Metabolites of 8 species 53 strains of Fusarium isolated from domestic animal feeds were tested in Salmonella mutagenicity test with and without metabolic activation. Mutagenic activity was detected on crude extracts of 5 species 13 strains of them; F. graminearum, F. nivale, F. tricinctum, F. semitectum, and F. oxysporum. From the result of thin layer chromatography, it seems that those mutagenic metabolites were the same compound. Moreover, the highest mutagenic activity extract was tested in chromosome aberrations in human lymphocytes and dominant lethal test in mice. It was detected chromatid type aberrations; gap and brake, in human lymphocytes, and induction of dominant lethality on late spermatids and sperms in mice.
To elucidate the underlying mechanism of nivalenol toxicity, we treated human promyelocytic leukemia HL60 cells with nivalenol for 24 h and investigated the toxin's effects on cell viability and interleukin (IL)-8 secretion. Whereas exposure to 1 μg/ml nivalenol led to slight morphologic damage, that after treatment with 3 or 10 μg/ml nivalenol was pronounced. In 5-bromo-2-deoxyuridine (BrdU) incorporation assays of the rate of cell proliferation, the mean 50% inhibitory concentration (IC50) of nivalenol was 0.16 μg/ml. The effect of nivalenol on mitochondrial succinic dehydrogenase activity was examined using the water-soluble tetrazolium-8 assay, in which the mean IC50 was 0.40 μg/ml. The activity of lactate dehydrogenase (LDH) released from the cytoplasm was very low, even at 10 μg/ml nivalenol, indicating that although treated cells had extensive morphologic damage, they did not burst. We used BrdU incorporation (the most sensitive indicator of cell viability) to assess the effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), which chelates intracellular calcium ion, on nivalenol toxicity. BrdU incorporation after concomitant treatment with nivalenol and BAPTA-AM was higher than that after treatment with nivalenol alone, indicating that the cytotoxicity of nivalenol is dependent in part on intracellular calcium ion. Further, the media of nivalenol-treated cells contained substantial amounts of IL-8, suggesting that this cytokine contributes to the nivalenol-caused pathologic phenomena.
Mold have been classified and determined according to morphological feature. From the morphological characteristics, an isolate is given species name. Many researchers have engaged in studying an important genus in society, and consequently there will have many eyes for observation. They describe the morphological differences of the isolate finely. From this reason, the social important genus of mold may include many species such as genus Aspergillus, Penicillium, and Fusarium. If the morphological character of the isolate to determine is genetically stable, there will be no problem. But, when the character to determine species is unstable and easy to change depending on environmental condition or growth phase, identification of an isolate often become confused. On the other hand, in the classification by base gene analysis, the number of bases exchange in the gene become great as the number of the base analyzed, and then, difference of the base arrangement also increase. Although it is useful DNA type to classify and identify species, it is a problem how you decide a target gene. If there is a molecular clock to reflect the process of evolution, gene analysis can be considered as an index for classification and identification. We have analyzed the mitochondrial cytochrme b gene of the pathogenic fungi and its related species, and found this gene is useful as an evolution clock.
Recent advances in molecular technology have allowed the rapid detection and identification of filamentous fungi. Ribosomal DNA (rDNA) has been utilized most frequently for phylogenetic studies because it is present and highly conserved in all organisms. This report described a new PCR-based method for heteroduplex panel analysis (HPA) for identification of Aspergillus section Flavi species. The HPA method involves formation of heteroduplexes with a set of reference fragments of the ITS regions amplified from A. flavus, A. parasiticus, A. tamarii and A. nomius, and subsequent minislab hydrolink-mutation detection enhancement (MDE) gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1 and N-1) generated by pairwise reannealing among four reference fragments. DNA sequencing revealed that the HPA method has the highest sensitivity for detecting single-nucleotide diversity within Aspergillus section Flavi species. Based on the present results, 19 HPA types were identified among a total of 351 section Flavi isolates: F-1 to F-6 of A. flavus/A. oryzae, P-1 and P-2 of A. parasiticus/A. sojae, T-1 of A. tamarii, T-2 of A. pseudotamarii, T-3 of A. caelatus, N-1 to N-5 of A. nomius, TN-1 and TN-2 of A. bombysis, and FP-1 of a new genotype that is potentially aflatoxigenic. Direct identification of section Flavi isolates can be accomplished by HPA without morphological observation. Recently, basic local alignment search tool (BLAST) analysis using rDNA has tentatively been employed for identification of filamentous fungi isolated from food and environmental sources. The advantages and the critical points of the method are also described.
