微囊藻毒素对鱼类的毒性效应

湖泊富营养化导致的蓝藻水华已成为国内外普遍关注的环境问题,它所带来的主要危害之一是产生的藻毒素对鱼类的影响。在已发现的藻毒素中,微囊藻毒素(microcystins,MCs)的分布广、毒性大、危害严重,而备受关注。阐述了MCs对鱼类的影响。微囊藻毒素能干扰胚胎的发育,降低孵化率,增加畸形率,影响存活率,胚胎孵化受微囊藻毒素影响还具有剂量依赖效应;野外室内实验均表明鱼类暴露于微囊藻毒素后不仅可在肝脏中富集还可在肌肉、肠道等组织器官中快速积累;对鱼类进行组织病理检测发现MCs可导致肝脏、肾脏、心脏、脑、鳃等组织受损;MCs在鱼体中的解毒过程可能开始于由谷胱甘肽S-转移酶催化的还原型谷胱甘肽的结合反应;MCs还可影响鱼类的生长、行为和血清生化指标,此外,还具有一定的免疫毒性。MCs的转运机制和分子作用机制以及在食物链中传递过程中对人类造成的潜在影响可能成为今后研究重点。     

一种检测微囊藻毒素LR诱导大鼠肾细胞凋亡的方法

微囊藻毒素(Microcystins)是一种具有生物活性的单环七肽毒素,主要由微囊藻产生。其中5个氨基酸为固定组成成分,另外2种氨基酸是可变的,由此衍生出众多的同类物,微囊藻毒素LR(Microcystin LR,MCLR)是其中具有代表性的一种。同位素示踪显示,静脉注射、腹腔注射和口服3种不同途径进入小鼠体内的125I-MCLR 70%以上分布在肝脏和肾脏,揭示这两个脏器是它的靶器官。本实验中采用正常大鼠的肾细胞来研究MCLR对细胞的影响,利用流式细胞仪PI/Annexin V双染色法检测MCLR能否引起细胞凋亡的改变,为进一步探讨MCLR对细胞的损伤及作用机制提供依据。       

鲤肝细胞抗氧化系统对微囊藻毒素毒性的反应

用10μg／L的微囊藻毒素LR(Microcystin—LR,MC—LR)处理鲤肝细胞培养物,检测鲤肝细胞抗氧化系统的6项指标。结果表明,MC—LR处理后活性氧(ROS)含量明显升高,还原型谷胱甘肽(GSH)含量迅速下降,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性明显升高,谷胱甘肽过氧化物酶(GSH—Px)活性在MC—LR处理15min后也有明显上升,但谷胱甘肽S-转移酶(GST)活性在MC—LR处理后没有明显变化。另外,还从氧自由基理论解释了微囊藻毒素造成鲤肝细胞损伤的可能机理。     

微囊藻毒素LR对草鱼肝脏超微结构影响的研究

关于微囊藻毒素对哺乳动物的影响已有较多的研究,指出微囊藻毒素LR是一种作用于肝脏的极强环肽毒素,并能引起肝细胞超微结构的改变［‘］,但是有关该毒素对鱼类影响的报道却很少,作者采用腹腔注射微囊藻毒素的方法,确定其在鱼体内作用的靶器官,观察超微结构的组织病理变化,并与对哺乳动物作用的结果相比较,进而探讨其对鱼类的致毒机理．回材料和方法微囊藻毒素LR由日本名城大学药学部提供。实验鱼为草鱼［Ctenopharyngodonidellus］,体重引一389,体长11—13cm,取自中国科学院水生生物研究所养殖场,在25℃下通以循环水驯化1周…     

微囊藻毒素LR诱导大鼠肾NRK细胞Bax蛋白表达

近年来水体富营养化已成为一个全球性关注的问题,过量的氮、磷使藻类过度生长并产生水华。微囊藻毒素是蓝藻的一些属产生的次生代谢产物,在发生水华的水体中这类毒素普遍存在。微囊藻毒素产生的原因和作用目前还不是很清楚,然而它们对动物和人类都能产生极为严重的影响。       

微囊藻毒素LR对大鼠肝细胞Caspase-3酶活性的影响

水体富营养化的加剧,导致藻类水华频频发生。在全球由于藻类水华所产生的毒素,对人和动物都产生了极为严重的影响,使之成为日益突出的环境问题。其中危害最严重的 是微囊藻毒素(Micnocystin,MCYST)。而微囊藻毒素LR(LR)是微囊藻毒素中存在最为普遍且毒性作用最明显的一种,是MCYST的主要代表物。       

