Metabolism of verruculogen in rats.

Radiolabeled verruculogen was detected in a wide range of body tissues 6 min after intravenous administration, but after a further 20 min it was mainly being excreted via the biliary route. In isolated liver perfusion, [14C]verruculogen was rapidly taken up by the liver and metabolized completely, principally to the related tremorgen TR-2 but also to a desoxy derivative of verruculogen. In addition, a smaller amount of an isomer of TR-2 was detected. These metabolic products were excreted in the bile.     

An intermediary role for the tremorgenic mycotoxin TR-2 in the biosynthesis of verruculogen.

14C-Labelled compound TR-2, a tremorgenic mycotoxin, was administered to Penicillium raistrickii in submerged fermentation. Half of the added radiolabel was taken up by the fungus during the 60 h incubation period and the secondary metabolites subsequently isolated, principally verruculogen but also fumitremorgin B, were found to be radiolabelled. The efficiency of biosynthetic incorporation of TR-2 into verruculogen within the mycelium was at least 35%, demonstrating for the first time an intermediary role for TR-2. Fumitremorgin B was also TR-2-derived but may not be an important intermediate in verruculogen biosynthesis.     

Tremorgenic Mycotoxin from Penicillium paraherquei

A tremorgenic mycotoxin was isolated from Penicillium paraherquei Abe ex G. Smith and identified as verruculogen. It was produced at the rate of approximately 1 mg/g of the dried fungal mycelium cultured on peptone-enriched Czapek-Dox medium at 28°C.     

Tremorgenic Toxin from Penicillium verruculosum

A new mycotoxin that produces severe tremors and acute toxicity when administered orally or intraperitoneally (ip) to mice and 1-day-old cockerels was obtained from a strain of Penicillium verruculosum Peyronel isolated from peanuts. The ip 50% lethal dose (LD(50)) of this tremorgen was 2.4 mg/kg in mice and 15.2 mg/kg in chickens. Orally administered LD(50) values for the toxin were 126.7 mg/kg in mice and 365.5 mg/kg in chickens. The trivial name "verruculogen" is proposed for this tremorgenic mycotoxin. Physical and chemical characteristics of the mycotoxin are described.     

Photoreaction of indole-containing mycotoxins to fluorescent products

Photochemical reaction of the non-fluorescent mycotoxin cyclopiazonic acid (CPA) to fluorescent products was recently reported. Because CPA contains an indole moiety, believed to contribute to the fluorescence, it was of interest to determine whether the effect might be more generally applicable to indole-containing mycotoxins. Three indole-containing tremorgens (penitrem A, paxilline, verruculogen) that have not previously been reported to be fluorescent were rendered fluorescent by exposure to ultraviolet light in a photoreactor. Naturally fluorescent ergot alkaloids, which also contain an indole-moiety, exhibited a diminished response after exposure. This suggests that the phenomenon may be most useful for detection of indole-containing tremorgens that are non-fluorescent, rather than for the enhancement of materials that are already fluorescent, such as the ergot alkaloids. The extent to which fluorescence enhancement was seen was strongly influenced by the reaction environment, in particular the solvent used and whether cyclodextrins were present. In an HPLC format, placement of the photoreactor post-column allowed for the fluorescence detection of penitrem A, paxilline, and verruculogen. The ability to photoreact indole-containing tremorgens and detect them by fluorescence may open up new avenues for detection of these mycotoxins alone or in combination.     

Tremorgenic Mycotoxins from Aspergillus Caespitosus

Two tremorgenic mycotoxins were isolated from Aspergillus caespitosus, and identified as verruculogen and fumitremorgin B. They were produced at the rate of 172 and 325 mg per kg, respectively, on autoclaved cracked field corn.     

Verruculogen Produced by soil fungi in England and Wales.

Soil fungi, including Aspergillus fischeri, Penicillium piceum, Penicillium nigricans, and Penicillium raistrickii, produced a tremorgenic toxin previously described as toxin X. Chemical analysis showed that this toxin was predominantly verruculogen.     

Structure of Porriolide,a New Metabolite from Alternaria porri

Compounds from a soil isolate of Penicillium sp. MG-11 showed remarkable convulsive activity against silkworms. Activity-based fractionation of an acetone extract of the fermentation products resulted in the isolation of four compounds. One was identified as verruculogen, which was an active principle. Another compound, acetoxydehydroaustin, was new and was structurally characterized by spectral data and a single-crystal X-ray analysis. The two other compounds were identified as dehydroaustin and austin on the basis of the spectral data. Although the latter three compounds were not active themselves, they enhanced the convulsive activity of verruculogen in a silkworm bioassay. Three additional compounds with no identifiable activity were also isolated. Two of them were identified as austinol and dehydroaustinol, and the third was determined to be a new related compound named neoaustin by an X-ray analysis of crystals.     

