Aflatoxin B1-2,3-oxide: evidence for its formation in rat liver in vivo and by human liver microsomes in vitro

Injection of [³H]aflatoxin B₁ into rats yielded covalently bound derivatives in hepatic DNA, rRNA, and protein. Mild acid hydrolysis of the DNA and rRNA adducts formed a derivative indistinguishable from 2,3-dihydro-2,3-dihydroxy-aflatoxin B₁. The data indicate that approximately 60% of the nucleic acid adducts were derived from reactions $\text{in vivo}$ with aflatoxin B₁-2,3-oxide. Acid hydrolysis of rRNA-[³Haflatoxin B₁ adduct formed by human liver microsomes $\text{in vitro}$ also liberated the dihydrodiol in significant amount. The 2,3-oxide of aflatoxin B₁ is a probable ultimate carcinogenic metabolite.     

Comparative metabolic conversion of aflatoxin B1 to M1 and Q1 by monkey, rat and chicken liver

We studied the $\text{in vitro}$ metabolish of flatoxin B₁ by liver microsomal preparations from monkey, rat and chicken. With all these species, both the previously recognized metabolite aflatoxin M₁ as well as the newly identified aflatoxin Q₁ were produced from the aflatoxin B₁ substrate. Aflatoxin Q₁ is an isomer of aflatoxin M₁ (with the hydroxyl on the carbon β to the carbonyl of the cyclopentenone ring) which we discovered recently in rat and monkey liver incubations with aflatoxin B₁. In our incubations we did not detect aflatoxin P₁ which has been reported as a major metabolite of aflatoxin B₁$\text{in vivo}$ in the monkey.In general the conversion to aflatoxin M₁ was comparable among the different species (1–3% of the substrate) except in the chicken in which it was lower (0.1–0.3%). Also the conversion to Q₁ was comparable to or slightly higher than the conversion to M₁ with rat and chicken liver but the conversion to Q₁ with the monkey liver was outstandingly high, accounting for 19–52% of the substrate in three species of monkeys tested.     

2,3-Dihydro-2,3-dihydroxy-aflatoxin B1: an acid hydrolysis product of an RNA-aflatoxin B1 adduct formed by hamster and rat liver microsomes in vitro

Fortified hamster and rat liver microsomes bind the carcinogen aflatoxin B₁ (AFB₁), but not its much less carcinogenic 2,3-dihydro derivative (aflatoxin B₂), to RNA in vitro. Mild acid hydrolysis of the RNA-AFB₁ adduct yields a product indistinguishable from synthetic 2,3-dihydro-2,3-dihydroxy-AFB₁ in (a) its UV absorption spectra in neutral and alkaline solution, (b) its thin layer chromatography in several solvent systems, and (c) the mass spectra of the acetonide and diacetyl derivatives. AFB₁-2,3-oxide is the probable reactive precursor of the RNA-AFB₁ adduct. This epoxide merits consideration as a candidate ultimate carcinogenic metabolite of AFB₁.     

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B₁ complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B₁ remained bound. Nonviable bacteria retained the highest amount of aflatoxin B₁. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B₁ from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B₁ to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B₁. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B₁ released. Binding of aflatoxin B₁ appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.     

Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s

We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test.     

The microsomal activation of aflatoxin B1 and 2-(N-ethylcarbamoyloxymethyl)furan in vitro using a novel diffusion apparatus

The activation by rat liver microsomal systems in vitro of a naturally occurring and a synthetic furan-containing toxin, aflatoxin B₁ and 2-(N-ethylcarbamoyloxymethyl)furan (CMF) has been examined. Both compounds are metabolised to form products which bind covalently to DNA and microsomal protein, Using a specially designed two-chamber diffusion apparatus it has been demonstrated that the active metabolite of CMF is able to bind covalently to DNA separated by a membrane barrier from the microsomal site of activation. In the case of aflatoxin B₁ the DNA must be in physical contact with the microsomal system for the active metabolite of aflatoxin B₁ to bind covalently. Differences between the activation of the two compounds have also been found with regard to their relative efficiencies in binding to DNA and also the effects of the nucleophile GSH. These results have suggested that if the molecular mechanisms of activation of the two compounds be similar, other factors, for example differences in lipid solubility, may play important roles in determining the relative biological activaties of the compounds. The results suggested that the subcellular site of activation of aflatoxin B₁, unlike that of CMF, may need to be adjacent to the target DNA. It is proposed that this site might be the outer nuclear membrane. Alternatively a carrier molecular might exist for the activated aflatoxin B₁ metabolite in vivo.     

