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It has been proved that clays were able to absorb aflatoxin B1. The amount of adsorbed aflatoxin depended on the nature of clay. The adsorbing power of the clays was checked in relation to some environmental factors. From this study, it is claimed that clays are suitable for détoxification of liquid food-stuffs poisoned by aflatoxin.
The effects of dietary administration of 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 2(3)-tert-butyl-4-hydroxyanisole (BHA), ethoxyquin (EQ) and 5-(2-pyrizinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) on aflatoxin B1 (AFB1) - DNA adduct formation in vivo in livers and kidneys of rats were investigated. Male F344 rats were treated with 1 mg/kg AFB1 by i.p. administration and nucleic acids isolated 2 h post dosing. Animals were fed a semipurified diet supplemented with either 0.5% EQ, 0.45% BHT, 0.45% BHA or 0.1% oltipraz for 2 weeks prior to AFB1 treatment. Analysis of nucleic acid bases by h.p.l.c. showed that several AFB1 metabolite-DNA adducts were formed in both tissues. The principal and related adducts of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 represented approximately 80-90% of all adducts in both tissues and in all treatment groups. However, inclusion of the antioxidants in the diet resulted in substantial reductions in overall AFB1 modified DNA levels. EQ, BHT, BHA and oltipraz reduced the covalent binding of AFB1 to liver DNA by 91, 85, 65 and 76% and to kidney DNA by 80, 35, 62 and 64%, respectively. Concordantly, the specific activities of hepatic enzymes of presumed importance to AFB1 detoxification, epoxide hydrase, and glucuronyl and glutathione transferases were significantly elevated by all antioxidants. Reduced glutathione levels were unchanged except by oltipraz, although activities of enzymes contributing to the maintenance of reduced glutathione pools, glutathione reductase and glucose-6-phosphate dehydrogenase, were elevated in most treatment groups. An excellent correlation (r = 0.95) was observed between the degree of inhibition of DNA binding by AFB1 and the induction of hepatic glutathione S-transferase activities by the four antioxidants.
Hepatocytes were prepared from rainbow trout by perfusion in situ with collagenase and hyaluronidase. Preparations normally showed high initial viability (95 +/- 5% dye exclusion, 92 +/- 5% lactate dehydrogenase retention) and gradually decreased in viability and glutathione concentration over 5 hours. Cellular metabolism of aflatoxin B1 (AFB1), a potent hepatocarcinogen, was characterized by an investigation of the following parameters: kinetics of AFB1 metabolism and DNA adduct formation, dose response, viabilities of detoxication and activation pathways with time, influence of organic solvents, and effect of variation in cell concentration. The AFB1 metabolites and DNA adducts were resolved and quantitated by high-performance liquid chromatography. From these results a standardized assay procedure was derived which we used to examine AFB1 metabolism and DNA adduct formation in hepatocytes from fish fed dietary substances known to alter the carcinogenic response to this mycotoxin. Dietary beta-naphthoflavone, which strongly inhibits AFB1 carcinogenesis in rainbow trout, dramatically and reproducibly altered AFB1 binding and metabolism in isolated hepatocytes. Overall rate of AFB1 metabolism and rates of detoxication reactions increased, whereas DNA binding decreased. Dietary cyclopropenoid fatty acids, powerful synergists and promoters of AFB1 carcinogenesis in trout, also repressed AFB1-DNA binding. Both dietary factors appeared to depress initial DNA damage by AFB1 but operated through different metabolic pathways to do so.
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We have developed a method to select for rat hepatoma cells that fail to express hepatocyte-specific functions. Well-differentiated cells descended from the H4IIEC3 hepatoma line express aldrin epoxidase (AE) activity, an indicator of the liver-specific forms of cytochromes P450 and, concurrently, are able to activate the procarcinogen aflatoxin B1 (AFB1) into highly toxic metabolites. Thus, differentiated hepatoma cells are highly sensitive to AFB1, while dedifferentiated derivatives, which fail to express AE activity, are resistant. Exposure of differentiated Fao cells to 10 microM AFB1 for 24 h permits the isolation, at a frequency of 5 x 10(-5), of resistant colonies that exhibit strongly reduced AE activity. Strikingly, various morphological types can be observed. In more than 90% of the colonies, cells are morphologically similar to the original differentiated cells and accumulate all liver-specific mRNAs examined in amounts comparable to Fao cells. Moreover, they are able to carry out gluconeogenesis, as judged by their capacity to grow in glucose-free medium. For a minor fraction of colonies, the cells exhibit nonhepatic morphology. These cells fail to express three or more of the liver functions and are not able to proliferate in glucose-free medium. Our results demonstrate that the use of AFB1 constitutes a simple and efficient single-step selective method for obtaining variant hepatoma cells of a wide variety of phenotypes.
