Chemical Constituents from Endophytic Fungus Annulohypoxylon cf. stygium in Leaves of Anoectochilus roxburghii.

The chemical investigation on endophytic fungus Annulohypoxylon cf. stygium in leaves of Anoectochilus roxburghii (Wall.) Lindl. has been performed. Sixteen compounds were isolated and their structures were identified as (-)-notoamide A, (-)-notoamide B, (+)-versicolamide B, notoamide C, notoamide D, stephacidin A, sterigmatocystin, dihydrosterigmatocystin, secosterigmatocystin, versiconol, averufanin, kipukasin D, kipukasin E, diorcinal, palmarumycin CP2 and (-)-(3R)-mellein methyl ether, respectively, by spectroscopic analysis and comparison with literature data. All the compounds were isolated from Annulohypoxylon genus for the first time. Sterigmatocystin and palmarumycin CP2 showed selective cytotoxic activities against HepG2, HeLa, MCF-7 and HT-29.

Unique Aggregation of Sterigmatocystin in Water Yields Strong and Specific Circular Dichroism Response Allowing Highly Sensitive and Selective Monitoring of Bio-Relevant Interactions.

We demonstrated the hitherto unknown property of the mycotoxin sterigmatocystin (STC) to provide homogeneous solutions in aqueous medium by forming a unique aggregate type (not formed by analogous aflatoxins), characterized by exceptionally strong circular dichroism (CD) bands in the 300-400 nm range. Results showed that these CD bands do not originate from intrinsic STC chirality but are a specific property of a peculiar aggregation process similar to psi-DNA CD response. Transmission electron microscopy (TEM) experiments revealed a fine fiber network resembling a supramolecular gel structure with helical fibers. Thermodynamic studies of aggregates by differential scanning calorimetry (DSC) revealed high reversibility of the dominant aggregation process. We demonstrated that the novel STC psi-CD band at 345 nm could be applied at biorelevant conditions (100 nanomolar concentration) and even in marine-salt content conditions for specific and quantitative monitoring of STC. Also, we showed that STC strongly non-covalently interacts with ds-DNA with likely toxic effects, thus contrary to the previous belief requiring prior enzyme epoxidation.

Three new sterigmatocystin analogues from marine-derived fungus Aspergillus versicolor MF359.

During the systematic screening of active compounds from marine-derived fungi, the extract of a strain of Aspergillus versicolor MF359 isolated from a marine sponge of Hymeniacidon perleve was identified for detailed chemical investigation. Three new secondary metabolites, named hemiacetal sterigmatocystin (1), acyl-hemiacetal sterigmatocystin (2), and 5-methoxydihydrosterigmatocystin (3), together with a known compound, aversin (4), were characterized. 1 represents a first structure of sterigmatocystin hemiacetal from nature. The antibacterial activities of these identified compounds were evaluated against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa. Compound 3 showed activity against S. aureus and B. subtilis with MIC values of 12.5 and 3.125 µg/mL, respectively.

Metabolic detoxification pathways for 5-methoxy-sterigmatocystin in primary tracheal epithelial cells.

1. The health effects of inhaled mycotoxins remain poorly documented despite their presence in bioaerosols. 5-methoxy-sterigmatocystin is produced in association with sterigmatocystin by some Aspergillus spp., sometimes in larger amounts than sterigmatocystin. Whereas sterigmatocystin can be metabolized through cytochromes P450 (CYP), UDP-glucuronosyltransferases and sulfotransferases in airway epithelial cells, little is known about 5-methoxy-sterigmatocystin. 2. The 5-methoxy-sterigmatocystin metabolites were analyzed using human recombinant CYP and porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. The induction of xenobiotic-metabolizing enzymes was examined by real-time quantitative PCR for mRNA expression and 7-ethoxyresorufin O-deethylation activity. 3. CYP1A1 metabolized 5-methoxy-sterigmatocystin into hydroxy-nor-methoxy-sterigmatocystin, nor-methoxy-sterigmatocystin and dihydroxy-methoxy-sterigmatocystin. CYP1A2 led to monohydroxy-methoxy-sterigmatocystin. In PTEC, 5-methoxy-sterigmatocystin metabolism resulted into a glucuroconjugate of 5-methoxy-sterigmatocystin, a sulfoconjugate and a glucuroconjugate of monohydroxy-methoxy-sterigmatocystin. The exposure of PTEC for 24 h to 1 µM 5-methoxy-sterigmatocystin induced a significant increase in the mRNA levels of CYP1A1, without significant induction of the 7-ethoxyresorufin O-deethylation activity. 4. These data suggest that 5-methoxy-sterigmatocystin is mainly detoxified in airway cells through conjugation, as sterigmatocystin. However, while CYP produced a reactive metabolite of sterigmatocystin, no such metabolite was detected with 5-methoxy-sterigmatocystin. Nevertheless, 5-methoxy-sterigmatocystin increases the CYP1A1 mRNA levels. The long-term consequences remain unknown.

[Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups].

Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were identified determined by enzyme immunoassay enzyme-linked immunosorbent assay. The following mycotoxins were identified found in these lichens in a broad concentration range with a frequency of 70-100%: sterigmatocystin (7-2090 ng/g), alternariol (20-6460 ng/g), and emodin (45-94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B1 was detected in only six species with a frequency of 2-42%, whereas roridin A was identified present in 10% of Hypogymnia physodes samples.

Contribution of uniformly 13C-enriched sterigmatocystin to the study of its pulmonary metabolism.

Mycotoxins are secondary metabolites of filamentous fungi which can cause a wide range of systemic effects. Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present, associated with air-borne particles. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Because its chemical structure is close to that of the aflatoxins, we studied its metabolism and its cellular consequences when in contact with the airway epithelium, using the mass spectral signature from the 10% (13)C uniformly enriched sterigmatocystin. The metabolism was studied in vitro, using recombinant cytochrome P450s enzymes, and in porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. The metabolites were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry detection. Expressed enzymes and PTECs were exposed to uniformly (13)C-enriched sterigmatocystin to confirm the relationship between sterigmatocystin and its metabolites because this isotopic cluster shape is conserved for all metabolites and their product ions. Incubation of sterigmatocystin with recombinant cytochrome P450 1A1 led to the formation of three metabolites identified as monohydroxysterigmatocystin, dihydroxysterigmatocystin and one glutathione adduct, the latter after the formation of a transient intermediate. In the PTEC cultures, sterigmatocystin metabolism resulted in a glucuro-conjugate. Two other products were detected, a sulfo-conjugate and a glucuro-conjugate of hydroxysterigmatocystin upon cytochrome P450 1A1 induction. This is the first study to report sterigmatocystin metabolism in airway epithelium, and it suggests that, contrary to the aflatoxins, sterigmatocystin is mainly detoxified into its conjugates and is unable to produce significant amounts of reactive metabolites in respiratory cells, at least in pigs.

Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus.

In aflatoxin biosynthesis, aflatoxins G(1) (AFG(1)) and B(1) (AFB(1)) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB(1), and conversion of HOMST to AFB(1) was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG(1) formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG(1) as well as AFB(1). In cell-free systems, HOMST was converted to AFG(1) when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG(1) as well as AFB(1), and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG(1).

Larvicidal activity of metabolites from the endophytic Podospora sp. against the malaria vector Anopheles gambiae.

In a screening for natural products with mosquito larvicidal activities, the endophytic fungus Podospora sp. isolated from the plant Laggera alata (Asteraceae) was conspicuous. Two xanthones, sterigmatocystin (1) and secosterigmatocystin (2), and an anthraquinone derivative (3) 13-hydroxyversicolorin B were isolated after fermentation on M(2) medium. These compounds were characterised using spectroscopic and X-ray analysis and examined against third instar larvae of Anopheles gambiae. The results demonstrated that compound 1 was the most potent one with LC(50) and LC(90) values of 13.3 and 73.5 ppm, respectively. Over 95% mortality was observed at a concentration 100 ppm after 24 h. These results compared farvorably with the commercial larvicide pylarvex® that showed 100% mortality at the same concentration. Compound 3 was less potent and had an LC(50) of 294.5 ppm and over 95% mortality was achieved at a concentration of 1,000 ppm. Secosterigmatocystin (2) revealed relatively weak activity and therefore LC values were not determined.

