Metabolism of tRNA in rats with aflatoxin B1-induced hepatomas

This study describes effects of aflatoxin B1-induced hepatomas on RNA metabolism in rats. At 4 and 24 hours after the administration of L-(14CH3)-methionine, tRNA was isolated from the livers and hydrolyzed enzymatically to nucleosides which were quantitatively measured by HPLC. Radioactivity of the nucleosides was also determined. The data indicate that although tRNA methylation may be more rapid in livers with hepatomas, catabolism of tRNA in tumorous tissue is slower than in control livers. The large increase in some radioactive methylated nucleosides and bases by the tumor-bearing rats during the 24-hour period following the administration of labeled methionine indicates increased turnover of mRNA and rRNA as well as tRNA. Since degradation of tumor tRNA appears to be delayed, the excessive amounts of the urinary methylated nucleosides must be derived from RNA in nonneoplastic tissue.     

Functional changes in rat liver tRNA following aflatoxin B1 administration

Administration of aflatoxin B₁ (3 mg/kg body wt) to rats leads to strong inhibition of the acceptor activity of liver tRNA as measured by charging with [¹⁴C]-chorella protein hydrolysate. The maximum inhibition occurs 2 h after treatment. At increasing intervals after treatment, the inhibition appears to be gradually relieved, till control values are restored by 72 h. The charging experiment using several [¹⁴C]-amino acids separately shows pronounced inhibition of acceptor activity of all tRNA species, although the degree of inhibition varies with individual species. Preliminary results seem to rule out the possibility of hypermethylation of tRNA or damage to the CCA terminus as probable causes. The resultant functional changes may be attributed to a covalent interaction of aflatoxin B₁-metabolite with tRNA.     

Dual effects of phloretin on aflatoxin B1 metabolism: activation and detoxification of aflatoxin B1

Typically, chemopreventive agents involve either induction of phase II detoxifying enzymes and/or inhibition of cytochrome P450 enzymes (CYPs) that are required for the activation of procarcinogens. In this study, we investigated the protective effects of phloretin against aflatoxin B1 (AFB1) activation to the ultimate carcinogenic intermediate, AFB(1)-8, 9-epoxide (AFBO), and its subsequent detoxification. Phloretin markedly inhibited formation of the epoxide with human liver microsomes in a dose-dependent manner. Phloretin also inhibited the activities of nifedipine oxidation and ethoxyresorufin O-deethylase (EROD) in human liver microsomes. These data show that phloretin strongly inhibits CYP1A2 and CYP3A4 activities, which are involved in the activation of AFB1. Phloretin increased glutathione S-transferase (GST) activity of alpha mouse liver 12 (AML 12) cells in a dose-dependent manner. GST activity toward AFBO in cell lysates treated with 20 μM phloretin was 23-fold that of untreated control cell lysates. The expression of GSTA3, GSTA4, GSTM1, GSTP1 and GSTT1 was induced by phloretin in a dose-dependent manner in AML 12 cells. GSTP1, GSTM1, and GSTT1 were able to significantly increase the conjugation of AFBO with glutathione. Concurrently, induction of the GST isozyme genes was partially associated with the Nrf2/ARE pathway. Taken together, the results demonstrate that phloretin has a strong chemopreventive effect against AFB1 through its inhibitory effect on CYP1A2, CYP3A4, and its inductive effect on GST activity.     

Metabolic effects of low aflatoxin B1 levels on broiler chicks.

