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PubMed1998

Investigations of the problems of assessing aflatoxin levels in peanuts.

In this study, a number of probability distributions that have been used to model the occurrence of aflatoxin in peanuts are compared. Two distributions, the compound gamma and the negative binomial, are shown to have special appeal in that both can be justified by reasoning from the fundamental biological and stochastic processes that generate the aflatoxin. Since method of moments and maximum likelihood give consistent estimates of parameters in both models, practical considerations suggest using the former. One hundred twenty data sets, each consisting of fifty observations, were not sufficient to provide goodness-of-fit tests to establish either as superior to the other as a model. Both models fit the data well, appreciably better than other models examined. An attractive aspect of the compound gamma and the negative binomial distributions is that, as a consequence of their theoretical underpinnings, both involve parameters that have meaningful interpretations. In the compound gamma, the alpha parameter reflects the shape of the kernel-to-kernel aflatoxin content distribution, the lambda parameter reflects the number (or frequency) of contaminated kernels in the sample, and the beta parameter is a scale parameter. In the negative binomial, the two parameters can be used as measures of mean or location and shape.

Aflatoxins↗

PubMed1998

Systemic aspergillosis caused by an aflatoxin-producing strain of Aspergillus flavus.

The first case of human systemic infection by an Aspergillus flavus isolate demonstrated to produce aflatoxins in vitro and in vivo is described. The patient, a 41-year-old man with acute myelogenous leukaemia, developed a complication of suspected pulmonary Aspergillus infection during remission induction therapy. Antifungal chemotherapy brought about a considerable degree of improvement, but remission of the underlying disease was not attained. Bone marrow transplantation was also not effective. The patient showed recovery from neutropenia but died despite aggressive antifungal chemotherapy. The autopsy revealed lesions in the lungs, myocardium, kidneys, brain, thyroid gland and skin due to a suspected Aspergillus sp. A fungus isolated from the right lung and the skin lesions was identified as A. flavus. Aflatoxins B1, B2 and M1 were detected in culture filtrates of the isolated A. flavus, and in an extract of lung lesions. These aflatoxins are considered to have played an important role in damaging the immune system of the patient through their toxic effects.

Adult↗

PubMed1998

[Systemic aspergillosis caused by an aflatoxin-producing strain of Aspergillus in a post-bone marrow transplant patient with acute myeloid leukemia].

We report a case in which an aflatoxin-producing strain of Aspergillus flavus (A. flavus) caused systemic aspergillosis in a post-transplant 41-year-old man with acute myeloid leukemia. The leukemia was initially resistant to two courses of induction chemotherapy. The third course of chemotherapy, however, induced complete remission. Thereafter, the patient underwent bone marrow transplantation from his HLA identical brother. Pulmonary aspergillosis was suspected as a complication during induction chemotherapy. Twenty days after the transplant, the patient's absolute neutrophil count had increased to 500/microliter. However, the symptoms of pulmonary aspergillosis were aggravated following neutrophil and monocyte recovery. The patient died of sinus arrest due to complete atrioventricular block 31 days after his transplant. At autopsy, we found that the fungus had invaded the brain, lungs, spleen, kidneys, skin, and myocardium, including the sinoatrial conduction system. There was no sign of acute graft-versus-host disease. A strain of A. flavus was isolated from cultured tissue samples of fungal lesions and shown by thin-layer chromatography to produce aflatoxins. To our knowledge, this is the first case report describing an infection by an aflatoxin-producing A. flavus.

Acute Disease↗

PubMed1978

[Some aspects relating to the aflatoxin generation during the self-heating of cereals].

In a stored batch of grain which was already affected by mould-formation tests were carried out with the known aflatoxin producer Aspergillus flavus. A significantly lower aflatoxin production ensued if the mould growth was not connected with self-heating of the stored product. However, in conformity with increasing mould formation the germinating power was adversely affected and the significant signs (fatty acid number, reductive and none-reductive sugars) were influenced in the grain, irrelevently whether or not this led to self-heating. It appears obvious that as the self-heating increases in the stored product, the optimal temperature range favourable for the Aspergillus flavus is rapidly exceeding and this caused restriction to the aflatoxin formation. This leads to the conclusion that the contamination on the outer layer by mycotoxines is more significant on the outer layers than the more or less strong mould formation in the interior (so-called-mouldly pockets) which ensued through the self-heating fenomena.

Aflatoxins↗

PubMed1976

Confirmation of results of rapid screening test for aflatoxins performed at corn elevator.

