封闭环境气载镰孢菌及其T-2毒素发生规律的研究进展

本文阐述了普遍存在的镰孢菌可增强封闭式环境中群体对疾病的易感性,并通过其次生代谢产物(如T-2毒素)对人和动物造成严重危害及预防和控制气载镰孢菌及T-2毒素危害的相关问题,包括气载真菌和毒素采集、毒素痕量富集技术、不同环境中的镰孢菌变化规律与产毒基因研究现状。提出研究封闭式环境中气载镰孢菌及T-2毒素发生规律研究的必要性。     

Effects of T-2 toxin on ethanol production by Saccharomyces cerevisiae.

A trichothecene mycotoxin, T-2 toxin, inhibits several aspects of cellular physiology in Saccharomyces cerevisiae, including protein synthesis and mitochondrial functions. We have studied growth of, glucose utilization by, and ethanol production by S. cerevisiae and show that they are inhibited by T-2 toxin between 20 and 200 micrograms/ml in a dose-dependent manner. At 200 micrograms/ml, T-2 toxin causes cell death. This apparent inhibition of ethanol production was found to be the result of growth inhibition. On the basis of biomass or glucose consumption, T-2 toxin increased the amount of ethanol present in the culture. This suggests that T-2 inhibits oxidative but not fermentative energy metabolism by inhibiting mitochondrial function and shifting glucose catabolism toward ethanol formation. As T-2 toxin does not directly inhibit ethanol production by S. cerevisiae, this system could be used for ethanol production from trichothecene-contaminated grain products.     

Effect of ochratoxin A and ochratoxin C on the monocyte and lymphocyte function

The effect of practically relevant mycotoxin concentrations on functions of immune cells was studied in in vitro experiments. Porcine mononuclear cells were exposed to a crudeAspergillus-ochraceus toxin containing OTA, a HPLC fraction identical with OTC derived from the crude toxin (RE2), as well as pure OTA and OTC in a concentration range from 0.46 to 3000 ng/ml. The influence of mycotoxin exposure on metabolic activity, mitogen induced proliferation, expression of the activation marker CD25 and the cell cycle of lymphocytes and on the formation of free oxygen radicals as well as the production of the cytokines IL-6 and TNF-α by monocytes was determined. Exposure to high concentrations of all mycotoxin preparations lead to non-specific suppression of the immune cell functions, which was related to cytotoxic effects. Low concentrations caused ambivalent reactions, especially on monocyte function. In general, the HPLC fraction RE2 had an up to 100-fold stronger effect than pure OTA. Ochratoxin-induced suppression of lymphocyte proliferation was not abrogated by phenylalanine or aspartame. The results indicate that immunomodulation can be caused by very low mycotoxin concentrations which are not related to clinical symptoms or loss of performance.     

Effect of γ-irradiation on F-2 and T-2 toxin production in corn and rice

Fusarium graminearum andF. tricinctum were grown on moistened corn and rice. After inoculation the substrates were exposed to γ-irradiation and growth rate together with mycotoxin production were measured. A delay in mycelium growth and an increase in F-2 and T-2 toxin production occurred after irradiation with 1 and 3 kGy. The maximum F-2 production was 10.7 mg/kg on rice at 3 kGy, whereas T-2 was 735 μg/kg on rice at 3 kGy. At 9 kGy neither growth nor toxin production could be detected in any inoculated corn and rice substrate.     

Production of the Hepatotoxic Chlorine-Containing Peptide by Penicillium islandicum Sopp

Twenty-six strains of Penicillium islandicum Sopp were tested for production of a chlorine-containing peptide (Cl-peptide), which is a hepatotoxin. Highest levels of the mycotoxin were produced in a modification of Wickerham medium. The mycotoxin was isolated by adsorption on charcoal and, after washing the charcoal with acetone-water (1:1), was eluted into n-butanol. Further purification was accomplished by gel filtration. Maximum yields were 10 to 20 mg of toxin per liter of culture filtrate.     

Inhibitory effects of spices on growth and toxin production of toxigenic fungi

The inhibitory effects of 29 commercial powdered spices on the growth and toxin production of three species of toxigenic Aspergillus were observed by introducing these materials into culture media for mycotoxin production. Of the 29 samples tested, cloves, star anise seeds, and allspice completely inhibited the fungal growth, whereas most of the others inhibited only the toxin production. Eugenol extracted from cloves and thymol from thyme caused complete inhibition of the growth of both Aspergillus flavus and Aspergillus versicolor at 0.4 mg/ml or less. At a concentration of 2 mg/ml, anethol extracted from star anise seeds inhibited the growth of all the strains.     

Inhibitory effects of spices on growth and toxin production of toxigenic fungi.

