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PubMed2000

Development of surface plasmon resonance-based immunoassay for aflatoxin B(1).

Aflatoxins are a group of highly toxic fungal secondary metabolites that occur in Aspergillus species and may contaminate foodstuffs and feeds. Two different anti-aflatoxin B(1) antibodies were examined to develop a surface plasmon resonance (SPR)-based immunoassay to aflatoxin B(1). A conjugate consisting of aflatoxin B(1)-bovine serum albumin (BSA) was immobilized on the dextran gel surface. Competition between immobilized aflatoxin B(1) conjugate and free aflatoxin B(1) in solution for binding to antibody injected over the surface formed the basis for the assay. Regeneration of the antibody from the immobilized conjugate surface is essential for the development of such an inhibitive immunoassay. Problems were encountered with the regeneration of the sensor surface, due to the high-affinity binding of the antibodies. Conventional regeneration solutions consisting of low concentrations of NaOH and HCl worked to a degree, but regeneration was at the expense of the integrity of the immobilized conjugate. A polyclonal anti-aflatoxin B(1) antibody was produced and was found to be regenerable using an organic solution consisting of 1 M ethanolamine with 20% (v/v) acetonitrile, pH 12.0. This combined high ionic strength and extreme pH, as well as chaotrophic properties and allowed the development of an inhibitive immunoassay. The assay had a linear range of 3.0-98.0 ng mL(-1) with good reproducibility.

Aflatoxin B1↗

PubMed2002

Structural refinement of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B(1) adduct in a 5'-Cp(AFB)G-3' sequence.

The structure of the cationic 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B(1) adduct embedded in a 5'-CpG-3' sequence context and paired with deoxycytosine in the oligodeoxynucleotide d(ACATC(AFB)GATCT) x d(AGATCGATGT) was refined using molecular dynamics calculations restrained by NOE data and dihedral angle restraints obtained from NMR data. The aflatoxin moiety intercalated above the 5' face of the modified guanine. It stacked between C(5) x G(16) and (AFB)G(6) x C(15). The AFB(1) H5, OCH(3), and methylene protons faced into the minor groove, with the methylene protons oriented between the C(15) and G(16) nucleobases. The aflatoxin B(1) H6a, H8, H9, and H9a protons faced the major groove, with H6a and H9a pointing toward the 5' direction from the lesion site. The refined structure was compared to the structure of the aflatoxin B(1) adduct embedded in a 5'-ATGCAT-3' sequence in the oligodeoxynucleotide d(TAT(AFB)GCATA)(2) [Jones, W. R., Johnston, D. S., and Stone, M. P. (1998) Chem. Res. Toxicol.11, 873-881]. The structure of the intercalated aflatoxin B(1) lesion in the ATC(AFB)GAT sequence is similar to its structure in the d(AT(AFB)GCAT) sequence. This is consistent with a mechanism in which the precovalent intercalation of aflatoxin-8,9-exo-epoxide on the 5' face of guanine places the epoxide in close proximity and in the proper orientation to the N7 position of guanine, thus facilitating an S(N)2 reaction. The data provides additional insight into the nature of the disruption of the B-DNA duplex induced by aflatoxin B(1) intercalation. Overall, the results suggest that sequence contributes a minor role in modulating the structure of the cationic guanine N7 AFB(1) lesion in duplex DNA. On the other hand, structural differences are observed when the correctly paired structure is compared to the structure of the cationic AFB(1) adduct mispaired with dA [Giri, I., Johnston, D. S., and Stone, M. P. (2002) Biochemistry 41, 5462-5472].

Aflatoxin B1↗

PubMed2003

Control of Aspergillus growth and aflatoxin production using antioxidants at different conditions of water activity and pH.

