Hiroko Furusawa

Kudoa septempunctata Invasion Increases the Permeability of Human Intestinal Epithelial Monolayer

Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) and causes a foodborne illness that affects more than 100 cases in Japan each year. We previously reported that the consumption of raw olive flounder meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhea and emesis through an unknown mechanism. Here, we demonstrate that K. septempunctata sporoplasm plays an important role in mediating the toxicity of K. septempunctata. When K. septempunctata spores were inoculated in Caco-2 human intestinal cells, K. septempunctata sporoplasms were released from spores, and they invaded the cells. Electron microscopic observations revealed that the sporoplasm invasion severely damaged the Caco-2 cells. The inoculation of K. septempunctata spores eliminated the transepithelial electrical resistance (TER) across the cell monolayer. Inhibiting the invasion of the sporoplasms prevented the observed loss in cell layer integrity, as illustrated by the rapid elimination of the TER. These results suggest that the invasion by sporoplasms severely damaged individual intestinal cells, resulting in a loss of cell monolayer integrity.

Comparative study of deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol on intestinal transport and IL-8 secretion in the human cell line Caco-2.

The effects of the trichothecene mycotoxin deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) on human intestinal cell Caco-2 were investigated by the studies of transepithelial transport, gene expression, and cytokine secretion. Permeability across a Caco-2 cell monolayer was evaluated by transport study. Transport rates were ranked as DON, 3ADON<15ADON in apical-basolateral direction. 15ADON showed the highest permeability, induced the highest decrease in transepithelial electrical resistance (TEER), and prompted significant Lucifer Yellow permeability. These results showed that 15ADON affect paracellular barrier function extremely. In addition, gene expressions induced by toxins were screened by DNA microarray for investigating cellular effect on Caco-2 cell. The most remarkable gene induced by DON and 15ADON was inflammatory chemokine IL-8 and thus mRNA expression and secretion of IL-8 were analyzed by PCR and ELISA. Both DON and acetylated DONs could induce mRNA expression and production of IL-8. In particular, ELISA assay showed that the ability to produce IL-8 was ranked as 3ADON<DON<15ADON. Our results indicated that 15ADON caused the highest permeability and highest IL-8 secretion among DON, 3ADON, and 15ADON in human intestinal cell.

Structural determination of a nivalenol glucoside and development of an analytical method for the simultaneous determination of nivalenol and deoxynivalenol, and their glucosides, in wheat.

Trichothecene mycotoxins such as nivalenol and deoxynivalenol frequently contaminate foodstuffs. Recently, several trichothecene glucosides have been found in trichothecene-contaminated foods, and information about their chemistry, toxicity, and occurrence is required. In this study, a glucoside of nivalenol was isolated from nivalenol-contaminated wheat and was identified as nivalenol-3-O-β-D-glucopyranoside. Analytical methods using a multifunctional column or an immunoaffinity column have been developed for the simultaneous determination of nivalenol, nivalenol-3-O-β-D-glucopyranoside, deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyranoside in wheat. The methods were validated in a single laboratory, and recovery from wheat samples spiked at four levels ranged between 86.4 and 103.5% for the immunoaffinity column cleanup. These mycotoxins in contaminated wheat samples were quantitated by the validated method. Nivalenol-3-O-β-D-glucopyranoside was detected in the nivalenol-contaminated wheat, and the percentage of nivalenol-3-O-β-D-glucopyranoside to nivalenol ranged from 12 to 27%. This result indicates that the analytical method developed in this study is useful for obtaining data concerning the state and level of food contamination by nivalenol, deoxynivalenol, and their glucosides.