The effects of daily ingestion of aflatoxin B1 (AFB1) on growth, feed intake, plasma glucose, plasma cholesterol, plasma amino acids, plasma albumin, plasma ceruloplasmin, muscle amino acids, liver lipid, and bone strength were studied. For 3 weeks, beginning at an age of 2 days, broiler chicks were dosed daily per os with 50 or 100 micrograms of AFB1 per kg of body weight. Body weight and feed consumption were recorded daily, and metabolic responses were determined at 3 weeks. Treatment with AFB1 did not significantly alter body weight or feed intake. Relative liver weight showed a significant increase at the highest dose, with a significant concomitant increase in liver lipid and decrease in hepatic zinc. Relative spleen and heart weights were not affected by the toxin. Plasma glucose and cholesterol were significantly elevated at the highest dose. AFB1 significantly decreased plasma lysine and histidine and significantly increased muscle histidine, arginine, and valine. AFB1 decreased plasma albumin and markedly increased plasma ceruloplasmin. Dimensions of the long bones (femur and tibiotarsus) were not altered by the toxin. However, AFB1 caused a significant linear decline in the resistance of bone to breakage ("bone breaking strength"). The results indicate that low levels of AFB1 reduced bone strength in broiler chicks. The alterations in blood parameters indicated that AFB1 can disrupt metabolism even at low levels.     

The identification of the tRNA substrates for the supK tRNA methylase.

Purified preparations of the tRNA methylase deficient in supK strains of Salmonella typhimurium transfer methyl groups from S-adenosylmethionine (SAM) to at least two tRNA species, an alanine tRNA and a serine tRNA. The identity of the tRNA substrates for this enzyme was determined by a change in the elution position of the methyl-labeled tRNA from BND-cellulose columns before and after aminoacylation with a specific amino acid followed by derivatization of the free primary amino group with phenoxy- or naphthoxyacetate. The radioactive methyl group enzymatically added to these tRNAs is both acid and base labile and can be hydrolyzed to a volatile product at pHs above 7.5 and also at pH 1. The methylated 3'-nucleotide isolated from digested tRNA is a pyrimidine derivative and chromatographs like a modified uridylic acid. Its identity has not been established, but it is likely that it corresponds to the methyl ester of V, uridin-5-oxyacetic acid.     

Solvent effects on the spectroscopic properties of aflatoxin B1

1. Solvent-induced changes in the spectral properties of aflatoxin B1 were investigated using protic and aprotic solvents. 2. The absorption data were less sensitive to solvent effects than the fluorescence emission data. 3. Stokes shifts in protic solvents were greater than those in aprotic solvents indicating hydrogen bond formation between solvent and the excited state of aflatoxin B1. 4. From the Stokes shift data for aprotic solvents, the dipole moment of aflatoxin B1 was estimated to increase by 15.7 Debye units upon excitation to the excited singlet state.     

Short-term effects of aflatoxin B1 on serum lipids in subhuman primates.

Aflatoxin B1 significantly depressed serum lipid levels in specimens of Cercopithecus aethiops, Cercopithecus mona, Erythrocebus patas and Papio anubis. Serum cholesterol, total phospholipids and total lipids were not affected to the same extent.     

Acute effect of aflatoxin B1 on different inbred mouse strains II

The acute effects of Aflatoxin B₁(AFB₁) were evaluated on C57B1/6, CBA/J, B10A and Balb/c mice challenged with a single intraperitoneal dose of the mycotoxin (60 mg/Kg animal weight). 90 mice per strain were divided into three groups of 30 animals each: the intoxicated group and control groups I and II. Intoxicated mice were injected intraperitonealy with AFB₁ dissolved in corn oil, while control I mice received corn oil only (0.01 ml/g) by the same route. Lots of 10 animals from the intoxicated and control groups were sacrificed 24, 72 and 168 hours after challenge. Control mice II remained untreated and were used as standards of normality for biochemical (hepatic and renal function) and hematological evaluation. AFB₁ was detected in the liver of C57B1/6 and CBA/J mice 24 hours (1.46 and 0.75 ng/g, respectively), 72 hours (2.30 and 0.08 ng/g, respectively), and 168 hours (2.18 and 0.25 ng/g, respectively) after challenge. The mycotoxin was also observed in the liver of B10A mice (6.20 ng/g) 72 hours post-injection. The most evident histological lesions were observed 168 hours after treatment in C57B1/6 and B10A mice. Serum levels of alkaline phosphatase in intoxicated C57B1/6 and B10A mice were significantly higher than those of control I and II animals. The histopathologic lesions and biochemical changes were very discrete in Balb/c and CBA/J mice. It is included that strains C57B1/6 and B10A are more susceptible than strains CBA/J and Balb/c to the acute effects of AFB₁. Such difference probably reflects each strains's ability to biotransform and eliminate AFB₁ and its metabolites.     

