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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↗

Application of a diphasic dialysis technique to the extraction of aflatoxins in dairy products.

A new method is described for the extraction of aflatoxins from milk and milk products based on a diphasic dialysis technique, followed by detection and quantification of aflatoxins by TLC. Recovery was 65 to 99% for aflatoxins B1, G1, and M1, and the detection limit in milk and yogurt was .01 to .02 ppb for all aflatoxins studied. In cheese, the detection limit was .03 to .04 ppb for aflatoxins B1 and G1 and .1 ppb for aflatoxin M1. The main advantages of this technique are its simplicity; sensitivity; very economical use of reagents, thereby minimizing environmental pollution; efficiency, allowing the quick processing of a high number of samples; use by laboratories with little equipment; and a sensitivity comparable with that of much more sophisticated techniques.

Aflatoxin M1↗

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

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

Aflatoxins↗

Survey of selected mycotoxins in food.

The contamination of a variety of foods with the mycotoxins aflatoxins B1, B2, G1, M1, ochratoxin A, patulin, and byssochlamic acid was investigated. With the developed methods, the identification was made possible by in situ fluorescence spectral analysis with a far reaching exclusion of substances which simulate sycotoxins. Aflatoxin B1 was detected in 2 samples of 105 spontaneously moulded food specimens and only in 1 of a group of 198 food samples that were obviously not moulded. Aflatoxin M1 was identified in 4 of 60 samples of the so-called wintermilk. Investigated dairy products did not contain aflatoxin M1. Ochratoxin A could be detected twice in 49 spontaneously moulded corn samples, aflatoxin, however, was not found. Further investigations of 50 samples of moulded food revealed ochratoxin A in 2 samples of raw coffee. Food samples obviously not moulded did not contain ochratoxin A. Patulin was detected in 19 of 110 samples of fruit and fruit products, especially in commercial apple juice, in rotten parts of apples, and in moulded fruit products. Byssochlamic acid was not detected in fruit juices or in fruit. The toxicological consequences of mycotoxins in food and possibilities to reduce mycotoxins are discussed.

Aflatoxins↗

Effect on calves of barley naturally contaminated with ochratoxin A and groundnut meal contaminated with low concentrations of aflatoxin B1.

Four groups of six 12-week-old male Friesian calves were fed diets containing 390 to 540 microgram ochratoxin A per kg, 320 to 500 microgram ochratoxin A plus 12 to 13 microgram aflatoxin B1 per kg, 10 to 13 microgram aflatoxin B1 per kg, or a control diet containing neither toxin. At the end of the 87-day experiment there was no evidence of kidney or liver damage in any group of animals as judged by serial plasma and urine enzyme assays, haematological examinations including the assay of blood coagulation factors, gross appearance at autopsy or histopathological assessment at the light microscopic level. Nor was there any significant alteration in serum IgA, IgM, IgG1 and IgG2 levels or in titres to environmental bacterial and viral antigens. Trace amounts of ochratoxin A were detected in kidneys of five out of the 12 calves exposed to this toxin but all 12 kidneys contained residues of the metabolite ochratoxin alpha (less than 5 to 10 microgram/kg). Traces of aflatoxin B1 and M1 were detected in the liver of one of the 12 calves exposed to aflatoxin B1 but nine kidneys contained the metabolite aflatoxin M1 (less than 0.01 to 0.03 microgram/kg). There was no evidence of interaction between the two toxins.

Aflatoxin B1↗

Comparative assessment of the aflatoxin B1, B2, G1, G2 and M1 embryotoxicity in the chick embryo.

The embryotoxicity of aflatoxins B1, B2, G1, G2 and M1 was investigated after administration to chick embryos on either Day 2, 3 or 4. Treatment resulted mainly in embryolethality and a rank order for embryotoxicity was established where B1 greater than G1 greater than M1 = B2 greater than G2. The sensitivity of embryos to aflatoxin administration decreased with their age. These results document the general cytotoxic character of aflatoxin action upon the embryonic morphogenetic systems, actions that apparently require neither specific metabolic activation nor any specific target.

