Decomposition of deoxynivalenol (DON) and nivalenol (NIV) during roasting of naturally contaminated barley was determined by GC-MS, and 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). As determined by GC-MS, DON and NIV were decomposed with the extent of reduction dependent upon temperature and processing time. By ELISA, a clear decrease in the toxin level was observed above 170 °C, whereas at 150 °C the toxin level increased after 5 and 30 min heating in spite of a slight decomposition observed by GC-MS. This phenomenon was also confirmed when the standard toxins were heated at 150 °C, indicating that heat-induced derivatives of the toxins may have a stronger cross-reactivity against the antibody.
Analysis of aflatoxins (AF) B1, B2, G1 and G2 in imported polished rice samples from Thailand, Pakistan and Bangladesh was performed by direct competitive ELISA (direct cELISA) and immunoaffinity column with high performance liquid chromatography (IAC-HPLC) methods using monoclonal antibody. Direct cELISA method with the detection limit of 2.8 μg/kg for AFB1 gave negative results in all of 20 samples analyzed. On the other hand, using IAC-HPLC with the detection limit of 0.1 μg/kg for each toxin, AFB1 was detected in five out of 20 (25 %) imported polished rice samples; in three Thailand and two Pakistan samples, with the concentrations ranging 0.1 - 0.3 μg/kg. In addition, AFB2 (0.1 μg/kg) was detected in one of the Pakistan rice sample contaminated with AFB1 (0.3 μg/kg). Even though the level of AF contamination in imported rice samples found to be well below the Japanese limit (10 μg/kg), AF contamination in rice as a staple food of Asian is an important issue for evaluation of human exposure due to the frequency of intake.
Aflatoxins are toxic and carcinogenic secondary metabolites that are produced primarily by certain strains of Aspergillus flavus and A. parasiticus. Contamination of crops with aflatoxins can have very serious effects on the health of both animals and humans. I have conducted research on aflatoxins for approximately 18 years in order to elucidate aflatoxin biosynthetic mechanisms; the present work is a review of this research.
Several human intoxications associated with Fusarium spp. and their mycotoxins, trichothecenes, have been evaluated by International programme on Chemical Safety (IPCS, WHO), FA/WHO Joint Expert Committee on Food Additives (JECFA), and International Agency for Research on Cancer (IARC, WHO). Of these episodes, alimentary toxic aleukia (ATA) in the former USSR, and red-mold intoxication in Japan, PR China and India are briefly reviewed.
It is well known that a part of mycotoxins induce various types of nephrotoxicity in livestocks and in humans. For example, ochratoxin A (OTA) is believed to induce Balkan endemic nephropathy (BEN). Fumonisin, citrinin, rubratoxin B and a few other mycotoxins are also considered to be nephrotoxic. It has been recently reported that nivalenol (NIV) and deoxynivalenol (DON), which belong to the trichothecene mycotoxin, experimentally, induce pathological changes in mouse kidney resembling human IgA nephropathy (IgAN), the most common chronic glomerulonephritis in Japan. Actually, we have created a reproducible IgAN model in mice which is orally induced by the mycotoxin, NIV, based on the hypothesis that IgAN is triggered by some exogenous antigen (s) which induces dysregulation of the mucosal immune system (the mucosal immunity-oriented hypothesis). Significant IgA deposition in glomeruli and remarkable elevation of serum IgA levels were reproducibly induced in all C3H/HeN, C3H/HeJ and BALB/c mice fed NIV 12 ppm for 8 wk (NIV model). The degree of pathological changes analogous to human IgAN and the elevation of serum IgA levels in mice were associated with the dose and duration of oral NIV presentation. Long-term administration of NIV for 12 months significantly increased serum IgA levels as well as the intensity of glomerular IgA deposition in mice over time. Furthermore, a significant increase in IgA-producing cells was demonstrated by an enzyme-linked immunospot procedure in Peyer' s patch (PP) lymphocytes (PPL) of the NIV model mice. Upregulation of CD4+ T cells was also revealed in PP of the model mice by a cytokinespecific reverse transcription-polymerase chain reaction which detected markedly high levels of mRNA specific for IL-4, IL-5, IL-6, IL-10 and TGF-β (Th2 type cytokines) as well as IFN-γ and IL-2 (Th1 type cytokines) in these cells. When NIV diluted in alcohol or saline phosphate buffer was injected into the duodenum in C3H/HeN mice to examine histopathological changes in the intestinal wall, 24 hours later not only the number of PAS-positive goblet cells (GC) looked decreased in the small intestine but also necrotic and degenerative damages were observed in Paneth cells and epithelial cells of the small intestinal villi especially around the crypts in the NIV-injected mice compared with those in controls. In conclusion, NIV reproducibly and strain-nonspecifically induces pathological changes in mice which resemble those in human IgAN. IgAN-like changes in mice were exacerbated over time in a period-dependent manner with NIV administration. It was suggested that PPL are immunologically dysregulated in NIV-induced IgAN, and that this kind of upregulation of the mucosal immune system might be associated with the pathogenesis of IgAN. We also suggest that mycotoxins such as NIV and DON might immunolocically and histopathologically damage the mucosal system such as the intestinal wall and have some etiologic role at least in some types of glomerulonephritis.
