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Treatment of freshly harvested 1980 Georgia dent corn samples collected for aflatoxin analysis.

In 1980, corn was harvested from six 15-ft rows in each of 67 fields in Georgia for aflatoxin analysis. Every sixth ear from each field was placed in a sample bag to be dried the day of collection. The rest of the corn was husked and shipped to Peoria in cardboard boxes. When undried ear samples arrived in Peoria, each sample was randomly separated into 5 equivalent subsamples. One set of 67 subsamples was shelled and dried as soon as possible to avoid further aflatoxin formation. Two other sets of 67 subsamples were stored 3 and 6 weeks before shelling and drying. The remaining 2 sets of ear samples were placed in plastic bags with 5% Monoprop (1 part propionic acid plus 1 part versite) and stored 3 and 6 weeks before shelling and drying. The samples dried in Georgia before shipping had an average total aflatoxin level of 217 ng/g. Samples shelled and dried immediately after arrival had an average level of 202 ng/g. Samples shelled and dried after 3 and 6 weeks of storage had average total aflatoxin levels of 417 and 387 ng/g, respectively. Samples stored 3 and 6 weeks in the presence of 5% Monoprop (2.5% propionic acid) had average total aflatoxin levels of 120 and 157 ng/g, respectively.

Aflatoxins↗

Growth of high aflatoxin B2 mutants on defined and complex media and with ethoxyquin.

The major naturally occurring aflatoxins are B1, B2, G1, and G2. A precursor-product relationship between B1 and the other toxins has been assumed by many workers, but experimental data are conflicting. Four independent reports, of mutants which produce aflatoxin B2, provide genetic evidence supporting an alternate origin for B2. Five high aflatoxin B2 mutants were obtained and grown in defined and complex liquid media and on a solid complex medium in order to determine the stability of the high B2 phenotype on different substrates and to define optimal levels of production. All the high B2 strains tested produced some aflatoxin yields were quite variable. The highest production was on complex liquid medium containing yeast extract. It was not possible to confirm reports that the antioxidant ethoxyquin was an inhibitor of G aflatoxins.

Aflatoxins↗

High performance liquid chromatographic determination of aflatoxins in animal tissues and products.

A method was developed for the determination of aflatoxins, M1, B1, G1, B2, and G2 in animal tissues, meat, eggs, and dairy products by high performance liquid chromatography (HPLC). Previous extraction procedures were modified to optimize the precision and recovery of the method. HPLC is performed isocratically with a 5 micrometer microparticulate octadecylsilica column and fluorometric detection. The analysis is based on treatment of the purified sample extract or standard with trifluoroacetic acid (TFA) to catalyze the hydration of aflatoxins M1, B1, and G1 to the highly fluorescent M2a, B2a, and G2a derivatives. Analysis by HPLC with and without TFA treatment of the extract provides quantitative and qualitative data. The recovery of added aflatoxins and the precision of the recovery depend on the type of sample; mean coefficient of variation for all recovery values was 19.7 +/- 11.0% for the analysis of liver, ground beef, dairy products, and eggs. The method was sensitive, with a detection limit of 0.05 + 0.10 ng/g for each aflatoxin. This procedure provides an alternative to existing thin layer chromatographic methods for determining aflatoxins in animal tissues and products

Aflatoxins↗

High pressure liquid chromatographic determination and fluorescence detection of aflatoxins in corn and dairy feeds.

A high pressure liquid chromatographic (HPLC) method is described for the determination of aflatoxins B1, B2, G1, and G2 in animal feeds at levels as low as 2.5 ppb. Samples are extracted with acetonitrile-water and initially purified by using a Sep-Pak silica cartridge. The aflatoxins are then reacted with trifluoracetic acid and acetonitrile-water (1 + 1). After filtration, the aflatoxins are completely resolved on a 10 micrometer C18 column using a radial compression separation system with an acetonitrile-water solvent system. Aflatoxins G2a, B2a, G2, and B2 were reported in less than 30 min, using fluorescence detection. The method was successfully applied to samples at levels of 20-2.5 ppb added aflatoxins with recoveries in the range of 82-99%.

Aflatoxins↗

Temporal patterns of covalent DNA adducts in rat liver after single and multiple doses of aflatoxin B1.