Aflatoxins↗

Fetal exposure to aflatoxins in the United Arab Emirates.

This is a prospective study in which aflatoxin levels were measured in umbilical cord blood from 201 women delivered consecutively in Tawam and Al Ain hospitals in order to determine whether the fetuses had been significantly exposed to the toxin. Aflatoxin B1, M1 and M2 were measured using high performance liquid chromatography. Aflatoxins were detected in 110 (54.7%) samples, 27 of which were positive for B1, 106 for M1 and 31 for M2. There was a significant negative correlation (p < 0.001) between birthweight and levels of aflatoxin. The high rate of detection of aflatoxins confirms that a significant number of infants in the UAE are exposed to these toxins which reflects maternal ingestion of aflatoxin-containing food. The presence of aflatoxin resulted in lower birthweights.

Aflatoxins↗

Mutagenicity of aflatoxins related to their metabolism and carcinogenic potential.

Aflatoxins and their animal biotransformation products were screened for carcinogenic potential using the Ames' in vitro microbial detection system for carcinogens as bacterial mutagens [B. N. Ames et al. (1973) Proc. Natl. Acad. Sci. USA 70,2281-2285]. Aflatoxicol, aflatoxins G1 and M1, aflatoxicol H1, and aflatoxins Q1, B2, P1, G2, B2a, and G2a, listed in order of decreasing mutagenic potency, were all less active than aflatoxin B1. No compound possesses activity in the absence of the rat liver preparation, and this indicates none of the animal metabolites are the ultimate mutagenic and/or carcinogenic species. The relative mutagenic potency observed with this in vitro system qualitatively correlates with in vivo carcinogenic data. Comparison of both methods indicates: (i) aflatoxin B1 possessed the structure optimal for both mutagenicity and carcinogenicity, (ii) the decreased carcinogenicity of various animal metabolites is associated with their decreased mutagenicity, and (iii) the 2,3-double bond is involved in both the mutagenic and carcinogenic activity of aflatoxins. The Ames' assay has been demonstrated to be an extremely promising (toxicological) tool for the analysis of mycotoxins for mutagenic and/or carcinogenic activity.

Aflatoxins↗

Detection of aflatoxins in autopsied materials from a patient infected with Aspergillus flavus.

We isolated two strains of Aspergillus flavus from a lung lesion and a skin lesion at autopsy from a patient with acute myelogenous leukemia complicated with fungal infection. An attempt was made to detect aflatoxins in culture filtrates of those isolates and the tissue extract of the lung lesion through the techniques of thin-layer chromatography (TLC), densitometry and high-performance liquid chromatography (HPLC). Aflatoxins B1, B2 and M1 were demonstrated in all of these materials qualitatively and quantitatively. The concentrations of aflatoxins in the cultures of the isolates and in the lung lesion extract determined by HPLC were aflatoxin B1: 11.715 microg/ml (lung isolate), 21.383 micro g/ml (skin isolate), 0.635 microg/g (lung extract), aflatoxin B2: 0.341 microg/ml (lung isolate), 0.577 micro g/ml (skin isolate), 0.0273 microg/g (lung extract) and aflatoxin M1: 0.277 microg/ml (lung isolate), 0.491 micro g/ml (skin isolate), 0.0525 microg/g (lung extract), respectively. B1, known as the most toxic among the aflatoxin group, showed the highest concentration through these experiments. This case may be considered as the first to detect aflatoxins in autopsied materials associated with A. flavus infection.

Adult↗

Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis.