微囊藻毒素LR对大鼠淋巴细胞DNA的损伤效应

微囊藻毒素是一类具生物活性的单环七肽,是蓝藻的一些属产生的次生代谢产物,在发生水华的水体中这类毒素普遍存在。由于水华的普遍发生,微囊藻毒素的存在已是一个令人瞩目的问题。流行病学调查发现人群原发性肝癌、大肠癌发病率与饮水源中的微囊藻毒素有关。微囊藻毒素LR(MCLR)是微囊藻毒素中最常见的一种,由于它们的高急性毒性、强促癌活性以及与人类癌症发生的相关性使其受到全球的关注。DNA是生物体内的遗传物质,它的稳定对于生命体的正常繁殖与生长是至关重要的。       

微囊藻毒素对鱼肝的毒性效应

研究已发现离体条件下微囊藻毒素时鱼的蛋白磷酸酶有极强的抑制作用’‘,”。有些研究证明活体致毒后细胞内蛋白的磷酸化水平增加,而毒素对生物活体致毒时蛋白磷酸酶的情况尚未见报道。本研究用鱼作为实验材料,活体致责时微囊藻毒素对鱼的蛋白磷酸酶及几种其它分子生态毒理学指标的影响。回材料与方法1．l微囊藻毒素L民根据HSYddd的方法卜‘,由日本SllWS湖水华样品中提取。l．2活体实验,对体重约259的鲫鱼腹腔注射LR,剂量分别为IO,50,100和400pg／kg。在lh和3h时取样,分别测定蛋白磷酸酶（Proteiphosphatase,简称PP）活性和…     

微囊藻毒素对鱼类毒性影响的研究进展

微囊藻毒素 (Microcystins,简称MC)是一些有毒蓝藻,如微囊藻、颤藻和鱼腥藻等,产生的一种具有肝毒性的环状七肽。       

Assessing the data quality in predictive toxicology using a panel of cell lines and cytotoxicity assays

In vitro cell viability assays have a central role in predictive toxicology, both in assessing acute toxicity of chemicals and as a source of experimental data for in silico methods. However, the quality of in vitro toxicity databanks fluctuates dramatically because information they contain is obtained under varying conditions and in different laboratories. The aim of this study was to identify the factors responsible for these deviations and thus the quality of the data extracted for predictive toxicology. Three cell viability assays measuring LDH leakage, WST-1 reduction, and intracellular ATP were compared in an automated environment using four mammalian cell lines: Caco-2, Calu-3, Huh-7, and BHK. Using four standard compounds--polymyxin B, gramicidin, 5-fluorouracil, and camptothecin--a significant lack of sensitivity in LDH assay compared with the other assays was observed. Because the viability IC(50) values for the standards were similar among the cell lines, the biochemical characteristics of different cell lines seem to play only a minor role, with an exception being the hepatocellular Huh-7 cell line. Toxicity assessment of new 1,2,4-triazoles revealed significant differences in their toxic potential, and the results indicate the same sensitivity profile among the assays as observed with the standard compounds. Overall, it can be argued that the assay selection is the most important factor governing the uniform quality of the data obtained from in vitro cell viability assays.     

乳酸菌对微囊藻毒素的清除

【目的】研究唾液乳杆菌(Lactobacillus salivarius BBE09-18)、发酵乳杆菌(L.fermentum BBE09-29)以及干酪乳杆菌(L.casei Zhang)对藻毒素的清除能力以及影响乳酸菌清除藻毒素的主要因素,以期为进一步解析乳酸菌清除藻毒素的作用机制提供理论基础,并为去除食品体系中的藻毒素提供新的思路。【方法】研究乳酸菌细胞的不同生理状态(活细胞与死细胞)对藻毒素清除能力的影响,考察菌浓差异、起始藻毒素浓度差异、葡萄糖的供给等对乳酸菌清除藻毒素效能的影响。【结果】3株实验乳酸菌均具有清除藻毒素的能力,其中,L.casei Zhang的藻毒素清除能力最强,当藻毒素初始浓度为150μg/L时,24h后残留藻毒素浓度为85.5μg/L,清除率可达43%。此外,研究还发现乳酸菌活细胞的清除能力显著高于热失活后的死细胞。添加外源物质葡萄糖能显著提高实验菌株清除藻毒素的效率,在初始浓度为1800μg/L的藻毒素溶液中,当添加5%(w/v)葡萄糖后,L.casei Zhang经24h可清除92%的藻毒素。【结论】3株乳酸菌均具有藻毒素清除能力;同时,菌体浓度以及菌体自身的生理状态对藻毒素的清除效率具有重要影响,乳酸菌对藻毒素的清除可能与菌体的代谢活性相关。     

Apoptotic events induced by synthetic naphthylchalcones in human acute leukemia cell lines

