Emericella venezuelensis, a new species with stellate ascospores producing sterigmatocystin and aflatoxin B1

Emericella venezuelensis is a new species, differing from two other species with stellate ascospores, E. variecolor and E. pluriseminata, by triangular flaps on the convex sides of the ascospores, and further from E. variecolor by producing an Aspergillus anamorph only on unconventional growth media. The three species also differ in their profiles of extrolites (secondary metabolites). Emericella venezuelensis produces aflatoxin B₁, sterigmatocystin, and terrein and compounds with chromophores of the shamixanthone, emerin and desertorin type of compounds. E. variecolor produces asteltoxin, shamixanthone, asperthecin, and terrein, in addition to metabolites unequivocally recorded in the literature or tentatively identified here as astellolide A & B, andibenin A, B, C, andilesin A, B, C, anditomin, astellatol, stellatic acid, stellatin, tajixanthone, radixanthone, najamxanthone, ajamxanthone, variecoxanthone A, B, C, isoemericellin, kojic acid, varitriol, varioxiran, dihydroterrein, 7-hydroxyemodin, avariquinone and stromemycin. E. pluriseminata produces several unknown specific extrolites. E. venezuelensis is the first organism of marine origin reported to produce aflatoxin. Aflatoxin production by E. venezuelensis makes this species an attractive model organism for the study of the regulation of this important type of carcinogenic mycotoxins in combination with the knowledge on sterigmatocystin production by E. nidulans, soon to be whole genome sequenced. The isolates were also analyzed cladistically using partial sequences of the β-tubulin gene. Since three species of Emericella have stellate ascospores, and the type material of E. variecolor is equivocal, this species is epitypified with CBS 598.65. Emericella species normally do not appear to cause problems for food safety, as they are most often found in litter and soil.     

Emericella astellata, a new producer of aflatoxin B, B and sterigmatocystin

AIMS: To report on aflatoxin B(1) and B(2) production from a species of Emericella. METHODS AND RESULTS: Aflatoxins and sterigmatocystin were determined by high-pressure liquid chromatography (HPLC) with diode array detection and confirmed by HPLC with mass spectrometry detection. Among 30 known species of Emericella only one species produced aflatoxin. Strains originating from the same geographical source material had different patterns of aflatoxin and sterigmatocystin production on different media, indicating that epigenetic factors may be involved in the regulation of aflatoxin production. However, two cultures from the same original genet were very similar. CONCLUSIONS: Emericella astellata can produce small amounts of sterigmatocystin and aflatoxin B(1) and B(2). SIGNIFICANCE AND IMPACT OF THE STUDY: Emericella has been used extensively in genetic studies and therefore the isolates producing aflatoxin can be used to elucidate the genetic, evolutionary and maybe ecological role of aflatoxins using molecular genetic methods.     

An aflatoxin biosynthesis cluster gene encodes a novel oxidase required for conversion of versicolorin a to sterigmatocystin

Disruption of the aflatoxin biosynthesis cluster gene aflY (hypA) gave Aspergillus parasiticus transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.     

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.     

The effect of aflatoxin B 1 , aflatoxin B 2 and sterigmatocystin on nuclear deoxyribonucleases from rat and mouse livers

Certain carcinogens have an effect on the activity of pancreatic deoxyribonuclease I (DNAase I, EC 3.1.4.5). The effect of two potent mycocarcinogens, viz. aflatoxin B₁ and sterigmatocystin, as well as the weak carcinogen, aflatoxin B₂, on the activity of two nuclear DNAases (DNAases I and II) from rat liver was therefore investigated.     

The conversion of sterigmatocystin to O-methylsterigmatocystin and aflatoxin B1 by a cell-free preparation

A cell-free system derived from a versicolorin A-accumulating mutant of Aspergillus parasiticus was found to convert sterigmatocystin to both O-methylsterigmatocystin and aflatoxin B1. It is suggested that the similarity in the chromatographic properties of these two metabolites has caused erroneous conclusions to be made with regards to the biosynthesis of aflatoxin B1.     

In vivo effect of selenium on the mutagenic activity of aflatoxin B1

Experiments were carried out to verify the effect of selenium on the mutagenic activity of AFB1. After 14 days of selenium administration to experimental animals (Chinese hamsters, Cricetulus griseus) in the form of 2 ppm Na2SeO3 solution available ad libitum the incidence of chromosomal aberrations in bone marrow cells due to a single p.o. administration of 5 mg AFB1 per 1 kg body weight was significantly reduced. The incidence of chromosomal aberrations was monitored till day 32 after AFB1 administration. A significant decrease in the frequency of aberrant cells, breaks and gaps was observed at almost any time during the investigation. 2 ppm Na2SeO3 solution itself did not enhance the frequency of chromosomal aberrations. The mechanism of the protective effect of selenium vis-a-vis the mutagenic and carcinogenic action of AFB1 remains obscure.     

