Sub-acute oral exposure to lowest observed adverse effect level of nivalenol exacerbates atopic dermatitis in mice via direct activation of mitogen-activated protein kinase signal in antigen-presenting cells.

This study investigated the immunotoxic effects of the mycotoxin nivalenol (NIV) using antigen-presenting cells and a mouse model of atopic dermatitis (AD). In vitro experiments were conducted using a mouse macrophage cell line (RAW 264.7) and mouse dendritic cell line (DC 2.4). After cells were exposed to NIV (0.19-5 µmol) for 24 h, the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNFα) was quantified. To further investigate the inflammatory cytokine production pathway, the possible involvement of mitogen-activated protein kinase (MAPK) pathways, such as ERK1/2, p-38, and JNK, in NIV exposure was analyzed using MAPK inhibitors and phosphorylation analyses. In addition, the pro-inflammatory effects of oral exposure to NIV at low concentrations (1 or 5 ppm) were evaluated in an NC/Nga mouse model of hapten-induced AD. In vitro experiments demonstrated that exposure to NIV significantly enhanced the production of TNFα. In addition, it also directly induced the phosphorylation of MAPK, indicated by the inhibition of TNFα production following pretreatment with MAPK inhibitors. Oral exposure to NIV significantly exacerbated the symptoms of AD, including a significant increase in helper T cells and IgE-produced B cells in auricular lymph nodes and secretion of pro-inflammatory cytokines, such as IL-4, IL-5, and IL-13, compared with the vehicle control group. Our findings indicate that exposure to NIV directly enhanced the phosphorylation of ERK1/2, p-38, and JNK, resulting in a significant increase in TNFα production in antigen-presenting cells, which is closely related to the development of atopic dermatitis.

Oral exposure to citrinin significantly exacerbates the pathophysiology of a mouse model of imiquimod-induced psoriasis via direct activation of dendritic cell.

Citrinin, a mycotoxin produced by Penicillium citrinum and Penicillium verrucosum, mainly contaminates cereals. The aim of study was to investigate the novel immunoreactive effect of citrinin using a mouse model of psoriasis. A mouse model of psoriasis was generated by topical application of 5% imiquimod in female BALB/c mice. Standard rodent diet and rice samples with 3 ppm of citrinin were mixed to obtain a final citrinin concentration of 0.3 ppm, and a citrinin-contaminated diet was fed to mice daily. Skin thickness, scratching behavior, and trans epidermal water loss (TEWL) were monitored continuously during the imiquimod application. Immediately after the final imiquimod application, ear skin and auricular lymph node (LN) were sampled for further analysis. Only a slight increase was observed in skin thickness in the citrinin exposure group; however, citrinin exposure significantly exacerbated hyperkeratinization and inflammatory cell infiltration in histological evaluation. TEWL, which is representative of cutaneous barrier function, was significantly increased by citrinin exposure. In terms of immune function, the number of immune cells in LN (T cells and dendritic cells) and gene expression of interleukin (IL)-17 in skin tissue were significantly increased by citrinin exposure. Direct interaction of dendritic cells (DCs) in citrinin-induced psoriasis development was further examined by proinflammatory cytokine determination in THP-1 cells and murine bone marrow derived DCs. IL-6 and/or tumor necrosis factor α were significantly increased by citrinin exposure. Taken together, our results imply that oral exposure to citrinin exacerbates the symptoms of a mouse model of psoriasis via direct activation of DCs.

Low-dose ethanol increases aflatoxin production due to the adh1-dependent incorporation of ethanol into aflatoxin biosynthesis.

Aflatoxins are toxic secondary metabolites produced by some aspergilli, including Aspergillus flavus. Recently, ethanol has attracted attention as an agent for the control of aflatoxin contamination. However, as aflatoxin biosynthesis utilizes acetyl coenzyme A, ethanol may be conversely exploited for aflatoxin production. Here, we demonstrated that not only the 13C of labeled ethanol, but also that of labeled 2-propanol, was incorporated into aflatoxin B1 and B2, and that ethanol and 2-propanol upregulated aflatoxin production at low concentrations (<1% and <0.6%, respectively). In the alcohol dehydrogenase gene adh1 deletion mutant, the 13C incorporation of labeled ethanol, but not labeled 2-propanol, into aflatoxin B1 and B2 was attenuated, indicating that the alcohols have different utilization pathways. Our results show that A. flavus utilizes ethanol and 2-propanol as carbon sources for aflatoxin biosynthesis and that adh1 indirectly controls aflatoxin production by balancing ethanol production and catabolism.

