Determination of aflatoxin levels in pistachio and cashew nuts using immunoaffinity column clean-up with HPLC and fluorescence detection

A technique for the detection of aflatoxins in pistachio and cashew nuts using immunoaffinity column clean-up with HPLC and fluorescent detection is presented. Recoveries were in the range of 79–99% for pistachio samples artificially contaminated with 10 g total aflatoxins kg–1 of food sample. For cashew samples recoveries ranged from 80–106%. This method is proposed as an accurate technique for aflatoxin detection in the range of g aflatoxins kg–1 nuts.     

Thermal stabilization of the DNA duplex by adducts of aflatoxin B1

The trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B(1) cationic guanine N7 adduct of aflatoxin B(1) thermally stabilizes the DNA duplex, as reflected in increased T(m) values upon adduction. The magnitude of the increased T(m) value is characteristically 2-3 degrees C. The major rotamer of the neutral guanine N7 adduct trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (the FAPY major adduct) exhibits a 15 degrees C increase in T(m) in 5'-d(CTAT(FAPY)GATTCA)-3'-5'-d(TGAATCATAG)-3'. Site-specific mutagenesis experiments reveal the FAPY major adduct induces G-->T mutations in Escherichia coli at a frequency six times higher than that of the cationic adduct (Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc Natl Acad Sci USA, 99, 6655-6660). Thus, the FAPY major lesion may account substantially for the genotoxicity of AFB(1). Structural studies for cationic and FAPY adducts of aflatoxin B(1) suggest both adducts intercalate above the 5'-face of the modified deoxyguanosine and that in each instance the aflatoxin moiety spans the DNA helix. Intercalation of the aflatoxin moiety, accompanied by favorable stacking with the neighboring base pairs, is thought to account for the increased thermal stability of the aflatoxin cationic guanine N7 and the FAPY major adducts. However, the structural basis for the large increase in thermal stability of the FAPY major adduct in comparison to the cationic guanine N7 adduct of aflatoxin B(1) is not well understood. In light of the site-specific mutagenesis studies, it is of considerable interest. For both adducts, the intercalation structures are similar, although improved stacking with neighboring base pairs is observed for the FAPY major adduct. In addition, the presence of the formamido group in the aflatoxin B(1) FAPY major adduct may enhance duplex stability, perhaps via intrastrand sequence-specific hydrogen bonding interactions within the duplex.     

Coordinate control of secondary metabolite production and asexual sporulation in Aspergillus nidulans

Microbial secondary metabolite production is frequently associated with developmental processes such as sporulation, but there are few cases where this correlation is understood. Recent work with the filamentous fungus Aspergillus nidulans has provided new insights into the mechanisms coordinating production of the toxic secondary metabolite sterigmatocystin with asexual sporulation. These processes have been shown to be linked through a common need to inactivate a heterotrimeric G protein dependent signaling pathway that, when active, serves to stimulate growth while blocking both sporulation and sterigmatocystin biosynthesis.     

Mycoflora and mycotoxins in Brazilian black pepper,white pepper and Brazil nuts

A wide range of field and storage fungi were isolated from black pepper, white pepper and Brazil nut kernels from Amazonia. A total of 42 species were isolated from both peppers. Aspergillus flavus and A. niger were isolated more frequently from black than from white pepper. Other potential mycotoxigenic species isolated included: A. ochraceus, A. tamarii, A. versicolor, Emericella nidulans and Chaetomium globosum, Penicillium brevicompactum, P. citrinum, P. islandicum and P. glabrum. Species isolated from pepper for the first time were Acrogenospora sphaerocephala, Cylindrocarpon lichenicola, Lacellinopsis sacchari, Microascus cinereus, Petriella setifera and Sporormiella minima. Seventeen species were isolated from Brazil nut kernels. A. flavus was the dominant species followed by A. niger. P. citrinum and P. glabrum were the only penicillia isolated. Species isolated for the first time included Acremonium curvulum, Cunninghamella elegans, Exophiala sp., Fusarium oxysporum, Pseudoallescheria boydii, Rhizopusoryzae, Scopulariopsis sp., Thielavia terricola and Trichoderma citrinoviride. Considerably more metabolites were detected from black than white pepper in qualitative analyses. Chaetocin, penitrem A, and xanthocillin were identified only from black pepper, and tenuazonic acid was identified from both black and white pepper. Aflatoxin G₂, chaetoglobosin C, and spinulosin were identified from poor quality brazil nuts. Aflatoxin B₁ and B₂ were also only detected in poor quality brazil nuts at concentrations of 27.1 g kg^–1 and 2.1 g kg^–1 respectively (total 29.2 g kg^–1).This revised version was published online in October 2005 with corrections to the Cover Date.     

Interactions of aflatoxin B1 with SRP components can disrupt protein targeting

Spectrofluorimetric studies have revealed that aflatoxin B1 (AFB1) interacts with signal recognition particle (SRP), which acts as an escort for polyribosomes with signal peptides to be transported and bound to the cytoplasmic face of the endoplasmic reticulum (ER). We further report that the binding of AFB1 to SRP is selective as it only binds to two (SRP9 and 14) out of its three constituent polypeptides studied. Binding of AFB1 to proteins is known to alter their conformations. Interactions of AFB1 with SRP polypeptides may generate structural and functional alterations in this particle and hinder secretory protein synthesis.     