We examined patterns of covalent modifications of DNA produced in rat liver after exposure to single and multiple doses of aflatoxin B1. The principal product, previously identified as 2,3-dihydro-3-hydroxy(N7-guanyl) aflatoxin B1, was removed rapidly from liver DNA in vivo after a 0.6-mg/kg dose was administered i.p. to male Fischer rats. This lesion had an apparent half-life of 7.5 hr. Similar kinetics of disappearance was seen for two other aflatoxin adducts, one of which was previously identified as an N7-guanine adduct of aflatoxin P1. The kinetics of formation and disappearance differed for two other products believed to be produced by scission of the imidazole ring of the 7-substituted guanine moiety of the principal adduct in the DNA molecule. These adducts were removed slowly, if at all, during the 72-hr period studied. Approximately 20% of the principal N7 adduct initially formed was converted to these products in 24 hr, at which time they were the predominant lesions in DNA. Administration of multiple doses of aflatoxin B1, using a regimen shown to produce a high incidence of hepatocellular carcinoma, caused accumulation of these persistent products in liver DNA over a 14-day period.

Aflatoxins↗

Aflatoxin and Reye's syndrome: a case control study.

Aflatoxin levels were determined in serum and urine of 17 patients with Reye's syndrome and in control subjects. No significant difference in aflatoxin levels was found for the two groups. However, 23% of all persons studied had levels of aflatoxin indicative of recent exposure. Aflatoxin levels were associated with ingestion of cornmeal and corn bread but not peanut-containing products. This prevalence of aflatoxin may be of public health importance.

Adolescent↗

[Changes of anaphylactic sensitivity to some food proteins and aflatoxin B1 in guinea pigs].

The study was made of aflatoxin B1 effect on guinea pig alimentary anaphylaxis to chicken ovalbumin (CO) and pasteurized cow milk (PCM), of CO-specific IgG antibody levels, some serum indices, sensitivity of the animals to LD50 histamine. In response to aflatoxin B1 alimentary anaphylaxis both to CO and PCM became more severe, lethality of the anaphylaxis to CO being in logarithmic relation to aflatoxin B1 dose (p < 0.05); specific IgG-antibodies to CO grew in number; serum total protein increased against unchanged levels of albumins; the activity of gamma-glutamyltransferase inhibited; histamine shock gained severity, its lethality being logarithmically related to the aflatoxin B1 dose. The discussion covers mechanisms underlying the animal allergic reactivity responses to aflatoxin B1.

Aflatoxin B1↗

Multifunctional column coupled with liquid chromatography for determination of aflatoxins B1, B2, G1, and G2 in corn, almonds, brazil nuts, peanuts, and pistachio nuts: collaborative study.

An AOAC/IUPAC collaborative study was conducted to evaluate the effectiveness of a multifunctional column for the determination of aflatoxins. The test portion is extracted with acetonitrile-water (9 + 1), the extract is filtered, and the filtrate is passed through the column. The aflatoxins in the eluate are determined by reversed-phase liquid chromatography after derivatization with trifluoroacetic acid. Naturally contaminated corn, almonds, Brazil nuts, peanuts, and pistachio nuts spiked with total aflatoxins at 5, 10, 20, and 30 ng/g were sent to 12 collaborators in the United States, Denmark, France, Japan, and Switzerland. Eleven collaborators completed the study. Average recoveries of total aflatoxins for each spike level for the various commodities (excluding Brazil nuts at 5 ng/g) were 93, 97, 95, and 95%, respectively; the repeatability relative standard deviation (RSDr) ranged from 6.0 to 23.2% and the reproducibility relative standard deviation (RSDR) ranged from 12.0 to 69.4%. The multifunctional column coupled with a liquid chromatographic method for determination of aflatoxins in corn, almonds, Brazil nuts, peanuts, and pistachio nuts has been adopted first action by AOAC INTERNATIONAL.

Aflatoxins↗

Cloning of cDNAs from fetal rat liver encoding glutathione S-transferase Yc polypeptides. The Yc2 subunit is expressed in adult rat liver resistant to the hepatocarcinogen aflatoxin B1.

