JSM Mycotoxins
Online ISSN : 1881-0128
Print ISSN : 0285-1466
ISSN-L : 0285-1466
Volume 2003 , Issue Suppl3
Showing 1-45 articles out of 45 articles from the selected issue
  • Hans P. van EGMOND, Marco A. JONKER
    2003 Volume 2003 Issue Suppl3 Pages 1-15
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    An international inquiry was held in 2002/2003 on worldwide limits and regulations for mycotoxins, under contract with the Food and Agriculture Organization. The survey has shown that, by the end of 2003, more than 100 countries (covering>90% of the world's inhabitants) had specific regulations or detailed guidelines for mycotoxins. Mycotoxins for which currently (proposed) limits and regulations exist include the naturally occurring aflatoxins, aflatoxin M1, agaric acid, deoxynivalenol, diacetoxyscirpenol, the fumonisins B1, B2 and B3, HT-2 toxin, ochratoxin A, patulin, phomopsins, sterigmatocystin, T-2 toxin and zearalenone. Insight will be provided in the mycotoxin regulatory situations in Africa, Asia/Oceania, Europe, Latin America and North America. Overviews will be given about limits that exist for the most regulated mycotoxins. Comparing the situations in 1995 (year of the last worldwide inquiry) and 2003 it appears that, at present, more mycotoxins are regulated in more commodities and products, whereas tolerance limits generally remain the same or tend to decrease. Regulations have become more diverse with newer requirements regarding official procedures for sampling and analytical methodology. At the same time, several regulations have been harmonised between countries belonging to economic communities, or they are in some stage of harmonisation. Nevertheless the regulatory requirements remain substantially different across many countries.
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  • James J. PESTKA, Hui-Ren ZHOU, Yuseok MOON, Yongjoo CHUNG, Zahidul ISL ...
    2003 Volume 2003 Issue Suppl3 Pages 17-31
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Macrophages, T cells, and B cells of the immune system are central targets of deoxynivalenol (DON) and other trichothecenes-mycotoxins that can be immunostimulatory or immunosuppressive depending on dose, exposure frequency and timing of functional immune assay. Notably, low dose trichothecene exposure transcriptionally and post-transcriptionally upregulates expression of cytokines, chemokines and inflammatory genes with concurrent immune stimulation, whereas high dose exposure promotes leukocyte apoptosis with concomitant immune suppression. DON and other trichothecenes, via a mechanism known as the ribotoxic stress response, bind to ribosomes and rapidly activate mitogen-activated protein kinases (MAPKs). The latter are important transducers of downstream signaling events related to immune response and apoptosis. Using cloned macrophages, our laboratory has identified two critical upstream transducers of DON-induced MAPK activation. One transducer is double-stranded RNA-(dsRNA)-activated protein kinase (PKR), a widely-expressed serine/theonine protein kinase that can be activated by dsRNA, interferon, and other agents. The second transducer is hematopoetic cell kinase (Hck), a non-receptor associated Src family kinase. Inhibitors and gene silencing studies have revealed that Hck and PKR play roles in DON induced gene expression and apoptosis. Future studies should focus on the molecular linkages between these kinases and trichothecene toxicity.
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  • histopathology and gene expression profiles
    Shinya SEHATA, Munehiro TERANISHI, Takashi YAMOTO, Naochika MATSUNUMA, ...
    2003 Volume 2003 Issue Suppl3 Pages 33-39
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    T-2 toxin is a trichothecene mycotoxin produced by various species of Fusarium spp. Single dose or subacute dose of T-2 toxin induces damage in the lymphoid and hematopoietic tissues, resulting in lymphopenia and immunosuppression in many species. Necrosis/apoptosis is also reported in the gastrointestinal tract and liver. As there are a few reports of T-2 toxin-induced toxicity in pregnant rats, we performed a study to examine T-2 toxin-induced morphological changes in pregnant rats. Single cell necrosis was observed in the thymus, liver, intestines, placenta and fetal liver in pregnant rats treated with T-2 toxin on day 13 of gestation. To investigate gene expression profiles in the liver, placenta and fetal liver in the pregnant rats treated with T-2 toxin, Wistar rats on day 13 of gestation were orally administered with T-2 toxin at a single dosage of 2 mg/kg. Twenty-four hours after treatment, rats were sacrificed. Microarray analysis in the liver, placenta and fetal liver was performed using the Affymetrix Rat Genome U34A chip. The results in these tissues showed the same changes in lipid metabolism-related genes, apoptosis-related genes and oxidative stress-related genes. From these results, the mechanism of T-2 toxin-induced toxicity is speculated that T-2 toxin caused oxidative stress, following the impairment of lipid peroxidation and metabolism-related enzymes. These changes may cause the changes in the intracellular environments, finally resulting in the induction of apoptosis. Further study on the gene expression profiles at the earlier time point should be done to clarify the whole mechanisms of T-2 toxin-induced toxicity.
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  • Kenneth A. VOSS, Ronald T. RILEY, Janee B. GELINEAU-VAN WAES, Charles ...
    2003 Volume 2003 Issue Suppl3 Pages 41-48
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Much has been learned about fumonisins since their discovery and investigations to further characterize their toxicity and mode of action continue. A recently developed mouse model for neural tube defects in fetuses of fumonisin-treated dams will be a valuable in vivo tool for elucidating the mechanistic relationships between fumonisin exposure, folate transport, and neural tube defects in this mouse model and for determining the relevance of the mouse model for humans. Determining how cooking and food processing affect fumonisins in food matrices and, using an approach combining bioassays and chemical analyses, their toxicity is another emerging research initiative. Ongoing research in these and other areas will contribute to better understanding of fumonisins and to the implementation of science-based mycotoxin management strategies.
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  • Manfred GAREIS
    2003 Volume 2003 Issue Suppl3 Pages 49-58
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Ochratoxin A (OTA) is known to occur throughout the food chain and has been predominantly found in cereal and products derived from cereals, coffee, beer, wine, dried fruits, spices, cocoa and nuts. Surveys carried out in several countries have demonstrated the presence of OTA in human blood and breast milk, indicating a permanent dietary exposure. Different hazard assessments based on carcinogenicity or nephrotoxicity led to Tolerable Daily Intake (TDI) values in the range of 1.2-14 ng OTA/kg b.w.. To estimate the intake of OTA from food, studies on the assessment of dietary intake of OTA were carried out in the recent years in European member states such as Germany and also for the entire European population. Twelve countries provided data on the occurrence of OTA in food products, on consumption of these food products and on occurrence of OTA in human blood and human milk. The exposure of the European consumers did not generally give rise to major health concerns. Evaluation of the dietary intakes revealed differences between countries and diets but showed that the intakes are quite below the TDI of 5 ng OTA/kg b.w. /day as suggested by the EU. Higher intakes approaching the TDI were noted for specific consumer groups, particular children, infants and babies. Public health measures therefore should focus on these groups of the population. The EU has set maximum limits for OTA of 5 μg/kg in raw cereal grains including rice and buckwheat, 3 μg/kg for derived cereal products or cereal grains for direct human consumption, and 10 μg/kg in dried vine fruits. Limits for other products are being considered. As most of the surveys represent the European situation and diet, it is difficult to transfer these data to other countries of the world. Thus, more surveys are needed in regions of the world other than Europe in order that intake in those regions may be assessed.
