Toxic Effects Of Toxins Present In Eutrophicated Water Body On Human Amnion Cells

With the rapid development of civilization, some unscientific human actions in life, industry and agriculture have caused a series of severe environmental problems. A plenty of waste water inpour into numerous stagnant waters including lakes, reservoirs and bays directly, and excessive nutrients (mainly phosphorus and nitrogen) accumulate incessantly in water. All of these lead to the water eutrophication and then result in the outbreak of the algal bloom. Depending upon the characteristic color of predominant species in algal bloom, the water presented different color such as red, blue, brown and so on. The phenomenon occurred in fresh water and marine water was named water bloom and red tide respectively. Since the nature toxins produced by the harmful algal bloom are the potential threats to human health, more and more attention has been paid to their various toxic effects and complicated toxic mechanism.Microcystins (MCs) are the most persistent and widely distributed cyanotoxins produced by freshwater cyanobacteria that belong to the genera Microcystis, Anabaena, Nodularia, Oscillatoria and Nostoc. Among the huge family of cyanobacteria, Microcystis aeruginosa is found to be the most common toxic species. Since 1980s, microcystins had been recognized as a family of monocyclic heptapeptide hepatotoxins. Up to now, more than 80 MC congeners have been found. Microcystin-LR (MCLR) is the most frequently studied as well as the most toxic in the group of MCs. During the recent decades, MCs have received increasing concern worldwide due to their high toxic potential. Okadaic acid (OA) is a marine toxin isolated originally from the sponges Halichondria okadai and H. melanodocia. Subsequently, the structure of OA established by X-ray crystallography showed that the natural product toxin was a polyether fatty acid with a 38 backbone carbon skeleton. Although OA is the major component of diarrhetic shellfish poisoning (DSP) toxins and DSP is widely distributed throughout the world, the toxicological effects of OA was not well studied originally, since clinical symptoms of DSP is usually resolved within 3 days and no human deaths have been reported in literature from cases of DSP. However since OA was found to be widely distributed in the bodies of mice after consumption and was a serious threat to human health, the toxin has gained an increasing interest among researchers because of its complicated biochemical mechanisms.Nowadays it has been well known that MCLR and OA are both potent tumor promoters and they may also act as tumor initiators. Interestingly, they also can induce apoptosis in many cell types. Although the reasons of above-mentioned opposite effects caused by MCLR and OA have not yet been given, it might be explained, at least in part, by their common ability to inhibit protein phosphatases, since a well-balanced network of protein kinases and protein phosphatases is crucial for cells to regulate many important cellular processes, such as proliferation, differentiation and apoptosis. However, to the authors' knowledge, few studies concerning the relationship between the alteration in protein expression of PP2A and the treatment with the inhibitor of protein phosphatases, such as MCLR and OA, have been published. Interestingly, upregulation of PP2A-A subunit was observed in our most recent study in FL cells treated with microcystin-RR (MCRR), one structural variant among MCs. In order to identify whether or not the upregulation is toxin-specific, the effects of MCLR and OA exposure on protein expression of PP2A-A subunit were studied in the same FL cell model.During recent years, there are numerous studies focused on the apoptosis induced by MCLR and OA. However, the exact mechanisms of their toxicity still have not been fully elucidated. It has been shown that following the MCLR-exposure, the increase of reactive oxidative species (ROS) precedes the onset of mitochondrial permeability transition (MPT) and loss of mitochondrial membrane potential, which lead to cytochrome-c release from the mitochondrion and caspases activation. At last, apoptosis was initiated. All the evidence indicates that mitochondria play a pivotal role in MCLR induced apoptosis. However, in addition to classical mitochondrial pathway, to date, mitochondria-independent pathways of apoptosis induced by many cytotoxin have gained an increasing interest and there has been an emerging view showing that disturbance of cell-anchorage and activation of endoplasmic reticulum stress related components can directly lead to apoptosis. So it was worth paying more attention to all kinds of changes induced by MCLR and OA to provide more evidence for complex apoptosis mechanism.The present study was designed to evaluate the ability of MCLR and OA to induce cytotoxicity. The cell viability was determined by MTT assay. The cell cycle was mensurated by PI staining using flow cytometric analysis. The apoptosis rates induced by MCLR and OA were evaluated by TUNEL assay and PI/Annexin V-FITC double staining respectively. The cell shape change