Fetal rat liver possesses substantial levels of glutathione S-transferase (GST) activity toward aflatoxin B1-8,9-epoxide. The enzyme responsible for this activity is an Alpha-class GST heterodimer comprising Yc1 and Yc2 subunits. The cDNAs encoding these polypeptides have been cloned and shown to share approximately 91% identity over 920 base pairs, extending from nucleotide -23 to the AATAAA polyadenylation signal. GST Yc2Yc2 expressed in Escherichia coli was found to exhibit 150-fold greater activity toward aflatoxin B1-8,9-epoxide than GST Yc1Yc1. Comparison between the structures of Alpha-class GST suggests that tyrosine at residue 108 and/or aspartate at residue 208 is responsible for the high aflatoxin B1 detoxication capacity of Yc2. Immunoblotting and enzyme assays have shown that liver from adult female rats contains about 10-fold greater levels of Yc2 than is found in liver from adult male rats. This sex-specific expression of Yc2 in adult rat liver may contribute to the relative insensitivity of female rats to aflatoxin B1. Dietary administration of oltipraz, a synthetic antioxidant which protects against aflatoxin-hepatocarcinogenesis, serves as an inducer of GST Yc2.

Aflatoxin B1↗

Selenium effect on the growth of carcinogenic fungi and cytotoxic action of aflatoxin B1 on lymphocyte culture and on embryonal development of Xenopus laevis.

The authors report the results of investigations on the protective effect of selenium against teratogenic and cytotoxic action of aflatoxin B1 and the inhibitory action of this element on the growth of fungi producing carcinogenic aflatoxins. Sodium selenate inhibited the growth of cultures of such fungi belonging to the class Fungi imperfecti as Aspergillus flavus, Aspergillus fumigatus, Penicillium meleagrinum and Penicillium rougolosum. Added to cell cultures of lymphocytes sodium selenate inhibited the cytotoxic action of aflatoxin B1 evidenced by inhibition of PHA-stimulated blastic transformation of these cells and reduction in the number of mitoses. Sodium selenate added to the medium in the culture of Xenopus laevis larvae poisoned with aflatoxin B1 reduced significantly the early mortality of embryos and counteracted the teratogenic action of aflatoxin B1. This action was observed, however, only in early phase of morulation. The authors discuss the importance of their observations for the studies on the role of bioelements deficiency in the environment for induction of malignant transformation.

Aflatoxins↗

[Reconsideration of the role of aflatoxins in public health].

The pathogenicity of aflatoxins for human has been largely demonstrated. Although aflatoxin contamination of some foodstuffs has been unquestionably established, stress should be laid upon a certain lack of information: some of the affected foodstuffs have been "forgotten" in the enumeration of contaminated products (especially soybeans); relations between mycotoxins and food balance have not yet been taken into account: for instance, the influence of some vitamins, of various compounds (carotenoids, methionine, ...), influence of deficiency in proteins (often very important in Africa) etc... aflatoxin bioavailability has not sufficiently been examined (particularly that of milk aflatoxin M1.); concerning aflatoxin evaluation in contaminated products, it should be known that their distribution is very irregular. Sampling and following analysis should thus be carried out most carefully; for these motives, some conditions and likewise regulations would can again scrutinized.

Aflatoxins↗

Effect of four pesticides on aflatoxin production by Aspergillus flavus IMI 89717.

The effect of three fungicides (Vitavax-Captan, Rizolex-T and Sumisclex) and one insecticide (Actellic), when incorporated into liquid medium or applied to corn grains and sunflower seeds, on the production of aflatoxin by A. flavus IMI 89717 was tested. In liquid medium, aflatoxin production was inhibited by 27%, 82%, 100% and 100% when Vitavax-Captan was added at 10, 25, 50 and 100 ppm, respectively. Sumisclex reduced to some extent the production of total aflatoxin, while Rizolex-T and Actellic did not affect quantitatively the mycotoxin production. Rizolex-T proved to be the most effective pesticides tested on aflatoxin production on both corn-grains and sunflower seeds, while Vitavax-Captan was the second most effective pesticide. Both Sumisclex and Actellic did not inhibit aflatoxin production on either corn grains or sunflower seeds at the levels tested.

Aflatoxins↗

Performance of two immunochemical assays in the analysis of peanuts for aflatoxin at 37 field laboratories.