Combined effects of aflatoxin B1 and citrinin on maize seedlings

Effect of two important mycotoxins, aflatoxin B1 and citrinin (concentration 2 g m-3) at various combinations (i.e., 1:1, 1:2, 2:1, 1:3 and 3:1, v/v) on seed germination, seedling growth, chlorophyll, carotenoid, starch, sugar, protein and nucleic acid contents, α-amylase activity, and respiration quotient was studied in maize cv. Suwan composite. The maximum and minimum inhibitions were recorded in most of the above parameters (except starch) at 3:1 and 1:3 combination ratios of these toxins, respectively. However, the inhibition rates varied with the treatments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Metabolic effects of low aflatoxin B1 levels on broiler chicks

The effects of daily ingestion of aflatoxin B1 (AFB1) on growth, feed intake, plasma glucose, plasma cholesterol, plasma amino acids, plasma albumin, plasma ceruloplasmin, muscle amino acids, liver lipid, and bone strength were studied. For 3 weeks, beginning at an age of 2 days, broiler chicks were dosed daily per os with 50 or 100 micrograms of AFB1 per kg of body weight. Body weight and feed consumption were recorded daily, and metabolic responses were determined at 3 weeks. Treatment with AFB1 did not significantly alter body weight or feed intake. Relative liver weight showed a significant increase at the highest dose, with a significant concomitant increase in liver lipid and decrease in hepatic zinc. Relative spleen and heart weights were not affected by the toxin. Plasma glucose and cholesterol were significantly elevated at the highest dose. AFB1 significantly decreased plasma lysine and histidine and significantly increased muscle histidine, arginine, and valine. AFB1 decreased plasma albumin and markedly increased plasma ceruloplasmin. Dimensions of the long bones (femur and tibiotarsus) were not altered by the toxin. However, AFB1 caused a significant linear decline in the resistance of bone to breakage ("bone breaking strength"). The results indicate that low levels of AFB1 reduced bone strength in broiler chicks. The alterations in blood parameters indicated that AFB1 can disrupt metabolism even at low levels.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Combination of solvent and radiation effects on degradation of aflatoxin B1

Summary Degradation of aflatoxin B₁ in chloroform, ethyl ccetate and coconut oil were examined by exposing the solvents in the form of thin layers to radiation from the sun and from UV and fluorescent tubes. Solar degradation of aflatoxin B₁ occurred in coconut oil leaving no residual aflatoxin B₁ or any new fluorescent derivatives. Other combinations of solvents and radiation produced up to four new fluorescent degradation compounds. Aflatoxin B₁ did not undergo solar degradation in the absence of moisture. Acidity enhanced solar degradation. A fluorescent derivative produced by solar degradation of aflatoxin B₁ in chloroform degraded further on solar irradiation in coconut oil but not in chloroform.

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Resumen Se examinó la degradación de la aflatoxina B1 en cloroformo, acetato de etilo y aceite de coco, exponiendo los disolventes en forma de capa fina a la radiación solar y a radiaciones procedentes de lámparas UV y fluorescentes. La degradación solar de la aflatoxina B1 tuvo lugar en aceite de coco sin dejar residuos de aflatoxina ni de ningún nuevo derivado fluorescente. Otras combinaciones de disolvente y radiaciones produjeron hasta 4 nuevos derivados fluorescentes. La degradación solar de la aflatoxina B1 no se produjo en ausencia de humedad. La acidez potenció la degradación solar. Uno de los derivados fluorescentes obtenidos en la degradación solar de la aflatoxina B1 en cloroformo continuó degradándose bajo la radiación solar en aceite de coco pero no en cloroformo.