Acute leukemia is a disorder of the hematopoietic system characterized by the expansion of a clonal population of cells blocked from differentiating into mature cells. Recent studies have shown that chalcones and their derivatives induce apoptosis in different cell lines. Since new compounds with biological activity are needed, the aim of this study was to evaluate the cytotoxic effect of three synthetic chalcones, derived from 1-naphthaldehyde and 2-naphthaldehyde, on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. Based on the results, the most cytotoxic compound (A1) was chosen for further analysis in six human acute leukemia cells and in a human colon adenocarcinoma cell line (HT-29). Chalcone A1 significantly reduced the cell viability of K562, Jurkat, Kasumi, U937, CEM and NB4 cells in a concentration and time-dependent manner when compared with the control group (IC₅₀ values between ∼1.5 μM and 40 μM). It was also cytotoxic to HL-29 cells. To further examine its effect on normal cells, peripheral blood lymphocytes collected from healthy volunteers were incubated with the compound. It has also been incubated with human fibroblasts cultured from bone marrow (JMA). Chalcone A1 is non-cytotoxic to PBL cells and to JMA cells. A1 caused significant cell cycle arrest in all phases according to the cell line, and increased the proportion of cells in the sub G0/G1 phase. To evaluate whether this chalcone induced cell death via an apoptotic or necrotic pathway, cell morphology was examined using fluorescence microscopy. Cells treated with A1 at IC₅₀ demonstrated the morphological characteristic of apoptosis, such as chromatin condensation and formation of apoptotic bodies. Apoptosis was confirmed by externalization of phosphatidylserine, which was detected by the Annexin V-FITC method, and by DNA fragmentation. The results suggest that chalcone A1 has potential as a new lead compound for cancer therapy.     

Studies on interactions of carbazole derivatives with DNA,cell image,and cytotoxicity

DNA-binding agents have been considered as an established opportunity for the development of anticancer drugs and DNA fluorescence probes. This work reported the synthesis of two novel carbazole derivatives (1 and 2) and investigated their DNA binding properties, living cell image, and cytotoxicity. The results demonstrated that both compounds presented the higher binding affinity to G-quadruplex than to duplex DNA by means of UV–Vis absorption titration and fluorescent intercalator displacement. Continuous variation analysis indicated that their binding stoichiometries of the compound/G-quadruplex were near 2 except the compound/bcl-2. Circular dichroism spectra showed that both compounds had no influence on the conformation of G-quadruplexes. Fluorescence titrations indicated that 2 had the potential to be a G-quadruplex selective fluorescent probe, while 1 could be used as a fluorescent probe for duplex DNA. Confocal fluorescence images indicated that both compounds could enter the living HepG2 cells, and 1 mainly located in nucleus whereas 2 mainly distributed in cytoplasm. DNase and RNase digest tests indicated that both compounds could enter into the nucleus and interact with duplex DNA, especially, 2 could interact with RNA and/or G-quadruplex DNA. They also exhibited an obvious antiproliferative activity to HepG2 by using MTT assays, with IC₅₀ values of 2.7 and 9.5?μM for 1 and 2, respectively.     

iTRAQ-based proteomic study of the effects of microcystin-LR on medaka fish liver

Microcystins are cyanotoxins that occur in ground water and thus pose a potential health risk. Microcystin-LR (microcystin-leucine-arginine) is a potent hepatotoxin, and is suspected of being a tumour promoter. Poisoning with this toxin causes several dysfunctions in hepatocytes by inhibiting protein phosphatases 1 and 2A, and notably produces oxidative stress, disrupts the cytoskeleton, and deregulates mitogen-activated protein kinase pathway. Medaka fish (Oryzias latipes) was chosen as a model for studying the effects of this cyanotoxin on liver proteins using a gel-free approach, iTRAQ. Fish were gavaged with microcystin-LR. Two hours later, 325 proteins could be identified by Scaffold Q+ and 32 proteins revealed statistically significant variations above a ∣0.2∣ threshold of log(2) ratio by comparison with control. These proteins are mostly involved in the translation and maturation of proteins, lipid metabolism and detoxification. Notably, apolipoproteins are deregulated which indicates a possible alteration of chylomicron-mediated transport.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR

Immunoassays are increasingly used to investigate the production, properties and fates of the cyanobacterial hepatotoxic microcystins in vitro and in vivo. Responses of an ELISA immunoassay to microcystins have been determined using the authentic toxin antigen, microcystin-LR, and conjugation products between the toxin and glutathione, cysteine-glycine and cysteine. The antibodies against microcystin-LR crossreacted with the toxin conjugation products with similar affinities (96-112%) to that of microcystin-LR, when assayed at a concentration of 1 microg l(-1). Toxicity assessment of the conjugates, in comparison to microcystin-LR, indicated a reduction according to mouse bioassay. In vitro protein phosphatase inhibition assay indicated that the conjugates possessed approximately 3-9-fold lower toxicity than microcystin-LR.     