Preparation of Monoclonal Antibodies Specifically Reacting with the Trichothecene Mycotoxins Nivalenol and 15-Acetylnivalenol via the Introduction of a Linker Molecule into Its C-15 Position.

Nivalenol (NIV) is a trichothecene mycotoxin that is more toxic than deoxynivalenol. It accumulates in grains due to infection with Fusarium species, which are the causative agents of scab or Fusarium head blight. An immunoassay, which is a rapid and easy analytical method, is necessary for monitoring NIV in grains. However, a specific antibody against NIV has not been prepared previously. To establish an immunoassay, we prepared NIV, introduced a linker, and generated antibodies against it. NIV was prepared from a culture of Fusarium kyushuense obtained from pressed barley through chromatographic procedures with synthetic adsorbents and silica gel. NIV was reacted with glutaric anhydride, and the reaction was stopped before mono-hemiglutaryl-NIV was changed to di-hemiglutaryl-NIV. 15-O-Hemiglutaryl-NIV was isolated via preparative HPLC and bound to keyhole limpet hemocyanin (KLH) using the active ester method. Two different monoclonal antibodies were prepared by immunizing mice with the NIV-KLH conjugate. The 50% inhibitory concentration values were 36 and 37 ng/mL. These antibodies also showed high reactivity in a direct competitive enzyme-linked immunosorbent assay and specifically reacted with NIV and 15-acetyl-NIV but not with deoxynivalenol and 4-acetyl-NIV.

Effects of Four Isothiocyanates in Dissolved and Gaseous States on the Growth and Aflatoxin Production of Aspergillus flavus In Vitro.

Aflatoxins (AFs), a class of toxins produced by certain species of the genus Aspergillus, occasionally contaminate food and cause serious damage to human health and the economy. AFs contamination is a global problem, and there is a need to develop effective strategies to control aflatoxigenic fungi. In this study, we focused on isothiocyanates (ITCs) as potential chemical agents for the control of aflatoxigenic fungi. We quantitatively evaluated the effects of four ITCs (allyl ITC (AITC), benzyl ITC (BITC), and methyl and phenylethyl ITCs) in dissolved and gaseous states on the growth and aflatoxin B1 production of Aspergillus flavus. In experiments using dissolved ITCs, BITC was found to be the strongest inhibitor of growth and aflatoxin B1 production by A. flavus. Meanwhile, in the gaseous state, AITC strongly inhibited the A. flavus growth. When the concentration of ITCs in the liquid medium was quantified over time, AITC levels decreased to below the detection limit within 24 h, whereas BITC levels remained stable even after 48 h. These results suggested that when ITCs are utilized to control aflatoxigenic fungi, it is necessary to use them in a dissolved or gaseous state, depending on their volatility.

A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing.

Members of the Fusarium graminearum species complex (Fg complex) are the primary pathogens that cause Fusarium head blight in wheat and barley. Fg complex members grow poorly on Fusarium oxysporum-selective media, such as Komada and Fo-G2, that have also been used for the isolation of other Fusarium species. Therefore, Komada medium was modified as FG medium for the isolation of Fg complex members. However, the production of pentachloronitrobenzene that is the most effective component of FG medium is discontinued and new media is required for the selective isolation of Fg complex members. In addition, the rapid diagnosis of isolated fungi is useful for the disease control. Novel tools have been developed for isolating and characterizing Fg complex members. FG21, a semi-selective medium for isolating Fg complex members, was developed using potato dextrose agar. Furthermore, a dipstick DNA chromatography assay was developed both to identify Fusarium graminearum sensu stricto and Fusarium asiaticum in the Fg complex and their trichothecene mycotoxin types. The easier isolation and characterization of Fg complex members in Japan was attained by the combined use of FG21 medium and the dipstick DNA chromatography assay.

Acute and Subacute Oral Toxicity of Deoxynivalenol Exposure in a Dermatophagoides farinae-Induced Murine Asthma Model.

