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Aflatoxin B1 formamidopyrimidine adducts in human hepatocarcinogenesis: a preliminary report.

The presence of aflatoxin-B1-formamidopyrimidine, a persistent aflatoxin-deoxyribonucleic acid (DNA) adduct, was investigated in vivo by immunohistochemical analysis in 14 paired hepatocellular carcinoma and nontumorous human liver tissue sections using a monoclonal anti-aflatoxin-B1-formamidopyrimidine antibody. Nuclear and cytoplasmic accumulations of adducts were found in 4 of 14 nontumorous specimens but in none of 14 tumorous tissues and in none of three normal control livers. In vitro adduct formation and cellular DNA was investigated with a modified DNA immunoblot assay. These studies revealed (a) no significant difference in the amount of adduct formed by DNA samples with or without integrated hepatitis B virus DNA, (b) no difference in the amount of adduct formed with DNA from either tumorous or nontumorous tissues from a given individual, and (c) remarkable and reproducible differences in the capacity of DNA from different individuals to form in vitro adducts. Our DNA immunoblot assay will facilitate further studies on the molecular role of aflatoxin-B1-form-amidopyrimidine adducts in human hepatocarcinogenesis.

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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.

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[Chemical composition of the meat from chickens treated with aflatoxin B1].

Studies were carried out to establish the effect of aflatoxin B1 on some qualitative indices of poultry meat. It was found that the use of aflatoxin B1 led to a decrease in the live weight as well as to changes in the chemical composition of meat-increase in the water content, decrease in the amount of fats and proteins, and drop of its biologic value. Most clearly manifested were the changes when aflatoxin B1 was applied at the rate of 37.5 micrograms daily, in the course of 30 days. It was also found that withdrawal periods of 7 days did not contribute to the elimination of the changes in the chemical composition of poultry meat.

Aflatoxin B1↗

Comparative mutagenicity of palmotoxin Bo and aflatoxins B1 and M1.

The mutagenicity of palmotoxin Bo and of aflatoxin M1 relative to that of aflatoxin B1, the potent mutagen, was studied in five Ames' Tester Strains of Salmonella typhimurium (TA-98, TA-100, TA-1535, TA-1537, TA-1538). Aflatoxins B1 and M1 are both highly mutagenic in a microsome-mediated system in TA-100. The prediction of the relative carcinogenicity of aflatoxin M1 to aflatoxin B1 posed by the mutation of TA-100 is probably more authentic than TA-87. The mutagenic potency of palmotoxin Bo is less than that of either aflatoxin B1 or M1.

Aflatoxins↗

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2.

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)

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Effects of maize weevils (Coleoptera: Curculionidae) on production of aflatoxin B1 by Aspergillus flavus in stored corn.

Insects play an important role as facilitators of the aflatoxin-producing fungus, Aspergillus flavus Link, in both preharvest and postharvest corn. The current study investigated the role of maize weevils, Sitophilus zeamais Motschulsky, in enhancing aflatoxin B1 content in stored corn. In laboratory experiments, aflatoxin B1 was quantified with an indirect enzyme-linked immunosorbent assay (ELISA) on corn following artificial infestation with adult weevils that had each been topically treated with 100 spores of A. flavus. Corn kernels infested with A. flavus-contaminated weevils had significantly higher levels of aflatoxin B1 than A. flavus-inoculated corn without weevils. The presence of maize weevils resulted in increased kernel moisture content during incubation, and grain moisture was positively correlated with aflatoxin content across treatments receiving spores. Aflatoxin B1 levels were higher in corn treated with fungus-contaminated weevils compared with corn that was mechanically damaged and inoculated with spores, which in turn had more aflatoxin than undamaged corn treated with spores. Aflatoxin B1 content in corn increased with time of weevil exposure from 7 to 21 d, but decreased after 28 d of exposure. Aflatoxin levels in infested corn increased significantly with increased numbers of A. flavus-contaminated weevils. Maize weevils carried spores both internally and externally; however, substantial numbers of spores were intimately associated with the exoskeleton of adult weevils. These findings indicate that maize weevils facilitate the growth of A. flavus and aflatoxin production in corn by increasing surface area susceptible to fungal infection and increasing moisture content as a result of weevil metabolic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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Drug resistance patterns and susceptibility to aflatoxin B1 of strains of Escherichia coli and Staphylococcus aureus.

