The effect of dietary fat on the anticoagulant activity of aflatoxin B.

A single i.p. dose of aflatoxin B1 had no significant effect on the thrombotest clotting times of monkeys subsisting on low-fat and high-fat dietary regimens, respectively. There was a significant interaction between aflatoxin and dietary fat level.     

Production and characterization of antibody against aflatoxin M1

Summary Antibody against aflatoxin M₁ was obtained after immunization of rabbits with bovine serum albumin-afla M₁ oxime conjugate. The antibody has greatest binding efficiency for afla M₁, and was less efficient for afla B₁. Cross-reaction of antibody with aflatoxin Q₁, aflatoxicol, and aflatoxin B_2a was weak. Aflatoxin B₂, G₁, and G₂ and afla B₁-guanine adduct showed almost no cross-reaction with the antibody. The sensitivity of the binding assay for aflatoxin M₁ detection is in the range of 1–10 ng per assay. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody determination are described.Supported by the College of Agricultural and Life Sciences, North Central Regional project NC-129, the University of Wisconsin-Madison, and by Public Health Service research grant number CA 15064 from the National Cancer Institute, NIH.The authors wish to thank Dr R.C. Garner for providing aflatoxin-B-guanine adduct, and Dr Dennis H. Hseih for providing aflatoxicol and aflatoxin Q₁.     

Chromosomenaberrationen in den Wurzelspitzen vonAllium cepa durch Aflatoxin B1

Summary After treatment of roots ofAllium cepa with aflatoxin B₁ in 0.3% dimethylformamide, chromosome bridges, C-mitose chromosomes and a reduction of the mitotic index were observed. The aberrations occurred especially frequently when the roots had grown in 200 µg/ml toxin for 48 h. In its cytotoxic effect onAllium cepa root tips, aflatoxin B₁ acts similarly to the chemically related coumarin.     

Reduction of aflatoxin B1 with zinc borohydride: An efficient preparation of aflatoxicol

Summary A simple and mild reduction of aflatoxin B₁, involving treatment of aflatoxin B₁ with ethereal zinc borohydride to give 57–65% yield of diastereomeric aflatoxicols, is described.This work was supported by the College of Agricultural and Life Sciences, the University of Wisconsin, Madison, and by Public Health Service Research, grant No. CA 15064, from the National Cancer Institute, and by funds from contributions of food industries to the Food Research Institute. We are grateful to Professor Dennis Hsieh for a gift of natural aflatoxicol, to Mr William Harder for technical assistance throughout this investigation, and to Mr Gary Girdaukas for taking mass spectra.     

The use of cell free extracts derived from fungal protoplasts in the study of aflatoxin biosynthesis

Summary A supernatant fraction derived from protoplasts ofAspergillus flavus was shown to be capable of converting both sterigmatocystin and versiconal hemiacetal acetate to aflatoxin B₁. Versicolorin A was not converted under the same conditions.     

Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity

Summary Cleavage of the lactone ring of aflatoxin B₁ results in a nonfluorescent compound that has greatly reduced biological activity. Mutagenicity, as measured by the Ames test, is reduced 450-fold compared to that of B₁, and toxicity, as measured by the chick embryo test, is reduced 18-fold.     

Aflatoxin B1 hepatotoxicity in rats pretreated with ethanol

Summary Ethanol pretreatment has the potentiation of the aflatoxin B₁-induced hepatotoxicity was indicated by an increase in the activities of plasma GPT, plasma GOT and in the severity of liver necrosis. The effect of ethanol pretreatment on an increase in the accumulation of liver triglycerides is additive in nature.     

