Fun S. Chu

Effects of temperature and time on mutagen formation in pan-fried hamburger

Mutagenic activity generated in hamburger during pan-frying is dependent upon both temperature and time, with temperature appearing to be the more important variable. Uniformly prepared frozen hamburger pattie (115 g; 19% fat) were fried under carefully controlled conditions at 143 degrees C, 191 degrees C and 210 degrees C. Mutagenic activity assayed with the Ames test was not detected in uncooked hamburger, and in hamburger fried at 143 degrees C mutagenic activity remained low at all times studied (4--20 min). In contrast, frying at 191 degrees C or 210 degrees C for up to 10 min resulted in the generation of considerably higher levels of mutagenic activity. Mutagenic activity in fried hamburgers sold at selected restaurants ranged from very low to moderately high. Evidence is also presented for mutagenic inhibitory activity in uncooked and fried hamburger. Mutagenic inhibitory activity decreased mutagenesis mediated by liver S-9 from normal rats but not from Aroclor 1254-treated rats.

Mycotoxins: food contamination, mechanism, carcinogenic potential and preventive measures

Mycotoxins constitute a large number of naturally occurring fungal secondary metabolites with very diversified toxic effects in humans and animals. Among many mycotoxins discovered, aflatoxins, ochratoxin A, sterigmatocystin and several others are identified as carcinogens; several others were found to be mutagenic. Nevertheless, aflatoxin B1 has been found to be one of the most potent carcinogens and contamination of aflatoxins in the food supply is still a major concern. Whereas extensive studies have been made on aflatoxins, little is known about the mode of action of other carcinogenic and mutagenic mycotoxins. Recent progress on research for the carcinogenic and mutagenic mycotoxins is presented in this review with emphasis on their contamination in foods, their carcinogenic potential to humans, and the mode of action as well as possible preventive measures.

Interaction of ochratoxin A with bovine serum albumin

Abstract The interaction of ochratoxin A (OA) with bovine serum albumin (BSA) has been demonstrated by spectrophotometric, spectrophotofluorometric, equilibrium dialysis, and Sephadex gel filtration analyses. Spectrophotometric analysis revealed that the absorption maximum of OA shifts to a longer wavelength (near 395–400 nm) as a result of interaction. Likewise, the fluorescence properties of OA are also altered in the presence of BSA. Sephadex gel filtration and equilibrium dialysis results indicate that 1 mole of BSA binds 1.87, 2.23, and 2.47 moles of OA with binding constants of 3.17 × 10 5 m −1 , 1.86 and 3.17 × 10 6 m −1 at 25 °, 12 °, and 6 °, respectively. No binding between OA and γ-globulin has been observed. Spectrophotometric and spectrophotofluorometric titrations of the OA:BSA complex indicate that the interaction occurs in the pH region where the protein structure is undergoing a profound change.

A sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitation of selected peanut proteins in foods

A sandwich-type, enzyme-linked immunosorbent assay (ELISA) was developed for the detection of selected peanut proteins in foods. Monoclonal antibodies against a series of allergenic peanut proteins were used as the capture antibody. Food sample extracts were then added, and polyclonal rabbit antibodies directed against roasted peanut proteins were employed as secondary antibodies. The amount of allergen bound to the solid-phase was determined by a biotin and streptavidin-peroxidase system. Radioallergosorbent assay (RAST) inhibition studies of the food extracts were done as a comparison. The coefficient of determination for the ELISA and RAST assays was 0.85. Selected food samples were tested by RAST inhibition at another laboratory for comparison. Skin tests were done with selected samples in peanut-allergic adults, and the results correlated to the ELISA and RAST inhibition results. In other studies, defatted peanut protein (0.01 to 5.0%) were added to vanilla ice cream, then extracted and analyzed using ELISA and skin tests. The sensitivity of the ELISA in ice cream was approximately 40 μg/ml. In six of seven peanut-sensitive adults tested, the lowest level of added peanut protein (0.01%, 21 μg/ml) still caused a positive skin test reaction.

A comparative study of the interaction of ochratoxins with bovine serum albumin.

Abstract The interaction of different ochratoxins with bovine serum albumin (BSA) has been studied by equilibrium dialysis, solubility and spectrophotometric analyses. Spectrophotometric analyses revealed that the absorption maxima of ochratoxin B (OB) and ochratoxin C (OC) shift to longer wavelengths (365–377 nm for OB. 380–390 nm for OC) as a result of interaction. The spectra of ochratoxins α (Oα) and β (Oβ) were not altered in the presence of BSA. Equilibrium dialysis results indicate that 1 mole of BSA binds 1.03, 1.02, 1.93 and 3.24 moles of Oα, Oβ, OB and OC, with binding constant of 1.5 × 10 6 M −1 , 5.1 × HF M −1 . 7.1 and 8.9 × 10 5 M −1 respectively. Spectrophotometric titrations of the 1:1 molar ratio of BSA ochratoxin complexes indicate that the p K values of OB and OC decrease on binding, whereas the p K values of Oα and Oβ remain unaltered. The dissociation of the phenolic hydroxyl group in the isocoumarin ring of ochratoxins A, B and C appears responsible for the binding of the second molecule of these toxins with albumin. The overall results suggest that both ionic and hydrophobic forces are important in the binding of ochratoxins with BSA.