A screening method for corn and corn products, based on a minicolumn, was modified slightly to assay 60 lots of corn at one elevator to determine whether they could be sold as animal feed. To be salable, the lots had to contain less than 20 ppb total aflatoxin. Aflatoxin levels in the lots were later determined by the official AOAC method for corn to check effectiveness of the screening. No lot had been designated for sale that contained 20 or more ppb total aflatoxin.

Aflatoxins↗

PubMed1977

Regulation of aflatoxin biosynthesis. III. Comparative study of adenine nucleotide pool in aflatoxigenic and non-aflatoxigenic strains.

The adenine nucleotide pools were studied in toxigenic (NRRL 3240) and non-toxigenic (NRRL 3537) strains of Aspergillus flavus group in relation to aflatoxin biosynthesis. The levels of ADP and AMP were comparatively low in the non-toxigenic strain throughout the growth period. In the toxigenic strain the levels of ADP and AMP increased significantly during the stationary phase. The ATP content of the toxigencic strain had higher ATP levels during the stationary phase compared to the toxigenic strain. The inorganic phosphate levels of the non-aflatoxigenic strain were higher when compared with the aflatoxigenic strain. High levels of inorganic phosphate have been reported to be inhibitory for secondary biosynthesis including aflatoxins. The energy charge values of the non-toxigenic strain, compared to the toxigenic strain, were lower during exponential phase but higher during stationary phase. The observed changes have been correlated with aflatoxin biosynthesis.

Adenine Nucleotides↗

PubMed1984

Effect of butylated hydroxytoluene on the disposition of [14C]aflatoxin B1 in the lactating rat.

The distribution and metabolism of an ip dose of [14C]aflatoxin B1 (AFB1) were studied in lactating Sprague-Dawley rats fed for the previous 13 days on a diet containing 0.5% butylated hydroxytoluene (BHT). Compared with ingestion of a BHT-free diet, treatment with BHT increased the biotransmission of AFB1 metabolites, predominantly aflatoxin M1 (AFM1), into the mammary gland and its content of milk, decreased AFB1 binding to liver nuclear DNA and enhanced the excretion of water-soluble metabolites of AFB1, all measured 6 hr after an oral dose of [14C]AFB1. These changes are related to the induction by BHT of hepatic enzymes involved in the transformation and detoxification of AFB1. The results suggest that exposure to BHT may protect the lactating animal from the carcinogenic effect of AFB1 but may increase the risk of exposure of the newborn infant to the carcinogenic metabolite AFM1.

Aflatoxin B1↗

PubMed1982

Aflatoxin B1 metabolism and DNA binding in isolated hepatocytes from rainbow trout (Salmo gairdneri).

The metabolism of aflatoxin B1 (AFB1) was examined in freshly isolated hepatocytes from rainbow trout. Intracellular DNA adduct formation was linearly related to AFB1 dose, and qualitatively similar to adducts formed in vivo. The rate of adduct accumulation was constant during the first hour following completion of the preparation, after which an increase and gradual decrease in rate routinely occurred. The relative rates of production of the major unbound AFB1 metabolites aflatoxicol, aflatoxin M1, and polar conjugates, also remained constant over the first hour of preparation age, but subsequently changed in a manner consistent with the changes in DNA binding. The common solvent vehicles ethanol and dimethyl sulfoxide were shown to seriously perturb AFB1 metabolism and DNA binding even at levels less than 1%. A simple method is reported for removal of ethanol prior to introduction of hepatocytes for incubation with AFB1. The influence of cell concentration was also examined. DNA binding and relative distribution of AFB1 metabolites showed little or no dependence in the range 1-6 x 10(6) cell/ml, but were substantially altered above 10(7) cells/ml. Under defined conditions, studies in isolated hepatocytes appear to reflect in vivo cell capabilities for AFB1 metabolism.

Aflatoxin B1↗

PubMed1986

Mechanisms of anti-carcinogenesis: the distribution and metabolism of aflatoxin B1 in rainbow trout fed aroclor 1254.

