at2.86mg/L. GSSG content showed an increasing change with the increasing Cd2+concentration, and the ratio of GSH/GSSG was decreased.
(4) Both exposure schemes induced downregulation of cco-1gene expression and lowered CCO activity. For acute exposure, tissue ATP level was increased, in association with increased IDH activity and decreased LDH activity, whereas subchronic exposure caused decreased IDH activity accompanied with increased ldh gene expression and LDH activity, resulting in lowered ATP level, and the mitochondrial cristae were ruptured even vacuolized.
(5) ATP content was found not changed during acute exposure, accompanied by the unchanged IDH, CCO and LDH activity, however, cco-1and ldh mRNA expression were upregulated. ATP content was decreased by subchronic exposure, in accordance with the decreased activities of IDH, CCO and LDH, and down-regulations of cco-1and ldh mRNA expression.
(6) X organ is made up of many nerve endocrine cells and no difference on microstructure was observed between control and treated group. In normal group, sinus gland has the central hemolymph sinus, surrounded by the wall tightly arranged by the axon, whose enlarged terminal and glial cell, making sure structure of the sinus gland is fixed and solid. Large numbers of neurosecretory granules wrapped in the enlarged anox terminal from different neuroendocrine cell were distributed around the small hemocoel. A small amount of mitochondria was contained in the terminal of axon. After the exposure of cadmium, the tightly arranged structure became porous and sinus gland structure near collapsed. Nerve secretory granules around the hemolymph sinus were decreased, and a large number of vacuoles and damaged mitochondrial were found in axon terminal. A few of secretory granules were observed in the axon.
Conclusions:
(1) A compensatory metabolism, such as breakdown of reserved energy substance and enhanced gluconeogenesis, is used by crabs to overcome the energy stress caused by low and short-term Cd2+exposure and that the higher and long-term Cd2+exposure may disturb the endocrine, impair the energy metabolism and lead to the accumulation of toxic metabolites in S. henanense. These biological effects shed light on the early assessment of Cd2+pollution.
(2) Oxygen consumption was related to oxygen uptake capacity of gill and oxygen-binding and transport capacity of hemocyanin. Lower MT content and higher lipid peroxidation level in subchronically exposed group may be responsible for the more profound gill damage.
(3) Acute cadmium exposure inhibited the PPP, lowering the production of reducing power NADPH and GSH, as well as the cellular antioxidant capacity. Subchronic cadmium exposure induced the stress responses of increased NADPH, however, it is difficult to maintain the cellular reduced state, possibly due to the large consumption of GSH during the cellular antioxidant response or impaired GSH synthesis.
(4) Both acute and subchronic Cd2+exposures led to the inhibition of the mitochondrial electron transfer chain in S. henanense heart. Acute Cd2+exposure caused increased oxygen consumption and enhanced aerobic respiration, which could increase the Cd2+intake, transport and accumulation, further resulting in serious damages within a short period of time. Under subchronic Cd2+exposure, the oxygen consumption decreased, and the impairing mitochondria function made the aerobic metabolism rate lower, which led to insufficient energy production but could provide survival of crabs under the condition of lower oxygen uptake.
(5) Both aerobic and anaerobic respiratory metabolism in S. henanense muscle was inhibited by subchronic cadmium exposure, as well as the energy production. For acute exposure, respiratory gene mRNA expression was more sensitive, reflecting the dynamic changes of respiratory manner from aerobic to anaerobic in muscle with the increasing exposure concentration.
(6) Cadmium treatment can lead to changes in the structure of sinus gland, affect the storage and release of neurohormone, may further disturb the physiological activities of the crabs.
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