An experimental group of female rats was exposed to aflatoxin B1 at a dose of 7.5 mg/kg body weight daily for 14 days through oral intubation. A deleterious effect on the gonads and embryotoxicity in the experimental animals were observed. There were reductions in ovarian and uterine sizes, increases in fetal resorption, implantation loss, and intra-uterine death. The data showed significant disturbances in oestrus cycle, inhibition of lordosis, and reductions in the conception rates and litter sizes, suggesting severe impairment of fertility in the aflatoxin exposed rats. An aflatoxin blood level of 86.2 +/- 1.3 ppb was recovered in the experimental animals.
Weanling rats were kept on a synthetic vitamin A free diet for 9 weeks, and subsequently on the same diet but with oral supplementation of vitamin A palmitate at different doses for 2-3 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 (AFB1) leading to its activation and DNA adduct formation was measured after each period of experimental feeding. An increased molecular reactivity of AFB1 was evident during vitamin A deficiency such that the degree of its conversion to its reactive metabolite and subsequent DNA adduct formation was higher with microsomes from vitamin A-deficient rats as compared to rats maintained on normal supply of vitamin A. These activities of microsomes returned to normal after the rats had adequate supplements of vitamin A. The vitamin A status was also found to play a role in regulating glutathione S-transferase activity of the liver cytosol fraction, the activity being low in deficiency but increased progressively with increasing supplementation of vitamin A. Results suggest that vitamin A can afford protection against adverse effects of the potent hepatocarcinogen AFB.
The reaction of chemical carcinogens with cellular macromolecules, particularly DNA, is considered to be an important event during chemical carcinogenesis. Until recently, most studies have dealt primarily with nuclear DNA (nDNA). Mitochondrial DNA (mtDNA) is increasingly being investigated for its possible role during chemical carcinogenesis. Using monoclonal antibodies against aflatoxin B1 (AFB1)-modified guanosine and ultrastructural immunocytochemistry, we have localized the AFB1-guanosine adducts predominantly in mitochondria and nuclei. Morphometric analysis of the electron micrographs demonstrates that localization of the AFB1-guanosine adducts is several-fold greater in mitochondria than in the nuclei. However, biochemical analysis demonstrates that 77% of the covalent binding was detected in nucleic acids of the mitochondria relative to the nuclear fractions. In RNA-free preparations, the extent of covalent modification of mt circular DNA was less than 50% of that of nuclear DNA. Our results indicate that 67% of AFB1 binding to mitochondrial nucleic acids is associated primarily with mtRNA.
A study was conducted to investigate the selective cleanup and determination of aflatoxin B1 (AfB1) from contaminated media. Composite adsorbents were formulated from calcium montmorillonite clay, which possesses a high affinity and enthalpy of adsorption for AfB1. Nanostructuring techniques were used to construct various formulations of the clay-based composite media. In AfB1 adsorption studies with prototypical affinity columns, these composites offered narrowly defined, reproducible capacity ranges. Composite recoveries of AfB1 from spiked grains exhibited linear trends that correlated well with the range of spike levels. Composite columns provided lower recoveries of AfB1 from naturally contaminated corn than did immunoaffinity columns; however, recoveries were consistent and purified extracts were free of interfering compounds, as determined by liquid chromatography with fluorescence detection.
Lung cells isolated from AC3F1 (A/J x C3H/HeJ) mice 7 wk after treatment with the carcinogenic mycotoxin aflatoxin B1 (AFB1) were examined for point mutations in the Ki-ras oncogene. Ki-ras mutant allele frequencies in fractions enriched with nonciliated bronchiolar epithelial (Clara) cells were consistently higher than in alveolar type II cell fractions. Mutant alleles were undetectable or minimal in macrophage- and polymorphonuclear leukocyte-enriched fractions. In cells from vehicle (dimethyl sulfoxide)-treated mice, small proportions of mutant Ki-ras alleles were found in the Clara cell-enriched fraction but not in other cell fractions. The results indicated that Clara cells are particularly susceptible to AFB1-induced Ki-ras mutation, an early event in AFB1-induced mouse lung tumorigenesis.
Dehydroepiandrosterone (DHEA) a naturally occurring steroid, has been reported to inhibit the binding of N-dimethylnitrosamine and 7,12-dimethylbenz[a]anthracene to DNA in vivo and to increase glutathione transferase activity. In this study, we have investigated if DHEA could protect hepatic DNA from damage by the potent hepatocarcinogen aflatoxin B1 (AFB1). Young male Fischer 344 (2-month-old) rats were fed a diet containing 0.8% DHEA for 14 days. Control rats were pair-fed the same diet without DHEA. The rats were then administered a single i.p. dose of [3H]AFB1 in dimethylsulfoxide (0.6 mg/kg body weight; 200 mCi/mmol) and killed after 3 h. Liver weight, mitochondrial, microsomal and cytosolic protein, cytochrome P450 content and glutathione transferase activity increased significantly (P less than 0.001) in DHEA-fed rats; however, the hepatic DNA content was not altered. DHEA feeding increased the total amount of AFB1 bound to hepatic protein but decreased the extent of DNA binding. In in vitro experiments, there was less total binding to DNA and protein by AFB1 when using microsomes from DHEA-fed rats. These results suggest that DHEA inhibits the binding of AFB1 to DNA by modifying the biotransformation of the carcinogen.
