| Cadmium (Cd) is a biotoxic element and one of the major metals that are ubiquitously distributed in aquatic systems, which causes a series of toxic effects in aquatic organism. This research systematically studied the effects of acute Cd toxicity on Anodonta woodiana, and explored the mechanism of Cd toxicity from the point of view of tissue injury and cell apoptosis. To explore the effects of acute Cd toxicity on freshwater mussel Anodonta woodiana, this thesis investigated the median lethal concentration of Cd by using the method of probability unit regression, and calculated the non-toxic concentration of Cd at first. Then, the mussels were exposed to Cd (4.21, 8.43,16.86,33.72 and 67.45 mg/L) for 24,48,72 and 96 h. After Cd treatment, the gills, mantle, foot, visceral mass and digestive gland tissues were collected for analyses. We examined the in vivo distribution of Cd and the protein level of metallothionein (MT) using the flame atomic absorption method and Cd-hemoglobin saturation method respectively, and assessed the relationship of Cd accumulation and the MT concentration in five tissues of Anodonta woodiana. Then, we selected two tissues (gills and mantle) that Cd accumulation was significant, and the MT expression was positively correlated with the Cd accumulation for experiments. To explore the mechanism of apoptosis induced by Cd in gills and mantle cells, we applied acute toxicity experiment at 0,8.43,16.86,33.72 and 67.45 mg/L Cd for 96 h. Activities of Caspase-3, Caspase-8 and Caspase-9 as well as H2O2 level of the gills and mantle were measured by enzymatic assays. At the same time, the changes of DNA ladder in gills and mantle were detected by DNA electrophoresis technique. Moreover, the changes in submicroscopic structures of gills and mantle in Cd treatment groups (0,16.86 and 67.45 mg/L) were documented.The results showed:1. The effects of acute Cd toxicity on Anodonta woodiana:Upon Cd treatment, Cd caused different poisoning symptoms in Anodonta woodiana, symptoms of Cd poisoning could occur both rapidly and over the periods of Cd treatment time. At the beginning of the experiment, double shell closed tightly, the water inlet and outlet tube stopped filtering and feeding. In later period of the experiment, the symptoms of Cd poisoning were shown by the following features:1. double shell opened gradually; 2. foot extended, and shrank slowly after receiving the external stimuli; 3. massive mussel deaths occurred; 4. there were a lot of white flocculent material in water. The values of LC50 for 24,48,72 and 96 h of treatment were 562.3,331.1,182.0 and 134.9 mg/L, respectively. The non-toxic concentration of Cd for Anodonta woodiana was 1.35 mg/L Cd.2. Cd accumulation and MT biosynthesis in Anodonta woodiana:We found that, in the controls, Cd distributed in all tissues in the concentration order of gills>mantle>foot>visceral mass>digestive gland, ranging from 0.012 mg/g to 0.032 mg/g. Upon Cd treatment, Cd concentration significantly increased in all tissues. The highest Cd accumulation was found in the digestive gland, which was 0.142 mg/g (P<0.05). MT levels in the gills and mantle of the mussels increased significantly to 4.13 μg/g and 4.69 μg/g respectively (P<0.05), which were in positive correlation with Cd accumulation in the tissues (P<0.05).3. Cd causes ultrastructure changes and induces apoptosis in the gill cells of Anodonta woodiana:In the gills, activities of Caspase-3 and Caspase-9 were increased by Cd in a dose-dependent manner, high doses of Cd (16.86,33.72 and 67.45 mg/L) increased their activities significantly (P<0.05). With the increase of Cd concentration, Caspase-8 activity decreased initially and increased subsequently, the low dose Cd (8.43 mg/L) reduced Caspase-8 activity, but the high doses of Cd (16.86,33.72 and 67.45 mg/L) increased its activity significantly (P<0.05). The higher dose Cd (16.86 mg/L) increased H2O2 level to the peak, which was significantly higher than in the control group (P < 0.05). Cd treatment was able to induce DNA fragmentation in a dose-dependent manner, as evidenced through agarose gel electrophoresis. Moreover, after Cd treatment (0,16.86 and 67.45 mg/L) for 96 h, ultrastructural alterations were observed by electronic microscopy in the gill cells. The histological abnormalities were shown by the following features:1. microvilli of epithelial cells arranged disorderly, broke off, decreased and disappeared gradually; 2. chromatin condensed near nuclear membrane and extremely irregular nuclei with fingerlike were apparent. Nuclear membrane was unclear, swelled, invaginated and ruptured gradually, and even some apoptotic bodies formed; 3. mitochondria became swelling and vacuolating gradually, and some mitochondrial crisae disintegrated and disappeared; 4. rough endoplasmic reticulum disintegrated, and formed many different sizes of vesicles; 5. the shrinking and deformation of golgi body were observed, golgi body vesicles decreased.4. Cd causes ultrastructure changes and induces apoptosis in the mantle cells of Anodonta woodiana:In the mantle, the low dose Cd (8.43 mg/L) reduced the activity of Caspase-3, but the higher dose Cd (33.72 mg/L) increased its activity significantly (P<0.05). The change in Caspase-9 activity was in the similar pattern. The activity of Caspase-8 was increased significantly after the mussels exposed to Cd in a dose-dependent manner (P<0.05). With the increase of Cd concentration, H2O2 level was increased gradually in a dose-dependent manner (P< 0.05). Meanwhile, DNA ladder could be observed in agarose gel electrophoresis with increasing concentration of Cd, which showed a dose-dependent feature. Moreover, after Cd treatment (16.86 and 67.45 mg/L) for 96 h, histological abnormalities were observed by electronic microscopy in the mantle cells. The histological abnormalities were shown by the following features:1. chromatin condensed near nuclear membrane. Nuclear membrane swelled and ruptured; 2. mitochondria became swelling and vacuolating gradually, and some mitochondrial crisae disintegrated; 3. microvilli arranged disorderly, broke off and were decreased gradually. The vacuole appeared; 4. rough endoplasmic reticulum disintegrated, and formed many different sizes of vesicles.The results indicated that:1. Cd has significant acute toxic effects on Anodonta woodiana. Upon Cd treatment, the mussels have presented various symptoms of Cd poisoning. The ability to accumulate Cd in different tissues is in the order of digestive gland>gills>mantle>foot>visceral mass. These tissues are all targets of Cd toxicity. Moreover, our results demonstrate a correlation between Cd accumulation and MT up-regulation in gills and mantle of the mussels after Cd treatment, which suggest that the protein level of MT in gills and mantle of Anodonta woodiana is a good biomarker for Cd contamination.2. Cd-exposure induces oxidative stress in the mantle, and the mantle produces a lot of H2O2. Cd increases H2O2 level in a dose-dependent manner, and Cd activates Caspase-3 and Caspase-9 activities in the same pattern in the mantle. It is possible that Cd induces apoptosis through the mitochondrial pathway, and initiates H2O2-dependent apoptosis in the mantle.3. In the gills, Cd-treatment does not increase H2O2 level in a dose-dependent manner, and Cd raises Caspase-3, Caspase-8 and Caspase-9 activities in the same pattern. It is suggested that Cd induces apoptosis through the pathway of cell surface death receptor and mitochondrial in the gills. |
PDF Full Text Request