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  • Masao HIROSE, Toshio IMAI, Kunitoshi MITSUMORI
    2003 Volume 2003 Issue Suppl3 Pages 59-67
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Kojic acid, 5-hydroxy-2-(hydroxymethyl)-4-pyrone, is a secondary fungal metabolite commonly produced by Aspergillus and Penicillium. It is used in skin care product as a whitening agent, as a protectant against u.v. light, and as a food additive to prevent enzymatic browning of raw crabs and shrimps. Carcinogenicity of kojic acid in the thyroid gland of male and female B6C3F1 mice (at doses of 1.5% and 3% in diet) and weak tumorigenicity in the liver of female mice (at a dose of 3% in diet) was demonstrated on 1998. Since that time many experiments have been conducted for the risk assessment of this compound. Kojic acid, at doses higher than 0.03% in diet, promoted rat thyroid carcinogenesis in the 2-stage carcinigenesis assay initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). It induced diffuse hypertrophy and focal hyperplasia in rat follicular cells without prior initiation treatment, also indicative of tumorigenicity. Since genotoxicity of kojic acid is highly suspected, mechanistic approaches are important for the regulation of this compound. Several experiments have shown serum T4 levels to be decreased and TSH levels were markedly increased by the kojic acid treatment, pointing to a negative feedback mechanism through the pituitary-thyroid axis exists. This was shown to be due to the inhibition of iodine uptake and iodine organification in the thyroid rather than to increase in the liver T4-UDP-GT. Treatment with 2 % kojic acid for 8 weeks in the initiation period, followed by the thyroid promoter sulfodimethoxine (SDM) failed to demonstrate any initiation activity in the rat thyroid. Therefore, it is suggested that thyroid tumors were induced through non-genotoxic mechanisms. Kojic acid induced adenomas in heterozygous p53-deficient CBA [p53(+/-)] mice, susceptible to genotoxic carcinogens, as well as in wild mice at dietary doses of 1.5% and 3% for 26 weeks, in addition to cell proliferation, which is a marker for non-genotoxic carcinogens. The incidences of neoplastic as well as preneoplastic lesions were significantly greater in the p53(+/-) than in the wild-type mice. Kojic acid at a dose of 2% in diet promoted rat hepatocarcinogenesis in the medium-term liver assay and induced GST-P positive foci without initiation treatment. Therefore, kojic acid is suggested to be a hepatocarcinogen in both mice and rats, with probable involvement of genotoxicity in addition to non-genotoxic mechanism.
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  • Philip JENNINGS, Mary E. COATES, Judith A. TURNER, Paul NICHOLSON
    2003 Volume 2003 Issue Suppl3 Pages 69-75
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Levels of FHB have been monitored in England and Wales since 1986, with the species responsible for symptoms determined since 1998. Between 1998 and 2001 the predominant species responsible for the FHB symptoms alternated between Microdochium nivale and Fusarium poae. In 2002 and 2003, no single FHB pathogen predominated. The frequency of isolation of F graminearum has steadily increased since 1998 and, at the same time isolation of F culmorum has declined. Chemotyping F culmorum and F graminearum by PCR analysis showed that both deoxynivalenol (DON) and nivalenol (NIV) chemotypes were present in UK populations. DON chemotypes predominated in both populations, however high numbers of NIV producers were also identified. Regional differences in chemotype were observed for F culmorum where a higher proportion of NIV producers were found in the south and west of England, and a higher proportion of DON producers in the north and east. No differences were seen in chemotype distribution for F graminearum. In separate fungicide trials differential control of the FHB pathogens was apparent depending on the product applied. Several fungicides based on triazole chemistry reduced the level of toxin-producing species and the subsequent levels of DON in the grain. These fungicides had little or no effect on the level of M nivale. The application of fungicides based on strobilurin chemistry effectively controlled M nivale but not the Fusarium species. There was evidence that the application of some fungicides, may lead to an increase in the level of DON in the grain.
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  • Jun SOUMA
    2003 Volume 2003 Issue Suppl3 Pages 77-82
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    The establishment by the Japanese government of a tentative guideline level (1.1 mg/kg) for deoxynivalenol (DON) in wheat has drawn new attention to Fusarium head blight control. This report describes the urgent and tentative recommendations set out by the Hokkaido government for DON reduction. First, the use of the intermediate-resistant cultivar Haruyokoi is recommended. The second recommendation regards chemical control; i.e., the timing and frequency of fungicide application and the selection of fungicide effective for DON control. Four serial applications, the first of which started at the beginning of flowering, was found to be effective. We selected the five following fungicides: tebuconazole, propiconazole, kresoxim-methyl, iminoctadine-acetate, and thiofanate-methyl. We demonstrated that sieve sorting and gravity sorting could separate the grain exceeding the guideline level into grains having different DON levels. Thus, we recommend gravity sorting and sieve sorting. We encourage producers to use all the recommendations comprehensively.
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  • Toshitsugu TANAKA, Takumi YOSHIZAWA, Hiroki TANAKA, Yoshitsugu SUGIURA ...
    2003 Volume 2003 Issue Suppl3 Pages 83-88
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    The provisional regulation level for deoxynivalenol in wheat is 1.1mg/kg in Japan. However, the regulation level for nivalenol and the occurrence of trichothecenes in rice still remain as matters to be settled. In this paper, we described a risk assessment for daily exposure of trichothecene mycotoxin based on the data obtained for wheat and rice harvested in 2002. By empoloying the weighted mean of trichothecene contamination levels, the trichothecenes risk in Japan was evaluated. The tolerable daily intake of the trichothecenes in Japanese dietary intake was also described.
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  • Gary A. PAYNE, Ahmad M. FAKHOURY, Kimberly A. SCHEIDEGGER, Michael S. ...
    2003 Volume 2003 Issue Suppl3 Pages 89-97
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Aflatoxins are toxic and carcinogenic secondary metabolites produced only by a few species of Aspergillus. Growth of these fungi on a number of plant host species can result in aflatoxin contamination and an unmarketable product. No plant genotype with adequate resistance to aflatoxin contamination is commercially available. The application of genomic sciences to better understand the factors that regulate aflatoxin biosynthesis and describe the interaction of A. flavus with its hosts holds promise for identifying new target sites for control of aflatoxin contamination. Gene expression analysis, along with gene disruption and protein-protein interaction studies, is being used in our laboratory to identify new regulatory circuits in aflatoxin biosynthesis. A targeted DNA microarray composed of 753 elements is being used to identify genes expressed during aflatoxin biosynthesis under a number of physiological conditions. A comparison of conducive and non-conducive conditions for aflatoxin production based on nitrogen source, time, and genetic regulation in A. f lavus has identified several genes differentially expressed during aflatoxin biosynthesis. Many of the differently expressed genes have no known function. Two genes are currently being examined for a role in aflatoxin biosynthesis, an alkyl hydroperoxide reductase (afahpl) and a 14-3-3 gene homolog (mafl). Disruption of (mafl) resulted in loss of aflatoxin production. Yeast two-hybrid analysis identified several genes (including some members of our EST library) that potentially interact with mafl. The function of these genes in aflatoxin biosynthesis is being evaluated.
