Cytotoxic effects of DON and T-2 toxin on plant cells

The effect of deoxynivalenol (DON) and T-2 toxin on mitotic index (MI) and relative division rate (RDR) in actively dividing onion (Allium cepa L.) root-tip cells was studied. Both these toxins resulted in decline of mitotic activity which was inversely proportional to the concentrations of these toxins. T-2 was more effective resulting in 59% RDR value at 2.5 ppm whereas DON treated root cells had 78% RDR at the same concentration as compared to respective sets of controls.     

T-2 toxin induces apoptosis in differentiated murine embryonic stem cells through reactive oxygen species-mediated mitochondrial pathway

T-2 toxin, a member of the trichothecene mycotoxin family produced by the Fusarium fungi, has been shown to exert a variety of toxic effects on multiple targets in vivo. However, the embryonic toxicity of T-2 toxin in vitro remains unclear. In the present study, two permanent cell lines, embryonic stem cells (ES cells D3) and fibroblast 3T3 cells, were used to evaluate T-2 toxin toxicity. Differentiated mouse ES cells were cultivated as embryoid bodies along with T-2 toxin at different concentrations (0.5, 1, and 2 ng/ml) for 24 h. The increases in cellular reactive oxygen species (ROS), lipid and DNA oxidative damage, and loss of mitochondrial transmembrane potential were observed at 1 and 2 ng/ml concentrations. Flow cytometry showed that T-2 toxin induced cell cycle arrest and apoptosis. Furthermore, T-2 toxin opened the mitochondrial permeability transition pore, caused the release of cytochrome c from mitochondria and induced the upregulation of p53, caspase-9, caspase-3 expression and increased the ratio of Bax/Bcl-2. However, T-2 toxin-induced oxidative damage and apoptosis in differentiated ES cells decreased significantly in the presence of the antioxidant Trolox. Taken together, these results demonstrate that T-2 toxin induces oxidative stress and apoptosis in differentiated murine ES cells, and ROS-mediated mitochondrial pathway plays an important role in T-2 toxin induced apoptosis.     

Cross-reactivity of antibodies against T-2 with deepoxide T-2 toxin

Two types of antibodies raised against T-2 toxin, namely anti-T-2-HS-BSA and anti-3 -Ac -NEOS-HS -BSA, showed good cross-reactivity with deepoxy T-2 toxin. Our results indicate that the epoxide is not an important epitope for the production of antibody against T-2 toxin     

Effect of T-2 toxin on feed intake, digestion and pathology of rabbits

Feed containing sublethal T-2 toxin concentrations (12.5 and 25 ppm) was fed to adult rabbits. The animals ate 60-70% less toxin-containing food. The dry matter content of their feces decreased significantly (on an average by 10%). The nutrient digestibility of the feed containing 12.5 ppm T-2 toxin, was increased by 2-6% and that of the 25 ppm T-2 toxin level decreased by 4-11% as compared to the control values. The rabbits showed emaciation, subacute catarrhal gastritis, necrosis of the lymphoid cells of the intestinal mucosa, depletion and necrosis in the lymphoid follicles of the ampulla ilei, spleen and lymph nodes. Necrosis of the cells of mononuclear phagocyte system and myeloid hemacytogenesis was characteristic. The toxin concentration of feces, cecotroph and urine was proportional to intake.     

Effect of T-2 toxin on growth, performance and haematobiochemical alterations in broilers

Administration of dietary T-2 toxin in 120 days old broiler chicks led to significant lower body weights and increase in feed conversion ratio from 2nd week of age. There was significant reduction in haemoglobin and packed cell volume in T-2 toxicated birds at 4 ppm level only. The other hematological parameters like TEC, TLC and absolute leucocyte count did not showed any variation due to T-2 toxin in feed. Significant reduction in serum total protein and cholesterol levels and rise in serum uric acid and LDH levels of broilers were observed due to dietary T-2 toxin. The result suggests that T-2 toxin is toxic to broilers even at very low concentrations.     