Tremorgenic mycotoxins from Aspergillus fumigatus as a possible occupational health problem in sawmills.

Wood-trimmers' disease, generally called extrinsic allergic alveolitis, which affects workers in sawmills, is thought to be caused by fungal diaspores. The importance of Aspergillus fumigatus on the surface of wood dried in kilns is accentuated by its ability to produce tremorgenic mycotoxins. Eight strains of A. fumigatus from five different sawmills were isolated and cultivated on liquid media, and one of the strains was also cultivated on wood blocks. Extracts were prepared, and the tremorgenic reactions were induced by oral administration of extracts to rats. Extracts of the strain grown in liquid medium and on wood blocks induced very strong tremorgenic reactions when administered orally to rats. Four other strains induced mild tremorgenic reactions. High-performance liquid chromatography analysis revealed two tremorgenic mycotoxins, verruculogen and fumitremorgen C, in the five toxic strains. One nontoxic strain produced detectable levels of verruculogen. These results, coupled with the known resemblance of the acutely toxic phase of wood-trimmers' disease to the symptoms produced by these tremorgens, imply that wood-trimmers' disease and similar occupational diseases are, at least in part, mycotoxicoses.     

Simple screening method for molds producing intracellular mycotoxins in pure cultures.

A simple screening method for molds producing the intracellular mycotoxins brevianamide A, citreoviridin, cyclopiazonic acid, luteoskyrin, penitrem A, roquefortine C, sterigmatocystin, verruculogen, viomellein, and xanthomegnin was developed. After removing an agar plug from the mold culture, the mycelium on the plug is wetted with a drop of methanol-chloroform (1:2). By this treatment the intracellular mycotoxins are extracted within seconds and transferred directly to a thin-layer chromatography plate by immediately placing the plug on the plate while the mycelium is still wet. After removal of the plug, known thin-layer chromatographic procedures are carried out. The substrate (Czapek yeast autolysate agar) and growth conditions (25 degrees C for 7 days) used by Penicillium taxonomists proved suitable for the production of the mycotoxins investigated when 60 known toxigenic isolates and 865 cultures isolated from foods and feedstuffs were tested with this screening method.     

Temporal separation of steps in the biosynthesis of verruculogen

A mixture of 2[3H] mevalonate and U[14C] proline, given to Penicillium simplicissimum on days 2, 3, 4, 5 or 6 of stationary liquid culture, gave rise to radiolabelled verruculogen in which the ratio of 3H to 14C measured on day 8 increased from 2.0:1 to 9.5:1 over the period of administration of radiolabelled precursors. This suggests temporal separation of the formation of the diketopiperazine moiety and its subsequent prenylation. The putative precursor diketopiperazine, cyclo-L-tryptophanyl-L-proline, was isolated from 21 day culture broth.     

Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity.

The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days. Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography. Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C. The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively. The production of fumitremorgin C by N. fischeri has not been previously reported. Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C. Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C). Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose. Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose. Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C.     

Tremorgenic mycotoxins from Aspergillus fumigatus as a possible occupational health problem in sawmills

Wood-trimmers' disease, generally called extrinsic allergic alveolitis, which affects workers in sawmills, is thought to be caused by fungal diaspores. The importance of Aspergillus fumigatus on the surface of wood dried in kilns is accentuated by its ability to produce tremorgenic mycotoxins. Eight strains of A. fumigatus from five different sawmills were isolated and cultivated on liquid media, and one of the strains was also cultivated on wood blocks. Extracts were prepared, and the tremorgenic reactions were induced by oral administration of extracts to rats. Extracts of the strain grown in liquid medium and on wood blocks induced very strong tremorgenic reactions when administered orally to rats. Four other strains induced mild tremorgenic reactions. High-performance liquid chromatography analysis revealed two tremorgenic mycotoxins, verruculogen and fumitremorgen C, in the five toxic strains. One nontoxic strain produced detectable levels of verruculogen. These results, coupled with the known resemblance of the acutely toxic phase of wood-trimmers' disease to the symptoms produced by these tremorgens, imply that wood-trimmers' disease and similar occupational diseases are, at least in part, mycotoxicoses.     

Meroterpenes from Penicillium sp found in association with Melia azedarach

A Penicillium sp was isolated from the root bark of Melia azedarach and cultivated over sterilized rice. After chromatographic procedures, two meroterpenes, named preaustinoid A and B, were obtained in addition to the known alkaloid verruculogen. Their structures were identified by extensive spectroscopic studies, and they exhibited moderate bacteriostatic effects on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus sp.     

