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Effect of some food preservatives on aflatoxin production.

The effect of some food preservatives, such as sorbic (SA) and propionic (PA) acids, on aflatoxin production in synthetic media or in moistened (20%) wheat seeds, was investigated. The preservatives tested, added to synthetic media at sublethal concentrations both at the inoculum and after 5 days of incubation, stimulated aflatoxin production by Aspergillus parasiticus. Sorbic and propionic acids are metabolized by the fungus in vivo and in vitro. Lower concentrations of PA and SA (0.05 to 0.1% w/w) in wheat seeds are ineffective against both fungal growth and aflatoxin production, whilst the combined use of butylated hydroxy toluene (BHT), and PA or SA was more effective in controlling aflatoxin production than their use as single components.

Aflatoxins↗

The development of five animal feed reference materials, certified for their aflatoxin B1 content.

The development of three peanut meal and two compound feed reference materials and the certification of their aflatoxin B1 content is described. The materials were prepared and certified within the Measurements and Testing Programme of the Commission of the European Communities as part of a broad activity to improve accuracy and agreement of results of measurements on food and agriculture. RM 262 (peanut meal) was prepared from uncontaminated peanut products. RM 263 and RM 264 (peanut meals) were prepared from naturally contaminated peanuts which were blended with uncontaminated ones, to achieve the desired aflatoxin B1 mass fractions. RM 375 and RM 376 (compound feeds) were made by blending decontaminated dairy feed together with commercial feed ration and contaminated dairy feed with several feed compounds, respectively. Details are given of the preparation and the investigations to verify homogeneity and stability of the materials. The certification exercise was carried out by 17 laboratories using a variety of extraction and clean-up procedures. Most laboratories used liquid chromatography as the determinative step, although operating under a variety of chromatographic conditions. A few laboratories applied thin layer chromatography with densitometric quantification. Peanut meal RM 262 was certified as containing aflatoxin B1 at a mass fraction of < 3 micrograms/kg, RM 263 at 43.3 +/- 2.1 micrograms/kg and RM 264 at 204 +/- 10 micrograms/kg. Compound feed RM 375 was certified as containing aflatoxin B1 at a mass fraction of < 1 micrograms/kg and RM 376 at 9.2 +/- 0.5 micrograms/kg. The materials can be employed either to establish or confirm a calibration curve, or to check the performance of a method.

Aflatoxin B1↗

Exposure of infants to aflatoxin M1 from mothers' breast milk in Abu Dhabi, UAE.

During a survey of the occurrence of aflatoxins in mothers' breast milk carried out in Abu Dhabi, involving 445 donors at the Cornich Hospital and the Al Nehyan Clinic for Maternity and Childhood, 99.5% of samples were found to contain aflatoxin M1 at concentrations ranging from 2 pg ml-1 to 3 ng ml-1. The mothers represented a wide range of nationalities, ages and health status and the opportunity was used to seek any correlation between these factors and the concentration of aflatoxin M1 in breast milk. The protein, fat and lactose contents of milk samples were also determined and the possibility of any correlation between any of these and aflatoxin M1 was studied.

Adolescent↗

Occurrence of aflatoxin M1 in milk and dairy products commercialized in Campinas, Brazil.

One hundred and fifty-two samples of pasteurized milk, powdered milk, cheese and yoghurt, marketed in Campinas, Brazil in 1989-1990, were analysed for aflatoxin M1 by the AOAC TLC method (visual quantitation) 980.21. Fifty-two pasteurized milk samples were also analysed in 1992 by the AOAC HPLC method 986.16. Aflatoxin M1 was not detected in the 1989-1990 samples. Four milk samples of the 1992 batch were contaminated at 73-370 ng/1. Except for the sample with 370 ng/l, which would have been also found positive by the TLC method, the detection of aflatoxin M1 in 1992 reflects the higher sensitivity of the HPLC method, not a greater occurrence of the toxin. Contamination of milk and milk products with aflatoxin M1 does not appear to be a serious public health problem in the city of Campinas at the moment.

Aflatoxin M1↗

Occurrence of aflatoxin M1 in commercial pasteurized milk determined with ELISA and HPLC.