Aflatoxins (AF) are contaminants of improperly stored foods; they are potent genotoxic and carcinogenic compounds, exerting their effects through damage to DNA. They can also induce mutations that increase oxidative damage. The goal of this study was to evaluate the possibility that a third mechanism could be involved in the carcinogenic action of aflatoxins, namely, direct binding to key enzymes involved in the regulatory pathways of the cell cycle, thereby modulating enzyme functionality. The 20S constitutive and immunoproteasome peptidase and proteolytic activities were assayed in the presence of aflatoxins B1, G1 and M1. All three toxins activated multiple peptidase activities of the proteasome. Aflatoxin (AF) M1 was the most potent activator of proteasome activity, while the constitutive 20S proteasome was specifically stimulated by AFG1. Furthermore, the effects of AFB1 on cultured hepatoma cells were investigated and the various proteasomal activities determined with cell lysates were differently affected. Taking into account the key role of the proteasome in cellular defense against oxidative stress, the carbonyl group content and the activities of antioxidant enzymes in cell lysates were analyzed. The proapoptotic effect of AFB1 was also investigated by measuring caspase-3 activity and cellular levels of p27 and IkappaBalpha.

Aflatoxin B1↗

Determination of selected mycotoxins in mould cheeses with liquid chromatography coupled to tandem with mass spectrometry.

A simple and feasible method is described for analysing nine mycotoxins in cheese matrix. The method involves liquid extraction followed by high performance liquid chromatographic separation and mass spectrometric detection of the analytes, and allows the determination of aflatoxins B1, B2, G1, G2 and M1, ochratoxin A, mycophenolic acid, penicillic acid and roquefortine C simultaneously. Average recoveries of the mycotoxins from spiked samples at concentration levels of 5-200 microg kg(-1) ranged from 96-143%. Within-day relative standard deviations at these concentration levels varied from 2.3-12.1%. The limit of quantification for aflatoxin M1 was 0.6 microg kg(-1) and for the other compounds 5 microg kg(-1). The method developed was applied for analysing these mycotoxins in blue and white mould cheeses purchased from Finnish supermarkets. Roquefortine C was detected in all of the blue mould cheese samples in concentrations of 0.8-12 mg kg(-1). One blue cheese contained also 0.3 mg kg(-1) mycophenolic acid. The other investigated mycotoxins were absent in the samples.

Aflatoxin B1↗

Effect of feeding corn naturally contaminated with aflatoxin on feed efficiency, on physiologic, immunologic, and pathologic changes, and on tissue residues in steers.

Two of 3 groups of Holstein-Friesian steers (groups II and III; n = 5 each) were fed a ration containing corn naturally contaminated with 800 ng of aflatoxin/g. The other group of steers (group I; n = 5) was fed a ration containing noncontaminated corn. The respective rations were fed for 17.5 weeks, except the ration given to group III; the latter's first diet (contaminated with aflatoxin) was changed to a noncontaminated diet after 15 weeks, continuing for the remaining 2.5 weeks. All steers were killed and tissues and fluids were obtained for aflatoxin analysis. Although aflatoxin B1 and M1 could be detected in blood and urine at several sampling times during the experimental period in groups II and III steers (given the diets containing aflatoxin), there appeared to be no effects on body weight gains and immune phenomena, such as lymphoblastogenesis and antibody production, but there was a waning of the delayed cutaneous hypersensitivity in steers given aflatoxin-contaminated diets. In group III animals (diet was changed to noncontaminated ration at 15 weeks), aflatoxin B1 and M1 disappeared from urine before they were slaughtered. All tissues and fluids, except the rumen contents from these group III steers, were void of detectable aflatoxins B1 and M1 at necropsy. The concentrations of aflatoxin B1 in the rumen content of the latter steers were low. All tissues collected at necropsy from the group II steers fed the aflatoxin diet throughout the 17.5 weeks had detectable aflatoxins B1 or M1 present.

Aflatoxin B1↗

Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography.

A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with [14C]aflatoxin B1, we identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

Aflatoxin B1↗

Species susceptibility to aflatoxin B1 carcinogenesis: comparative kinetics of microsomal biotransformation.

