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PubMed2002

Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin m(1) in milk and milk-based confectionery.

High-titer rabbit polyclonal antibodies to aflatoxin M(1) (AFM1) were produced by utilizing AFM1-bovine serum albumin (BSA) conjugate as an immunogen. An indirect competitive enzyme-linked immunosorbent assay was standardized for estimating AFM1 in milk and milk products. To avoid the influence of interfering substances present in the milk samples, it was necessary to prepare AFM1 standards in methanol extracts of certified reference material (CRM) not containing detectable AFM1 (< 0.05 ng/g). The reliability of the procedure was assessed by using CRM with AFM1 concentrations of < 0.5 and 0.76 ng/g. Also, assays of milk samples mixed with AFM1 ranging in concentration between 0.5 and 50 ng/L gave recoveries of > 93%. The relative cross-reactivity with aflatoxins (AF) and ochratoxin A, assessed as the amount of AFM1 necessary to cause 50% inhibition of binding, was 5% for AFB1 and much less for AFB2, AFG1, and AFG2; there was no reaction with ochratoxin A. AFM1 contamination was measured in retail milk and milk products collected from rural and periurban areas in Andhra Pradesh, India. Of 280 milk samples tested, 146 were found to contain < 0.5 ng/mL of AFM1; in 80 samples it varied from 0.6 to 15 ng/mL, in 42 samples from 16 to 30 ng/mL, and in 12 samples from 31 to 48 ng/mL. Most of the milk samples that contained high AFM1 concentrations were obtained from periurban locations. The results revealed a significant exposure of humans to AFM1 levels in India and thus highlight the need for awareness of risk among milk producers and consumers.

Aflatoxin M1↗
PubMed2003

ELISA and HPLC determination of the occurrence of aflatoxin M(1) in raw cow's milk.

Raw cow's milk collected from dairy farms in the province of Leon, Spain, was examined for aflatoxin M(1) (AFM(1)). The samples were analysed with a commercial competitive enzyme-linked immunosorbent assay (ELISA) kit and high-performance liquid chromatography (HPLC). The concentrations of AFM(1) in the milk extracts were initially estimated by ELISA, with recovery rates of 74.6-109% for artificially contaminated milk at levels of 10-80 ng x l(1). Samples found to contain more than 10 ng x l(1) were further quantified with HPLC. The mean recovery for this method was 89.3%. The quantification limit was 10 ng x l(1) for both ELISA and HPLC. Although AFM(1) was confirmed in only 3.3% of the samples, the concentrations in all these cases were lower than the maximum limit applicable to these products pursuant to European Union legislation. Both methods were validated with reference material certified by the Community Bureau of Reference.

Aflatoxin M1↗
PubMed2002

Occurrence of aflatoxin M(1) in raw and market milk commercialized in Greece.

From December 1999 to May 2000, 114 samples of pasteurized, ultrahigh temperature-treated (UHT) and concentrated milk were collected in supermarkets, whereas 52 raw milk samples from cow, sheep and goat were obtained from different milk producers all over Greece. Sample collection was repeated from December 2000 to May 2001 and concerned 54 samples of pasteurized milk, 23 samples of bulk-tank raw milk and 55 raw milk samples from cow, sheep and goat. The total number of samples analysed for aflatoxin M(1) (AFM(1)) contamination by immunoaffinity column extraction and liquid chromatography was 297. In the first sampling, the incidence rates of AFM(1) contamination in pasteurized, UHT, concentrated and cow, sheep and goat raw milk were 85.4, 82.3, 93.3, 73.3, 66.7 and 40%, respectively, with only one cow raw milk and two concentrated milk samples exceeding the EU limit of 50 ng l(-1). In the second sampling, the incidence rates of AFM(1) contamination in pasteurized, bulk-tank and cow, sheep and goat raw milk were 79.6, 78.3, 64.3, 73.3 and 66.7%, respectively, with only one cow and one sheep raw milk samples exceeding the limit of 50 ng l(-1). The results suggest that the current regulatory status in Greece is effective.

Aflatoxin M1↗
PubMed2005

Aflatoxin M(1) and ochratoxin A in a human milk bank in the city of São Paulo, Brazil.

Because infants are more susceptible to the adverse effects of mycotoxins, this work was carried out to determine aflatoxin M(1) (AFM(1)) and ochratoxin A (OA) in milk from the Human Milk Bank of the Southern Regional Hospital, São Paulo, Brazil. Analytical methods were first established and evaluated. The methods involved the extraction of AFM(1) with methanol and OA with 1% aqueous sodium bicarbonate solution and methanol, clean-up with immunoaffinity columns having antibodies specific for each mycotoxin and quantification by high performance liquid chromatography (HPLC) with fluorescence detection. The method established for AFM(1) had mean recovery percentages of 94, 77 and 82% and coefficients of variation of 17.5, 3.4 and 4.2% at 0.01, 0.03 and 0.05 ng ml(-1), respectively. For the OA method, the corresponding values were 84, 84 and 75% for recovery and 14.1, 3.7 and 4.0% for the coefficient of variation. The limit of quantification for both methods was 0.01 ng ml(-1). Of a total of 50 samples analysed, only one was contaminated with AFM1, at 0.024 ng ml(-1), and two with OA, at 0.011 and 0.024 ng ml(-1). Although the incidence observed was low, it is recommended that the study be extended to other milk banks of the city of São Paulo.