Metabolism and acute toxicity of aflatoxin B1 in rats

Female rats are more resistant to the acute oral toxicity of aflatoxin B₁ and have higher tissue levels of aflatoxin M₁, a fluorescent hydroxylated metabolite, than males. These differences are reduced by castration, as castrated animals of either sex are more resistant and have higher tissue levels than entire males and females. The toxicity of a single oral dose of aflatoxin B₁ appears to be inversely related to levels of aflatoxin M₁ in the tissues.     

Interaction of aflatoxin B1 with electron-donating organic molecules,including aromatic amino acids

The interaction of the carcinogenic mycotoxin, aflatoxin B₁, with some electrondonating organic compounds including aromatic hydrocarbons, dimethylaniline, and aromatic amino acids, was studied. Spectrophotometric analysis of aflatoxin B₁ revealed that hypochromicity in the absorption around 360 nm and hyperchromicity around 385 nm were induced by dimethylaniline, hexamethylbenzene, tryptophan, and imidazole. A similar shifting of aflatoxin B₁ absorption was observed in benzene, toluene, and xylene in the presence of ZnCl₂. The interaction of aflatoxin B₁ with polystyrene was observed in a biphasic system. The association constants of aflatoxin B₁: DMA⁴ (1:1) and of aflatoxin B₁: tryptophan (1:1) were found to be 0.64 and 22.6 liters per mole, respectively. The results suggest that charge-transfer interaction occurs between aflatoxin B₁ and these π-electron donors. Since the spectral changes on aflatoxin B₁ absorption induced by these π-electron donors are similar to those induced by nucleic acids and proteins, it is postulated that charge-transfer interaction also occurs between aflatoxin B₁ and these macromolecules. The role of such interaction in the biological activity of aflatoxin B₁ is discussed.     

Qualitative and quantitative detection of aflatoxin B1 in poultry sera by enzyme-linked immunosorbent assay

An indirect competitive inhibition type enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of aflatoxin B₁, in poultry sera. Preincubation of aflatoxin B₁, samples with the antibody prior to competition yielded better results in terms of higher sensitivity. After competition, amount of antibody bound to solid phase was measured by incubation with anti-rabbit immunoglobulins coupled with horse raddish peroxidase. Intensity of colour decreased as the amount of free aflatoxin B₁, increased. Final detection of aflatoxin B₁, was made by (i) visual comparison with standard aflatoxin B₁ using dot-ELISA (qualitative) and (ii) by plate-ELISA, where optical density was measured at 492 nm (quantitative). Plate-ELISA was more sensitive than dot-ELISA, with sensitivity limits being 100 fg and 1 pg per 10 μl, respectively. However, due to ease and speed of performance, dot-ELISA has greater potential as a test for the diagnosis of mycotoxicosis at the field level.     

Aflatoxins in mutants of Aspergillus flavus

Mutants ofAspergillus flavus were recovered following the irradiation of conidia with ultraviolet light. Analysis of the mutants for aflatoxins B₁, B₂, G₁, and G₂ indicated a wide range of variability in aflatoxin levels. None of the isolates produced the G toxins, and four produced little or no aflatoxin B₂. Production of B₁ and B₂ by the mutants ranged from 1.3 µ;g/ml to 967 µg/ml and zero to 30 µg/ml, respectively. The correlation between production of B₁ and B₂ was statistically significant. There was no apparent correlation between nutritional requirement or conidial color and aflatoxin production.     

Enhancement of aflatoxin B1 cytotoxicity in differentiated rat hepatoma cultures by a prior glucocorticoid treatment of the monolayer.

