Previously we isolated a cancer cell line AFB-1 from a hepatocellular carcinoma that had been induced by aflatoxin B1 in a male Fischer 344 rat. In the present study, we constructed the subtracted cDNA libraries between mRNAs from AFB-1 and rat normal liver cell line BL9, and identified a 650 bp cDNA which hybridized with mRNA of AFB-1 but not with that of BL9 in Northern blot analysis. We transfected the 650 bp cDNA/vector recombinant into AFB-1 and BL9, and then inoculated either transfected cells or parental cells at 1×107 cells/rat subcutaneously at the dorsal region of syngenic rats. The transfection of AFB-1 with the 650 bp cDNA/vector recombinant increased the incidence of subcutaneous tumor growth (14 of 14 in male rats inoculated with transfected AFB-1 versus 8 of 13 in those with parental AFB-1, P<0.02) and that of lung metastasis (9 of 14 in male rats inoculated with transfected AFB-1 versus 0 of 13 in those with parental AFB-1, P<0.001). The transfection of the 650 bp cDNA/vector recombinant transformed normal liver cell line BL9 into cancer cells i.e. the transfected BL9 formed subcutaneous tumor nodules in 9 of 10 male rats and in all 10 female rats and formed metastatic foci in the lungs in 6 of 10 female rats. This is the first instance where a cDNA from a subtracted cDNA library not only enhanced subcutaneous tumor growth and lung metastasis of cancer cells but also induced those activities in normal cells.
Aspergillus was dominant in 5 koji samples collected from kecap and tauco factories in Java, Indonesia, whereas Mucorales was dominant in one sample. Although, no aflatoxin was detected in all the 6 koji samples, but two out of 24 Aspergillus strains isolated from them were identified as a producer of aflatoxins. Aspergillus sp. 2-5 isolated from the tauco koji produced aflatoxins during 2-day of cultivation on cooked soybeans. The mash prepared by the kecap koji inoculated with the tempe starter contained lower formol nitrogen (FN) than those inoculated with Aspergillus strains. The kecap koji prepared by inoculating with white-spored mutants, induced from koji molds by UV irradiation, could be distinguished from those inoculated with their original koji strains or the aflatoxin-producing strain by developing white conidia (spore) during koji making. FN contents of the kecap mashes prepared using whitespored mutants were not different from those prepared using original strains. These results show that white-spored mutant can be used as a starter for preparation of kecap koji from the standpoint of preventing aflatoxin contamination.
Possible anti-atherogenic activities of emodin were found. In this study, we investigated the effects of emodin on monocyte chemotactic protein (MCP)-1 and tissue inhibitor of metalloproteinases (TIMP)-2 secretion using aortic smooth muscle cell A-10. Both of them are considered to play important roles in the pathogenesis of atherosclerosis. Emodin had a dose-dependent inhibitory effect on tumor necrosis factor-αinduced MCP-1 secretion. Exposure to 50 and 100 μM emodin caused significant inhibition of TIMP-2 secretion. These results suggested that emodin has an anti-atherogenic potential. Also, the estrogenic activity of emodin was investigated. Emodin competed against estrogen in estrogen receptor (ER) binding, indicating that emodin is an estrogenic mycotoxin. Seeing that estrogens also inhibit the secretion of MCP-1, ER(s) may be a mediator of the emodin′s transducing signal.
In the European Union the major concerns relating to mycotoxins are from aflatoxins in imported foods, and ochratoxin A and patulin in home-produced commodities. Whilst other mycotoxins such as deoxynivalenol do frequently occur, for example in cereals, they are not presently regulated and not regarded as a high priority. Commodities such as animal feed containing peanut meal, dried figs, pistachios and various spices from specific countries have from time-to-time presented problems and the approach has been to control imports through tough regulatory limits. To back-up these controls the EU has funded a multi-national project which developed and validated several mycotoxin methods for regulatory purposes. HPLC methods with good performance characteristics for aflatoxins, ochratoxin A and patulin at ng/g and sub-ng/g limits in a range of commodities were established. In Europe, ochratoxin A and patulin are two mycotoxins that contaminate home-produced products. Exposure to ochratoxin A has always been of some concern, and whilst there are a range of foodstuffs (cereals, meat products, dried fruit, coffee, wine and beer) that can give rise to that exposure, in the area of home-grown cereals there is the most scope to reduce contamination. In the UK and in Europe, research has focussed on what might be done to prevent or reduce ochratoxin A occurrence in cereals and to develop a HACCP approach to production, identifying and controlling mould growth and toxin formation. Factors such as cereal varieties, farming methods (cultivation and storage), pesticide treatments and the effect of climate are being examined in relation to mould and toxin occurrence. This paper reviews some UK and European funded mycotoxin projects covering the areas indicated above, and presents a view of priorities in the mycotoxin area.
Environmental factors in the house have an opportunity to give some kind of human health risk during staying at home. Among important environmental problems, air pollution, water pollution, noise pollution, and UV damage remain to be solved. Because, it is hard to control such factors by ourselves. In our house such as living room, bedroom, and kitchen, we are exposing ourselves to fungal spores, house dust, mycotoxins, and volatile organic chemicals. Most of people do not believe that those may cause allergic symptoms to us. In this symposium, I organized to make a discussion concerning the view of human health hazard in indoor environments. On the basis of the presentation by four panelists, I focussed on the indoor problems with respect to mycological and chemical risk.