[The secondary metabolites of the HS-3 Alternaria sp. fungus associated with holothurians].

OBJECTIVE: The metabolites of HS-3 associated with holothurians were studied, which was identified by molecular biology as Alternaria sp.. METHODS: The holothurians were gathered from the Sea of Zhifu Islet, Shandong Province. HS-3 Alternaria sp. was culternitived in potato medium, and four compound was got by TLC, chromatography and HPLC, and 1-hydroxyl-3-methylanthracene-9,10-dione (1), chrysophanol (2), sterigmatocystin (3) and cerebroside (4) were elucated by modern spectrum. CONCLUSION: All of this provides scientific data for further study of holothurians, and the four coumpouns are isolated from the microbe associated with holothurians for the first time.

Cytotoxic effects of individual and combined sterigmatocystin and nivalenol on liver hepatocellular carcinoma cells.

Since humans are exposed to different mycotoxins through daily intake, there is increasing concern about the adverse effects of the interactions between them. Cytotoxicity of sterigmatocystin (STE) and nivalenol (NIV) alone and in combination in human hepatocarcinoma (HepG2) cells was evaluated by MTT assay. Furthermore, ROS production and alteration of ΔΨm as mechanisms of action were assessed. Cells were treated with concentrations ranging from 0.15 to 5 µM for NIV and from 0.78 to 50 µM for STE individually and in binary combinations. The combination ratio between the mixture STE + NIV was 10:1. The IC50 values of NIV ranged from 0.96 to 0.66 µM, whereas no IC50 values were obtained for STE at any time tested. For the combinations studied, synergistic, antagonistic and addictive effects were obtained with the two type of analyses performed, the isobologram analysis and the Combenefit method. No relevant effects on ROS and ΔΨm were observed. In conclusion, predictive models based on combination data could help to better understand the interactions between mycotoxins and their implications in food safety assessment. However, a further analysis of the molecular mechanism underlying these interactive effects is required.

Aflatoxin B1 and Sterigmatocystin Binding Potential of Lactobacilli.

Due to global climate change, mould strains causing problems with their mycotoxin production in the tropical-subtropical climate zone have also appeared in countries belonging to the temperate zone. Biodetoxification of crops and raw materials for food and feed industries including the aflatoxin B1 (AFB1) binding abilities of lactobacilli is of growing interest. Despite the massive quantities of papers dealing with AFB1-binding of lactobacilli, there are no data for microbial binding of the structurally similar mycotoxin sterigmatocystin (ST). In addition, previous works focused on the detection of AFB1 in extracts, while in this case, analytical determination was necessary for the microbial biomass as well. To test binding capacities, a rapid instrumental analytical method using high-performance liquid chromatography was developed and applied for measurement of AFB1 and ST in the biomass of the cultured bacteria and its supernatant, containing the mycotoxin fraction bound by the bacteria and the fraction that remained unbound, respectively. For our AFB1 and ST adsorption studies, 80 strains of the genus Lactobacillus were selected. Broths containing 0.2 µg/mL AFB1and ST were inoculated with the Lactobacillus test strains. Before screening the strains for binding capacities, optimisation of the experiment parameters was carried out. Mycotoxin binding was detectable from a germ count of 107 cells/mL. By studying the incubation time of the cells with the mycotoxins needed for mycotoxin-binding, co-incubation for 10 min was found sufficient. The presence of mycotoxins did not affect the growth of bacterial strains. Three strains of L. plantarum had the best AFB1 adsorption capacities, binding nearly 10% of the mycotoxin present, and in the case of ST, the degree of binding was over 20%.

Cottoquinazoline A and cotteslosins A and B, metabolites from an Australian marine-derived strain of Aspergillus versicolor.