The cytotoxic effect of aflatoxin B₁ on cultures of a differentiated rat hepatoma cell line, Faza 967, has been evaluated by scoring the surviving colonies two weeks after briefly exposing the freshly plated cells to the mycotoxin. At the lowest concentration, aflatoxin B₁ exhibits no toxicity, unless the cultures have been pretreated with dexamethasone. HF-1, an hepatoma hybrid cell line exhibiting extinction of the hepatic functions and HF1-4, its subclone, that reexpresses all of these functions, have been compared. A 6hrs exposure to 60ng/ml aflatoxin B₁ is not toxic for HF₁ even after an hormonal treatment, while dexamethasone enhances the effect on HF₁-4. Glucocorticoïds have been shown previously to induce, in the differentiated clones, the hydroxylation of bile acid - a cytochrome P-450-mediated reaction ; in contrast, 3-methylcholanthrene, an inducer of benzopyrene hydroxylase in hepatoma cultures, is without effect on bile acid metabolism and on aflatoxin B₁ cytotoxicity. These results suggest that in the differentiated hepatoma cells, aflatoxin B₁ is converted into a cytotoxic metabolite by a glucocorticoïd-induced monooxygenase belonging to the cytochrome P-450-related group.     

Carcinogen-Nucleic Acid Interactions: Equilibrium Binding Studies of Aflatoxins B1 and B2 with DNA and the Oligodeoxynucleotide d(ATGCAT)2

Abstract

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B₁ and B₂ with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B₂ with the oligodeoxynucleotide d(ATGCAT)₂. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B₁ binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin ? 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels ? 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a “two-site” binding model [L.S. Rosenberg, M J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002–1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B₂ binding to d(ATGCAT)₂ indicates that the aflatoxin B₂/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1–0.5 ppm are observed for the aflatoxin B₂ 4-OCH₃, H5, and H6a protons. Much smaller chemical shift changes ? 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T₁ experiments demonstrate a 15–25 % decrease in the relaxation time for the adenine H2 protons when aflatoxin B₂ is added to the solution. This result suggests that aflatoxin B₂ protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B₁ and B₂ complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B₁,-guanine N7 DNA adducts.     

In vivo regulation of adipose tissue protein kinase by adenosine 3',5'-monophosphate mediated glucagon stimulation.

The cytochrome P-450 content of primary hepatocyte cultures was maintained at levels close to those found $\text{in vivo}$ by using a defined medium containing testosterone, thyroxine, hydrocortisone, estradiol, glucagon, insulin, linoleic acid and oleic acid. Using these cultures, [¹⁴C]aflatoxin B₁, a potent liver carcinogen, was metabolized primarily to water-soluble metabolites. In agreement with $\text{in vivo}$ results, aflatoxin M₁ was the only nonpolar metabolite detected. In addition, a significant portion of radioactivity was covalently bound to cell constituents. These results suggest that primary hepatocyte cultures may be a good model of the liver for studying the metabolism and mechanism of action of toxic chemicals.     

Effect of temperature on production of aflatoxin on rice by Aspergillus flavus

Summary The effect of temperature on formation of aflatoxin on solid substrate (rice) byAspergillus flavus NRRL 2999 has been studied in some detail. The optimum temperature for production of both aflatoxin B₁ and G₁ under the conditions employed is 28° C. Comparable yields of B₁ were obtained at 32° C, but considerably less G₁ was produced at this temperature. Both B₁ and G₁ were found in lesser amounts at temperatures above 32° C, and the aflatoxin content of rice incubated at 37° C was low (300–700 ppb) even though growth was good.Reducing the temperature from 28° to 15° C resulted in progressively less aflatoxin, but 100 ppb of B₁ was detected in cultures incubated 3 weeks at 11° C. No aflatoxin was produced at 8° C.The ratio of the four aflatoxins is affected by temperature. At the lower temperatures, essentially equal amounts of aflatoxin B₁ and G₁ were produced, whereas at 28° C, approximately four times as much B₁ was detected as G₁. At the higher temperatures, relatively less G was formed, until at 37° C, less than 10 ppb was detected.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Degradation products from aflatoxin B1 byCorynebacterium rubrum,Aspergillus niger,Trichoderma viride andMucor ambiguus

Summary Degradation of aflatoxin B₁ byCorynebacterium rubrum and byAspergillus niger was analysed by adding¹⁴C-labeled aflatoxin B₁ to cultures of these microorganisms. Two blue fluorescent compounds, formed byA. niger from aflatoxin B₁ with R_f-values 0.42 and 0.48 (R_f of aflatoxin B₁=0.54) were accumulated and characterized by UV-, fluorescence and mass spectrometry. Based on their properties both products were identified to be aflatoxin R_o. Under the same conditionsMucor ambiguus andTrichoderma viride also produced aflatoxin R_o.     