Effect of T-2 toxin on organ ultrastructure and organelle-specific enzyme activity in rats

The time-course of the ultrastructural changes and activities of 6 marker enzymes of subcellular particles (succinate dehydrogenase, beta-glucosidase, beta-N-acetylglucosaminidase, acid RNAse, glucose-6-phosphatase and 5'-nucleotidase) has been studied in the liver, spleen and thymus in rats administered T-2 toxin (mycotoxin produced by some Fusarium species). A pronounced difference in the effect of T-2 toxin on the organs has been found. In the liver, the toxin induced a destruction of rough endoplasmic reticulum membranes, reduced ribosome number and progressively decreased activities of most enzymes. In the spleen, early and significant ultrastructural disturbances of all the cell membrane components and simultaneous lysosomal activation were observed. The changes in the thymus were characterized by a fast development of cell hydratation, organelle swelling and necrosis of some thymocytes with parallel increase in repair processes, infiltration by phagocytes and a selective activation of lysosomal hydrolases in the end of experimental time (72 h.). The results obtained emphasize an importance of cellular and subcellular membrane alterations in the mechanism of T-2 toxin action.     

Metabolic effects of trichothecene T-2 toxin

Cereals and other agricultural products contaminated with trichothecene mycotoxins are unfit for consumption. Until recently, the metabolic effects of T-2 toxin (T-2) were thought to reside in its ability to inhibit protein synthesis. It is now clear that trichothecenes have multiple effects, including inhibition of DNA, RNA, and protein synthesis in several cellular systems, inhibition of in vitro protein synthesis, inhibition of mitochondrial functions, effects on cell division, normal cell shape, and hemolysis of erythrocytes. It is argued that these effects are pleiotropic responses of the cell's biosynthetic network to protein synthesis inhibition. However, in studies with erythrocytes, which lack nuclei and protein synthesis, changes in cell shape and lytic response towards T-2 are observed. Susceptibility to lysis is species dependent and correlates with the presence of phosphatidylcholine. Owing to their amphipathic nature, T-2 and other trichothecenes could exert their cytotoxicity by acting on cell membranes. As for cell energetics, T-2 inhibits the mitochondrial electron transport system, with succinic dehydrogenase as one site of action. Although initial investigations of the metabolic effects of T-2 mediated cytotoxicity suggested the inhibition of protein synthesis as the principal site of action, current thought suggests that the effects of trichothecenes are much more diverse.     

四逆散对人肝癌HepG2细胞增殖、凋亡的影响及其机制

目的:探讨含四逆散药液血清对人肝癌HepG2细胞增殖、凋亡的影响及机制。方法:将人肝癌HepG2细胞分为5组,每组3个复孔。实验组细胞用五氟尿嘧啶(5-FU)或不同浓度的含四逆散药液血清处理48 h后,用倒置显微镜观察含四逆散药液血清处理后人肝癌HepG2细胞形态的变化; MTT法检测含四逆散药液血清对HepG2细胞生长的抑制作用;荧光染色和流式细胞术分别分析含四逆散药液血清对HepG2细胞凋亡的影响。Rho123染色法检测线粒体膜电位变化,Western blot检测细胞凋亡相关蛋白的表达。结果:与对照组比较,含四逆散药液血清处理人肝癌HepG2细胞后,细胞数量显著减少(P<0.01),形态发生改变,呈现典型的凋亡细胞形态; G1期细胞数明显增加,而G2期细胞数量显著减少(P<0.05); Bax、Caspase-3、-9和Cyt-c的表达显著升高,而Bcl-2的表达显著降低(P<0.05);随着含四逆散药液血清浓度增大,HepG2细胞线粒体膜电位显著下降(P<0.05)。结论:四逆散可以抑制HepG2细胞增殖,并通过线粒体途径诱导细胞凋亡。     

Effects of ovarian theca cells on apoptosis and proliferation of granulosa cells: changes during bovine follicular maturation

We have investigated the role of theca cells in the control of apoptosis and proliferation of granulosa cells during bovine ovarian follicular development using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. A DNA fluorescence flow cytometry was used to determine the extent of apoptosis and proliferation in populations of granulosa cells. When granulosa cells were isolated from small follicles (3-5 mm), the percentage of apoptotic cells gradually increased by 1.8-fold during the 3 days of culture. This change was reduced (3.1-fold) by the presence of theca cells. When the cells were isolated from large follicles (15-18 mm), the percentage of apoptotic granulosa cells was gradually reduced (3.4-fold) during the 3 days of culture in single-cultured groups. The percentage of apoptosis on Day 1 was reduced (1.6-fold) by the presence of theca cells. However, such an effect was not detected on Days 2 and 3 of the culture. Theca cells did not affect the proliferation of granulosa cells obtained from either small or large follicles. The present study suggests that theca cells regulate the fate of granulosa cells throughout the follicular maturation process by secreting factors that suppress apoptosis.     

