Characterization of the aflatoxin B1-binding site of rat albumin

A fluorescence-enhancement method was used to investigate the non-covalent interaction between aflatoxin B1 and rat albumin. Solvent-induced shifts in the emission spectrum of aflatoxin B1 provided evidence that the aflatoxin B1-binding site of rat albumin is a highly nonpolar environment. A dissociation constant of 20 microM was determined at 20 degrees C. The possibility that aflatoxin B1 binds one of the three major drug sites of albumin was investigated by ligand-displacement experiments. Mechanisms whereby marker ligands displace aflatoxin B1 were further investigated by comparing the experimental binding parameters with those derived theoretically, assuming competitive binding. The results indicate that: aflatoxin B1 and phenylbutazone compete for a common high-affinity site on rat albumin; high-affinity binding of aflatoxin B1 and site-II marker ligands takes place independently; aflatoxin B1 does not compete with either cholate or warfarin for the same high-affinity site, but the simultaneous binding of warfarin or cholate negatively modulates the binding of aflatoxin B1 to albumin. Fluorescence energy-transfer studies show that the lone tryptophan residue, Trp-214, is not associated with the aflatoxin B1-binding site.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Metabolism of aflatoxin B1 by rat hepatic microsomes induced by polyhalogenated biphenyl congeners

The metabolism of aflatoxin B1 to aflatoxins M1 and Q1 by rat liver microsomes from animals pretreated with polychlorinated or polybrominated biphenyl congeners depended on the structure of the halogenated biphenyl inducers. Microsomes from rats treated with phenobarbital (PB) or halogenated biphenyls that exhibit PB-type activity preferentially enhanced the conversion of aflatoxin B1 to aflatoxin Q1. In contrast, microsomes from rats treated with 3-methylcholanthrene (MC) or halogenated biphenyls that exhibit MC-type induction activity increased the metabolism of aflatoxin B1 to aflatoxin M1. The coadministration of PB and MC produced microsomes that exhibited both types of induction activity (mixed type) in catalyzing the oxidative metabolism of diverse xenobiotic agents. However, PB-plus-MC-induced hepatic microsomes from immature male Wistar rats preferentially increased the metabolism of aflatoxin B1 to aflatoxin M1 but did not enhance the conversion of aflatoxin B1 to aflatoxin Q1. Comparable results were observed with microsomes from rats pretreated with halogenated biphenyls classified as mixed-type inducers; moreover, in some cases there was a significant decrease in the conversion of aflatoxin B1 to aflatoxin Q1 (compared with that of controls treated with corn oil).     

Effect of antiserum against aflatoxin B1-bovine serum albumin complex on aflatoxin B1-induced lysogenesis

Escherichia coli K12 bacteria lysogenic for the lambda phage were used to study the effect of antiserum against aflatoxin B1-induced lysogenesis. The antiserum was obtained from rabbits immunized with water in oil emulsion of aflatoxin B1-bovine serum albumin complex (AFB1-BSA). A marked reduction in the degree of lysogenesis was observed when the antiserum was added to the reaction medium prior to microsomal enzyme activation of aflatoxin B1. There was no detectable effect when the antiserum was added after aflatoxin B1 activation. The result presented suggests that the antibodies in the AFB1-BSA antiserum can interact with aflatoxin B1 prior to its activation. This implies that an immune-protective effect can only be exerted if the antibodies intervene before activation.     

Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.

Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.     

Mechanism of action of aflatoxin B1 in Bacillus megaterium

Bacillus megaterium cells from various growth phases were equally susceptible to the lethal effects of aflatoxin B1. Known surfactants (EDTA and Tween-80) accentuated the effects of aflatoxin B1. Viability and inulin uptake in aflatoxin B1-exposed cells decreased considerably. The effect was concentration dependent. A straight-line relationship observed in the death curve indicated a single target for aflatoxin B1 action in B. megaterium. Leakage of intracellular constituents in B. megaterium was also concentration dependent, and this can be related to the extent of cell membrane damage.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

Mechanism of action of aflatoxin B1 in Bacillus megaterium.

Bacillus megaterium cells from various growth phases were equally susceptible to the lethal effects of aflatoxin B1. Known surfactants (EDTA and Tween-80) accentuated the effects of aflatoxin B1. Viability and inulin uptake in aflatoxin B1-exposed cells decreased considerably. The effect was concentration dependent. A straight-line relationship observed in the death curve indicated a single target for aflatoxin B1 action in B. megaterium. Leakage of intracellular constituents in B. megaterium was also concentration dependent, and this can be related to the extent of cell membrane damage.     

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.     

葡萄糖氧化酶解除黄曲霉毒素 B1应用研究

黄曲霉毒素是由真菌产生的一类剧毒物质,含黄曲霉毒素B1的饲料对小鸡的危害很大。研究中发现,用5％o25U／g（25U／mL）葡萄糖氧化酶完全可以解除饲料中500ppb浓度黄曲霉毒素B1的毒性。用含2000ppb浓度黄曲霉毒素B1和添加5‰ 25U／g葡萄糖氧化酶的饲料喂小鸡,相比对照组,实验组存活数量提高39．5％,在小鸡饮用水中加入5％e25U／mL葡萄糖氧化酶,存活数量可提高28．5％。     

Immunoenzyme test system for detection of aflatoxin B1

Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B1 carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B1 with a low relative cross-reactivity against aflatoxin B2, G1, G2, M1, B2a and G2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.     