An Australian marine-derived isolate of Aspergillus versicolor (MST-MF495) yielded the known fungal metabolites sterigmatocystin, violaceol I, violaceol II, diorcinol, (-)-cyclopenol, and viridicatol, along with a new alkaloid, cottoquinazoline A (1), and two new cyclopentapeptides, cotteslosins A (2) and B (3). Structures for 1-3 and the known compounds were determined by spectroscopic analysis. The absolute configurations of 1-3 were addressed by chemical degradation and application of the C(3) Marfey's method. The use of "cellophane raft" high-nutrient media as a device for up-regulating secondary metabolite diversity in marine-derived fungi is discussed. The antibacterial properties displayed by A. versicolor (MST-MF495) were attributed to the phenols violaceol I, violaceol II, and diorcinol, while cotteslosins 2 and 3 were identified as weak cytotoxic agents.

Selective inhibition of acyl-CoA:cholesterol acyltransferase 2 isozyme by flavasperone and sterigmatocystin from Aspergillus species.

Five known fungal metabolites, aurasperone A, aurasperone D, averufanin, flavasperone and sterigmatocystin, were isolated from the culture broths of Aspergillus species as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT) in the cell-based assay using ACAT1- and ACAT2-expressing CHO cells. These compounds share a similar polycyclic skeleton. Among them, flavasperone and sterigmatocystin, having an angular skeleton, showed selective inhibition toward ACAT2 isozyme, while the others having a linear one had no selectivity in inhibition.

Aflatoxin B1 and sterigmatocystin in wheat and wheat products from supermarkets in China.

Wheat is an important cereal but it is often contaminated with mycotoxins. The natural occurrence of aflatoxin B1 (AFB1) and sterigmatocystin (STC) was determined in 178 food samples (32 wheat samples and 146 wheat products) purchased from Chinese supermarkets. The methodology was validated, the wheat and wheat products samples were treated with a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). From these samples 18.8% of wheat and 8.2% of cracker samples were contaminated with AFB1. Mean levels were 0.06 µg/kg and 0.05µg/kg, respectively. There was no AFB1 contamination in white bread or whole meal bread. Meanwhile 53.1% of wheat, 59.2% of crackers, 20.8% of white bread and 16% of whole meal bread samples were contaminated with STC. The mean levels were 0.07, 0.79, 0.12 and 0.12 µg/kg respectively. Although the levels were low, this demonstrates the need for more comprehensive surveys for these two mycotoxins in wheat and wheat products from China.

A novel biosensor for sterigmatocystin constructed by multi-walled carbon nanotubes (MWNT) modified with aflatoxin-detoxifizyme (ADTZ).

Sterigmatocystin, ST, is carcinogenic mycotoxin with toxicity second to aflatoxins, contaminated in foods- and feeds-stuff widely. A three-electrode system was employed to examine the response character of the covalently united ADTZ-MWNTs electrode to ST, and the results indicated that an oxidation peak of ST was observed at about +400 mv, the linear detection range of ST was from 4.16 x 10(-5) mg/ml (0.13 microM) to 1.33 x 10(-3) mg/ml (4.29 microM) with the detection limit at 0.13 microM. Compared to the corresponding results obtained from the MWNTs modified electrode that ADTZ was directly sediment (adsorbed) on it, the sensitivity of ours had been improved by two orders of magnitude, which could provide some important data to further research.

Adsorption of sterigmatocystin by montmorillonite and inhibition of its genotoxicity in the Nile tilapia fish (Oreachromis nilaticus).

Sterigmatocystin (Stg) is closely related to the mycotoxin aflatoxin as a precursor in aflatoxin biosynthesis and classified as an IARC Group-2B carcinogen. The aim of this study was to investigate the efficacy of Egyptian montmorillonite (EM), a clay mineral, to adsorb Stg, to test the stability of the resulting complex under different conditions in vitro, and to utilize the Nile tilapia fish as an in vivo model to evaluate the protective effect of EM against Stg-induced toxicity and clastogenicity. In the in vitro study, four concentrations of EM (0.5, 1, 2 and 4 mg/L aqueous solution) and three concentrations of Stg (5, 10 and 50 microg/ml) were tested. The results show that EM had a high capacity of adsorbing Stg at different concentrations tested. The adsorption ranged from 93.1 to 97.8% of the available Stg in aqueous solutions. The complex was stable at different pHs at 37 degrees C in different organic solvents. An in vivo experiment was conducted to evaluate the ability of EM to prevent the toxicity and chromosomal aberrations induced by Stg in the Nile tilapia fish. Fish received an intragastric dose of EM in corn oil (0.5 mg/kg bw) with or without Stg (1.6 microg/kg bw) twice a week for 4 weeks. Body weight was recorded during dosing, and blood and tissue samples were collected at the end of treatment. Stg residues were determined in fish tissue. The results show that Stg was toxic and clastogenic to fish as indicated by the significant decrease of body weight and the increase in frequencies of micronucleated red blood cells (MN RBC) and chromosomal aberrations in the kidney. The intragastric administration of EM combined with Stg to fish resulted in a reduction of the number of MN RBC and the frequency of chromosomal aberrations in the kidney compared with the group treated with Stg alone. It could be concluded that EM itself was safe and successful in the prevention of Stg toxicity and clastogenicity.