Previously, researchers have demonstrated that mycotoxin deoxynivalenol (DON) significantly enhances immunocyte activation. However, the interaction between DON exposure and immune disorders remains unclear. In this study, we aimed to investigate whether acute and subacute oral exposure to DON exacerbates the development of respiratory allergy using a mite allergen (Dermatophagoides farina, Derf)-induced mouse model of asthma. The direct relationship between DON exposure and asthma development was examined following acute oral DON administration (0, 0.1, or 0.3 mg/kg body weight), immediately before the final mite allergen challenge. Simultaneously, the influence of subacute oral exposure via low dose DON contaminated wheat (0.33 ppm) was evaluated using the same settings. To detect the proinflammatory effects of DON exposure, we examined the total and Derf-specific serum IgE levels, histology, number of immunocytes, and cytokine and chemokine secretion. Acute oral DON significantly enhanced the inflammatory responses, including cellular infiltration into bronchoalveolar lavage fluid, infiltration of immunocytes and cytokine production in local lymph nodes, and cytokine levels in lung tissues. Corresponding proinflammatory responses were observed in a mouse group exposed to subacute oral DON. In vivo results were validated by in vitro experiments using the human bronchial epithelial (BEAS-2B) and human eosinophilic leukemia (EOL-1) cell lines. Following exposure to DON, the secretion of interleukin (IL)-1ß, IL-6, IL-8, and/or tumor necrosis factor-α in BEAS-2B cells, as well as EoL-1 cells, increased significantly. Our findings indicate that DON exposure is significantly involved in the proinflammatory response observed in respiratory allergy.

Acute and subacute oral administration of mycotoxin deoxynivalenol exacerbates the pro-inflammatory and pro-pruritic responses in a mouse model of allergic dermatitis.

Deoxynivalenol (DON) contamination in food is a public health concern; however, the effect of DON exposure on immune disorders including allergies remains unclear. The aim of this study is to elucidate the effect of oral exposure to DON on pro-inflammatory and pro-pruritic responses in a mouse model of allergic dermatitis, which was generated by topical application of toluene-2,4-diisocyanate (TDI), a hapten that induces type-2 helper T cells. To evaluate acute exposure to DON, the mice were orally administered vehicle alone, 0.1 mg/kg DON, or 0.3 mg/kg DON 48, 24, and 1 h before the final challenge with TDI. To study subacute exposure, the mice were fed DON-contaminated rodent diet (0.3 ppm) during the experimental period. After the itch behavior and ear-swelling response were monitored, the serum, auricular lymph node, and skin tissue were collected for analyzing immunocyte differentiation, cytokine determination, and histological changes. Acute oral administration of DON significantly enhanced pro-inflammatory responses including ear-swelling response, immunocyte infiltration, and cytokine productions. Histological evaluation supported the occurrence of pro-inflammatory responses. In contrast, acute DON exposure only slightly increased itch behavior. Subacute oral exposure to DON significantly up-regulated the inflammatory responses, but showed almost no effect on pruritic response. In vitro evaluation in dendritic cells and keratinocytes indicated that DON pre-exposure induced a dose-dependent significant increase in cytokine production. Our results imply that both acute and subacute exposures to DON are associated with pro-inflammatory responses in cutaneous allergy.

Improvement of the Culture Medium for the Dichlorvos-Ammonia (DV-AM) Method to Selectively Detect Aflatoxigenic Fungi in Soil.

The dichlorvos-ammonia (DV-AM) method is a simple but sensitive visual method for detecting aflatoxigenic fungi. Here we sought to develop a selective medium that is appropriate for the growth of aflatoxigenic fungi among soil mycoflora. We examined the effects of different concentrations of carbon sources (sucrose and glucose) and detergents (deoxycholate (DOC), Triton X-100, and Tween 80) on microorganisms in soils, using agar medium supplemented with chloramphenicol. The results demonstrated that 5⁻10% sucrose concentrations and 0.1⁻0.15% DOC concentrations were appropriate for the selective detection of aflatoxigenic fungi in soil. We also identified the optimal constituents of the medium on which the normal rapid growth of Rhizopus sp. was completely inhibited. By using the new medium along with the DV-AM method, we succeeded in the isolation of aflatoxigenic fungi from non-agricultural fields in Fukui city, Japan. The fungi were identified as Aspergillus nomius based on their calmodulin gene sequences. These results indicate that the new medium will be useful in practice for the detection of aflatoxigenic fungi in soil samples including those from non-agricultural environments.

