Study On Toxicological Effects Of Microcystins On Typical Aquatic Organisms And Related Molecular Toxic Mechanisms

In recent years cyanobacterial blooming has occurred frequently in lakes, which can bring about water pollution induced by its various derivate pollutants and even lead to great ecological disasters in severe cases. Cyanobacterial blooming in lakes has certainly become challenge to the sustainable development of regional economy. Microcystins (MCs) have become common potential hazardous pollutant in aquatic environments for their great toxicity, widespread distribution and structural stability. Previous studies focused on the acute toxicology and toxic mechanisms of single species of MCs at high doses on model organisms. However, the reports about the MCs-induced toxicological effects on typical lake aquatic organism are few. Therefore pepole can not explain clearly the toxic mechanisms of MCs on aquatic organisms in practical environment and develop the early warning system for cyanobacterial blooms. In this thesis, emphasis placed on the toxic mechanism of microcystins on aquatic organisms and their early ecotoxicological effects on Vallisneria natans (Lour.) Hara and Cyprinus carpio L. Based on our studies, the role of oxidative stress on the toxic mechanisms of cyanobacterial derivate pollutants on aquatic organisms was discussed in order to reveal the actual toxic mechanism of MCs on aquatic organisms under low-doses and long-term exposure and to select sensitive biomarkers for cyanobacterial blooms pollution. The feasibility of application of oxidative stress indexes in early warning the cyanobacterial blooming in Lake Taihu was also assessed. In additional,2D DIGE was performed to screen and quantify the differentially expressed proteins in the liver of C. carpio exposed to canobacterical blooms, which can extend a better understanding of canobacterial blooms-induced toxicological effects on fishes. The main results are listed as follows:1. MC-LR could be absorbed and accumulated in leaves of V. natans seedling and the possible maximum concentration of MC-LR absorbed under environmentally relevant concentrations exposure was 13.9 ng g-1 DW.O2·- intensity could be induced by MC-LR and therefore launched the oxidative stress in V. natans, which was evidenced by the responses of antioxidant system and elevation of lipid peroxidation. A significant positive correlation between the MDA content and ROS intensity could be observed (r=0.817, p< 0.05). CAT activity and the GSH/GSSG ratio was sensitive to low concentration of MC-LR (0.1?g L-1), showing their potential to be biomarkers to indicate the MCs pollution. The induction of GST and inhibition of GSH revealed the conjugation reaction of MC-LR with GSH under the catalysis of GST is the detoxification of MC-LR in V natans. MC-LR at 1.0-25.0?g L-1 lead to a significant decrease of Chl a content; MC-LR at 1.0?g L-1, the drinking water guide recommended by WTO, could induc the oxidative damage, decrease the soluble protein content, and alter the mesophyll cells'ultrastructure in leaves of V. natans, suggesting that the lowest non-effect concentration of MC-LR for V. natans at the subcellular and molecular level is between 0.5 and 1.0?g L-2. Direct evidence of ·OH generation in the liver of C. carpio exposed to MC-LR was provided by the spin trapping technique, carried out by electron paramagnetic resonance (EPR) followed by analysis of the resulting adducts. The hyperfine splitting constants for the PBN-·OH adducts are g value=2.0058,?N= 13.7 G and ?H= 1.8 G. Intraperitoneal injection (i.p.) of MC-LR in C. carpio at sublethal doses suggested that ·OH was sensitively induced at 120?g/kg MC-LR for only 1 h. A significant positive correlation between the MDA content and ·OH intensity could be observed in the liver of C. carpio exposed to 50?g/kg MC-LR (r 0.970, p< 0.01). Changes of antioxidant enzymes activities, GSH content and HSP70 expression were found in response to the ROS elevation and most significant changes of these parameters was showed between 5-12 h. MC-LR administration (i.p.) brought about a significant reorganization of cytoskeleton and histopathological damage in fish liver. Futhermore, MC-LR administration (i.p.) activated the mRNA p38 and JNKa exporession, which have close relationship with apoptosis and may regulated by ROS in vivo, and subsequently induced the hepatocyte apoptosis at 12-48. The decreased apoptosis rate at later stage might be related to the expression of Bcl-2. Toxicological studies on C. carpio immersed in MC-LR at environmentally relevant concentrations (0.1-10?g L-1) were also carried out in this study. Results showed that MC-LR might be taken up through the gastrointestinal tract or through gill epithelium cells and therefore accumulated in various fish tissues/organs. The maximum accumulation of MC-LR was found in fish liver. However, MC-LR contents in fish gills was higher than that in liver or intestine at 0.1?g L-1 MC-LR, which might be related the direct absorption and the histopathological alterations in gills structure exposed immediately to MC-LR in water. Immersion in MC-LR at common environmental concentrations seems likely to induce only a mild