AIMS: The effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone (THB) and propyl paraben (PP) (at concentrations of 1, 10 and 20 mmol l(-1)) on germination, growth and aflatoxin B1 production by Aspergillus section Flavi was evaluated. METHODS AND RESULTS: Studies on the percentage of spore germination, elongation rate, growth rate and aflatoxin B1 production were carried out in vitro in relation to water activity (aw) at 0.982, 0.937, 0.809 and 0.747 values. At 0.809 and 0.747aw values none of the isolates was able to germinate. Overall, PP and BHA were the antioxidants most effective at inhibiting germination of both species. In the presence of the lowest concentration of BHA and PP (1 mmol l(-1)) the conidial germination percentage ranged from 2 to 19% after 15 h of incubation at the highest water activity tested. BHA and PP at 10-20 mmol l(-1) completely inhibited conidial germination. The antioxidants more efficient in controlling Aspergillus elongation rate were PP, BHT and BHA. All strains were much more sensitive to all antioxidants tested on the percentage of spore germination and growth rate at 0.937aw. The antioxidants PP and BHA completely inhibited aflatoxin B1 production by all strains when added at 1 mmol l(-1). Decreased aflatoxin B1 levels in comparison with the control, were observed with BHT at 1, 10 and 20 mmol(-1) with the strain T20 at 0.982aw. In contrast, stimulation was observed with the antioxidant THB at 10 and 20 mmol l(-1) at 0.937aw with the strains T20 and T23. The effect of BHA and PP at 1 mmol l(-1) on lag phase and growth rate was maintained in the pH range between 6 and 8. At all pH values the inhibitory effect of BHA was higher than PP. No aflatoxin B1 was detected at all pH values. CONCLUSIONS: The data show that BHA and PP could be considered as effective fungitoxicants for A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained show promise for controlling growth and aflatoxin B1 in stored maize. Futher studies should be carried out to examine the potential for antioxidants, such as BHA and PP to effectively control both growth and aflatoxin production.

Aflatoxin B1↗

PubMed2001

Inhibitory effect of acetosyringone on two aflatoxin biosynthetic genes.

AIMS: The objective of this study was to determine if acetosyringone affected the expression of aflatoxin biosynthetic genes. METHODS AND RESULTS: Two genes, nor1 and ver1, representing genes whose products are involved in early and late steps in aflatoxin biosynthesis, were examined. Two GUS (beta-glucuronidase) reporter constructs, nor1:GUS (pGAP12) and ver1:GUS (pGAP13), were used to study the effect of acetosyringone on expression of aflatoxin biosynthetic (AF) genes, nor1 and ver1. The product of nor1 is involved in the formation of norsolorinic acid, the first stable intermediate in the aflatoxin pathway. The ver1 gene codes for the enzyme catalyzing the formation of demethylsterigmatocystin, an intermediate late in the AF pathway. GUS activities of these two reporter constructs were inhibited by 80% in the presence of 2 m mol l-1 acetosyringone. CONCLUSION: Aflatoxin production in a toxigenic strain 42-12 was also shown to be inhibited by acetosyringone to the same level. The levels of inhibition in aflatoxin production and gene transcription are congruous in these three strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Recent studies have indicated that some phenolics act as signal molecules in plant microbial interactions. Concentration of acetosyringone is shown to increase about ten fold when certain metabolically active plant tissues are wounded. The knowledge gained can be applied to develop strategies in plant breeding programs. The compound may be useful for studying molecular mechanism of modulating aflatoxin biosynthesis.

Acetophenones↗

PubMed1998

Aflatoxins in autopsy kidney specimens from children in Nigeria.

Autopsy kidney specimens from 24 children with kwashiorkor and 21 with other miscellaneous diseases, at the Obafemi Awolowo Teaching Hospital Complex, Ile-Ife, Nigeria, were analyzed for the presence of aflatoxins using high-performance liquid chromatography. Aflatoxins were detected in 14 children who died of kwashiorkor and in 13 of those who died from miscellaneous diseases. Aflatoxicol was detected in 10 specimens, 7 of which had severe gastroenteritis. Seven kidney specimens demonstrated the presence of more than one type of aflatoxin; four of these were kidneys of patients with kwashiorkor and the remaining three died from renal failure. No difference was found between the frequency of detection, type of aflatoxin detected, or mean concentrations of total aflatoxins in the kidney specimens of the kwashiorkor children when compared to the kidney specimens of children who died from miscellaneous diseases. These findings demonstrate that aflatoxins can be detected in the kidneys of children exposed to aflatoxins.