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  • Shohei SAKUDA, Tatsuhiko KONDO, Tomoya YOSHINARI, Hiromichi NAGASAWA
    2003 Volume 2003 Issue Suppl3 Pages 99-105
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Aflastatin A (AsA) and blasticidin A (BcA), metabolites of Streptomyces with similar structures, are inhibitors of aflatoxin production by Aspergillus parasiticus and they can inhibit production of other fungal polyketide metabolites such as melanin produced by Colletotrichum lagenarium. We have clarified that they inhibit a very early step involved in aflatoxin and melanin production, which may be present in primary metabolism of the fungi. In this study, to elucidate the mode of action of AsA and BcA, we examined their effects on pH control and carbon metabolism in A. parasiticus. As a result, they did not affect pH of culture broth and phosphatase expression pattern in the mycelia, but they drastically changed carbon metabolism in the fungus by elevating glucose consumption and ethanol accumulation. We also prepared a photoaffinity probe of BcA to identify its target molecule. A specific binding protein of the probe has not been obtained, but we found that biotinylated proteins endogenously present in the fungus became undetectable when BcA was added to the culture. This suggested that BcA affected metabolism of biotinylated proteins of the fungus.
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  • Makoto KIMURA, Takeshi TOKAI, Naoko TAKAHASHI-ANDO, Isamu YAMAGUCHI
    2003 Volume 2003 Issue Suppl3 Pages 107-115
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Trichothecenes and zearalenone (ZEN) are major mycotoxins produced by fungi in several genera including phytopathogenic Fusarium species. This short overview outlines molecular and genetic mechanisms of trichothecene biosynthesis. We also describe a biotechnological method for ZEN detoxification.
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  • Yin-Won LEE, Jae-Jin JEON, Hun KIM, In-Young JANG, Hye-Sun KIM, Sung-H ...
    2003 Volume 2003 Issue Suppl3 Pages 117-122
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Gibberella zeae (anamorph: Fusarium graminearum) is an important pathogen of cereal crops in many areas of the world. Infected grain with this fungus is often contaminated with mycotoxins. Phylogenetic trees of G. zeae isolates from maize and rice were constructed by using amplified fragment length polymorphism (AFLP). AFLP showed polymorphic bands and the bands, haplotypic loci, were used to analyze genetic diversity of G. zeae population. Population structure of maize isolates consisted of four lineages. Lineage 7 was the major group followed by lineage 3, lineage 6, and lineage 2. On the other hand, the population structure of rice isolates consisted of only lineage 6. Maximum parsimony trees based on sequencing data from Tri101, Tri7 and MAT-1-1 genes were found to be concordant with AFLP data. Trichothecene production of maize isolates was variable; lineage 7 and 3 isolates were deoxynivalenol (DON) producers and most lineage 6 and 2 isolates were nivalenol (NIV) producers. All lineage 6 isolates from rice were mostly NIV producers with a few DON chemotypes. This result suggests that genetic variations exist in G. zeae population from maize and rice in Korea.
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  • Koji YOKOYAMA, Haruo TAKAHASHI, Li WANG, Masakatsu ICHINOE
    2003 Volume 2003 Issue Suppl3 Pages 123-131
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Distribution of Aspergillus flavus and A. parasiticus in sugarcane field soils, air and harvested sugarcane stems was studied in Okinawa Prefecture, the southernmost in Japan. Using a combination of dilution plate and plant debris plate techniques, the fungi were detected on all seven islands tested and in 74% of 53 soil samples. The fungi were also found on the cut surface of sugarcane stems from one of the islands. A. parasiticus was the predominant fungus although many atypical isolates of this fungus that produced metulated conidial heads were also detected. The aflatoxin producing strains were approximately 89% (146/164) of all the isolates and approximately 69% of A. flavus isolates. More than 40% of A, flavus isolates also produced G aflatoxin. SEM observation of conidial wall texture was useful for distinguishing A. parasiticus from A. flavus. Cyclopiazonic acid, an indole mycotoxin, was never synthesized by any of the A. parasiticus or G aflatoxin-producing A. flavus isolates tested. The digest this study is as follows: (1) A. parasiticus was isolated from sugarcane field soils, air and harvested sugarcane; (2) all strains of A. parasiticus isolated in these areas were different from type strain of A. parasiticus in terms of cytochrome b gene sequence and divided into 3 DNA types; and (3) the strains of afratoxin G-producing A. flavus were all A. nomius and divided into 2 DNA types. Molecular analysis of isolates showed that the strains isolated from sugarcane field soil, air and harvested sugarcane stems from the southernmost part of Japan are highly diverse and differ in terms of aflatoxin-producing ability.
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  • Kimiko YABE, Hiromitsu NAKAJIMA
    2003 Volume 2003 Issue Suppl3 Pages 133-138
    Published: March 30, 2004
    Released: January 25, 2010
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    Aflatoxins are highly toxic and carcinogenic metabolites, which are produced by some strains of common fungi, mainly Aspergillus flavus and A. parasiticus. Aflatoxins B1, B2, G1 and G2 are the major aflatoxins produced by the fungi. Sterigmatocystin is a penultimate precursor of aflatoxins produced by many species including A. nidulans and also toxic and carcinogenic. Recently, the majority of enzyme reactions in aflatoxin/sterigmatocystin biosynthesis have been clarified, and the genes encoding the enzymes have been isolated. However, several unknown steps still remain. We have studied these unknown steps by using mainly enzymological methods. We finally clarified the detailed pathways of some of the unknown steps. We will summarize the newest information about the enzyme steps involved in aflatoxin/sterigmatocystin biosynthesis.
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  • Tami McDONALD, Thirumala DEVI, Kiminori SHIMIZU, Sungchur SIM, Nancy K ...
    2003 Volume 2003 Issue Suppl3 Pages 139-147
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Secondary metabolite production and sporulation are tightly co-regulated in the Aspergilli and Fusaria. Here we discuss two conserved pathways, including a G-protein/cAMP/Protein kinase A cascade and an oxylipin-mediated signalling process, that genetically link sporulation and secondary metabolism in these genera. The G-protein alpha subunit FadA negatively regulates aflatoxin and cyclopiazonic acid production in Aspergillus, and positively regulates penicillin production in A. nidulans and tricothecene production in Fusarium sporotrichiodes. Inactivation of FadA eliminates or decreases asexual spore production in both genera. The G-protein cascade is conserved throughout eukaryotes, and regulation of sporulation and secondary metabolism by this signal transduction pathway appears to be conserved within filamentous fungi. On the other hand, the oxylipin-mediated signalling pathway appears to be restricted to filamentous fungi. We have identified novel genes encoding putative dioxygenases likely to be responsible for secreted oxylipins which act as sporulation factors. Deletion of these genes affects asexual sporulation and secondary metabolite production in A. nidulans and F. sporotrichiodes.
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  • Prapeuk TANGMUNKHONG, Worawith WAJJAWALKU, Prakorn JARA, Susumu KUMAGA ...