Biosynthetic origin of aflatoxin G1: confirmation of sterigmatocystin and lack of confirmation of aflatoxin B1 as precursors

The origin of aflatoxin G1 was studied using mutant strains of Aspergillus parasiticus blocked early in the pathway and by tracing 14C-labelled aflatoxin B1 (AFB1) in wild-type A. flavus and A. parasiticus strains. Sterigmatocystin (ST) was a precursor of AFB1, AFG1 and AFG2 in the four mutants examined. The identity of AFG1 was confirmed by mass spectrometry. No evidence for conversion of AFB1 to AFG1 was found. A rigorously controlled study of conversions of radioactivity based on preparative thin-layer chromatography of aflatoxins demonstrated that low levels of aflatoxin interconversions previously reported in the literature might actually be artifacts.     

Role of versicolorin A and its derivatives in aflatoxin biosynthesis

The involvement of various anthraquinone metabolites in the biosynthesis of aflatoxin B1 was investigated by using a labeled double-substrate technique in a cell-free system. The results showed that both versicolorin A hemiacetal and versicolorin A hemiacetal acetate were converted to aflatoxin B1, whereas versicolorin A was not, even though it was added to the same cell-free system. Thus, versicolorin A hemiacetal, versicolorin A hemiacetal acetate, or both were implicated as key intermediates, whereas versicolorin A and C became side shunt metabolites. These latter compounds reentered the pathway depending on the availability of the appropriate enzymes and suitability of conditions. Dichlorvos, a specific inhibitor of aflatoxin biosynthesis, is considered to have its primary action on either an oxygenase or dehydrogenase involved in the pathway and to act in a secondary capacity as an inhibitor of an esterase which may also be involved in the pathway.     

Role of versicolorin A and its derivatives in aflatoxin biosynthesis.

The involvement of various anthraquinone metabolites in the biosynthesis of aflatoxin B1 was investigated by using a labeled double-substrate technique in a cell-free system. The results showed that both versicolorin A hemiacetal and versicolorin A hemiacetal acetate were converted to aflatoxin B1, whereas versicolorin A was not, even though it was added to the same cell-free system. Thus, versicolorin A hemiacetal, versicolorin A hemiacetal acetate, or both were implicated as key intermediates, whereas versicolorin A and C became side shunt metabolites. These latter compounds reentered the pathway depending on the availability of the appropriate enzymes and suitability of conditions. Dichlorvos, a specific inhibitor of aflatoxin biosynthesis, is considered to have its primary action on either an oxygenase or dehydrogenase involved in the pathway and to act in a secondary capacity as an inhibitor of an esterase which may also be involved in the pathway.     

Effects of aflatoxin B, aflatoxin B, aflatoxin G and sterigmatocystin on viability, rates of development, and body length in two strains of Drosophila melanogaster (Diptera)

Two wild-type laboratory populations of Drosophila melanogaster, Florida-9 (sensitive to aflatoxin (AF) B₁-induced toxicity) and Lausanne-S (resistant to AFB₁-induced toxicity) were tested to determine relative degress of sensitivity to growth from the egg stage on media containing 0.2, 0.6, 2.0, and 4.0 ppm AFB₁, AFG₁, AFB₂, or sterigmatocystin (ST). Data indicate that strain Florida-9 is quite sensitive to AFG₁ toxicity at both the egg-pupa and egg-adult stages of development while Lausanne-S is quite resistant to such toxic effects. For Lausanne-S, AFB₁ > AFG₁ in relative toxicity, while for Florida-9, AFG₁ > AFB₁. The latter is noteworthy since vertebrate studies consistently show that AFB₁ is a significantly stronger carcinogen and mutagen than AFG₁. Possible explanations are discussed. Neither strain tested displayed toxic responses to the presence of AFB₂ or ST in the culture media; however, the 4.0-ppm Lausanne-S treatment displayed a significantly lower adult mortality rate than the control, indicating that Lausanne-S flies may benefit from the presence of ST in the culture medium.     

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.     

stcS, a putative P-450 monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in Aspergillus nidulans.