Detection of Aflatoxigenic and Atoxigenic Mexican Aspergillus Strains by the Dichlorvos⁻Ammonia (DV⁻AM) Method.

The dichlorvos⁻ammonia (DV⁻AM) method is a sensitive method for distinguishing aflatoxigenic fungi by detecting red (positive) colonies. In this study, the DV⁻AM method was applied for the isolation of aflatoxigenic and atoxigenic fungi from soil samples from a maize field in Mexico. In the first screening, we obtained two isolates from two soil subsamples of 20 independent samples and, in the second screening, we obtained two isolates from one subsample of these. Morphological and phylogenic analyses of the two isolates (MEX-A19-13, MEX-A19-2nd-5) indicated that they were Aspergillus flavus located in the A. flavus clade. Chemical analyses demonstrated that one isolate could produce B-type aflatoxins, while the other produced no aflatoxins. These results demonstrate that the DV⁻AM method is useful for the isolation of both aflatoxigenic and atoxigenic Aspergilli.

Analysis of the Masked Metabolite of Deoxynivalenol and Fusarium Resistance in CIMMYT Wheat Germplasm.

Fusarium head blight (FHB) causes significant grain loss and contamination of grains with harmful mycotoxins, especially deoxynivalenol (DON). Fusarium resistance and DON accumulation have been extensively investigated in various cultivars; however, the level of DON-3-O-glucoside (D3G) has not been as carefully studied. In this study, we measured accumulated DON and D3G levels in CIMMYT wheat elite germplasm using an analytical method validated in-house. Co-occurring nivalenol (NIV) and ergostrerol (ERG) were also analyzed. LC-MS/MS and LC-UV analyses were applied to the 50 CIMMYT elite wheat lines. D3G showed rather high correlation with DON (r = 0.82), while FHB symptoms showed slight correlation with DON and D3G (r = 0.36 and 0.32, respectively). D3G/DON ratio varied widely from 8.1 to 37.7%, and the ratio was not related with FHB resistance in this dataset.

Detection of N-(1-deoxy-D-fructos-1-yl) Fumonisins B2 and B3 in Corn by High-Resolution LC-Orbitrap MS.

The existence of glucose conjugates of fumonisin B2 (FB2) and fumonisin B3 (FB3) in corn powder was confirmed for the first time. These "bound-fumonisins" (FB2 and FB3 bound to glucose) were identified as N-(1-deoxy-D-fructos-1-yl) fumonisin B2 (NDfrc-FB2) and N-(1-deoxy-D-fructos-1-yl) fumonisin B3 (NDfrc-FB3) respectively, based on the accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) analysis. Treatment on NDfrc-FB2 and NDfrc-FB3 with the o-phthalaldehyde (OPA) reagent also supported that D-glucose binding to FB2 and FB3 molecules occurred to their primary amine residues.

Development of the dichlorvos-ammonia (DV-AM) method for the visual detection of aflatoxigenic fungi.

Aflatoxins (AFs) are carcinogenic and toxic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus. To monitor and regulate the AF contamination of crops, a sensitive and precise detection method for these toxigenic fungi in environments is necessary. We herein developed a novel visual detection method, the dichlorvos-ammonia (DV-AM) method, for identifying AF-producing fungi using DV and AM vapor on agar culture plates, in which DV inhibits the esterase in AF biosynthesis, causing the accumulation of anthraquinone precursors (versiconal hemiacetal acetate and versiconol acetate) of AFs in mycelia on the agar plate, followed by a change in the color of the colonies from light yellow to brilliant purple-red by the AM vapor treatment. We also investigated the appropriate culture conditions to increase the color intensity. It should be noted that other species producing the same precursors of AFs such as Aspergillus nidulans and Aspergillus versicolor could be discriminated from the Aspergillus section Flavi based on the differences of their phenotypes. The DV-AM method was also useful for the isolation of nonaflatoxigenic fungi showing no color change, for screening microorganisms that inhibit the AF production by fungi, and for the characterization of the fungi infecting corn kernels. Thus, the DV-AM method can provide a highly sensitive and visible indicator for the detection of aflatoxigenic fungi.

A single nucleotide polymorphism in the translation elongation factor 1α gene correlates with the ability to produce fumonisin in Japanese Fusarium fujikuroi.

