|Other Abstract||Toxic cyanobacterial blooms in fresh water bodies have been becoming a kind of ecological disaster worldwide. One of the most harmful effects of cyanobacteria blooms is typically from the toxins produced by the algae, which present a major threat to the health of human, livestock and wildlife. Of all the algal toxins, microcystins are the most widely distributed toxic and abundant species. Most investigations into the toxicity of microcystins are focused on animals. In this paper, the toxicological effects of microcystins on terrestrial higher plants, Nicotiana tabacum L., and its possible mechanism were studied. The main results are as follows:
1. Microcystins from Microcystis bloom in Lake Dianchi were extracted and purified with high performance liquid chromatography (HPLC). MC-RR reagent with the purity over 95% was finally obtained by preparative HPLC.
2. When tobacco BY-2 suspension cells were exposed to 2 µg/mL MC-RR with 0.5% DMSO or 2 mmol/L ASA, the formation of ROS and MDA were prevented, and the contents of GSH, ASA and activities of SOD and POD all decreased in contrast to MC-RR treatment. It is indicated that tobacco BY-2 suspension cells suffered oxidative stress after MC-RR treatment and the exogenous ASA and DMSO can protect the cells from MC-RR induced oxidative stress.
3. Adopting 0.1, 1, and 10 µg/mL microcystin-RR (MC-RR) to treat tobacco BY-2 suspension cells, the cell viability, contents of protein, soluble sugar, nitrate nitrogen (Nitrate-N), and total phosphorus were determined, and also the activity of acid phosphatase (ACP) was detected. The results showed that the cell viability and protein content were markedly decreased in both the 1 and 10 µg/mL exposure groups after 2 d compared with the control. The soluble sugar content significantly decreased in the 10 µg/mL treated cells to a value 45.57% of the control, while the soluble sugar content was elevated in the later period of low concentration MC-RR exposure. After 4 d exposure, a significant decrease in the nitrate-N content was only observed in the 10 µg/mL treatment group, while a further reduction in nitrate-N content was observed in all the treated groups after 7 d treatment. MC-RR also decreased the total phosphorus content and after 9 days exposure the total phosphorus content in 0.1, 1, and 10 µg/mL MC-RR treated cells accounted for 74.98%, 76.47% and 84.00% of control, respectively. ACP activity in 3 treated groups first decreased and then increased compared with the control. It can be concluded that when the toxin concentration was over 1 µg/mL, the nutritional metabolism were significantly restrained by MC-RR.
4. Tobacco BY-2 cells were exposed to microcystin-RR (MC-RR) at two concentrations, 60 and 120 µg/mL, to study the changes in morphology and ultrastructure of cells as results of the exposure. Exposure to the lower concentration for 5 d led to typical apoptotic morphological changes including condensation of nuclear chromatin, formation of a characteristic ‘half moon’ structure, and cytoplasm shrinkage and decreased cell volume, as revealed through light microscopy, fluorescence microscopy, and transmission electron microscopy respectively. Exposure to the higher concentration, on the other hand, led to morphological and ultrastructural changes typical of necrosis, such as rupture of the plasma membrane and the nuclear membrane and a marked swelling of cells. The presence of many vacuoles containing unusual deposits points to the involvement of vacuoles in detoxifying MC-RR. Results of the present study indicated that exposure of tobacco BY-2 cells to MC-RR at a lower concentration (60 µg/mL) results in apoptosis and that to a higher concentration (120 µg/mL), in necrosis.
5. Tobacco BY-2 cells were exposed to 10 µg/mL MC-RR. PTP, mitochondrial permeability transition pores, was studied and a time-dependent opening was observed, the loss of mitochondrial transmembrane potential (ΔΨm) was also induced. Changes in activities of Ca2+-, Na+-K+- and H+-ATPase on mitochondrial membrane of tobacco BY-2 cells were studied. Results showed that all these three ATPases increased under the treatment. Mitochondrial morphological changes were observed by TEM. The mitochondria in untreated cells had high electronic density with abundant and acerose cristae. In the MC-RR treated group, the mitochondria were slightly swollen, with lower electronic density and fewer cristae than those in the control group. These results suggested that the toxicity of microcystin-RR caused the damage in mitochondria of tobacco BY-2 cells.
6. When tobacco BY-2 cells were treated with MC-RR of 60 μg/mL for 5 d, time-dependent effects of MC-RR on the cells were observed. Morphological changes such as abnormal elongation, evident chromatin condensation and margination, fragmentation of nucleus and formation of apoptotic-like bodies suggest that 60 μg/mL MC-RR induced rapid apoptosis. Moreover, there was a significant and rapid increase of ROS level before the loss of mitochondrial transmembrane potential (ΔΨm) and the onset of cell apoptosis. Ascorbic acid (AsA), a major primary antioxidant, prevented the increase of ROS generation, reversed the decrease in ΔΨm and subsequent cell apoptosis, indicating a critical role of ROS in MC-RR-induced loss of ΔΨm and apoptosis. In addition, a specific mitochondrial permeability transition pores (PTP) inhibitor, cyclosporin A (CsA), significantly blocked the MC-RR-induced ROS formation, loss of ΔΨm, as well as cell apoptosis, suggesting that PTP is involved in 60 μg/mL MCRR-induced tobacco cell apoptosis signalling process. Thus, it could be concluded that the MC-RR-induced ROS formation may lead to the opening of PTP, inducing the decrease of ΔΨm and subsequent apoptosis in tobacco BY-2 cells. Furthermore, it was found that MC-RR caused activation of caspase 3 and 9. Caspase-3 inhibitor AC-DEVD-CHO significantly prevented apoptosis of tobacco BY-2 cells at the 5th day, suggesting that MC-RR induced apoptosis mainly through caspase-dependent pathway. Based on the results using mitochondrial inhibitors, it is suggested that complexs Ⅰand Ⅲ of respiratory chain are potentially involved in MC-RR induced ROS generation in mitochondria. The finding of our study that MC-RR induced tobacco cell apoptosis was abrogated by mitochondrial inhibitors such as Rot and AA supports the hypothesis that mitochondria provide a link between MC-RR, ROS generation, and tobacco cell apoptosis.
7. This study was undertaken to investigate the role of the glutathione-involved detoxifying mechanism in defending the tobacco BY-2 suspension cells against microcystin-RR (MC-RR). Analysis showed that exposure of the cells to different concentrations of MC-RR (0.1, 1 and 10 µg/mL) for 0-6 d resulted in a time and concentration-dependent decrease in cell viability and increase in reactive oxygen species (ROS) content. Reduced glutathione (GSH) and total glutathione (tGSH) content as well as glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities significantly increased after 3-4 d exposure in the highest two concentration treated groups, while decreased until reaching the control values except for GPX at day 6. Oxidized glutathione (GSSG) content markedly increased compared with control in high concentration MC-RR treated group after 6 d exposure. The GSH/GSSG ratio was much higher than control in 10 µg/mL MC-RR treated group at day 4, but after 6 d exposure, the ratios in all treated groups were lower than that of the control group. It can be concluded from all the data that the inherent GSH-involved detoxifying mechanism might constitute the first line of defense against MC-RR stress. The GSH synthesis would be significantly induced and GSH-related enzymes such as GST, GR and GPX would be activated to compensate the defensive system.|