Polychlorinated biphenyls (PCBs) have previously been shown to be inhibitors of carcinogenesis in trout. The mechanism of this inhibition was investigated by studying the effects of PCBs on aflatoxin B1 (AFB1) distribution, metabolism and DNA adduct formation, both in vivo and in vitro. A 24 h distribution study of injected tritiated AFB1 showed more radioactivity in blood, liver and bile in fish fed PCBs, but less in residual carcass. The metabolites of AFB1 found in vivo in blood plasma and liver homogenates were shifted by PCB pretreatment towards greater production of the polar metabolite aflatoxin M1 (AFM1) and glucuronide conjugates. The major metabolite in bile of PCB fish was the glucuronide of aflatoxicol M1 (AFL-M1), which was enhanced 15-fold over controls. Levels of aflatoxicol (AFL) glucuronide, the major conjugate in controls, were unaltered by PCBs. The pattern of AFB1 metabolism in isolated hepatocytes from PCB-prefed fish was consistent with in vivo metabolism. AFB1--DNA adduct formation in a 1 h assay was similar in hepatocytes from PCB-fed and control fish. However, the total rate of AFB1 metabolism was significantly elevated in hepatocytes from PCB-fed fish such that the degree of AFB1--DNA adduct formed per unit AFB1 metabolized was 42% lower than control. Similarly, adduct formation in vivo during the first 24 h post-AFB1 injection in PCB fish was not significantly different from controls. However, over a longer 21 day period, adduct levels in PCB fish were only 48-69% of controls (P less than 0.005, analysis of variance), once peak adduct formation was reached. Thus, initial rates of adduct formation may be misleading in the absence of further information on rates of carcinogen metabolism in vitro and/or pharmacokinetics of peak adduct formation in vivo. These results indicate that PCB inhibition of AFB1 carcinogenesis in trout involves dramatic initial changes in carcinogen distribution, metabolism and elimination which, over time, results in a net reduction of DNA adduct formation.

Aflatoxin B1↗

PubMed1984

Production and characterization of monoclonal antibodies against aflatoxin M1.

By using an indirect enzyme-linked immunosorbent assay, four monoclonal antibodies were selected after fusion of mouse P3-NS1-Ag4-1 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin M1 (AFM1) conjugated to bovine serum albumin. Two of these antibodies were found to be specific for AFM1 and were designated AMW-1 and AMW-4. The specificities of AMW-1, which had higher affinity to AFM1, were determined by a competitive direct enzyme-linked immunosorbent assay with peroxidase-AFM1 as the marker. The relative cross-reactivity of each toxin (relative to AFM1) with AMW-1, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 12, greater than 40, 12, and greater than 40 for B1, B2, G1, and G2, respectively.

Aflatoxin B1↗

PubMedDate not supplied

Surveillance and control of aflatoxins B1, B2, G1, G2, and M1 in foodstuffs in the Republic of Cyprus: 1992-1996.

Aflatoxins (AFs) B1, B2, G1, and G2 in locally produced and imported foodstuffs (nuts, cereals, oily seeds, pulses, etc.) were monitored and controlled systematically and effectively from 1992-1996. Samples (peanuts, pistachios, etc.) with total AFs above the Cyprus maximum level (ML) of 10 micrograms/kg fluctuated between 0.7 and 6.9%. The results indicate the effectiveness of monitoring, as well as the need for constant surveillance and control, especially at critical control points (sites of import, primary storage, etc.), to prevent unfit products from entering the Cyprus market. The control included sampling, retainment, analysis, and destruction of foodstuff lots with AF levels above MLs. The highest incidence of aflatoxin contamination was observed in peanut butter (56.7%) and the highest level of AF B1 was found in peanuts (700 micrograms/kg). Levels of AF M1 in raw and pasteurized milk analyzed in 1993, 1995, and 1996 were within both the Cyprus ML (0.5 microgram/L) and the lower ML (0.05 microgram/L) of some European countries. Only 12% of samples had detectable levels of AF M1. Analyses were performed by immunochemical methods. When recoveries were lower than 80%, the AF levels were corrected for recovery.

Aflatoxin B1↗

PubMed1988

Comparative study of aflatoxins M1 and B1 production in solid-state and shaking liquid cultures.

Production of aflatoxins M1 (AFM) and B1 (AFB) by Aspergillus flavus NRRL 3251 in solid-state and shaking liquid cultures using rice as the carbon source was compared. In general, solid-state cultures produced more aflatoxins than shaking liquid cultures on an equal rice weight basis. Solid-state cultures with continuous shaking yielded higher levels of toxins than those with intermittent shaking. However, intermittent shaking is a feasible replacement for the continuous shaking method for AFM production. A typical solid rice culture supplemented with yeast extract produced 30 and 2600 mg per kg rice of AFM and AFB, respectively, in 8 days at 29 degrees C. The optimal culture conditions for toxin production in a shaking liquid culture were also studied. Parameters under consideration included the amount of carbon (rice) and nitrogen source, initial medium pH, and aeration rate. At optimum conditions, a representative shaking liquid culture produced 18 and 1680 mg per kg rice of AFM and AFB, respectively, in 5 days at 29 degrees C. This shaking liquid culture appears feasible for scaling up and routine production of AFM and AFB for toxicological investigations.

