| 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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