A bioassay for the simultaneous measurement of metabolic activity, membrane integrity, and lysosomal activity in cell cultures

The aim of this study was the development of an in vitro bioassay that combines several endpoints of general cytotoxicity for the screening of compounds or mixtures of compounds with potential bioactivity. The Alamar Blue assay was employed to assess metabolic activity, the Neutral Red assay was used for the assessment of membrane function and lysosomal activity, and the lactate dehydrogenase leakage assay was employed for the assessment of membrane integrity. Each assay was performed separately and in combination using a human fibroblast cell line (MRC-5). Three fungal secondary metabolites of different chemistry that affect different cellular targets were tested as model compounds: deoxynivalenol, enniatin B1, and 2-amino-14,16-dimethyloctadecan-3-ol. The obtained inhibitive compound concentrations for the assays performed separately and in combination were not significantly different (P < 0.05, n = 9). The combination of several cytotoxicity endpoints in a single assay increases the chance that potential bioactive/cytotoxic compounds are discovered during the screening of mixtures of natural compounds (e.g., extracts from fungal cultures or plants) when one endpoint fails and, at the same time, might give some basic information on the cellular target.     

Evaluation of antioxidant and neuroprotective effect of Hippophae rhamnoides (L.) on oxidative stress induced cytotoxicity in human neural cell line IMR32

Aim and objectiveHippophae rhamnoides is an edible, nutrient rich plant found in the northern regions of India. It belongs to the family Elaeagnaceae and is well known for its traditional pharmacological activities. The present study was aimed to investigate the antioxidant and neuroprotective activities of H. rhamnoides.MethodologyThe hydroalcoholic extract of H. rhamnoides was evaluated for free radical scavenging activity using DPPH, hydroxyl radical scavenging and ferric thiocyanate assays. In vitro neuroprotective activity was assessed on human neuroblastoma cell line-IMR32 against hydrogen peroxide (H₂O₂) induced cytotoxicity. The neuroprotective effect was determined by measuring the cell viability through tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reducing assay and propidium iodide (PI) staining. Also the intracellular reactive oxygen species (ROS) activity was assessed using dichloro-dihydro-fluorescein diacetate (DCFDA) assay by flowcytometer.ResultsThe results of the study demonstrated that H. rhamnoides extract possesses potential free radical scavenging activity. The IC₅₀ value for DPPH and OH radical scavenging assay was 70.92 μg/ml and 0.463 mg/ml, also the extract was also found to have considerable level of lipid peroxidation activity. The neuroprotective effect of H. rhamnoides was confirmed by its cell viability enhancing capacity against hydrogen peroxide induced cell cytotoxicity. The extract acted on IMR32 cells in a dose dependent manner as observed through PI and MTT assays. The percentage intracellular ROS activity was reduced by 60–70% in treated cells compared to H₂O₂ control.ConclusionThus the outcome of the study suggests that H. rhamnoides acts as a neuroprotectant against oxidative stress induced neurodegeneration.     

In vitro cytotoxicity on human ovarian cancer cells by T-type calcium channel blockers

The growth inhibition of human cancer cells via T-type Ca²⁺ channel blockade has been well known. Herein, a series of new 3,4-dihydroquinazoline derivatives were synthesized via a brief SAR study on KYS05090 template and evaluated for both T-type Ca²⁺ channel (Ca_v3.1) blockade and cytotoxicity on three human ovarian cancer cells (SK-OV-3, A2780 and A2780-T). Most of compounds except 6i generally exhibited more potent cytotoxicity on SK-OV-3 than mibefradil as a positive control regardless of the degree of T-type channel blockade. In particular, eight compounds (KYS05090, 6a and 6c–6h) showing strong channel blockade exhibited almost equal and more potent cytotoxicity on A2780 when compared to mibefradil. On A2780-T paclitaxel-resistant human ovarian carcinoma, two compounds (KYS05090 and 6d) were 20-fold more active than mibefradil. With respect to cell cycle arrest effect on A2780 and A2780-T cells, KYS05090 induced large proportion of sub-G₁ phase in the cell cycle progression of A2780 and A2780-T, meaning the induction of cancer cell death instead of cell cycle arrest via blocking T-type Ca²⁺ channel. Among new analogues, compounds 6g and 6h induced cell cycle arrest at G₁ phase of A2780 and A2780-T cells in dose-dependent manner and exhibited strong anti-proliferation effects of ovarian cancer cells by blocking T-type Ca²⁺ channel. Furthermore, 6g and 6h possessing strong cytotoxic effects could induce apoptosis of A2780 cells, which was detected by confocal micrographs using DAPI staining.