A study was conducted to measure the precision of 2 rapid aflatoxin assay systems in use at 37 peanut buying points during the 1991 harvest season. Aflatoxin laboratories were established at the 37 buying points to analyze peanut samples from all incoming farmers' stock loads as part of a joint project sponsored by various segments of the U.S. peanut industry and the U.S. Department of Agriculture. Eighteen laboratories were equipped with Neogen's veratox FSP rapid assay system, whereas 19 laboratories used Vicam's Aflatest rapid assay system. To monitor the performance of the field laboratories during the project, 3 portions of each of six 27 kg samples of ground peanuts were sent to each laboratory for analysis over a period of 6 weeks. Aflatoxin concentrations ranged from 0 to 300 ng/g when eight 200 g subsamples of each sample were analyzed by liquid chromatography (LC). For the 5 samples contaminated with aflatoxin, relative standard deviations for repeatability (RSDr) for laboratories using veratox FSP ranged from 18.66 to 53.29%, and the relative standard deviations for reproducibility (RSDR) ranged from 22.79 to 59.29%. For laboratories using the Aflatest system, RSDr values ranged from 18.70 to 41.48%, and RSDR values ranged from 23.84 to 47.56%. Horwitz ratios < 2.0 were found for 4 of the 5 contaminated samples for both methods, indicating that the overall precision of the 2 methods used in the project was good. Mean aflatoxin concentrations, as determined with the rapid assay systems, were generally lower than those determined by LC, particularly for more highly contaminated samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxins↗

Efficacy of zeolitic ore compounds on the toxicity of aflatoxin to growing broiler chickens.

Commercially available zeolitic ore compounds, when incorporated into the diets at 0.5%, were evaluated for their ability to reduce the deleterious effects of 3.5 mg aflatoxin/kg feed on growing broiler chickens from 1 day to 3 weeks of age. In a series of four experiments, the compounds used included the following: mordenite (particle size of -20 mesh; Zeomite); clinoptilolite (particle size of -20 mesh; Zeobrite); SC Zeolite (particle size of -20 mesh); and clinoptilolite (particle size of -35 mesh; Clino 1) or clinoptilolite (particle sizes of -20 plus +35 mesh; Clino 2). Results demonstrated that 0.5% Zeobrite, Clino 1, or Clino 2 added to aflatoxin-contaminated diets did not significantly (P < 0.05) diminish the toxicity of high concentrations of aflatoxin to growing broiler chicks. Zeomite mordenite ore reduced the toxicity of aflatoxin to growing chicks by 41%, as indicated by weight gains, liver weight, and serum biochemical measurements, which compares favorably with its in vitro binding capacity to aflatoxin. SC Zeolite reduced weight-gain toxicity of aflatoxin by approximately 29%.

Adsorption↗

Mechanism of aflatoxin B1 inhibition of rat hepatic nuclear RNA synthesis.

This paper reports the evidence for the existence of multiple sites of action of aflatoxin B1 in relation to its inhibition of rat hepatic nuclear RNA synthesis. Two hours after aflatoxin B1 injection (0.3 mg/100 g body weight), rat hepatic nuclear and nucleolar RNA synthesis, in vitro, were inhibited 70 and 90% respectively. When total nuclear free and engaged RNA polymerases were solubilized and assayed in the presence of alpha-amanitin (3.2 micrograms/ml), only alpha-amanitin-sensitive activity was reduced (50 to 70%) by aflatoxin B1. DEAE-Sephadex column chromatography confirmed this finding and further demonstrated that RNA polymerase II was the activity selectively inhibited. Since aflatoxin B1 dramatically inhibited nucleolar RNA synthesis, but had little effect on RNA polymerase I activity per se, it is concluded, therefore, that, in addition to its direct inhibitory effect on the enzymic function of RNA polymerase II, aflatoxin B1 must also cause impairment of the nucleolar DNA template function.

Aflatoxins↗

Temporal patterns of aflatoxin-albumin adducts in hepatitis B surface antigen-positive and antigen-negative residents of Daxin, Qidong County, People's Republic of China.