Eighty-one samples of commercial pasteurized milk from Athens market were analysed for the presence of aflatoxin M1 (AFM1). A combination of a commercial ELISA kit and a modified HPLC method was applied for the rapid and reliable determination of AFM1. AFM1 concentrations in milk extracts were initially estimated by ELISA. Samples found to contain more than 5 ng/l were further quantitated by HPLC. Determination was performed after derivatization of AFM1 to its hydroxylated product AFM2a. The recovery of the HPLC method used was found to be close to 100%. Thirty-two samples contained aflatoxin M1 at levels of 2.5-5 ng/l, none contained more than 5 ng/l, while 31 contained only traces of aflatoxin (0.5-1 ng/l). In nine samples no AFM1 was detected. There was no seasonal influence on the aflatoxin content of the milk samples analysed.

Aflatoxin M1↗

Detection of naphthols and aflatoxins in Nigerian cord blood.

Widespread use of napthol-containing compounds and frequent contamination of foods by aflatoxins occurs in Nigeria. Napthols cause haemolysis and aflatoxins are hepatotoxic. A study was carried out to determine the extent of fetal exposure to these compounds and their influence on birthweight. Cord blood samples were collected at delivery from 625 babies and their sera were analysed for aflatoxins and naphthols. Mothers' histories and babies' weights were recorded. Naphthols were detected in 6.9% and aflatoxins in 14.6% of serum samples. No correlation was found between the presence of either compound and birthweight. Reported exposure to naphthalene-containing compounds was not related to detection of serum naphthol. Results show considerable fetal exposure to these potentially toxic compounds in Ibadan, Nigeria.

Aflatoxins↗

Aflatoxins and ochratoxin A in the weaning food of Nigerian children.

A total of 48 samples of maize-based gruels, used as weaning food for children, were taken daily from hospital meals and from patients' mothers while their children were on admission at the Wesley Guild Hospital, Ilesha, Nigeria. These samples were analysed for aflatoxins and ochratoxin A. Twelve samples (25%) were positive for aflatoxins (concentration range 2-19,716 pg/g) but only four (8%) were positive for ochratoxin (concentration range 142-6516 pg/g). In the majority, the concentrations were relatively low, only two having aflatoxin and one ochratoxin A levels greater than 1000 pg/g. It is concluded that, unlike aflatoxin, ochratoxin A contamination is unlikely to present a major health hazard to the newly weaned child in this area of Nigeria.

Aflatoxins↗

Measurements of airborne aflatoxins during the handling of contaminated corn.

Sample of airborne dust generated the handling of aflatoxin-contaminated corn were collected and analyzed to assess potential exposures of farmers and other agricultural workers to these mycotoxins. Using high volume total dust samplers and a high volume Andersen sampler, downwind dust samples were collected on glass filter when contaminated corn was transferred by augers from a storage bin into a wagon and back into the storage bin. The aflatoxin B1 content of the 15 dust samples ranged from 12.5 to 204.3 ppb, with an average of 138 ppb; the aflatoxin B2 content ranged from 1.1 to 41.6 ppb, with an average of 24.6 ppb. The B1 and B2 levels of contamination in the bulk corn were 223.9 and 17.5 ppb, respectively. The gravimetric dust concentration in the air ranged from 7 mg/m3 to 417 mg/m3. The samples taken with an Andersen sampler indicate the dust is relatively coarse with only approximately 17% less than 7 micrometer. An analysis of the dust from each stage showed higher levels of aflatoxins in the larger first-stage particles than in the finer particles on the succeeding stages. The results of this study indicate that the dust generated when handling contaminated commodities also may be contaminated and represent a potential inhalation hazard. This fact, coupled with the extreme toxicity and carcinogenicity previously demonstrated in animal studies, suggests that appropriate measures be taken to prevent worker exposure during handling of contaminated materials.

Aflatoxins↗

Airborne aflatoxin in corn processing facilities in Georgia.

