Aflatoxin Binders I: In vitro binding assay for aflatoxin B1 by several potential sequestering agents

Nine potential proprietary sequestering agents consisting of 4 activated charcoals, 3 sodium bentonites, a calcium bentonite, and an esterified glucomannan were compared in a novel in vitro assay for aflatoxin B1 (AFB1) binding. Agents were evaluated in 10% methanol prepared as 1% stirred suspensions at pH 3, 7, 10 and pH-unadjusted, with or without AFB1 at 5 g/ml. All nine agents bound more than 95% of the 5 g of AFB1 in solution, regardless of pH. The sodium bentonites bound 98, 95, and 98% of the AFB1. The four activated charcoals bound over 99%, the calcium bentonite bound 98%, and the esterified glucomannan bound 97% of the AFB1 in solution. The results suggested that the sequestering agents tested here had sufficient potential to bind AFB1 at pH values commonly found in the gastrointestinal tracts of ruminants and other animals.This revised version was published online in October 2005 with corrections to the Cover Date.     

Carryover of aflatoxin from feed to milk in dairy cows with low or high somatic cell counts

Aflatoxin M1 (AFM1) residues in milk are regulated in many parts of the world and can cost dairy farmers significantly due to lost milk sales. Additionally, due to the carcinogenicity of this compound contaminated milk can be a major public health concern. Thirty-four lactating dairy cows were utilised to investigate the relationship between somatic cell counts (SCC), milk yield and conversion of dietary aflatoxin B1 (AFB1) into milk AFM1 (carryover (CO)). The AFM1 in milk increased as soon as the first milking after animal ingestion with a pattern of increment up to the observed plateau (between 7th and 12th days of AFB1 ingestion). There was a significant (P < 0.01) effect of the milk yield whereas no effect could be attributed to the SCC levels or to the milk yield × SCC interaction. Similarly, the main effect of milk yield was also observed (P < 0.01) on the total amount of AFM1 excreted during the ingestion period. Although the plasma concentration of gamma-glutamyl transferase was significantly affected by aflatoxin administration, levels of this liver enzyme were within the normal range for lactating dairy cows. The current data suggest that milk yield is the major factor affecting the total excretion of AFM1 and that SCC as an indicator of mammary gland permeability was not related to an increase in AFM1 CO.     

Aflatoxin binders I: in vitro binding assay for aflatoxin B1 by several potential sequestering agents

Nine potential proprietary sequestering agents consisting of 4 activated charcoals, 3 sodium bentonites, a calcium bentonite, and an esterified glucomannan were compared in a novel in vitro assay for aflatoxin B1 (AFB1) binding. Agents were evaluated in 10% methanol prepared as 1% stirred suspensions at pH 3, 7, 10 and pH-unadjusted, with or without AFB1 at 5 microg/ml. All nine agents bound more than 95% of the 5 microg of AFB1 in solution, regardless of pH. The sodium bentonites bound 98, 95, and 98% of the AFB1. The four activated charcoals bound over 99%, the calcium bentonite bound 98%, and the esterified glucomannan bound 97% of the AFB1 in solution. The results suggested that the sequestering agents tested here had sufficient potential to bind AFB1 at pH values commonly found in the gastrointestinal tracts of ruminants and other animals.     

Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.     

Comparison of urinary aflatoxin M1 and aflatoxin albumin adducts as biomarkers for assessing aflatoxin exposure in Tanzanian children

Purpose: To determine levels of urinary aflatoxin M1 (AFM1) in children and correlate the concentrations with previously reported aflatoxin albumin adduct (AF-alb) levels in these children.

Materials and methods: Matched urine and blood samples were collected from 84 Tanzanian children aged 6–14 months old. From 31 children in one village (Kigwa), samples were collected at three time points six months apart. Samples were collected from 31 and 22 children from two different regions at the second time point only. Urinary AFM1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol to improve sensitivity. AF-alb was measured using an established ELISA method.

Results: The relative ranking of the three villages for exposure to aflatoxin based on either AFM₁ or AF-alb biomarker measurements was the same. In Kigwa village, both AFM1 and AF-alb levels were higher at six months post-harvest compared to baseline. However, at the next visit, the AFM1 levels dropped from a GM (interquartile range) of 71.0 (44.7, 112.6) at visit two to 49.3 (31.5, 77.3) pg/ml urine, whereas AF-alb levels increased from 47.3 (29.7, 75.2) to 52.7 (35.4, 78.3) pg/mg albumin between these two visits, reflecting the fact that AFM1 measures short-term exposure, whereas AF-alb measures longer term exposure. There was a correlation between AFB1 intake and AFM1 excretion (r=?0.442, p?≤?0.001).

Conclusions: Urinary AFM1 is a good biomarker for AFB1 exposure in Tanzanian children, reflecting geographical and temporal variations in exposure to this foodborne toxin.     

