A survey was carried out to obtain data on the mycoflora and the occurrence of aflatoxigenic mold contamination of hay cube (cube-shaped alfalfa hay). As the mycoflora of 24 samples, Eurotium spp. (Aspergillus glaucus), Scopulariopsis spp., Aspergillus flavus and A. versicolor were predominant. A. flavus was isolated from 3 of 24 samples in colony counts per g from 1.0×10¹ to 1.0×10⁷, but aflatoxins (AF) could not be detected even on the sample which was contaminated with 1.0×10⁷ cfu/g of A. flavus. Furthermore, inoculation test of aflatoxigenic A. flavus on hay cube medium or on glucose-added hay cube medium was found that growth of aflatoxigenic A. flavus was well without any accumulation of AF in the media. These findings were subsequently proven that addition of hay cube water extract (equivalent amounts of 2.50g of hay cube) in 50 ml of yeast extract sucrose medium completely inhibited the AF production by aflatoxigenic A. flavus while this inhibitory effect was not pronounced on its mycelial growth. The pH of medium did not substantially influence the inhibitory action of hay cube. Attempts to find similar inhibitory effect of hay cube water extract against ochratoxin A (OCT-A) production by A. ochraceus, sterigmatocystin (ST) production by A. versicolor and citrinin (CI) production by Penicillium citrinum were unsuccessful. Therefore, data reported here indicate that hay cube can support the production of OCT-A, ST and CI by these mycotoxigenic contaminants.

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Averufin, an intermediate in aflatoxin B₁ biosynthesis, has been found to be a potent uncoupler of oxidative phosphorylation in mitochondria. At higher concentrations than those for uncoupling activity, averufin, strongly depresses state 3 respiration of mitochondria. In the present study, the mode of inhibition of averufin on the state 3 respiration has been investigated by using electron transport particles (ETP) which are reverted membrane vesicles of mitochondrial inner membranes. Averufin was observed to strongly inhibit succinate oxidase in ETP, suggesting a direct interaction of averufin with the respiratory chain of mitochondria. Succinate dehydrogenase (succinate→complex II→PMS) and cytochrome c oxidase (ascorbate→TMPD→cytochrome c→complex IV→O₂) were not affected but succinate cytochrome c reductase (succinate→complex II→CoQ→complex III→cytochrome c) was markedly depressed by averufin. These results apparently indicate that averufin selectively interfere with complex III of the respiratory chain in mitochondria, by which averufin depresses the state 3 respiration of mitochondria. None of these enzyme complexes in ETP were affected by averufin dimethylether which was able to depress the state 3 respiration of intact mitochondria.

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In vitro culture cell line, Kagura-1, was established from a rat hepatocellular carcinoma induced by aflatoxin B₁ (AFB₁). When the cells at passage 15 were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum in collagencoated dishes, the population doubling time and saturation density were 13.2 hr and 1.9×10⁵ cells/cm², respectively. The established cells formed colonies in soft agar.We examined the expression of proto-oncogenes in two liver tumors induced by AFBI and Kagura-1 cell. Both c-myc and c-Ha-ras expressions were activated at high levels in both tumors and Kagura-1 cells.

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The effects of aflatoxin B₁ (AFB₁) and sterigmatocystin (STC) on the hormonal induction of liver-specific enzymes and the binding capacity of glucocorticoid receptor (GR) were observed with cultured mammalian cells, H4-II-E, which possess many functional enzymes of mature liver cells. The liver-specific enzymes such as tyrosine aminotransferase (TAT), phosphoenol-pyruvate carboxykinase (PEPCK), glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT), were induced by various hormones such as hydrocortisone (HC), insulin, and dibutyryl cAMP (Bt₂ cAMP) in various degrees. The 50% inhibi-tion concentrations (IC₅₀, μg/ml) of AFB₁ to HC-dependent induction of TAT, GPT, and PEPCK were 0.2, 0.055, and 2.0, respectively. Whereas, insulin-dependent induc-tion of TAT was slightly inhibited by AFB₁. As for STC, the IC₅₀ for HC induction of TAT was 3.5 μg/ml, and no inhibitory effect on insulin-dependent induction was observed. Quick-blot analysis, which showed a marked reduction of the HC-induced synthesis of TAT mRNA, indicated that AFB₁ inhibited a transcriptional step. In the cell-free system, both mycotoxins showed no direct inhibitory effect on the formation of [³H]triamcinolone acetonide (TA)-GR complex, its activation and binding to nuclear acceptor sites. The inhibition of this GR-complex formation was observed only when the mycotoxins were activated by S9 system. In the whole cell system pretreated with AFB₁, the formation of cytosolic [³H]TAGR complex was greatly reduced, and the release of nuclear GR complex was promoted. STC caused no marked reduction of the cytosolic GR complex. Kinetic analysis revealed that the cytosolic GR receptor was markedly impaired by AFBI, and the capacity of nuclear GR acceptor sites was greatly reduced by STC. The present data indicated marked differences between the carcinogenic bisfuranoids, AFB₁ and STC, in regards to the hormonal induction of liver-specific enzymes and the function of cytosolic/nuclear GRs, and the impairment of cellular factors (enzymes) which regulates GR recycling system was suggested.

