Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse.

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.     

Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds.

The treatment of germinating maize seeds (cv. Ganga 2) with aflatoxin B1 resulted in suppression of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) synthesis at 3, 4, and 5 h, respectively. At or below the concentrations inhibitory for these in vivo syntheses, the toxin inhibited chromatin-bound DNA-dependent RNA polymerase activity. The synthesis of both polyadenylated and non-polyadenylated RNA was inhibited, but the effect on the former was more pronounced. Equilibrium dialysis and difference spectral and viscometric analyses showed a binding of aflatoxin B1 to DNA isolated from the seeds. It is proposed that the inhibition of RNA synthesis in maize seeds by the toxin is due to the interference with the RNA polymerase activity, which seems, at least partially, due to the impairment of DNA template functions.     

Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds

The treatment of germinating maize seeds (cv. Ganga 2) with aflatoxin B1 resulted in suppression of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) synthesis at 3, 4, and 5 h, respectively. At or below the concentrations inhibitory for these in vivo syntheses, the toxin inhibited chromatin-bound DNA-dependent RNA polymerase activity. The synthesis of both polyadenylated and non-polyadenylated RNA was inhibited, but the effect on the former was more pronounced. Equilibrium dialysis and difference spectral and viscometric analyses showed a binding of aflatoxin B1 to DNA isolated from the seeds. It is proposed that the inhibition of RNA synthesis in maize seeds by the toxin is due to the interference with the RNA polymerase activity, which seems, at least partially, due to the impairment of DNA template functions.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

The effect of aflatoxin B1 on normal and cortisol-stimulated rat liver ribonucleic acid synthesis

1. Aflatoxin B(1), administered in vivo, inhibits the incorporation of [(14)C]orotic acid in vivo into rat liver nuclei, and also inhibits both Mg(2+)- and Mn(2+)-dependent RNA polymerase activities in nuclei assayed in vitro. 2. Aflatoxin B(1) inhibits the cortisol-induced increase in incorporation of [(14)C]leucine in vivo, but does not affect the control value of this activity. 3. Aflatoxin B(1) administered in vivo inhibits the increase in nuclear Mg(2+)-dependent RNA polymerase activity, assayed in vitro, which results from the treatment with cortisol. 4. Adrenalectomy causes a decrease in Mg(2+)-dependent RNA polymerase activity. The effect on this enzymic activity of adrenalectomy plus treatment with aflatoxin B(1) is no greater than that of treatment with aflatoxin B(1) alone. 5. These results suggest that the inhibition of cortisol-stimulated biochemical pathways by aflatoxin B(1) is due to an inhibition of cortisol-stimulated RNA synthesis. 6. The cytoplasmic action of aflatoxin is thought to be due to a competition for receptor sites on the endoplasmic reticulum between steroid hormones and aflatoxin B(1). No evidence was obtained for a similar competition for nuclear receptor sites between [(3)H]cortisol and aflatoxin B(1). 7. No differences were observed between the activities of RNA polymerase preparations solubilized from control or aflatoxin-inhibited nuclei. 8. No differences in ;melting' profiles were observed between DNA and chromatin preparations isolated from control nuclei or from aflatoxin-inhibited nuclei. 9. It is suggested that aflatoxin B(1) exerts its effect on RNA polymerase by decreasing the template capacity of the chromatin and that the aflatoxin ;target' area of the chromatin includes that region which is stimulated by cortisol. This process, however, does not involve inhibiting the movement of cortisol from the outside of the hepatic cell to the nuclear chromatin.     

Binding of [-14C]aflatoxin B1 to cellular macromolecules in the rat and hamster.

The uptake and binding of ring-labelled [-14C]aflatoxin B1 (AFB1) by rat and hamster liver and kidney has been studied, the former species being extremely sensitive to the carcinogenic action of AFB, whereas the latter is resistant. In contrast to an earlier report (Lijinsky et al, Cancer Res., 30 (1970) 2280-2283, binding of the carcinogen to nucleic acids was far greater than that to protein. Rat liver DNA bound ten times and rRNA twenty times more carcinogen than protein. There were also differences in the amount of carcinogen bound to rat liver nucleic acids compared to those of the hamster, the latter species binding lower amounts of the carcinogen. Rat liver DNA bound four times and rRNA ten times as much AFB1 6 h after carcinogen administration whereas liver protein bound AFB1 was similar for the two species. Not only was there a difference in the amount of AFB1 bound but whereas in the rat, liver nucleic acid bound carcinogen decayed with time, no such fall was seen in the hamster, this remaining at a low level throughout the 48-h time period studied. In contrast, reaction of the carcinogen with kidney macromolecules was similar for the two species. The much higher binding of AFB1 to nucleic acids than to protein might account for the potent carcinogenicity of this compound in the rat, particularly since liver protein binding does not differ between a susceptible and a resistant species. A further important factor in determining carcinogenic sensitivity may be the removal of nucleic acid bound radioactivity with time, a possible repair process.     

