Aflatoxin biosynthesis: detection of transient, acetate-dependent intermediates in Aspergillus by kinetic pulse-labeling.

A simple technique was developed for the detection of intermediary metabolites of Aspergillus versicolor that are putative precursors of aflatoxin. Minicolony populations were allowed to metabolize [1,2-14C]acetate over various time intervals. The biosynthetic reactions were quenched by quick-freezing the minicolonies, the cells were disrupted, and the metabolites were extracted into acetone. Small silica thin-layer chromatographic plates were then used to separate any radioactive metabolites present. Elution in two or three different directions was often necessary. Radioautography of the thin-layer chromatography plates provided a sensitive assay for the appearance of the various intermediates in a timing pattern which implicated the sequence of formation. Transient intermediates were distinguished from dead-end metabolites by the rapid formation and disappearance of the former. At least five unknown precursors of versicolorin A, a dead-end metabolite, were recognized. The kinetic pulse-labeling technique should be generally applicable to other fungal species whenever the entrapment of intermediary metabolites in the mycelium poses and technical problem.     

Conversion of sterigmatocystin to aflatoxin B 1 by Aspergillus parasiticus

¹⁴C-Sterigmatocystin isolated from cultures of $\text{Aspergillus}$$\text{versicolor}$ supplemented with (1-¹⁴C)acetate was shown to be efficiently converted to aflatoxin B₁ by the resting mycelium of $\text{A. parasiticus}$. The experimental results may indicate a biosynthetic pathway leading from 5-hydroxysterigmatocystin to sterigmatocystin and then to aflatoxin B₁.     

Mutant of Aspergillus flavus producing more aflatoxin B2 than B1.

A mutant of Aspergillus flavus having a high and relatively stable aflatoxin B2/B1 ratio was recovered after treatment with nitrosoguanidine.     

Production of aflatoxin B1 by Aspergillus ruber THOM and CHURCH

Production of aflatoxins by Aspergillus ruber THOM and CHURCH was first reported by KULIK and HOLADAY (1967), although these results have lacked confirmation. In this paper we provide evidence that this fungal strain produces aflatoxins. This finding has implications for food hygiene, especially in countries where such moulds are used in the preparation of foodstuffs.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus.

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.     

Enzymatic conversion of sterigmatocystin into aflatoxin B1 by cell-free extracts of Aspergillus parasiticus.

A cell-free extract, prepared from Aspergillus parasiticus ATCC 15517 grown in synthetic medium, was active in converting [14C]sterigmatocystin into aflatoxin B1 in the presence of reduced nicotinamide adenine dinucleotide phosphate. The activity was demonstrated by the time course of conversion and the linear dependence of the yield of product on enzyme concentrations. Optimum activity was obtained at pH 7.5 to 7.8 at 27 C. The results confirm sterigmatocystin as a biogenetic precursor of aflatoxin B1. Techniques were developed for enzymatic studies on aflatoxin biosynthesis.     

Epoxidation of aflatoxin B1 by Aspergillus flavus microsomes in vitro: interaction with DNA and formation of aflatoxin B1-glutathione conjugate

Metabolism of aflatoxin B1 (AFB1) by subcellular preparations of Aspergillus flavus is least understood. The results reported here have demonstrated for the first time the epoxidation of AFB1 and subsequent conjugation with glutathione (GSH). Microsomes prepared from toxigenic mycelia catalysed [3H]AFB1 to calf thymus DNA to a greater extent (approximately 2-fold) as compared to that of non-toxigenic. The binding of [3H]AFB1 to exogenous and A. flavus nuclear DNA catalyzed by A. flavus microsomes was found to be comparable with that of mammalian extrahepatic tissue such as lung. Addition of phenobarbitone to the growing cultures resulted in 1.5-fold increase in [3H]AFB1-DNA binding mediated by microsomes prepared from either of the two strains. Tolnaftate, an inhibitor of aflatoxin synthesis enhanced the epoxidation rate in a dose-related manner. The binding of [3H]AFB1 to DNA catalyzed by A. flavus microsomes was significantly reduced (50% of control) upon addition of hamster liver cytosol, thereby substantiating the formation of the carcinogen adduct with DNA as reported in mammalian tissues. The metabolite formed by subcellular preparation of A. flavus was found to be AFB1-GSH having Rf value (6.5) similar to that obtained for mammalian liver preparations.     