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat,mouse and pig

[¹⁴C]Aflatoxin B₁ (AFB₁) was isolated from cultures of Aspergillus parasiticus grown on [1-¹⁴C]sodium acetate. Covalent binding of AFB₁ to liver DNA of rat and mouse was determined 6–8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a ‘Covalent Binding Index’ (CBI), (μmol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors.The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable.Aflatoxin M₁ (AFM₁) is a metabolite found in the milk of cows that have been fed AFB₁-contaminated diet. [¹⁴C]AFM₁ was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM₁ must also be regarded as a strong hepatocarcinogen. It is concluded that AFB₁ contaminations should be avoided in dairy feed.     

Automated column-switching high-performance liquid chromatography for the determination of aflatoxin M1

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFM1 recovery reached 97% in the 10–100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.     

Effectiveness of sodium bentonite and two commercial products as aflatoxin absorbents in diets for broiler chickens

Three hundred day-old commercial broiler chicks were reared in 10 groups to examine the effects of indigenous sodium bentonite (SB) as a sodium bentonite simple, sodium bentonite + gention violet and sodium bentonite + acetic acid on two levels 0.5 and 1% of feed, along with two market products Sorbatox and Klinofeed on two levels of aflatoxin 0 and 100 mcg/kg. Each group was replicated three times. The aflatoxins (AF) were incorporated in a standard commercial broiler ration and fed to chicks for 6 week. Body weight and feed consumption was recorded weekly. At the end of the experiment two birds from each group were slaughtered to note the relative weight of heart, liver, gizzard and dressing percentage. Titre against Newcastle disease (ND) was monitored at day 21st, 28th, 35th and 42nd of the experiment.Statistical analysis indicated that aflatoxin in diet negatively affected (P<0.05) all the parameters. Chicks receiving aflatoxin-contaminated diet had suppressed body weight, feed consumption and FCR value, which was significantly improved with the addition of 0.5% simple sodium bentonite. The relative weight of liver (5.34%), heart (0.72%), gizzard (2.05%) and mortality (40%) increased significantly with the addition of 100 mcg/kg aflatoxin and were restored to 3.57, 0.57, 1.43 and 16.6%, respectively, with the dietary inclusion of 0.5% simple sodium bentonite. However, dressing percentage significantly decreased (51.7%) when diet (B) was supplemented with 100 mcg/kg aflatoxin as compared to the diet where no aflatoxin was added (diet A). The titre against Newcastle disease (ND) also showed poorest results by addition of aflatoxin which also improved by inclusion of 0.5% simple sodium bentonite. Addition of indigenous sodium bentonite gave overall better results than the market products and between two levels of indigenous sodium bentonite, 0.5% level gave better results than 1%.     

Immunodetection and immunopurification of aflatoxins using a high affinity monoclonal antibody to aflatoxin B1

A monoclonal antibody was obtained from BALB/c mice immunized with aflatoxin Bl (AFB1) conjugated to bovine serum albumin. This IgG2a antibody, ASCI, with K light chain has a high specificity for AFB1. In an indirect enzyme-linked immunosorbent assay the antibody litre in ascites fluid was 1: 6000 for 50% binding to plates coated with aflatoxin-poly-L-lysine. The assay is sensitive to 2.5 pg aflatoxin/assay. ASCI cross-reacts with closely related aflatoxin metabolites such as AFB2, AFM1 and AFG1. However, ASCI displays negligible cross-reactivity with other related aflatoxin analogues such as AFM2, AFP1, AFQ1 and aflatoxicol. An immunoabsorbent was prepared by coupling ASCI antibody to Ultrogel AcA 22. This immunomatrix was used to purify aflatoxins at 0–1 ng/ml levels from contaminated body fluids such as bovine milk. The antibody affinity column was regenerated and re-used several times. Owing to its high specificity for AFB1 and AFM1, ASCI will be of value in immunodetection and immunopurification of these toxins in various foodstuffs.     

Mycoflora and incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina

In Argentina, there is rather little information about the natural occurrence of mycotoxins in feedstuffs. The aim of this work was to determine the fungal flora and natural incidence of aflatoxin B1 (AFB1), zearalenone (ZEA) and deoxynivalenol (DON) in poultry feeds from 5 factories of Río Cuarto, Córdoba. Three hundred samples were taken from May 1995 to May 1996. Fungal counts of poultry feeds ranged 104 to 106 CFU g-1. The lowest counts were obtained on the first months from the sampling (May to September 1995) with mean values significantly different from those found at the last of the sampling (October 1995 to April 1996). The most prevalent species isolated of poultry feed samples belonged to the genera Penicillium that was present in 98% of the samples, Fusarium (87%) and Aspergillus (52%). Fusarium species isolated were: F moniliforme in 73% of the samples, F subglutinans (35%), F graminearum (20%) and within Aspergillus species: A. parasiticus (33%) and A. flavus (8%) were identified. In poultry feeds aflatoxin B1 (AFB1) was the most significant mycotoxin with levels ranging from 17 to 197 ng/g. For deoxynivalenol (DON) the levels ranged from 240 to 410 ng/g. Only three out of 300 samples were contaminated with zearalenone (ZEA) in concentrations of 30, 120 and 280 ng/g. These are preliminary data on this subject in our region. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular modeling studies on the interactions of aflatoxin B1 and its metabolites with the peripheral anionic site of human acetylcholinesterase

Aflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus. Among them, aflatoxin B1 (AFB1) is the most frequent type in nature and the most carcinogenic for mammals. It can contaminate many kinds of food like seeds, oil, olives, milk, dairy products, corn and meat, causing acute and chronic damages to the organism, especially in the liver, being, for this reason, considered highly hepatotoxic. AFB1 is also a mixed inhibitor of the enzyme acetylcholinesterase (AChE). This fact, together with its high toxicity and carcinogenicity, turns AFB1 into a potential chemical and biological warfare agent, as well as its metabolites. In order to investigate this, we performed inedited molecular modeling studies on the interactions of AFB1 and its metabolites inside the peripheral anionic site of human AChE (HssAChE), to verify their stability, suggest the preferential ways of inhibition, and compare their behavior to each other. Our results suggest that all metabolites can be better inhibitors of HssAChE than AFB1 and that AFBO and AFM1, the most toxic and carcinogenic metabolites of AFB1, are also the most effective HssAChE inhibitors among the AFB1 metabolites.

Communicated by Ramaswamy H. Sarma     

A comparison of aflatoxin B1-induced cytotoxicity,mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains.Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538.In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains.These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.     

Occurrence of aflatoxin B1 in dairy cow feed over 10 years in Portugal (1995-2004)

In Portugal, there is rather little information about the natural occurrence of aflatoxin in feedstuffs. The aim of this work was to report the results of screening the natural incidence of aflatoxin B1 (AFB1) in samples of cattle feed collected from seven dairy cow's farms from Portugal distributed in several locations of the country. One thousand and one samples were taken from 1995 to 2004. High performance liquid chromatography (HPLC) was used for separation, identification and quantification of the compound. Detection limit was 1 microg/kg. Aflatoxin B1 was detected in 374 (37.4%) of the samples. The incidence and mean content of AFB1 was generally low. Levels of aflatoxin B1 above the maximum limit established in Portugal (5 microg/kg) for dairy cattle feed samples were observed in 62 samples (6.2%) with levels ranging from 5.1 to 74 microg/kg. Out of those 62 samples, 3.7% had levels between 5.1 to 10 (mean 7.8); 1.8% had a contamination level of 10.1 to 20 (mean 12.0), and 0.7% exceeded 20.1 microg/kg (mean 50.4). On the last two years (2003-04) none of the samples exceeded the maximum permissible level of the toxin.     

The effects of rumen fluid on the in vitro aflatoxin binding capacity of different sequestering agents and in vivo release of the sequestered toxin

In an in vitro experiment, aluminosilicates (Atox^® and Novasil™ Plus) and a yeast cell wall derivate (Mycosorb^®) were used as sequestering agents (SAs) to verify their capacity for binding aflatoxin B1 (AFB1) in vitro. SAs were individually mixed at three different ratios with AFB1 (1:5000, 1:50,000 and 1:500,000, w/w) in water (CTR), rumen fluid from a lactating cow with a low rumen pH (LRS) or rumen fluid from a dry cow with a high rumen pH (HRS), and then used in a 3 × 3 × 3 factorial arrangement of a completely randomized design. At the 1:500,000 AF:SA ratio Atox^® and Novasil™ Plus sequestered over 0.87 and 0.98 of the AFB1 in the CTR and rumen solutions (LRS and HRS), respectively. This efficacy decreased when the amount of clays was reduced, with higher values (P<0.001) for Atox^® compared with Novasil™ Plus (0.50 vs. 0.28 in CTR; 0.58 vs. 0.16 in LRS and 0.44 vs. 0.27 in HRS). Mycosorb^® had a lower sequestering efficacy (P<0.001) in all of tested experimental conditions, with 0.34 being the maximum value obtained in the CTR solution.     

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.     