Estimated Dietary Exposure to Mycotoxins after Taking into Account the Cooking of Staple Foods in Japan

Mycotoxins are commonly present in cereal grains and are not completely destroyed during their cooking and processing. When mycotoxins contaminate staple foods, the risk for exposure becomes serious. In East Asia, including Japan, rice is consumed as a staple food, and with the increasingly Westernized lifestyle, the consumption of wheat has increased. The mycotoxins commonly associated with rice and wheat are total aflatoxin (AFL) and ochratoxin A (OTA), respectively. This study examined the retention of AFL and OTA during the cooking of rice and pasta. AFL was retained at 83%–89% the initial level after the cooking of steamed rice. In pasta noodles, more than 60% of the OTA was retained. These results show that AFL and OTA are relatively stable during the cooking process, suggesting that a major reduction in the exposure to these mycotoxins cannot be expected to occur by cooking rice and pasta. The estimated exposure assessment at the high consumer level (95th percentile) and the mycotoxin contamination level determined by taking into account these reductions in the present study should be useful for the establishment of practical regulations for mycotoxins in staple foods.

Formulation of a pectin gel that efficiently traps mycotoxin deoxynivalenol and reduces its bioavailability.

We aimed to develop a new food-processing approach using pectin to reduce gastrointestinal absorption of mycotoxins. When Ca(2+) is added to low-methoxyl pectin, a gel resembling an egg box-like structure forms that is able to trap certain molecules. We examined whether or not low-methoxyl amidated pectin (LMA) and low-methoxyl non-amidated pectin (LMNA) trapped the mycotoxin deoxynivalenol (DON) after being ingested. We first determined the trapping effects of LMA and LMNA on DON in vitro under conditions similar to those in the human stomach, with results showing that LMA gel trapped DON to a greater extent than the LMNA gel. We then performed in vivo experiments and demonstrated that the LMA gel containing DON reduced DONs absorption from the gastrointestinal tract. This new food-processing technique holds great promise for reducing the bioavailability of DON in contaminated food and may be useful in mitigating the effects of other mycotoxins.

Molecular epidemiological analysis of Kudoa septempunctata by random amplified polymorphic DNA analysis

Molecular epidemiological analysis of Kudoa septempunctata isolates from 34 olive flounders associated with foodborne disease outbreaks and from 6 reference samples was performed using random amplified polymorphic DNA (RAPD) analysis. The K. septempunctata isolates analyzed in this study were divided into 8 groups. Eight isolates obtained from the large Ehime Prefecture outbreak in Japan that had occurred on October 8, 2010, were further divided into 4 groups. Eight isolates obtained from Korean samples were divided into 3 groups. These groups included isolates that had been identified from the large Ehime Prefecture outbreak. These results indicated that the Korean isolates had similar genetic backgrounds to those involved in the Ehime Prefecture outbreak. Isolates associated with outbreaks with similar dates of onset tended to be classified in the same group, suggesting that the strains involved in these incidents were genetically related. These results demonstrated that RAPD analysis is a useful molecular epidemiological analysis method for K. septempunctata.

Kudoa septempunctata was recognised by Toll-like receptor 2 produced by a RAW 264 macrophage-like cell line

Kudoa septempunctata is a myxosporean parasite that infects Paralichthys olivaceus (olive flounder). Previously, we reported that the consumption of raw P. olivaceus meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhoea and emesis. In this study, we investigated the cytokine production of mouse macrophage-like RAW 264 cells stimulated with K. septempunctata. When the RAW 264 cells were incubated with the spores of K. septempunctata for 24 h, they secreted tumour necrosis factor α (TNF-α) and several chemokines, such as IP-10, MIP-1β, and MIP-2. The secretion of TNF-α was induced in a dose-dependent manner in a bioassay using L929 cells and mouse TNF-α-specific enzyme-linked immunosorbent assay (ELISA). To identify the macrophage receptor of K. septempunctata, activation of HEK 293 cells expressing one of the Toll-like receptors (TLR) was measured using an NF-κB-dependent reporter assay. TLR2-expressing HEK 293 cells were strongly activated following stimulation with the spores. These results suggested that K. septempunctata was recognised by TLR2 on the macrophages, which were then activated and produced TNF-α.