Sensitive Quantification of Aflatoxin B1 in Animal Feeds,Corn Feed Grain,and Yellow Corn Meal Using Immunomagnetic Bead-Based Recovery and Real-Time Immunoquantitative-PCR

Aflatoxins are considered unavoidable natural mycotoxins encountered in foods, animal feeds, and feed grains. In this study, we demonstrate the application of our recently developed real-time immunoquantitative PCR (RT iq-PCR) assay for sensitive detection and quantification of aflatoxins in poultry feed, two types of dairy feed (1 and 2), horse feed, whole kernel corn feed grains, and retail yellow ground corn meal. Upon testing methanol/water (60:40) extractions of the above samples using competitive direct enzyme linked immunosorbent assay, the aflatoxin content was found to be <20 μg/kg. The RT iq-PCR assay exhibited high antigen hook effect in samples containing aflatoxin levels higher than the quantification limits (0.1–10 μg/kg), addressed by comparing the quantification results of undiluted and diluted extracts. In testing the reliability of the immuno-PCR assay, samples were spiked with 200 μg/kg of aflatoxin B₁, but the recovery of spiked aflatoxin was found to be poor. Considering the significance of determining trace levels of aflatoxins and their serious implications for animal and human health, the RT iq-PCR method described in this study can be useful for quantifying low natural aflatoxin levels in complex matrices of food or animal feed samples without the requirement of extra sample cleanup.     

Exposure of Rat Offsprings to Aflatoxin Risks Through Suckling Mothers Dosed with aflatoxin B1

Abstract

One source of human exposure to aflatoxins is by ingestion of aflatoxins carried over from feeds into milk and milk products such as cheese and powdered milk, where they appear mainly as aflatoxin M. We carried out a study using rats to establish whether milk from lactating rat mothers dosed with aflatoxin B₁ carried over potent aflatoxin metabolites capable of inducing liver cancer in their offsprings. The results of this study demonstrated that the presence of such metabolites of aflatoxin B₁ in milk and milk products presents serious health hazards especially to children consuming milk from dairy cows feeding on aflatoxin B₁ contaminated animal feeds. As much emphasis, therefore, should be placed on the control regulations on the presence of aflatoxin in animal feeds as is placed on its direct contamination of cereals and nuts.     

Effect of ammoniation on the toxicity of corn artificially contaminated with aflatoxin B1

The effect of an aqueous ammonia treatment of aflatoxin B₁-containing corn was determined on toxicity of the corn administered orally to male Fischer rats. Corn was contaminated with aflatoxin B₁ (5 mg/g corn) and administered per se (2.0 or 4.0 g corn/kg) or after treatment with ammonium hydroxide. Body weight changes, liver weight, hexobarbital sleeping times, hepatic microsomal concentrations of protein and cytochromes P-450 and b₅, and mortality were determined 72 hr after dosing with corn, and serum alkaline phosphatase activity was determined 96 hr after dosing. The ammonia treatment of contaminated corn prevented the changes in these parameters caused by aflatoxin B₁. Ammoniation of corn free of aflatoxin had no adverse effect on the parameters, although ammoniation per se did raise the concentrations of hepatic microsomal protein. The results of the study indicate that ammoniation may prevent acute aflatoxicosis produced by aflatoxin-contaminated corn.     

A Survey Using Normal Phase Hplc of Aflatoxins in Domestic and Imported Foods and Dairy Products in the Ussr

Abstract

An improved normal phase HPLC method has been developed to study the occurrence of aflatoxins B₁, B₂, G₁, G₂ and M₁ in domestic and imported foods. Ether-methanol-water (95:5:1) mobile phase and fluorometric detector with silica gel packed flow cell were used. The detection limit of the method was 0.1 ug/kg for aflatoxin B₁, coefficients of variation were 11% and 8.5% at contamination levels 10 and 100 ug/kg of aflatoxin B₁ respectively. Recoveries of added aflatoxins B₁, B₂, G₁, G₂ for corn ranged from 78% to 88%. This method allows the determination of aflatoxins B₁, B₂, G₁, G₂, and M₁, B_2a, M_2a as well as other aflatoxin metabolites. The method is used in monitoring aflatoxin contamination of foods, the first stage of which is a preliminary screening of samples by TLC method (the detection limit is 1.0 ug/kg for aflatoxin B₁). A survey of the occurrence of aflatoxins B₁, B₂, G₁, G₂, in Soviet domestic and imported cereals, nuts, beans, and oil seeds harvested in 1985-87 (over 4300 samples) as well as aflatoxin M₁ in domestic dairy products (over 250 samples) was carried out using HPLC and TLC methods. It was shown that 26.9% of imported peanuts, 2.2% of corn and 6.2% of barley were contaminated by aflatoxins at levels exceeding the maximum tolerated level established in the USSR (5 ug/kg for aflatoxin B₁ in foods of all kinds excluding baby foods). Maximum concentrations were 3600, 155 and 8.0 ug/kg respectively. As much as 28.3% of domestic cotton seed samples, which were chosen for the analysis due to toxic effects on animals, were also contaminated by aflatoxins. The USSR aflatoxin monitoring system was demonstrated to be effective during this study.     