was observed directly by light microscope. The cytoskeleton modification was observed by fluorescent microscope after FITC-phalloidin staining. The Caspase 3 activation was detected by the fluorochrome-labeled inhibitor of Caspase 3. The alteration of protein expression on PP2A-A, Chop, Bax, Bcl-2 and p53 were determined by the western blot. The expression level of PP2A-A mRNA after MCLR exposure was detected by reverse transcription-polymerase chain reaction. Finally the DNA damage was measured by the single cell gel electrophoresis method.The results were shown as below:1. MCLR and OA exposure could induce the proliferation instead of fatal effect in human amnion FL cells.2. There was no obvious impact on cell cycle after MCLR exposure. However the influence of OA to the cell cycle of FL was mainly on the increase of cells that in the S period.3. MCLR and OA exposure induced apoptosis of FL cells in a dose-dependent manner.4. There was no distinct change of cell shape and F-actin distribution after MCLR exposure. However, the FL cells incubated with OA showed loss of cell-to-cell contact, detachment from the substratum and cytoplasmic rounding. In addition, cells showed extensive F-actin depolymerization and focal aggregations of F-actin around the cell periphery.5. MCLR exposure induced the increase of Caspase 3 activation of FL cells and significant differences were observed in all treated groups with respect to control. However, the activation of Caspase 3 induced by OA was significantly increased only in 20 nM, 40 nM and 100 nM OA-treated groups.6. After treatment with MCLR, the expressions of PP2A-A, Bax, and p53 proteins were significantly increased and had statistical significance from the concentration of 10 or 50 nM. In addition, the Chop protein was also upregulated at most treatment groups, although it decreased at the highest concentration group. In contrast, the expression of Bcl-2 seemed to be a concentration-dependent decrease. There were significant differences on its protein levels in 500 and 1000 nM MCLR-treated FL cells compared with control. However, PP2A-A mRNA level of treated groups was not obviously changed compared with that of control group.7. The dose-effect study after OA exposure showed that the levels of PP2A-A and Chop proteins in FL cells were both significantly increased from 60 nM groups. However, the protein expressions of Bax, Bcl-2 and p53 were a dose-dependent decrease. In addition, the time-effect study also showed that the alteration of the three apoptotic-related proteins, Bax, Bcl-2 and p53 were a time-dependent decrease.8. Comet assay showed that both MCLR and OA exposure could induce the DNA damage of FL cells. Both tail length and tail moment increased in a dose-dependent manner.Conclusions:1. MCLR exposure could induce the DNA damage of FL cells. It initiated both proliferation and apoptosis of cells.2. MCLR exposure could increase the expression of p53 protein which is responsible for DNA damage and then triggered apoptosis via a mitochondrial pathway by up-regulation of Bax protein and down-regulation of Bcl-2 protein. The up-regulation of PP2A-A and Chop proteins after MCLR exposure suggested that MCLR might induce apoptosis though more than one pathway.3. The activation of Caspase 3 in MCLR-induced FL cells might be one of the direct events in the mitochondrial pathway of apoptosis process. On the other hand, it also might be due to the indirectly regulation of Chop, one protein involved in the process of apoptosis associated with ER stress, since it has been reported that over-expression of Chop can lead to translocation of Bax protein from the cytosol to the mitochondria and decrease in Bcl-2 protein and finally trigger the mitochondrial pathway.4. OA exposure could induce the DNA damage of FL cells. It initiated both proliferation and apoptosis of cells.5. The change of cell shape and the destruction of F-actin in OA-treated FL cells suggested that OA might trigger a specific form of apoptosis, termed anoikis via the cytoskeletal damage. During the process, the increase of Caspase 3 activation after OA exposure was not in concentration-dependent. It indicated that the response of Caspase 3 activation caused by OA was complicated and there might be multiple caspase isoforms contributed to the apoptosis induced by OA.6. The up-regulation of Chop proteins after OA exposure might play an important role in OA-induced apoptosis, since it has been reported that Chop can directly induce apoptosis as well as can decrease Bcl-2 protein and finally trigger the mitochondrial pathway. And the alteration of p53 protein expression suggested that: the severe DNA damage induced by OA could not been well repaired in time and in effect and many cells were arrested at S phase. Following the change of p53, Bax protein was also decrease, which clued on an anti-apoptotic factor other than Bax might be involved in the apoptosis induced by OA. Finally, the dualistic characteristics of OA, namely it could inhibit PP2A activity as well as increase the expression of PP2A-A might attribute to, at least in part, the complicated apoptosis mechanism induced by OA.