    2003 Volume 2003 Issue Suppl3 Pages 149-152
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Mycotoxin contamination of feedstuffs used in Thailand was studied by analyzing aflatoxin B1, zearalenone and deoxynivalenol in raw materials of feeds and complete feeds collected in 2000-2003 with TLC method. For raw material samples, 1, 373 imported and domestic samples of corn, soybean mill, peanut product, cassava and rice bran were examined, and for complete feed, 915 domestic samples were examined. The samples were extracted with acetonitril-water (84:16, v/v), and the extracts were then cleaned up with Mycosep 226 cleanup columns and developed on TLC with tolueneacetone (1:1, v/v). The plate was visually examined under ultraviolet light using pure standard toxins as references. For raw materials, the contamination level of aflatoxin B1 higher than 20μg/kg was found in 24.02 %, 10.96 %, 5.68 % and 4.29 % of the samples collected in 2000, 2001, 2002 and 2003, respectively. The zearalenone level higher than 200 μg/kg was found in 8.11 %, 1.33 %, 6.18 % and 0.71 % in 2000, 2001, 2002 and 2003, respectively. The deoxynivalenol level higher than 1, 000 μg/kg was found in 7.50 %, 1.33 %, 0.83 % and 0 % in 2000, 2001, 2002 and 2003, respectively. The contamination in complete feeds was observed to change roughly from year to year in parallel with that in raw materials. The aflatoxin B1 level higher than 20 μg/kg was found in 14.29 %, 13.39 %, 7.53 % and 1.39 % in 2000, 2001, 2002 and 2003, respectively. The zearalenone level higher than 200μ/kg was found in 11.70 %, 1.79 %, 6.63 % and 1.39 % in 2000, 2001, 2002 and 2003, respectively. The deoxynivalenol level higher than 1, 000 μg/kg was found in 12.33 %, 1.79 %, 2.11 % and 0 % in 2000, 2001, 2002 and 2003, respectively.
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  • Nguyen Huong TRA, Le Thien MINH, Bui Thi HUONG, Nguyen Thuy CHAU
    2003 Volume 2003 Issue Suppl3 Pages 153-158
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    One hundred eighty one samples of food and feed products were collected from some provinces in northern Vietnam. Natural occurrence of aflatoxin B1 and ochratoxin A were surveyed in 26 samples of peanut kernel, 55 samples of animal feed, 20 samples of peanut products including peanut candy and fried peanut, 15 samples of soybean products including soybean powder, compressed soybean cake and soybean meat, 15 samples of maize products including nutrient corn powder and fried maize, 18 samples of coffee products including instant coffee and coffee berry, 16 samples of cashew nut products including cashew nut powder, cashew nut cake and cashew nut pigment powder, and 16 samples of meat products including crustaceous meat. Aflatoxin B1 and ochratoxin A were analyzed according to an AOAC method and the modified AOAC method of Doronina and Makshimenko. Two of nine samples of peanut products in Hanoi city were contaminated with aflatoxin B1 at an average concentration of 6μg/kg. One of five samples of peanut products in Haiphong city and one of six of those in Thanh hoa province were contaminated with aflatoxin B1 at the level of 4 and 5μg/kg, respectively. Ochratoxin A was not detected in 20 peanut product samples. Neither aflatoxin B1 nor ochratoxin A was detected in 80 samples including 15 soybean product samples, 15 maize product samples, 18 coffee product samples, 16 cashew nut product samples and 16 meat product samples. Thirteen of 26 peanut kernel samples and 35 of 55 animal feed samples were contaminated with aflatoxin B1 at average concentrations of 43μg/kg and 12μg/kg, respectively.
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  • Hassan YAZDANPANAH, Parvin E. GHEIDARI, Seyed N. S. HOMAMI, Seyed K. M ...
    2003 Volume 2003 Issue Suppl3 Pages 159-165
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Fumonisin B1 (FB1) is the most abundant of the fumonisin mycotoxins, mainly produced on maize by the Fusarium species (F. verticillioides and F. proliferatum). A previous study on fumonisin contamination of maize harvested in Mazandaran and Isfahan Provinces in Iran during 1998 demonstrated a high level of fumonisin contamination in the maize from Mazandaran Province. This study was done to confirm the high FB1 levels found in maize of Mazandaran Province, to further investigate annual variations in levels of contamination and to verify if there is any risk for the population with regard to presence of FB1 in corn. Contamination of maize with FB1 was assayed in healthy corn samples collected from Mazandaran Province during 2000. In Mazandaran Province, the mean level of FB1 in maize harvested during 2000 (5.67mg/kg) was higher than that found during 1999 (3.18mg/kg, maximum level 7.7mg/kg), with a maximum observed level of 13mg/kg in 2000. These results confirm the relatively high levels of FB1 contamination in Mazandaran Province as opposed to the much lower levels found in Isfahan Province, as were previously noted for the 1998 and 1999 harvests. But, exposure assessment study showed that there is no risk for both urban and rural populations regarding presence of FB1 in healthy corn of Mazandaran and Isfahan Provinces.
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  • Rosario H. ARIM
    2003 Volume 2003 Issue Suppl3 Pages 167-173
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Mycotoxins are fungal metabolites which cause adverse effects in humans and animals when contaminated foods are ingested. In the Philippines, the most studied mycotoxin is the aflatoxin. There is very limited data on other mycotoxins such as fumonisins, ochratoxin, zearalenone, T-2 toxin and deoxynivalenol. In the country, problems on the export commodity copra meal is one of the major concerns due to its high aflatoxin levels which cause rejection of the oil mill by-product by importing countries, particularly in the European Union. National problem involved the local corn produce which shows aflatoxin levels beyond the 20 μg/kg tolerance limit. Previous studies showed that corn and peanuts are the most susceptible agricultural commodities that contain toxic levels of aflatoxin.
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  • Norhayati ALI, Noor Hasani HASHIM
    2003 Volume 2003 Issue Suppl3 Pages 175-183
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Food safety became an important issue in Malaysia following the Healthy Living and Food Safety Campaigns launched in 2002. Malaysia will also be involved in recent development of global interest to study the risk assessment of food contaminants such as mycotoxin in dietary staples. Mycotoxin research in Malaysia started in 1965 and dealt mainly with aflatoxins (AF) contamination in foods. Screening work for the other mycotoxins was carried out in recent years. This paper reviewed all the previous and recent data (1966-2003) available in Malaysia with regard to mycotoxin research. The reliability of analytical results and the possible cause of contamination in foods and feeds were briefly discussed. It is concluded that a further systematic, comprehensive and well-coordinated surveillance and research program on important mycotoxins should be carried out in Malaysia to get the latest and accurate data for risk assessment study.
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  • Feng-Qin LI, Gan-Rong XU, Yu-Wei LI, Tao JIANG, Yun CHEN, Rong JI, Hui ...
    2003 Volume 2003 Issue Suppl3 Pages 185-192
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Thirty-five Monascus strains used in food processing were selected to conduct the effect of cultivation conditions and the medium composition on citrinin production with the aim of screening for the strains with less or nearly non-citrinin production. The results indicated that all strains produced citrinin on solid-state culture (rice) with the levels ranged from 0.28 to 2460 mg/kg (average 202 mg/kg and median 62 mg/kg), while 30 strains (86 %) in submerged fermentation medium produced citrinin between 0.09 and 56 mg/kg (average 12 mg/kg and median 3.5 mg/kg). In addition, the red pigment production on rice was found to be 3-510 (average 93) times higher than that in the liquid. One strain with the highest red pigment yields (color value, 1130 U/g) but lower citrinin production was obtained. These results suggest that it is necessary to make the safety evaluation of microorganisms used for the production of foods and food ingredients, to investigate the ability of citrinin production by Monascus strains preserved by either the food manufacturers or the national culture collection units, and to survey for the citrinin contamination in Monascus products countrywide. It is urgent for China to establish the tolerance limit for citrinin in Monascus products.