Effect of Corn Steep Liquor on Mycelial Growth and Aflatoxin Production in Aspergillus parasiticus

Total aflatoxin concentrations produced by Aspergillus parasiticus, isolate 64-R8, in Czapek's broth fortified with corn steep liquor increased proportionately as the concentration of corn steep was increased from 0.5 to 8.0% (v/v) until maximal growth, as measured by dry mycelial weight, was reached. Thereafter, aflatoxin concentrations declined more rapidly than the rate of autolysis of mycelial material. Data are presented which indicate that the concentration of corn steep liquor also affects the ratio of production of aflatoxin B(1) and B(2) to that of aflatoxin G(1) and G(2). Further, this ratio also varies with time of incubation. Although both growth of the fungus and aflatoxin production are stimulated by the addition of corn steep to the basic medium, the stimulation of toxin production is much greater than fungus growth.     

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxins B1 and B2 with DNA and the oligodeoxynucleotide d(ATGCAT)2

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 less than 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 less than 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 (less than or equal to 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.     

Phage-displayed peptides that mimic aflatoxin B1 in serological reactivity

AIMS: To test phage-displayed random peptide libraries as sources of peptides that mimic the binding of aflatoxin B1 to monoclonal antibodies raised against the toxin. METHODS AND RESULTS: For two of the three MAbs tested, clones were obtained by panning, producing phage that bound specifically to MAb 13D1-1D9 (MAb 24; specific for aflatoxins B1 and G1) and MAb 6E12-1E9 (MAb 13; specific for aflatoxins B1, G1 and B2) in ELISA. The amino acid sequences of the binding peptides varied. Those binding to MAb 24 contained the sequence of '...YMD...', and those that bound to MAb 13 contained the dipeptide 'PW'. Mimotope phage was used in a competition ELISA format for assaying aflatoxin concentrations. CONCLUSION: The results show that mimotope preparations are effective substitutes for pure toxin in these ELISA procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: These results should contribute significantly to enhancing the safety and diminishing the costs of aflatoxin assays.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid.

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Stimulation of aflatoxin B1 and T-2 toxin production by sorbic acid

Aspergillus flavus grown on yeast extract-sucrose medium produced higher amounts of aflatoxin B1 in the presence of 0.025% sorbic acid than without this chemical with a maximum at 17 days of incubation. Addition of 0.05 to 0.0125% sorbic acid stimulated T-2 toxin production of Fusarium acuminatum cultures grown on maize meal. The highest amounts of the mycotoxin were detected in 14-day-old cultures containing 0.025% sorbic acid. It is assumed that certain amounts of sorbic acid near the minimal inhibitory concentration reduce the activity of the tricarboxylic acid cycle; this may lead to an accumulation of acetyl coenzyme A, which is an essential intermediate in the biosynthesis of aflatoxin B1 and T-2 toxin.     

Aflatoxin B1 Uptake by Flavobacterium aurantiacum and Resulting Toxic Effects

Removal of aflatoxin B(1) from liquid cultures by resting and growing cells of Flavobacterium aurantiacum NRRL B-184 was studied. Spectrophotometic and thin-layer techniques served as aflatoxin assays. Cells grown in the presence of 5 ppm or higher levels of aflatoxin developed aberrant morphological forms. These toxin concentrations partially inhibited growth, and the nature of the inhibition suggested that aflatoxin interfered with cell wall synthesis. Incubation of 1.0 x 10(11) resting cells per milliliter with 7.0 mug/ml of aflatoxin B(1) during a 4-hr period facilitated complete toxin removal from a buffered aqueous medium. Autoclaved cells and cell wall preparations could remove a fraction of the aflatoxin of a test system. However, the toxin removed by autoclaved cells and cell walls could be extracted by washing with water but the aflatoxin B(1) removed by intact cells could not be extracted into the liquid phase. The uptake of aflatoxin B(1) by resting cells was sensitive to temperature and pH. Ruptured preparations of F. aurantiacum were not able to remove or modify the aflatoxin in an aqueous solution.     

Fungal degradation of aflatoxin B1

A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.     

