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PubMed2004

Determination of aflatoxins and zearalenone in different culture media.

Some molds produce desirable changes in food, but most are merely esthetically undesirable. There has also been an increasing awareness that certain metabolic products of some molds commonly found on foods and feed are dangerous to humans and animals. These toxin substances, mycotoxins, are secondary metabolites produced by different fungi, especially Aspergillus, Penicillium, Fusarium, and, to a lesser degree, Alternaria. The most important toxins for humans are aflatoxins, ochratoxin A, fumonisins, certain trichothecenes, and zearalenone. Aflatoxins are fungal metabolites produced by different Aspergillus species: A. flavus, A. parasiticus, and A. nomius. The most commonly encountered aflatoxins are B1, B2, G1, G2, M1, and M2, but aflatoxin B1 is the most frequently found in contaminated samples, and aflatoxins B2, G1, and G2 are generally not reported in the absence of AFB1. The International Agency for Research on Cancer (IARC) considers that aflatoxins are carcinogenic (hepatocarcinogenic) to humans (group 1) and animals. Zearalenone is an estrogenic mycotoxin produced by several species of Fusarium (F. acuminatum, F. culmorum, F. equiseti, F. graminearum, F. verticilliodes, F. oxysporum, F. poae, F. rosum, F. solani, F. semitectum, and F. sporotrichioides) that primarily colonize different cereal grains. Several reports were noted on the occurrence of ZEA along with various combinations of group B trichothecenes, fumonisins, aflatoxins, and ochratoxins. In most cases, the levels of ZEA were considered to be low; however, the toxicological significance is not known. This toxin is not classifiable as to carcinogenicity in humans (group 3) by the IARC, but ZEA was implied in precocious sexual development in children in Puerto Rico and a breast enlargement in young boys in Italy.

Aflatoxins↗
PubMed2001

Genetic polymorphisms of glutathione S-transferases M1 and T1 associated with susceptibility to aflatoxin-related hepatocarcinogenesis among chronic hepatitis B carriers: a nested case-control study in Taiwan.

This study was conducted to investigate the modifying effect of glutathione S-transferase (GST) M1 and T1 polymorphisms on aflatoxin-induced hepatocarcinogenesis among chronic hepatitis B virus surface antigen (HBsAg) carriers. A total of 79 HBsAg-positive cases of hepatocellular carcinoma (HCC) diagnosed between 1991 and 1997 were identified and individually matched to one or two HBsAg-positive controls on age, gender, residence and date of recruitment from the same cancer screening cohort in Taiwan. Blood samples were tested for hepatitis B and C viral markers by enzyme immunoassay and for aflatoxin B(1) (AFB(1))-albumin adducts by competitive enzyme-linked immunosorbent assay. GSTM1 and GSTT1 genotypes were determined by PCR. There was a statistically significant relationship between detectable levels of AFB(1)-albumin adducts in serum and risk of HCC among chronic HBsAg carriers, with an adjusted odds ratio (OR) of 2.0 [95% confidence interval (CI) 1.1-3.7]. In addition, the effect of aflatoxin exposure on HCC risk was more pronounced among chronic HBsAg carriers with the GSTT1 null genotype (OR 3.7, 95% CI 1.5-9.3) than those who were non-null (OR 0.9, 95% CI 0.3-2.4). The interaction between serum AFB(1)-albumin adduct level and GSTT1 genotype was statistically significant (P = 0.03). For GSTM1 the effect of aflatoxin exposure on HCC risk in those with the null genotype was also greater (adjusted OR 2.8, 95% CI 1.0-7.8) than in those with the gene present (adjusted OR 1.8, 95% CI 0.8-4.5), but the difference was not significant (P = 0.91). Notably, when the interaction between aflatoxin exposure and GSTT1 genotype was considered, aflatoxin exposure by itself was not a significant determinant of HCC risk among chronic HBsAg carriers. These results demonstrate the importance of gene-environment interactions in the multifactorial development of HCC.

Adult↗
PubMed1975

Excretion of carbon-14-labeled aflatoxin B1 via bile, urine, and intestinal contents of the chicken.