Impedimetric nanoimmunosensor platform for aflatoxin B1 detection in peanuts

This article developed a novel electrochemical immunosensor for the specific detection of aflatoxin B1 (AFB1). Amino-functionalized iron oxide nanoparticles (Fe₃O₄-NH₂) were synthesized. Fe₃O₄-NH₂ were chemically bound on self-assembly monolayers (SAMs) of mercaptobenzoic acid (MBA). Finally, polyclonal antibodies (pAb) were immobilized on Fe₃O₄-NH₂-MBA. The sensor system was evaluated through atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). A reduction in the anodic and cathodic peak currents was observed after the assembly of the sensor platform. The charge transfer resistance (R_ct) was increased due to the electrically insulating bioconjugates. Then, the specific interaction between the sensor platform and AFB1 blocks the electron transfer of the [Fe(CN)₆]^3−/4− redox pair. The nanoimmunosensor showed a linear response range estimated from 0.5 to 30 μg/mL with a limit of detection (LOD) of 9.47 μg/mL and a limit of quantification (LOQ) of 28.72 μg/mL for AFB1 identification in a purified sample. In addition, a LOD of 3.79 μg/mL, a LOQ of 11.48 μg/mL, and a regression coefficient of 0.9891 were estimated for biodetection tests on peanut samples. The proposed immunosensor represents a simple alternative, successfully applied in detecting AFB1 in peanuts, and therefore, represents a valuable tool for ensuring food safety.     

Hyperplasia, partial hepatectomy, and the carcinogenicity of aflatoxin B1

Generalized cellular hyperplasia has long been associated as a factor in the causation of liver cancer. Parenchymal cell hyperplasia resulting from hepatotoxins, viruses, parasites, or malnutrition is exceedingly variable as to when it occurs, its extent, and its duration. Partial hepatectomy has been used as an experimental tool precisely because the timing and extent of hyperplasia can be known and controlled. With regards to aflatoxin B1 (AFB1) carcinogenesis, partial hepatectomy has produced variable results. An explanation appears to reside in the hepatotoxic properties of AFB1 that enhance the early stages of carcinogenesis.     

The toxicity and decreased concentration of aflatoxin B in natural lactic acid fermented maize meal

AIMS: Aflatoxin B(1) (AFB(1)) is a mycotoxin which is known to frequently contaminate poorly stored food products destined for human consumption. This study was carried out to investigate the potential activity of lactic acid fermentation in reducing AFB(1) level in fermented maize meal products. METHODS AND RESULTS: Maize meal was spiked with 60 mug g(-1) AFB(1) and fermented, with or without starter culture, for 4 days at 25 degrees C. Unbound AFB(1) in solution and the pH of the media were monitored daily. A significant decrease (P < 0.05) in the level of unbound AFB(1) was observed (75% in the fourth day). Simultaneously, a progressive decrease in the pH of the media from 6.5 to 3.1 was also observed. AFB(1) was below the detection limit in commercial fermented porridge (amahewu) samples. Cytotoxicity tests on AFB(1)-spiked fermented extracts showed that those with a starter culture were comparatively less toxic (30-36%) than those with no added starter culture (24-30%). However, this difference was not significant (P > 0.05). CONCLUSIONS: These results indicate that lactic acid fermentation can significantly reduce the concentration of AFB(1) in maize to trace levels. However, the safety of fermented products has not been well studied, as the mechanism of AFB(1) removal is not well understood. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural fermentation may potentially reduce exposure to natural toxins occurring in food.     

假单胞菌胞外酶降解黄曲霉毒素B1的酶学性质

[背景]黄曲霉毒素B1(AflatoxinB1,AFB1)毒性强、污染普遍,目前尚无有效的防治办法.[目的]为了发掘高效的AFB1降解菌并探索其降解特性,对红树林污泥样品中一株AFB1降解菌株(HAI2)的酶学性质进行分析.[方法]以AFB1结构类似物为唯一碳源,筛选出一株高效的AFB1降解菌,利用16SrRNA基因测...     

Inhibition of the biosynthesis of SRP polypeptides and secretory proteins by aflatoxin B1 can disrupt protein targeting

Cell culture and western blotting studies revealed that aflatoxin B(1) (AFB(1)) inhibits the biosynthesis of two of the constituent polypeptides of signal recognition particle (SRP) (SRP54 and 72). SRP escorts polyribosomes carrying signal peptides from free form in the cytosol to the bound form on endoplasmic reticulum (ER) membrane during protein targeting. These effects of AFB(1) on SRP biosynthesis may inhibit the formation of functional SRP. Our experiments have further shown that AFB(1) also inhibits the biosynthesis/translocation of a secretory protein, preprolactin, which fails to appear in the lumen of ER consequent to the treatment with this hepatocarcinogen. The results of the experiments presented in this article therefore enable us to infer for the first time that aflatoxin B(1) may inhibit the functioning of SRP as an escort and deplete the ER of polyribosomes for secretory protein synthesis. As these secretory proteins are important components of the plasma membrane, gap junctions and intercellular matrix, their absence from these locations could disturb cell to cell communication leading to tumorigenesis.