Natural toxins最新文献

The present work has been designed to study the effect of feeding on transgenic potatoes, which carry the CryI gene of Bacillus thuringiensis var. kurstaki strain HD1, on the light and electron microscopic structure of the mice ileum, in comparison with feeding on potatoes treated with the 'delta-endotoxin' isolated from the same bacterial strain. The microscopic architecture of the enterocytes of the ileum of both groups of mice revealed certain common features such as the appearance of mitochondria with signs of degeneration and disrupted short microvilli at the luminal surface. However, in the group of mice fed on the 'delta-endotoxin', several villi appeared with an abnormally large number of enterocytes (151.8 in control group versus 197 and 155.8 in endotoxin and transgenic-treated groups, respectively). Fifty percent of these cells were hypertrophied and multinucleated. The mean area of enterocyte was significantly increased (105.3 microm(2) in control group versus 165.4 microm(2) and 116.5 microm(2) in endotoxin and transgenic-treated groups, respectively). Several forms of secondary lysosomes or auotophagic vacuoles were recognized in these cells. These changes were confirmed with the scanning electron microscope which revealed a remarkable increase in the topographic contour of enterocytes (23 microm in control group versus 44 microm and 28 microm in endotoxin and transgenic-treated groups, respectively) at the divulged surface of the villi. The basal lamina along the base of the enterocytes was damaged at several foci. Several disrupted microvilli appeared in association with variable-shaped cytoplasmic fragments. Some of these fragments contained endoplasmic reticulum, as well as ring-shaped annulate lamellae. In addition, the Paneth cells were highly activated and contained a large number of secretory granules. These changes may suggest that delta-endotoxin-treated potatoes resulted in the development of hyperplastic cells in the mice ileum. Although mild changes are reported in the structural configuration of the ileum of mice fed on transgenic potatoes, nevertheless, thorough tests of these new types of genetically engineered crops must be made to avoid the risks before marketing.

Pure ochratoxin A (OA) was added to buffered rumen fluid collected from fistulated cows and incubated under anaerobic conditions. The kinetic pattern of the disappearance of OA and the appearance of ochratoxin alpha (Oalpha) was principally the same with four diets fed to donor animals and with three donor animals. The concentration of OA declined to a very low or non-detectable level with half-lives at 0.17-1.84 h; its rate of disappearance was first order throughout. The concentration of Oalpha increased to a constant level under all conditions examined. The average amounts of Oalpha formed relative to the disappearance of OA were not different (p > 0.05) and ranged between 94 +/- 8 and 98 +/- 8 %. The rate of disappearance of OA differed (p < 0.001) between diets and animals. It was accelerated by increasing the content of concentrate in the diet. Ochratoxin alpha was not metabolized when added in pure form to buffered rumen fluid and incubated under the same conditions as OA. Pure OA was also added to buffered fluids from the forestomach and abomasum of a slaughtered cow. It disappeared exponentially in a mixture of fluids from the rumen and reticulum and in fluid from the omasum, with the average amounts of Oalpha formed relative to OA disappearance being 107 and 109 %, respectively. Ochratoxin A also was not metabolized in fluid from the abomasum. These studies demonstrate that OA is hydrolysed in the rumen via first order kinetics, diet and animal affect the rate of hydrolysis, OA is quantitatively converted to Oalpha and Oalpha is not degraded.

Previously we isolated prymnesin-1 (PRM1) and prymnesin-2 (PRM2) as the major hemolytic and ichthyotoxic agents in the red tide organism Prymnesium parvum and disclosed the structure of PRM2 as a novel glycoside with unusual multiple functionality. PRM2 caused 50% hemolysis of a 1% suspension of dog red blood cells at 0.5 nM. The potency exceeded that of plant saponin by 50000 times. The lethality of PRM2 on freshwater fish Tanichthys albonubes was comparable to that of brevetoxin and also the ichthyotoxicity was markedly enhanced by Ca2+ and by a slight elevation of pH: LC50 in a Ca2+ free medium (pH 7.0) was 300 nM and in the presence of 2 mM Ca2+ (pH 8.0) was 3 nM. The hemolytic activity of PRM2 was not affected by Ca2+ but was markedly affected by blood cell origin. Also, the observation of competitive inhibition by the PRM2 analogues allowed us to assume the presence of a specific binding site on the blood cell surface.

