Mechanism Study of Endothelial Protection and Inhibits Platelet Activation of Low Molecular Weight Fucoidan from Laminaria japonica

Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline(0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. v WF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce v WF level in vascular endothelial injury rats and also significantly reduce v WF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and v WF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.     

Establishing Gene Delivery Systems Based on Small-Sized Chitosan Nanoparticles

Chitosan is a natural cationic polysaccharide,which is often used for preparing biomedical materials because of its high biocompatibility.In this study,chitosan with a molecular weight of 160 k Da was chosen to prepare chitosan nanoparticles(CSNPs) as gene vectors by ionic cross-linking with tripolyphosphate(TPP).CSNPs were characterized in terms of particle size,zeta potential,and polydispersity index(PDI) using a Zetasizer,and morphology was evaluated by transmission electron microscopy(TEM).Furthermore,the cytotoxicity and biocompatibility of CSNPs were correspondingly examined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and histological examination.Agarose gel electrophoresis and UV spectrophotometric methods were performed to measure the loading capacity.The cell transfection efficiency of CSNPs loaded with plasmids or si RNA was analyzed by fluorescence microscopy or laser scanning confocal microscopy.The results showed that CSNPs were prepared successfully by the ionic gelation method,which had a smaller particle size(100 nm-200 nm),stable dispersibility,low cytotoxicity,good tissue-biocompatibility,and high gene-loading efficiency.These CSNPs could transfer the plasmids or si RNA to cells.However,CSNPs might have a much higher transfection efficiency for si RNAs than for plasmids,which implies that CSNPs might be a safer and more efficient vector for delivering si RNAs rather than plasmids.     

Studies on Antiviral and Immuno-Regulation Activity of Low Molecular Weight Fucoidan from Laminaria japonica

The antiviral activity in vitro and in vivo and the effect of the immune system of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica(LMW fucoidans) were investigated in order to examine the possible mechanism. In vitro, I-type influenza virus, adenovirus and Parainfluenza virus I were used to infect Hep-2, Hela and MDCK cells, respectively. And 50% tissue culture infective dose was calculated to detect the antiviral activity of two LMW fucoidans. The results indicated that compared with the control group, 2 kinds of LMW fucoidans had remarkable antiviral activity in vitro in middle and high doses, while at low doses, the antiviral activity of 2 kinds of LMW fucoidans was not statistically different from that in the blank control group. And there was no statistically difference between two LMW fucoidans in antiviral activity. In vivo, LMW fucoidans could prolong the survival time of virus-infected mice, and could improve the lung index of virus-infected mice significantly, which have statistical differences with the control group significantly(p 0.01). However, the survival time of the two LMW fucoidans was not statistically significant(p 0.05). In this study, it was shown that both of two LMW fucoidans(LF1, LF2) could increase the thymus index, spleen index, phagocytic index, phagocytosis coefficient and half hemolysin value in middle and high doses, which suggested that LMW fucoidans could play an antiviral role by improving the quality of immune organs, improving immune cell phagocytosis and humoral immunity.     

菲律宾蛤仔糖胺聚糖抗肿瘤活性研究

从菲律宾蛤仔全脏器中提取得到糖胺聚糖粗品(CRG),通过DEAE-52离子交换柱层析得到纯品(RG),分别采用MTT法和动物移植性肿瘤模型检测其体外和体内抗肿瘤活性,同时测量荷瘤小鼠的胸腺指数的脾指数;建立小鼠免疫抑制模型,并测量正常小鼠和免疫抑制小鼠的胸腺指数、脾指数和碳粒廓清指数。结果显示,菲律宾蛤仔糖胺聚糖可以抑制体外培养的人肝癌Bel7402肿瘤细胞的生长,能显著抑制移植性S180肉瘤的生长,可抑制荷瘤小鼠的脾肿大现象,对环磷酰胺造成的小鼠胸腺和脾脏萎缩有抵抗和修复作用。     

A Comparative Study About the Neuroprotective Effects of EPA-Enriched Phosphoethanolamine Plasmalogen and Phosphatidylethanolamine Against Oxidative Damage in Primary Hippocampal Neurons