The inhibitory effects of 29 commercial powdered spices on the growth and toxin production of three species of toxigenic Aspergillus were observed by introducing these materials into culture media for mycotoxin production. Of the 29 samples tested, cloves, star anise seeds, and allspice completely inhibited the fungal growth, whereas most of the others inhibited only the toxin production. Eugenol extracted from cloves and thymol from thyme caused complete inhibition of the growth of both Aspergillus flavus and Aspergillus versicolor at 0.4 mg/ml or less. At a concentration of 2 mg/ml, anethol extracted from star anise seeds inhibited the growth of all the strains.     

A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes, monoclonal antibodies, and complement

The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate agricultural products and cause intoxication of humans and animals. We have developed a homogeneous competition inhibition assay for T-2 mycotoxin based on complement-mediated lysis of liposomes. The T-2 mycotoxin was converted to an acid chloride derivative, subsequently coupled to the amino group of phosphatidylethanolamine, and incorporated with the phospholipid into unilamellar liposomes. Carboxyfluorescein, which is self-quenched at high concentrations, was entrapped in the liposomes as a release marker. We used a monoclonal IgG1 antibody specific for T-2 mycotoxin and a polyclonal anti-mouse Ig as a secondary antibody since the anti-T-2 IgG1 does not activate complement. In the absence of free T-2, the liposomes were lysed within 30 min after the addition of complement, releasing carboxyfluorescein into the surrounding buffer. In the presence of free T-2 toxin, the binding of antibodies to the liposomes was reduced, causing a corresponding decrease in lysis. This assay proved to be sensitive to T-2 toxin levels as low as 2 ng, which is 10-fold more sensitive than the present enzyme immunoassay using the same antibodies.     

一株产T-2毒素镰孢菌的分离、鉴定及其产毒条件的研究

【目的】从青海大骨节病区小麦麦穗中分离的内生真菌中筛选产T-2毒素的菌株,并研究影响其合成该毒素的条件。【方法】采用种子胚芽抑制试验和抑菌试验从分离所得的菌株中筛选产毒菌株;利用薄层层析和高效液相检测待测菌株产物,复筛出产T-2毒素的菌株。通过显微形态学观察及ITS序列分析对筛选出的菌株5-5m-1进行鉴定。应用单因素筛选方案研究固体培养时间、温度以及液体培养转速、初始p H等对其产T-2毒素的影响,并采用正交试验进一步优化。【结果】菌株5-5m-1的显微形态与梨孢镰孢菌(Fusarium poae)相似;ITS序列分析显示,该菌株与F.poae的相似度也较高。其产T-2毒素的最佳条件为:玉米固体培养基、日温25°C/夜温15°C、光暗交替。【结论】5-5m-1菌株为梨孢镰孢菌,培养条件对其产T-2毒素能力有很大影响。实验结果将为进一步研究T-2毒素产生的机制和防止真菌毒素污染提供参考。     

Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities

Of 10 Penicillium species reported to cause blue-eye disease of corn, four (P. martensii, P. palitans, P. cyclopium, P. puberulum) were found capable of producing the mycotoxin penicillic acid on various agricultural commodities. Commodities with high protein contents did not support toxin synthesis. The extent of toxin production varied with the strain of mold, the commodity, and the temperature; low temperatures (1 to 10 C) favored toxin accumulation.     

Alternaria metabolites in sunflower seeds

The alternariol and alternariol monomethyl ether contamination in sunflower seeds was determined. The levels of alternariol found ranged between 35 to 792 µg/kg and 90 to 630 µg/kg for alternariol monomethyl ether. The fungicides showed different effect on the mycotoxin production depending on the substrate, strains and toxin analysed. Mercury phenyl acetate, Octave, Metiram, Thiram and Orthene inhibited alternariol monomethyl ether production while for alternariol production only Thiram, Metiram and Octave were effective.     

A gastrointestinal function bioassay evaluation of the mycotoxin,T-2 trichothecene

The Fusarium-produced mycotoxin T-2 trichothecene toxin was administered to two groups of young CD-1 mice to test the effects on three parameters of intestinal motility. The criteria selected included composite motility (cm²/min), peak amplitude (mm) and contraction frequency (recorded peaks/min). T-2 treated mice showed an increase in composite motility in response to low dosage (0·085 mg/kg), and at the higher dosage (0·250 mg/kg) a decreased motility. In the lowest treatment group there was a mean decrease of 39·54% in contraction amplitude while contraction frequency increased by 24·84%. The motility measurements were obtained by perfusing 2 cm sections of small intestine, including the entire duodenum excised from mice pre-treated with mycotoxin. Contractions were recorded with a physiograph and the composite motility measurements were taken using a computer program to determine the area of the data curves. T-2 toxin caused an alteration in the amplitude and frequency of motility measurements, but no overall concentration-related changes were noted. T-2 toxin causes measurable responses in the duodenum which may be one of the sites-of-action for this mycotoxin.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid.