Variation in regulation of aflatoxin biosynthesis among isolates ofAspergillus flavus

Two new phenotypes ofAspergillus flavus which exhibit novel patterns of aflatoxin production have been identified and characterized. In one of the new variants ofA. flavus, aflatoxin is made in the absence of carbohydrate and concomitantly with growth, without a lag period. A second variant did not produce aflatoxin in the presence or absence of carbohydrate. Chemical mutagenesis of this nonaflatoxigenic strain resulted in mutant strains which produced aflatoxin on carbohydrate-containing media. The aflatoxin production pattern observed in these mutants resembled the typical production scheme, with a lag period through log phase growth.     

The use of fungal protoplasts in the study of aflatoxin biosynthesis

Summary Protoplasts derived fromAspergillus flavus are shown to be capable of synthesizing aflatoxins when incubated in a chemically defined medium.¹⁴C-Acetate and¹⁴C-Versicolorin A, added to protoplasts from 3-day-old mycelium, are incorporated into aflatoxin B₁.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B₃ to bovine serum albumin. Aflatoxin B₃ was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B₁ (AFB₁) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B₁, B₂, G₁, and G₂ when tritiated AFB₁ was used as the marker ligand. The concentrations causing 50% inhibition of binding of³H-AFB₁ to the antibodies by unlabeled aflatoxins B₁, B₂, G₁, G₂ and B₃ were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B₁ and G₁, two of the most important toxic metabolites produced byAspergillus flavus andA. parasiticus.     

Covalent binding of aflatoxin B1 to liver DNA in rats pretreated with ethanol

Summary Male Fischer F-344 rats were given ethanol in the drinking water and/or by single oral administration. Following this, the animals received p.o. 100 ng/kg of the hepatocarcinogen [³H]aflatoxin B₁ (AFB₁). 24 h later, the level of DNA-bound AFB₁ was determined in the liver and was found not to be affected by any type of ethanol pretreatment. A cocarcinogenic effect of ethanol in the liver is therefore unlikely to be due to an effect on the metabolic activation and inactivation processes governing the formation of DNA-binding AFB₁ metabolites.To whom correspondence should be addressed.Acknowledgment. We thank the European Science Foundation for the Toxicology Research Fellowship awarded to M.M.     

Aflatoxin metabolism and absence of cytochrome P-450 in rat colon tissue during vitamin A malnutrition

Summary Homogenized mucosal linings prepared from vitamin A adequate and deficient male rats were used in metabolic studies of aflatoxin B₁ (AFB₁). Cytochrome P-420 was identified in both groups which metabolized AFB₁ to 4 metabolic products in vitro. The implications of this observation are discussed in relation to colon carcinoma.Acknowledgments. This work was supported by USPHS (NIEHS), grant R01 ES 00336 and R01 CA 00270. Hoffman-La Roche Foundation Research Corporation awarded to T. C. C., Research Career Development Awardee of the NIEHS. Present address: Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA.     

Variation in regulation of aflatoxin biosynthesis among isolates of Aspergillus flavus.

Two new phenotypes of Aspergillus flavus which exhibit novel patterns of aflatoxin production have been identified and characterized. In one of the new variants of A. flavus, aflatoxin is made in the absence of carbohydrate and concomitantly with growth, without a lag period. A second variant did not produce aflatoxin in the presence or absence of carbohydrate. Chemical mutagenesis of this nonaflatoxigenic strain resulted in mutant strains which produced aflatoxin on carbohydrate-containing media. The aflatoxin production pattern observed in these mutants resembled the typical production scheme, with a lag period through log phase growth.     

Sclerotial and low aflatoxigenic morphological variants from haploid and diploidAspergillus parasiticus

Summary Serial transfer of mycelial macerates of a wild type, haploid, aflatoxigenic strain ofAspergillus parasiticus in a defined liquid medium resulted in the production of three new morphological classes: a sclerotial form with high aflatoxin production, and two variant forms (fan andfluff) with lowered sporulation, no sclerotia, and attenuated levels of aflatoxin. A genetically marked diploid containing mutant markers for aflatoxin pathway intermediates yielded the same three morphological classes upon serial transfer of macerated mycelia. When these diploid variants were treated with a haploidization agent, and the phenotypes of the resultant segregants scored, a low frequency of colonies producing aflatoxin pathway intermediates was recovered. These genetic data indicate that the structural genes for the aflatoxin pathway are present but somehow attenuated in thefan andfluff strains.This work was supported by a Cooperative Agreement from the U.S. Department of Agriculture, (58-7B30-3-556).     