Growth of and fumitremorgin production by Neosartorya fischeri as affected by temperature, light, and water activity

The effects of temperature, light, and water activity (aw) on the growth and fumitremorgin production of a heat-resistant mold, Neosartorya fischeri, cultured on Czapek Yeast Autolysate agar (CYA) were studied for incubation periods of up to 74 days. Colonies were examined visually, and extracts of mycelia and CYA on which the mold was cultured were analyzed for mycotoxin content by high-performance liquid chromatography. Growth always resulted in the production of the tremorgenic mycotoxins verruculogen and fumitremorgins A and C. The optimum temperatures for the production of verruculogen and fumitremorgins A and C on CYA at pH 7.0 were 25, 30, and 37 degrees C, respectively. The production of fumitremorgin C by N. fischeri has not been previously reported. Fumitremorgin production was retarded at 15 degrees C, but an extension of the incubation period resulted in concentrations approaching those observed at 25 degrees C. Light clearly enhanced fumitremorgin production on CYA (pH 7.0, 25 degrees C), but not as dramatically as did the addition of glucose, fructose, or sucrose to CYA growth medium (pH 3.5, 25 degrees C). Growth and fumitremorgin production was greatest at aw of 0.980 on CYA supplemented with glucose or fructose and at aw of 0.990 on CYA supplemented with sucrose. Growth and fumitremorgin production were observed at aw as low as 0.925 on glucose-supplemented CYA but not at aw lower than 0.970 on CYA supplemented with sucrose. Verruculogen was produced in the highest amount on all test media, followed by fumitremorgin A and fumitremorgin C.     

Production of the Tremorgenic Mycotoxins Verruculogen and Fumitremorgin B by Penicillium piscarium Westling

The tremorgenic mycotoxins verruculogen and fumitremorgin B were isolated from Penicillium piscarium Westling. The coexistence of these tremorgens in culture has previously been reported for one other unrelated fungal species, Aspergillus caespitosus Raper and Thom, and lends further support to the suggestion that the tremorgens have a common biosynthetic origin.     

Action of tremorgenic mycotoxins on GABAA receptor

The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on gamma-aminobutyric acid (GABAA) receptor binding and function in rat brain and on binding of a voltage-operated Cl- channel in Torpedo electric organ. None of the mycotoxins had significant effect on [3H]muscimol or [3H]flunitrazepam binding to the GABAA receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced 36Cl- influx and [35S] t-butylbicyclophosphorothionate [( 35S]TBPS) binding in rat brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of [35S]TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABAA receptor function by binding close to the receptor's Cl- channel. On the voltage-operated Cl- channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of [35S]TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABAA receptor function.     

Patulin produced by an Aspergillus clavatus isolated from feed containing malting residues associated with a lethal neurotoxicosis in cattle

A severe neurotoxicosis, comprising tremors, ataxia, paresis, recumbency and death, occurred simultaneously among several herds of beef cattle in the region of Flanders (Belgium). After a first multi-toxin screening of some suspected diet elements, verruculogen was detected in a sample of a common feed ingredient. However, when the first animal necropsies revealed serious nervous lesions, including neuronal degeneration of the central nervous system and axonal degeneration in the peripheral nervous system, further investigations focused on fungal isolation. As expected from the pathological lesions, Aspergillus clavatus was found to be the dominant fungal species in a sample of compacted fodder, containing malting residues, consumed by all the affected herds. The isolated fungus appeared to produce patulin in culture medium. Traces of patulin were also detected in the fodder. These findings and their possible role in the intoxication are discussed.     

Nonribosomal peptide synthetase genes pesL and pes1 are essential for Fumigaclavine C production in Aspergillus fumigatus

The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H(2)O(2), and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.     

Differentiation and secondary metabolism in some prokaryotes and fungi

Relationships between growth and differentiation and secondary metabolism in microorganisms are reviewed and discussed. The trophophase/idiophase concept is illustrated by patulin biosynthesis. Other features of the biosynthesis of secondary metabolites (such as β-lactams, mycophenolic acid, rubratoxin, enniatin, tentoxin and citrinin) are also presented. Examples of secondary metabolism in relation to differentiation of selected prokaryotes (bacteria and actinomycetes) are also given. The major part of the review concerns relationships of differentiation and secondary metabolite production in fungi. The following metabolites are discussed: cephalosporin C, brefeldin A, ergot alkaloids, solanopyrone phytotoxins, verruculogen, griseofulvin, penitrems, paxilline, patulin, anthraquinones, mycosporines, P-factor, cyclopenin, cyclopenol, zearalenone, and sex hormones of lower fungi (trisporic acid, antheridiol and sirenin). Extended and referenced version of a lecture given at the124th Meeting of the Society for General Microbiology, University of Kent at Canterbury, United Kingdom 5–7 January 1993.