Divergence of West African and North American Communities of Aspergillus Section Flavi

West African Aspergillus flavus S isolates differed from North American isolates. Both produced aflatoxin B₁. However, 40 and 100% of West African isolates also produced aflatoxin G₁ in NH₄ medium and urea medium, respectively. No North American S strain isolate produced aflatoxin G₁. This geographical and physiological divergence may influence aflatoxin management.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

Regional Differences in Production of Aflatoxin B1 and Cyclopiazonic Acid by Soil Isolates of Aspergillus flavus along a Transect within the United States

Soil isolates of Aspergillus flavus from a transect extending from eastern New Mexico through Georgia to eastern Virginia were examined for production of aflatoxin B₁ and cyclopiazonic acid in a liquid medium. Peanut fields from major peanut-growing regions (western Texas; central Texas; Georgia and Alabama; and Virginia and North Carolina) were sampled, and fields with other crops were sampled in regions where peanuts are not commonly grown. The A. flavus isolates were identified as members of either the L strain (n = 774), which produces sclerotia that are >400 μm in diameter, or the S strain (n = 309), which produces numerous small sclerotia that are <400 μm in diameter. The S-strain isolates generally produced high levels of aflatoxin B₁, whereas the L-strain isolates were more variable in aflatoxin production; variation in cyclopiazonic acid production also was greater in the L strain than in the S strain. There was a positive correlation between aflatoxin B₁ production and cyclopiazonic acid production in both strains, although 12% of the L-strain isolates produced only cyclopiazonic acid. Significant differences in production of aflatoxin B₁ and cyclopiazonic acid by the L-strain isolates were detected among regions. In the western half of Texas and the peanut-growing region of Georgia and Alabama, 62 to 94% of the isolates produced >10 μg of aflatoxin B₁ per ml. The percentages of isolates producing >10 μg of aflatoxin B₁ per ml ranged from 0 to 52% in the remaining regions of the transect; other isolates were often nonaflatoxigenic. A total of 53 of the 126 L-strain isolates that did not produce aflatoxin B₁ or cyclopiazonic acid were placed in 17 vegetative compatibility groups. Several of these groups contained isolates from widely separated regions of the transect.     

Aflatoxin contamination in insect damaged seeds of horsegram under storage

Studies on one of the protein rich pulses, horsegram (Dolichos biflorus L.) were carried out to know how far these low risk pulses are free from aflatoxin contamination under severe insect infestation in storage. A total of 150 stored seed samples of horsegram were analyzed for the presence of aflatoxins by collecting 25 samples each of undamaged and insect damaged seeds of all the three varieties (PDM-1, PHG-1 and HG-96). More than 33% of insect damaged seed samples were contaminated with aflatoxin B₁ and B₂, whereas less than 8% of the undamaged seed samples contain only low levels of aflatoxin B₂. Higher levels of aflatoxin B₁ (up to 130 μg/kg) were reported in insect damaged seed samples of all the three varieties under study. The levels of aflatoxin B₂ were always lower than aflatoxin B₁ of the corresponding seed samples with insect damage. Aflatoxin B₁ was reported in both the undamaged and insect damaged seed samples of all the three varieties of horsegram. It is evident from the varietal response studies that PDM-1 and HG-96 varieties of horsegram are highly vulnerable to aflatoxin contamination whereas, PHG-1 variety is relatively less susceptible to it. In general, insect infestation leads to increase in fungal invasion (including aflatoxigenic fungi) and this further enhances the levels of aflatoxin contamination in horsegram seeds.     

Aflatoxin inhibition of glucocorticoid binding capacity of rat liver nuclei

The effect of aflatoxin B₁ on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [³H]dexamethasonccytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B₁. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 36 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B₁ dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant.Administration of [³H]dexamethasone to aflatoxin B₁-treated rats produced a similar pattern of glucocortocoid binding distribution in vivo to that observed in vitro. No differences in [³H]dexamethasone-cytoplasmic receptor binding between control and aflatoxin B₁-treated rats were found, whereas nuclear [³H]dexamethasone binding was reduced 34% by aflatoxin B₁ treatment.