The furofuran-ring selectivity, hydrogen peroxide-production and low Km value are the three elements for highly effective detoxification of aflatoxin oxidase.

AFO (aflatoxin oxidase), an enzyme from Armillariella tabescens previously named aflatoxin detoxifizyme, exhibits oxidative detoxification activity toward aflatoxin B1 and sterigmatocystin. Bioinformatics reveals that AFO is a newly discovered oxidase because AFO does not share any significant similarities with any known oxidase. It is critically important to understand how AFO acts on aflatoxin B1. In this study, in addition to aflatoxin B1 (AFB1) and sterigmatocystin (ST), five other chemicals that have furan or pyran structures were investigated. The results indicated that in addition to AFB1 and ST, AFO is also able to act on versicolorin A, 3,4-dihydro-2H-pyran and furan. These results suggested that 8,9-unsaturated carboncarbon bond of aflatoxin B1 is the potential reactive site for AFO. Further findings indicated that the action of AFO is oxygen-dependent and hydrogen peroxide-producing. The simultaneously produced-hydrogen peroxide possibly plays the essential role in detoxification of AFO. In addition, the extremely low Km value of 0.33 µmol/l for AFO-AFB1 and 0.11 µmol/l for AFO-ST signifies that AFO is highly selective for AFB1 as well as ST.

Solubility and stability of sterigmatocystin in different organic solvents.

In the present work we studied the solubility of sterigmatocystin (ST) in different organic solvents and the stability of these solutions during cold and frozen storage. ST was more soluble in chloroform and in pyridine. In cold storage ST was more stable in chloroform after both 7 and 30 days. In pyridine, ST was stable on day 7 but not on day 30. In frozen storage, 90% recovery was achieved only in chloroform. We conclude that chloroform is the most suitable organic solvent of those studied to solubilize and store standard solutions of ST.

Biological reactive intermediates of bisfuranoid mycotoxins.

Based on the mode of action of AFB1 and the activities of its biologically active intermediates, one may conclude that: 1. The mode of toxic action of the bisfuranoid mycotoxin is through epoxidation of the vinyl ether double bond of their dihydrobisfuran functionality. 2. The DNA and plasma albumin adducts formed in vivo may be useful in the molecular dosimetry of these environmental carcinogens. 3. Monitoring of these adducts of AFB1 in biological samples so far indicates that aflatoxin is likely involved in the etiology of human liver cancer.

Production and characterization of sterigmatocystin.

Fourteen strains of Aspergillus versicolor and 2 strains of A. nidulans were screened for sterigmatocystin (ST) production on a semi-synthetic solid substrate by high performance liquid chromatography (HPLC) analysis. Two strains of A. versicolor producing ST at 550.5 mg.kg-1 substrate and 1160.8 mg.kg-1 substrate were selected to inoculate 4 kg solid ST-producing media. After 30 days stationary incubation at 28 degrees C in the dark, 2271.6 mg of pale-yellow needle-shaped crystals were isolated and purified from the culture with a procedure applying column chromatography and recrystallization method. The crystal was proved to be sterigmatocystin by spectroanalysis and some physico-chemical analysis. The purity of the final material obtained were more than 99.9% as shown by HPLC and TLC detection. With this procedure, ST was obtained at about one tenth of its commercial cost.