Correlation of dietary aflatoxin B1 levels with excretion of aflatoxin M1 in human urine.

Corn and peanut oil (total, 253 samples) were collected from 32 households in Fushui county of the Guangxi autonomous region of the People's Republic of China, where high liver cancer incidence has been reported, every day over a period of 1 week and analyzed for aflatoxin B1 (AFB). A total of 252 urine samples were collected simultaneously from the residents in the households which were shown to have consumed AFB and were analyzed for aflatoxin M1 (AFM) by a competitive direct enzyme-linked immunosorbent assay. A good correlation between total dietary AFB intake and total AFM excretion in human urine was observed during a 3-day study. A regression equation of 0.143 plus 0.0135 multiplied by the amount of AFB consumed was observed. Between 1.23 and 2.18% of dietary AFB was found to be present as AFM in human urine. A good correlation was also observed between the AFB concentration in corn and the AFM concentration in human urine. The results suggest that analysis of AFM in urine by enzyme-linked immunosorbent assay could be used as an index for human exposure of AFB in an extensive epidemiological study.

Characterization of byssochlamyopeptidase A.

The enzyme properties of byssochlamyopeptidase A, a chymosin-like enzyme produced by Byssochlamys fulva were studied. The enzyme was shown to be electrophoretically and immunochemically pure. Most metallic cations had negligible effect, whereas Hg2+ greatly suppressed the enzyme activity. N-Bromosuccinimide and I2 completely inactivated the enzyme. For milk clotting at pH 4.6-6.6, the enzyme was less sensitive to pH than pepsin. The substrate specificity of the enzyme was studied by incubation of the enzyme at 37 degrees C with whole and individual casein fractions at pH 6.6 and pH 3.0, respectively. The proteolysis by the enzyme was found to be most extensive for alphas-, less for kappa-, and least for beta-casein. Studies with different synthetic dipeptides and tripeptides revealed that byssochlamyopeptidase A exhibits specificity for Phe-Tyr and Gly-Phe-Phe, but the enzyme did not hydrolyze Ac-Phe-Tyr(I2).

Preparation of gold-labeled antibody probe and its use in immunochromatography assay for detection of aflatoxin B1.

Preparation of an antibody-colloidal gold probe (conjugate) specific to aflatoxin B1 (AFB1) and its use in developing a rapid AFB1 diagnostic method was presented in this paper. Monodispersional nanogold colloid was synthesized and preparation of nanogold-labeled polyclonal antibody probe to aflatoxin B1 under friendly and optimal condition. Combination of antibody with nanogold particles was also characterized by UV-visible (UV-vis) light absorption spectra, transmission electron microscopy (TEM), fluorescence spectroscopy, titers, cross reactivity and stability measurements. Furthermore, nanogold-labeled probe was used to develop an immunochromatographic (IC) method for aflatoxin B1 analysis. With this method, analysis could be completed in less than 10 min. Detection time was reduced 6-10 times comparative with ELISA. With visual observation, lower test limit was found to be around 2.5 ng/ml aflatoxin B1 standard solution, which was increased to two times of ELISA.

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.

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.

Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements.

The presence of blue-green algae (BGA) toxins in surface waters used for drinking water sources and recreation is receiving increasing attention around the world as a public health concern. However, potential risks from exposure to these toxins in contaminated health food products that contain BGA have been largely ignored. BGA products are commonly consumed in the United States, Canada, and Europe for their putative beneficial effects, including increased energy and elevated mood. Many of these products contain Aphanizomenon flos-aquae, a BGA that is harvested from Upper Klamath Lake (UKL) in southern Oregon, where the growth of a toxic BGA, Microcystis aeruginosa, is a regular occurrence. M. aeruginosa produces compounds called microcystins, which are potent hepatotoxins and probable tumor promoters. Because M. aeruginosa coexists with A. flos-aquae, it can be collected inadvertently during the harvesting process, resulting in microcystin contamination of BGA products. In fall 1996, the Oregon Health Division learned that UKL was experiencing an extensive M. aeruginosa bloom, and an advisory was issued recommending against water contact. The advisory prompted calls from consumers of BGA products, who expressed concern about possible contamination of these products with microcystins. In response, the Oregon Health Division and the Oregon Department of Agriculture established a regulatory limit of 1 microg/g for microcystins in BGA-containing products and tested BGA products for the presence of microcystins. Microcystins were detected in 85 of 87 samples tested, with 63 samples (72%) containing concentrations > 1 microg/g. HPLC and ELISA tentatively identified microcystin-LR, the most toxic microcystin variant, as the predominant congener.