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The ability of 207 strains of 122 species of Ascomycetes, to produce a monoamine oxidase inhibitor in solid cultures on rice was examined. The production of monoamine oxidase inhibitors by members of the following genera was confirmed: Achaetomium, Anixiella, Apiosordaria, Arxiomyces, Chaetomium, Cordyceps, Diplogelasinospora, Emericella, Eupenicillium, Kernia, Lophotrichus, Microascus, Neocosmospora, Onygena, Petriella, Pithoascus, Pseudallescheria, Sphaerodes, Talaromyces and Trichocoma.

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Contamination by aflatoxins B₁ and B₂ in shelled peanuts harvested from Shizuoka Prefecture, Japan, was investigated. The extraction with methano1-1% NaCl solution (55:45, v/v), followed by purification on a Sep-Pak silica cartridge column, and HPLC analysis revealed the contamination of 5.9mg/kg of aflatoxin B1 and 0.3mg/kg of aflatoxin B₂ in the shelled peanuts. Aflatoxin B₁ was confirmed by mass spectral analysis. Mycological survey detected aflatoxigenic strains of Aspergillus flavus on peanut kernels in the infested lot.

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To assess toxic effects of the purified satratoxins G and H, 4 week-old ddY male mice were given a single intraperitoneal injection and were evaluated for histopathological evidence of the organ injuries. Intraperitoneal LD₅₀ of satratoxins G and H were estimated as 1.23±0.08 and 5.69±0.43mg/kg body weight, respectively. Both toxins caused extensive ulceration of the small intestine and a relatively mild damage of the lymphoid and hematopoietic tissues in lethal animals. Furthermore, satratoxin G caused vacuolar and fatty degeneration of the hepatic cells.

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The distribution of Aspergillus flavus, a fungus that causes aflatoxin contamination of maize, was examined for soil samples collected from several maize production areas during rainy season in Thailand. Some strains of A. flavus appeared to be isolated geographically with different frequency.

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The distribution of airborne Aspergillus flavus spores was determined at several maize production areas during rainy season in Thailand. The higher distribution was detected in the atmosphere at middleman's, natural drying places, compared with maize fields where little or no spores were found.

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Re-evaluation of Aspergillus flavus/parasiticus agar (AFPA) was made after incubation test of 90 strains of A. flavus group and 6 strains of other Aspergillus and Penicillium species. Most of the tested strains of A. flavus and A. parasiticus were isolated from imported foods in Japan. It may be concluded from the plate cultures of the tested strains on AFPA that the previously reported procedure is reproducible and suitable for use in quality control as an index to the presence of aflatoxin-producing fungi in imported foods.

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Mycoflora of pine and cedar trees were investigated on collection of about 400 samples from allover Japan since 1980. A total of 31 species were isolated. In these fungi, 12 species were pathogenic by inoculation experiments. Apiocarpella sp., Phomopsis pini-densiflora, Phomopsis sp. (Akita), Haplosporella sp., Phlyctaena picea and Phlyctaena sp. (Hakodate) were generally detected on the wilted pine trees. Haplosporella sp., Phomopsis sp. (Fusa) and Phomopsis sp. (Kawaguchi) were encountered in wilted cedar trees, while Cercospora sequoiae was not able to isolate during this investigation.Haplosporella sp., Phomopsis pini-densi flora, and Phlyctaena picea were detected on both pine and cedar trees.

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