Aflatoxin B1↗

PubMed1988

Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase.

Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.

Aflatoxin B1↗

PubMed1981

Improved method for confirmation of identity of aflatoxins B1 and M1 in dairy products and animal tissue extracts.

A method is described for confirming the identity of aflatoxins B1 and M1 in dairy products and liver extracts on a thin layer plate. Extracts and standards containing aflatoxins B1 and M1 are spotted on 10 x 10 cm plates, which are developed 2-dimensionally in mixtures of isopropanol-acetone-chloroform. After the first development, trifluoroacetic acid-hexane (1 + 4) is sprayed on that part of the plate containing the separated extract components and the underdeveloped standard spots of B1 and M1, and the plate is heated 6-8 min at 75 degrees C. Then the plate is developed in a second direction, and the reaction products of B1 and M1 with trifluoroacetic acid from the extract are compared with the same derivatives of the respective standards. The method has been used successfully on extracts of milk, cheese, and liver containing 0.1 ng B1 or M1/g and can be completed in 35-45 min.

Aflatoxin B1↗

PubMed1982

Determination and thin layer chromatographic confirmation of identity of aflatoxins B1 and M1 in artificially contaminated beef livers: collaborative study.

An international collaborative study involving 13 laboratories was conducted to test methods for the determination and thin layer chromatographic (TLC) confirmation of identity of aflatoxins B1 and M1 in beef liver. For the determination, each collaborator furnished fresh or frozen beef liver. Samples were artificially contaminated by adding solutions containing various concentrations of aflatoxins B1 and M1 (0.032-0.69 ng/g). Two TLC confirmation methods were tested with extracts obtained from the determination. Two measurement methods using 2-dimensional TLC were evaluated. In the first, sample extracts were compared directly with B1 and M1 standards on TLC plates; in the second, internal standards plus sample extracts were compared with B1 and M1 standards on the plates. Average within-laboratory coefficients of variation (CV) for the direct method were 26% for B1 and 26% for M1 compared with 24 and 26%, respectively, for the internal standard method. The average between-laboratory CV values were 39% for B1 and 41% for M1 by the direct method and 36% for B1 and 39% for M1 by the internal method and 36% for B1 and 39% for M1 by the internal standard method. Recoveries ranged from 64 to 90% for B1 and from 72 to 86% for M1. These data indicate that the more convenient direct method was sufficient, and internal standards were unnecessary. An analysis of variance was calculated from combined sample data to determine components of variance. The within-laboratory CV values were 27.0 and 32.3% for B1 and M1, respectively, and the between-laboratory CV values were 47.1 and 53.2%, respectively. Both TLC confirmation methods gave satisfactory results and have been adopted official first action, along with the determination method.

Aflatoxin B1↗

PubMed1987

Aflatoxin B1-4-hydroxylase is associated with cytochrome P3-450 in C57BL/6 mouse liver.

Messenger RNA from the livers of Aroclor 1254 treated mice was used to produce a cDNA library. cDNA clones corresponding to cytochromes P1-450 and P3-450 were isolated from this library by screening with a probe for the rat cytochrome P-450c gene. Specific non-cross hybridizing probes for P1-450 and P3-450 were prepared from unique restriction fragments. The radiolabeled probes were hybridized to RNA from mice treated with a low (15 mg/kg) and high (150 mg/kg, 400 mg/kg) doses of beta-naphthoflavone. The low dose of beta-naphthoflavone was found to induce only P3-450 mRNA, whereas higher doses induced both P1-450 and P3-450 mRNA. Similarly, a low dose of beta-naphthoflavone induced aflatoxin B1-4-hydroxylase, whereas higher doses induced both aflatoxin B1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. These results suggest that P3-450 mRNA codes for the cytochrome that is associated with aflatoxin B1-4-hydroxylase activity.

Animals↗

PubMed2006

The aflatoxin biosynthesis cluster gene, aflX, encodes an oxidoreductase involved in conversion of versicolorin A to demethylsterigmatocystin.

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA, ver-1, and aflY, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX (ordB), is also required for this conversion. A homolog of this gene, stcQ, is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated approximately 4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO (avfA). Some of domains in the protein are similar to those of epoxide hydrolases.

Amino Acid Sequence↗

PubMed1966

Effect of Aeration on Growth and Aflatoxin Production by Aspergillus flavus in Submerged Culture.

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.

Journal Article↗

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