Structure elucidation of ostreocin D, a palytoxin analog isolated from the dinoflagellate Ostreopsis siamensis.

The structure of ostreocin D, a palytoxin analog isolated from the marine dinoflagellate Ostreopsis siamensis, was found to be 42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin by detailed 2D NMR analyses of intact ostreocin D and its ozonolysis products. Partial stereochemical assignments were done. This result indicates that the dinoflagellate O. siamensis is one of the biogenetic origins of palytoxin.     

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.     

甲基莲心碱抗乳腺癌 MCF-7细胞的研究

目的:观察甲基莲心碱对乳腺癌细胞系MCF-7增殖和凋亡的影响,并探讨其诱导乳腺癌细胞系MCF-7凋亡的可能作用机制。方法:采用体外培养人乳腺癌细胞系MCF-7,CCK-8实验检测不同浓度甲基莲心碱对MCF-7细胞增殖抑制作用;乳酸脱氢酶(LDH)试剂盒(微板法)检测细胞上清液LDH含量;流式细胞术分析甲基莲心碱对MCF-7细胞周期及凋亡的影响;实时定量PCR(RT-PCR)检测线粒体凋亡相关基因Bax和Bcl-2的表达水平。结果:CCK-8、LDH结果显示甲基莲心碱以时间、浓度依耐性的方式抑制乳腺癌MCF-7细胞的增殖及促进细胞毒性的增加;流式细胞术结果表明不同甲基莲心碱作用下MCF-7的平均凋亡率分别为(15.44±0.52)、(18.81±2.24)、(24.26±2.84)、(36.90±3.15)、(59.27±5.86),且使其周期阻滞于G0/G1期;RT-PCR检测结果证明甲基莲心碱可上调乳腺癌细胞中促凋亡基因Bax的表达,而下调抑制凋亡基因Bcl-2。结论:甲基莲心碱以时间和浓度依赖的方式抑制乳腺癌细胞增殖、细胞毒性增加,导致细胞周期于G0/G1阻滞并促进癌细胞凋亡。甲基莲心碱抗乳腺癌的可能作用机制是激活线粒体凋亡途径。     

An inhibitor specific for the mouse T-cell associated serine proteinase 1 (TSP-1) inhibits the cytolytic potential of cytoplasmic granules but not of intact cytolytic T cells

We have investigated a proteinase inhibitor, designed according to the preferred amino acid sequence that is cleaved by the murine T-cell specific serine proteinase 1 (TSP-1) for its effect on the cytolytic potential of cloned cytotoxic T-cell lines (CTLL) and of cytoplasmic granules, derived from these cells. Pretreatment of effector cells with H-D-Pro-Phe-Arg-chloromethyl-ketone (PFR-CK) prior to the cytotoxicity assay did not result in inhibition of cytolytic activity of three independent CTLL and did not effect their granule-associated TSP-1 activity after extraction with Triton X-100. Furthermore, PFR-CK did not interfere with cytolysis of target cells by CTLL when present for the entire incubation period. In contrast, PFR-CK inhibited in a dose-dependent manner both TSP-1 activity and the hemolytic/cytolytic potential of isolated cytoplasmic granules after their pretreatment with high-salt concentration. We interpret these results to mean that cytolysis of target cells by CTLL involves the granule-associated proteinase TSP-1, which probably becomes active upon exocytosis following effector-target cell interactions.     

Human monocyte cytotoxic factor mediates cytolysis of WEHI 164 cells

Human monocytes can be activated to release a 40,000-Da cytostatic protein factor (CF). In this report we have investigated the cytolytic activity of CF on WEHI 164 cells which are sensitive to monocyte-mediated cytolysis. Monocyte supernatants containing CF induced cytolysis of murine WEHI 164 sarcoma cells, as determined in a 51Cr-release assay. Preincubation of WEHI 164 cells with actinomycin D enhanced cytolysis induced by supernatants containing CF, suggesting that CF may be involved in drug-dependent cellular cytotoxicity. The cytolytic activity was profoundly inhibited by a rabbit antiserum raised against purified CF, indicating that the cytolytic activity in the supernatants was in fact mediated by CF. These results indicate that CF may be an important effector molecule in monocyte-mediated cytostatic and cytolytic reactions.     