Oxidative stress is involved in the progression of neurodegenerative diseases. Previous evidences showed that plasmalogens could improve neurodegenerative diseases. In this study, we investigated the function of phosphoethanolamine plasmalogens enriched with EPA(EPA-p PE) and phosphatidylethanolamine enriched with EPA(EPA-PE) on oxidative damage prevention after hydrogen peroxide(H_2 O_2) and tert-butylhydroperoxide(t-BHP) challenge in primary hippocampal neurons. Results showed that neurons pretreated with EPA-p PE and EPA-PE demonstrated the ability to alleviate oxidative damage, which was proved by the increased cell viability. Moreover, the shape and number of neurons were more similar to those of the control group. Antioxidant activity, apoptosis, as well as Trk B/ERK/CREB signaling pathway were investigated to explore the mechanisms. The results suggested that EPA-PE was superior to EPA-p PE in regulating mitochondrial apoptosis. EPA-p PE was more prominent than EPA-PE in upregulating Trk B/ERK/CREB signaling pathway. Phospholipids with EPA exerted neuroprotective effects via inhibiting oxidative stress, suppressing apoptosis, and regulating Trk B/ERK/CREB signaling pathway. Therefore, the results provide a scientific basis for utilization of phospholipids enriched with EPA on the treatment of neurodegenerative disease.     

Fascaplysin sensitizes cells to TRAIL-induced apoptosis through upregulating DR5 expression

This study investigated the molecular mechanism of anti-tumor effect of fascaplysin, a nitrogenous red pigment firstly isolated from a marine sponge. Microarray analysis show that the TNF and TNF receptor superfamily in human umbilical vein endothelial cells (HUVEC) and human hepatocarcinoma cells (BEL-7402) were significantly regulated by fascaplysin. Western Blot results reveal that fascaplysin increased the expression of cleaved caspase-9, active caspase-3, and decreased the level of procaspase-8 and Bid. Flow cytometry and cytotoxicity tests indicate that fascaplysin sensitized cells to tumor necrosis-related apoptosis-inducing ligand-(TRAIL) induced apoptosis, which was markedly blocked by TRAIL R2/Fc chimera, a dominant negative form of TRAIL receptor DR5. Therefore, our results demonstrate that fascaplysin promotes apoptosis through the activation of TRAIL signaling pathway by upregulating DR5 expression.     

Characterization,tissue distribution,and expression of neuropeptide Y in olive flounder Paralichthys olivaceus

Neuropeptide Y(NPY) is a 36-amino acid peptide of the neuropeptide Y family that plays key roles in the regulation of food intake. In this study,we focused on NPY m RNA expression changes around feeding time and during food deprivation in olive flounder. The olive flounder NPY m RNA levels were analyzed in different tissues and a high level of expression was detected in the brain. We also demonstrated a correlation between NPY expression levels in the brain and feeding schedule. NPY expression levels in olive flounder maintained on a daily scheduled feeding regimen increased shortly before feeding and decreased after the scheduled feeding time. Compared with the-1 h group before feeding,NPY expression in the 3 h group after feeding decreased significantly( P <0.05). Food deprivation led to an 81.7% decrease in NPY m RNA levels in the 24 h fasted group(P <0.05) and a 91.7% decrease in the 48 h fasted group(P <0.05). Therefore,our study demonstrates that NPY expression is associated with food intake in olive flounder. This result reveals the function of NPY in regulating food intake and its potential importance in olive flounder aquaculture.     

Comparative Study on Antineoplastic Activity of Polysaccharide from Four Kinds of Sea Cucumber

The antineoplastic activity of polysaccharide was investigated in Stichopus chlorontus, Isostichopus badionotus, Stichopus horrens and Holothuria lessoni massin. Crude polysaccharide was prepared with enzyme hydrolyzation method and purified by anion exchange chromatography using DEAE-sepharose fast flow column. The effect of polysaccharide on cells apoptosis of SiHa and U87 was examined with cell counting kit-8 colorimetry method. Western blotting was used to analyze related proteins of cellular apoptosis including p53 and Bcl-2. Results showed that there were two main components in each sea cucumber polysaccharide, which could be eluted down by 1.0 mol/LNaCl solution. The four types of polysaccharide in the second component were named as SC-2, IB-2, HLM-2 and SH-2, respectively. They were used for comparing the antineoplastic activity. Results showed that SC-2, IB-2, HLM-2 and SH-2 could promote apoptosis of U87 and SiHa cells. SH-2 and HLM-2 were selected for the subsequent experiment to explore the additional effect of U87 and SiHa cells, The protein expressions of Bcl-2 and p53 decreased considerably with the increase of polysaccharide concentration in U87 cells. In SiHa cells, protein expressions of Bcl-2 and high dosage group of p53 decreased significantly, whereas no obvious decrease was observed in other groups. The polysaccharides are more effective in promoting apoptosis of U87 and SiHa cells from S. horrens and H. lessoni massin than from the rest species.     

The antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3),2,2',3,3'-tetrabromo-4,4',5,5'-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTr assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S180-bearing mice.All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC50 below 10 μg/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%,respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenoi derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer.     

赤魟组织粗提物的抗肿瘤作用

采用四甲基偶氮唑盐(MTT)微量酶反应比色法检查赤魟三种组织(皮肤、软组织和软骨)粗提物对体外培养的人癌细胞生长的影响,用腹腔注射或灌胃法检查赤魟三种组织粗提物对小鼠移植性肉瘤180(S180)和肝癌(H22)生长的影响。结果表明,赤魟组织粗提物对体外培养的人早幼粒白血病细胞(HL-60)、人子宫颈癌细胞(HeLa)和人低分化鼻咽癌细胞(CNE-2Z)的生长无明显影响,赤魟组织粗提物无论腹腔注射还是灌胃均显著抑制小鼠肉瘤和小鼠肝癌的生长,且对体重无明显影响;当三种组织粗提物以500mg/kg/d(相当生药量,下同)的剂量连续腹腔注射(ip)15d时,对S180的抑制率分别为63.9%、47.2%和80.3%;以3500mg/kg/d连续灌胃(ig)15d时,对S180的抑制率分别为66.9%、61.5%和66.9%;以500mg/kg/d连续腹腔注射15d时,对H22的抑制率分别为43.3%、43.3%和80.4%。     

Antithrombotic effects of bromophenol,an alga-derived thrombin inhibitor

Thrombin,the ultimate proteinase of the coagulation cascade,is an attractive target for the treatment of a variety of cardiovascular diseases.A bromophenol derivative named (+)-3-(2,3-dibromo-4,5-dihydroxy-phenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroiso-benzofuran 1,isolated from the brown alga Leathesia nana exhibited significant thrombin inhibitory activity.In this study,we investigated the inhibition of human thrombin in vitro with this bromophenol derivative,and its antithrombotic efficacy in vivo using th...     

The hydrogen peroxide impact on larval settlement and metamorphosis of abalone Haliotis diversicolor supertexta

Abalone Haliotis diversicolor supertexta is an important economic mollusk.The settlement and metamorphosis are two critical stages during its development period,which has direct influence on abalone survival and production.The influence of reactive oxygen species(hydrogen peroxide) on abalone embryo and juvenile development were examined in this study.Larvae of Haliotis diversicolor supertexta were induced to settlement and metamorphose by exposure to seawater supplemented with hydrogen peroxide.They had the best performance at 800 μmol/L.The concentration of 1 000 μmol/L or higher was toxic to the larvae,as the larvae could settle down only at benthic diatom plates without complete metamorphosis.In addition,H2O2 adding time was critical to the larval performance.24h after two-day post-fertilization was proved to be the optimal adding time.In this paper,two action mechanisms of hydrogen peroxide are discussed:(1) hydrogen peroxide has direct toxicity to ciliated cells,thus cause apoptosis;(2) hydrogen peroxide,as a product from catecholamines' autoxidation process in vivo,can reverse this process to produce neuro-transmitters to induce abalone metamorphosis.     

Expression of the Phycoerythrin Gene of Gracilaria lemaneiformis (Rhodophyta) in E. coli and Evaluation of the Bioactivity of Recombinant PE

Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E.coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bod- ies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were col- lected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indi- cated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein con- centration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) (P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mg mL-1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially de- creased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This re- search showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.     

Expression of the Phycoerythrin Gene of Gracilaria lemaneiformis （Rhodophyta） in E. coil and Evaluation of the Bioactivity of Recombinant PE

Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) (P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples, except for the 0.2 mg mL⁻¹ BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological activity was still retained and conspicuous. This research showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value.     

Characterization of Bacterial Communities Associating with Larval Development of Yesso Scallop(Patinopecten yessoensisis Jay, 1857) by High-Throughput Sequencing