During investigation of fungal flora in imported and domestic cereal products many isolates of Penicillium were obtained. Of the representative 20 Penicillium strains, the MicroLog system was tested for species identification. The MicroLog system is a 96 well microplate with 95 different carbon sources selected to include all of the important biochemical groups: sugers, carboxylic acids, amino acids, fattyacids, etc. The FF MicroPlate was used in this study and purchased from BIOLOG, Inc. One plate is inoculated with one isolate conidial suspension. After incubation, plates were evaluated with a microplate reader, which measures the turbidity in each well. The microplate reader connected to a computer, using the MicroLog software. During the incubation of the MicroPlates it was observed that most of the isolates produced colored soluble pigments on some of the carbon sources. Some species of the genus Penicillium are difficult to separate based on morphology alone, and the differentiation between species of this genus is difficult without the aid of physiological tests, i.e, assimilation of carbon sources and secondary metabolic profiles. We found that the MicroLog system could distinguish between the limited number of mycotoxigenic Penicillia such as P. citreonigrum, P. citrinum, P. expansum, P. islandicum and P. verrucosum. In addition, we examined to distinguish among the members of aflatoxigenic section Flavi species, including Aspergillus flavus, A. parasiticus and A. nomius isolates from imported peanuts and sugarcane field soil collected from Okinawa by the MicroLog system. Our results indicate that difficult of separate of these species.
Phytoalexins, compounds produced by plants in response to pathogens, and biologically active metabolites produced by endophyte are beneficial to plant growth. There are many trials to utilize these biologically active substances for plant disease control. On the other hand, some of these substances are toxic to mammals. We experienced ryegrass staggers of cattle caused by endophyte infected ryegrass straw. In this review, toxicities of phytoalexins and endophyte related substances to animals are overviewed. Furthermore, toxic substances found in false smut balls in rice plant are cited.
The secondary metabolites produced by genus Alternaria; dibenzo-α-pyrone derivatives alternariol (AOH), alternariol monomethyl ether(AME), and altenuene (ALT), tetramic acid derivative tenuazonic acid (TeA), perylene derivatives altertoxin-I (ATX-I) and relative compounds (altertoxin II and III) were known as Alternaria mycotoxins. Alternaria mycotoxins have been detected in crops and fruits harvested in field affected with Alternaria and also their products including apple juice and tomato products. The metabolites had the toxicity to mammalian cell, bacteria and experimental animals. Alternaria may become a potential biohazard of the Alternaria to humans and animals concerning mycotoxicosis. Alternaria had been isolated frequently from atmosphere, therefore the risk of human and animal health evoked by Alternaria exposure could be came high.
As mycotoxins can never be completely removed from commodities, many countries have regulated their levels in food according to their known adverse health effect. For defining the regulation level, an assessment of the health risk is required. To understand the toxic properties of mycotoxins, it is necessary to perform short-term, sub-chronic, and chronic studies at various doses using experimental animals. Also needed are carcinogenic studies in vivo and in vitro using bacteria. From each adequately performed animal study, a threshold dose, such as the NOAEL or NOEL, is defined. The value is essential for the estimation of a provisional tolerable daily intake (PMTDI). I present toxicological data and information of mycooxins that have been evaluated by JECFA.