Histamine regulates the generation of human cytolytic T lymphocytes

Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.     

Presence of IgM Antibodies Which Sensitize HIV-1-Infected Cells to Cytolysis by Homologous Complement in Long-Term Survivors of HIV Infection

Although human cells are resistant to homologous human complement due to the presence of species-specific membrane inhibitors, a naturally occurring IgM antibody which recognizes an asialo-oligosaccharide can sensitize HIV-1-infected cells for complement-mediated cytolysis. Therefore, we investigated whether long-term survivors of HIV-1 infection harbor such antibodies in their sera. Thirty of 31 sera from HIV-1 seropositive hemophilia patients who have survived HIV-1 infection 10 years or more showed appreciable cytolytic activity, while only 2 sera of 10 seropositive patients presumed to have been infected with HIV-1 (due to sexual contact) more recently showed cytolytic activity. On the other hand, only 7 out of 43 sera from seronegative hemophilia patients showed cytolytic activity. Immunofluorescence staining for IgM on HIV-L -infected cells essentially correlated with the cytolytic capacity of the sera. Therefore, naturally occurring IgM antibodies and/or generated IgM antibodies reactive with the HIV-L -infected cells in patients might have been responsible for long-term survival due to complement-mediated immune cytolysis which may, in conjunction with cytotoxic T lymphocytes, synergistically suppress the infected cells in vivo. Therefore, the transfusion of such IgM antibodies could be effective for the treatment of HIV-L -infected individuals.     

Staurosporine-induced death of MCF-7 human breast cancer cells: a distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions

To test the role of caspase 3 in apoptosis and in overall cell lethality caused by the protein kinase inhibitor staurosporine, we compared the responses of MCF-7c3 cells that express a stably transfected CASP-3 gene to parental MCF-7:WS8 cells transfected with vector alone and lacking procaspase-3 (MCF-7v). Cells were exposed to increasing doses (0.15-1 microM) of staurosporine for periods up to 19 h. Apoptosis was efficiently induced in MCF-7c3 cells, as demonstrated by cytochrome c release, processing of procaspase-3, procaspase-8, and Bid, increase in caspase-3-like DEVDase activity, cleavage of the enzyme poly(ADP-ribose) polymerase, DNA fragmentation, changes in nuclear morphology, and TUNEL assay and flow cytometry. For all of these measures except cytochrome c release, little or no activity was detected in MCF-7v cells, confirming that caspase-3 is essential for efficient induction of apoptosis by staurosporine, but not for mitochondrial steps that occur earlier in the pathway. MCF-7c3 cells were more sensitive to staurosporine than MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to killing by staurosporine when evaluated by a clonogenic assay. A similar distinction between apoptosis and loss of clonogenicity was observed for the cancer chemotherapeutic agent VP-16. These results support our previous conclusions with photodynamic therapy: (a) assessing overall reproductive death of cancer cells requires a proliferation-based assay, such as clonogenicity; and (b) the critical staurosporine-induced lethal event is independent of those mediated by caspase-3.     

Reversal of phorbol ester-mediated reduction of cloned T lymphocyte cytolysis by interleukin 2

During previous studies on the regulation of cloned T lymphocyte function, we observed that murine cytotoxic T lymphocyte (CTL) clones progressively lose the ability to lyse appropriate target cells during prolonged (24 to 48 hr) incubation with the tumor promoter phorbol myristic acetate (PMA). We further observed that the cytolytic function of PMA-treated CTL clones can be restored by incubation with secondary MLC supernatant (2 degrees MLC SN), a potent source of cytokines. We now report our observations on the nature of the cytokine(s) responsible for recovery of CTL activity. Like 2 degrees MLC SN, the lectin-induced SN from a cloned helper T cell and the lectin-induced SN from a T cell hybridoma can restore cytolytic activity to cloned CTL treated with PMA. In contrast, supernatants from L929 cells, WEHI-3 cells, and P388D1 cells fail to restore cytolytic activity to similarly treated cloned CTL. These data suggest that IL 2 and/or gamma-IFN, but not CSF-1, CSF-GM, IL 3, or IL 1, can influence expression of cytolysis by cloned CTL. Furthermore, highly purified IL 2 can restore cytolytic activity, even when cytosine arabinoside is present to inhibit clonal expansion. Our studies indicate that cytolysis is a reversible function of cloned CTL, and that cytolysis may not necessarily represent an end-stage feature of CTL maturation. Our studies further show that IL 2 is both necessary and sufficient for resumption of cytolytic function by "deactivated" CTL. As such, these observations suggest that IL 2 can regulate not only T cell proliferation but also the expression of cytolysis by some cytolytic T cell populations.     