Putaminoxin and pinolidoxin, two structurally related nonenolides, isolated respectively from organic extracts of Phoma putaminum and Aschochyta pinodes cultures, together with some of their natural analogs and synthetic derivatives, were used in a structure-activity relationship study. Their phytotoxic, antifungal and zootoxic activities were assayed with the aim to find compounds with potential herbicidal properties. The strongest phytotoxic compounds proved to be putaminoxin and pinolidoxin, whose activity appeared to be correlated to the integrity of the nonenolide ring and to the presence of both the hydroxy groups and the unmodified propyl side chain. None of the assayed nonenolides showed antifungal activity, whereas pinolidoxin analogs and derivatives showed high to weak zootoxicity.

The mycotoxin ochratoxin A (OTA) is a chlorinated dihydroisocoumarin derivative connected through an amide-bond to L-phenylalanine. In a previous study we could show that competition with L-phenylalanine-dependent processes does not play a role in OTA neurotoxicity. To test whether the isocoumarin part is responsible for the neurotoxic effects, we determined in the present study the effects of the hydrolysis product of OTA, ochratoxin-alpha (OTalpha), and of ochracin on embryonic chick brain cell cultures. In addition, we investigated the interaction between OTA and ochracin regarding the neurotoxic effects. We report here that OTalpha did not affect brain cell cultures at concentrations up to 15 microM. With the exception of a small (20%) but significant reduction in cell culture, cellular protein at concentrations above 0.3 microM, in our cell cultures' cell function, as defined by neutral red uptake and MTT-dehydrogenase activity, was only reduced by high OTalpha concentrations (1 mM). Addition of 0.1 microM OTA increased ochracin cytotoxicity as defined by latter parameters. No effects on cell culture NF68kD content could be detected. The results are discussed with regard to the existence of an OTA target interaction binding site.

In this work we have demonstrated for the first time in any Venezuelan Crotalus, haemorrhagic activities that are present in the Neotropical Uracoan rattlesnake (Crotalus vegrandis) venom. This venom has been little studied, perhaps because the snake is restricted to a small habitat located in the dry savannahs of northeastern Venezuela. In our experiments Crotalus vegrandis venom caused a very evident haemorrhagic area consisting of approximately 2/3 diameter of the area caused by a positive control Bothrops lanceolatus venom. Crotalus vegrandis venom affects blood coagulation and causes intense haemorrhages. It does not clot fibrinogen, therefore it has neither thrombin-like activity which transforms fibrinogen to fibrin nor procoagulant enzymatic function which produces thrombin. On the other hand, it degrades fibrinogen making it incoagulable to thrombin. The venom, when injected in the animals, resulted in a high increase of the Partial Time of Thromboplastin (PTT) tests. It is interesting to observe that the haemorrhagic capacity in the Crotalus genus (widely distributed in the American continent) increases from south to the north, being present in North American Crotalus, a venom with wide haemorrhagic activities, and almost non-existent in most of the South American Crotalus species and subspecies.

The biosynthesis of the neurotoxin domoic acid (DA) in the diatom Pseudo-nitzschia multiseries was investigated using 13C- and 14C-labelled precursors. The labelling pattern determined by NMR spectroscopy following incorporation of [1,2-13C2]-acetate showed enrichment of every carbon of DA. The enrichment levels were consistent with a biosynthetic pathway involving two different intermediate precursor units. Addition of labelled acetate either early or late during exponential growth gave similar patterns and levels of incorporation. Analysis of the labelling pattern indicated that DA is biosynthesised by condensation of an isoprenoid intermediate with another intermediate derived from the tricarboxylic acid (TCA) cycle. The absence of deuterium at C2 in DA following incorporation of [2-13C, 2H3]-acetate is consistent with alpha-ketoglutarate or a derivative as the TCA cycle-derived intermediate. The different incorporation efficiencies of acetate into the putative precursor intermediates suggest that either each unit is biosynthesized in a different part of the diatom cell, or that the isoprene chain is not assembled by the usual acetate-mevalonate pathway. The latter proposal is supported by the complete absence of deuterium retention in the isoprenoid-derived portion following incorporation of [2-13C, 2H3]-acetate.