Prolonged sublethal exposure to the protein phosphatase inhibitor microcystin-LR results in multiple dose-dependent hepatotoxic effects.

The purpose of this study was to relate dose-dependent hepatotoxicity stemming from prolonged exposure to sublethal concentrations of the cyclic heptapeptide microcystin-LR (Mcyst) to hepatic Mcyst concentrations and protein phosphatase activity. Mcyst is a potent inhibitor of protein phosphatase types 1 and 2A (PP1 and PP2A). Twenty male Sprague-Dawley rats were infused continuously with 0, 3, 6, or 9 micrograms Mcyst/day for 28 days using intraperitoneal mini-osmotic pumps containing highly purified toxin or saline. At the end of 28 days, dose-dependent increases in several serum biochemical tests including sorbitol dehydrogenase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, and bile acids had occurred. Serum albumin decreased in a dose-dependent fashion. Liver activity of both PP1 and PP2A decreased in a dose-dependent manner, but with a relatively greater effect on PP2A than PP1. Liver cytosol Mcyst concentrations, measured by direct competitive ELISA, also increased in a dose-dependent manner, although at a higher rate than would be predicted from the incremental increase in dose given. This disproportional increase is suggestive of the bioaccumulation of Mcyst with increasing dose. Histopathological abnormalities included hepatocellular apoptosis and cytosolic vacuolation of principally zone 3 hepatocytes. Immunohistochemical stains revealed Mcyst predominantly within pericanalicular regions of zone 3 hepatocytes. It was concluded that prolonged exposure to sublethal concentrations of Mcyst results in multiple dose-dependent hepatotoxic effects that correspond to decreased hepatic serine/threonine protein phosphatase activity and increasing cytosolic Mcyst concentrations. The disproportional increase of hepatic Mcyst concentrations observed may suggest the bioaccumulation of toxin and an increasing relative risk of hepatotoxicity with increasing dose.

In vitro neutralization of the inhibitory effect of Microcystin-LR to protein phosphatase 2A by antibody against the toxin.

Microcystin-LR (MCYST-LR) was found to be a very potent protein-phosphatase 2A (PP2A) inhibitor at an ID50 as low as 0.1 nM. Comparing to calyculin A and okadaic acid, MCYST-LR was found to be about 100 times stronger than calyculin A and okadaic acid for their inhibition to PP2A. The inhibitory effect on PP2A by MCYST-LR was abolished when antibodies against MCYST-LR were present in the assay system. Polyclonal antibodies were more effective than monoclonal antibodies in reversing the inhibitory effect. A dose-dependent neutralization of the inhibitory effect of MCYST to PP2A by anti-MCYST polyclonal antibodies were observed. Almost 80% of the enzyme activity was restored when as low as 0.012 micrograms of Pab was present. The specific reaction caused by the antibody was evident from an analysis that the antibodies had no effect on reversing the inhibition of PP2A caused by okadaic acid and calyculin A.

Monitoring of microcystin-protein phosphatase adduct formation with immunochemical methods.

Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 microg/kg) of MCYST-LR into mice, approximately 0-27% of the injected toxin was found covalently bound while 0.2-9.2% existed as free form in liver cytosol.

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.

Production and characterization of a monoclonal anti-anti-idiotype antibody against fumonisin B(1).