Selective depletion and enrichment of alloreactive cytolytic effector lymphocytes using anti-fluorescein affinity columns

Peritoneal exudates from BALB/c mice rejecting C57BL ascites lymphoma EL4 are a rich source of cytolytic effector lymphocytes (CL); however, these preparations are still contaminated even after removal of adherent cells with other mononuclear cells which do not appear to be cytolytic. The relationship of the cytolytic and “non-cytolytic” cells to graft rejection in vivo is not completely understood. We have used anti-fluorescein (α-FL) columns to separate sensitized lymphoid populations into fractions enriched or depleted in cytolytic activity. EL4 were directly labeled with fluorescein isothiocyanate (FL-EL4), centrifuged with CL and the mixture was applied to a column of horse α-FL antibody conjugated to Sepharose 4B. FL-EL4 and lymphocytes bound to them were retained on the column, while non-bound lymphocytes were collected in a medium wash (passed cells). CL bound to FL-EL4 were then eluted with EDTA (eluted cells). Cytolytic activity of the two fractions was compared to that of the unfractionated population in an in vitro⁵¹Cr release assay. Passed cells were consistently depleted in cytolytic activity compared to unfractionated cells or manipulation controls reaching 100% depletion in some experiments. Enrichment of cytolytic activity in eluted populations was frequently but not invariably observed. Rate of cytolysis was used as a measure of cytolytic activity in fractionated populations. Specificity of binding was investigated in reciprocal experiments using CS7BL/6J effectors raised against BALB/c lymphoma RL♂ 1. Viability of recovered cells was high and the procedure was rapid, efficient and versatile. In contrast to monolayer cellular immunoabsorbents, contamination of fractions with absorbing cells was consistently less than 5%. Both enriched and depleted populations are available for further study of surface markers and function.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Comparative effects of 17 beta-estradiol, progestin R5020, tamoxifen and RU38486 on lactate dehydrogenase activity in MCF-7 human breast cancer cells

The effects of 17 beta-estradiol (estradiol), synthetic progestin R5020 and their antagonists, tamoxifen (Tam) and synthetic RU38486 on lactate dehydrogenase (LDH) activity in MCF-7 human breast cancer cells during the growth period were studied. A specially developed quantitative cytochemical assay was used; LDH activity is expressed per cell, and is thus independent of the positive and negative growth effects of the hormones and antagonists. Estradiol and R5020 stimulated LDH activity after similar exposures (6-48 h) and the stimuli were concentration dependent over the range 10(-7) M to 10(-10) M. As for the antagonists, RU38486 stimulated LDH activity in much the same way as estradiol and R5020; Tam alone, on the other hand, does not stimulate LDH, but when added to estradiol, Tam inhibits estradiol mediated LDH activation. When present at half-stimulant concentration, estradiol + R5020 and estradiol + RU38486 exhibit additive effects on LDH activity. Thus LDH appears to be an interesting tool for the study of hormone and antagonist effects in MCF-7 breast cancer cells.     

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.     