Crotalus durissus envenomation is treated using antivenins produced in horses. During production, animals have problems, sometimes followed by death, due to the high toxicity of the main toxin, crotoxin. Several methods tested to detoxify this toxin often resulted in decreased immunogenicity. Gamma irradiation has proved to be a successful method for crotoxin detoxification without loss of immunogenicity. We have studied the biodistribution of 2 kGy 60Co irradiated crotoxin (iCTX) in mouse tissues. We used both 125I-labeled iCTX or its detection by a specific immunohistochemistry assay (IHA). Both approaches showed similar early excretion of toxins by the kidneys. Higher iCTX uptake was seen in spleen and liver, which are rich in immune responder cells. In contrast to previous reports concerning native crotoxin (nCTX), we failed to detect iCTX in the neuromuscular junction, but both toxins were found on the kidney tubular cell surface, with rapid excretion that was more intense for iCTX. Kupffer cells and splenocyte macrophages presented IHA staining, as shown by the increased uptake of 125I toxin by these organs. No staining was observed in the brain, lung or heart, which also showed very low 125I counts. Allied to reduced toxicity, irradiation induced early endocytosis of crotoxin by phagocytic cells, improving antigen processing.

Production of domoic acid (DA) by the pennate diatom Pseudo-nitzschia multiseries is associated with physiological stress caused by silicate (Si) and/or phosphate (P) limitation. Such limitation may promote DA synthesis by (1) reducing primary metabolic activity, thus making available necessary precursors, high energy compounds, and cofactors, and (2) favoring the expression of genes involved in the biosynthesis of this toxin. In the case of Si and P-limitation, DNA synthesis and the progression through the cell division cycle are slowed, perhaps prolonging or arresting the cells in the stage of the division cycle which is most conducive to DA production. However, N-limitation results in an insufficient pool of cellular free N, which restricts synthesis of this nitrogenous toxin. A continuous supply of photophosphorylated high-energy intermediates (e.g., ATP and NADPH) is necessary for DA synthesis. In order to better understand the mechanism(s) of DA production, more studies are needed to elucidate: (1) the details of the biosynthetic pathway, (2) the regulation of enzymes involved in the pathway, (3) the relation between DA synthesis and the cell division cycle, (4) the cellular compartmentalization of DA biosynthesis, and (5) other environmental factors that may trigger DA production. Finally, these studies should be extended to include toxigenic Pseudo-nitzschia species other than P. multiseries, to confirm the commonality of these mechanisms.

A thin-layer chromatography (TLC) method has been developed for the semi-quantitative analysis of domoic acid (DA) in shellfish tissues. Tissues were extracted in a single-step homogenization of tissue with 50 % aqueous methanol and then taken through a selective strong anion exchange cleanup. Cleaned extracts were applied directly to silica gel TLC plates and developed with a butanol-acetic acid-water mixture (3:1:1, Rf = 0.45 for DA). As little as 10 microg DA per gram of tissue could be detected after chromatography using a hand-held short-wave UV lamp to detect fluorescence quenching. Confirmation was provided by spraying the plate with ninhydrin, which reacts with the secondary amine of DA to give a distinctive yellow colored product. The extraction, cleanup and TLC procedures are fast and simple, and do not require the use of expensive equipment. This method should prove useful for the routine screening of shellfish tissues in those laboratories not equipped with an LC system. It should also be useful as a chemical confirmation method for DA in samples tested positive by assay methods such as immunoassay.