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.     

In vivo effect of selenium on the mutagenic activity of aflatoxin B1

Experiments were carried out to verify the effect of selenium on the mutagenic activity of AFB1. After 14 days of selenium administration to experimental animals (Chinese hamsters, Cricetulus griseus) in the form of 2 ppm Na2SeO3 solution available ad libitum the incidence of chromosomal aberrations in bone marrow cells due to a single p.o. administration of 5 mg AFB1 per 1 kg body weight was significantly reduced. The incidence of chromosomal aberrations was monitored till day 32 after AFB1 administration. A significant decrease in the frequency of aberrant cells, breaks and gaps was observed at almost any time during the investigation. 2 ppm Na2SeO3 solution itself did not enhance the frequency of chromosomal aberrations. The mechanism of the protective effect of selenium vis-a-vis the mutagenic and carcinogenic action of AFB1 remains obscure.     

Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1.

The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear. In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo. Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes. Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks. The AFB1-treated rats were injected i.p. with 1 mg/kg body weight. In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups. However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals. For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively). DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test. These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo. Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo. However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism. Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.     

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.     

The mutagenic activity of aflatoxin B1 in the Cricetulus griseus hamster and Macaca mulatta monkey

Chromosome aberrations were scored in bone marrow cells of Cricetulus griseus hamsters and Macaca mulatta monkeys given a single i.p. injection of aflatoxin B1 (AFB1). The mutagenic activity of AFB1 was assessed by the percentage of cells bearing aberrations and by the total frequency of chromosome and chromatid breaks. Chinese hamsters were treated with five different doses of AFB1 ranging from 1 microgram to 5 mg/kg (LD50/30 = 12.2 mg/kg) and the aberration yields at each AFB1 dose level tested were determined at 24 h intervals for 5 consecutive days. Compared to controls the increase in the two types of chromosome abnormalities was significant in all tests. At 5 mg/kg of AFB1 the tests were carried out over a period of 92 days to assure the analysis of aberration yields with time. All chromosome aberration assays conducted during this period showed significant increases in the frequencies of aberrant cells and chromosome and chromatid breaks in comparison to controls. Macaque monkeys were treated in the same fashion using 0.1 and 1.0 mg/kg of AFB1 and the dynamics of chromosome aberration yields was analyzed for a period of 730 days. Similarly as in the case of Chinese hamsters the percentage of cells with aberrations and the frequency of chromosome and chromatid breaks were always higher in this period than the control value. Long-term aberration yield data obtained experimentally were expressed in the form of analytical curves which allowed to establish the time when the yields of aberrant cells reached their maxima and when they returned to the control level. In both animal species tested the courses of analytical curves had a similar dynamics. Factors that might be responsible for a long-term persistence and relatively great fluctuations of the chromosome aberration yields encountered after a single injection of AFB1 are discussed in detail.     

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.     

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.     

Acute effects of aflatoxin B1 on tRNA methylase function.

Transfer RNA methylase activity and capacity were measured in relation to the acute effects of aflatoxin B₁ on rat liver. Dose-independent methylase activity was elevated approx. 40% within 3 days after dosing, and gradually declined towards control values over a 3-week period. Transfer RNA methylase capacity, in contrast, exhibited a linear dose-response relationship with values elevated as much as 100% over control levels.     

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.     

Effect of dietary aflatoxin B1 on the growth response and haematologic changes of young Japanese quail

Feeding of aflatoxin B₁ at the rate of 0.5 ppm to young Japanese quail resulted in significant (p<0.01) decrease in body weight gain that became apparent on the third week. There was no significant difference in the mean values of haemoglobin, packed cell volume and total erythrocyte counts of quail chicks given aflatoxin B₁ in feed in comparison to those fed on a similar diet without aflatoxin. However, the total leucocyte count revealed an increase on the third week which was due to an increase in the percentage of heterophils and decrease in lymphocytes.     

Genetic analysis of aflatoxin B1 activation in rat hepatoma cells

Summary We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.Abbreviations AFB1 aflatoxin B1 - AE aldrin epoxidase - AHH aryl hydrocarbon hydroxylase - PAH polycyclic aromatic hydrocarbons - PB phenobarbital     

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations. The p53 gene-specific adduct frequencies in DNA, modified in cells with 40-400 microM AFB1, were 0.07-0.74 adducts per kilobase (kb). In vitro modification with 0. 1-4 ng AFB1-8,9-epoxide per microgram DNA produced 0.03-0.58 lesions per kb. The adduct patterns obtained with the epoxide and the different microsomal systems were virtually identical indicating that adducts form with a similar sequence-specificity in vitro and in vivo. The lesions were detected exclusively at guanines with a preference towards GpG and methylated CpG sequences. The methods utilizing QPCR and LMPCR thus provide means to assess gene-specific and sequence-specific AFB1 damage. The results also prove that microsomally-mediated damage is a suitable method for avoiding manipulations with very unstable DNA-reactive metabolites and that this damage can be detected by QPCR and LMPCR.     

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.