Failure of plant tissues to metabolize aflatoxin B1?

After exposure of peas and wheat kernels to aflatoxin B₁ solutions the following aflatoxins could not be detected in seed extracts: aflatoxins M₁, B_2a, Q₁, aflatoxicol and tetrahydrodeoxyaflatoxin B₁.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

Synergistic effect ofSacoglottis gabonensis bark extract,

Aflatoxin B₁ metabolism was studied using microsomal and cytosolic fractions isolated from weanling male Fischer F344 rats given in drinking water for 7 days an aqueous extract ofSacoglottis gabonensis bark, 0.1% ethanol solution, or a solution containing both extract and ethanolad libitum. Microsomal production of aflatoxin B₁-dihydrodiol, aflatoxin Q₁, aflatoxin M₁, aflatoxin Pi and proteinaflatoxin adduct formation, and cytosolic aflatoxin B₁-glutathione conjugation were assayed. Pretreatment with the extract alone or together with ethanol caused significant increases in aflatoxin M₁ production as compared to controls given only water, but aflatoxin Q₁ production was enhanced only by pretreatment with both extract and ethanol. All the three treatments caused significant reductions in liver glutathione content. The highest aflatoxin B₁ metabolising activity as determined by aflatoxin M₁ and aflatoxin Q₁ production was observed in rats pretreated with both ethanol and the extract, suggesting synergism. The findings suggest that at relatively mild doses,S. gabonensis extract alone or in concert with ethanol may influence response to aflatoxin.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Reduction of aflatoxin B1 levels by sheep saliva

This study determined the decrease of aflatoxin B₁ by sheep saliva at concentrations of 150 and 300 μg aflatoxin B-₁/L saliva. Analyses for aflatoxins B₁, M₁, and aflatoxicol (R₀) were performed after 2, 4, 6, 24, and 48 hours of incubation. Aflatoxin M₁ and R₀ were not detected and only residues of aflatoxin B₁ were found. 4 to 13% of aflatoxin B₁ were decomposed by sheep’s saliva within 2 hrs and 33 to 43% of aflatoxin B₁ after 24 hrs. Decomposition was affected by the aflatoxin concentration. Decrease of aflatoxin B₁ at 2, 4, 6 hrs was nearly three times higher at the low concentration (150 ppb) compared to the high concentration (300 ppb). After 48 hrs incubation more than 80% of the initial aflatoxin B₁ had been decomposed by the saliva.     

An enzyme immunoassay system for aflatoxin B1

Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B₁ carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B₁ with a low relative cross-reactivity against aflatoxin B₂, G₁, G₂, M₁ B_2a, and G_2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.     

Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei

This study evaluated the ability of the microorganisms Rhizopus oryzae (CCT7560) and Trichoderma reesei (QM9414), producers of generally recognized as safe (GRAS) enzymes, to reduce the level of aflatoxins B₁, B₂, G₁, G₂, and M₁. The variables considered to the screening were the initial number of spores in the inoculum and the culture time. The culture was conducted in contaminated 4 % potato dextrose agar (PDA) medium, and the residual mycotoxins were determined every 24 h by HPLC-FL. The fungus R. oryzae has reduced aflatoxins B₁, B₂, and G₁ in the 96 h and aflatoxins M₁ and G₂ in the range of 120 h of culture by approximately 100 %. The fungus T. reesei has reduced aflatoxins B₁, B₂, and M₁ in the 96 h and aflatoxin G₁ in the range of 120 h of culture by approximately 100 %. The highest reduction occurred in the middle of R. oryzae culture.     

Liquid Chromatography of Aflatoxins

There are numerous reports on studies of aflatoxins, but there are only a few reports on the isolation and mutual separation of aflatoxins by liquid chromatography. Following the previous reports on the liquid chromatography of four kinds (B₁, B₂, G₁ and G₂) of aflatoxins, the authors carried out the chromatography of six kinds of aflatoxins including aflatoxins B_2a and G_2a. Various kinds of adsorbents and eluting solvents were examined, and then the good mutual separation of aflatoxins was obtained by using the Sephadex G-10 (CM), a newly prepared adsorbent containing carboxymethyl group, and pure water for eluting solvent. Aflatoxins G_2a, B_2a, G₂, B₂, G₁ and B₁ were eluted in this order.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

Aflatoxins B1 and G1 solubility in standard solutions and stability during cold storage

In the present work we study the use of different solvents to store aflatoxins B₁ and G₁ standard solutions. We have obtained significant differences between aflatoxin B₁ and G₁ In ethyl acetate, methanol and water, with aflatoxin G₁ being less stable. We recommend chloroform as the election solvent to store the aflatoxin solutions. The fact that aflatoxins are highly stable in water may have a potential use in experiments of biological activity.     