Adolescent↗

PubMed2002

Occurrence of aflatoxin in commodities imported into Qatar, 1997-2000.

The occurrence of aflatoxin in commodities imported into Qatar was investigated from 1999 to 2000. During the 4 years, 351 samples of susceptible commodities were analysed. Aflatoxin was detected in 71 (20%) samples in the range 0.1-20 microg kg(-1) and in 50 (14%) samples above the permitted level of 20 microg kg(-1). The highest incidence and levels of aflatoxin contamination were recorded in pistachio without shell followed by pistachio with shell. Aflatoxin levels >20 microg kg(-1) in the pistachio samples varied from 8.7 to 33%. The highest level of total aflatoxin found in pistachio without shell was 289 microg kg(-1). A few samples of corn and corn products (three of 54 analysed), peanut and peanut products (nine of 42 analysed) and other nuts like almond, walnut and cashew (one of 40 analysed) were found contaminated with low levels (0.1-20 microg kg(-1)) of aflatoxins. Only one sample of custard powder and one sample of roasted peanut were found with aflatoxin >20 microg kg(-1)

Aflatoxins↗

PubMedDate not supplied

Use of immunoaffinity chromatography as a purification step for the determination of aflatoxin M1 in cheeses.

A method using immunoaffinity as a purification step for the determination of aflatoxin M1 in cheese is described. A simple solvent extraction with dichloromethane followed by a washing step with N-hexane gives a prepurified extract. A comparison between two ways of aflatoxin M1 purification, by solid-phase extraction clean-up and by immunoaffinity, was carried out. The use of immunoaffinity columns containing monoclonal antibodies against aflatoxin M1 gives the best result, i.e. a very clean preparation containing purified aflatoxin M1. The quantification of aflatoxin M1 is then performed by high performance liquid chromatography using fluorometric detection. This method was successfully carried out on naturally-contaminated and spiked cheeses. Recoveries are about 75%. The limit of quantification is 0.020 microgram of aflatoxin M1 per kg of cheese. This method seems suitable for use in monitoring programmes for aflatoxin M1 contamination in dairy products such as cheese.

Aflatoxin M1↗

PubMedDate not supplied

Influence of bag materials on the moisture loss and final aflatoxin content of in-shell peanuts stored moist. First studies.

In the present work the influence of bag materials on the moisture loss and final aflatoxin content of stored moist in-shell peanuts (MIP) was studied in the rainy season of 1990, in Marília, São Paulo, and in the rainy season of 1991, in Jaboticabal, São Paulo. In each season, MIP were ventilated, as they arrived from the field, to get rid of extraneous materials and then put into 120 bags of jute and into 120 bags of polypropylene, and stored in stacks (12 bags base x 10 bags high). In the rainy season of 1990 (February-April) moisture and aflatoxin were determined at the beginning (average moisture = 14.31%; aflatoxin not detected). Subsequently, moisture was determined twice a week in samples taken from the external part of the stacks for 80 days, when the stacks were dismantled and moisture and aflatoxin were determined in six samples of each stack. In the rainy season of 1991 (February-March), because of operational difficulties, closed mesh jute bags were used (green coffee type) and the experiment lasted only 30 days. Moisture and aflatoxin were determined only at the beginning (average moisture = 15%; aflatoxin not detected). At the end of the storage period three samples were taken from each lot, for moisture and aflatoxin analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxins↗

PubMedDate not supplied

Aflatoxin in respirable corn dust particles.