oxidative stress in fish liver which could be regulated through the induction of antioxidant system and HSP70 expression. Results from dynamic/static exposure experiment showed that ·OH and GSH/GSSG ratio were sensitive to the MC-LR exposure period, while ·OH, GSH and PP activity were sensitive to MC-LR exposure concentrations, suggesting their potential to be biomarkers in fish to indicate the MCs pollution. No note increase of hepatocellular apoptosis was found in fish exposed to fish at 0.1-10?g L-1. However, the PP activities in liver were significantly inhibited in treated groups in a dose-dependent way which could lead to hyperphosphorylation of a diverse array of cellular proteins and therefore induce dose-dependent histopathological alterations (like vacuolar degeneration, partial hepatic cells necrosis and parenchymal architecture dissolving, etc.) in fish livers. In addition, a series of dose-dependent histopathological alterations, such as the increased distance between the secondary gill lamellae and the distorted and shrank taste bud structure, were also observed in gills of C. carpio, which suggest that exposure to MC-LR could affect the effciency of respiratory in fish.3. Results from the in-situ study in Lake Taihu (2009.7.11-2009.7.24) showed that the occurrence of blue-green algae had significant correlation with temperature, TP and MC-LR/RR concentrations in water. Both ELISA and ESI-LC-MS analyses showed the presence of MC-LR in ogans/tissues of the C. carpio exposed cyanobacterial blooms and the order of MCs accumulation was as follows:liver> intestine> gill> muscle. The presence of MCs in carnivorous fishes collected from Lake Taihu indicated the potential risk on human health via food chains. The intensity of oxidative stress in C. carpio in Meiliang Bay was well correlated with the environmental factor (temperature and pH value), pollution level of cyanobacterial blooming (like blue-green algae density and MC-LR/RR concentrations, etc.) and the internal MCs exposure in fish tissues. There was positive correlation between the LPO/PCO contents and the ROS intensity in fish (p< 0.05). The GSH and GSSG content were found sensitive to diffenent in-situ spots and the GSH/GSSG ratio was significantly decreased in C. carpio in Meiliang Bay. Results from in-situ experiment suggested that the ROS, GSH, GSH/GSSG ratio might be potential biomarkers to indicate the cyanobacterial blooms.148 differentially expressed proteins were found in C. carpio in Meiliang Bay with 2D DIGE and 57 proteins were identified unambiguously using MALDI TOF/TOF. These proteins were involved in various biological pathways and the toxic mechanism of cyanobacterial blooms on fish was in similar way with MCs. Exposure to cyanobacterial blooms could induce the oxidative stress, mitochondrial stress and endoplasmic reticulum stress in liver of C. carpio, and therefore disturb the metabolism pathways. In the liver, exposure to cyanobacterial blooms mainly induced the expression of proteins related to amino acid metabolism and TCA cycle and suppressed the expression of proteins related to glucose metabolism. The MCs and ammonian might be the main factors of cyanobacterial blooms'toxicological effects on fish.4. Results from the study on combined pollution of MC-LR and atrazine showed that single MC-LR and its combination with atrazine were both able to enhance the ·OH in vivo and induce mild oxidative stress in the liver of C. carpio, which could be regulated through the self-restoring through antioxidative system or other cleaning mechanisims. The effects of combined pollutants are not the simple synergistic effects. The order of effects of MC-LR on different indexes in combined pollution was as follows:MDA> PP> ROS> GSH> SOD> GST> CAT. In the single MC-LR treatment groups or the combined pollution groups, a series of histopathological changes in the liver or gills of C. carpio were observed. More attention should be paid to the observation that the combination of pollutants (1?g L-1 MC-LR+5?g L-1 Atrazine) could make the histopathological damages more serious. Futhermore, this combination of pollutants could induce the significant hepatocyte apoptosis in C. carpio.5. By standardization of the ROS intensity and the MDA in difference species of ogransims under low-level pollution, a significant positive correlation was found between LPO in organisms and ROS in vivo, which seems to have no explicit relationship with the organism species or the exposure routes. Therefore, the LPO in the organisms was directly induced by ROS in vivo.These results suggest that oxidative stress play an important role in the toxic mechanisms of cyanobacterial blooms-producing derivate pollutants on typical aquatic organisms. However, the application of oxidative stress biomarkers to indicate the cyanobacterial blooms pollution should take more other characteristic factors into consideration. Our studies imply that even at relatively low MCs concentrations the aquatic plants may still suffer a negative ecological impact. The results will help to establish early diagnosis system for lake ecological safety and provide scientific evidence for evaluation of safety threshold values of related pollutants.