| In the21st century, Karenia mikimotoi Hasen has become the prevalent algae of red tide in Chinese offshore sea, especially in Fujian waters, causing massive mortaliy of bivalves. It produces numerous toxins, such as the hemolytic toxin and cytotoxin, which have strong toxic effects on marine lives and lead to huge economic losses.In order to uncover toxicology of Karenia mikimotoi on marine lives, we cultured Karenia mikimotoi and tested its toxic effects on abalones as they prey on Karenia mikimotoi. We collected algas and utilized an organic extraction method with Chloroform, methanol and water to obtain the toxic fractionations of it. We harvested series of crude methanol extracts and proceeded to toxicity assay of Zebrafish embryos. The crude methanol extracts were further fractionated through the positive phase silica gel column, HPLC and TLC to gain the hemolytic toxin and cytotoxin. Since our previous study showed that the methanol extracts from Karenia mikimotoi have hemolytic activity, hemolytic assay of fractioned component on Rabbit erythrocytes was conducted. We also used the Human tumor cell lines and fish cell lines for cyotoxicity assay. Upon these studies, we preliminarily lay the root in study of Karenia mikimotoi in red tide.Our results showed that the density of Karenia mikimotoi cultured in the laboratory was up to5.1x107per liter, which exceeded the maximum density of Karenia mikimotoi in red tide. The abalone feeding experiments showed that abalones were lethal when they were cultured in the environment containing algas up to1.0x106/L. Based on toxicity assay against Zebrafish embryos, we found that low concentrations of K. mikimotoi methanol crude extracts could inhibit zebrafish embryo hatching rate, its IC50was216μg/mL. The methanol crude extracts also caused growth retardation, somites shortening and other malformations of zebrafish embryos. Zebrafish embryo mortality was100%within24hours when the crude extracts’concentration over2.16mg/mL. By liquid chromatography—mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analysis, we confirmed structure of the fractioned hemolytic toxin MS-2-17from K. mikimotoi and found it was a kind of oil amides with a M.W of281. Hemolysis test against rabbit erythrocytes indicated that hemolytic activity of toxin would be up to80%when its concentration was over12ng/mL.In this study, we also isolated and purified the cytotoxin MS-2-3from K. mikimotoi. Through MTT cytotoxicity assay, the apoptosis assay and cell cycle tests by the Flow Cytometory, we found that the cytotoxin could inhibit proliferation of human tumor cells, such as HeLa cells, when the concentration of cytotoxin was80ng/mL. The apoptosis assay and cell cycle tests demonstrated that HeLa proliferation was blocked in G1phase by the cytotoxin, being excluded from S, G2-M phases.In summary, this study simulate the early and late of Karenia mikimotoi in laboratory at the first, proved that feeding K. mikimotoi could lead the death of abalone.Using zebrafish as experimental subjects,first proved low concentrations of dissolved material in the cell have a strong biological toxicity to embryos and larvae of marine organisms. We also isolated and purified hemolytic toxin, then identified the structure of this compound. Hemolytic activity assay was done at the same time. The paper for the first time analyzed the mechanism of cytotoxic of K. mikimotoi that it inhibited the tumor cell cycle. |
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