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Résumé La dégradation de l'aflatoxine B1 dans le chloroforme, l'acétate d'éthyle et l'huile de noix de coco a été examinée en exposant les solvents sous la forme de films minces à l'irradiation du soleil, des uv et de tubes fluorescents. La dégradation solaire de l'aflatoxine B1 s'est produite dans l'huile de noix de coco en ne laissant ni aflatoxine B1 résiduelle ni nouveaux dérivés fluorescents. D'autres combinaisons de solvents et d'irradiation ont produit jusqu'à 4 nouveaux dérivés fluorescents de dégradation. L'aflatoxine B1 ne subit pas de dégradation solaire en absence d'humidité. L'acidité augmente la dégradation solaire. Un dérivé fluorescent produit par dégradation solaire de l'aflatoxine B1 dans le chloroforme, s'est dégradé davantage sous l'irradiation solaire dans l'huile de noix de coco mais non dans le chloroforme.

     

The effects of aflatoxin B1 on growth hormone regulated gene-1 and interaction between DNA and aflatoxin B1 in broiler chickens during hatching

Many types of aflatoxin cause problems for both public and animal health. Aflatoxin B₁ (AFB₁) is the most toxic and commonly encountered fungal toxin that appears in poultry feed and in feeds stored under unsuitable conditions. AFB₁ decreases feed quality, egg production and fertility of hatching eggs. Also, AFB₁ alters the development of embryos by infecting eggs. We investigated using sequence analysis the changes caused by different concentrations of AFB₁ on the promoter sequences of the growth hormone regulated gene-1 (GHRG-1) in chick embryo at 13, 17, 19 and 21 days incubation. DNA isolated from the liver of chick embryos treated with different concentrations of AFB₁ was separated using agarose gel electrophoresis to detect apoptosis, and DNA interaction with AFB₁ was investigated using plasmids to detect changes in electrophoretic mobility and their effects on DNA. Base changes of the promoter sequences of GHRG-1 in 5 ng/egg, 15 ng/egg and 40 ng/egg doses of AFB₁ were increased on day 19 compared to base changes of the same AFB₁ doses on day 13. We also found that AFB at different concentrations changed the mobility of DNA by binding to it, and that high doses of AFB1 destroyed DNA. The DNA interaction study using plasmid demonstrated that AFB₁ at high doses was bound to plasmid DNA, slowed its mobility and inhibited restriction cuts.     

Production of antibody against aflatoxin B1.

Antibody against aflatoxin B1 was obtained after one multiple-site injection of bovine serum albumin-aflatoxin B1 conjugate into rabbits. The antibody has greatest binding efficiency for aflatoxin B1, less efficiency for B2, G1, and Q1, and least for aflatoxicol, G2, and M1. Sterigmatocystin, coumarin, and 4-hydroxycoumarin did not give a cross-reaction with the antibody. The sensitivity of the binding assay for detection of aflatoxin B1 is in the range of 0.2 to 2.0 ng per 0.5-ml sample. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody titer determination are described.     

Purification and characterization of a tRNA methylase from Salmonella typhimurium.

A tRNA methylase, in which supK strains of Salmonella typhimurium are deficient, was purified from strain LT2 and characterized. Column chromatography of protein extracts from wild-type cells on phosphocellulose, diethylaminoethyl-Sephadex A-50, and hydroxlapatite resulted in an enzyme that was estimated to be about 50% pure. tRNA from S. typhimurium which had been incubated at pH 9.0 served as a substrate for this methylase. The enzyme has a molecular weight of about 50,000 as estimated by gel chromatography and by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The optimal assay conditions, as well as the kinetics and stability of the enzyme, were studied. As with other tRNA-methylating enzymes, S-adenosylhomocysteine is a potent inhibitor.     

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.     

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.