Problems affecting the health of agricultural workers in processing facilities where grains are contaminated with aflatoxin have been noted previously. Airborne particulates produced during processing are reported to produce various carcinomas when inhaled by factory workers. Two corn processing plants within Georgia were surveyed during the fall of 1984 and 1985 with utilization of an Andersen 6-stage air sampler, a high-volume air sampler and a slit sampler. No airborne aflatoxin was found; however, 10% of settled dust samples were contaminated with aflatoxin. The average particles were found to be globular in shape with an effective diameter between 2 to 3 microns. Only 30% of the bulk corn samples contained aflatoxin, which was present at low levels (0.15 to 8 ppb).

Aflatoxins↗

Aflatoxin-albumin adducts in human sera from different regions of the world.

An immunoassay now permits the determination of human exposure to aflatoxin at an individual level and consequently allows a better assessment of the role of aflatoxin, and its interaction with hepatitis B virus infection, in the aetiology of liver cancer. Measurements of aflatoxin bound to serum albumin in children and adults from various African countries show that between 12 and 100% contain aflatoxin-albumin adducts, with levels up to 350 pg AFB1-lysine equivalent/mg albumin. In Thailand, lower levels and prevalence of this adduct were observed, while no positive sera were detected from France or Poland. Data are presented showing that exposure to this carcinogen can occur throughout life and the relevance of these observations to the understanding of the multifactorial aetiology of liver cancer in these countries is discussed.

Adult↗

Potent inhibition of aflatoxin-induced hepatic tumorigenesis by the monofunctional enzyme inducer 1,2-dithiole-3-thione.

1,2-Dithiole-3-thiones are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, radioprotective and chemoprotective properties. Several substituted 1,2-dithiole-3-thiones are used medicinally and one of these, oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione], has been recently shown to be an inhibitor of aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Structure-activity studies have been undertaken to probe the mechanisms by which dithiolethiones inhibit carcinogenesis. Such studies revealed that unsubstituted 1,2-dithiole-3-thione was more effective than oltipraz at inhibiting aflatoxin-DNA adduct formation in vivo and at inducing electrophile detoxication enzymes in cell culture. In the present studies the effects of dietary administration of 1,2-dithiole-3-thione on the induction of xenobiotic metabolizing enzymes and inhibition of aflatoxin-induced hepatic tumorigenesis were examined. Male F344 rats were fed graded doses of 1,2-dithiole-3-thione (0.001-0.03%) for 4 weeks. During the second and third weeks of 1,2-dithiole-3-thione feeding, rats were dosed by gavage with 250 micrograms of AFB1/kg five times a week. Rats were then restored to control AIN-76A diet 1 week after cessation of AFB1 dosing. At 4 months, focal areas of hepatocellular alteration were identified and quantified by staining sections of liver for gamma-glutamyltranspeptidase (GGT) activity and glutathione S-transferase P (GST-P) expression. Treatment with 1,2-dithiole-3-thione at the lowest dose (0.001%) reduced by greater than 80% the volume of liver occupied by GGT or GST-P foci; higher dietary concentrations provided greater than 98% reductions in the volume per cent of these markers for presumptive preneoplastic lesions. All dietary concentrations of 1,2-dithiole-3-thione resulted in significant elevations in hepatic GST activities. In accord with the protective effects against tumorigenesis, 4- to 6-fold increases in the specific activities of aflatoxin-glutathione conjugation were observed in cytosols prepared from livers of animals fed 1,2-dithiole-3-thione. By contrast, 1,2-dithiole-3-thione did not have any detectable inductive effects on hepatic microsomal cytochrome P450 levels or activities. Dietary administration of 1,2-dithiole-3-thione also elevated activities of GSTs and other phase II enzymes in several extrahepatic organs. This broad pattern of induction of detoxication enzymes by 1,2-dithiole-3-thione supports the potential widespread use of this compound as a protective agent against chemical carcinogenesis and other forms of electrophile toxicity.

Aflatoxin B1↗

Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their secretion into breast milk.