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  • Setsuko TABATA
    2003 Volume 2003 Issue Suppl3 Pages 193-198
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Patulin contamination in apple-based products in Japan was surveyed, and the risk of patulin in Japan was estimated. A total of 252 samples of apple-based products, collected from market or juice plants in Japan during the years 1996 to 2001, were examined for patulin with the method of analysis using GC/MS; the recovery was 101 %, and limit of confirmation was 5μg/kg. Patulin was detected in 15.6 % of commercial apple juices in the range of 5 to 42μg/kg. No commercially available apple juice contained more than 50μg/kg of patulin, the proposed future regulatory limit in Japan. In 81 % of apple juice concentrates, patulin was detected in the range of 5 to 670μg/kg. The daily intake of patulin for mean of all population in Japan is quite below the provisional maximum tolerable daily intake (PMTDI) indicated by JECFA, 0.4μg/kg body weight/day. The risk of patulin is considered to be negligible in Japan.
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  • Masahiro NAKAJIMA, Setsuko TABATA, Hiroshi AKIYAMA, Yoshinori ITOH, To ...
    2003 Volume 2003 Issue Suppl3 Pages 199-208
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    A systematic survey of aflatoxin M1 (AFM1) in commercial pasteurized milk was carried out for the first time in Japan using immunoaffinity column (IAC) and reversedphase high performance liquid chromatography (HPLC) with fluorescence detection (FLD). A total of 208 milk samples were randomly purchased from retail outlets in 11 regions during the period of December 2001 to February 2002 and analyzed for AFM1 in 4 independent laboratories. Each milk sample was filtered, and applied to the IAC. After washing with water, the AFM1 was eluted with acetonitrile and then detected by HPLC-FLD. The identity of the putative AFM1 was confirmed by the formation of aflatoxin M1 hemiacetal with trifluoroacetic acid (TFA) and in one sample, the liquid chromatography-mass spectrometry (LC-MS) was performed. Based on the results of spiked samples (0.05μg AFM1/kg), the mean recovery, relative standard deviation for repeatability (RSDr) and relative standard deviation for reproducibility (RSDR) among 4 independent laboratories were found to be as 91.4%, 4.6% and 8.0%, respectively. AFM1 was detected in 207 (99.5%) out of 208 milk samples ranged 0.001-0.029μg/kg, with a mean level of 0.009μg/kg. No significant difference of the level of AFM1 contamination was observed among the regions. Based on these data, the risk of development of liver cancer due to AFM1 contamination in milk was estimated to be lower than 10-9 per year in Japan.
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  • Jean Marc FREMY, Sylviane DRAGACCI
    2003 Volume 2003 Issue Suppl3 Pages 209-214
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    The quality and reliability of the analytical results which should estimate as close as possible the “true value” of any mycotoxin contamination depend of several factors. The availability and reliability of analytical procedures used to estimate that value are more and more considered before any value or maximal limit in any legislation is developed. It is now considered that validated methods and/or standards validated through inter laboratory studies organised by recognised organisations are the most reliable analytical procedures. The quality and reliability of the analytical results depend not only of the performance on the methodology but also on the performance of the laboratory. Analytical laboratories involved in official programmes focused on mycotoxin occurrence may be assumed to be “in control”. The current recommendations indicate that such laboratories should be formally accredited, participate in proficiency testing schemes, use internal quality control procedures and use appropriately validated methods of analysis. The way of expressing results by the analysts is very important also. More and more countries or regulation bodies are recommending that the “recovery factor” should be considered and consequently included in the expression of results as “estimated value”. Analytical results actually take more and more the form “a±2u” where “a” is the best estimate of the true value and “u” is known as the “standard measurement uncertainty”. The measurement uncertainty takes into account both the “trueness” (average departure from a true value) and the “precision” (the degree to which successive results agree with each other).
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  • Chris M. MARAGOS
    2003 Volume 2003 Issue Suppl3 Pages 215-223
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Accurate and sensitive methods for detection of the major mycotoxins have been developed and improved for many years. Immunoassays, first introduced for mycotoxins in the 1970's, have found widespread use as screening tools. Despite the success of the enzyme-linked immunoassay (ELISA) format, new formats for immunoassays continue to be explored with the goal of improving upon the speed and ease-of-use of the assays. Recent advances in analytical technologies have allowed the adaptation of immunoassays to a wide variety of new formats. This review focuses on the recent developments in detection technologies for mycotoxins, in particular those using antibodies as the recognition element (immunosensors). Recent developments in surface plasmon resonance, fiber optic sensors, membrane-based immunoassays, fluorescence-based assays, and capillary-based immunoassays, are presented. The challenge remaining for many of these new technologies is one of making the transition from laboratory-based assays to validated field-portable screening assays.
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  • Elisabeth FUCHS, Josy HANDL, Eva M. BINDER
    2003 Volume 2003 Issue Suppl3 Pages 225-232
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    A quick and efficient method for the determination of type-A and -B trichothecenes is described employing high performance liquid chromatography with UV (ultra violet) and MS (mass spectrometry) detection. Using Mycosep multifunctional clean-up columns multi-toxin clean-up was done within one procedure and one solvent system, thus saving considerable time in comparison with other clean-up methods. The method was tested and validated in corn, wheat and barley with MS detection for the most common A-Trichothecenes (T-2 toxin, HT-2 toxin and DAS), using T-2 toxin-d3 as internal standard. For most prevalent B-Trichothecenes, i.e. deoxynivalenol (DON), nivalenol (NIV), 3-acetyl-deoxynivalenol, and fusarenon X (FUS-X), the method was validated with UV absorption and calibrated for MS detection.
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  • Kazuyuki IKEDA, Yuriko MUNAKATA, Kazuki MORITA, Osamu KUSADA, Takumi Y ...
    2003 Volume 2003 Issue Suppl3 Pages 233-239
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Trichothecene mycotoxins such as deoxynivalenol (DON), nivalenol (NIV), and T-2 toxin (T-2) are representative mycotoxins that contaminate major cereal crops. Methods such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and high performance liquid chromatography (HPLC) are generally used to measure these toxins. These methods are considered unsuitable for routine use when many samples must be treated within a short time. Enzyme-linked immunosorbent assay (ELISA) would be equivalent to a method of measurement at high throughput, such as screening. An antibody that distinguishes NIV from DON was required to precisely measure the above three kinds of toxins by an ELISA method. We were successful in developing a monoclonal antibody for 3, 4, 15-triacetyl-NIV, which was generated under conditions where an acetyl group was introduced at the C-4 position of NIV without an acetylation at the C-7 position. Simultaneously, we obtained a monoclonal antibody for both 3, 4, 15-triacetyl-NIV and 3, 15-diacetyl-DON as well as one for acetyl-T-2. Three kinds of ELISA kits for the individual measurement of DON, NIV, and T-2+HT-2 were constructed using these monoclonal antibodies. The components derived from wheat extracts did not influence our kits. An investigation of the performance of the kits revealed high reproducibility and a good correlation with GC-MS. Thus, we could offer simpler and more useful ELISA systems as alternative methods to GC-MS, LC-MS, and HPLC for screening contaminated cereals and foods.