Carbon-14-labeled aflatoxin B1 was prepared by growing Aspergillus parasiticus in medium containing sucrose-14-C as the sole carbon source. The 14-C-labeled aflatoxin B1 was extracted with chloroform and purified by thin-layer chromatography (TLC) followed by precipitation from solution and washing. The 14-C-labeled aflatoxin B1 was intravenously given to anesthetized dwarf White Leghorn or Rhode Island Red hens with cannulated ureters and bile ducts. The 14-C had a calculated plasma half-life of 1.5 minutes and rapidly appeared in the bile. The 14-C concentration in the bile reached values approximately 7 times maximum for plasma, and most could no longer be extracted with chloroform. This finding indicated that metabolites of the 14-C-labeled aflatoxin B1 had been excreted against a concentration gradient into the bile. The quantities of 14-C excreted via bile, urine, and intestinal contents remained at a fairly constant ratio of 70:15:15, respectively, over the 315-minute experimental period. Six major chloroform-soluble compounds containing 14-C were isolated. Three of these were tentatively identified as aflatoxin B1, B2, and M1. A 4th was believed to be aflatoxin B2a. None of the early chloroform-soluble metabolites were as cytotoxic as aflatoxin B1.

Aflatoxins↗
PubMed1990

Distribution of aflatoxins in tissues of growing pigs fed an aflatoxin-contaminated diet amended with a high affinity aluminosilicate sorbent.

The effect of hydrated sodium calcium aluminosilicate (HSCAS) added to the diet of swine fed an aflatoxin-contaminated diet on tissue aflatoxin levels was investigated. Pigs were fed control (less than 10 ng/g B1 + B2), contaminated (500-600 ng/g B1 + B2), and contaminated +0.5% HSCAS diets. Tissues analyzed for the presence of aflatoxin B1, B2, and M1 residues included liver, muscle, kidney, and adipose. Addition of HSCAS to the contaminated diet significantly reduced the amount of M1 in liver, kidney, and muscle tissue. Aflatoxin B1 was not reduced in liver or kidney, but was decreased in muscle.

Aflatoxins↗
PubMed1997

Immunotoxicological effects on piglets of feeding sows diets containing aflatoxins.

Three groups of four Large White sows were fed diets containing either 800 ppb purified aflatoxin B1 (group 1), 800 ppb purified aflatoxin G1 (group 2) or 400 ppb B1 and 400 ppb G1 (group 3) throughout gestation and lactation. A control group of four sows was fed a diet free of aflatoxins. Aflatoxins B1 and M1 were found in milk samples taken five and 25 days after parturition from the sows of group 1, aflatoxin G1 was present in the milk of the sows of group 2 and all three aflatoxins were present in samples from the sows of group 3. The concentration of aflatoxin in the milk was about 1000-fold lower than that in the feed, but increased over the 25 days after parturition. The piglet suckling on a central teat was selected from each sow, given sow milk until the fourth day of age, and was then free to eat prepared feed while suckling. At the 25th day of age the selected piglets were removed from the sow and sacrificed. Blood samples were collected from each piglet and cellular populations were separated for immunological measurements: an in vitro lymphocyte proliferation test, and tests to derive the phagocytic activity, phagocytic index and superoxide anion production of monocyte-derived macrophages were carried out along with studies on the motility, differential chemotaxis and chemotactic index of circulating granulocytes. The lymphoproliferative response to mitogens was reduced and monocyte-derived macrophages failed to efficiently produce superoxide anions after oxidative burst stimulation in vitro, while their ability to phagocytose red blood cells was not compromised. Granulocytic cells showed a reduction of chemotactic response in vitro to chemoattractant bacteria factor and casein.

Aflatoxins↗
PubMed1986

Measurement of aflatoxin B1, its metabolites, and DNA adducts by synchronous fluorescence spectrophotometry.

Sensitive and specific methods are needed for measuring human exposure to carcinogens. Synchronous fluorescence spectrophotometry can be used to measure fmol of aflatoxins, their metabolites, and DNA adducts. Computer-assisted analysis of spectra of these agents obtained by synchronous fluorescence spectrophotometry can be displayed as contour maps which are highly specific for each agent. Individual agents in mixtures, e.g. aflatoxins B1 and M1, can be identified by fourth derivative spectral analysis. This physical method should complement immunological and other methods to measure aflatoxin B1, its metabolites, and nucleic acid adducts.