Sterigmatocystin (ST) and aflatoxin are carcinogenic end point metabolites derived from the same biochemical pathway, which is found in several Aspergillus spp. Recently, an ST gene cluster, containing approximately 25 distinct genes that are each proposed to function specifically in ST biosynthesis, has been identified in Aspergillus nidulans. Each of these structural genes is named stc (sterigmatocystin) followed by a consecutive letter of the alphabet. We have previously described stcU (formerly verA) as encoding a keto-reductase required for the conversion of versicolorin A to ST. We now describe a second A. nidulans gene, stcS (formerly verB), that is located within 2 kb of stcU in the ST gene cluster. An stcS-disrupted strain of A. nidulans, TSS17, was unable to produce ST and converted ST/aflatoxin precursors to versicolorin A rather than ST, indicating that stcS functions at the same point in the pathway as stcU. Genomic sequence analysis of stcS shows that it encodes a cytochrome P-450 monooxygenase and constitutes a novel P-450 family, CYP59. Assuming that StcU activity mimics that of similar P-450s, it is likely that StcU catalyzes one of the proposed oxidation steps necessary to convert versicolorin A to ST. These results constitute the first genetic proof that the conversion of versicolorin A to ST requires more than one enzymatic activity.     

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.     

Individual reaction requirements of two enzyme activities, isolated from Aspergillus parasiticus, which together catalyze conversion of sterigmatocystin to aflatoxin B1

Individual reaction requirements were determined for each of two enzyme activities present in Aspergillus parasiticus mycelia which together catalyze conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1). A postmicrosomal activity (PMA) catalyzed conversion of ST to O-methylsterigmatocystin (OMST) and a microsomal activity (MA) catalyzed conversion of OMST to AFB1. PMA was stimulated two- to three-fold in the presence of S-adenosylmethionine. Addition of NADPH promoted the maximum MA; this activity was not detected when FAD, FMN, NAD, or NADH were utilized individually as cofactors in reaction mixtures. A substantial amount (62%) of MA was lost during isolation of the microsomal fraction, but the activity was completely restored by reconstitution with a heat-treated (100 degrees C) postmicrosomal fraction. The reaction catalyzed by MA was optimum at pH 7.0 and at 17-23 degrees C, whereas the PMA reaction was optimum at pH 8.0-8.5 and at 35-40 degrees C. Apparent Km values of approximately 2.6 X 10(-6) M (for ST) and 6.6 X 10(-7) M (for OMST) were determined for PMA and MA, respectively.     

Thermal stability of aflatoxin B1 and ochratoxin A

Within this research project, the LCI (Lebensmittelchemisches Institut des Bundesverbandes der Deutschen Süßwarenindustrie e. V.) conducted systematic studies to determine the thermal stability of the mycotoxins ochratoxin A (OTA) and aflatoxin B₁, as the available literature provides contradictory data. Firstly, the said mycotoxins in pure form were subjected to thermal treament. Secondly, tests were conducted to determine the influence of certain matrix substances, including carbohydrates and proteins, on the thermal decomposition behaviour of the mycotoxins. As a result it can be said that OTA seems to be stable up to 180 °C; however aflatoxin B₁ was almost completely degraded at heating temperatures of 160 °C and above. In several model assays it could further be shown that the degradation of mycotoxins is improved by the existence of certain matrix compounds.     

The aflatoxin biosynthesis cluster gene, aflX, encodes an oxidoreductase involved in conversion of versicolorin A to demethylsterigmatocystin

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion. A homolog of this gene, stcQ, is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated approximately 4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO (avfA). Some of domains in the protein are similar to those of epoxide hydrolases.     

Comparative binding and sequence interaction specificities of aflatoxin B1, aflatoxicol, aflatoxin M1, and aflatoxicol M1 with purified DNA

The covalent binding of the activated forms of several aflatoxins to N-7 of guanine residues on purified DNA has been studied. The aflatoxins include aflatoxin B1 (AFB1) and two human metabolites, aflatoxicol and aflatoxin M1, along with aflatoxicol M1, a rabbit and trout metabolite. DNA binding studies using tritiated [3H]aflatoxins indicate that equimolar solutions of each aflatoxin upon activation with chloroperoxybenzoic acid readily react to produce covalently bound adducts. These reactions produce alkali-labile sites which can be identified using a simple variation of the Maxam-Gilbert sequencing procedure. Two DNA fragments were exposed to each aflatoxin, and the reaction intensities at 33 guanine residues were determined. As much as 10-fold variation in reaction intensities was observed for various guanyl sites. Data indicate that none of the aflatoxins had identical reaction profiles, although AFB1 and aflatoxicol M1 were similar, as were aflatoxicol and aflatoxin M1. Hence, the frequency with which the various aflatoxin epoxides might damage specific sites critical for tumor initiation in vivo would not be predictable from total covalent binding indices. The frequency of occurrence of modifications at particular sites for AFB1 was also compared with the empirical "rules" established for AFB1 by Misra et al. (Misra, R. P., Muench, K. F., and Humayun, M. Z. (1983) Biochemistry 22, 3351-3359). Identical sites within fragments were compared for each aflatoxin, and the data showed that the attacking frequency for some such sites varied significantly. These results indicate that binding intensity rules based on nearest neighbor nucleotides do not reliably predict guanyl-AFB1 binding frequencies.