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  • Danrey W. TOTH, Nancy A. ZABE, Barb A. COHEN
    2003 Volume 2003 Issue Suppl3 Pages 241-247
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Immunoaffinity column chromatography was identified as the best method for mycotoxin analysis due to great sensitivity, wide application, ease of use, and operator safety. Very recently, manufacturers and testing laboratories have begun to investigate ways to improve the efficiency of testing with Immunoaffinity columns by performing analyses with columns capable of isolating multiple mycotoxins, which are then all detected in a single HPLC run. These improvements make testing easier while still keeping all the advantages of Immunoaffinity analysis. Also, trichothecene testing methods have been developed that broaden the number of trichothecenes that can be easily analyzed from a wide variety of samples. Groups of scientists from the European Union and the United States have been experimenting with immunoaffinity methods capable of purifying up to ten different mycotoxins with a single column. The work of Lusky and Gobel demonstrated that aflatoxins, ochratoxin A and zearalenone could be analyzed effectively in samples of rice, barley, rye and feed, with recovery of toxins averaging in the 90 % range with the exception of aflatoxin G2. Recovery of aflatoxin G2 was subsequently boosted as high as 93.4 % with a modification of the immunoaffinity column. Using gradient and wavelength switching for detection, all toxins could be measured with baseline resolution in a single run of 31 minutes. Recoveries were similar across matrices, a typical advantage of immunoaffinity chromatography. In a complex analysis involving isolating and detection of ten mycotoxins from beer and sake, scientists from the United States Alcohol and Tobacco Tax and Trade Bureau and their industry collaborators found recovery of the following toxins using multiple mycotoxin column analyses of beer and sake: aflatoxins B1, B2, G1, and G2; fumonisin B1, B2, B3; ochratoxin A; zearalenone and, deoxynivalenol. Another recent development in mycotoxin testing has increased interest in analysis of different trichothecenes. Pascale and coworkers recently developed an HPLC method for T-2 toxin (T-2), which is generally difficult to analyze because it lacks a chemical signature. In the improved method, T-2 is isolated from wheat, corn, oats, sorghum or rice with an immunoaffinity column. This renders the T-2 sufficiently pure that it can be derivatized with a fluorophore and detected by HPLC analysis, where it elutes at about 10 min with baseline resolution from other peaks. Recoveries from spiked cereals were from 80 to 99 %, and the relative standard deviation with these samples is excellent, being below 6 % in all cases. For deoxynivalenol, an earlier method by Cahill and coworkers produced recoveries from wheat of 90 % at relative standard deviations of 8.3 %. Detection was through absorbance during an isocratic HPLC run of 5.5 min. Both of these methods for trichothecenes provide the ease and accuracy needed for analysis of this class of toxins.
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  • Masahiro NAKAJIMA
    2003 Volume 2003 Issue Suppl3 Pages 249-254
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    The immunoaffnity column (IAC) methods using specific antibodies against mycotoxins have been developed. The use of IACs in the cleanup step of mycotoxin analysis provides a number of advantages over the conventional chemical methods, such as highly specific, simple and rapid, and saving toxic solvents. Recently, with the availability of commercial IACs for several mycotoxins, the IACs have become the powerful tools in the cleanup stage of mycotoxin analysis. However, there are some disadvantages of commercial IACs also. One of the major disadvantages is cost of IAC. To solve this problem, methods of regenerating IACs for reuse have been investigated. Other disadvantages are low and varied recoveries because of low mycotoxin affinity of IACs. In general, aflatoxins (AFs) are eluted with methanol from IAC. However, AFs are decomposed during evaporation of methanol eluate. To avoid this decomposition, acetonitrile elution from IAC was very effective. In the case of IAC for ochratoxin A (OTA), low recoveries result from low OTA affinity of antibody at pH condition other than neutral pH. By introducing 10 mM ammonium acetate buffer (pH 6.6) into the washing steps of IAC, high and stable recoveries of OTA could be achieved. Because of low toxin affinity of antibody against deoxynivalenol, only a small portion of sample solution can be applied onto IAC. To solve this problem, the combination of solid phase extraction column and IAC was effective.
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  • Masahiro NAGASE, Junichi KIMURA, Takumi YOSHIZAWA, Nobuo SAKATO
    2003 Volume 2003 Issue Suppl3 Pages 255-262
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Satratoxins, which are produced Stachybotrys species, have been recognized as potential agents associated with sick building syndromes. In spite of the potential importance of satratoxins, the molecular mechanism induced by them remains unknown. Recently, we have found that satratoxin G induced potent apoptosis in HL-60 human promyelotic leukemia cells and both caspase-8 and caspase-9 are activator of caspase-3. However, the detail activation profile of caspases and role of the other caspases including caspase-6, caspase-7 and a novel initiator caspase, caspase-2 remain to be clear. Here we report detail caspase cascade in satratoxin G-induced apoptosis in HL-60 cells. Caspase assay with DEVD-AMC, a fluorogenic substrate, and western blot analysis of caspase-7 suggested that caspase-3 was involved, but casapase-7 was not directly involved in satratoxin-induced apoptosis. We investigated in-depth time courses of caspase activation. Caspase-3 was activated after 1 hour from satratoxin treatment. Activation of caspase-2 was observed apparently as early as 1 h. Caspase-9 was also activated significantly at the 1 hour time point. Caspase-6 and caspase-8 were activated at the 3-hour time point. Western blot analysis showed that cleavage of bid and activation of caspase-12 were not involved in satratoxin-mediated apoptosis. These findings suggest that the initial activators of caspase-3 are caspase-2 and casapse-9 in satatoxin-induced apoptosis. Because caspase-8 and casapse-9 were activated independently, caspase-8 is also involved in activation of caspase-3 in the late stage of the apoptosis. Moreover, caspase-6 appears to be associate with activation of caspase-8. Both caspase-7 and caspase-12 may be not potential caspases in the apoptosis.
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  • Amnart POAPOLATHEP, Susumu KUMAGAI, Kunio DOI
    2003 Volume 2003 Issue Suppl3 Pages 263-275
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Nivalenol (NIV) is a trichothecene mycotoxin mainly produced by Fusarium nivale, and our previous study clarified that NIV is able to induce apoptosis in lymphoid tissues of mice in a dose dependent manner. In this study, NIV was orally administered to ICR mice at the dose of 15 mg/kg b.w., then thymus was taken from 30 min to 12 hours after inoculation (HAI), in order to investigate the expression of apoptosis-related genes (fas, c-fos, c-fun, p53, bcl-2 and c-myc) by RT-PCR method. The results indicated that c-fos and c-jun might play a critical role in NIV-induced thymocyte apoptosis because the levels of both c-fos and c-jun mRNAs were prominently elevated from 30 min and peaked 1 HAI prior to the development of apoptosis. In addition, the development of early apoptosis and changes in lymphocyte subsets were examined by FACS analysis in lymphoid tissues of BALB/c mice orally administered with 15 mg/kg b.w, of NIV, and also in thymocyte primary cultures treated with NIV at the dose levels of 0.25, 0.5 and 1μg/ml. The results indicated that NIV attacked Peyer's patches (PP) first and thymus most severely. In thymus, selective damage in CD4+CD8+ cells was observed at 12 and 24 HAI, following the peak of apoptosis at 9 HAI. CD4+ cells were clearly suppressed at 3 HAI in PP, at and after 9 HAI in mesenteric lymph nodes (ML), and from 3 to 12 HAI in spleen (SP), respectively. CD8+ cells were also suppressed at 24 HAI in ML and at 12 HAI in SP, respectively. As a result of in vitro experiment, the apoptotic cell index significantly increased in all groups at and after 3 hours after treatment (HAT) generally in a time-dependent manner. The number of CD4+CD8+ cells was prominently depleted in all groups in a time-dependent manner. These results also indicated that NIV directed affected thymocytes and induced apoptosis mainly in CD4+CD8+ cells. As to changes in B cell subsets, IgG+ cells significantly decreased from 3 to 12 HAI and all B cell subsets at 24 HAI in ML. In SP, IgM+ cells were suppressed at 9 HAI. On the other hand, in PP, following clear decrease in the numbers of pan-T and pan-B cells and viable cells at 3 HAI, all B cell subsets, especially IgA+ cells, showed a significant increase in their numbers at 9 HAI, and the numbers of IgA+ and IgM+ cells remained higher values than controls thereafter. Taken together, in the course of recovery from NIV-induced prominent damage in PP at 3 HAI, interaction of NIV with PP might result in in vivo stimulation of interleukin production at this site and result in increased proliferation and differentiation of IgA-secreting B cells at and after 9 HAI.
