| The freshwater crab Sinopotamon yangtsekiense (Bott,1967) is a common endemic species in China. In the preset study, S. yangtsekiense were selected as experiment animals and exposed in the laboratory to a wide range of sublethal concentrations of cadmium (Cd)(7.25,14.50,29.00,58.00,116.00mg·L-1), the histopathological alternations of hepatopancreas, gills, heart and testis were investigated. To explore the toxic effects and stress responses of Cd exposure on heart of S. yangtsekiense, we next detected the accumulation of Cd in the heart and the effects of Cd on the histopathology, antioxidant enzymes and lipid peroxidation. Then, to verify if apoptosis is one of the cardiomyocyte death pathways upon Cd administration, the effects of Cd on cardiomyocyte apoptosis in S. yangtsekiense were assayed. The present study applied morphological, biochemical, cellular, molecular and toxicological methods and technology comprehensively. The aim of this work was to clarify the mechanism of toxic effects of Cd on the heart of S. yangtsekiense and provide useful references on the healthy aquaculture and the effective evaluation of Cd pollution. The present study will enrich the database of Cd's cardiotoxicity in crustaceans. The main results and conclusions in this thesis are summarized as follows:1. Effects of cadmium on hepatopancreas, gills, heart and testis in the freshwater crab Sinopotamon yangtsekienseThe freshwater crab S. yangtsekiense was exposed in the laboratory to a wide range of sublethal concentrations of cadmium (Cd)(7.25,14.50,29.00,58.00,116.00mg·L-1) for4days to explore the acute toxic effects. The results showed Cd caused progressive histopathological damages to the principal tissues, including hepatopancreas, gills, heart and testis. Hepatopancreas alterations included increases in the number of R-cells, extensive vacuolation, detachment of necrotic cells from basal laminae and then tubular necrosis lesion. The gills showed disordered interlamellar spaces, breakdown of pillar cell system, an increased number of nephrocytes and haemocytes, increased lamellae width, ruption of lamella epithelium as well as telangiectasia. The main histopathological alternations in the heart were characterized by degeneration and/or necrosis of focal areas accompanied with infiltration of inflammatory cells. In the testis, vacuolar degeneration, nuclear condensation, a reduction in the number of sperms lined with degeneration and/or necrosis of spermatogenic cells, atrophied seminiferous epithelia as well as tubular necrosis were noticed. These findings provide baseline information on the deleterious effects of Cd on S. yangtsekiense and suggest structural alternations of vital tissues following elevated Cd exposure, which could affect vital physiological functions, such as absorption, storage and secretion of the hepatopancreas, respiration, osmotic and ionic regulations of the gills, as well as spermatogenesis of testis. Such alternations could eventually affect the survival and reproduction of S. yangtsekiense. Thus, all possible remedial measures should be adopted to prevent Cd contamination in aquatic environments.2. Morphologic changes and oxidative damage of the heart induced by cadmium exposure in the freshwater crab Sinopotamon yangtsekienseCadmium (Cd) is a highly toxic element in water. Its toxicity has been attributed to oxidative stress mediated by free radicals. Here we investigated the effects of Cd on histopathology, antioxidant enzymes and lipid peroxidation of crustacean heart. The freshwater crabs S. yangtsekiense were exposed to different concentrations of Cd for1,3,5and7days. After exposure, histological abnormalities were discovered, including myocardial edema, vacuolar and vitreous degeneration, and infiltration of inflammatory cells. Additionally, alterations in nuclei, mitochondria, rough endoplasmic reticulum as well as myofibrils were observed. Meanwhile, superoxide dismutase (SOD) activity was significantly increased after Cd exposure. Catalase (CAT) activity was only increased in the group exposed to14.50mg·L-1Cd on day5and decreased with increasing Cd concentration and exposure time. Glutathione peroxidase (GPx) activity was increased in groups treated with29.00,58.00and116.00mg·L-1on days1and3, and decreased thereafter. Besides, malondialdehyde (MDA) levels were significantly increased after3days of Cd exposure at all the indicated concentrations. These results showed that acute Cd exposure led to harmful effects on the histology of crab heart, which are most likely linked to Cd-induced oxidative stress.3. Effects of cadmium on apoptosis of cardiomyocyte in the freshwater crab Sinopotamon yangtsekienseThe effects of cadmium (Cd) on cardiomyocyte apoptosis in the freshwater crab S. yangtsekiense were investigated by acute systemic toxicity testing methods. To verify if apoptosis is one of the cardiomyocyte death pathways upon Cd administration, crabs were exposed to different concentrations of Cd (7.25,14.50,29.00,58.00and116.00mg·L-1) for1,3,5and7days. The results showed the characteristic apoptotic morphologic changes and "ladder" pattern were not observed by fluorescence microscopy with Hoechst33258staining and agarose gel electrophoresis. Flow cytometry assay indicated that the percentage of apoptotic cells was increased with the increasing of exposure time and concentrations of Cd compared with control, but there was no significant difference. Moreover, the activity of Caspase-3was not affected by Cd treatment after7days of exposure. However, these findings suggest that apoptosis was not induced by Cd in cardiomyocytes according to the result of the present study. While previous studies in our lab showed that Cd induced apoptosis in the hepatopancreas and testis cells of crabs after2days and7days of exposure. In the crab cardiomyocyte, the lack of Cd-induced apoptosis may be attributed to different Cd accumulation levels in the three tissues.4. Expression analysis of heat shock protein70and metallothionein genes in heart of the freshwater crab Sinopotamon yangtsekiense in response to cadmiumIn order to elucidate the molecular mechanisms of the freshwater crab S. yangtsekiense response to cadmium (Cd) stress, we cloned and sequenced a partial cDNA encoding a70kDa heat shock protein (SyHSP70) from the heart of S. yangtsekiense. Next we investigated its time-and dose-related effects by quantitative real-time PCR on mRNA levels of SyHSP70and metallothionein (MT) in the heart of crabs. The results showed the expression of SyHSP70mRNA decreased significantly after exposure to58.00mg·L-1,116.00mg·L-1Cd for1day or7.25mg·L-1,14.50mg·L-1for3days. While the expression of MT mRNA increased significantly with increasing Cd concentration and exposure time. These results indicate that MT plays an important role in the physiological changes related to metabolism and cell protection that occur in S. yangtsekiense exposed to Cd. This is the first report on the expression of HSP70induced by Cd in S. yangtsekiense. |