Protective Effect of Ebselen on Aflatoxin B1‐Induced Cytotoxicity in Primary Rat Hepatocytes

Recent studies have shown that aflatoxin B₁ enhances reactive oxygen species formation and causes oxidative damage, which may ultimately contribute to the cytotoxicity and carcinogenic effect of aflatoxin B₁. Ebselen, 2‐phenyl‐1,2‐benzoisoseleazol‐3(H)‐one, a synthetic seleno‐organic compound has been shown to possess glutathione peroxidase‐like activity and free radical scavenging ability. Thus present study was designed to investigate the protective effect of ebselen on aflatoxin B₁‐induced cytotoxicity in primary rat hepatocytes. Aflatoxin B₁‐induced cytotoxicity and lipid peroxidation were determined by lactate dehydrogenase leakage and malondialdehyde generation, respectively. Intracellular reactive oxygen species level was measured using the fluorescent probe 2′,7′‐dichlorofluorescin diacetate, and the intracellular reduced glutathione concentration was determined with a fluorometric method. Ebselen was found to display a dose‐dependent protective effect on lactate dehydrogenase leakage and malondialdehyde generation caused by aflatoxin B₁ exposure. The results also demonstrate that ebselen efficiently inhibits the intracellular reactive oxygen species formation in aflatoxin B₁‐treated hepatocytes in a dose and time‐dependent manner. It was also noted that ebselen was able to increase the intracellular reduced glutathione concentration, both in the control and in aflatoxin B₁‐treated hepatocytes. The protection of ebselen against aflatoxin B₁ cytotoxicity, however, was not affected by lowering the concentration of intracellular reduced glutathione. The overall data indicate that ebselen possesses a potent protective effect against aflatoxin B₁‐induced cytotoxicity, and the main mechanism involved in the protection may be its strong capability in inhibiting intracellular reactive oxygen species formation and preventing oxidative damage.     

Survey of aflatoxin contamination in peanut products in Taiwan from 1997 to 2011

Aflatoxins are toxic, mutagenic, and carcinogenic compounds that contaminate various types of foods and feedstuffs. The aflatoxin levels in various kinds of peanut products in Taiwan were surveyed during the recent decade. A total of 1827 commercial peanut products were collected from different regions of Taiwan from 1997 to 2011. The samples were cleaned by immunoaffinity columns and analyzed for aflatoxins B₁, B₂, G₁, and G₂ by high-performance liquid chromatography with fluorescence detection. Aflatoxins were detected in 32.7% of samples with levels ranging from 0.2 μg/kg to 513.4 μg/kg. Peanut butter had the highest aflatoxin-positive incidence, followed by peanut flour, peanut candy, rice syrup, and peanuts. In addition, 6.8% of the samples had total aflatoxin concentrations (i.e., the sum of B₁ + B₂ + G₁ + G₂) that were higher than the Taiwan regulatory limit of 15 μg/kg. Among the aflatoxin-positive samples, aflatoxin B₁ had the highest frequency of detection, followed by aflatoxin B₂, aflatoxin G₂, and aflatoxin G₁. This longterm survey provides valuable information on aflatoxin contamination in peanut products marketed in Taiwan.     

Distribution of [14C]-Labelled Aflatoxin B1 in Mice

Abstract The distribution of [¹⁴C]-labelled aflatoxin B₁ has been studied in mice with the aid of whole-body autoradiography. In addition to the localisation of labelled aflatoxin B₁ and/or its metabolites in the liver, bile, kidney, lung and urine an uptake of ¹⁴C in the pigment of the Harderian gland and the eye was observed. Uptake of radioactivity was also found in the eyes of the foetuses although their livers did not accumulate radioactivity.     