Structural requirements for ochratoxin intoxication

Abstract The toxicity of several ochratoxin derivatives and the apparent dissociation constant of the phenolic hydroxyl group in these derivatives have been studied. Ochratoxin C failed to induce toxic effect after the phenolic hydroxyl group was chemically modified. A direct correlation between the dissociation constant for the phenolic hydroxyl group and the acute toxicity was found. It is suggested that the phenolic group in the dissociated form is necessary for orchatoxin intoxication.

Ethylenediamine modified bovine serum albumin as protein carrier in the production of antibody against mycotoxins

The efficiency of conjugation of mycotoxin derivatives containing a carboxylic group or other related functional groups to bovine serum albumin (BSA) by water soluble carbodiimide method, mixed anhydride method and reductive alkylation technique, improved considerably when a modified BSA was used in which the carboxylic groups in the protein were blocked by ethylenediamine (EDA). Due to the increased efficiency, smaller amounts of the haptens were satisfactory for conjugation to EDA-BSA as compared to amounts for the unmodified protein. The mycotoxin-EDA-BSA conjugates were shown to be highly immunogenic in rabbits.

Production and Characterization of Antibodies against Fumonisin B1

Polyclonal antibodies against fumonisin B1 (FmB1) were produced in rabbits after immunizing the animals with either FmBl-keyhole limpet hemocyanin (KLH) or FmB1 bovine serum albumin (BSA). A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were used for the characterization of the antibodies and for analysis of the toxin in corn samples. The antibody titers in the serum of rabbits immunized with FmBl-KLH were considerably higher than in those immunized with FmBl-BSA. The antibodies from the rabbits immunized with FmBl-KLH were further characterized. The concentrations causing 50% inhibition of binding of FmB1-horseradish peroxidase (HRP) to the antibodies by FmB1, FmB2 and FmB3 in the ELISA were found to be 0.45, 0.72, and 25 ng/ml, respectively. The detection limit of FmBl, based on 95% confidence at 5% of inhibition of binding of FmBl-HRP conjugate, in buffer of the dc-ELISA was found to be 0.05 ng/ml. In the presence of a matrix such as corn, the detection limit was less than 50 ppb. The overall analytical recoveries of FmBl (50 to 1,000 ng/g) added to the ground corn and then extracted with CH3CN/H2O (1/1, vol/vol) with cleanup and without cleanup in the dc ELISA were found to be 70.5 and 85.9%, respectively. A good correlation was found between the FmBl levels in 2 starch and 10 naturally contaminated corn samples analyzed by the dc-ELISA and the high-pressure liquid chromatography (HPLC) method. The correlation coefficients between ELISA and HPLC were found to be 0.955 (y [ELISA] = 1.3 1x [HPLC] + 77 ppb; P < 0.001) and 0.811 (y = 1.13x + 34 ppb; P < 0.01) for the sample without and with cleanup treatment, respectively.

Immunoassays of Selected Mycotoxins in Hay, Silage and Mixed Feed

Immunoassays, including radioimmunoassay (RIA) and direct competitive enzyme-linked immunosorbent assays (dc-ELISAs) were used for the analysis of the mycotoxins, produced by Alterneria alternata , AAL TA toxin (AAL), cyclopiazonic acid (CPA), deoxynivalenol (DON), fumonisin B1 (FmB1), PR toxin (PRT) and zearalenone (ZEA) in hay, silage and mixed feed. In the analysis, 10 g well-ground feed samples in 100 ml of 84% acetonitrile in water were shaken in an orbital shaker for 1 h. After filtration, the filtrates were diluted with buffer and directly subjected to dc-ELISA for AAL toxin, CPA, FmB1, PRT and ZEA. For total DON, including 15-acetyldeoxynivalenol (15-AcDON) and 3-acetyldeoxynivalenol (3-AcDON), the extracts were air-dried, acetylated, and subjected to both RIA and dc-ELISA using the Ridascreen DON enzyme immunoassay (EIA) kit. The average recoveries of toxins added to the hay at 0.3 and 1.0 ppm levels were found to be 77.5, 111, 95.8, 85.7, 95.5 and 138% for AAL toxin, DON, CPA, FmB1, PRT and ZEA,...

Enzyme-linked immunosorbent assay for T-2 toxin

An enzyme-linked immunosorbent assay (ELISA) was developed for the rapid quantitation of T-2 toxin, a tricothecene mycotoxin produced by members of the genusFusarium. T-2 toxin was first converted to the T-2 hemisuccinate (T-2 HS) and then conjugated by the water-soluble carbodiimide method to either bovine serum albumin for use as an immunogen or to horseradish peroxidase for use as an enzyme marker. T-2 antiserum was air-dried onto polystyrene microtissue culture plates and the ELISA conducted by simultaneously incubating standards of T-2 toxin and the T-2 HS-peroxidase conjugate. Competition curves were prepared by determining total bound enzyme. The ELISA took about 2 hr to complete and allowed minimal detection of T-2 at levels of 2.5 pg/assay. Average recoveries from samples of wheat flour spiked with T-2 toxin in the 1.0-30 ppb range were 95 ± 25% and those for corn meal spiked in the 5.0-30 ppb range were 98 ± 19%. The results suggested the ELISA is a simple and convenient alternative for the screening of T-2 toxin in food and feeds.