The biotransformation of the potential human carcinogen aflatoxin B1 (AFB1) was studied using hepatic microsomes from the rat, mouse, monkey, and human. Initial rates of AFB1 oxidation to aflatoxins Q1, M1, and P1, as well as the reactive intermediate AFB1-8,9-epoxide, were determined using a high performance liquid chromatography assay. The rates of generation of these AFB1 metabolites were investigated at low substrate concentrations (more representative of environmental exposures) and also at high ("saturating") concentrations commonly utilized in studies in vitro. Striking differences in ratios of the metabolites were observed. At an AFB1 concentration of 124 microM, mouse and monkey microsomes had the highest rates of AFB1-8,9-epoxide formation. Primate liver microsomes formed aflatoxin Q1 in large amounts but failed to produce detectable aflatoxin P1. Determination of the rates of formation over initial AFB1 concentrations ranging from 15 to 475 microM revealed that the proportion converted to AFB1-8,9-epoxide increased at lower substrate concentrations in the case of the rat and human microsomes but not with mouse or monkey microsomes. The differences in patterns of metabolite formation with varying concentrations have implications for interspecies comparisons of carcinogenic potency of AFB1.

Aflatoxin B1↗

Sample size and statistical power assessing the effect of interventions in the context of mixture distributions with detection limits.

Often in randomized clinical trials and observational cohort studies, a non-negative continuously distributed response variable is measured in treatment and control groups. In the presence of true zeros for the response variable, a two-part zero-inflated log-normal model (which assumes that the data has a probability mass at zero and a continuous response for values greater than zero) is usually recommended. However, in some environmental health and human immunodeficiency virus (HIV) studies, quantitative assays for metabolites of toxicants, or quantitative HIV RNA measurements are subject to left-censoring due to values falling below the limit of detection (LD). Here, a zero-inflated log-normal mixture model is often suggested since true zeros are indistinguishable from left-censored values due to the LD. When the probabilities of true zeros in the two groups are not restricted to be equal, the information contributed by values falling below LD is used only to estimate the probability of true zeros in the context of mixture distributions. We derived the required sample size to assess the effect of a treatment in the context of mixture models with equal and unequal variances based on the left-truncated log-normal distribution. Methods for calculation of statistical power are also presented. We calculate the required sample size and power for a recent study estimating the effect of oltipraz on reducing urinary levels of the hydroxylated metabolite aflatoxin M(1) (AFM(1)) in a randomized, placebo-controlled, double-blind phase IIa chemoprevention trial in Qidong, China. A Monte Carlo simulation study is conducted to investigate the performance of the proposed methods.

Aflatoxin M1↗

Production of certified reference materials for mycotoxins: IRMM's view on the assessment of uncertainties.

Analytical difficulty and the economic importance of controlling mycotoxin levels in food and feed led the Community Bureau of Reference (BCR) to prepare a series of certified reference materials (CRM) for various mycotoxins. Because of the wide acceptance of these CRM and the need to ensure the comparability and traceability of measurements in the future it is necessary to prepare and certify new batches of mycotoxin reference materials (RM). In the following text two different approaches for evaluation of the characterisation uncertainty of CRM will be compared using the certification of aflatoxin M(1) (AfM(1)) in milk powder as an example. The conventional approach is based on evaluation of characterisation exercise data; the alternative approach is based on measurement uncertainties of the employed analytical methods. Because laboratories are using totally different approaches to estimate the measurement uncertainties, combination of the uncertainties obtained from the participating laboratories was not recommended. Therefore, a new integrated approach for assessment of the measurement uncertainties of the analytical methods on the basis of additional data collected during the characterisation exercise will be described. The conventional approach was found to be the most appropriate and economical approach to evaluate the characterisation uncertainty as a characterisation exercise must be performed anyway to establish the property values of candidate (C)RM, irrespective of whether or not reliable measurement uncertainties can be provided by the laboratories. An integrated approach for assessment of measurement uncertainties based on additional characterisation data as applied here to enable use of an uncertainty-based approach provides more information but is too time-consuming and cost-intensive to become common practice.