Characterisation of hemolysis induced by T-2 toxin

The erythrocyte constitutes a good model system for the study of membrane-associated toxicity events caused by the trichothecene mycotoxin, T-2. This study confirms that T-2 has a direct lytic effect on erythrocytes. Lysis of guinea pig red cells requires approx. 10(10) molecules/cell and reaches plateau values after 4-6 h. An activation energy, Ea approximately equal to 4.5 kcal was derived from the Arrhenius equation. By use of osmotic blockers of differing Stokes' radii, the functional size of the membrane lesion caused by T-2 toxin was shown to be smaller than 5.5 A. It is concluded that T-2 toxin may exert its toxic effects via the cell membrane.     

Production of antibody against T-2 toxin.

Antibody against T-2 toxin was obtained after immunization of rabbits with bovine serum albumin-T-2 hemisuccinate conjugate. The antibody had greatest binding efficiency for T-2 toxin, less efficiency for HT-2, and least for T-2 triol. Cross-reaction of antibody with neosolaniol, T-2 tetraol, and 8-acetyl-neosolaniol was very weak. Diacetoxyscirpenol, trichodermin, vomitoxin, and verrucarin A essentially gave no cross-reaction with the antibody. The sensitivity of the binding assay for T-2 toxin detection was in the range of 1 to 20 ng per assay. Detailed methods for the preparation of the conjugate and the production of immune serum and methods for antibody determination are described.     

Interaction of T-2 toxin with murine lymphocytes

T-2 toxin is taken up by lymphocytes in 10–15 min in a saturable manner. Uptake is dependent on temperature and partially on the availability of energy. Approx. 10⁵ molecules of T-2 toxin are bound per cell, having a mean affinity constant, K_a = 1.6·10⁷ M^?1. The toxin is rapidly dissociated from the cell to leave approx. 10–15% of the original loading in 1 h. It is concluded that T-2 toxin uptake and release do not follow conventional mechanisms.     

BCG对乳腺癌细胞增殖和凋亡的影响

目的从细胞增殖和凋亡两方面观察BCG对乳腺癌细胞MDA—MB-231的抑制作用。方法用MTT法检测BCG作用后MDA—MB-231细胞的增殖能力,采用TUNEL法检测凋亡。结果BCG能明显降低MDA-MB-231细胞的增殖代谢活性而抑制其增殖。TUNEL法染色显示BCG可显著增加凋亡细胞。结论BCG不仅能显著抑制MDA—MB-231细胞增殖,还能有效诱导其凋亡。     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

T-2 toxin and diacetoxyscirpenol metabolism by Baccharis spp

Hybrids resulting from crosses between Baccharis sarothroides and B. pilularis (FS1), B. sarothroides (FS2) and B. megapotamica (FS3) were tested for their tolerance to trichothecenes as well as their ability to metabolize the toxins. B. sarothroides (desert broom) was placed in an aqueous solution containing 500 ppm of T-2 toxin and showed visible signs of toxicity on the twigs at 21 h after exposure but not at 6 h, indicating some resistance. Samples of the twigs harvested 6 and 21 h after treatment contained, respectively, T-2 (0.03 and 2.2 micrograms/g), HT-2 (0.09 and 7.6 micrograms/g), and T-2-tetraol (2.1 and 2.6 micrograms/g). The hybrid FS1 showed no signs of toxicity 6 h after treatment, and its twigs contained T-2 (0.8 micrograms/g), HT-2 (10.2 micrograms/g), and T-2-tetraol (10.8 micrograms/g). The leaves at 6 h contained 0.5 micrograms of T-2, 1.7 micrograms of HT-2, 0.01 microgram of 3'-hydroxy-HT-2, and 41 micrograms of T-2-tetraol per g. At 21 h, toxic signs were apparent and the twigs contained T-2 (39 micrograms/g), HT-2 (62 micrograms/g), 3'-hydroxy-HT-2 (0.8 microgram/g), and T-2-tetraol (22 micrograms/g).(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryotoxicity of T-2 toxin and secalonic acid in embryonic chicks varies with the site of administration.

A crucial role of the site of administration in the sensitivity of the alternative system using chick embryo for testing embryotoxicity was demonstrated by morphological evaluation of the effects of T-2 toxin and secalonic acid D, and by incorporation of [14C]sodium acetate radioactivity. Secalonic acid D, administered to 2-, 3-, and 4-day-old embryos in doses higher than 1 microgram produced mostly malformations of the face (bilateral cleft beak, microphthalmia) while the teratogenic effects of T-2 toxin were being limited to the embryonic trunk of 2-day-old embryos (rumplessness) after administering doses higher than 0.001 microgram. In case of subgerminal and intraamniotic injections, the doses of both mycotoxins needed for producing embryotoxic effects comparable to those obtained with the more commonly used yolk sac injections appeared to be lower by one and two orders of magnitude, respectively. The results stress the need of using the shortest transport channel of test substances from the site of application to the target tissues of the embryo, when the maximum sensitivity and reproducibility of the test system are to be expected.     