Increasing the sensitivity of in vitro transformed hamster embryo cells (STHE strain) to cytolysis by resident and activated macrophages

The sensitivity of low-malignant spontaneously in vitro transformed hamster embryo cells (STHE strain) to cytolysis of both resident and activated macrophages has been examined with cytolytic 3H-thymidine release assay. Activated macrophages were obtained from Syrian hamster peritoneal exudate cells 5 days following priming with 3% thioglycollate-broth and subsequent in vitro activation with proper-myl, levan, LPS, MDP, 1,4-dihydropyridine-derivate PP-256 and PMA. It has been shown that the STHE strain cells were sensitive to cytolysis by only fully activated macrophages. Both resident and non-activated (Thioglycollate-elicited) macrophages have not developed significant levels of the cytolytic activity against STHE cell targets. Short-term treatment of STHE cells with actinomycin D result in augmentation of their sensitivity to cytolysis by resident and activated macrophages.     

Sprouty2蛋白与人乳腺癌MCF-7细胞增殖与迁徙的关系

目的：探讨Sprouty2蛋白与人乳腺癌MCF-7细胞增殖与迁徙的关系。方法：通过siRNA技术干扰MCF-7细胞sprouty2基因的表达,通过qPCR,细胞免疫荧光和westernblotting检测sprouty2基因的干扰效果,MTT检测细胞增殖活力,划痕实验观察细胞迁徙能力,westernblotting检测MMP-2,MMP-9和MMP-13的表达。结果：qPCR,细胞免疫荧光和westernblotting检测sprouty2基因,发现sprouty2基因的下调很明显,MTT实验发现siRNASprouty2基因后的MCF-7细胞比对照组细胞活力明显提高,沉默组细胞的迁徙能力也明显强于对照组,且沉默sprouty2基因的MCF-7细胞,MMP-2,MMP-9和MMP-13蛋白相对对照组均上调。结论：sprouty2基因下调后,MCF-7细胞的细胞活力和迁徙能力明显提高。     

Facile synthesis of triterpenoid saponins bearing β-Glu/Gal-(1→3)-β-GluA methyl ester and their cytotoxic activities

Convenient synthetic strategy toward spinasaponin A methyl ester 1 and calenduloside G methyl ester 2, two natural oleanane-type triterpenoid saponins bearing an unique β-D-glucosyl/galactosyl-(1→3)-β-D-glucuronic acid methyl ester disaccharide moiety, was established. Based on this facile approach, four structurally modified congeners 3-6 with ursolic acid and glycyrrhetinic acid as aglycones were efficiently synthesized. MTT assay revealed the cytotoxicities against cancer cells of the synthesized saponins were varied with the change of aglycones and sugar units. Saponin 2 possessing the most potent cytotoxic effects could induce apoptosis of MCF-7 cells, which was detected by confocal micrographs using DAPI staining and flow cytometry using Annexin V and PI double staining. Furthermore, 2-induced apoptosis in MCF-7 cells was associated with ROS generation and loss of the mitochondria membrane potential (Δψ(m)).     

腺病毒载体AdING4 对MCF-7乳腺癌细胞的增殖抑制及化疗增敏作用

目的:研究腺病毒载体AdING4对人MCF-7乳腺癌细胞的生长抑制及化疗增敏作用。方法:将搭载有ING-4基因的重组腺病毒载体AdING4感染人MCF-7乳腺癌细胞,用荧光显微镜观察感染后的MCF-7细胞形态学变化;RT-PCR和Western-Blot法检测ING-4基因在MCF-7细胞中的转录和表达;RT-PCR法检测凋亡相关基因在MCF-7细胞中的表达;CCK法测定Ad-ING4感染MCF-7乳腺癌细胞后所发挥的细胞增殖抑制作用。流式细胞技术检测ING-4对MCF-7乳腺癌细胞的促凋亡作用。CCK-8法分别测定病毒感染前后的MCF-7乳腺癌细胞的药物半数抑制浓度IC50,并观察Ad-ING4与化疗药物合用后对MCF-7细胞增殖抑制和化疗增敏现象。结果:MCF-7细胞在转染ING-4基因后,明显出现变圆、脱落、皱缩、聚集等现象;外源性ING-4基因在MCF-7细胞中获得成功表达;外源性ING-4基因作用下MCF-7细胞的增殖受到了明显抑制,凋亡率有所升高,凋亡相关基因Bax的表达水平明显上调,Bcl-2、Survivin的表达水平明显下调。ING-4基因感染MCF-7细胞后,使MCF-7细胞对相关化疗药物的敏感度更高;ING-4基因与化疗药物合用后对MCF-7细胞的增殖抑制作用,较之单用化疗药物更为明显。结论:MCF-7细胞在转染ING4基因后其增殖受到了明显抑制并更易凋亡,该现象可能是通过改变Bax,Bcl-2及Survivin表达水平来实现的,且对化疗药物的敏感性更高。     