A monoclonal anti-anti-idiotype antibody (mAb3) against fumonisin B(1) (FmB1) was produced from the hybridoma cell line 7C7F4, which was generated by the fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse that had been immunized with the Fab fragments of affinity-purified anti-idiotype antibodies. The mAb3 belongs to the immunoglobulin M, kappa light chain. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were established for antibody characterization and toxin analysis. In an idc-ELISA using FmB1-ovalbumin (OVA) as the coating antigen, the concentrations causing 50% inhibition of binding (IC(50)) of mAb3 to the solid-phase FmB1-OVA by free FmB1, FmB2, and FmB3 were found to be 75, 95, and 450 ng/mL, respectively. In the dc-ELISA, the concentration causing IC(50) of FmB1-horseradish peroxidase to the solid-phase mAb3 by free FmB1 was found to be 233 ng/mL. Analysis of 12 samples naturally contaminated with fumonisins with mAb3-based idc-ELISA and polyclonal-based dc-ELISA showed a good correlation between these two methods with a correlation coefficient of 0.76 at p < 0.02. The linear regression slope was found to be y[polyclonal ELISA] = 0.87x[mAb3 ELISA] - 52 ppb.

Development of a polyclonal antibody-based sensitive enzyme-linked immunosorbent assay for fumonisin B(4).

Polyclonal antibodies (PAb) against fumonisin B(4) (FmB(4)), which have good cross-reactivity with four major fumonisins, were produced by immunizing a rabbit with FmB(4)-keyhole limpet hemocyanin conjugate. A sensitive competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for fumonisins was developed. Because of the limited supply of FmB(4), both FmB(1)-horseradish peroxidase conjugate (HRP) and FmB(3)-HRP were tested as the toxin-enzyme markers in the CD-ELISA. In the FmB(1)-HRP-based CD-ELISA, the concentrations of FmB(1), FmB(2), FmB(3), and FmB(4) causing 50% inhibition of binding of enzyme marker (IC(50)) were 9.0, 2.1, 9.0, and 6.5 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 58.5, 309.5, 58.5, and 100%), respectively. In the FmB(3)-HRP-based CD-ELISA, the IC(50) values for FmB(1), FmB(2), FmB(3), and FmB(4) were 7.1, 1.9, 7.6, and 5.3 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 74, 280, 70, and 100%), respectively. The FmB(3)-HRP-based CD-ELISA was then used in a series of analytical recovery experiments using Fusarium moniliforme corn culture material spiked with FmB(1) and with clean corn spiked with a FmB(3)/FmB(4)-containing extract. The overall recovery of FmB(1) from culture material in the range of 10-100 ppm was 65%. The detection limit for FmB(1) with clean corn as matrix was between 100 and 500 ppb. F. moniliforme cultures were analyzed with the developed CD-ELISA and a well-established FmB(1) antibody-based ELISA, which is not sensitive to FmB(4). Differences in the fumonisin levels found by the two assays were used as an indication of the presence of FmB(4) in the culture material and, therefore, as a method to identify FmB(4)-producing strains. Using ELISA in combination with HPLC individual B-series fumonisins were quantified. The ELISA developed in the present study would be a useful supplement to FmB(1) antibody-based ELISA for screening of Fusarium strains for the production of major fumonisins.

Indirect immunoperoxidase localization of aflatoxin B1 in rat liver.

Aflatoxin B2a (AFB2a) antibody was used as a histochemical probe in the indirect immunoperoxidase localization of aflatoxin B1 (AFB1) bound to rat liver. The efficacy of the indirect method was initially demonstrated by detecting AFB1 covalently bound to DNA in an enzyme-linked immunosorbent assay. AFB1-modified DNA was attached to a polystyrene microtissue culture plate (solid phase) and then subjected to sequential incubation with AFB2a antiserum followed by goat anti-rabbit peroxidase conjugate. Assays for bound peroxidase revealed that the AFB2a antiserum could be diluted 200,000-fold and still yield a signal-to-noise ratio of 10 when compared to an unmodified DNA control. When the same indirect immunoperoxidase protocol was applied to the light-microscopic localization of AFB1 in liver sections of rats treated in vivo with the mycotoxin, bound toxin could be identified in excellent detail in tissues fixed with periodate-lysine-paraformaldehyde and embedded in glycol methacrylate, but was detectable with only poor resolution in unfixed cryostat sections. Peroxidase-positive reactions in hepatocytes typically exhibited strong nuclear and relatively lighter cytoplasmic staining. Greater concentrations of peroxidase-positive hepatocytes were detected in the periportal area than in the area of the central vein, suggesting a circulatory pattern for AFB1 binding in the liver.