Specific Features of Albumin Interactions with Hemiacetals of Aflatoxins and Sterigmatocystin

Aflatoxin B_2a (AB_2a), aflatoxin G_2a (AG_2a), and the hemiacetal of sterigmatocystin have been shown to form immunoreactive conjugates with albumin. The conjugates were formed following incubation of solution mixtures at room temperature for 1 h, as demonstrated by spectrophotometry and enzyme immunoassay. Anti-AB_2a antibodies reacted with AB_2a, aflatoxin B₁, and aflatoxin B₂ (100, 8.8, and 5.9%, respectively); a similar result was obtained for anti-AG_2a antibodies reacting with AG_2a, aflatoxin G₁, and aflatoxin G₂ (100, 2.5, and <1.0%, respectively). Binding of anti-AB_2a and anti-AG_2a antibodies to solid-phase conjugates of AB_2a or AG_2a exhibited similar analytical characteristics.     

Evaluation of the mycological status of luncheon meat with special reference to aflatoxigenic moulds and aflatoxin residues

The luncheon meat samples analyzed, which were produced locally by the two main luncheon meat producing companies in Egypt were relatively highly contaminated either by moulds and yeasts in general, aflatoxigenic species and aflatoxin residues in particular. The most frequently encountered fungi from the samples were yeasts, Aspergillus niger, A. flavus, Penicillium chrysogenum, Rhizopus stolonifer, Mucor circinelloides. Less common were Cladosporium sphaerospermum, Alternaria alternata, Mycosphaerella tassiana, P. aurantiogriseum and P. oxalicum. The most important aflatoxigenic species, A. flavus, was isolated frequently. It was 10% of the total fungal isolates from both samples of the two companies. Seven luncheon meat samples out of 50 analyzed were positive for aflatoxin B₁ or B₁ and G₁, while all samples were negative for aflatoxins B₂, G₂, M₁ and M₂. Aflatoxin B₁ was detected only in 4 and 3 samples out of 25 analyzed from each of company A and B, respectively. The highest detectable level, 11.1 ppb, was recorded in a sample from company B and the least, 0.5 ppb, in a sample from company A. Aflatoxin G₁, at concentration of 3.2 ppb, was detected in only one sample of the aflatoxin B₁ – contaminated 3 samples of company B: this sample also had the highest level of aflatoxin B₁. Some luncheon meat samples had higher numbers of aflatoxigenic A. flavus than others, however these samples were negative for aflatoxins. The hazardous potential of such contamination will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid On-Site Sensing Aflatoxin B1 in Food and Feed via a Chromatographic Time-Resolved Fluoroimmunoassay

Aflatoxin B₁ poses grave threats to food and feed safety due to its strong carcinogenesis and toxicity, thus requiring ultrasensitive rapid on-site determination. Herein, a portable immunosensor based on chromatographic time-resolved fluoroimmunoassay was developed for sensitive and on-site determination of aflatoxin B₁ in food and feed samples. Chromatographic time-resolved fluoroimmunoassay offered a magnified positive signal and low signal-to-noise ratio in time-resolved mode due to the absence of noise interference caused by excitation light sources. Compared with the immunosensing performance in previous studies, this platform demonstrated a wider dynamic range of 0.2-60 μg/kg, lower limit of detection from 0.06 to 0.12 µg/kg, and considerable recovery from 80.5% to 116.7% for different food and feed sample matrices. It was found to be little cross-reactivity with other aflatoxins (B₂, G₁, G₂, and M₁). In the case of determination of aflatoxin B₁ in peanuts, corn, soy sauce, vegetable oil, and mouse feed, excellent agreement was found when compared with aflatoxin B₁ determination via the conversational high-performance liquid chromatography method. The chromatographic time-resolved fluoroimmunoassay affords a powerful alternative for rapid on-site determination of aflatoxin B₁ and holds a promise for food safety in consideration of practical food safety and environmental monitoring.     

Destruction of aflatoxins B1 and G1 in bread making

The effect of processing steps as well preservatives used in French bread making namely propionic acid and/or potassium sorbate (0.2%) on the destruction of aflatoxins B₁ and G₁ was studied. Mixing and baking processes showed marked destruction of aflatoxins B₁ and G₁; being 71.2% and 52.5% for aflatoxin B₁ after mixing and baking steps, while reaching 73.9% and 54.5% for aflatoxin G₁. Fermentation step caused additional 15.3% and 15.0% destruction of aflatoxins B₁ and G₁. On the other hand, aflatoxin B₁ destruction was 79.2% and 50.7% when propionic acid was used and 75.3 and 56.7% in the presence of potassium sorbate and after mixing and baking steps respectively. Concerning aflatoxins G₁ it was found that mixing and baking steps showed destruction of 81.9% and 53.4% in the presence of propionic acid and 75.1 and 49.4% in the presence of potassium sorbate in this respective order. Generally, it can be concluded that using propionic acid as preservative appeared to be more effective on the destruction of aflatoxins B₁ and G₁ than potassium sorbate in French bread making.     

Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

We detected biosynthetic activity for aflatoxins G₁ and G₂ in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G₁ and G₂ were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B₁, aflatoxin B₂, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G₂ from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G₁ was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G₁ and G₂. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.     

Biotransformation of sterigmatocystin and absence of aflatoxin biotransformation by blocked mutants ofAspergillus parasiticus

Four blocked aflatoxin mutants were used to test biotransformation of sterigmatocystin and aflatoxins. Three blocked anthraquinone-accumulating mutants (avn-1, avr-1, ver-1) were able to convert sterigmatocystin into both B and G aflatoxins. No conversions of sterigmatocystin were observed with autoclaved controls or with the fourth blocked mutant (fan). Under equivalent resting cell conditions, no interconversion of aflatoxins B₁, B₂, G₁, or G₂ was observed byver-1, fan, or autoclaved controls.     

Aflatoxin contamination of pearl millet during field and storage conditions with reference to stage of grain maturation and insect damage

Aflatoxin contamination in five varieties of pearl millet (ICMH-451, ICMP-50I, ICTP-8203, WCC-75 and ICMV-155) was studied from field and storage conditions in three districts of Andhra Pradesh State, India and the inter-relationships between various parameters such as stage of grain maturation in the field and insect pest infestation in storage in relation to aflatoxin production were evaluated. Aflatoxin contamination was more frequent in the seed samples collected from the fields during rainy season than winter season. All major aflatoxins were isolated from one or the other varieties of pearl millet, whereas aflatoxin G₂ was not commonly observed in the seed samples collected during winter. Among all the varieties tested, ICMH-451 was vulnerable to aflatoxin contamination whereas ICMV-155 was the least susceptible variety. The higher amount of aflatoxins was observed in the matured seed samples followed by pre-matured and milky stage. Among all the toxins reported in the field, aflatoxin B₁ was found in higher concentration (185 (μg/kg) followed by B₂ (105 μg/kg). The four major types of aflatoxins with higher levels (35, 40, 140, 190 μg/kg of G₁, G₂, B₂, B₁ were reported in the rainy season seed samples after six months of storage, whereas aflatoxin G₁ was not observed in any variety of stored seed sample from winter. Statistical analysis revealed that the aflatoxin incidence in relation to different parameters studied was significantly different for each factor. The relationship between aflatoxin contamination and insect damaged-grain clearly indicated that the seed samples with 16-40% of insect damage contained higher amounts of aflatoxins (758 μg/kg). Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007     

Aflatoxins in the autopsy brain tissue of children in Nigeria

Autopsy brain (cerebrum) specimens from 18 kwashiorkor children and 19 children who had died from a variety of other diseases, at the Obafemi Awolowo Teaching Hospital complex, Ile-Ife, Nigeria, were analysed for the presence of aflatoxins using high-performance liquid chromatography. Aflatoxins were detected in 81%, 15 specimens in each group. More than one type of aflatoxin was detected in 14 (37.8%) of all the specimens. Aflatoxin B₁ and its reversible metabolite, aflatoxicol, were detected in 11 brain specimens of patients with kwashiorkor and 6 of those who died of other miscellaneous diseases; out of these 6, two died from measles and its complications. The frequent detection of aflatoxins in the brains of these children and sometimes in multiple forms may suggest that aflatoxins are stored in the brain tissue which could be related to the lipophilic nature of these compounds. These findings also suggest that although many children in the tropics are exposed to aflatoxins, the accumulation of aflatoxin B₁ and aflatoxicol in the brains of kwashiorkor children may be a result of an impaired metabolism of these compounds by these children.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Mycotoxins as human carcinogens—the <Emphasis Type="Italic">IARC Monographs</Emphasis> classification

Humans are constantly exposed to mycotoxins (e.g. aflatoxins, ochratoxins), mainly via food intake of plant and animal origin. The health risks stemming from mycotoxins may result from their toxicity, in particular their carcinogenicity. In order to prevent these risks, the International Agency for Research on Cancer (IARC) in Lyon (France)—through its IARC Monographs programme—has performed the carcinogenic hazard assessment of some mycotoxins in humans, on the basis of epidemiological data, studies of cancer in experimental animals and mechanistic studies. The present article summarizes the carcinogenic hazard assessments of those mycotoxins, especially aflatoxins (aflatoxin B₁, B₂, G₁, G₂ and M₁), fumonisins (fumonisin B₁ and B₂) and ochratoxin A (OTA). New information regarding the genotoxicity of OTA (formation of OTA-DNA adducts), the role of OTA in oxidative stress and the identification of epigenetic factors involved in OTA carcinogenesis–should they indeed provide strong evidence that OTA carcinogenicity is mediated by a mechanism that also operates in humans–could lead to the reclassification of OTA.