The breast cancer resistance protein (BCRP/ABCG2) usually protects the body from a wide variety of environmental and dietary xenotoxins by reducing their net uptake from intestine and by increasing their hepatobiliary, intestinal and renal elimination. BCRP is also highly expressed in lactating mammary glands in mice, and this expression is conserved in cows and humans. As a result, BCRP substrates can be secreted into milk. We investigated whether different classes of dietary carcinogens are substrates of Bcrp1/BCRP and the implications for systemic exposure and breast milk contamination. Using polarized cell lines, we found that Bcrp1 transports the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and the potent human hepatocarcinogen aflatoxin B1, and decreases their cellular accumulation up to 10-fold. In vivo pharmacokinetic studies showed that [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin B1 plasma levels were substantially lower in wild-type compared with Bcrp1-/- mice, after both oral and intravenous administration, demonstrating that Bcrp1 restricts systemic exposure to these carcinogens. Moreover, Bcrp1 mediates transfer of [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin into milk, with 3.4+/-0.6, 2.6+/-0.3 and 3.8+/-0.5-fold higher milk to plasma ratios, respectively, in lactating wild-type versus Bcrp1-/- mice. We have thus identified Bcrp1/BCRP as one of the molecular mechanisms by which heterocyclic amines and aflatoxin are transferred into milk, thereby posing a health risk to breast-fed infants and dairy consumers. Paradoxically, Bcrp1/BCRP appears to have both protective and adverse roles with respect to exposure to dietary carcinogens.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

Determination of aflatoxins in peanut products using reverse phase HPLC.

A high performance liquid chromatographic (HPLC) method is described for the determination of the four major aflatoxins, B1, B2, G1 and G2, in peanut products. The aflatoxins are extracted by adapting a procedure developed by Pons (1) at the SRRC, USDA, and quantitated utilizing a new 5 mum reverse-phase column with NaCl/acetontrile/methanol mobile phase (3 + 1 + 1). The 5 mum column achieved baseline resolution of each of the four aflatoxins. Retention times and peak heights were reproducible. The procedure was successfully applied to several types of peanut products and was applicable to both roasted and unroasted peanuts, which is a decided advantage over the current CB and BF extraction methods. Additionally, it can be used for sweetened peanut matrixes with no interferences in the chromatography. The total time required for sample preparation and aflatoxin determination is less than 1.5 hours.

Aflatoxins↗

Aflatoxin B; specific antibodies and their use in radioimmunoassay.

New Zealand White rabbits immunized with covalent conjugates prepared from polylysine and the O-carboxy-methyloxime derivatives of either aflatoxin B1 (AFB1) or an analogue, 5,7-dimethoxycyclopentenon (2,3-c) coumarin, produced antibodies that bind 3H-AFB1. The specificities of the antisera with respect to aflatoxins BI, B2a, G1, G2, Q1, P1, and some other structually related compounds were determined. Radioimmunoassays that can detect levels as low as 0.27 pmoles (0.06 ng) of AFB1-were used to analyze serum, urine, and crude extracts of corn and peanut supplemented with aflatoxin. In the foodstuffs, as little as 1 mug AFB1/kg was measured. The immunoassay was at least as sensitive and specific as other available analytic methods, but did not require the purification of samples by chromatography before analysis. The technique may be particularly useful in epidemiologic studies designed to study the possible relationship between chronic aflatoxin ingestion and cancer.

Aflatoxins↗

Effects of black tea theafulvins on aflatoxin B(1) mutagenesis in the Ames test.

Black tea theafulvins, a fraction of thearubigins isolated from black tea aqueous infusions, potentiated the mutagenic activity of the mycotoxin aflatoxin B(1) in the Ames test, in the presence of a hepatic S9 activation system derived from Aroclor 1254-treated rats. In contrast, when the S9 activation system was replaced with isolated microsomes, theafulvins suppressed the mutagenicity of the mycotoxin. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, incorporation of the theafulvins into the activation system reduced the mutagenic activity, whereas if it was added before termination of microsomal activity a potentiation of mutagenic response was observed. In in vitro studies, theafulvins inhibited epoxide hydrolase and glutathione S-transferase activities in a concentration-dependent manner. Finally, the mutagenicity of aflatoxin B(1) was much more pronounced in bacteria that were pre-exposed to theafulvins but from which they were subsequently washed off. It may be inferred from the above studies that the genotoxic synergy between aflatoxin B(1) and black tea theafulvins does not occur during the bioactivation of the carcinogen, but may partly be due to decreased deactivation of the reactive intermediate, aflatoxin B(1) 8,9-oxide, by conjugation with glutathione.