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  • Akira KITAGAWA, Yoko ITO, Yoshitake ITO, Kiyoshi KAWAI
    2003 Volume 2003 Issue Suppl3 Pages 277-280
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Xanthoascin, a hepato- and cardio-toxic metabolite of Aspergillus candidus, has been examined for in vitro toxicity to respiratory function of isolated rat liver mitochondria to elucidate the molecular mechanism of its in vivo toxicity. It was found that xanthoascin exerted dual deteriorate effects to mitochondrial respiration, exhibiting an uncoupling effect and an electron transport inhibition at NADH-UQ oxidoreductase (complex I) and cytochrome c oxidase (complex IV).
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  • Hidetake ESAKI, Susumu KUMAGAI
    2003 Volume 2003 Issue Suppl3 Pages 281-287
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Objectives: To study the liver glutathione S-transferase (GST) activity toward aflatoxin B1 (AFB1) exo-epoxide in mastomys in comparison with rat, mouse and hamster, we performed in vitro studies of the liver enzymes activity. Materials and Methods: AFB1 metabolism by liver microsomes including formation of AFB1-DNA adducts was studied using [3H] AFB1. The liver cytosolic GST activity of mastomys and other rodents toward AFB1-exo-epoxide were measured using HPLC with chiral column. Results. Cytosolic GST activity toward AFB1-exo-epoxide was highest in mastomys liver, and higher in the hamster and mouse livers than in the rat liver, correlating well with the differences of the sensitivity of a given species to the toxicity of AFB1. However, no relationship was noted between the sensitivity of a given species to the toxicity of AFB1 and the microsomal activity of binding of AFB1 to DNA or metabolizing AFB1 to AFM1, AFQ1, and AFP1. The importance of GST mediated AFB1-exo-epoxide conjugation with glutathione in ditermining the differing sensitivities of these species to AFB1 toxicity was demonstrated. The extremely high activity of GST in mastomys seems to be related to the tolerance to AFB1 toxicity and indicates that this species would be a good model animal for studying the relationship between hepatic GST activity and AFB1 toxicity.
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  • Tetsuhisa GOTO, Krishan D. SHARMA, Hitoshi NAGASHIMA
    2003 Volume 2003 Issue Suppl3 Pages 289-293
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    A polyclonal antibody for Rubratoxin B (RB) was raised in rabbit and the competitive direct ELISA method to detect RB was set. Optimum concentration of antibody and enzyme conjugate were 1:1000 and 1:1500, respectively. Fifty % inhibition for RB at these conditions were about 5 ng/ml. This ELISA system can be used up to 50% methanol or 20% acetonitrile for solvent of sample solution. Aflatoxins (B1, B2, G1 and G2), cyclopiazonic acid, kojic acid, patulin, ochratoxin A, sterigmatocystin and zearalenone did not inhibit this reaction. However, unknown interferences from various agricultural commodities were observed and further more study is needed to apply this method to real samples.
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  • Zhongming ZHENG, Donna HOUCHINS, Jacqueline UNG, John L. RICHARD
    2003 Volume 2003 Issue Suppl3 Pages 295-302
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    An ELISA Microtiter Plate, Deoxynivalenol (DON) Test called AgraQuant® was validated to measure DON in a range from 0.25-5.0mg/kg. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, DON is extracted from ground sample with distilled water and diluted sample extract plus conjugate are mixed and then added to the antibody-coated microwells. After 15 minutes incubation at room temperature, the plate is washed and substrate is added and allowed to incubate for an additional 5 minutes. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability, accuracy, precision and limit of detection in wheat and other grains and grain products, comparison of method to HPLC and ruggedness of the test kits determined this test to be rugged, sensitive, accurate, precise and effective comparable to HPLC for measuring DON ranging from 0.25-5.0mg/kg in several commodities.
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  • Keiko SUGA, Naoki MOCHIZUKI, Koichi HARAYAMA, Hiroshi YAMASHITA
    2003 Volume 2003 Issue Suppl3 Pages 303-308
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    A simple method for the analysis of trichothecenes [Type A: diacetoxyscirpenol (DAS), neosolaniol (NEO), HT-2 toxin (HT-2), and T-2 toxin (T-2), Type B: deoxynivalenol (DON), nivalenol (NIV), fusarenon-X (F-X)] using liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) was developed. Trichothecenes was extracted with ethyl acetate/methanol and diluted with water/methanol for injection into the LC/MS/MS. The LC separation was performed with an octadecylated silica column (3μm particles, 150 mm length, 2 mm i.d.) with a flow-rate of 0.2 mL/min, using a mobile phase consisting of water, methanol, and acetonitrile. MS/MS was used in multiple reaction monitoring, employing electrospray ionization (ESI-MRM). The recoveries of trichothecenes from beer and barley tea at 1μg/L (Type A), 10μg/L (Type B) were 68.1-127.5 % (beer), 52.5-115.2 % (barley tea). The lower limits of quantification were 0.07-1.0μg/L and 0.01-0.8μg/L. The method was applied to ten domestic beers and five commercial barley teas. No trichothecenes was detectable. This method may have applications in quality assurance.
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  • Yoshiki HAYASHI, Takumi YOSHIZAWA
    2003 Volume 2003 Issue Suppl3 Pages 309-313
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    The method for determination of CPA using silica solid phase extraction was evaluated in this study. This method failed to remove interferences from corn and peanut samples. Both CPA and interferences were affected in the same way by ZnSO4 in the mobile phase during HPLC analysis. Modifications were done on the HPLC conditions in order to separate CPA from interferences, but were unsuccessful. CPA determination without using toxic solvents such as chloroform was investigated. A clean-up procedure consisting of diethyl ether liquid-liquid extraction was effective in removing interferences.
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  • Gerhard BUTTINGER, Hermann KNAPP, Elisabeth FUCHS, Eva-Maria BINDER, R ...