Samples of airborne grain dust representing a variety of grains were examined for the presence of aflatoxins. Although aflatoxins could not be detected in most of the samples, one corn sample contained 130 ppb aflatoxin B1. When the components of this sample were separated according to aerodynamic diameter and analyzed for aflatoxins, it was found that particles with diameters 7-11 micrometers and less than 7 micrometers had much higher levels of aflatoxins than the sample as a whole. These findings suggest that measurements of aflatoxin in whole corn might underestimate aflatoxin levels in corn dust aerosols.

Aflatoxins↗

PubMed1998

Assessing airborne aflatoxin B1 during on-farm grain handling activities.

The presence of aflatoxin in corn and corn dust during relatively normal years and the increased risk of Aspergillus flavus infestation during drought conditions suggest that airborne agricultural exposures should be of considerable concern. Liquid extraction, thin layer chromatography, and high pressure liquid chromatography were used for the analysis of aflatoxin B1 in grain dust and bulk corn samples. A total of 24 samples of airborne dust were collected from 8 farms during harvest, 22 samples from 9 farms during animal feeding, and 14 sets of Andersen samples from 11 farms during bin cleaning. A total of 14 samples of settled dust and 18 samples of bulk corn were also collected and analyzed. The airborne concentration of aflatoxin B1 found in dust collected during harvest and grain unloading ranged from 0.04 to 92 ng/m3. Higher levels of aflatoxin B1 were found in the airborne dust samples collected from enclosed animal feeding buildings (5-421 ng/m3) and during bin cleaning (124-4849 ng/m3). Aflatoxin B1 up to 5100 ng/g were detected in settled dust collected from an enclosed animal feeding building; however, no apparent correlation was found between the airborne concentration of aflatoxin B1 and its concentration in settled dust or bulk corn. The data demonstrate that farmers and farm workers may be exposed to potentially hazardous concentrations of aflatoxin B1, particularly during bin cleaning and animal feeding in enclosed buildings.

Aflatoxin B1↗

PubMed1993

Quantitative analysis of aflatoxin-albumin adducts.

Quantitative exposure assessments made using biologically relevant markers will facilitate epidemiological studies of risk from environmental carcinogens. Blood proteins are readily accessible macromolecules that have been shown to be targets for activated chemical carcinogens. Serum albumin is quantitatively the most abundant target for aflatoxin B1 and the measurement of aflatoxin-serum albumin adducts has been used to detect exposed individuals. The goal of these experiments was to devise an analytical procedure that would increase the overall recovery of aflatoxin adducts in serum albumin, and thereby improve the accuracy of exposure monitoring. The method developed consisted of the following procedures. Proteins were precipitated from serum (< or = 100 microliters) with 80% ammonium sulfate, with incubation at 4 degrees C for 2 h. Following dialysis against phosphate-buffered saline (pH 7.0 for 3 h at 4 degrees C), the proteins were digested with protease (Pronase) (1:4.1 w/w enzyme:protein) for 15 h at 37 degrees C with shaking. Enzyme and other undigested proteins were precipitated with acetone (1:2 v/v, 40 min, 4 degrees C). After evaporation of the acetone under vacuum, levels of aflatoxin B1-albumin adducts were determined by radioimmunoassay carried out on 300 microliters fractions. This procedure obviated the isolation of albumin prior to analysis and reduced interference in the radioimmunoassay. High recoveries of aflatoxin B1 adducts were achieved together with a low limit of detection. The applicability of the procedure in epidemiological studies of human aflatoxin exposure was illustrated by results of analysis of aflatoxin-albumin adducts in serum samples from residents of Chongming Island, People's Republic of China.

Aflatoxin B1↗

PubMed2000

Aflatoxin B1-induced mitotic recombination in L5178Y mouse lymphoma cells.