PCR-RFLP based on the translation elongation factor 1α (TEF) gene was developed to identify Fusarium fujikuroi in the Fusarium (Gibberella) fujikuroi species complex. Ninety-three strains, most of which were obtained from various sources in Japan, were identified as F. fujikuroi and their capability to produce fumonisin was investigated using an in vitro assay. Fumonisin production was detected in 50 strains isolated from maize, strawberry, wheat, and rice, whereas it was undetectable in 43 strains derived from rice seeds and rice seedlings carrying the bakanae disease, and from unknown sources. A single nucleotide polymorphism in the TEF gene (T618G) correlated with the ability to synthesize fumonisin.

Opposite effects of two trichothecene mycotoxins, deoxynivalenol and nivalenol, on the levels of macrophage inflammatory protein (MIP)-1α and MIP-1ß in HL60 cells.

To elucidate the mechanisms underlying the toxicities of the trichothecene mycotoxins deoxynivalenol and nivalenol, their effects on the secretion of anti-hematopoietic chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1ß in human promyelocytic leukemia cell line HL60 were investigated. Exposure to deoxynivalenol for 24h significantly induced the secretion of chemokines. The induction of these chemokines may account for the leukopenia after exposure to trichothecene mycotoxins. Treatment with nivalenol decreased the secretion of these chemokines. Our finding that deoxynivalenol induces the secretion of these chemokines, whereas nivalenol has the opposite effect, clearly indicates that the toxicity mechanisms of deoxynivalenol and nivalenol differ.

Experimental infection of Fusarium proliferatum in Oryza sativa plants; fumonisin B1 production and survival rate in grains.

Fusarium proliferatum is a plant pathogenic fungus associated with crops such as asparagus and corn, and it possesses the ability to produce a range of mycotoxins, including fumonisins. In Asia, rice (Oryza sativa) is a staple cereal and is occasionally colonized by this fungus without obvious physiological changes. F. proliferatum is closely related to Gibberella fujikuroi (anamorph F. fujikuroi) responsible for Bakanae disease in rice; however there are few reports of F. proliferatum as a rice pathogen. In this study, we examined the pathogenic potential of F. proliferatum in rice plants with respect to browning, fumonisin production, and survival rates in rice grains. Fungal inoculation was conducted by spraying a conidial suspension of F. proliferatum onto rice plants during the flowering period. Browning was found on the stalk, leaf, and ear of rice. Fumonisin B(1) was detected at levels from trace to 21 ng/g grains, using tandem mass spectrometry. Fungal recovery after 6 months indicated that F. proliferatum had high affinity to rice plants being still viable in grains. From this study, it can be concluded that F. proliferatum is a possible pathogen of rice and possesses a potential to produce fumonisin B(1) in rice grains in the field.

Nuclear factor-κB inhibitors alleviate nivalenol-induced cytotoxicity in HL60 cells.

Tricothecene mycotoxins, such as nivalenol, are toxic to leukocytes. To elucidate the molecular mechanism of nivalenol toxicity, we investigated the involvement of nuclear factor-κB (NF-κB) in nivalenol-induced cytotoxicity in HL60 cells using the NF-κB inhibitors pyrrolidinedithiocarbamate (PDTC) and dexamethasone. Cells were treated with the chemicals for 24h before assays were performed. Nivalenol elicited interleukin (IL)-8 secretion. IL-8 secretion was lower in cells concomitantly treated with nivalenol and NF-κB inhibitors than with nivalenol alone. Nivalenol reduced monocyte chemotactic protein (MCP)-1 secretion. MCP-1 secretion was higher in cells concomitantly treated with nivalenol and NF-κB inhibitors than with nivalenol alone. NF-κB inhibitors thus alleviated the effects of nivalenol, indicating that NF-κB is important for nivalenol-caused changes in cytokine secretion. Nivalenol hindered cell proliferation, and dexamethasone reduced this effect, suggesting that NF-κB contributes to cell proliferation. Thus, it appears that NF-κB is involved in nivalenol-induced toxicity in HL60 cells.

Distinct distribution of deoxynivalenol, nivalenol, and ergosterol in Fusarium-infected Japanese soft red winter wheat milling fractions.