Aflatoxin M1↗

Determinants of aflatoxin levels in Ghanaians: sociodemographic factors, knowledge of aflatoxin and food handling and consumption practices.

Aflatoxins are among the most potent of carcinogens found in staple foods such as groundnuts, maize and other oil seeds. This study was conducted to measure the levels of aflatoxin B(1) (AFB(1)) albumin adducts in blood and aflatoxin M(1) (AFM(1)) metabolite in urine of people in a heavy peanut and maize consuming region of Ghana and to examine the association between aflatoxin levels and several socio-demographic factors and food handling and consumption practices. A cross-sectional study was conducted in four villages in the Ejura Sekyedumase district of Ghana. A socio-demographic survey was administered to 162 participants. Blood samples were collected from 140 and urine samples from 91 of the participants and AFB(1) albumin-adduct levels in blood and AFM(1) levels in urine were measured. High AFB(1) albumin-adduct levels were found in the plasma (mean+/-SD=0.89+/-0.46pmol/mg albumin; range=0.12-3.00pmol/mg; median=0.80pmol/mg) and high AFM(1) levels in the urine (mean+/-SD=1,800.14+/-2602.01pg/mg creatinine; range=non-detectable to 11,562.36pg/mg; median=472.67pg/mg) of most of the participants. There was a statistically significant correlation (r=0.35; p=0.007) between AFB(1)-albumin adduct levels in plasma and AFM(1) levels in urine. Several socio-demographic factors, namely, educational level, ethnic group, the village in which participants lived, number of individuals in the household, and number of children in the household attending secondary school, were found to be significantly associated with AFB(1) albumin-adduct levels by bivariate analysis. By multivariate analyses, ethnic group (p=0.04), the village in which participants live (p=0.02), and the number of individuals in the household (p=0.01), were significant predictors of high AFB(1) albumin-adducts. These findings indicate strongly that there is need for specifically targeted post-harvest and food handling and preparation interventions designed to reduce aflatoxin exposure among the different ethnic groups in this region of Ghana.

Aflatoxin B1↗

Cytochrome P450-mediated metabolism and cytotoxicity of aflatoxin B(1) in bovine hepatocytes.

Aflatoxin B(1) (AFB(1)) biotransformation comprises cytochrome P450-mediated reactions resulting in hydroxylated and demethylated metabolites as well as AFB(1) epoxides. As the latter are highly nucleophilic, the species-specific rate of epoxidation and the ability for rapid conjugation to glutathione by glutathione S-transferase determines the individual susceptibility to AFB(1). Here we show the time- and dose-dependent rate of AFB(1)-metabolism in bovine hepatocytes. Aflatoxin M(1) (AFM(1)) is the most prominent metabolite formed within the first 2-8 hr of incubation, whereas AFB(1)-dhd is detectable in medium mainly after a prolonged incubation period. The delayed formation of AFB(1)-dhd corresponds to the cytotoxicity demonstrated by the MTT assay. alpha-Naphthoflavone and ketoconazole, inhibitors of CYP1A and CYP3A, respectively in humans, were used to evaluate the contribution of specific P450 isoenzymes in bovine biotransformation of AFB(1). Initial experiments confirmed that alpha-naphthoflavone and ketoconazole inhibited ethoxyresorufin O-deethylation and testosterone 6beta-hydroxylation also in bovine hepatocytes. Both inhibitors reduced AFM(1) and AFB(1)-dhd formation concentration dependently, suggesting that both enzyme groups contribute to the formation of these metabolites. However, the formation of AFM(1) was less inhibited by both compounds than the formation of AFB(1)-dhd.

Aflatoxin B1↗