The effect of hydrocortisone on lymphocyte-mediated cytolysis

This paper reports the results of experiments designed to analyze the mechanism by which hydrocortisone suppresses the cell-mediated cytolysis produced by sensitized lymphocytes. We used an in vitro system in which rat lymph node cells were sensitized to, and caused cytolysis of mouse fibroblasts.We found that hydrocortisone probably suppresses cytolysis by preventing the primary activation of the cytolytic mechanism by target cell antigens. Suppression was most efficient when hydrocortisone was added at the beginning of the cytolytic reaction. The cytolytic mechanism itself appeared to remain intact, and could be activated by the lectin concanavalin A (con A) despite the presence of hydrocortisone.Suppression of cytolysis could not be related to any general inhibition of DNA, RNA, or protein synthesis. The influence of hydrocortisone on cytolysis was not modified by vitamin A (retinol), an agent which antagonizes the effect of hydrocortisone on lysosome membranes.Hydrocortisone was found to be less effective in suppressing the activity of lymphocytes that had been sensitized initially in the presence of hydrocortisone.     

Augmentation of human monocyte-mediated cytolysis by interferon

Human monocytes, separated by either plastic adherence or adherence to microexudatecoated surfaces, from the peripheral blood of most normal donors were shown to have significant cytolytic activity against TU5, a mouse SV40-transformed target cell. Spontaneous cytolysis ranged from 0 to 32% at a 40:1 effector:target (E:T) ratio. Augmentation of cytolysis was usually seen when human fibroblast interferon (IF) (10³–10⁴ units/ml) was cultured with the effector and target cells for the duration of the assay. The mean increase in percentage cytolysis at 40:1 and 20:1 E:T ratios was greater with monocytes obtained by a microexudate method (24.1 and 22.4%) than with monocytes obtained by a plastic adherence method (16.0 and 8.1%). Only a slight augmentation of cytotoxicity was observed when the effector cells were pretreated with IF for 1-hr. The increased levels of cytotoxicity observed when IF was present during the assay did not appear to be due to the toxic effects of IF on the target cells or to a stable increase in the susceptibility of the target cells to lysis.     

Lactate dehydrogenase release assay from Vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains

The Vero cell assay presently used for virulence testing of verotoxigenic Escherichia coli (VTEC) requires at least 48-96 h where cytotoxicity effects are examined under a microscope. Here, a complimentary rapid assay was developed that measures endogenous lactate dehydrogenase (LDH) release from Vero or HEp-2 cells as an indicator of cytotoxicity. Toxin preparations from 24 VTEC strains induced 36-89% LDH from Vero cells and 15-62% LDH from HEp-2 cells in 12-16 h. A verotoxin-positive but enterohemolysin negative strain also showed a similar cytotoxicity effect. In contrast, three VT-negative strains caused only 13-16% LDH from Vero cells and 1-7% LDH from HEp-2 cells. Five presumptive E. coli isolates from naturally contaminated food and clinical sources did not induce significant LDH release from either cell lines. PCR analysis confirmed the presence of vt1 or vt2 genes in E. coli showing positive LDH values. Similarly, RiboPrinter analysis confirmed and identified the test strains as E. coli except for two meat isolates, which were identified as Hafnia alvei. Cytopathic effects of toxin preparations from VTEC revealed severe lysis, vacuole formation and death in Vero cells and multiple vacuoles and cell elongation in HEp-2 cells. The colorimetric cytotoxicity assay described here can provide quantitative data for determining the virulence potential of verotoxigenic E. coli in 12-16 h.