Molecular epidemiological studies of populations at high risk for liver cancer have shown that hepatitis B virus (HBV) and aflatoxin B1 (AFB1) exposure are two major risk factors for this disease. These etiological agents, combined with nutritional deficiencies, are important for the initiation and promotion of liver cancer in various parts of the world. In Qidong, People's Republic of China, liver cancer accounts for 10% of all adult deaths, and both HBV and AFB1 exposures are common. To study temporal and possible chemical-viral interactions in people, serum samples were collected during a longitudinal study designed to measure aflatoxin molecular biomarkers in residents of Daxin Township, Qidong City, People's Republic of China. In this study, the temporal modulation of aflatoxin adduct formation with albumin over multiple lifetimes of serum albumin was examined in both HBV-positive and HBV-negative people in two periods: September-December 1993 (wave 1) and June-September 1994 (wave 2). During the 12-week monitoring period of wave 1, 120 individuals (balanced by gender and HBV status) provided a total of 792 blood samples. AFB1-albumin adducts were detected in all but one of the serum samples. The range of binding detected by RIA in the Daxin population was 0.17-4.39 pmol AFB11/mg albumin with an overall mean +/- SD of 1.51 +/- 0.21 pmol AFB11/mg albumin. The mean +/- SD for weeks 0, 2, 4, 6, 8, 10 and 12 of wave 1 were 1.21 +/- 0.41, 1.58 +/- 0.70, 1.36 +/- 0.52, 1.71 +/- 0.44, 1.18 +/- 0.60, 2.00 +/- 0.59, and 1.68 +/- 0.34 pmol AFB1/mg albumin, respectively. During wave 2, 103 individuals from wave 1 provided a total of 396 blood samples collected monthly over wave 2, with mean +/- SD aflatoxin-albumin adduct levels of 1.19 +/- 0.37, 0.85 +/- 0.45, 0.89 +/- 0.28, and 0.61 +/- 0.15 pmol AFB1/mg albumin. Using linear regression models, the mean aflatoxin-albumin adduct levels increased (P < 0.05) during the 12 weeks of wave 1 and decreased (P < 0.05) over the 4 months of wave 2. Neither HBV surface antigen status nor gender modified either the baseline mean or the temporal trend. High-performance liquid chromatography confirmation was done on a subset of serum samples, and the results show an excellent association between the immunoassay data and high-performance liquid chromatography. Taken together, these data demonstrate that AFB1-albumin is a sensitive and specific biomarker for assessing exposure to this carcinogen in the population in Qidong.

Adult↗

Aflatoxin B1 in common Egyptian foods.

Samples of common Egyptian foods (17 nuts and seeds, 10 spices, 31 herbs and medicinal plants, 12 dried vegetables, and 28 cereal grains) were collected from markets in Cairo and Giza. A portion of each sample was extracted with chloroform, and the concentrated extract was cleaned by passing through a silica gel column. Aflatoxin B1 was determined by reversed-phase liquid chromatography with UV detection. The highest prevalence of aflatoxin B1 was in nuts and seeds (82%), followed by spices (40%), herbs and medicinal plants (29%), dried vegetables (25%), and cereal grains (21%). The highest mean concentration of aflatoxin B1 was in herb and medicinal plants (49 ppb), followed by cereals (36 ppb), spices (25 ppb), nuts and seeds (24 ppb), and dried vegetables (20 ppb). Among nuts and seeds, the prevalence of aflatoxin B1 was highest (100%) in watermelon seeds, inshell peanuts, and unshelled peanuts. The lowest prevalence and concentrations were in hommos (garbanzo beans). The highest concentrations of aflatoxin B1 were detected in foods that had no potential for field contamination but required drying during processing and storage, such as pomegranate peel, watermelon seeds, and molokhia.

Aflatoxin B1↗

Thin layer chromatographic determination of aflatoxin B1 in eggs.

A method for determining aflatoxin in dairy products was modified for eggs, initially by additional cleanup steps. The modified method proved practical for use in survey analyses and was sufficiently sensitive to detect aflatoxin B1 at levels less than 1 ng/g in eggs from hens on experimentally contaminated feed and in spiked eggs; interfering compounds in eggs received from many different producing areas were eliminated. Although recovery was greater than 75% when the aflatoxin was extracted immediately after spiking, recovery was usually less than 30% when the aflatoxin was extracted after more than 24 hr. Adding sodium chloride, sodium sulfate, or urea to the extracting solvent remedied this defect, which is thought to be related to the binding of aflatoxin to egg protein.

Aflatoxins↗