The effect of aflatoxins on mammalian acid deoxyribonucleases

1. Difference spectroscopy studies indicated that tetrahydrodeoxyaflatoxin B1 and aflatoxicol bind slightly to DNA, whereas aflatoxins B2a, G2a, G2 and aflatoxicol bind to bovine and porcine spleen DNAse II but aflatoxins B1, B2, G1 and tetrahydrodeoxyaflatoxin did not. 2. Kinetic studies showed that aflatoxins B1, G1 and B2 activated bovine and porcine spleen DNAse II while aflatoxins B2a, G2a and G2 had an inhibiting effect. 3. Dissociation constants for the enzyme: substrate-aflatoxin complexes (KAS) as well as the inhibition constants (Ki) were obtained from kinetic studies.     

Stereochemical effect in the mutagenicity of the aflatoxicols toward Salmonella typhimurium

The mutagenicities of [1R] and [1S] aflatoxicol were measured using the Salmonella microsome test. In strain TA100 the [1R] form (unnatural aflatoxicol, aflatoxicol B) had a mutagenic potency approximately four times that of the [1S] epimer (natural aflatoxicol, aflatoxicol A, Ro) in the presence of S-9 liver microsomal fraction. The order in mutagenic potency compared to some other toxicologically important aflatoxins was as follows: B1 greater than [1R] approximately equal to G1 much greater than [1S] much much greater than B2. Thus, the trans relationship between the vinyl ether and hydroxyl groups leads to greater mutagenicity than the cis relationship. This may be important in the elucidation of stereochemical structure-activity relationships for the aflatoxins.     

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.     

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.     

Inhibition of aflatoxin production by selected insecticides

The insecticide naled completed inhibition production of aflatoxins B1, B2, G1, and G2 by and growth of Aspergillus parasiticus at a 100-ppm (100 microgram/ml) concentration. The insecticides dichlorvos, Landrin, pyrethrum, Sevin, malathion, and Diazinon significantly (P = 0.05) inhibited production of aflatoxins at a 100-ppm concentration. However, at a concentration of 10 ppm, significant inhibition in production of aflatoxins was found only with naled, dichlorvos, Sevin, Landrin, and pyrethrum. Dichlorvos, Landrin, Sevin, and naled inhibited growth of A. parasiticus by 28.9 , 18.9, 15.7, and 100%, respectively, at 100 ppm. Stimulation of growth was observed when diazinon was added to cultures. Aflatoxin B1 was most resistant to inhibition by insecticides, followed by G1, G2, and B2, respectively.     

Inhibition of aflatoxin production by selected insecticides.

The insecticide naled completed inhibition production of aflatoxins B1, B2, G1, and G2 by and growth of Aspergillus parasiticus at a 100-ppm (100 microgram/ml) concentration. The insecticides dichlorvos, Landrin, pyrethrum, Sevin, malathion, and Diazinon significantly (P = 0.05) inhibited production of aflatoxins at a 100-ppm concentration. However, at a concentration of 10 ppm, significant inhibition in production of aflatoxins was found only with naled, dichlorvos, Sevin, Landrin, and pyrethrum. Dichlorvos, Landrin, Sevin, and naled inhibited growth of A. parasiticus by 28.9 , 18.9, 15.7, and 100%, respectively, at 100 ppm. Stimulation of growth was observed when diazinon was added to cultures. Aflatoxin B1 was most resistant to inhibition by insecticides, followed by G1, G2, and B2, respectively.     

Effect of aflatoxins on rat peritoneal macrophages

Phagocytosis, intracellular killing of Candida albicans, and superoxide production by rat peritoneal macrophages exposed to aflatoxins B1, B2, G1, G2, B2a, and M1 at several times and concentrations were analyzed to evaluate the intensity of a depressive effect for each mycotoxin. All aflatoxins used at very low concentrations had a depressive effect on the functions of macrophages. The biggest impairment of phagocytosis, intracellular killing, and spontaneous superoxide production was observed in macrophages exposed to aflatoxins B1 and M1.     