Leptin directly controls proliferation, apoptosis and secretory activity of cultured chicken ovarian cells

The aim of our in-vitro experiments was to examine, whether leptin can directly control functions of avian ovarian cells and to outline potential intracellular mediators of its effects. Granulosa cells or fragments of ovarian follicular wall were cultured with leptin (0, 1, 10 or 100 ng/mL medium). The expression of peptides involved in apoptosis (TdT, bax, its binding protein, bcl-2, ASK-1 and p53), cell cycle-related peptides (PCNA and cyclin B1), release of hormones (progesterone, testosterone, estradiol, arginine-vasotocin), as well as the expression of protein kinases (PKA, MAPK/ERK1,2 and CDK/p34) in the ovarian cells were examined by using immunocytochemistry, TUNEL, SDS-PAGE-Western immunoblotting, EIA and RIA. It was found that leptin inhibited expression of all markers of cytoplasmic apoptosis (bax, ASK-1 and p53), stimulated expression of anti-apoptotic peptide bcl-2, but did not affect nuclear DNA fragmentation (TdT). Furthermore, leptin inhibited expression of PCNA (marker of S-phase of mitosis), but not of cyclin B1 (marker of G phase of cell cycle). Moreover, it promoted release of progesterone and estradiol, suppressed release of testosterone, but did not affect arginine-vasotocin. Finally, leptin inhibited expression of MAPK/ERK1,2 and CDK/p34 and stimulated expression of PKA. The present observations demonstrate that leptin can directly control basic chicken ovarian functions - inhibit cytoplasmic apoptosis and proliferation (S-phase, but not G-phases of mitosis), regulate secretory activity (release of steroids, but not nonapeptide hormone) and expression of MAPK, PKA and CDC2, which might be potential intracellular mediators of leptin action.     

The hemolytic activity of deoxynivalenol and T-2 toxin.

The hemolytic effects of deoxynivalenol (DON) and T-2 toxin (T-2) individually on rat erythrocytes were studied at different concentrations. Sodium azide was used as an enzyme inhibitor to prevent T-2 toxin metabolism. The concentration of T-2 was controlled by GC-MS and no decrease of the toxin was found during the time of the experiment. In spite of the much higher toxicity of T-2 toxin to eucaryotic cells, DON and T-2 showed similar lytic activity toward erythrocytes at high and low concentrations. Neither of these toxins at a concentration of 130 micrograms/ml, produced significant hemolysis even after 11 hr incubation. This finding suggests that there is a threshold level for both T-2 and DON, below which the lytic reaction does not occur. An additional hemolysis test was conducted in the presence of mannitol, glutathione, ascorbic acid, alfa-tocopherol, and histidine. The assay demonstrated that all the compounds inhibited to some extent the hemolytic reaction of the toxins. It is suggested that DON and T-2 exert their toxicity on procaryotic cells in three different ways: by penetrating the phospholipid bilayer and acting at the subcellular level, by interacting with the cellular membranes, and by free radical mediated phospholipid peroxidation. Most probably, more than one mechanism operates at the same time.     

The effect of T-2 toxin on bone microstructure in rabbits

T-2 toxin is the most toxic of the trichothecene mycotoxins. Its effect on bone microstructure is still unknown. This study focuses on acute effects of the T-2 toxin on compact and trabecular bone tissues structure of rabbits after a single intramuscular administration. Experimental E group (n?=?4) consisted of animals which were intramuscularly injected with T-2 toxin at dose 0.08 mg.kg^?1 body weight 72 h before slaughter. Group C (n?=?4) without T-2 toxin application served as a control. An absence of primary vascular longitudinal bone tissue near endosteal surfaces, its deposition on periosteal surfaces and a lower density of secondary osteons in the middle part of the substantia compacta were observed in both females and males injected with T-2 toxin. On the contrary, morphometrical analysis of the compact bone showed no demonstrable alternations in the sizes of primary osteons’ vascular canals, Haversian canals or secondary osteons between rabbits from E and C groups. Also, no significant effects of the T-2 toxin on trabecular bone morphometry and cortical bone thickness were observed between rabbits of either sex. The single intramuscular application of T-2 toxin at the dose used in our study affects only qualitative histological characteristics of the compact bone in rabbits.