Morphological alterations in toxigenic <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> exposed to neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) leaf and seed aqueous extracts

The mode of action of the extracts prepared from neem plant i.e., Azadirachta indica on aflatoxin formation in toxigenic Aspergillus species is not well understood. Aflatoxin production by A. parasiticus was suppressed depending on the concentration of the plant aqueous extract (0, 1.56, 3.12, 6.25, 12.5, and 50% v/v) added to the culture media at the time of spore inoculation. Aflatoxin production in fungal mycelia grown for 96 h in culture media containing 50% neem leaf and seed extracts was inhibited by approximately 90 and approximately 65% respectively. Under similar conditions, culture media amended with 1.56% of leaf or seed extract caused approximately 23 and approximately 7% inhibition respectively. Mycelial samples exposed to selected concentrations of the plant extract (1.56 or 50% v/v) collected and processed for morphological studies. Semi-thin longitudinal and cross sections prepared from control (untreated) and treated mycelia (1.56% v/v) revealed that alterations are limited to the vacuolation of the mycelial cytoplasm. Nevertheless, exposure to high concentration i.e., 50% v/v of the extract resulted in vacuolation of the mycelial cytoplasm and vesicle deformation causing attenuation of cell wall at variable intervals. Herniation of the cytoplasmic contents that was protruding from the mycelium was associated with deformation of the mycelium. Some mycelia showed a cleft between the cell wall and cytoplasm. Association of aflatoxin production with morphological changes suggest that probably integrity of the cell barriers particularly cell wall is critical in regulation of aflatoxin production and excretion.     

Effect in rats of simultaneous prenatal exposure to ochratoxin A and aflatoxin B1. I. Maternal toxicity and fetal malformations

Ochratoxin A (OA) and Aflatoxin B1 (AFB1), the food borne mycotoxins are produced by several fungal species of the genera Aspergillus and Penicillium. To determine the teratogenic effects, these mycotoxins were administered orally either individually or in combination to the pregnant Wistar rats on days 6-15 of gestation. OA and AFB1 were dissolved in corn oil and different doses of OA (0.125, 0.25, 0.50, and 0.75 mg/kg), AFB1 (0.125, 0.25, 0.50, and 1.00 mg/kg), and a combination of OA+AFB1 (0.125+0.125; 0.25+0.50; 0.50+0.25 mg/kg) were given by gastric intubation to rats. During dosing period, the body weight and body weight gains significantly decreased at a higher dosage, in both individual and combined treatments. In all the combination treatments, the percent implants resorbed, fetal body weights, and crown-rump lengths were comparable to those of controls and with the individual mycotoxin treatment. The number of dead fetuses was significantly increased in the high OA combination (OA+AFB1 0.50+0.25) group as compared with the other two combinations. OA and AFB1 alone and in combination caused various gross, skeletal, and visceral anomalies. The occurrence was considerably less pronounced in fetuses of AFB1 and combination groups as compared with those of OA group fetuses. The exencephaly, incomplete closure of skull, wavy and fused ribs, agenesis of the ischium bone, and enlarged renal pelvis, recorded in OA treatment and ear abnormality and incomplete ossification of skull bones observed in AFB1 when given individually, were not seen in combination groups. However, new manifestations, such as gastroschisis and syndactyly were observed and the incidence of cardiac defects was increased in fetuses due to the combined treatment. The results of the present study indicated that there is some interaction between these mycotoxins that resulted in reduced teratogenic activity of OA in the presence of AFB1. Apparently, new manifestations observed in combination treatment points to the potential threat of teratogenicity in terms of public health hazards.     

Phylogenetic analysis of homologous fatty acid synthase and polyketide synthase involved in aflatoxin biosynthesis

The first two steps of aflatoxin biosynthesis are catalyzed by the HexA/B and by the Pks protein. The phylogenetic analysis clearly distinguished fungal HexA/B from FAS subunits and from other homologous proteins. The phylogenetic trees of the HexA and HexB set of proteins share the same clustering. Proteins involved in the synthesis of fatty acids or in the aflatoxin or sterigmatocystin biosynthesis cluster separately. The Pks phylogenetic tree also differentiates the aflatoxin-related polypeptide sequences from those of other kinds of secondary metabolism. The function of some of the A. flavus Pks homologues may be deduced from the phylogenetic analysis. The conserved sequence motifs of protein domains shared by HexA/B and Pks - namely, β-polyketide synthase (KS), acetyl transferase (AT) and acyl carrier protein (ACP) - have been identified, and the HexA/B and Pks involved in aflatoxin biosynthesis have been distinguished from those involved in primary metabolism or other kinds of secondary metabolism.     

Prevention of preharvest aflatoxin contamination through genetic engineering of crops

Current practices on prevention of aflatoxin contamination of crop species include time consuming, expensive agronomic practices. Of all the methods available to-date, conventional breeding and/or genetic engineering to develop host plant-based resistance to aflatoxin-producing fungi appear to be valuable for several reasons. However, breeding for disease-resistant crops is very time consuming, especially in tree crops, and does not lend itself ready to combat the evolution of new virulent fungal races. Moreover, availability of known genotypes with natural resistance to mycotoxin-producing fungi is a prerequisite for the successful breeding program. While it is possible to identify a few genotypes of corn or peanuts that are naturally resistant toAspergillus we do not know whether these antifungal factors are specific toA. flavus. In crops like cotton, there are no known naturally resistant varieties toAspergillus. Availability of transgenic varieties with antifungal traits is extremely valuable as a breeding tool. Several antifungal proteins and peptides are available for genetic engineering of susceptible crop species, thanks to the availability of efficient modern tools to understand and evaluate protein interactions by proteomics of host, and genomics and field ecology of the fungus. Transgenic approaches are being undertaken in several industry and academic laboratories to prevent invasion byAspergillus fungi or to prevent biosynthesis of aflatoxin. Recent trends in reducing aflatoxin contamination through genetic engineering of cultivated crop species with antifungal proteins are summarized in this report. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005     

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.