Aflatoxin M1↗
PubMedDate not supplied

Mycotoxin methodology.

Sensitive, specific, accurate and precise methods of analysis are needed for enforcement of mycotoxin regulations, other monitoring programmes, and research studies. Rapid screening tests are useful for control at all stages of food and feed production. There is a wide choice of both quantitative and qualitative methods for the more well known mycotoxins. Those at present covered by method standardization organizations such as AOAC International are aflatoxins (including M1), Alternaria toxins, citrinin, cyclopiazonic acid, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes, and zearalenone. Methodology for mycotoxins is selectively reviewed in this paper with emphasis on the procedures comprising the analytical method--sampling, extraction of naturally contaminated samples, clean-up, detection and determination, and confirmation. Also covered are automation, method comparison, and method assessment.

Chromatography, High Pressure Liquid↗
PubMed1996

Inhibition of aflatoxin Ml excretion in rat urine during dietary intervention with oltipraz.

4-Methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (oltipraz) is an effective chemopreventive agent against several classes of carcinogens in many target organs. Induction of carcinogen detoxication enzymes, particularly glutathione S-transferases, appears to be an important component of the protective actions of oltipraz. It has recently been observed that addition of oltipraz to rat liver microsomes or to cultured human hepatocytes blocks the oxidative metabolism of aflatoxin B1 (AFB1) to its 8,9-oxide and the hydroxylated derivative aflatoxin Ml (AFM1). 0ltipraz is a competitive and perhaps irreversible inhibitor of cytochromes P450 1A2 and 3A4. To determine whether oltipraz can affect cytochrome P450-dependent metabolism of AFB1 in vivo we have assessed the effect of oltipraz on the urinary excretion of oxidative metabolites of AFB1 before, during and after a transient intervention. Male F344 rats, housed individually in glass metabolism cages, were gavaged daily with 25 microg [3H]AFB1 for 28 consecutive days. Starting on day 6 and extending to day 16 half of the rats were fed a diet supplemented with 0.075% oltipraz. Sequential 24 h urine samples were collected and a subset analyzed for AFB1 metabolites. AFM1 was the major metabolite detected in all urine samples, accounting for 2-6% of the administered dose. The excretion of AFM1 was greatly reduced (77%) during the active phase of the intervention, when oltipraz was added to the diet, but rapidly returned to control levels after cessation of oltipraz administration. This inhibition of AFM1 excretion was not seen in animals receiving oltipraz by gavage 24 h prior to dosing with AFB1. Collectively these data are consistent with the view that oltipraz or a short-lived metabolite inhibits cytochrome P450 1A2 in vivo.

Aflatoxin B1↗
PubMed2004

Morbidity in neonates of mothers who have ingested aflatoxins.

This study was undertaken to assess whether aflatoxin M(1) concentrations in newborn infants correlated with those of their mothers and to determine whether the presence of aflatoxin M(1) in cord blood was associated with an increase in morbidity in the newborn. There was a strong correlation (r =0.797, p <0.0001) between mothers' and cord blood levels of aflatoxin. There was also a strong negative correlation between aflatoxin levels and birthweight (r =-0.565, p <0.001) but there was no association between aflatoxin M(1) concentration in maternal or cord blood and rates of jaundice or infection.

Aflatoxin M1↗
PubMed2002

Effects of dimethyl diphenyl bicarboxylate on the metabolism and hepatotoxicity of aflatoxin B1 in rats.

AIM: To study the effect of antihepatitis drug, dimethyl diphenyl bicarboxylate (DDB) on the metabolism and hepatotoxicity of aflatoxin B1(AFB1) in rats. METHODS: Rats were given orally DDB 300 mg.kg-1.d-1 for 3 days and then injected intraperitioneally with AFB1 1.5 mg.kg-1. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were examined 16 hours after the injection of AFB1. The in vitro metabolism of AFB1 by DDB-pretreated rat liver microsome was investigated by HPLC assay. RESULTS: DDB (300 mg.kg-1) pretreatment provided significant protection against AFB1 hepatotoxicity as evidenced by the decrease of AFB1-elevated serum marker enzymes in rats. Pretreatment with DDB was shown to slightly increase the level of AFM1, the less toxic metabolite. DDB significantly increased the liver cytochrome P450 content, P450 isozyme 2B1-mediated 7-pentoxyresorufin O-dealkylase (PROD) activity, cytosolic glutahione (GSH) level and GSH S-transferase (GST) activities. In addition, DDB slightly increased P450 isozymes, 3A-mediated erythromycin-demethylase and 1A-mediated 7-ethoxyresorufin O-deethylase (EROD) activities. CONCLUSION: The results indicate that DDB protected rats against AFB1 hepatotoxicity by increasing the detoxifying metabolism of AFB1 in the liver.

