In the course of searching for new antifungal substances in Malbranchea spp., two new furanone glycosides, malfilamentosides A (1) and B (2), were isolated from Malbranchea filamentosa along with a furanone derivative, 4-benzyl-3-phenyl-5H-furan-2-one (3) and a dioxopiperazine derivative, amauromine, and an anthraquinone, erythroglaucine. The structures of 1 and 2 were established by spectroscopic and chemical investigation. Malfilamentosides A (1) and B (2) are the first examples of the glycosides of the furanone derivative from the fungal sources.
A method of analysis for determination of aflatoxins (AFs) in chocolate was optimized and evaluated its within laboratory precision. The method uses acetonitrile-methanol-water (60+10+40, v/v/v) as extraction solvent and an immunoaffinity column (IAC) for the cleanup, and HPLC with fluorescence detection for the determination of AFs. The method was validated by testing replicate analyses of the finally ground chocolate samples spiked at 0.1 and 10.0 μg/kg for each AFB1, AFB2, AFG1 and AFG2. The average recovery of AFs at 0.1 and 10.0 μg/kg spiking levels ranged from 90 to 97 % for all AFs. The relative standard deviation (RSD) for within day and between day ranged from 1.7 to 3.3 %, and 0 to 4.1 % for all AFs, respectively. The HorRat for the total RSD at each level was within 0.2 for all AFs. These results show that this method is reliable for the analysis of AFs in chocolate in the range of 0.1 and 10.0 μg/kg.
Many human and animal diseases other than Ergotism, Alimentary Toxic Aleukemia and Turkey X Disease have been postulated to be caused by mycotoxins. Such diseases are represented by human hepatic cancer occurring in some regions in China, South-East Asia and Africa by intake of aflatoxin, acute aflatoxicoses in human and animals occurring in India and Kenya, ELEM (equine leuco-encephalomalacia) by fumonisin in South Africa and United State of America, human esophagal cancer by fumonisin in South Africa and China, and Balkan Endemic Nephropathy (BEN) and urinary tract tumor by ochratoxin A in Balkan area. In recent years, fumonisin has been postulated to be involved in human neural tube defect. However, such diseases described even in textbooks as mycotoxin induced diseases are not fully supported by epidemiological and toxicological evidence. Some of toxicity mechanisms of mycotoxins that are now accepted widely might be accepted with many unresolved problems, i.e. mechanisms of acute toxicity of aflatoxin in relation to its chemical structure, involvement of ochratoxin A in BEN and urinary tract tumor, a large difference in fumonisin toxicity among animals, etc.
Trichothecenes, such as deoxynevalenol (DON), nivalenol (NIV) and T-2 toxin are typical immunotoxic mycotoxins. Contamination of trichothecene mycotoxins in wheat is a serious world-wide problem affecting human health. In Japan, in particular, it has been reported that relatively high levels of DON and NIV are frequently found in domestic wheat. I have extensively conducted studies on the toxicity and control of trichothecene mycotoxins in order to assess their risk, and this paper is the summary of those findings.
A novel method for heteroduplex panel analysis (HPA) utilizing fragments of the PCR amplified ITS regions of rDNA was developed. The method involves formation of heteroduplexes with a set of reference fragments amplified from A. flavus, A. parasiticus, A. tamarii and A. nomius, and subsequent minislab gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1 and N-1), generated by pairwise reannealing among four reference fragments. Application of HPA to a field study demonstrated that a new genotype of FP-1 has been predominantly distributed throughout sugarcane field soil in the southernmost islands of Japan. The high prevalence of type FP-1 may reflect its adaptation to the soil environment. The type FP-1 isolates were also isolated from the sugarcane fields in Vietnam, but not find them in our culture collection derived from other sources. CBS 108.30 originated from sugarcane in Egypt was the only one strain identified as type FP-1. These results imply that type FP-1 may be associated with sugarcane fields in Asia. Type FP-1 is consisted of the morphologically intergrading isolates between A. parasiticus and A. flavus. The phylogenetic analysis based on the ITS sequences indicated that type FP-1 formed an independent clade positioned between A. parasiticus and A. flavus, and was more closely related to the former species. HPA also demonstrated that A. nomius has been distributed in sugarcane field soil and sugarcane stems in Japan and Vietnam. All of the tested isolates of A. nomius and type FP-1 were able to produce aflatoxins B and G. These fungi are probably responsible for aflatoxin contamination of crude sugar produced from sugarcane.
Stachybotrys chartarum as an indoor fungus and its macrocyclic trichothecene mycotoxins, satratoxins, are noted as possible etiological agents of illnesses associated with water-damaged buildings especially in USA. The following topics are overviewed briefly in this paper: 1) indoor fungi and infant pulmonary hemorrhage/hemosiderosis, 2) stachybotryotoxicoses in animals and the toxicity of satratoxins, and 3) a human intoxication case by poisonous mushroom, Podostroma cornu-damae, and satratoxins.
Various fungi are present in the indoor air. Many of them are known to produce mycotoxins, but the relation of the toxin of these fungi to human diseases is not well known. The only exception is the recent finding that gliotoxin produced by Aspergillus fumigatus play a significant role as a virulence factor in the development of the infection, i.e. aspergillosis. Stachybotrys chartarum is a ubiquitous fungus commonly found in our living environment. Although inhalation of the fungus has been suspected as a possible cause of acute idiopathic alveolar hemorrhage in infants, the definite relation is yet to be known. The fungus produces various secondary metabolites such as trichothecenes, we suspected that the repeated inhalation of the fungus may cause some damage to the human lung. To learn the effect of the long-term inhalation of the fungus, we repeatedly injected the spores of the fungus into mouse trachea. The histopathological examination of the mice disclosed the development of pulmonary hypertension. When isolates of S. chartarum with or without trichothecene production were used, the isolate with trichothecene production solely developed pulmonary hypertension, which fact suggests the possible role of the trichothecene in the development of the vascular changes. Further investigation is now under way.
Trichothecenes are a family of over 200 sesquiterpenoid mycotoxins produced by Fusarium, Stachybotrys, Myrothecium and other molds. Satratoxin G (SG) is a macrocyclic trichothecene mycotoxins produced by Stachybotrys chartarum, the "black mold" reported to illnesses associated with water-damaged buildings. We found that single instillation of macrocyclic trichothecenes SG or Roridin A (RA) exposure in C57Bl/6 female mice specifically targeted olfactory sensory neurons (OSNs) in the olfactory epithelium in the mouse nose. The OSN death was verified by imuunohistochemial staining of activated caspase-3, electronmicroscopic analysis, Real Time PCR method for the expression of pro-apoptotic genes. The results suggested that SG-induced OSN death was via apoptosis. The death of OSN caused the atrophy of the olfactory epithelium. The maximum atrophy of the epithelium was observed at 3 days after single instillation of SG (500 μg/kg bw). After 7 days postinstillation (PI), the thickness of the olfactory epithelium recovered partially with the recruitment of fresh OSNs in the epithelium. When the mice are instilled with lower doses of SG (100 μg/kg bw) for 5 consecutive days it also induces same degree of atrophy and apoptosis, suggesting that these effects are cumulative. SG also induced an influx of mucosubstances filled with neutrophils (neutrophilic rhinitis) in the airways at 24 hr PI. Proinflammatory cytokines and the chemokine genes were upregulated at 24 hr PI in both the ethmoid turbinates of the nasal airways and the adjacent olfactory bulb of the brain. Marked atrophy of the olfactory nerve and glomerular layers of the olfactory bulb was also detectable by 7 days PI along with mild neutrophilic encephalitis.