Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3537 in relation to aflatoxin production

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.     

Nitrogen metabolism in Aspergillus parasiticus NRRL 3240 and A. flavus NRRL 3537 in relation to aflatoxin production

The relationship between nitrogen assimilation, metabolism and aflatoxin formation has been investigated in a toxigenic and a non-toxigenic strain of Aspergillus parasiticus. Ammonia from the medium is mainly assimilated via NADP-requiring glutamate dehydrogenase. During growth NAD-requiring glutamate dehydrogenase followed an inverse pattern of activity with respect to NADP glutamate dehydrogenase. Alpha-ketoglutarate, the product of NAD glutamate dehydrogenase, stimulated acetate incorporation into aflatoxins. Glutamine synthetase, ornithine transcarbamylase, both utilizing glutamate as substrate were assayed under different growth conditions. An important regulatory role for glutamine synthetase is suggested. The metabolic route of asparagine utilization was also investigated. Both the known pathways, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase are operative simultaneously.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucroselow salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Pyridine nucleotides and redox state regulation of aflatoxin biosynthesis in Aspergillus parasiticus NRRL 3240

In vivo regulation of lipid and aflatoxin biosynthesis by pyridine nucleotides and their derived functions was studied in Aspergillus parasiticus NRRL 3240. Aflatoxins, total lipids and pyridine nucleotide content were estimated under different growth conditions. Aflatoxin formation was highest in cultures grown in sucrose-low salts medium followed by asparagine- and zinc-deficient media. The lipid content of the cultures followed an inverse pattern. The levels of oxidized nucleotides decreased with age under all culture conditions employed. Concentrations of NADPH peaked before the onset of aflatoxin biosynthesis. For each medium used, the estimated catabolite reduction charge was constant at all stages of growth whereas the anabolic reduction charge varied. A direct relationship between the level of extracellular ammonium ions and anabolic reduction charge was established. A high anabolic reduction charge was associated with increased lipid biosynthesis rather than aflatoxin biosynthesis.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Inhibition of aflatoxin production by trifluoperazine in Aspergillus parasiticus NRRL 2999

Trifluoperazine, an anti-calmodulin agent, inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999, without affecting the growth significantly. Culturing the organism for 3 days in the presence of 0.14mm trifluoperazine resulted in a generalized decrease in the production of all aflatoxins; the production of aflatoxin B1, a potent hepatocarcinogen, was inhibited to 88% under such conditions. Culturing 7-day-old preformed cultures in the presence of higher concentrations of trifluoperazine (>1mm) completely abolished production of all aflatoxins including AFB1. The inhibitory influence of trifluoperazine on aflatoxin production was accompanied by calmodulin-dependent phosphorylation of an 85kDa cytoplasmic calmodulin-binding protein. While the functions of calmodulin in mediating primary events of germination, growth and differentiation in fungi have earlier been reported, the present results indicate a possible role for calmodulin in the production of fungal toxins.     

Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus

Six isolates of Bacillus pumilus were tested for their ability to inhibit aflatoxin production of Aspergillus parasiticus NRRL 2999 in yeast extract sucrose (YES) broth. Aflatoxin production was inhibited in both simultaneous and deferred antagonism assays, suggesting that the inhibitory activity was due to extracellular metabolite(s) produced in cell-free supernatant fluids of cultured broth. The inhibition was not due to organic acids or hydrogen peroxide produced by B. pumilus since the inhibitory activity was not lost after pH adjustment or treatment of supernatant fluids with catalase. A range of media tested for the production of inhibitory metabolite(s) in supernatant fluids showed that all media supported bacterial growth and production of the metabolite(s). The metabolite(s) were produced over a wide range of temperature (25 to 37°C) and pH (4 to 9) of growth of B. pumilus. They were stable over a wide range of pH (4 to 10) and were not inactivated after autoclaving at 121°C for 30 minutes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of blastospores in protecting Aspergillus parasiticus NRRL 3240 from high levels of aflatoxins.

The role of blastospores in the protection of Aspergillus parasiticus from high levels of aflatoxins was studied. The strain protects itself from aflatoxicity by forming thick-walled blastospores. The formation of blastospores was not observed under conditions of reduced aflatoxin formation, e.g., under zinc and asparagine deficiencies. The germination of blastospores coincided with an increase in the specific activity of glutamate dehydrogenase (NADP) and a simultaneous decrease in the specific aflatoxin production.     