Uptake and metabolism of T-2 toxin in relation to its cytotoxicity in lymphoid cells.

The sensitivity of lymphoid cells to the cytotoxic effects of T-2 toxin (T-2) varies according to their degree of differentiation. To understand the mechanisms of these variations, the uptake and the metabolism of T-2 in susceptible (human lymphoma Daudi and phytohaemagglutinin-stimulated murine lymphocytes) and resistant (human leukaemia KE37 and REH) cells were studied in culture. When cells were incubated with [3H]T-2 a significant increase in the quantity of T-2 associated with the cell occurred during the first 30 min, this increased further from 10-16 hr, and decreased after 24 hr. Daudi and REH cells took up 20 and 3% of the T-2 present in the medium, respectively. Metabolites, extracted from the culture medium and from cells, were analysed by the thin-layer chromatography. The products were identified by comparison with standards for T-2 tetraol, T-2 triol, HT-2 toxin, neosolaniol and T-2. Qualitatively, similar metabolic pathways were found in all cells examined. The presence of these metabolites demonstrated that T-2 was taken up by these cells. A correlation existed between the relative sensitivities of the cells toward T-2 and the amount of intracellular T-2 and/or metabolites. It is thought that differences in the kinetics of uptake and processing of T-2 account for the known differences in cellular sensitivities to the toxin.

Immunochemical methods for fumonisins.

Both monoclonal and polyclonal antibodies have been raised against fumonisins (Fm) and the hydrolyzed toxin in several laboratories. They have been used in several immunoassays and can detect 5 to 5000 ng of FmB1/mL in a "clean" standard solution. However, sample matrices still give severe interferences. Thus, without a cleanup treatment, most immunoassays can only be used as a screening tool for Fm in corn/feed in the 0.5 to 10 ppm range. The antibodies have also been used to generate anti-idiotype antibodies, to use as immunohistological reagents and to make affinity columns that are being used as a cleanup tool. In the present symposium, approaches used to generate antibodies, antibody specificity, sensitivity of various immunoassays and problems encountered in the immunoassays will be reviewed. Approaches used to overcome such problems and methods to improve the sensitivity and specificity of immunoassays of Fm are discussed.

Painful right iliac fossa mass caused by a migrating left ovary.

A young woman presenting with right iliac fossa pain was found to have a palpable mass. Ultrasound and computed tomography demonstrated a calcified solid mass, which was extraintestinal on barium enema. Laparotomy confirmed an infarcted left ovarian cyst due to torsion of an attenuated but intact fallopian tube. To our knowledge, this is the first documented case of ovarian autoamputation in evolution. A migrating left ovary should be added to the differential diagnosis of a painful right iliac fossa mass.

Recent progress on analytical techniques for mycotoxins in feedstuffs.

Analysis of mycotoxins in feedstuffs is a difficult task because only trace amounts of the toxins are present in the sample. However, rapid progress in the area of mycotoxin analysis has been made during the last few years. Simplified sample cleanup protocols and new chromatographic methods, especially HPLC, have been developed. New, more sensitive and versatile instruments such as high-resolution mass spectrometry (MS) and gas chromatography/tandem MS/MS are coming to the market. After 15 yr of laboratory research, immunoassay techniques have gained more acceptance as analytical tools for mycotoxins. Several immunoassay kits for mycotoxins are currently available. The development of these new techniques and their application for monitoring various mycotoxins in foods and feeds are described in this review.

The effect of cyclopiazonic acid on the development of pale, soft, and exudative pork from pigs of defined malignant hyperthermia genotype.