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Penicillium roqueforti PR toxin gene cluster characterization

PR toxin is a well-known isoprenoid mycotoxin almost solely produced by Penicillium roqueforti after growth on food or animal feed. This mycotoxin has been described as the most toxic produced by this species. In this study, an in silico analysis allowed identifying for the first time a 22.4-kb biosynthetic gene cluster involved in PR toxin biosynthesis in P. roqueforti. The pathway contains 11 open reading frames encoding for ten putative proteins including the major fungal terpene cyclase, aristolochene synthase, involved in the first farnesyl-diphosphate cyclization step as well as an oxidoreductase, an oxidase, two P450 monooxygenases, a transferase, and two dehydrogenase enzymes. Gene silencing was used to study three genes (ORF5, ORF6, and ORF8 encoding for an acetyltransferase and two P450 monooxygenases, respectively) and resulted in 20 to 40% PR toxin production reductions in all transformants proving the involvement of these genes and the corresponding enzyme activities in PR toxin biosynthesis. According to the considered silenced gene target, eremofortin A and B productions were also affected suggesting their involvement as biosynthetic intermediates in this pathway. A PR toxin biosynthesis pathway is proposed based on the most recent and available data.

     

Targeting of mycotoxins to reticuloendothelial system of mice with carrier erythrocytes

The tricothecene mycotoxin, T-2 toxin, was encapsulated in bovine erythrocytes for in vivo delivery of T-2 toxin to macrophages. Intraperitoneal injection of bovine carrier erythrocytes (5 X 10(8) cells) containing T-2 toxin saturated mouse liver uptake of erythrocytes by 6 h postinjection. At 24 h postinjection, 20% of the injected carrier cells containing toxin were localized in the liver of mice. Saturation of the liver uptake of bovine carrier cells was independent of encapsulated or free T-2 toxin. A dose of T-2 toxin sufficient to inhibit 50% of the macrophage protein synthesis was targeted to the liver via the carrier erythrocytes. A methodology for delivery of highly toxic molecules to liver macrophages is described.     

Decreased resistance to mycobacterial infection in mice fed a trichothecene compound (T-2 toxin)

The effect of T-2 toxin, a trichothecene compound, on bacterial infection was examined in mice infected intravenously with mycobacteria. T-2 toxin dissolved in olive oil was given orally in a dose of 0.1 mg, six to 12 times, at various stages of infection. The resistance-decreasing effect of the toxin was judged by two different criteria, the mouse survival period and the fate of tissue viable counts. This effect was accompanied by a decreased spleen weight. T-2 toxin was found to be a more potent immunosuppressing agent in this model than 5 mg of cortisone given intraperitoneally according to a similar schedule. In view of these observations, the potential importance of this mycotoxin was considered in relation to food hygiene.     

Detection and quantitation of T-2 mycotoxin with a simplified protein synthesis inhibition assay.

We describe a simple, rapid, and sensitive bioassay for the detection and quantitation of T-2 mycotoxin by using a protein synthesis assay in cultured cells. Increased sensitivity of the cells to the mycotoxin occurred with time up to ca. 60-min. Time and dose response curves show that an average of 10 to 20 ng of T-2 per ml was sufficient to cause 50% inhibition of protein synthesis in tissue culture cells. A wide range of tissue culture cells with varied type, tissue, and species sources and growth characteristics were tested by this system. All showed approximately the same sensitivity to the mycotoxin. A slight modification of the procedure was used for suspended cultures of mitogen-stimulated lymphocytes, which also showed an equal degree of sensitivity to the mycotoxin. By simply changing the labeled precursor, the inhibition of RNA, DNA, and protein synthesis by T-2 mycotoxin can be compared. Although T-2 mycotoxin had little effect on RNA synthesis, DNA and protein synthesis were equally inhibited. Because of its sensitivity and its capacity to quickly assay a large number of samples, this technique has been a valuable tool in screening samples for the presence of active toxin and has been used to help establish laboratory safety standards for the inactivation of T-2 mycotoxin by chemical agents. It is presently being used in studies of mycotoxin mechanism of action and approaches toward in vivo neutralization of the toxic effects of mycotoxins.     

Studies on the PR toxin of penicillium roqueforti

A mycotoxin, confirmed by chemical, physical and spectroscopic data as the PR toxin described by Ru-Dong Wei and coll. (15) has been isolated from culture filtrates of Penicillium roqueforti Thom. Factors affecting the toxin and mycelium production, acute and chronic toxicity in experimental animals and the frequency of toxinogenesis of 21 isolates of P. roqueforti (including a brown mutant) isolated from different materials, foods especially, were also studied. An hypothesis on the absence of PR toxin in cheeses fermented with P. roqueforti is also advanced.