Cytotoxicity and phytotoxicity of trichothecene mycotoxins produced by Fusarium spp.

Trichothecenes, a major class of mycotoxins produced by Fusarium, Myrothecium, and Stachybotrys species, are toxic to both plants and mammals. Simple trichothecenes, including type A (e.g., T-2 toxin) and type B (e.g., deoxynivalenol), are generally less toxic than macrocyclic trichothecenes. We sought to determine if simple trichothecenes are a potential source of candidates for development as bioherbicides, which require high phytotoxicity and low mammalian toxicity. We examined 28 simple trichothecenes in vitro for phytotoxicity using a small aquatic plant, Lemna pausicostata, and for mammalian toxicity using four cultured mammalian cell lines. Several structure-activity relationships were identified, including the following two, which may be relevant to bioherbicide development: peracetylation of type B trichothecenes and de-epoxidation of type A trichothecenes both substantially reduced mammalian toxicity with little effect on phytotoxicity. It was concluded that simple trichothecenes possessing strong phytotoxicity and minimal mammalian toxicity in vitro can be identified.

[Mycological examination of domestic unpolished rice and mycotoxin production by isolated Penicillium islandicum].

Fungi growing on domestic rice were examined from April to June, 2003. One hundred samples of rice, which had been harvested in the autumn of 2002, were collected from the local market, and 15 samples of stored rice, which had been harvested in 2001 and stored in warehouses under government control, were used as samples. From each sample, 50 grains (100 grains in total) were plated on potato-dextrose agar (PDA) and malt yeast 40% sucrose agar (M40YA) containing chloramphenicol after being washed with sterile distilled water to remove any microorganisms on the surface, and incubated at 25 degrees C for a week. For most of the rice samples harvested in the preceding year, the proportion of grains infected with fungi was less than 20% of the total grains tested. In about half the samples of rice stored for one and half years, more than 80% of the grains were infected with fungi that grew on M40YA. The major genera of fungi isolated from the rice harvested in the preceding year were Penicillium and Alternaria, and those from the rice stored for one and a half years were Aspergillus, Penicillium and Eurotium. P. islandicum, A. versicolor, A. ochraceus and others were isolated as possible mycotoxin-producers in the mycoflora of domestic rice. P. islandicum was isolated from 3 samples, and 82% of the grains were infected with this fungus in one sample. All three isolates from these samples appeared to produce luteoskyrin on Czapek yeast extract agar, based on TLC and HPLC analysis.

Updated profile of aflatoxin and Aspergillus section Flavi contamination in rice and its byproducts from the Philippines.

Our goal was to develop an updated profile of aflatoxin (AF) and AF-producing fungi contamination in rice and its byproducts from the Philippines. The total AF levels in 78 samples of polished and brown rice, determined by an immunoaffinity column clean-up method coupled with HPLC (detection limit: 25 ng/kg), ranged from <0.025-2.7 microg/kg (mean of positive samples: 0.37 microg/kg) and 0.03-8.7 microg/kg (mean of positive samples: 2.7 microg/kg), respectively. The incidence (% of positive samples) of AF in polished and brown rice were 94% and 100%, respectively. The AF levels in polished rice imported from Thailand and Vietnam were approximately 20% of the levels found in locally produced polished rice. AF levels decreased as the rice progressed through the various stages in milling. Fungi recovered include toxigenic Aspergillus flavus and A. parasiticus with an incidence ranging from 14% in rice bran to 78% in rough rice and producing <0.025-6200 microg/kg total AF in in vitro cultures on rice. All samples of rice bran and rice hull contained AF at levels ranging from 0.27-11 microg/kg. The estimated potential daily intake of AFB(1) from rice is between 0.1 and 7.5 ng/kg of body weight/day, the mean of which is 1.0 ng representing 9.1-5.3 times the estimated tolerable daily intake for AFB(1) reported to date for Asia. Thus, Filipinos have a potentially high risk of exposure to AF that can be easily controlled through proper post-harvest handling and storage of rice and its byproducts.

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines.