Aflatoxin B1 epoxidation catalysed by partially purified human liver lipoxygenase

(1) This study demonstrates for the first time the human liver lipoxygenase-mediated co-oxidation of aflatoxin B₁ to the reactive metabolite, aflatoxin B₁-8,9-epoxide, which rapidly hydrolyzes to dihydrodiol and preferentially binds to Tris. (2) The Tris- diol complex formed was quantitated fluorimetrically, based on its characteristic excitation at λ_ex 395 nm and emission at λ_em 435 nm. (3) The incubation of partially purified human liver lipoxygenase for 30?min under optimum assay conditions (3.5 mM linoleic acid and 50 μM aflatoxin B₁ in Tris buffer atpH 7.2) resulted in the formation of 10 ± 1.7 nmol Tris- diol/mg protein. (4) In addition to linoleic acid, other unsaturated fatty acids namely γ-linolenic acid, cis-11,14-eicosadienoic acid and arachidonic acid also supported the lipoxygenase mediated epoxidation of aflatoxin B₁. (5) The enzymatic Tris- diol formation was significantly inhibited by all the lipoxygenase inhibitors tested in a concentration-dependent manner. (6) These results strongly suggest that lipoxygenase is capable of aflatoxin B₁ metabolism and this may represent yet another pathway for the bioactivation of this hepatocarcinogen in the human liver.     

Determination of aflatoxin B1 levels in Iranian rice by ELISA method

This study was carried out to detect the presence of aflatoxin B₁ (AFB₁) in 40 samples of Tarom rice from Iran. Enzyme-linked immunosorbent assay (ELISA) was applied to analyze AFB₁ in the samples. All the analyses were conducted twice. Aflatoxin B₁ was found in all rice samples, the concentration of AFB₁ ranged from 0.29 to 2.92?µg/kg. The AFB₁ concentration mean in the rice samples produced in 2013 was higher (P < 0.05) than the findings in rice in 2012.. However, 25 of the 40 samples exceeded the maximum prescribed limit, i.e. 2?µg/kg of European Union Regulations and also none of the samples reached the maximum prescribed limit 5?µg/kg of the Institute of Standards and Industrial Research of Iran (ISIRI) for aflatoxin B₁. Although, rice is ranked the second among cereal staples consumed food in Iran and many countries, it can make a serious health problem for people even for a small amount of aflatoxin.     

Biochemical Changes Induced in Liver and Serum of Aflatoxin B1‐Treated Male Wistar Rats: Preventive Effect of Picroliv

Abstract: Administration of aflatoxin B₁ to rats (2 mg/kg intraperitoneally) caused significant increase in the activities of γ‐glutamyl transpeptidase, 5′‐nucleotidase, acid phosphatase, acid ribonuclease as well as content of lipid peroxides in liver after six weeks. However, the activities of succinate dehydrogenase, glucose‐6‐phosphatase, catalase, superoxide dismutase, glutathione‐S‐transferase, glutathione peroxidase and glutathione reductase in liver were decreased. The levels of glycogen and reduced glutathione were also decreased. There were significant elevations in the levels of serum transaminases, phosphatases (acid and alkaline), dehydrogenases (sorbitol, lactate and glutamate) and bilirubin following aflatoxin B₁ administration. Picroliv (25 mg/kg/day orally for six weeks), an iridoid glycoside isolated from the roots and rhizomes of Picrorhiza kurroa, significantly prevented the biochemical changes induced by aflatoxin B₁.     

Pharmacokinetic evaluation and studies on the clinical efficacy of guar gum‐–based oral drug delivery systems of albendazole and albendazole‐β‐cyclodextrin for colon‐targeting in human volunteers

The present investigation assessed the in vivo properties of the guar gum–based colon‐targeted matrix tablets of albendazole‐β‐cyclodextrin (KA), albendazole matrix tablet (GA), and an immediate‐release albendazole tablet (CA) in humans. A single oral dose was administered in healthy human volunteers and a completely randomized, two‐way, three‐period crossover design was adopted. The data were statistically analyzed and a value of P<0.05 was considered statistically significant. In healthy human volunteers, guar gum colon‐targeted tablets showed delayed t_max and absorption time, decreased absorption rate constant, and unaltered t_1/2indicating that albendazole is not released in stomach and small intestine, but is delivered to the colon, resulting in a slow absorption of the drug and making the drug available for local action in the colon. The increase in C_max and AUC_0‐∞ of KA shows that the bioavailability of albendazole in humans could be definitely improved by complexing the drug with β‐cyclodextrin. Furthermore, an open, parallel, single‐blind clinical study was conducted in patient volunteers who had helminthiasis. Clinical studies showed that the guar gum colon‐targeted tablets could reduce eggs per gram faster than conventional tablets could, resulting in improved clinical symptoms, Hb content, and decreased total count as compared to conventional tablets of albendazole. The investigation shows that albendazole, when complexed with β‐cyclodextrin, improves in vivo bioavailability of the drug and colon targeting using guar gum ensures drug delivery in the colonic fluids, and therefore improves clinical efficacy in human beings with helminthiasis. Drug Dev. Res. 67:154–165, 2006. © 2006 Wiley‐Liss, Inc.     

Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells

To screen cost‐effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC₅₀ values by the alamar blue assay in the DEF cells had a high correlation (R² = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC₅₀ values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC₅₀ for aflatoxin B₁, fumonisins B₁ and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml^–1, respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC₅₀ values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species‐specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Effects of Dietary β‐Cyclodextrin in Hypercholesterolaemic Rats

Abstract: β‐Cyclodextrin is a compound that forms inclusion complexes with a variety of molecules, specially bile acids and sterols. This study examines the effects of β‐cyclodextrin on cholesterol and bile acid metabolism in hypercholesterolaemic rats. Male Wistar rats were divided into 4 groups that received during 7 weeks: control diet, 2% cholesterol diet (A), A+2.5% β‐cyclodextrin (B) and A+5% β‐cyclodextrin (C). The cholesterol‐rich diet induced hepatomegaly and fatty liver and significantly reduced cholesterol, bile acid and phospholipid secretion. Addition of β‐cyclodextrin normalised biliary lipid secretion. Moreover, when compared to A, β‐cyclodextrin significantly lowered plasma phospholipid concentration (B: ?21%; C: ?29%) and the liver free/total cholesterol molar ratio (B: ?40%; C: ?38%), increased bile acid faecal output (B: +17%; C: +62%) and enhanced cholesterol 7α‐hydroxylase activity (B:+50%; C: +100%) and mRNA levels (B: +14%; C: +29%). 5% β‐cyclodextrin also reduced plasma triglycerides concentration (?38%). However, ALT and AST activities were significantly increased (B: +140% and +280%; C: +72% and +135%) and there was a high incidence of cell necrosis with portal inflammatory cell infiltration. Addition of β‐cyclodextrin to a cholesterol‐rich diet results in a triglyceride‐lowering action, enhancement of bile acid synthesis and excretion, and normalization of biliary lipid secretion, but produces a marked hepatotoxic effect.     

Effects of a Calcium Bentonite Clay in Diets Containing Aflatoxin when Measuring Liver Residues of Aflatoxin B1 in Starter Broiler Chicks

Research has shown success using clay-based binders to adsorb aflatoxin in animal feeds; however, no adsorbent has been approved for the prevention or treatment of aflatoxicosis. In this study, growth and relative organ weights were evaluated along with a residue analysis for aflatoxin B₁ in liver tissue collected from broiler chickens consuming dietary aflatoxin (0, 600, 1200, and 1800 µg/kg) both with and without 0.2% of a calcium bentonite clay additive (TX4). After one week, only the combined measure of a broiler productivity index was significantly affected by 1800 µg/kg aflatoxin. However, once birds had consumed treatment diets for two weeks, body weights and relative kidney weights were affected by the lowest concentration. Then, during the third week, body weights, feed conversion, and the productivity index were affected by the 600 µg/kg level. Results also showed that 0.2% TX4 was effective at reducing the accumulation of aflatoxin B₁ residues in the liver and improving livability in birds fed aflatoxin. The time required to clear all residues from the liver was less than one week. With evidence that the liver’s ability to process aflatoxin becomes relatively efficient within three weeks, this would imply that an alternative strategy for handling aflatoxin contamination in feed could be to allow a short, punctuated exposure to a higher level, so long as that exposure is followed by at least a week of a withdrawal period on a clean diet free of aflatoxin.     

Toxicological study of aflatoxin P 1 using the fertile chicken egg

Aflatoxin P₁, the desmethyl metabolite of aflatoxin B₁ in the monkey, was injected into fertile chicken eggs in a parallel experiment with aflatoxin B₁, using dose levels at which aflatoxin B₁ was known to be highly toxic and teratogenic. No significant change in embryo viability or teratogenic effect attributable to aflatoxin P₁ was observed, whereas the response of the embryos to aflatoxin B₁ was the same as observed in all previous experiments.     