Aflatoxin B1↗
PubMed1992

[Aflatoxins and primary liver cancer--a population based case-control study].

A case-control study of primary liver cancer (PLC) was conducted in 1989 at Fusui County, Guangxi Province. There were 99 PLC cases and 99 age-sex-residence matched controls. There were no difference of age, sex, race, cultural, level, marital status and annual income distribution (P > 0.05). The mean dietary AFB1 intakes of cases and controls were 117.72 + 105.68 mg/d, the mean outputs of AFM1 were 22.56 and 21.62 ng per morning urine respectively. The mean sera aflatoxin-albumin adducts in both groups were 24.96 and 18.72 pg per mg albumin respectively. These indicators were of no statistical significance. Conditional Logistic regression showed that HBV infection (OR = 5.33), drinking pond-ditch water (OR = 3.70), family history of PLC (OR = 2.88), and total alcohol intakes (OR = 1.002) were statistically significant as risk factors (P > 0.05) rather than aflatoxins. This may be due to the fact that the samples selected in hyperendemic area both took high quantities of aflatoxin.

Aflatoxin B1↗
PubMed1992

[The aflatoxins and liver cancer in Guangxi, China].

The AFB1 intake and the AFM1 excretion of 81 households in 10 villages, Guanxi were investigated using the ELISA method. The results showed that there was positive correlation between PLC mortality and AFB1 intake from corn and peanut oil, but not from rice. The results of stepwise regression showed that main factors were AFB1 intake of males, AFM1 excretion of females and consumption of corn. The results showed that aflatoxins were correlated with mortality rates of liver cancer. Further investigation needs to be carried out in case-control and cohort studies.

Aflatoxin B1↗
PubMed1981

Production and characterization of aflatoxin B2a antiserum.

The specificity and sensitivity of antiserum elicited from rabbits against aflatoxin B2a-bovine serum albumin conjugates were characterized with a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA). Aflatoxin B1 was first converted to aflatoxin B2a and then conjugated to bovine serum albumin and horseradish peroxidase by a reductive alkylation method. The antiserum was developed in New Zealand white rabbits by multiple-site injection with the aflatoxin B2a-bovine serum albumin conjugate. Antibody titers were determined by both RIA and ELISA. Competitive RIAs with various aflatoxin analogs indicated that the antiserum was most reactive with aflatoxin B1 and slightly cross-reactive with aflatoxins B2a, B2, and M1. Competitive ELISAs showed the antiserum to be equally specific for aflatoxins B2a and B12 and less reactive with aflatoxins B2 and M1. The relative sensitivities of RIA and ELISA for aflatoxin B1 quantitation were 100 and 10 pg per assay, respectively.

Aflatoxins↗
PubMed1985

[The effect of the preparation Kurasan (ethoxyquin) on the toxigenic properties of the mold Aspergillus flavus].

The effects of Kurasan antioxidant (min. 78% ethoxyquin) were studied on toxin formation of the fungus Aspergillus flavus (strain AF 1982/M1). Aflatoxins mostly of type B1, G1, M1 are produced by the mentioned fungal strain. The strain was cultivated for seven days at high relative humidity on a substrate mostly of cereals (Karlovarské suchary--Carlsbad rusks). Application of 25 mg Kurasan per 1 kg substrate reduced type G1 aflatoxin formation by 90%. The total production of aflatoxins decreased expressively (by 50-60% after addition of 100-200 mg Kurasan per 1 kg substrate). It was only at high amounts of Kurasan that B1 and M1 aflatoxin formation was inhibited and it was correlated. Positive effects of Kurasan antioxidant applied to feed mixtures were demonstrated--aflatoxin formation by toxicogenic fungi is inhibited.

Aflatoxins↗
PubMed1979

Determination of mycophenolic acid, penicillic acid, patulin, sterigmatocystin, and aflatoxins in cheese.