Aflatoxin B1↗
PubMed2001

Variability in aflatoxin-albumin adduct levels and effects of hepatitis B and C virus infection and glutathione S-transferase M1 and T1 genotype.

Exposure to aflatoxin B1 (AFB1), an important cofactor in the etiology of hepatocellular carcinoma in Taiwan, is influenced by dietary and other factors. The present study examined the intraindividual variability in AFB1-albumin adducts, the most reliable long-term biomarker of AFB1 exposure, and whether the baseline or follow-up adduct levels and the intraindividual variability in adduct levels are modified by endogenous and environmental factors. The study measured AFB1-albumin adduct levels among 264 healthy male residents of three townships (Hu-Hsi, Ma-Kung, and Pai-Hsa) of Penghu Islets, Taiwan, at two different time points with a median interval of 1.68 years (range 1.00-3.17 years). There was a generalized reduction in the adduct levels, with the median values being 22.1 pmol/mg (range 5.0-355.8 pmol/mg) at time 1 and 14.3 pmol/mg (range 5.0-205.2 pmol/mg) at time 2. This intraindividual variability in adduct levels was inversely associated with the age of subjects and the time interval between the two blood draws. The variability in adduct levels was lower among subjects in Hu-Hsi and Pai-Hsa townships as compared to those in Ma-Kung. No significant association was observed for the intraindividual variability in AFB1-albumin adducts with regard to the season when blood was drawn. There was also no significant association between intraindividual variability and hepatitis B surface antigen, anti-hepatitis C virus (anti-HCV), glutathione S-transferase (GST) M1, or GSTT1 status. In conclusion, we found substantial intraindividual variability in the AFB1 exposure (as determined by AFB1-albumin adducts) in Taiwan, which was probably more likely related to dietary or other environmental influences rather than to endogenous factors (e.g., hepatitis B/C viral infection or GST M1/T1 genetic status).

Adult↗
PubMed1983

Metabolite distribution and rate of residue clearance in turkeys fed a diet containing aflatoxin B1.

The retention of aflatoxin residues in tissues of turkey poults fed a diet containing aflatoxin B1 (500 ppb) for 18 days was determined. Free and conjugated aflatoxin metabolites were quantified using high-pressure liquid chromatography. Aflatoxin residue levels were greater in liver than muscle tissues, although all levels were low (range 0.01-1.19 ng/g tissue). Free and conjugated aflatoxins B1 and M1, a metabolite of B1, were the principal tissue residues. Other metabolites investigated included aflatoxicol, which was detected in certain samples, and aflatoxin Q1 which was not detected. Conjugated aflatoxins, hydrolysed and extracted from the aqueous tissue fractions, comprised 55-91% of the total detected aflatoxin residues. All aflatoxin residues were rapidly cleared following discontinuation of the dietary aflatoxin B1 (half-life 1.4 days for total aflatoxin clearance from liver). These results provide further evidence that tissues from animals maintained on diets containing aflatoxins do not provide a major source of aflatoxins when consumed by man.

Aflatoxin B1↗
PubMed2001

New reversed-phase liquid chromatographic method to detect aflatoxins in food and feed with cyclodextrins as fluorescence enhancers added to the eluent.