Effect of miconazole on growth and aflatoxin production by Aspergillus parasiticus

At 5 M, miconazole prevented the growth of Aspergillus parasiticus Speare in a number of media. Sensitivity to miconazole was increased approximately 10-fold in a medium containing glycerol. At sub-inhibitory concentrations, miconazole stimulated aflatoxin synthesis on media which normally support toxin formation. Miconazole inhibited respiration and altered mitochondrial ultrastructure, suggesting that miconazole inhibits growth and stimulates aflatoxin production by depressing mitochondrial activity.     

Chemoprevention by thyme oils of Aspergillus parasiticus growth and aflatoxin production

The essential oils from Thymus eriocalyx and Thymus X-porlock obtained by hydrodistillation were analyzed by GC/MS. The major components of T. eriocalyx and T. X-porlock oils were thymol (63.8, 31.7%), beta-phellandrene (13.30, 38.7%), cis-sabinene hydroxide (8.1, 9.6%), 1,8-cineole (2, 1.7%), and beta-pinene (1.31, 2%), respectively. Antifungal activities of the oils were studied with special reference to the inhibition of Aspergillus parasiticus growth and aflatoxin production. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oils were determined. Static effects of the above oils against A. parasiticus were at 250 ppm and lethal effects of T. eriocalyx and T. X-porlock were 500 and 1000 ppm of the oils, respectively. Aflatoxin production was inhibited at 250 ppm of both oils with that of T. eriocalyx being stronger inhibitor. Transmission electron microscopy (TEM) of A. parasiticus exposed to MIC level (250 ppm) of the oils showed irreversible damage to cell wall, cell membrane, and cellular organelles. It is concluded that the essential oils could be safely used as preservative materials on some kinds of foods at low concentrations to protect them from fungal infections.     

Requirement of Ca2+ for aflatoxin production: inhibitory effect of Ca2+ channel blockers on aflatoxin production by Aspergillus parasiticus NRRL 2999

Aflatoxin production by Aspergillus parasiticus NRRL 2999 was inhibited when Ca2+ channel blockers, i.e., verapamil and diltiazem (> 1 mmol 1(-1)), were included in the culture medium. Inhibition was not accompanied by growth inhibition, nor was the [14C]-glucose uptake by the organism altered. However, both the compounds inhibited [14C]-acetate incorporation into aflatoxin B1 in a dose-dependent manner and decreased sporulation of the organism. Even though a nutritional role for Ca2+ has not been demonstrated unequivocally in fungi, the present study suggests the importance of Ca2+ in the production of these secondary metabolites.     

Decrease of growth and aflatoxin production in Aspergillus parasiticus caused by spices

Non-commercial spices and herbs Tetrapleura tetrapetra, Triumfetta cordifolia, Garcina kola, Monodora myristica and Xylopia aethiopica at 0.08 to 0.32% (w/v) decreased the mycelial weight of Aspergillus parasiticus NRRL 2999 in yeast extract/sucrose broth by up to 68%. Aflatoxin production, monitored with ELISA, was most effectively decreased, from 97 to 23 g/ml, when the extract of G. kola was added at 0.32% (w/v).     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

Factors affecting aflatoxin production by Aspergillus parasiticus in a chemically defined medium

The optimum levels of sucrose, (NH4)2SO4, MgSO4, KH2PO4 and ZnSO4 for aflatoxin production in a chemically defined medium have been established. The last two were found to be essential for fungal growth and aflatoxin production. The effect of various carbon sources on aflatoxin production was tested using the defined medium. Asparagine was found to be essential for aflatoxin production. Very little aflatoxin was produced in the absence of asparagine with any of the other inorganic nitrogen sources tested. Supplementation with yeast extract, Casamino acids, Casitone and peptone increased the aflatoxin yield, but omission of asparagine led to decreased aflatoxin yields even when complex nitrogen sources were present. Asparagine could be replaced by aspartic acid or alanine.     

Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus

The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.     

Effect of ultraviolet light irradiation on viability and aflatoxin production by Aspergillus parasiticus

The effect of ultraviolet light irradiation (254 nm) on both the viability and the aflatoxin-producing ability of the fungus Aspergillus parasiticus NRRL 2999, a good aflatoxin-producing strain, was studied. This strain showed noticeable resistance and irradiation for more than 10 min was necessary to reduce survival to under 10%, while the white mutants were more susceptible (5 min of irradiation reduced survival to under 1%). Induction of mutants with complete loss of aflatoxigenicity was rare and only 3 of the 1463 survivors tested were aflatoxinless.