A method has been developed for detection of aflatoxins, mycophenolic acid, patulin, penicillic acid, and sterigmatocystin in cheese. It is based on selective extraction with a mixture of equal volumes of 5% sodium chloride, methanol, and aceton, precipitation of caseins at -25 C, defatting with hexane, and removal of extraneous matter by transfer of mycotoxins to chloroform and ethyl acetate. The extract is purified further by column chromatography. Mycotoxins are quantitated on thin layer chromatograms by fluorescence comparisons. Mycophenolic acid, patulin, and penicillic acid are visualized with diethylamine. The limits of detection in cheese are about 20 micrograms/kg for mycophenolic acid, patulin, and sterigmatocystin, 30 microgram/kg for pencillic acid, and 1 microgram/kg for aflatoxins B1 and M1.

Aflatoxins↗
PubMed1984

Liver carcinogenesis in tropical Africa.

The geographical pathology of hepatocellular carcinoma (HCC) was essentially anecdotal until systematic cancer registration was introduced, but it is now clear that sub-Saharan Africa is a high-incidence area. The disease is multifactorial in etiology, the possible etiological agents including hepatitis B virus and a number of chemical carcinogens, among which the most important appear to be the aflatoxins and N-nitroso compounds. Medicinal plants and herbal teas used in the tropics also contain compounds such as furocoumarins that are mutagenic and/or carcinogenic. A modified form of the Ames Salmonella typhimurium assay was used to study the mutagenicity of aflatoxins B1 and M1 and palmotoxin B0, a co-metabolite of aflatoxin B1, and also of chamuvaritin and chamuvarin, two benzyldihydrochalcones derived from the roots of Uvaria chamae, which are used for medicinal purposes in West Africa. The mutagenicity of a number of furocoumarins isolated from Nigerian medicinal plants was studied by means of the same assay as well as with Chinese hamster V79 cells and C3H 10T1/2 cells; in the last two systems the studies were carried out both with and without photoactivation. The same compounds, and 8-methoxypsoralen, were also investigated by means of cell transformation studies.

Aflatoxins↗
PubMed1995

Functional impairment of rat Kupffer cells induced by aflatoxin B1 and its metabolites.

Contamination of food with mycotoxins is a major health problem. Impairment of several immune functions has been repeatedly reported in animals fed with contaminated fodder. Since the liver is a major target of toxicity by aflatoxins, the effects of aflatoxins B1, and its hepatic metabolites Q1 and M1 on Kupffer cell function was investigated in vitro. Aflatoxin B1 induced significant (P < 0.05) inhibition of phagocytosis, intracellular killing of Candida albicans, and intrinsic anti-Herpes virus activity at concentrations as low as 0.01 pg ml-1. Aflatoxin Q1 and M1 had similar effects on phagocytosis and microbicidal activity, but were two- to ten-fold less potent than aflatoxin B1.

Aflatoxin B1↗
PubMed1992

Distribution of aflatoxin-producing moulds and aflatoxins in dairy cattle feed and raw milk.

Distribution of aflatoxigenic moulds and aflatoxin B1 in Yugoslav dairy cattle feeds as well as the presence of aflatoxin B1 and M1 in raw milk, was tested. The experiments were carried out through three years (in all seasons). Samples were taken from state and private farms in Vojvodina. Feeds were contaminated in 83-100% with moulds. Fungi of farms in Vojvodina. Feeds were contaminated in 83-100% with moulds. Fungi of Aspergillus flavus-oryzae group were present permanently and the highest incidence of them was noticed during the third research year. Aflatoxin B1 was not found in the first year, but malt spent grains used for cows' feeding in summer of the second research year was contaminated with it (50.0 micrograms/kg). The same feed and pelleted sugar beet pulp were contaminated with aflatoxin B1 in winter, spring and summer of the third research year (5.0 to 16.0 micrograms/kg). Aflatoxin B1 and M1 were not found in raw milk through three-years investigations.

Aflatoxin B1↗
PubMed1982

High pressure liquid chromatographic determination of aflatoxins by using radial compression separation.

A rapid method for the determination of aflatoxins was developed using high pressure liquid chromatography and a radial compression separation system. A standard solution of aflatoxins B1, B2, G1, G2, and M1 was analyzed solution of aflatoxins B1, B2, G1, G2, and M1 was analyzed at flow rates of 2.0 and 6.0 mL/min. Retention times, peak heights, and peak areas were reproducible over a 3-day period. Coefficients of variation for aflatoxin B1 at 2.0 and 6.0 mL/min were, respectively, 1.04 and 0.87% (retention time); 2.9 and 4.7% (peak height); and 8.2 and 4.7% (peak area). At 6.0 mL/min there was an approximate 25% loss in sensitivity but a greater than 50% reduction in retention time. Separation of all the aflatoxins was excellent using a dual flow rate of 2.0 mL/min with a change to 8.0 mL/min at 15 min post-injection. The applicability of the radial compression separation system for the rapid determination of aflatoxins in human tissues was also tested. Spiked samples of liver, serum, and urine-showed good resolution of all aflatoxin peaks at the higher flow rates.

Aflatoxins↗