The antibacterial properties of aflatoxin B1 have been evaluated against antibiotic-resistant clinical isolates of Escherichia coli and Staphylococcus aureus. The inhibition of growth ranged from 11.5 to 60.0% and 4.5 to 18.5% in the strains of S. aureus and E. coli, depending on the extent of drug resistance. Aflatoxin-B1 binding varied with toxin concentration, the presence of surfactants (Tween-80 or EDTA) as well as with the antibiotic-resistance pattern; binding was maximal in antibiotic-sensitive strains and least in the most resistant strains. Binding of aflatoxin B1, correlated with growth inhibition. Aflatoxin B1 also caused leakage of cell contents and decrease in inulin uptake, effects which were also concentration dependent.

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Effect of alpha-tocopherol supplementation on the impact of aflatoxin B1 on the testes of rats.

Rats were given aflatoxin B1 (7.5 microg/200 g b.w.) and alpha-tocopherol (0.9 mg/200 g b.w.) for 14 days oral intubation to investigate the effect of alpha-tocopherol on the toxicity of aflatoxin B1 on rat's testes. There were significant alterations in testicular sorbitol dehydrogenase, lactic dehydrogenase, glucose-6-phosphate dehydrogenase, gamma glutamyl transpeptidase; reduced quality of sperm and marked pathological changes in the testis of rats given aflatoxin B1 alone. Alpha-tocopherol supplementation increased the aflatoxin blood level. A reduction in the toxicity of free radicals by alpha-tocopherol in association with a reduction in aflatoxin metabolism seem to be responsible for the protective influence observed.

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In vitro effect of aflatoxin B1 on rat liver macrophages (Kuffer cells).

The effect of aflatoxin B1 on the uptake and incorporation of [3H]leucine and [3H]uridine and on phagocytosis of latex particles was studied using cultures of rat liver macrophages (Kuffer cells). Aflatoxin B1 inhibited the incorporation of both isotopes, but inhibition of uridine incorporation was greater than that of leucine, suggesting that RNA synthesis was a major site of inhibition. Aflatoxin B1 also inhibited phagocytosis of latex particles in a time- and dose-dependent manner.

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Liquid chromatographic method for determination of aflatoxins B1, B2, G1, and G2 in corn and peanut products: collaborative study.

A collaborative study of a liquid chromatographic method for the determination of aflatoxins B1, B2, G1, and G2 was conducted in laboratories located in the United States, Canada, South Africa, and Switzerland. Twenty-one artificially contaminated raw peanuts, peanut butter, and corn samples containing varying amounts of aflatoxins B1, B2, G1, and G2 were distributed to participating laboratories. The test portion was extracted with methanol-0.1N HCl (4 + 1), filtered, defatted with hexane, and then partitioned with methylene chloride. The concentrated extract was passed through a silica gel column. Aflatoxins B1 and G1 were derivatized with trifluoroacetic acid, and the individual aflatoxins were determined by reverse-phase liquid chromatography with fluorescence detection. Statistical analysis of the data was performed to determine or confirm outliers, and to compute repeatability and reproducibility of the method. For corn, relative standard deviations for repeatability (RSDr) for aflatoxin B1 ranged from 27.2 to 8.3% for contamination levels from 5 through 50 ng/g. For raw peanuts and peanut butter, RSDr values for aflatoxin B1 were 35.0 to 41.2% and 11.2 to 19.1%, respectively, for contamination levels from 5 through 25 ng/g. RSDr values for aflatoxins B2, G1, and G2 were similar. Relative standard deviations for reproducibility (RSDr) for aflatoxin B1 ranged from 15.8 to 38.4%, 24.4 to 33.4%, and 43.9 to 54.0% for corn, peanut butter, and raw peanuts, respectively. The method has been adopted official first action for the determination of aflatoxins B1, B2, G1, and G2 in peanut butter and corn at concentrations greater than or equal to 13 ng total aflatoxins/g.

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Influence of gamma-irradiation and maize lipids on the production of aflatoxin B1 by Aspergillus flavus.