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.     

Neonatal diethylstilbestrol treatment alters aflatoxin B1-DNA adduct concentrations in adult rats

Aflatoxin B1-DNA adduct concentrations were measured in the livers of adult Sprague-Dawley CD rats treated on days 2, 4, and 6 postnatally with 1.45 mumols of diethylstilbestrol and in adulthood with phenobarbital, 3-methylcholanthrene, or vehicle prior to treatment with aflatoxin B1. Aflatoxin B1 (1 mg/kg) was injected 5 hr prior to killing the rats. Female rats exposed neonatally to diethylstilbestrol had significantly higher aflatoxin B1-DNA adduct concentrations (three- to sixfold) than adult female rats treated neonatally with propylene glycol. Liver aflatoxin B1-DNA adduct concentrations were slightly higher in control males as compared to adduct concentrations in neonatally diethylstilbestrol-treated males, as compared to adduct concentrations in control females (not significant [NS]). Phenobarbital and 3-methylcholanthrene treatment followed by aflatoxin B1 injection resulted in decreased aflatoxin B1-DNA adduct concentrations in all rats. Our results demonstrate that neonatal exposure to diethylstilbestrol alters the capacity of adult female rats to form and/or dispose of carcinogen-DNA adducts following a single dose of aflatoxin B1 (increased adduct concentration). This alteration may be a consequence of altered imprinting mechanisms with diethylstilbestrol causing developmental modifications early in life. The animals were, however, able to respond to cytochrome P-450 and P-448 inducers as evidenced by decreased aflatoxin B1-DNA adduct concentrations.     

Synthesis and degradation of aflatoxins by Aspergillus parasiticus. II. Comparative toxicity and mutagenicity of aflatoxin B1 and its autolytic breakdown products

A crude mycelial protein extract from a 16-day-old culture of A. parasiticus, on purification, lost 50% of its ability to degrade aflatoxin B1. The addition of hydrogen peroxide increased this activity to 97% of that of the crude extract. Ducklings dosed orally with aflatoxin extracts from 14- and 20-day-old cultures containing 46 micrograms or more of aflatoxin B1 developed enlarged livers, haemorrhaged and died in less than 10 days, giving and LD50 of 17.5 and 17.1 micrograms aflatoxin B1 per 50 g body weight respectively for each extract. When pure aflatoxin B1 was mixed with either the crude or purified mycelial protein extract the aflatoxin B1 level was decreased by 29% as was the toxicity of the mixture. The main breakdown product of aflatoxin B1 was isolated and was shown to have an RF value of 0.34, was non-fluorescent, and was non-toxic for ducklings at oral doses as high as 400 micrograms per 50 g body weight. The mutagenic effect of aflatoxin B1 on Salmonella typhimurium was relative to its concentration. The main breakdown product of aflatoxin B1 was non-mutagenic.     

Enzyme-linked immunosorbent analysis for aflatoxin B1.

An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B2alpha, and least specific for aflatoxin G1.     

Fluorescence energy transfer measurements of the complexes of aflatoxin B1 and cytochromes P-450

The distances between the heme of cytochrome P-450 and the substrate, aflatoxin B1, in the complex of aflatoxin B1 and each of two species of cytochrome P-450 were determined by fluorescence energy transfer measurements. Cytochromes P-450 used were cytochrome P-450 I-d and cytochrome P-450 II-a prepared from hepatic microsomes of polychlorinated biphenyl-treated rats; the main metabolic products of aflatoxin B1 were aflatoxin Q1 and aflatoxin M1, respectively. The distances between the heme and the substrate were calculated to be 6.9nm and 4.7nm in cytochrome P-450 I-d and cytochrome P-450 II-a, respectively. The results suggest that the difference in the metabolic products of aflatoxin B1 is due to the difference in the conformation of the enzyme-substrate complexes.     

Alleviation of Aflatoxin B1‐Induced Genomic Damage by Proanthocyanidins via Modulation of DNA Repair

In order to study the mechanisms underlying the alleviation of aflatoxin B1‐induced genomic damage by proanthocyanidins (PAs), we examined the modulation of oxidative DNA damage induced by aflatoxin B1 in PAs‐pretreated animals. The effects of PAs on changes in the expression of DNA damage and repair genes induced by aflatoxin B1 were also evaluated in rat marrow cells. Administration of PAs before aflatoxin B1 significantly mitigated aflatoxin B1‐induced oxidative DNA damage in a dose‐dependent manner. Aflatoxin B1 treatment induced significant alterations in the expression of specific DNA repair genes, and the pre‐treatment of rats with PAs ameliorated the altered expression of these genes. Conclusively, PAs protect against aflatoxin B1‐induced oxidative DNA damage in rats. These protective effects are attributed to the antioxidant effects of PA and enhanced DNA repair through modulation of DNA repair gene expression. Therefore, PAs are a promising chemoprotective agent for averting genotoxic risks associated with aflatoxin B1 exposure.     

Enzyme-linked immunosorbent analysis for aflatoxin B1.

An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B2alpha, and least specific for aflatoxin G1.     

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.