The sterols of the khapra beetle,Trogoderma granarium everts

Summary The khapra beetle,Trogoderma granarium Everts, does not dealkylate and convert dietary C₂₈- or C₂₉-phytostorols to C _2T sterols such as cholesterol. There is, however, an increase in the concentration of cholesterol and campesterol in its tissues relative to the dietary concentrations of these sterols, presumably as a result of selective uptake.We thank Dr S. R. Dutky of the Insect Physiology Laboratory, Agricultural Research, SEA, USDA, Beltsville, Maryland for providing GC-MS analyses.     

Kinetics of transformation of aflatoxin B1 into aflatoxin M1 in lactating mouse: an ELISA analysis

A new enzyme-linked immunosorbent assay (ELISA) was used to study the kinetics of transformation of aflatoxin B1 into aflatoxin M1 in lactating mice. Aflatoxin M1 concentration in the milk samples reached a maximum 30 min after injection of aflatoxin B1 and decreased thereafter. At the maximum time, the levels of aflatoxin M1 in the samples were proportional to the dosages administered. Aflatoxin B1 was also detected in the milk samples but at a lower concentration.     

Production and characterization of antibody against aflatoxin M1

Antibody against aflatoxin M1 was obtained after immunization of rabbits with bovine serum albumin-afla M1 oxime conjugate. The antibody has greatest binding efficiency for afla M1, and was less efficient for afla B1. Cross-reaction of antibody with aflatoxin Q1, aflatoxicol, and aflatoxin B2a was weak. Aflatoxin B2, G1, and G2 and afla B1-guanine adducts showed almost no cross-reaction with the antibody. The sensitivity of the binding assay for aflatoxin M1 detection is in the range of 1-10 ng per assay. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody determination are described.     

Electrophoretic variation in esterases in 3 wild-type and respective mutant strains ofAspergillus flavus

Summary Wild-type strains and auxotrophic mutants ofAspergillus flavus, differing regarding aflatoxin production, were tested for esterases isozymes. Esterases variation was found in all strains used, and a possible correlation between the pattern of esterase bands and aflatoxin production is suggested.Acknowledgment. The authors are thankful to the National Council for the development of Science and Technology (CNPq) and Assistance to Research for São Paulo State (FAPESP) for financial assistance.     

Electrophoretic variation in esterases in 3 wild-type and respective mutant strains of Aspergillus flavus.

Wild-type strains and auxotrophic mutants of Aspergillus flavus, differing regarding aflatoxin production, were tested for esterases isozymes. Esterases variation was found in all strains used, and a possible correlation between the pattern of esterase bands and aflatoxin production is suggested.     

Vitamin D receptors in heart: Effects on atrial natiuretic factor

We report that receptors for vitamin D exist in distinct regions of the heart in female and male mice, predominantly in the right atrium where most of the cardial atrial natriuretic peptide (ANF) is produced. Tritiated 1,25-dihydroxyvitamin D₃ (1,25-D₃, vitamin D, soltriol) and ANF are colocalized in nuclei and cytoplasm respectively in identical cardiomyocytes. Changes of ANF tissue and blood levels under dietary deficiency and treatment with 1,25-D₃ suggest direct genomic actions of vitamin D on myoendocrine cells of the atrium for the regulation of ANF manufacture and secretion. These results were obtained by combining thaw-mount autoradiography with immunocytochemistry using tritiated 1,25-D₃ and an antibody against rat ANF. This antibody was also used in a radioimmunoassay to determine atrial natriuretic factor in plasma, atria and ventricles of normal or vitamin D-deficient mice.