Study On The Toxicity Mechanism Of Microcystin On Protein Level

The outbreak of the cyanobacerial (specifically Microcystin aeruginosa) blooms due to eutrophication has been a worldwide threat since the late 1970s, it exerts severe adverse health effects on human and livestock by virtue of their ability to produce the heptapeptide toxin, microcystin. The general structure of microcystin (MC) is cyclo-(D-Ala-L-X-erythro-β-methyl-D-isoAsp-L-Y-Adda-D-isoGlu-N-Methyldehydr o-Ala), where Adda stands for 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, and X and Y are two variable amino acids. Among which, Adda is an unusual amino acid, which is essential for expression of biological activity. Till now, over 60 structural variants have been recognized. The XY variable amino acids for MC-LR and MC-RR are leucine (L), arginine (R), which are the two most predominant microcystins.Using tritium-labled MC, it has been demonstrated that the liver is the prime target organ affected, but to some extent, MC also concentrated in the intestine and the kidneys. The toxic effects of MC on different types of mammalian cells including hepatocytes, embryo kidney cells, fibroblast, endothelial, and epithelial cells or lymphocytes, were observed. Recently, it has been reported that MC could accumulate on the eggs of the shrimps except in the hepatopancreas and gonads,indicating that it can be transferred to offspring from their adults. MCs are poisonous to higher levels of the food chain due to the bioaccumulation of MCs in aquatic animals of natural waters.Microcystins are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promotion activity have not been fully elucidated. One of the most extensively studied mechanisms is that microcystins are potent inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), leading to increased protein phosphorylation. The hyperphosphorylation of proteins have been attributed to the reorganization of cytoskeleton which related to its cytotoxicity, moreover, which also attributed to its tumor promotion activity.Some previous result showed that MC can initiate apoptosis in a variety of cell types characterized by cell membrane blebbing, cytoplasmic shrinkage, nuclear chromatin condensation, DNA fragmentation and formation of apoptotic bodies. Although it has been implicated that protein phosphorylation status and caspases acyivation may play an important role in MC-induced apoptosis, and the induction of oxidative stress and mitochondrial alteration were involved in this process, the exact mechanisms of MC-induced apoptosis are still unknown.The role of apoptosis-related proteins, Bcl-2 family protein and p53 in the microcystin induced apoptosis were studied in our lab. The results showed that administration of MC-LR in vivo and in vitro can both significantly increases the Bax and p53 expression level, the Bcl-2 expression level is significantly decreased in vitro, but interestingly, there is no significant difference emerged in vivo. The difference between the protein expression in vitro and in vivo suggested that the mechanisms of microcystin toxicity were complicated.A number of studies which investigated individual proteins such as ATP-synthetase beta subunit protein, Bcl-2 and p53, which attempted to explain effects observed at the cell level in response to microcysyin. However, the effects of microcystin on the cellular biological function must be complicated. In order to clarify the biological responses machinery followed exposure to toxins, using high throughput method ofchoice to detect differentially expressed proteins between profiles after exposure to toxicants is preferred. Proteomic analysis, which combining 2-DE and mass spectrometry, allows simultaneous monitoring of the expression of hundreds and even thousands of proteins in a sample following exposure to toxicant. Therefore, proteomic analysis is becoming a popular high throughput method of choice to detect differentially expressed proteins between profiles after exposure to toxicants.Therefore, the apoptosis alteration induced by microcystin was detected by PI/Annexin V-FITC double staining using flow cytometry. And the activation of caspase-3 in hepatocytes and renal cells were detected directly by fluorochrome-labeled inhibitor of caspase-3. Proteomic analysis combinind 2-DE and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was undertaken to identify differentially expressed proteins following exposure to microcystin, meanwhile, the dose response and time course of the cellular response to microcytin was studied. Following, western blot analysis was employed to validate the deregulated expression of proteins. The differentially expressed were classified according to their function through bioinformatics analysis and the functional implications of these proteins were discussed.Main results:1. The key protein of apoptosis-caspase-3 is involved in the apoptosis induced by microcystin: Microcystin can induce apoptosis in a dose-dependent manner. Caspase-3, which plays a key role in the apoptotic process, can be activated in