The effect of gamma-irradiation and maize lipids on aflatoxin B1 production by Aspergillus flavus artificially inoculated into sterilized maize at reduced water activity (aw 0.84) was investigated. By increasing the irradiation doses the total viable population of A. flavus decreased and the fungus was completely inhibited at 3.0 kGy. The amounts of aflatoxin B1 were enhanced at irradiation dose levels 1.0 and 1.5 kGy in both full-fat maize (FM) and defatted maize (DM) media and no aflatoxin B1 production at 3.0 kGy gamma-irradiation over 45 days of storage was observed. The level in free lipids of FM decreased gradually, whereas free fatty acid values and fungal lipase activity increased markedly by increasing the storage periods. The free fatty acid values decreased by increasing the irradiation dose levels and there was a significant enhancement of fungal lipase activity at doses of 1.0 and 1.50 kGy. The ability of A. flavus to grow at aw 0.84 and produce aflatoxin B1 is related to the lipid composition of maize. The enhancement of aflatoxin B1 at low doses was correlated to the enhancement of fungal lipase activity.

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[The effect of aflatoxin B1 on the human lymphocyte blast transformation reaction].

The effect of various aflatoxin B1 concentrations on human peripheral blood lymphocyte blast-transformation reaction was studied. There was aflatoxin B1-induced dose-dependent inhibition of DNA synthesis in the lymphocytes stimulated by phytohemagglutinin. The aflatoxin B1 concentrations causing 50% decreases in DNA synthesis were defined by the regression method. (ID50). Judging from the ID50 values, the treatment of cultured lymphocytes with cytochrome P-448 inductor and lymphocytic exposure in the presence of toxin and hepatic microsomes of mice showed a 6-7-fold increase in the inhibitory action of aflatoxin B1. This indicates that the metabolism of aflatoxin B1 brought about by the cytochrome P-450-dependent monooxygenase system plays an important role in the action of aflatoxin B1 on human lymphocytes.

Aflatoxin B1↗

Risk assessment for aflatoxin: I. Metabolism of aflatoxin B1 by different species.

A comparison of the generation and detoxification of reactive aflatoxin B1 metabolites in different species may elucidate why animal species vary widely in sensitivity to aflatoxin B1 carcinogenicity, in addition to offering some perspective on how sensitive man may be to the carcinogenic effects of this mycotoxin. Scientific literature comparing the ability of cellular fractions from different species to metabolize aflatoxin B1 is reviewed. However, in vitro studies exclude components for multiple metabolic pathways, eliminating the possibility of competition between metabolic activation and detoxification. Quantification of metabolic activation by different species from these studies is therefore limited. The species-specific carcinogenic potency of aflatoxin B1 may be reflected in levels of aflatoxin B1-DNA adducts generated in vivo. The current paucity of quantitative information on human DNA adduct levels does not permit comparisons between different species on this basis.

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The 1995 Pharmacological Society of Canada Merck Frosst Award. Cellular and molecular targets in pulmonary chemical carcinogenesis: studies with aflatoxin B1.

Although most notorious as a liver carcinogen, the mycotoxin aflatoxin B1 targets other tissues as well, including those of the respiratory system. Because the biotransformation of aflatoxin B1 to toxic and nontoxic metabolites has been fairly well characterized, it serves as a useful and relevant model carcinogen for studying the biochemical (i.e., balance between bioactivation and detoxification) and molecular (i.e., mutations in target genes) mechanisms of pulmonary chemical carcinogenesis. Because of the cellular diversity of the lung, it is of particular interest to assess these processes in different lung cell types, if we are to identify the target cells for carcinogen action. This review summarizes studies that have been aimed at identifying the basis for susceptibility of the lung to aflatoxin B1.

Aflatoxin B1↗

Solvent-efficient thin-layer chromatographic method for the determination of aflatoxins B1, B2, G1, and G2 in corn and peanut products: collaborative study.