Aflatoxin B1 is a human hepatocarcinogen. It is also a known point mutagen in bacteria and mammalian cells. This mutagenic activity may be at least partly responsible for its carcinogenic activity. However, recent studies show that aflatoxin B1 induces mitotic recombination in the yeast Saccharomyces cerevisiae. Because numerous reports have implicated mitotic recombination in mechanisms leading to carcinogenesis and because no one has shown that aflatoxin B1 induces recombination in mammalian cells, we decided to examine the ability of aflatoxin B1 to induce recombination in a mammalian cell line. We used a combination of methods, analysis for loss of heterozygosity and whole chromosome in situ hybridization, to identify mechanisms of chromosome mutation, including mitotic recombination in the mammalian L5178Y mouse lymphoma cell system. Our experiments revealed that mitotic recombination caused approximately 60% or more of the aflatoxin B1-induced mutagenic lesions in this cell system. Thus, mitotic recombination plays an important role in aflatoxin B1-induced mutagenesis in mammalian cells and possibly in chemically induced mutagenesis and carcinogenesis. This work suggests that multiple genetic lesions may be involved in aflatoxin B1-induced pathology.

Aflatoxin B1↗

PubMed2006

In vitro and in vivo assessment of humic acid as an aflatoxin binder in broiler chickens.

The in vitro affinity and adsorption capacity of a humic acid, oxihumate, for aflatoxin B1 (AFB1) was evaluated, utilizing Langmuir and Freundlich adsorption isotherms. Oxihumate showed a high in vitro affinity for AFB1. The Freundlich isotherm fitted the data better than the Langmuir isotherm, and binding capacities of 10.3, 7.4, and 11.9 mg of AFB1/g of oxihumate at pH 3, 5, and 7, respectively, were calculated. The in vivo efficacy of oxihumate as an aflatoxin binder in male broiler chickens exposed to aflatoxin-contaminated feed from 7 to 42 d of age was also assessed. The efficacy of oxihumate was compared with a commercially available product with a brewers dried yeast (BDY) and brewers fermentation solubles as main active ingredients. A total of 420 birds were assigned to 28 pens, with 15 birds per pen. The following treatments were applied: 1) 0 mg of AFB1 + 0 additives, 2) 1 mg of AFB1/kg of feed + 0 additives, 3) 1 mg of AFB1/kg of feed + 3.5 g of oxihumate/kg of feed, 4) 1 mg of AFB1/kg of feed + 3.5 g of BDY/kg of feed, 5) 2 mg of AFB1/kg of feed + 0 additives, 6) 2 mg of AFB1/kg of feed + 3.5 g of oxihumate/kg of feed, and 7) 2 mg of AFB1/kg of feed + 3.5 g of BDY/kg of feed. Each treatment consisted of 4 replicates. Oxihumate was effective in diminishing the adverse effects caused by aflatoxin on BW of broilers (P < 0.05). Oxihumate also showed protective effects against liver damage, stomach and heart enlargement, as well as some of the hematological and serum biochemical changes associated with aflatoxin toxicity (P < 0.05). Results indicated that oxihumate, but not BDY, could alleviate some of the toxic effects of aflatoxin in growing broilers. Oxihumate might, therefore, prove to be beneficial in the management of aflatoxin-contaminated feedstuffs for poultry when used in combination with other mycotoxin management practices. Additional studies are warranted to assess its efficacy under a wide variety of circumstances.

Adsorption↗

PubMed1979

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.

Aflatoxins↗

PubMed1995

The plant growth regulator methyl jasmonate inhibits aflatoxin production by Aspergillus flavus.