Retinol (vitamin A alcohol) at the doses tested neither induced an increase of frequency of sister chromatid exchanges (SCE) nor cell cycle delay in Chinese hamster V79 cells with or without the metabolic activation of S-9 mix. However, it inhibited SCE frequencies and cell cycle delay in V79 cells induced by the indirect mutagen cyclophosphamide or aflatoxin B1. The inhibition was found to be dose and time dependent. These results suggest that retinol itself may have no direct effect on the genetic materials but rather exert its effects possibly by inhibiting the metabolic activation of an indirect mutagen or carcinogen. Thus, the antitumor activities of retinoids may not be limited to the widely accepted role of preventing the promotion step but also the initiation step of carcinogenesis.
The worldwide human exposure to aflatoxin B1 (AFB1), particularly in developing countries, remains to be a serious public health concern. Although AFB1 is best known as a hepatocarcinogen, epidemiological studies have shown a positive association between human lung cancer occurrence and inhalation exposure to AFB1. Cytochrome P450 (CYP)-catalyzed metabolic activation is required for AFB1 to exert its carcinogenicity. Previous studies have identified CYP1A2 and CYP3A4 as the major enzymes for AFB1 activation in human liver. However, the key CYP enzymes in human lung that can efficiently activate AFB1 in situ are unknown. In the present study, we demonstrate that CYP2A13, an enzyme predominantly expressed in human respiratory tract, has a significant activity in metabolizing AFB1 to its carcinogenic/toxic AFB1-8,9-epoxide and AFM1-8,9-epoxide at both low (15 microM) and high (150 microM) substrate concentrations. Under the same conditions, there was no detectable AFB1 epoxide formation by CYP2A6, which was also reported to be involved in the metabolic activation of AFB1. Consistent with the activity data, there was an approximately 800-fold difference in LC50 values of AFB1 (48-hr treatment) between Chinese hamster ovary (CHO) cells expressing CYP2A13 and CYP2A6 (50 nM versus 39 microM). We further demonstrate that amino acid residues Ala117 and His372 in CYP2A13 protein are important for AFB1 epoxidation and its related cytotoxicity. Our results suggest that CYP2A13-catalyzed metabolic activation in situ may play a critical role in human lung carcinogenesis related to inhalation exposure to AFB1.
A systematic search for feed contamination by aflatoxin-B1 as well as for lesions indicating an acute or chronic aflatoxicosis was made in three groups of industrially raised hens in Côte-d'Ivoire. The contamination rates were low although they were 6 to 10 times higher than the official standards. No lesion due to an acute or chronic intoxication by aflatoxins was observed. However, the repeated intake of small amounts of aflatoxins might eventually have repercussions on the technics-economical performance either by a direct action of the toxin or via the development of an immune depression in the animals.
A cDNA encoding a novel member of the cytochrome P-450 superfamily, Cyp3a-13, has been isolated from mouse liver cDNA library by hybridization screening. The Cyp3a-13 encoded 503 amino acid residues and shared 71% amino acid identity with Cyp3a-11. When Cyp3a-13 cDNA was expressed in CR119 cells which had been established as a cell line stably expressing NADPH-cytochrome P-450 reductase cDNA of guinea pigs, aflatoxin B1-dependent cytotoxicity was observed. This cytotoxicity was enhanced by alpha-naphthoflavone (7,8-benzoflavone), which is known to augment the CYP3A enzymatic activity. The results indicate that CYP3A in mice, which are relatively insensitive to aflatoxin B1, can activate aflatoxin B1 to a genotoxic product.
The BALB/3T3 clone A31-1-1 mouse embryo cell line at passages 7 to 13 was selected for morphologic studies of neoplastic transformation by carcinogens of different chemical classes, in the absence of any added extracellular metabolic activation. Dose-related transforming activity was demonstrated for the carcinogens aflatoxin B1 (AFB) and benzidine (BZ) not previously reported in this system, and was confirmed for benzo[a]pyrene (BP), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Spontaneous transformation per cells at risk was low (0.14 type III foci x 10(-4), while chemically induced transformation was 2 to 3 orders of magnitude higher with all compounds. The molar concentration of carcinogens in complete medium, required to induce a transformation frequency of 1.0 type III foci x 10(-3) showed the highest level of activity for BP (0.04 microns), an intermediate level for AFB (0.2 to 1.4 microns), MCA (1.1 micron), and MNNG (2.3 microns), and the lowest level of activity for BZ (30.0 microns). The dose-related induction of morphological transformation in this clone by carcinogens of different classes indicates the potential value of this biological system in quantitative studies of carcinogen combinations, especially at low dose levels.
The method proposed by the Federal Department of Health for the determination of aflatoxin B1,B2,G1, and G2 was tested for additional determination of aflatoxin M1. With relatively small changes of the original method, all aflatoxins including. B1, B2,G1,g2, and M1 can be determined quantitatively in milk, milk powder, butter, cheese, quark, cream, yoghurt and fruit yoghurt.