Mold counts and Aspergillus section Flavi populations in rice and its by-products from the Philippines were examined. The average mold counts of rough rice, brown rice, and locally produced polished rice were 4.1 x 10(3), 1.0 x 10(3), and 1.1 x 10(3) CFU/g, respectively. Average Aspergillus section Flavi counts of the same samples were 3.0 x 10(2), 1.1 x 10(2), and 2.6 x 10(2) CFU/g, respectively. Twenty-seven percent of mold isolates from rough rice, polished rice, and brown rice were section Flavi spp., 31% of which were toxigenic. No section Flavi isolates were obtained from imported rice samples from Thailand and Vietnam. Aspergillus section Flavi was also isolated from rice hull, rice bran, and settled dust from rice milling operations. Toxigenic isolates of both Aspergillus flavus and Aspergillus parasiticus were present in at least one sample of each type of rice and rice by-product except settled dust. Aflatoxins produced in vitro by the isolates ranged from <1 microg/kg to 6,227 microg/kg. A. flavus isolates produced only B aflatoxins, whereas A. parasiticus isolates produced both B and G aflatoxins. Although total mold counts of Philippine rice and its by-products are within tolerable limits, the establishment of maximum limits in counts of potentially aflatoxigenic species in foods and feeds is important because the mere presence of toxin producers is considered a possible risk factor. The results of this research illustrate the need for strict monitoring of rice during both storage and marketing, especially in warm and humid seasons when infestation and consequent production of aflatoxins by Aspergillus section Flavi is expected.

A practical method for measuring deoxynivalenol, nivalenol, and T-2 + HT-2 toxin in foods by an enzyme-linked immunosorbent assay using monoclonal antibodies.

We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9-6.2%), accuracy (recovery: 93.8-112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography-mass spectrometry (r=0.9146-0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or liquid chromatography-ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.

Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3.

Satratoxins have been recognized as potential immunomodulatory agents in outbreaks of building-related illness. Here we report that satratoxin G-treated human leukemia HL-60 cells underwent apoptosis through the action of caspase-3 which was activated by both caspase-8 and caspase-9. Western blot analysis of caspase-3 in the satratoxin G-treated cells apparently indicated the appearance of a catalytically active fragment of 17 kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, exposure to satratoxin G led to cleavage of PARP from its native 116 kDa form to a 85 kDa product. Moreover, DFF-45/ICAD were cleaved into a 12.5 kDa fragment via satratoxin G treatment. Enzymic assay on IETD-AMC revealed that caspase-8 is strongly activated by exposure to satratoxin G while T-2 toxin (T-2) could not activate caspase-8 at an early stage of apoptosis. Furthermore, satratoxin G caused a release of cytochrome c from mitochondria into the cytosol and increased the activity of caspase-9 against LEHD-AMC. These findings indicate that satratoxin G-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through both activation of caspase-8 and cytosolic accumulation of cytochrome c along with activation of caspase-9.

Fusarium toxins in wheat from an area in Henan Province, PR China, with a previous human red mould intoxication episode.

Wheat samples of the 1998 and 1999 crops from Puyang, an area in Henan Province, PR China with a previous human red mould intoxication episode, were analysed for trichothecenes and zearalenone (ZEA). For the 1998 Puyang crop, deoxynivalenol (DON) was the predominant toxin detected abundantly and frequently at a level of up to 14,000 microg kg(-1) (mean 2850 microgkg(-1)) in 30 of 31 (97%) wheat samples. Among these were 21 (70%) with a DON level that exceeded the Chinese regulation of 1,000 microg kg(-1). Nivalenol (NIV) and 15-acetyl-DON (15-ADON) were also found at 578 microg kg(-1) (one sample) and 59-1,800 microg kg(-1) (mean 365 microg kg(-1), 20 samples), respectively. ZEA co-occurred in 21 samples at 9-1,400 microg kg(-1) (mean 209 microg kg(-1)). Twenty-five (89%) wheat samples from Zhumadian, a region without a history of human red mould intoxication in the same province, contained low levels of DON (53-1240, mean 223 microg kg(-1)) with seven (25%) co-contaminated with ZEA (10-217, mean 108 microg kg(-1)). All were free from 15-ADON and NIV. Significant differences in DON, 15-ADON and ZEA concentrations between both areas were found. DON (<1000 microg kg(-1)) and ZEA (5-111 microg kg(-1)) were also detected in the 1999 Puyang wheat. Proper environmental conditions for Fusarium species surviving winter combined with unusual high precipitation during wheat flowering were responsible for a high concentration of Fusarium mycotoxins in the 1998 Puyang wheat.