    2003 Volume 2003 Issue Suppl3 Pages 315-318
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    High Performance Liquid Chromatography (HPLC) with fluorescence detection (FLD) and Thin Layer Chromatography (TLC) are widely used analytical methods for the determination of Ochratoxin A (OTA). As normally the crude extract cannot be used directly for these analyses, a clean-up step is necessary. Commonly used clean-up procedures comprise immunoaffinity columns (IAC), liquid liquid extraction (LLE) and solid phase extraction (SPE). The performance of a new single step clean-up column Mycosep® 229 Ochra from Romerlabs® was tested in matrices often contaminated with OTA like green coffee beans, raisins and various kinds of cereals. In all these matrices an average recovery of more than 90% (range 2.6-92μg/kg; n=24) of the added OTA could be obtained. Additionally, a certified reference material (CRM), OTA in wheat, was tested with the column. An average recovery of 96.2% (n=9) was achieved for the CRM proving the applicability and high accuracy of the developed method.
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  • Rosario H. ARIM, Lyn A. ESTEVES, Concepcion A. FEROLIN, Victoria M. LU ...
    2003 Volume 2003 Issue Suppl3 Pages 319-324
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Two enzyme-linked immunosorbent assay (ELISA)-based screening and a developed minicolumn test kits were compared with a quantitative method for aflatoxin determination in corn. The samples analyzed included one set of artificially-contaminated corn containing different aflatoxin levels (0, 10, 20 and 50μg aflatoxin B1/kg sample), and a set of naturally-contaminated corn samples containing the same levels of aflatoxin contamination. Data of the analysis with each test kit were evaluated and compared with the HPLC data of the test samples. Results showed that both the two ELISA-based test kits gave positive responses of 71 % and the “lahar” minicolumn test kit, 83.3 %. The performance of the locally-developed aflatoxin test kit which utilizes a “lahar” minicolumn is comparable to that of the commercial test kits.
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  • Anthony C. SALES, Patricia V. AZANZA, Takumi YOSHIZAWA
    2003 Volume 2003 Issue Suppl3 Pages 325-329
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Aspergillus Section flavi populations, natural and in vitro aflatoxin (AF) production in dried Cavendish bananas (Musa cavendishii), a major agricultural commodity in Southern Philippines, were evaluated. Aspergillus Section flavi was isolated from fresh and dried Cavendish bananas, food-contact surfaces, and the air surrounding the production area. Isolates were identified to be Aspergillus flavus based on spore type and conidial structure. Of 20 Aspergillus Section flavi isolates tested, 8 (40 %) produced AFB1+AFB2 ranging from 1 μg/kg to 16 mg/kg in rice, Cavendish banana and Yeast Extract Sucrose medium. Six (75 %) of the 8 isolates produced >20μg/kg total AF which is the Philippine regulatory limit for AF in food and processed peanut products. No AF was detected in 65 samples of dried Cavendish bananas. The possible role of competitive microorganisms and microbial metabolites is presented to explain the low incidence of AF in dried Cavendish bananas. With this research, we ascertained that Cavendish banana is suitable as substrate for growth and AF production by Aspergillus Section flavi. Although AF levels in the commodity may be negligible, the development and application of measures to control populations of fungi at all levels of production, particularly those that are mycotoxigenic, is vital to ensure safety of the commodity.
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  • LIMAY-RIOS Victor, YOSHIZAWA Takumi
    2003 Volume 2003 Issue Suppl3 Pages 331-336
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    A total of 194 wheat and barley spikes showing head blight symptoms were collected randomly in May 2002 from fields located at Kagawa University Farm, Miki, Japan. The presence of NIV-and DON-producing strains of F. graminearum was confirmed in a subset of the samples. Based on the distribution of NIV and DON contamination in the spikes as analyzed by GC-MS, it is likely that under low disease incidence and severity, spikes are commonly infected by one and rarely by both producers in the field. A gradual decrease in the frequencies of 4-ANIV and 3-ADON contamination, compared to those of their unacetylated forms, was observed when spikes were stored at 2°C and analyzed over a period of 110 days after sampling. It is possible that deacetylation was induced by host-plant enzymes during the maturation of cereal grains.
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  • E. Blanca, YUMBE GUEVARA, YOSHIZAWA Takumi
    2003 Volume 2003 Issue Suppl3 Pages 337-343
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    As a part of a study on the heat stability of deoxynivalenol (DON) and nivalenol (NIV), the decomposition patterns of both compounds were determined by ELISA using a monoclonal antibody which recognizes the partially acetylated derivatives of DON and NIV (3, 15-diacetyl-DON and 3, 4, 15-triacetyl-NIV, respectively). In that investigation it was found that heat-induced derivatives of the toxins have a stronger cross-reactivity with the antibody. Considering the nature of the antibody, it was speculated that a structural change due to the rearrangement of the A-ring in the trichothecenes probably occurred. To corroborate this hypothesis. DON and NIV were heated in acetic anhydride to determine which compounds are formed during acetylation. Two new compounds were isolated from the reaction mixture: 3, 8, 15-triacetoxy-12, 13-epoxytrichothec-8-en-7-one (TAisoDON) and 3, 4, 8, 15-tetraacetoxy-12, 13-epoxytrichothec-8-en-7-one (TeAisoNIV), indicating that during heating at least part of the decrease in DON and NIV level is due to isomerization.
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  • Mihoko TOMINAGA, Yun Hae LEE, Risa HAYASHI, Osamu YAMADA, Kazutoshi SA ...
    2003 Volume 2003 Issue Suppl3 Pages 345-348
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    Koji mold, Aspergillus oryzae has been used in the traditional fermentation industry for sake, shoyu (soy sauce), miso and other beverages and foods, and its safety is proved historically. However, A. oryzae taxonomically related to aflatoxin (AF) producer A. flavus and some strains possess the entire AF biosynthetic pathway gene cluster. In case of using A. oryzae in food processing, it is desirable to select the strains which do not possess AF biosynthetic genes. The simple method which can confirm the presence of these genes is needed to select these strains. In this study, we designed PCR primers which specifically amplify AF biosynthetic genes and analyzed the presence of these genes in 210 RIB strains (A. oryzae culture collection in NRIB). The classification and the characteristic evaluation of these strains were also examined. From results of PCR amplification patterns of these genes, RIB strains were classified into four groups, but most of the strains were classified into main two groups. Group 1 conserves AF biosynthetic gene cluster entirely (122 of 210 strains, 58.1 %) and group 2 possesses a part of its cluster which is from vbs to avnA (76 of 210, 36.2 %). The differences between group 1 and group 2 on mycological characters were recognized statistically in length of stalk, production of kojic acid, and so on.
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  • Emi SAKUNO, Hiromitsu NAKAJIMA, Kimiko YABE
    2003 Volume 2003 Issue Suppl3 Pages 349-352
    Published: March 30, 2004
    Released: January 25, 2010
    JOURNALS FREE ACCESS
    In aflatoxin biosynthesis 5'-hydroxyaverantin (HAVN) is converted to averufin (AVR). We demonstrated that this step is composed of two enzymatic steps: an enzyme, HAVN dehydrogenase, catalyzes the reaction from HAVN to a new intermediate and another new enzyme catalyzes the following reaction from the intermediate to AVR. The intermediate was assigned to be 5'-oxoaverantin (OAVN) by spectroscopic methods. We purified HAVN dehydrogenase and OAVN cyclase from the cytosol fraction of A. parasiticus and characterized. MALDI-TOFMS analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme is encoded by adhA gene in the aflatoxin gene cluster of A. parasiticus.
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