Aflatoxin B1↗

Aflatoxin-induced toxicity and depletion of hepatic vitamin A in young broiler chicks: protection of chicks in the presence of low levels of NovaSil PLUS in the diet.

Aflatoxin contamination of foods and livestock feeds is an ongoing problem. In this research, NovaSil PLUS (NSP), a calcium montmorillonite clay that is commonly used as an anticaking agent in feeds, was evaluated for its ability to bind aflatoxin B1 (AfB1) in vitro and to prevent the onset of aflatoxicosis and vitamin A depletion in broiler chicks in vivo. Isothermal analyses were conducted with NSP and AfB1 to quantitate and characterize critical sorption parameters at equilibrium, i.e., ligand saturation capacities, affinity constants, and thermodynamics of the sorption process. In vitro results indicated that AfB1 was tightly sorbed onto the surface of NSP, which provided a high capacity and high affinity for the ligand. Thermodynamics favored sorption of AfB1 to NSP. The process was exothermic and spontaneous with a mean heat of sorption equal to approximately -50 kJ/mol, suggesting chemisorption (or tight binding). In addition to the in vitro studies, the effectiveness of NSP as an aflatoxin enterosorbent to attenuate the onset of aflatoxicosis in broiler chicks was determined at 3 different inclusion levels in the diet (0.5, 0.25, and 0.125%). NSP alone was not toxic to chicks at a level as high as 0.5% in the total diets (based on body and organ weights, feed intake, and hepatic vitamin A levels). NSP in the diet significantly protected chicks from the effects of highlevel exposure to aflatoxins (i.e., 5 mg/kg) and preserved hepatic vitamin A levels, even at lower dietary intake of clay.

Aflatoxins↗

Effects of dietary oltipraz and ethoxyquin on aflatoxin B1 biotransformation in non-human primates.

Following aflatoxin B1 (AFB) exposure, rats readily develop liver tumors. However, treatment of rats with a variety of compounds, including the synthetic dithiolthione oltipraz and the antioxidant ethoxyquin, protects these rodents from AFB-induced hepatocarcinogenesis. Several epidemiological studies strongly suggest that AFB is also a causative agent of liver cancer in humans. However, relatively little is known about the efficacy of cancer chemoprevention in human and non-human primates. To this end, we examined the effects of chemopreventive agents on AFB metabolism in non-human primates. Hepatic aflatoxin B1 metabolism profiles of macaque (Macaca nemestrina) and marmoset (Callithrix jacchus) monkeys were determined and compared to humans. Quantitatively, the oxidative metabolism of this mycotoxin was similar in the three primate species. In contrast to macaques, both humans and marmosets lacked AFB-glutathione conjugating activity. It was concluded that marmosets resembled human AFB metabolism more closely than the macaques, and therefore, marmoset monkeys were chosen for this study. Eleven adult male marmosets were randomly assigned to three groups. Animals received the synthetic dithiolthione oltipraz, the antioxidant ethoxyquin, or vehicle only. In addition, two single doses of AFB were also administered orally before and after animals were treated with aforementioned compounds. Both oltipraz and ethoxyquin induced aflatoxin B1-glutathione conjugating activity in the livers of some but not all marmosets. In addition, 10 microM oltipraz inhibited cytochrome P450-mediated activation of AFB to the ultimate carcinogenic metabolite, aflatoxin B1-8,9-epoxide, in vitro, up to 51%. Furthermore, animals treated in vivo with oltipraz, but not ethoxyquin, exhibited a significant reduction (53% average) in AFB-DNA adduct formation relative to the control animals (p < 0.05). Together, our data suggest that chemoprevention is also effective in primates; however, most likely to a lesser degree than in rodents.

Aflatoxin B1↗

Aflatoxin exposure and its relationship to kwashiorkor in African children.

The urinary excretion of aflatoxins in normal children from a periurban area, as well as those hospitalized with kwashiorkor and marasmus was assessed. In no case was aflatoxin isolated from the urine. It is concluded that aflatoxin exposure is unusual in this population and that aflatoxins do not play a primary role in the pathogenesis of kwashiorkor.

Aflatoxins↗