The effect of succynil-beta-cyclodextrin (beta-CD-Su), dimethyl-beta-cyclodextrin (DIMEB) and beta-cyclodextrin (beta-CD) on the fluorescence of aflatoxins B1, B2, G1, G2 and M1 (AFB1, AFB2, AFG1, AFG2 and AFM1) was studied: beta-CD-Su promoted the largest fluorescence enhancement for AFB1 and AFM1 while DIMEB showed better results for AFG1 . On the basis of the fluorescence enhancement, a new RP-HPLC method for detecting aflatoxins B1, B2, G1, G2 and M1 was developed using cyclodextrins directly dissolved in the LC eluent. Aflatoxins B1, B2, G1 and G2 were resolved using a MICRA NPS ODS-1 column using methanol-water as mobile phase to which 6 x 10(-3) M beta-CD-Su or beta-CD were added. Chromatographic responses of AFB1 and AFG1 achieved using beta-CD dissolved in the mobile phase were enhanced, respectively, 8 and 12 times, and 10 and 15 times with beta-CD-Su. Detection limits lower than 0.3 microg/kg were achieved for all the four aflatoxins. Aflatoxin M1 was analysed using a Spherisorb S3 ODS-2 Narrow Bore column and methanol-water as mobile phase with added 2 x 10(-3) M beta-CD-Su. An area enhancement of 1.5 was detected for the toxin and the detection limit achieved under these analytical conditions was lower than 0.0005 microg/kg. Both methods were statistically validated showing a linear response for all the aflatoxins tested (R2 > or = 0.99), and applied to the analysis of spiked and naturally contaminated food samples.

Aflatoxins↗
PubMed1987

Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3-thiones and 1,2-dithiol-3-ones.

1,2-Dithiol-3-thiones, reported constituents of cruciferous vegetables, are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, and chemoprotective activities. The effects of dietary administration of a substituted 1,2-dithiol-3-thione, oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione], a potent antischistosomal agent, on aflatoxin B1 (AFB1) metabolism, DNA adduct formation, and hepatic tumorigenesis were examined in male F344 rats. Rats were fed graded doses of oltipraz (0.01-0.1%) for 4 wk. During the second and third wk of oltipraz feeding rats were gavaged with 250 micrograms of AFB1/kg five times a wk. Rats were finally restored to control diet 1 wk after cessation of AFB1 dosing. At 4 months focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transpeptidase activity. Treatment with oltipraz at all doses reduced by greater than 90% the volume of liver occupied by gamma-glutamyl transpeptidase-positive foci. Levels of AFB1 bound to hepatic DNA were reduced between 40 and 80% in animals fed increasing doses of dietary oltipraz (0.01-0.1%) for 1 wk prior to a single exposure to AFB1. Feeding of the higher levels of oltipraz led to marked increases in the specific activity of glutathione S-transferases, presumably serving to facilitate the detoxication of the ultimate electrophilic form of AFB1, the 8,9-oxide. At low dietary concentrations of oltipraz (0.01%), the only inductive effects seen were on the activities of selected cytochrome P-450 monooxygenases. Therefore, the protection afforded by oltipraz may be due to both the enhancement of electrophile detoxication pathways as well as modified oxidative metabolism of AFB1. In in vitro metabolism studies with hepatic post-mitochondrial supernatant, low-dose oltipraz pretreatment facilitated the oxidative production of aflatoxins P1 and Q1, but not M1, from AFB1. High-dose (0.1%) oltipraz pretreatment enhanced the primary metabolism of AFB1 to aflatoxins P1, M1, and Q1 as well as the formation of chloroform-insoluble metabolites. Feeding studies with a series of 1,2-dithiol-3-thione and 1,2-dithiol-3-one derivatives of oltipraz demonstrated that the inductive activity for cytochrome P-450-dependent monooxygenases and electrophile detoxication enzymes, such as glutathione S-transferases, could be readily separated by minor modifications of the 1,2-dithiol-3-thione structure. The unsubstituted 1,2-dithiol-3-thione nucleus strongly induced electrophile detoxication enzymes, but not the monooxygenases, and was the most effective inhibitor of the binding of AFB1 to hepatic DNA in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Aflatoxin B1↗
PubMed1996

Chronic hepatitis B carriers with null genotypes of glutathione S-transferase M1 and T1 polymorphisms who are exposed to aflatoxin are at increased risk of hepatocellular carcinoma.