The neurotoxicity of aflatoxin B1 in the rat

The effects of repeated intraperitoneal administration of aflatoxin B₁ on the peripheral and central nervous systems of rats were investigated. Biochemical markers of neurotoxicity were monitored in nervous tissues following aflatoxin B₁ dosage and after the cessation of aflatoxin B₁ administration. Aflatoxin B₁ increased the activities of β-glucoronidase and β-galactosidase in the central and peripheral nervous systems. Repeated exposure of rats to aflatoxin B₁ also activated Na⁺ K⁺-ATPase and inhibited Mg²⁺-ATPase. Nervous tissue levels of DNA and total protein increased while the concentrations of RNA and phospholipid were depressed by aflatoxin B₁. The alterations in these parameters were specific for each of the tissues examined during the recovery of the rats. The findings indicate that the repeated administration of aflatoxin B₁ to rats results in degeneration in the central and peripheral nervous systems that may be related to the overt toxicity observed following aflatoxin administration.     

Binding of aflatoxins B1 and G1 to rat liver chromatin

Direct evidence for aflatoxin-chromatin interaction was obtained using fluorescence quenching as a measure of the interaction. Interaction of aflatoxin B₁ with components of chromatin increased in the following order: nonhistone residue, DNA, histone, histone-free chromatin, and intact chromatin. Fluorescence change due to interaction of aflatoxin B₁ with various complexes of chromatin constituents was not significantly different from that obtained with DNA. Association of aflatoxin B₁ with chromatin components was very weak whereas that with reconstituted deoxyribonucleoprotein (consisting of histone, nonhistone residue, and DNA) and with native chromatin was strong as shown by increase in fluorescence polarization of aflatoxin. Such data suggest the importance of structural integrity of chromatin in the binding with aflatoxin. Interaction of aflatoxin G₁ with chromatin was less than that of aflatoxin B₁.     

Comparative Oxidative Metabolism of Aflatoxin B1 and Palmotoxins B0 and G0 by Rat Liver Microsomal Fractions

Abstract

1. The enzymic transformations of aflatoxin B₁ and palmotoxins B₀ and G₀ by cell fractions of rat liver have been studied. Metabolism involves NADPH-dependent enzymes with max. activity in the presence of nicotinamide, MgCl₂, glucose-6-phosphate and NADP.

2. The substances all undergo demethylation with production of formaldehyde. With both aflatoxin B₁ and palmotoxin B₀, two metabolites were detected by t.l.c. The two metabolites of aflatoxin B₁ correspond to aflatoxin M₁ and possibly aflatoxin B_2a. The two derivatives of palmotoxin B₀ have not been characterized but, like those of aflatoxin B₁, are probably hydroxylation products, as they are more polar than the parent compound. Only one derivative of palmotoxin G₀ was detected.

3. Aflatoxin B₁ showed a higher rate of metabolism than the two other substances.     

Microinjection of rainbow trout at the sac-fry stage: a modified trout carcinogenesis assay

Liver tumors were induced in rainbow trout (Salmo gairdneri) 1 yr after trout were injected with aflatoxin B₁ in the sac-fry stage of development. In this modification of the trout embryo microinjection carcinogenesis assay, there are fewer mortalities than in earlier embryo stage injection protocols. Injection efficiency, as measured by retention of [¹⁴C]benzo[a]pyrene in the embryo, and sensitivity to the carcinogenic effects of aflatoxin B₁ is not reduced by injection at this later stage of development.     

Aflatoxin exposure during the first 1000 days of life in rural South Asia assessed by aflatoxin B1-lysine albumin biomarkers

Aflatoxin B₁ is a potent carcinogen, occurring from mold growth that contaminates staple grains in hot, humid environments. In this investigation, aflatoxin B₁-lysine albumin biomarkers were measured by mass spectrometry in rural South Asian women, during the first and third trimester of pregnancy, and their children at birth and at two years of age. These subjects participated in randomized community trials of antenatal micronutrient supplementation in Sarlahi District, southern Nepal and Gaibandha District in northwestern Bangladesh. Findings from the Nepal samples demonstrated exposure to aflatoxin, with 94% detectable samples ranging from 0.45 to 2939.30 pg aflatoxin B₁-lysine/mg albumin during pregnancy. In the Bangladesh samples the range was 1.56 to 63.22 pg aflatoxin B₁-lysine/mg albumin in the first trimester, 3.37 to 72.8 pg aflatoxin B₁-lysine/mg albumin in the third trimester, 4.62 to 76.69 pg aflatoxin B₁-lysine/mg albumin at birth and 3.88 to 81.44 pg aflatoxin B₁-lysine/mg albumin at age two years. Aflatoxin B₁-lysine adducts in cord blood samples demonstrated that the fetus had the capacity to convert aflatoxin into toxicologically active compounds and the detection in the same 2-year-old children illustrates exposure over the first 1000 days of life.