Effect of aflatoxins on rat peritoneal macrophages.

Phagocytosis, intracellular killing of Candida albicans, and superoxide production by rat peritoneal macrophages exposed to aflatoxins B1, B2, G1, G2, B2a, and M1 at several times and concentrations were analyzed to evaluate the intensity of a depressive effect for each mycotoxin. All aflatoxins used at very low concentrations had a depressive effect on the functions of macrophages. The biggest impairment of phagocytosis, intracellular killing, and spontaneous superoxide production was observed in macrophages exposed to aflatoxins B1 and M1.     

Development and validation of a method for the quantitative determination of aflatoxin contaminants in Maytenus ilicifolia by HPLC with fluorescence detection

A method for the quantification of aflatoxins B1, G1, B2 and G2 in the medicinal herb Maytenus ilicifolia was developed and validated. The method used immunoaffinity columns for sample clean-up and HPLC with fluorescence detection without any derivatisation step. The method showed good inter-day accuracy (bias values in the range 4.5-10.7%) and precision (5-16% RSD) when applied to the determination of levels of aflatoxins ranging from 7 to 20 ppb in the plant material. The detection limits for samples of the plant material spiked with aflatoxins were 3.5 ng/g for B1 and G1 and 0.1 ng/g for B2 and G2. The method was successfully applied to commercial samples of Maytenus ilicifolia for the screening of aflatoxin contaminants.     

Aflatoxin-selective molecularly-imprinted polymer membranes based on acrylate-polyurethane semi-interpenetrating polymer networks

Synthetic analogs of biological receptors able to group-selective recognition of aflatoxins were obtained using the combination of the technique of molecular imprinting with the method of computer modeling. The synthetic receptors were obtained in a form of thin and porous membranes based on semi-interpenetrating polymer networks. The selection of functional monomers able to noncovalent interactions with aflatoxins B1, B2, and G2 was based on the data of computer modeling. Allylamine, diethylaminoethylmethacrylate, and N,N'-methylenebisacrylamide, providing high binding energies with aflatoxins B1, B2, and G2 were selected as functional monomers for the formation of aflatoxin B1-imprinted polymer membranes. It was shown that aflatoxin-B1-imprinted polymeric membranes synthesized using N,N'-methylenebisacrylamide as a functional monomer were characterized with good physico-mechanical properties as well as good adsorbtion capability towards aflatoxin B1. Neglidgible levels of aflatoxin B1 adsorbtion on the surface of blank membranes were observed. High adsorbtion capability of the MIP membranes towards mycotoxins affiliated to the group of aflatoxins was demonstrated, while negligible adsorbtion of ochratoxin A was observed. Therefore, synthetic analogs of biological receptors able to group-selective recognition of aflatoxins in the range 1-1000 ppb were developed.     

Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

The interaction of aflatoxins with purines and purine nucleosides

From measurements of thermal hyperchromicity and the behaviour of an aflatoxin-DNA mixture on a Sephadex column it was concluded that aflatoxin B(1) is capable of weak binding to single-stranded DNA. The interactions of the aflatoxins (B(1), G(1) and G(2)) with nucleosides result in difference spectra and suggest that the purine bases and the amino group play a role in the binding of all the aflatoxins to DNA.     

Products of spontaneous conjugation of aflatoxins with bovine serum albumin: immunochemical properties

Products of spontaneous conjugation of aflatoxins B1, G1, and G2 with bovine serum albumin (BSA) were shown to interact with antibodies against aflatoxins. Solid-phase BSA conjugates inhibited the binding of aflatoxins by anti-aflatoxin antibodies. Antisera against BSA-B1, BSA-G1, and BSA-G2 were obtained and their specificity, determined. The mechanisms of spontaneous binding of aflatoxins by proteins are discussed.     

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.     

