Cytotoxic Effect And Hemolytic Characteristic Of Aureococcus Anophagefferens (Qinhuangdao Strain)

In the year 2009,for the first time in China,brown tide has broken out above the sea area near Qinhuangdao,Hebei Province,and till now,there are still brown tides of various scales breaking out in the wasters every year.Brown tides had threatened to destory the marine resources and shellfish mariculture industry.Up to now,there was a systematic understanding of the impact of the brown tide on marine organisms,but the mechanism has so far been inconclusive.Whether it produces toxins is an outstanding question and its physical and chemical properities and action mode are also need to further explore.Adrenal gland chromaffin tumor cell(PC 12)was employed to study the toxicity of the extraction of Aureococcus anophagefferens(causative species of brown tide).Firstly,the cytotoxic effects of A.anophagefferens on cell proliferation,leakage of latic dehydrogenase,morphological changes and apoptosis/necrosis were investigated.According to the results of cytotoxic effects,we hypothesized that the cytotoxicity of A.anophagefferens was attributed to hemolytic activity.The interaction of the extraction of A.anophagefferens and rabbit erythrocytes was observed by microscope and atomic force microscope(AFM),and the components of hemolysin was preliminarily analysed by thin layer chromatography.Effects of temperature,pH,different divalent cations,oxidation-reductant on the hemolysis induced by A.anophagefferens were observed.Moreover,the interaction between hemolysin and erythrocytes were also studied as an attempt to explore the hemolysis mechanism of the hemolysin.Main conclusions are as follow:The chloroform extraction of A.anophagefferens inhibited the proliferation of PC 12 significantly in a time and dose dependent manner with IC50 of extraction being 79.94μg·mL-1 when the exposure time was 36h.However the methanol extraction had not shown any inhibition.Lower level of the chloroform extraction of A.anophagefferens could induce the leakage of lactic dehydrogenase,suggesting that the chloroform extraction could destroy the integrity of cytomembrane.The chloroform extraction could result in the cells rounding up,detaching from the substratum,swollen and breaking,these indexes indicated that the cells were undergone necrosis other than apoptosis under the action of A.anophagefferens extract.This speculation was further proved by Annexin V&PI double-staining.Under optical microscope we observed that the erythrocytes treated with the chloroform extraction were almost dissociation indicating that the chloroform extraction have intense hemolytic activity.Severe deformation of erythrocytes treated with the extraction was also observated by AFM.The hemolytic activity of A.anohagefferens was 21.33 ±0.45 HU·L-1 and(1.42±0.03)× 10-8 HU·cell-1 respectively.Components analysis using thin layer chromatography showed that the chemical natural of hemolysin from A.anophagefferens might be glycolipid and consist of at least two components and the Rf value were 0.90 and 0.76 respectively.The hemolysis was strengthen with the increasing of temperature in 4～37℃.The hemolytic activity was enhanced in acid condition and inhibited in alkaline.When the concentration was 5mmol·L-1,the divalent cations such as Cu2+、Mg2+、Nn2+、Ca2+、Co2+、Zn2+ and Hg2+ had different inhibition on the hemolysis,in which Hg2+ was the strongest inhibitor.High concentrations of Hg2+ induced a rapid and cooperative aggregation and prevent penetration of Hg2+ into the cells,which did not only avoid more hemolysis induced by the Hg2+ entrance,but also inhibited the destruction by the hemolytic toxin.However,this phenomenon could be eliminated by addition of EDTA.Beyond that the hemolytic activity was inhibited by oxidant of hydrogen peroxide and enhanced by reducers of cysteine hydrochloride indicating that the hemolysin was a thiol-activated toxin.The characteristic spectrum of erythrocytes sensitivity to hemolysin indicated that erythrocyte of sea bream was of highly sensitive to this hemolysin,whereas chicken and human erythrocytes were insensitive.The fact that sphingomyelin and D-xylose inhibited the hemolysis significantly while other membrane lipid and saccharides showed no inhibition suggesting that sphingomyelin and D-xylose might be acting as potential receptors of the hemolysin on the erythrocyte membrane.The finding that none of the osmotic protectants with different hydrodynamic diffusion diameter can inhibited hemolysis effectively implying that the hemolysin might not act as a pore-forming toxin.The fact that the extraction of A.anophagefferens could not formed a clear ring on the egg yolk emulsion plate showing that the hemolysin had not any phospholipase activity.These characteristics of the hemolysin differ from that of pore-forming toxins and phospholipase-like toxins suggesting that the hemolysin produced by A.anophagefferens might act through a detergent mechanism.