物质和能量代谢是生物体最基本的特征之一。本项目在7项国家自然科学基金资助的基础上，在前期研究积累的前提下，综合利用透射电镜和激光共聚焦、免疫细胞化学与ELISA、双向电泳与质谱、基因克隆、实时荧光定量PCR等方法，以溪蟹为材料，研究镉对糖代谢五条途径中关键酶活性的影响和高血糖激素基因表达调控糖代谢的细胞分子机制；蛋白质代谢酶活性及氨基酸含量和热休克蛋白HSP70、自由基清除蛋白SOD、GST基因的分子保护机制；脂肪代谢中脂肪消化、转运和合成重要酶活性；ATP能量需求和血蓝蛋白mRNA表达、CCO-1基因转录及线粒体膜电位。通过研究，阐明镉对高血糖激素合成和分泌造成干扰或抑制的机理，对蛋白质合成及氨基酸代谢的主要防御机制，在脂肪组成、储备及其功能发挥的生化机理，线粒体功能和血蓝蛋白对氧气利用的调节机制。筛选敏感性强且便于检测的生物效应分子，完善生物评价方法，为水产品健康养殖提供科学依据。

Alteration or failure of metabolism has been considered as the initial symptoms of sublethal poisoning. To explore the metabolic responses of freshwater crab Sinopotamon henanense to cadmium (Cd) exposure, key indexes of carbohydrate, protein and lipid metabolisms would be measured and mechanisms would be explored. Enzymes involved in main pathways of carbohydrate metabolism would be assayed. In situ detection of crustacean hyperglycemic hormone (CHH) in X organ-sinus gland and CHH quatitative in hemolymph of the sample will be carried out with the methods of immunohistochemistry and ELISA. Before these detections, CHH gene cloning, preparations of CHH recombinant protein and anti-serum should be completed. For protein metabolism, amino acids mobilization and detoxification protein synthesis will be studied. Real-time PCR is used to analyze the gene expression of detoxificated proteins, i.e., HSP70, SOD and GST. Then, two-dimensional gel electrophoresis and mass spectrometry are used to isolate and identificate the differentially expressed proteins during Cd stress. For lipid metabolism, elements related to lipid storage and key enzymes related to digestion, transfer and synthesis of lipid will be measured. Pyruvate, amino acids and fatty acid derived from the three metabolisms are translated into acetyl coenzyme A, where the cellular respiration begins. Firstly, oxygen consumption and parameters involved in oxygen combining and transport, i.e., oxyhemocyanin and its ratio in hemolymph will be measured. Scondly, ATP levels in tissues will be detected. Thirdly, proteic and gene expression of key enzymes involved in oxygen transport, the citric acid cycle, mitochondrial respiratory chain will be analyzed by spectrophotometry and RT-PCR. Lastly, mitochondrial structure and membrane potential will be observed with transmission electron microscopy and laser scanning confocal microscope. This work would lead to understand Cd-induced high energy demand in the crab and the contributions of carbohydrate metabolism pathway changes to hypoglycemia, then illuminate the interference of Cd with CHH synthesis and excretion. The results also lead to find differentially expressed proteins during Cd stress and are helpful to clarify the toxic mechanisms of Cd and the defence mechanisms of the crab against the toxicity. Biochemical mechanisms focused on Cd-induced alterations of lipid constitutes, storage and function performance would be illustrated by analyzing the relationship between lipid content and transport. In the end, the hypothesis that impairment of mitochondrial energy production derived from carbohydrate, protein and lipid metabolism and the direct damage of Cd to mitochondrial would be testified by the physiological, biochemical and histopathological alterations. This work would be significant for selecting the sensitive biomarker, and improving the biological-evaluated process, further for the bio-monitoring of Cd pollution.