The occurrence of mycotoxins in small grain cereals and their retention in final products are serious concerns for food safety. Previously, we investigated the fate of deoxynivalenol and nivalenol in a Japanese soft red winter wheat cultivar during milling and we found that deoxynivalenol and/or nivalenol was readily distributed among flours for human consumption. In the present study, we analyzed the ergosterol concentrations in the milling fractions as an index of fungal biomass to elucidate the relationship between deoxynivalenol/nivalenol accumulation and fungal invasion into the grain, after the in-house validation of an analytical method for quantifying ergosterol in the resulting milling fractions (patent flour, low-grade flour, bran, and shorts). Using three samples with different levels of deoxynivalenol and/or nivalenol contamination, the contents of deoxynivalenol/nivalenol and ergosterol in the resulting milling fractions were evaluated. The concentration of ergosterol was always lowest in patent flour and highest in bran or shorts, indicating that most of the fungi is retained in the outer layers of grain (bran and shorts) even in highly contaminated grain. On the other hand, the concentrations of deoxynivalenol and nivalenol were similar in the low-grade and patent flours and only slightly lower than in the medium-level and high-level contaminated grains. Moreover, the percentage distribution of ergosterol was higher in bran than in other fractions in all cases, which differed from that of deoxynivalenol/nivalenol. This result indicates the diffusion of deoxynivalenol/nivalenol inside the grain that is independent of fungal invasion.

Distribution of deoxynivalenol and nivalenol in milling fractions from fusarium-infected Japanese wheat cultivars.

The fate of the Fusarium mycotoxins deoxynivalenol and nivalenol during the milling of Japanese wheat cultivars artificially infected with Fusarium was investigated. Grain samples with different mycotoxin concentrations were milled using a laboratory-scale test mill to produce eight fractions: three breaking flours (1B, 2B, and 3B), three reduction flours (1M, 2M, and 3M), wheat bran, and wheat shorts. Patent flour for human consumption was made from the 1B, 2B, 1M, and 2M flours, and low-grade flour was made from 3B and 3M flours. The four resulting samples (patent flour, low-grade flour, bran, and shorts) were analyzed for deoxynivalenol and/or nivalenol with an in-house validated analytical method using high-performance liquid chromatography with UV absorbance detection. In samples with different mycotoxin concentrations, the distribution of those toxins differed among the milling fractions. Grains with a lower level of contamination produced bran and shorts samples with a high relative concentration of nivalenol. A high percentage of nivalenol was found in patent flour, followed by bran. Contrary to the less-contaminated sample, the concentration of nivalenol in moderately contaminated grain was high only in the shorts sample. The highest percentage of deoxynivalenol and nivalenol was observed in the patent flour. The results of this study indicate that the distribution of deoxynivalenol and nivalenol in milled Japanese wheat could be influenced by the contamination level of the original grain, and the milling process is not always effective for removal of toxins from wheat grains.

Limited surveillance of fumonisins in brown rice and wheat harvested in Japan.

Fumonisins are mycotoxins mainly produced by Fusarium verticillioides, which is a major contaminant of corn. However, there are sporadic reports of fumonisin contamination in wheat worldwide. The rice adherent fungus Gibberella fujikuroi is taxonomically closely related to F. verticillioides. Therefore, the potential risk of fumonisin contamination in rice and wheat is significant. Previously, a sensitive detection method utilizing liquid chromatography with tandem electrospray mass spectrometry (LC-ESI-MS-MS) was developed for the determination of fumonisins in brown rice. In the present study, the incidence of fumonisins in brown rice and wheat harvested in Japan was investigated using LC-ESI-MS-MS. Forty-eight rice samples and 47 wheat samples were screened and analyzed for the major B-type fumonisins: fumonisin B1 (FB1) and fumonisin B2 (FB2). About 1 kg of rice or wheat seed was divided into three subsamples, and 10 g from each subsample was used for the analysis. The limits of detection were 0.012 and 0.011 mg/kg for FBt and FB2, respectively, in rice samples and 0.010 and 0.008 mg/kg for FB1 and FB2, respectively, in wheat samples. The mean (standard deviation) recoveries of FB1 spiked at 0.50 mg/kg into toxin-free rice and wheat samples were 77.6 (4.2)% and 84.5 (3.1)%, respectively. One of the wheat samples was positive for FBt with a value greater than the limit of detection,but no fumonisin was found in any of the rice samples. This is the first report of fumonisins detected in Japanese wheat.