1. A total of 19,716 strains of microfungi were isolated and identified from 533 samples of house dust collected in nine homes. The dust samples were collected monthly for one year using the home-owner′s vacuum cleaner fitted with a new collection bag. Molds belonging to the Deuteromycota were the most frequent group detected in dust (59.4% of the total fungi). Penicillium (1.2 × 104 CFU/g), Phoma (7.7 × 103 CFU/g) and Cladosporium (7.0 × 103 CFU/g ) were frequently isolated and revealed important concentrations in the dust samples. Fungal colony counts in the house dust were higher in spring and midsummer, the peak periods being March-April and August. 2. Fungal propagules of 98 house dust samples from asthmatic patients, dwellings were compared by three isolation methods: dilution plate,ethanol pretretment and Warcup & Bakers ′ methods. Hydrophilic and mesophilic Deuteromycota in which growth can develop only at water activity values (Aw) greater than 0.80 were detected on PDA by dilution plating,while the microfungi developed on DG-18 plates by the same method were xerophilic and xerotolerant. The latter were Eurotium spp., Aspergillus restrictus and Wallemia sebi. After the ethanol treatment, Chaetomium spp. and Nigrospora oryzae were predominantly encountered in the same samples. The application of Warcup & Bakers′ method (treatment with ethanol and heating at 60°C) resulted the isolation of heatresistant Ascomycota such as species of Talaromyces, Eurotium, Nigrospora and Eupenicillium. 3. To study moisture contents of house dust and fungal growth relations,laboratory experiments under fiver levels of water activity (Aw 0.901, 0.807, 0.752, 0.708 and 0.577) at steady-state temperture of 25°C. In the total counts on PDA plate,there was mostly a decrease in the numbers of viable fungi in the Aw 0.807, 0.752, 0.708 and 0.577 chambers. However, at Aw 0.901, the number of fungi increased rapidly until three months. In the total counts on DG-18 plate, the experiments at Aw 0.807 to 0.752 show a remarkable growth of xerophilic fungi such as Aspergillus restrictus, Eurotium spp. and Wallemia sebi. In the experiments,high moisture count (Aw 0.901) was shown to be an important factor for the growth of Aspergillus versicolor. On the other hand, the optimal growth of Aspergillus restrictus had been observed on the house dust samples in Aw 0.752 or even 0.708 chambers.
Human dwellings are seldom sources of invasive mycotic disease in healthy persons but may harbour microfungi capable of causing opportunistic infection disease. Proliferation of microfungi in indoor environments may be connected to invasive fungal disease in an immunocompromised patient, especially in the aged person. With the exception of pathogenic yeasts, the most important agents of mycotic infection in Japan are dermatophytes, some Aspergillus species, some species of the Mucoraceae, and phaeohyphomycetes such as Exophiala species. From our recent papers, case reports of fatal Aspergillus and Fusarium infections of hospitalized patients included other outpatient with aspergillosis in the hospital are focused.
Human health damage by exposure of toxic chemicals in indoor environments is called as “sick house syndrome” or “chemical super-sensitivity”, which is caused by toxic principles with low or detectable concentrations. Recently, this disease has gradually increased in Japan. The causal toxic chemicals are considered to come from several house materials, such as wallpaper, ceiling, curtains, carpets, furniture, electrical appliances, and so on. Thus, the Minister of Health, Labour and Welfare manages to introduce air quality guidelines for indoor air pollutants in order to prevent human health damage from such toxic chemicals.
Effects of the revised sampling plans for the aflatoxin (AF) inspection of raw shelled peanuts and pistachios were examined by statistical evaluation. Instead of the present method for preparing a single sample that is composed of 1 kg from a lot, the multi-sub-sampling plans were prepared as follows. For small kernel peanuts, three 1 kg sub-samples from a lot imported from countries where low incidence of AF contamination might be expected to occur, and five 1 kg sub-samples from a lot imported from countries where high incidence of AF contamination might be expected to occur were prepared. If all of the 3 or 5 subsamples are not over the regulation level of AFB1 (10 μg/kg), the lot should be accepted. For large kernel peanuts, 2 kg of sub-samples were prepared as the same manner of small kernel peanuts. The detoxifying percentage (DP), the consumer′s risk (CR), and the processor′s risk (PR) of these multisub- sampling plans were evaluated by Whitaker′s negative binominal distribution method. Statistical indexes were estimated at 56.2% (DP), 3.5% (CR) and 6.1% (PR) in the case of 3 sub-sampling plan, and were 66.6%, 2.0% and 9.3%, respectively, in the case of 5 sub-sampling plan. For the inspection of pistachios from Iran, the present method of 8 sub-sampling method should be kept because of its maximum limit as routine work. Furthermore, it is necessary to harmonize the sampling methods in the National government (Mycotoxin Research Association, MRA) and the local governments. In the case of the inspection by local governments, the inspected result should be effective on limited part of the lot presenting at markets or food shops, because sampling size are smaller than MRA. If a large scale of the lot is existent, the local governments should carried out the multi-sub-sampling plans like as MRA.
The Japanese official method for analysis of aflatoxins, Kanshoku No. 128, requires large volumes of solvents, is time consuming, and performed by silica gel column chromatography (column clean-up method). Therefore, to clean-up and measure aflatoxins, thin layer chromatography (TLC clean-up method) was included in the modified Kanshoku No. 128. In this study, TLC and column clean-up method were compared with respect to treatment time, clean-up effect and the volumes of solvents for aflatoxins naturally contaminating whole lentils, white millet flour, buckwheat flour and raw shelled peanuts. The concentrations of aflatoxins were higher with the TLC clean-up method than with the column clean-up method for all samples. Recovery (%, N=3) of aflatoxin B1 added to non-contaminated samples (whole lentils, white millet flour, buckwheat flour and raw shelled peanuts) was 91.3% - 96.6% and 88.6 % - 93.6 % with the TLC and column clean-up method, respectively. The volume of solvent used with TLC and column clean-up method per sample was 50.5 ml and 652.5 ml, and the treatment time was 77 min.- 87 min. and 102 min. -125 min. respectively.