An interlaboratory study of a solvent-efficient thin-layer chromatographic (TLC) method for the determination of aflatoxins B1, B2, G1, and G2 was conducted in laboratories located in the United States, France, Tunisia, and Denmark. Eighteen artificially contaminated samples plus blanks of raw peanuts and peanut butter and corn containing varying amounts of aflatoxins B1, B2, G1, and G2 were distributed to participating laboratories. The method consists of elements of the U.S. Food and Drug Administration (FDA), Contaminants Branch (CB) (AOAC Method 968.22) and FDA, Best Foods (BF) (AOAC Method 970.45) methods with reduced requirements for solvents. Participating laboratories used either visual or densitometric techniques during the final determinative step. Statistical analysis of the data was performed to determine or confirm outliers and to compute repeatability and reproducibility of the method using either visual or densitometric techniques for the determinative step. Reported results from laboratories using a densitometer showed that, for corn, the relative standard deviation for repeatability (RSDr) for aflatoxin B1 ranged from 56.6 to 41.7% for contamination levels ranging from 5 to 50 ng/g. For raw peanuts and peanut butter, the RSDr values for aflatoxin B1 ranged from 21.3 to 37.3% and 65.9 to 42.1%, respectively, for the contamination levels ranging from 5 to 25 ng/g. RSDr ranges for aflatoxins B2, G1, and G2 were similar. For reproducibility (R), the RSDR ranges for aflatoxin B1 were 41.7-56.6%, 56.6-84.8%, and 26.4-37.3% for corn, peanut butter, and raw peanuts, respectively. Average recoveries for all aflatoxins at all levels were 95.3, 139.0, and 95.6% for corn, peanut butter, and raw peanuts, respectively. When analysts determined aflatoxin concentrations in corn by visual comparison to standards, the RSDr values for aflatoxin B1 were 47.8-11.4% for contamination levels ranging from 5 to 50 ng/g. For raw peanuts and peanut butter, the RSDr values for aflatoxin B1 were 76.3-12.6% and 33.4-8.8%, respectively, for the contamination levels ranging from 5 to 25 ng/g. RSDr values for aflatoxins B2, G1, and G2 were similar. The RSDR values for aflatoxin B1 were 34.6-90.2%, 45.5-59.3%, and 31.8-78.3% for corn, peanut butter, and raw peanuts, respectively. Average recoveries for all aflatoxins at all levels were 111.0, 157.6, and 92.3% for corn, peanut butter, and raw peanuts, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

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Activation of aflatoxin B1 by mouse CYP2A enzymes and cytotoxicity in recombinant yeast cells.

The ability of three highly homologous mouse liver CYP2A enzymes to activate aflatoxin B1 was studied by expressing them in recombinant AH22 Saccharomyces cerevisiae yeast cells. The reconstituted monooxygenase complex with CYP2A5 purified from yeast cell microsomes produced epoxide at a rate of 17.2 nmol/min per nmol P450 in the presence of 50 microM aflatoxin B1 while CYP2A4 had about 10% and P4507 alpha only 1.5% of this activity. However, Km values were 530 and 10 microM and Vmax values 12.5 and 14.3 nmol/min per nmol P450 for CYP2A4 and CYP2A5, respectively. When recombinant yeast cells were exposed to aflatoxin B1 LC50 concentrations were 7.5 +/- 5.5 microM for CYP2A4, 0.45 +/- 0.10 microM for CYP2A5 and > 320 microM for P4507 alpha expressing yeast cells. Aflatoxin B1-DNA adduct levels in the same yeast cells were 50, 890 pmol/mg DNA and below detection limit when 3.0 microM aflatoxin B1 was used in the incubation mixture. Coumarin an inhibitor for CYP2A4 and a substrate for CYP2A5 diminished the toxicity of aflatoxin B1 in a dose-dependent manner for these recombinant yeast cells. These data demonstrate that (1) highly homologous mouse CYP2A enzymes activate aflatoxin B1 in a different manner and (2) that recombinant yeast cells expressing mammalian CYP enzymes are a useful and inexpensive system to test the role of different enzymes in aflatoxin B1 toxicity. The data also indicate that mouse CYP2A5 and its counterpart in other species could have a significant role in aflatoxin B1 toxicity in organs where it is expressed at high levels.

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DNA binding, adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal, Kupffer and endothelial cells.

In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B1, DNA binding and adduct formation. In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP+ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B1 to a metabolite mutagenic to Salmonella typhimurium TA 98. In the case of nonparenchymal cells (i.e. Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B1 had to be used to obtain a similar number of revertants to that observed with parenchymal cells. Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B1 in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B1. Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2 alpha-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2 alpha-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B1 to its epoxide. When freshly isolated, intact cells were incubated with tritiated aflatoxin B1, a dose-dependent aflatoxin B1 binding to DNA in parenchymal and nonparenchymal cells was observed. HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1.

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Preferential attack of mitochondrial DNA by aflatoxin B1 during hepatocarcinogenesis.

Administration of the hepatic carcinogen aflatoxin B1 to experimental animals results in covalent binding to liver mitochondrial DNA at concentrations three to four times higher than nuclear DNA. The concentration of carcinogen adducts in mitochondrial DNA remains unchanged even after 24 hours, possible because of lack of excision repair. Similarly, mitochondrial transcription and translation remain inhibited up to 24 hours suggesting long-term effects of aflatoxin B1 on the mitochondrial genetic system.

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