hepatocytes and renal cells. There is correlation between apoptosis alteration and the activation of caspase-3. Therefore, the results indicate that caspase-3 may play an important role in the apoptosis induced by microcystin.2. Screening of the microcystin targted proteins using proteomic anaysis: Based on proteomic analysis of the dose response, eighty-nine proteins showed significant differential expression in different doses MC-RR treated cells compared with control, and 66 proteins were identified with high confidence. Meanwhile, 119 proteins showed significant differential expression and 103 proteins were identified in the time course study. The proteins identified can be assigned into several functional groups,such as signal transducer, enzyme and enzyme regulator, structure molecule and transporter et al. There are comprehensive and complex responses in the FL cells after exposure to microcysyin.3. Verification of differential expressed protein by Western blot analysis: From the candidates, ERK and p53 were selected for Western blot analysis. The expression change of the selected protein was consistent with the 2-DE and silver-staining results. Such result demonstrated that the proteomics analysis of cellular response to microcystin was convincing.4. Microcystin can regulate the protein level of PP2A in addition to inhibition of its activity: The result obtained from the time course study indicates that the peotein level of PP2A regulatory subunit A can be regulated by microcystin.5. Microcystin can regulate the protein level of some key proteins in addition to modulation their phosphorylation status: Some studies suggested that Microcystin can modulate the phosphorylation status of ERK, p53 and tubulin. Moreover, the resulted obtained indicate Microcystin can regulate the expression level of these important proteins.6. Some important proteins which have not been reported previously to be involved in cellular processes responded to microcystin: the finding of the differential expression of eIF5A, CDK4, tau protein and peroxiredoxin which have not been reported previously to be involved in cellular processes responded to Microcystin offered the prospect of identifying new toxic mechanisms.Main conclusion:1. Caspase-3 may take part in the apoptotic process induced microcystin. However, there are must be some other proteins which involved in the microcystin induced apoptosis except for caspase-3. Whether or not does caspase-3 take part in the apoptosis induced by microcystin seems to be dependent on the cell type and the exposure time.2. A proteomic approach was carried out to evaluate the cellular response to microcystin. Eighty-nine proteins show the differentially expressed followed MC-RRtreatment. Similarity, the number in the time course study was 119. The indentifeid proteins out of the differentially expressed proteins which can't be assigned into the same functional group indicated that they take part in a variety of cellular processes.3. There was a little dose-dependent or time-dependent manner could be observed in some proteins in the dose response study or time course study. Most of the differentially expressed proteins were dose-independent or time-indenpedent. Therefore, the biological responses of high dose exposure can't be considered as only an amplification of low dose response although changes can occur after low and high dose exposure. Microcystin may activate the independent pathway to regulate the cell response machinery followed exposure to different doses for different times.4. The finding about Microcystin can inhibit PP2A activity play an important role in the study of Microcystin toxicity mechanism. Moreover, the differential expression of PP2A obtained from in this study further indicated that the toxicity mechanism of Microcystion is complicated. Therefore, the dual effects of microcystin on PP2A suggested that the toxicity of microcystin was complicated.5. In the present study, both the 2D analysis and western blot demonstrate that MC-RR can up-regulate the p53 level in all the three doses used in the study compared with control and in the time course study. And there was a dose-dependent up-regulation of p53 in the present and the previous studies. Moreover, it was noteworthy that heat shock protein 70 and retinoblastoma binding protein encoded by p53-targeted gene were both up-regulated by microcystin. These results suggest that up-regulation of p53 play a key role in the cellular responses to Microcystin.6. The expression level and phosphorylation status of p53, Bcl-2 and Bax may be involved in the Microcysyin induced cell response machinery.7. Most of the differentially expressed proteins detected in this study have not been reported previously to be involved in cellular processes responded to Microcysyin. Therefore, more work is needed to evaluate the physiological relevance of particular proteins such as eIF5A, CDK4, 14-3-3 and peroxiredoxin. This work provides thenew insight into the mechanisms of MC-RR and the possibility of new biomarkers for evaluating the exposure to MC-RR.