Incorporation of32P-orthophosphate into phospholipids by a toxigenic and a nontoxigenic strain of Aspergillus flavus

The incorporation of³²P-orthophosphate into phospholipids by a toxigenic and a nontoxigenic strain ofAspergillus flavus was compared on a glucose-salts medium (AM medium) and a sucrose-yeast extract medium (YES medium). AM medium gave higher incorporation of³²P than YES medium. In AM medium the highest specific activities were found in phosphatidyl choline and phosphatidyl ethanolamine but in YES medium phosphatidyl inositol and phosphatidyl serine also had specific activities of the same order as phosphatidyl choline and phosphatidyl ethanolamine. Mycelia of the toxigenic strain grown and resuspended in AM medium yielded 1.4 times specific activities given by nontoxigenic strain. The two strains did not differ very much in the incorporation obtained in the other media combination tried. These results are in contrast to the large differences obtained in the incorporation of¹⁴C-acetate in earlier studies. The significance of these findings are discussed.These results were presented at the Symposium on the Chemistry and Metabolism of Lipids and Related Subjects held at Vallabhbhai Patel Chest Institute, Delhi on October 3–5, 1969.     

Influence of aflatoxin B1 on a bacteriocinogenic strain ofSerratia marcescens

The ability of aflatoxin B₁, the most potent hepatocarcinogen known, to induce bacteriocin synthesis inSerratia marcescens strain Nima was studied. The optimal induction dose of aflatoxin B₁ was found to be 20μg/ml, which on the average increased the number of bacteriocinproducing cells by a factor of 11.5. Doses less than 5 μg or more than 40 μg per ml did not have a pronounced stimulating action. The optimum incubation time for maximum bacteriocin synthesis was found to be 1.5 h.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Identification of O-methylsterigmatocystin as an aflatoxin B1 and G1 precursor in Aspergillus parasiticus.

An isolate of Aspergillus parasiticus CP461 (SRRC 2043) produced no detectable aflatoxins, but accumulated O-methylsterigmatocystin (OMST). When sterigmatocystin (ST) was fed to this isolate in a low-sugar medium, there was an increase in the accumulation of OMST, without aflatoxin synthesis. When radiolabeled [14C]OMST was fed to resting mycelia of a non-aflatoxin-, non-ST-, and non-OMST-producing mutant of A. parasiticus AVN-1 (SRRC 163), 14C-labeled aflatoxins B1 and G1 were produced; 10 nmol of OMST produced 7.8 nmol of B1 and 1.0 nmol of G1, while 10 nmol of ST produced 6.4 nmol of B1 and 0.6 nmol of G1. A time course study of aflatoxin synthesis in ST feeding experiments with AVN-1 revealed that OMST is synthesized by the mold during the onset of aflatoxin synthesis. The total amount of aflatoxins recovered from OMST feeding experiments was higher than from experiments in which ST was fed to the resting mycelia. These results suggest that OMST is a true metabolite in the aflatoxin biosynthetic pathway between sterigmatocystin and aflatoxins B1 and G1 and is not a shunt metabolite, as thought previously.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Study of aflatoxin B1 production by Aspergillus parasiticus in bee pollen of Greek origin

Aflatoxin B₁ (AFB₁) is a carcinogenic metabolite produced by certain Aspergillus species such as A. parasiticus and A. flavus. The beneficial properties of bee pollen have transformed this commodity into an increasingly frequent component of the human diet. As bee pollen is a substrate on which aflatoxigenic fungi can grow, AFB₁ production is likely. In the present study, we describe a method for aflatoxin B₁ determination in bee pollen utilising high pressure liquid chromatography (HPLC) with a fluorescence detector (FD). The recovery factor of the method was found to be 111% (RSD% 1.61), while the detection limit (LOD) was 0.08 ng AFB₁/g. An additional aim of this study was to investigate the growth of A. parasiticus and AFB₁ production in bee pollen. Results indicated that no mycelial growth was observed and no AFB₁ was detected in bee pollen samples containing natural microbiota throughout the entire observation period (20 days). In contrast, AFB₁ production in treated bee pollen samples (15 g pollen/flask) inoculated with A. parasiticus was significantly higher (p?≤?0.05) compared to control samples (treated but not inoculated) throughout the entire incubation period, while no mycelial growth was apparent. Maximum production was observed on the 12th day (79.29 ng AFB₁/flask and 32.44 ng AFB₁/flask for inoculated and non-inoculated bee pollen, respectively). As a result, AFB₁ production in bee pollen is likely even following a minor contamination, which could occur randomly.     

The affinity purification and characterization of a dehydrogenase from Aspergillus parasiticus involved in aflatoxin B1 biosynthesis.

A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to omega-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140,000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25 degrees to 35 degrees C. The Km of the enzyme for NA and NADPH was determined to be 3.45 microM and 103 microM respectively.     

假单胞菌胞外酶降解黄曲霉毒素B1的酶学性质

[背景]黄曲霉毒素B1(AflatoxinB1,AFB1)毒性强、污染普遍,目前尚无有效的防治办法.[目的]为了发掘高效的AFB1降解菌并探索其降解特性,对红树林污泥样品中一株AFB1降解菌株(HAI2)的酶学性质进行分析.[方法]以AFB1结构类似物为唯一碳源,筛选出一株高效的AFB1降解菌,利用16SrRNA基因测...