Some physiological abnormalities induced by aflatoxin B1 in mung seeds (Vigna radiata variety Pusa Baishakhi)

Physiological processes of mung seeds (Vigna radiata variety Pusa Baishakhi) and their germination were found to be affected by different concentrations of aflatoxin B₁. Inhibition in seed germination, seedling growth, chlorophyll, protein and nucleic acid syntheses was found to be due to aflatoxin B₁. The range of inhibition varied with the concentration of the toxin added.     

Comparison of aflatoxin B1 and aflatoxin G1 binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterisation of the major nucleic acid adducts.

A comparison between [14C]aflatoxin B1 (AFB1) and [14C]aflatoxin G1 (AFG1) binding to rat liver and kidney cellular macromolecules has shown AFG1-DNA and-ribosomal RNA binding to be lower in both organs. For both mycotoxins more was bound to nucleic acids than to protein. Two hours after intraperitoneal injection (60 microgram/100 g) of [14C] AFB1, 40 ng, 151 ng/mg. Loss of radioactivity bound to liver DNA for both [14C]AFB1 and protein respectively and for [14C]AFG1 the respective figures were 10, 7 and 1 ng/mg. Loss of liver bound radioactivity to DNA for both [14C]AFG1 and [14C]AFG1 appeared to be biphasic indicating that an enzymic DNA repair process may be operating. In vitro binding studies also showed less AFG1 was bound to exogenous DNA after microsomal activation than AFB1. This difference was not a result of differences in the chemical reactivity of the "ultimate" electrophilic species, the respective expoxides, since chemical activation studies using 3-chloroperbenzoic acid showed similar amounts of AFG1 and AFB1 to be converted to the epoxides and to bind to DNA. Studies on the distribution coefficients of the two mycotoxins showed AFB1 to be more lipophilic than AFG1 and this may be an important factor in determining the weaker carcinogenicity of the latter compound. Characterisation of the major AFG1-DNA adduct formed in vitro, in vivo and after peracid oxidation showed it to have the structure trans-9,10-dihydro-9-(7-guanyl)-10-hydroxy-aflatoxin G1. This adduct is similar to that obtained from AFB1 by activation in vivo, in vitro and after peracid oxidation.     

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.     

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.     

Inhibition by diacylmethane derivatives of mutagenicity in Salmonella typhimurium and tRNA-binding of chemical carcinogens

Effects of diacylmethanes on the mutagenicity of 2-naphthohydroxamic acid, methylnitrosourea, benzo[a]pyrene and aflatoxin B1 in S. typhimurium and the tRNA binding by benzo[a]pyrene and aflatoxin B1 were investigated. Acetylacetone, benzoylacetone and dibenzoylmethane inhibited the mutagenicity of 2-naphthohydroxamic acid, and dibenzoylmethane and 1,3-indandione inhibited that of methylnitrosourea, benzo[a]pyrene and aflatoxin B1. The binding to tRNA of benzo[a]pyrene and aflatoxin B1 was inhibited by benzoylacetone and dibenzoylmethane, and dibenzoylmethane, 1,3-indandione and 1,1,1-trifluoroacetylacetone, respectively. The inhibition of methylnitrosourea mutagenicity was observed when the bacteria were exposed concomitantly to the inhibitors and the mutagen, but not when they were exposed to the inhibitors 1 h after exposure to the mutagen. These results demonstrate that active methylene compounds can inhibit mutagenicity and nucleic acid-binding of chemical carcinogens presumably by trapping carcinogenic electrophiles, and they are potential anti-carcinogenic agents during the initiation stage.     

Reverszal of grwoth inhibition of Bacillus megaterium dut to aflatoxin by coffee and tea extracts

Growth inhibition of Bacillus megaterium by aflatoxin B₁ is employed in semi-quntitative assay of the toxin,. Aqueous extracts of tea and coffee were found in interfere with this assay, which indicated that tea and coffee contain factors(s) which can reverse the aflatoxin toxicity on B. megaterium     

A comparative study of the effect of aflatoxin B1 and actinomycin D on HeLa cells

1. The cytotoxic effects of aflatoxin B(1) on HeLa cells were examined and effects of short exposures of the cells to the toxin were found to be reversible. 2. Aflatoxin B(1) inhibited the synthesis of both ribosomal and heterodisperse RNA. It is proposed that the toxin's mechanism of action on ribosomal RNA synthesis is related to its inhibitory effect on the maturation of the 45s-ribosomal-RNA precursor. 3. Protein synthesis is inhibited to a greater extent by aflatoxin B(1) than by actinomycin D. In contrast with actinomycin D, aflatoxin B(1) was shown to disaggregate polyribosomes directly.