Aflatoxins are highly toxic and carcinogenic compounds produced by certain Aspergillus species on agricultural commodities. The presence of fatty acid hydroperoxides, which can form in plant material either preharvest under stress or postharvest under improper storage conditions, correlates with high levels of aflatoxin production. Effects on fungal growth and aflatoxin production are known for only a few of the numerous plant metabolites of fatty acid hydroperoxides. Jasmonic acid (JA), a plant growth regulator, is a metabolite of 13-hydroperoxylinolenic acid, derived from alpha-linolenic acid. The volatile methyl ester of JA, methyl jasmonate (MeJA), is also a plant growth regulator. In this study we report the effect of MeJA on aflatoxin production and growth of Aspergillus flavus. MeJA at concentrations of 10(-3)-10(-8) M in the growth medium inhibited aflatoxin production, by as much as 96%. Exposure of cultures to MeJA vapour similarly inhibited aflatoxin production. The amount of aflatoxin produced depended on the timing of the exposure. MeJA treatment also delayed spore germination and inhibited the production of a mycelial pigment. These fungal responses resemble plant jasmonate responses.

Acetates↗

PubMed1979

Aflatoxin inhibition of viral interferon induction.

The inhibitory effect of four basic aflatoxins on interferon induction by influenza virus in LLC-MK2 cell monolayers follows a structure-activity series with decreasing potency in the order aflatoxin B1 greater than G1 greater than B2 approximately G2. Of the four aflatoxins, B1 was the most deleterious to both cell growth and the viability of cells in confluent cultures. The fact that higher levels of influenza virus growth were attained in aflatoxin-treated cells than in normal cell monolayers was related to increased aflatoxin concentration in association with decreased interferon production. The ability of interferon to confer cellular resistance against viral infection, however, was not altered by aflatoxin. The inhibitory activity of aflatoxin on interferon production may be a factor contributing to impairment of host resistance to viral infections.

Aflatoxins↗

PubMed1978

Inhibition of aflatoxin biosynthesis by tolnaftate.

Tolnaftate [2-napthyl-N-methyl-N-(m-tolyl)thionocarbamate], an antifungal drug, is widely used to control superficial fungal infections in humans and other animals. In this study the effect of tolnaftate on aflatoxin biosynthesis by Aspergillus parasiticus NRRL 3240 was investigated. Tolnaftate changed the morphology of A. parasiticus to yeastlike forms and inhibited aflatoxin formation. The formation of aflatoxin G was blocked considerably, indicating a metabolic block in the conversion of aflatoxin B to aflatoxin G. The incorporation of [1-14C]acetate into aflatoxin was significantly inhibited at a concentration of 1 mM tolnaftate. The presence of zinc in the resuspension buffer resulted in reversal of the tolnaftate-induced inhibition of aflatoxin G1 biosynthesis.

Acetates↗

PubMed1987

Sodium bicarbonate reduces viability and alters aflatoxin distribution of Aspergillus parasiticus in Czapek's agar.

The potential of sodium bicarbonate to inhibit growth of and aflatoxin synthesis by Aspergillus parasiticus was examined in Czapek's agar (CA), a medium in which fluorescence under UV light indicates aflatoxin production. Incorporation of sodium bicarbonate (SB) into CA at 0.011, 0.022, and 0.033 mol% reduced cell viability 63-, 10(3)-, and greater than 10(7)-fold, respectively. Colonies resulting from surviving cells did not fluoresce under UV light, but thin-layer chromatography analysis of culture extracts detected aflatoxins. Potassium bicarbonate (KB) at 0.011 and 0.022 mol% produced inhibitory effects similar to those of SB, but NaCl and silica had no effect. After 7 days, control cultures had the normal aflatoxin distribution (B1 greater than G1 greater than B2 greater than G2), but this distribution shifted to B2 greater than B1 approximately equal to G2 greater than G1 during prolonged incubation. Cultures supplemented with SB and KB contained mostly aflatoxins B1 and G1 after 28 days. Both SB and KB raised the pH of CA to 7.5 to 8.5 at the time of growth. Culture growth on CA adjusted to pH 7.5 to 8.5 with NaOH was not inhibited but exhibited reduced fluorescence and elevated levels of aflatoxins B1 and G1. Thus, while bicarbonate inhibition of growth could not be attributed to pH elevation, the lack of culture fluorescence on CA-SB and CA-KB and the altered aflatoxin distribution were caused by the ability of SB and KB to elevate pH.

Aflatoxins↗