Bacterial community presumably plays an essential role in inhibiting pathogen colonization and maintaining the health of scallop larvae, but limiting data are available for Yesso scallop(Patinopecten yessoensisis Jay, 1857) larval development stages. The aim of this study was to characterize and compare the bacterial communities associating with Yesso scallop larval development at fertilized egg S1, trochophora S2, D-shaped larvae S3, umbo larvae S4, and juvenile scallop S5 stages by Illumina high-throughput sequencing. Genomic DNA was extracted from the larvae and their associating bactera, and a gene segment covering V3-V4 region of 16 S r RNA gene was amplified and sequenced using an Illumina Miseq sequencer. Overall, 106760 qualified sequences with an average length of 449 bp were obtained. Sequences were compared with those retrieved from 16 S r RNA gene databases, and 4 phyla, 7 classes, 15 orders, 21 families, 31 genera were identified. Proteobacteria was predominant phylum, accounting for more than 99%, at all 5 larval development stages. At genus level, Pseudomonas was dominant at stages S1(80.60%), S2(87.77%) and S5(68.71%), followed by Photobacterium(17.06%) and Aeromonas(1.64%) at stage S1, Serratia(6.94%), Stenotrophomonas(3.08%) and Acinetobacter(1.2%) at stage S2, Shewanella(25.95%) and Pseudoalteromonas(4.57%) at stage S5. Moreover, genus Pseudoalteromonas became dominant at stages S3(44.85%) and S4(56.02%), followed by Photobacterium(29.82%), Pseudomonas(11.86%), Aliivibrio(8.60%) and Shewanella(3.39%) at stage S3, Pseudomonas(18.16%), Aliivibrio(14.29%), Shewanella(4.11%), Psychromonas(4.04%) and Psychrobacter(1.81%) at stage S4. From the results, we concluded that the bacterial community changed significantly at different development stages of Yesso Scallop larvae.     

Anti-proliferative activity of phlorotannin extracts from brown algae Laminaria japonica Aresch

In this study, we evaluated the anti-proliferative activity of phlorotannins derived from brown algae Laminaria japonica Aresch extracts on the human hepatocellular carcinoma cell (BEL-7402) and on murine leukemic cells (P388) by MTT assay. Cells were incubated with 100 μg/mL of the phlorotannin extract (PE) for 48 h. The inhibitory rate of PE on BEL-7402 and P388 cells was 30.20±1.16% and 43.44±1.86%, respectively, and the half-inhibitory concentration of PE (IC₅₀) on P388 and BEL-7402 cells was 120 μg/mL and >200 μg/mL, respectively. Microscopic observation shows that the morphologic features of tumor cells treated with PE and 5-fluorouracil are markedly different from the normal control group. The inhibitory rate of fraction A₂ isolated from PE by sephadex LH-20 for BEL-7402 and P388 cells at the sample concentration of 70.42 μg/mL was 61.96±7.02% and 40.47±8.70%, respectively. The apoptosis peak for fraction A₂ was the most profound of all fractions used in the flow cytometry assay. The results indicate that the anti-proliferative of this algal extract is associated with the total phlorotannin content.     

Establishment of a spleen cell line from large yellow croaker Pseudosciaena crocea and its primitive application in foreign gene transfection

A large yellow croaker,Pseudosciaena crocea,spleen(LYCS)cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study.Primary culture of LYCS cells was initiated from spleen tissue pieces,which were cultured at 25℃ in Dulbecco’s modiced Eagle medium/F12 medium(DMEM/F12,1:1)(pH7.2),supplemented with 20% fetal bovine serum,carboxymethyl chitosan,chondroitin sulfate,basic fibroblast growth factor(bFGF)and insulin-like growth factor-I(IGF-I).The cultured LYCS cells,in fibroblast shape,proliferated to 100% confluency 20 days later.Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P.crocea(6m+6sm+36t,NF=60).A LYCS cell line,with a population doubling time of 48.7 h at passage 60,has been established and subcultured to passage 70.Transgenic feasibility test demonstrated that positive green fluorescence protein(GFP)expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection.In conclusion,a continuous foreign gene trans-fection feasible LYCS cell line has been established successfully.The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.     

The Toxic Mechanism of High Lethality of Herbicide Butachlor in Marine Flatfish Flounder,Paralichthys olivaceus

The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder,Paralichthys Olivaceus,was analyzed by histopathological examination,antioxidant enzymes activities and ATP content assay.Histopathological examination of gill,liver and kidney of exposed fishes showed that gill was a target organ of butachlor.The butachlor seriously impaired the respiration of gills by a series of lesions such as edema,lifting and detachment of lamellar epithelium,breakdown of pillar cells,and blood congestion.The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality.Antioxidant enzyme activity assay of the in vitro cultured flounder gill(FG) cells exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase(SOD),catalase(CAT) and glutathione peroxidase(GPX).Furthermore,along with the decline of antioxidant enzyme activities,ATP content in the exposed FG cells decreased,too.This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP.Similar decrease of ATP content was also observed in the exposed flounder gill tissues.Taken together,as in FG cells,butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells,which in turn resulted in the blood congestion and suffocation of exposed flounder.