| Cyanobacterial blooms have been documented worldwide ecological problem and have caused a great threat to the ecological environment and human health. Among cyanotoxins, the microcystins (MCs) are the most common and toxic group. Previous studies have shown that MCs are hepatotoxicity, renal toxicity, immune toxicity, reproductive toxicity, developmental toxicity and potential cancer-promoting activity. There were some studies about anemia caused by microcystin-LR (MC-LR) which are concentrated in the hematological indices, the proliferation of bone marrow cells, genetic toxicity and so on, but the mechanism research about anemia is still less. Therefore, we researched the toxic effects of crucian carp and mice erythrocytes exposed to the MC-LR using the acute experiments; evaluated the suppression of hematopoiesis function in mice after prolonged MC-LR exposure; observed the anemia and changes of cell cycle and apoptosis of rat bone marrow cells caused by MC-LR. The main results and conclusions are as follows:(1) Crucian carp erythrocytes were incubated in vitro with MC-LR at doses of 0,1, 10,100 and 1000 nM at 15℃. The antioxidative enzymes activities, lipid peroxidate level, biochemical biomarkers, haemolysis and erythrocytes morphology were detected. We found that the level of lipid peroxidate significantly increased in MC-LR treatment groups and showed some time-does effects. The activities of antioxidative enzymes, including superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase were significantly increased after exposure with MC-LR. The glutathione levels significantly decreased at 12 and 24h. While the activities of acetylcholinesterase, Na+-K+-ATPase and Ca2+-Mg2+-ATPase were significantly decreased. The haemolysis was significantly increased. In addition, pathological alterations of agglomerated and jagged erythrocytes were observed in blood smears. The findings indicated that oxidative damages of erythrocytes should also be responsible for anemia and hypotensive shock or even death.(2) In the present study, Kunming mice erythrocytes in vitro were incubated with 1,10, 100 and 1000 nM MC-LR at 37℃. Lipid peroxidation, haemolysis, cell morphology, antioxidative response and some biochemical biomarkers were measured. The results showed that the level of lipid peroxidation significantly increased in MC-LR treatment groups. The level of glutathione and activities of glutathione peroxidase, glutathione-S-transferase and superoxide dismutase were significantly increased after incubation with MC-LR. Also, significant decreases in activities of acetylcholinesterase, Na+-K+-ATPase and Ca2 + -Mg2 + -ATPase were observed. Obvious increases of haemolysis were determined in 10,100 and 1000 nM groups from 12 to 48 h. Additionally, abnormal erythrocytes with blebbing and notched cell membrane, dissolving cellular membrane were observed in both 100 and 1000 nM groups. It is presumed that MC-LR triggers lipid peroxidation of erythrocytes and oxidative stress destroys the structure of cell membrane, leading to alterations of antioxidative enzymes and biochemical indicators.(3) In the present study, Balb/c mice were intraperitoneally injected with MC-LR at the doses of 0.5,2 and 8 μg/kg bw every 48 h for 30 d. After the prolonged exposure of MC-LR, significant decreases of RBC and Ht were observed in 2 and 8 μg/kg bw groups, but MCV showed no significant change. While the ESR and WBC significantly increased. Significantly elevated micronucleus frequency was observed in bone marrow cells in all MC-LR treatments with 3.2-fold,5.1-fold and 6.7-fold. The bone marrow nucleated cells were significantly decreased in 2 and 8 μg/kg bw groups. And the proliferation of bone marrow cells significantly declined in both 2 and 8 μg/kg bw groups. Serum hematopoietic growth factors including granulocyte-macrophage, erythropoietin, interleukin-3 were significantly decreased and TNF-a was significantly increased. The transcriptional levels of granulocyte-macrophage, erythropoietin, interleukin-3 in bone marrow cells were significantly down-regluated and TNF-a was significantly up-regulated. The serum levels of some apoptosis-related factors (caspase-3, caspase-8, caspase-9 and p53) significantly increased. The present study indicates that prolonged exposure of MC-LR induces damages of hematopoietic function, and the disturbed hematopoietic growth factors and the apoptotic damage in bone marrow cells are responsible for this suppression of hematopoietic function.(4) In order to further clarity the relationship of apoptosis caused by MC-LR and hematopoietic function, we detected the peripheral blood indicators, bone marrow cell cycle, cell apoptosis factors mRNA expression level of bone marrow cells when the rats exposed with MC-LR at the doses of 1 and 10 μg/kg bw every day for 50 d. The results showed that RBC, Ht, Hb, PLT were significantly decreased while WBC significantly increased. Bone marrow cell cycle analysis showed that MC-LR significantly inhibited the G1 to S cell cycle progression and significantly promoted the cell apoptosis in 10 μg/kg bw group. The Real-Time PCR results showed that the transcription of Bcl-2 was significantly down-regulated while the transcription of Bax、p53、caspase-3、Fas， Fas-L were significantly up-regulated. These results indicated that MC-LR caused anemia, induced G1 arrest and apoptosis of bone marrow cells. In addition, MC-LR maybe regulate the apoptosis of bone marrow cells through regluateded the mRNA expression of Fas、Fas-L、Bax、Bcl-2、p53、caspase-3, ultimately inhibit hematopoietic function in rats. |
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