This study was carried out to elucidate the effect of glutathione S-transferase (GST) Ml and Tl polymorphisms on the aflatoxin-related hepatocarcinogenesis among chronic carriers of hepatitis B surface antigen (HBsAg). A total of 32 newly diagnosed hepatocellular carcinoma (HCC) cases and 73 age-matched controls selected from a cohort of 4,841 chronic HBsAg carriers who had been followed for 5 years were studied. The level of aflatoxin B1 (AFB1)-albumin adducts in their serum samples collected at the recruitment was examined by competitive enzyme-linked immunosorbance assay, and genotypes of GST M1 and T1 were determined by PCR. There was a dose-response relationship between serum level of AFB1-albumin adducts and risk of HCC. The biological gradients between serum AFB1-albumin adducts level and HCC risk were observed among chronic HBsAg carriers who had null genotypes of GST M1 and/or T1 but not among those who had non-null genotypes. The multivariate-adjusted odds ratios of developing HCC for those who had low and high serum levels of AFB1-albumin adducts compared with those who had a undetectable adduct level as the referent (odds ratio = 1.0) were 4.1 and 12.4, respectively, for HBsAg carriers with null GST M1 genotype (P < .01, on the basis of the significance test for trend); 0.7 and 1.4 for those with non-null GST Ml genotype (P = .98); 1.8 and 10.2 for those with null GST T1 genotype (P < .05); and 1.3 and 0.8 for those with non-null GST T1 genotype (P = .93). The interaction between serum AFB1-albumin adduct level and polymorphisms of GST M1 and T1 was at marginal statistical significance levels (.05 < P < .10).

Adult↗
PubMedDate not supplied

Enhanced surveillance of foodborne mycotoxins by immunochemical assay.

Mycotoxins are a chemically diverse group of fungal secondary metabolites with a wide range of toxic effects. Conventional thin-layer and instrumental methods of mycotoxin analysis are time-consuming and make routine safety and quality control screening of these compounds in agricultural commodities difficult. As an alternative, specific polyclonal and monoclonal antibodies have been raised against mycotoxin-protein conjugates and used in sensitive radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs). One of the simplest ELISA approaches involves competition for a solid-phase antibody between a mycotoxin-enzyme conjugate and an unconjugated mycotoxin in the sample extract. ELISAs have been developed for aflatoxins B1 and M1, zearalenone, T-2 toxin, and deoxynivalenol, which are highly specific, rapid (10 min), easily adaptable for analyzing large numbers of samples, and directly applicable to assaying methanol-water extracts of a wide range of foods. Several commercial mycotoxin ELISAs using this approach (most typically for aflatoxin B1) are currently being marketed. Since ELISAs will be used in large part by personnel with limited technical expertise, individual kits must be critically evaluated by analytical chemists for suggested sampling procedures, efficiency of extraction, cross-reactivity, mycotoxin recovery, assay reproducibility, and product shelf-life prior to routine use in food safety and quality control screening.

Food Contamination↗
PubMed1982

High performance liquid chromatographic quantitation of aflatoxin metabolites in animal tissues.

A previously reported reverse phase high performance liquid chromatographic (HPLC) procedure was modified to permit the determination of the parent aflatoxins and various free and conjugated metabolites in animal tissues. The modified procedure was based on HPLC analysis of duplicate portions of a sample extract which had been prepared with and without treatment with trifluoroacetic acid (TFA). TFA catalyzes the conversion of aflatoxins G1, B1, M1, and Q1 to the fluorescent derivatives G2a, B2a, M2a, and Q2a. Aflatoxicol, which exhibited a sharp fluorescent peak in its native state, eluted as a tailing peak with weaker fluorescence following TFA treatment. Acid hydrolysis of the aqueous phase of tissue samples permitted the analysis of water-soluble conjugated forms of the various aflatoxins. Analysis of fluorescence spectra of manually collected HPLC fractions qualitatively supported the accuracy of the method. Representative data on the distribution of aflatoxins in turkey liver following ingestion of an experimentally contaminated ration are presented.

Aflatoxins↗
PubMed1976

Growth of mink (Mustela vison) kits when nursing dams given aflatoxin G1.

During lactation, 5 female mink (Mustela vison) were given 50 mug of aflatoxin B1 orally twice, 2 weeks apart. Another group of 5 lactating mink were used as controls, Milk was collected 24 hours after each treatment with toxin. Aflatoxin B1 and M1 could not be detected in the milk. There was no appreciable difference in body weight among kits that nursed treated mink and those that nursed untreated mink.