To clarify the characteristics and mechanisms of T-2 toxin-induced cell death in the lymphoid and hematopoietic tissues, in vivo and in vitro studies were carried out. As a result, T-2 toxin-induced lesions in the thymus, spleen and bone marrow of mice were shown to be brought about by apoptosis of component cells. The sequence of T-2 toxin-induced apoptosis varied among tissues, and apoptosis was occurred earlier in the hematopoietic tissues than in the lymphoid tissues. By RT-PCR method, the expression of c-fos mRNA increased immediately after T-2 toxin-inoculation and remained high levels throughout the observation period. Cycloheximide, a protein synthesis inhibitor, blocked apoptosis in the thymus of T-2 toxin-inoculated mice. As observed in in vivo study, in Con A-stimulated mouse thymocyte cultures, T-2 toxin induced the elevation of c-fos mRNA expression prior to the development of apoptosis. BAPTA/AM and Quin-2/AM, intracellular calcium chelators, and H-7, a PKC inhibitor, blocked the increase in the level of c-fos mRNA expression after T-2 toxin-treatment. These intracellular calcium chelators also inhibited DNA fragmentation after T-2 toxintreatment. These results suggest that T-2 toxin attacks cells with high proliferating activity such as lymphoid and hematopoietic cells, c-fos gene plays an important role in the early phase of T-2 toxin-induced apoptotic cell death probably through sythesis of a certain protein such as heat shock protein, and the elevation of c-fos mRNA expression may require the mobilization of [Ca2+]i and partially involve a PKC-dependent pathway. The mobilization of [Ca2+]i seemed to activate calcium-dependent enzymes, resulting in internucleosomal DNA fragmentation.
The effects of trichothecenes on specific and non-specific host resistance were examined. A relative low dose of deoxynivalenol (DON) reduced host resistance to salmonellosis but not the non-specific host resistance. DON disturbed the production of pro-inflammatory cytokines such as TNF-α after infection in vivo. Experiments using human macrophage cell line U-937 revealed that DON and other 8-ketotrichothecens disturbed the synthesis of pro-inflammatory cytokines. When cells were cultured with these trichothecens in combination with lipopolissacharide (LPS), the production of TNF-α, IL-8 and IL-6 was superinduced. These results suggested that DON and other 8-ketotrichothecens have the potential to superinduce pro-inflammatory cytokines production after infection in the presence of lipopolysaccharide.
Deoxynivalenol, T2-toxin and HT-2 toxin were evaluated in the 56th JECFA (Joint FAO/WHO Expert Committee on Food Additives) held in February, 2001. The Committee proposed a PMTDI (provisional maximum tolerable daily intake) of 1 μg/kg bw/day for deoxynivalenol, based on the NOEL of 100 μg/kg bw/day in a 2-year feeding study using mice. It proposed a PMTDI of 60 ng/kg bw/day for T-2 toxin and HT-2 toxin, alone or in combination, based on the LOEL of 29 μg/kg bw/day in a 3-week dietary study in pigs. Carcinogenicity was not recognized for these trichothecens.