Malignant hyperthermia (MH) and the mycotoxin cyclopiazonic acid (CPA) are each associated with abnormal calcium homeostasis in skeletal muscle, a key underlying factor in the development of pale, soft, and exudative (PSE) pork. To determine whether the natural presence of CPA in livestock feed ingredients contributes to the varying incidence of PSE in the pork industry, various levels of CPA (.1 to 50 mg/kg of diet) were included in the diets of market weight hogs (n = 52) of defined malignant hyperthermia genotype (NN = normal, Nn = a MH carrier, and nn = MH-positive). Animals with two copies of the MH mutation (nn) displayed improved live animal performance compared with NN and Nn animals (increased feed intake, average daily gain, and feed efficiency) but yielded lower quality loin chops as indicated by lower 45-min pH (P<.01), higher Commission Internationale de l'Eclairage (CIE) L* color coordinate values (P<.05), and higher drip losses (P<.01). The effects of CPA varied. In the first feeding trial, conducted under normal outside temperatures (2 degrees C), CPA had no effect (P> .2) on either live animal performance or meat quality. During the second trial, conducted under extreme outside temperatures (-18 degrees C), CPA-dependent reductions (P<.05) in feed intake, average daily gain, and 45-min pH in nn hogs support the possibility of interactions between malignant hyperthermia and dietary CPA on skeletal muscle calcium homeostasis and the development of PSE pork. These results suggest that this interaction may require stressful environmental conditions or the ingestion of CPA doses much higher than occur under natural conditions.

Analysis and pharmacokinetics of cyclopiazonic acid in market weight pigs.

The pharmacokinetic behavior of cyclopiazonic acid (CPA) was determined in market weight pigs using a competitive indirect ELISA developed for the determination of the mycotoxin in various biological matrices. Sample preparation for corn and skeletal muscle was achieved with a single extraction and recoveries of 53+/-6% over the effective range of the standard curve. The detection limit of CPA was 1 ppb in plasma, which required no extraction, and 20 ppb in corn and skeletal muscle with average intra- and interassay CV of 11 and 23%, respectively. Levels of CPA contamination in corn grown and stored in Michigan were unremarkable compared with published toxicity thresholds; the highest level of CPA found in any sample was 47 ppb. In pigs given a 20-mg i.v. bolus, CPA distributed rapidly among three compartments, with an overall volume of distribution (49 L) nearly equivalent to total body water. Cyclopiazonic acid was eliminated with a half-life of 24 h. Estimates of these pharmacokinetic parameters were supported by the achievement of steady-state plasma CPA levels within 6 d in pigs consuming a diet containing 10 ppm CPA, and by measured concentrations of CPA in plasma (410+/-44 ng/mL) and skeletal muscle (469+/-86 ng/ g). From these and other data, we concluded that the threat of CPA toxicity to livestock from consumption of cereal grains or to humans from consumption of animal products is minimal.

Assessment of immunochemical methods for the analysis of trichothecene mycotoxins in naturally occurring moldy corn.

The efficacy of various immunochemical methods for simultaneous analysis of different trichothecene mycotoxins in corn samples naturally contaminated with various Fusarium molds was evaluated. Antibodies against either T-2 tetraol tetraacetate (T-2-4ol-4Ac) or deoxynivalenol triacetate (Tri-Ac-DON) were used in the enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) for total type A and total DON-related trichothecenes (TCTCs), respectively. Specific antibodies against T-2 toxin were used in an RIA for analysis of T-2 toxin alone. Analytical recoveries of T-2 toxin analyzed as T-2-4ol-4Ac by ELISA and T-2 toxin by RIA at 10-200 ppb were 83.5-123% and 81.8-110.6%, respectively. T-2 toxin accounts for about 30% of total type A TCTCs by both liquid chromatography (LC)-ELISA and RIA methods. The correlation coefficients (r) of total type A TCTC levels versus T-2 toxin level by RIA was 0.725 (p < 0.0001). A good correlation (r = 0.84 and p = 0.04) also was found for T-2 toxin levels obtained by LC-ELISA and RIA methods. In addition to T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and T-2 tetrol were found in samples containing high levels of type A TCTCs by LC-ELISA. For analysis of DON alone, samples were treated with a Sep-Pak C18 cartridge to remove other acetylated DONs before RIA. Recovery of DON after the C18 cartridge treatment was 90%. Overall analytical recoveries of DON as Tri-Ac-DON (sample with no C18 treatment) and DON alone (with C18 treatment) at 10-500 ppb were 81.3-94% and 72.7-104%, respectively. RIA revealed that DON accounts for about 50% of total DON-related TCTCs. The coefficient of correlation between total DON-related TCTCs and DON levels was 0.84 (p < 0.0001).