Aflatoxins↗
PubMedDate not supplied

An analytical survey of aflatoxins in tissues from swine grown in regions reporting 1988 aflatoxin-contaminated corn.

A joint project was undertaken by the Food Safety and Inspection Service (FSIS) and the Agriculture Research Service branches of the U.S. Department of Agriculture to determine the presence of aflatoxins in the U.S. meat supply during a drought year. In 1988, high incidences of aflatoxins occurred in corn grown in regions of the Midwest, Southeast, and South. Six states were identified as having serious aflatoxin contamination in their corn crop: Virginia, North and South Carolina, Texas, Iowa, and Illinois. Swine liver and pillars of diaphragm (muscle) tissues were sampled by federal FSIS Inspectors in plants located in these states. A worstcase sampling plan was conducted. Samples were taken in January 1989 from hogs fed corn soon after harvest and in April 1989 from hogs fed corn originally stored and then fed in the spring. A modification of the official AOAC method for the thin-layer chromatography (TLC) determination of aflatoxins in animal tissue was used to permit quantitation by LC with fluorescence detection. The official AOAC TLC confirmation of identity method was used to confirm all positive samples with B1 concentrations greater than 0.04 ppb and M1 concentrations greater than 0.1 ppb. Sixty samples in the January group and 100 samples in the April group were assayed. Concentrations of aflatoxins B1 and M1 in the first group of pig livers ranged from 0.04 to 0.06 ppb. The identity of aflatoxin B1 was confirmed in all positive samples. Aflatoxin M1 could not be confirmed in any of the positive liver samples because the method was insufficiently sensitive for this aflatoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗
PubMed1981

Determination of aflatoxins in animal tissues.

A method for the determination of aflatoxins in animal tissues has been developed, and applied successfully to beef, swine, chicken, and human livers, and to beef kidney, heart, spleen, muscle, and blood. Blended tissue is denatured with citric acid and extracted with dichloromethane on a wrist-action shaker. After filtration, the extract is partially purified on a silica gel column, and aflatoxins B1 and M1 are determined by 2-dimensional thin layer chromatography and densitometry. Recoveries of B1 and M1 added to meat tissues and blood were approximately 90 and 80%, respectively. The method gave results for a contaminated freeze-dried liver comparable to analyses by 3 other published meat tissue methods. The method is rapid and has a determination limit less than or equal to 0.1 ng/g. In addition, the method uses less toxic and smaller quantities of solvents and chemicals.

Aflatoxins↗
PubMed1983

Acute experimentally induced aflatoxicosis in the weanling pony.

Nineteen weanling ponies and 1 adult pony were given a single oral dose of aflatoxin B1 (AFB1). Dosages were: 0, 0.5, 1, 2, 4, 5, 6, and 7.4 mg of AFB1/kg of body weight. Vital signs were monitored, and whole blood and serum collected for analysis of serum enzymes, prothrombin time, blood cell counts, and serum urea nitrogen. Ponies that died were examined for gross lesions, and tissues were collected for histopathologic examination and analysis of AFB1 and AFM1 residues. Two of the 4 ponies given the 2 mg/kg dose and all ponies given the larger dosages died within 76 hours. Clinical signs included increased rectal temperature, faster heart and respiratory rates, abdominal straining, bloody feces, and tetanic convulsions. At necropsy, ponies that died of acute aflatoxicosis showed visceral petechiae and hepatic focal lesions. Histopathologic changes included severe hepatic necrosis, vacuolation, and bile duct hyperplasia. Aflatoxins B1 and M1 were recovered from liver, kidney, skeletal muscle, and gastrointestinal contents. One other pony given the 2 mg/kg dose died 32 days after dosing, and 1 control pony died after 70 days. Continuous elevations in prothrombin time and serum aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transpeptidase levels were observed in ponies dosed at 4 mg/kg or more. Significant (P less than 0.05) elevations in these values, which peaked 2 to 3 days after dosing, were seen in ponies given the 2 mg/kg dose. This group also had significant increases over controls in PCV and hemoglobin concentration 5 days after dosing.

Acute Disease↗