Aflatoxin inhibition of rat liver mitochondrial cytochrome oxidase activity

Aflatoxins B1, B2, G1, G2, and M1 have been evaluated for activity toward cytochrome oxidase in isolated rat liver mitochondria employing ferrocytochrome c and p-phenylene diamine as reductants. The aflatoxins inhibited the cytochrome oxidase activity to a greater extent when monitored by O2 uptake measurements than by substrate oxidation. AFG2 and AFM1 were the most potent (50-70%). Using oligomycin and 2,4-DNP as respiratory inhibitor and uncoupler, respectively, the aflatoxins appear to inhibit e- rather than energy transfer reactions. These toxins did not uncouple cytochrome oxidase activity.     

The influence of L-ascorbic acid on the antibacterial-toxic activity of aflatoxins on adsorbent layer

AIMS: To substantiate the role of formaldehyde (HCHO) and its reaction products in the mechanism of the antibacterial-toxic effect of aflatoxins B1 (AFB1), B2, G1 and G2. MATERIALS AND RESULTS: Toxins were separated by overpressured layer chromatography, which was followed by biological evaluation directly on the adsorbent layer (BioArena system with Pseudomonas savastanoi pv. phaseolicola indicator bacteria). HCHO formed in this system was eliminated with exogenously added capturer molecule dimedone and L-ascorbic acid (AA) and measured as the adduct of dimedone and HCHO. The amount of HCHO was higher in the toxin-containing spots, particularly in the most toxic AFB1 spot, compared to a toxinless background. 0.1 mg ml(-1)AA augmented, 0.2 mg ml(-1) dimedone or 0.5 and 1 mg ml(-1) AA reduced the antibacterial effect of all four aflatoxins. CONCLUSION: The antibacterial-toxic effect of aflatoxins may be mediated by HCHO (and/or its reaction products) generated from bound HCHO forms in the bacterial cells. Basis of antibacterial-toxic activity of the four aflatoxins appears the same. SIGNIFICANCE AND IMPACT OF THE STUDY: Involvement of HCHO as a key molecule in the effect of aflatoxins indicates a totally new mechanism of action of these dangerous molecules. The BioArena system is useful to dissect the mode of action of antimicrobial compounds from different biological matrices.     

Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts

An ultrasensitive immunochromatographic (IC) assay for simultaneous detection of total aflatoxins (AFB1, AFB2, AFG1, and AFG2) was developed to meet the requirement for rapidly monitoring aflatoxins in agro-products. The assay was based on a competitive format and its sensitivity was improved by using a novel criterion to screen the optimal amount of monoclonal antibody (MAb) labeled to nanogold particles. The visual detection limits (VDLs) for aflatoxins B1, B2, G1, and G2 in peanut matrix were 0.03, 0.06, 0.12, and 0.25 ng mL(-1), respectively, which were lower than those of published literatures. The results of IC assay were in good agreement with those of high performance liquid chromatography (HPLC) in the analysis of aflatoxins in peanuts, demonstrating the practical applicability of the developed assay in real samples. This qualitative test based on the visual evaluation of results did not require any equipment. Overall, to our knowledge, this is the first report of qualitative detection for total aflatoxins by immunochromatographic assay.     

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Aflatoxin Production in Meats. I. Stored Meats

Aflatoxins were produced on fresh beef (in which bacterial spoilage was delayed with antibiotics), ham, and bacon inoculated with toxinogenic fungi and stored at 15, 20 and 30 C. Meats stored at 10 C were spoiled by bacteria and yeast before detectable levels of aflatoxins were produced. High levels of aflatoxins were formed in meats stored at 20 C; one sample supported the production of 630 mug of aflatoxins per g of meat, the major portion (580 mug) of which was aflatoxin G(1). Meats stored below 30 C developed higher levels of aflatoxin G(1) than B(1), but at 30 C Aspergillus flavus produced equal amounts of B(1) and G(1), whereas A. parasiticus continued to produce more G(1) than B(1).