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.     

Non-toxicity to ducklings of solar-irradiated coconut oil contaminated with aflatoxin

Summary Pure aflatoxin Bl caused reduction in rate of weight gain, more than 50% mortality, bile duct hyperplasia and focal necrosis when fed to one-day-old veluvi ducklings for seven days. The same amount of aflatoxin B1 in coconut oil was solar irradiated in a pilot plant. The oil was then extracted for aflatoxins and the extracts fed to ducklings so that they would receive the same dose as birds fed with pure aflatoxin Bl. The pattern of weight gain in the birds fed with extracts of toxic oil after irradiation was the same as those of birds in control groups fed with non-toxic oil after irradiation or the vehicle, propylene glycol, alone. The birds showed no mortality or histopathological changes attributable to aflatoxins. Solar irradiation appeared to remove the toxicity of aflatoxin B1 in coconut oil, confirming TLC analysis of the irradiated oil.

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Aceite de coco contaminado con aflatoxinas, sometido a radiación solar, sin toxicidad para crias de pato

Resumen Se alimentaron crias de pato de un dia de edad de la raza veluvi durante siete dias con aflatoxina B1 pura. Como consecuencia se observó una reducción en la tasa de incremento de peso, más de un 50% de mortalidad e hyperplasia de los conductos biliares con necrósis focal. La misma cantidad de aflatoxina B1 en aceite de coco se sometió a radiación solar en una planta piloto. A continuación se extrajo el aceite para la obtención de aflatoxinas. Los extractos se dieron a crías de pato de forma que recibieran la misma dósis que las que se alimentaron con aflatoxina B1 pura. El patrón seguido por el incremento de peso de las crías alimentadas con los extractos del aceite tóxico irradiado fue el mismo que el obtenido por las crias alimentadas con aceite no tóxico irradiado y por otras que lo fueron con tan sólo el vehículo: propilenglicol. No se observaron variaciones en la mortalidad ni en la histopatología de las crías que fueran atribuíbles a aflatoxinas. La radiación solar parece eliminar la toxicidad de la aflatoxina B1 en aceite de coco, confirmando el análisis TLC (cromatografía de capa fina) del aceite irradiado.

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Non-toxicité pour les canetons de l'huile de coco contaminée par une aflatoxine et traitée par irradiation solaire

Résumé Administrée oralement à des canetons veluvi pendant sept jours, l'aflatoxine B1 pure détermine une réduction du gain de poids, une hyperplasie des canaux biliaires et des nécroses focales. La même quantité d'aflatoxine B1 dans de l'huile de coco a été irradiée par le soleil dans une usine pilote. Les aflatoxines ont été extraites et les extraits administrés à des canetons de façon à ce que ceux-ci reçoivent la même dose que des oiseaux traités par l'aflatoxine Bl pure. Les gains de poids des oiseaux traités par les extraits de l'huile toxique irradiée sont identiques à ceux des oiseaux témoins des groupes de contrôle ayant reçu de l'huile non-toxique irradiée, ou le véhicule seul (propylène glycol). Les oiseaux n'ont présenté aucune mortalité nimanifestation histopathologique attribuable aux aflatoxines. L'irradiation solaire parait éliminer la toxicité de l'aflatoxine B1 dans l'huile de coco, ce qui confirme l'analyse par TLC de l'huile irradiée.

     

B1-phytoprostanes trigger plant defense and detoxification responses

Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress. In this work, we show that the two naturally occurring B(1)-phytoprostanes (PPB(1)) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities. Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB(1)-I or -II revealed that both regioisomers triggered a massive detoxification and defense response. Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB(1) regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation. Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB(1) considerably prevented cell death caused by severe CuSO(4) poisoning. Several Arabidopsis genes induced by PPB(1), such as those coding for adenylylsulfate reductase, tryptophan synthase beta-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB(1) treated leaves of Arabidopsis. Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB(1). In three different plant species, PPB(1)-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate. PPB(1)-I was much weaker active or almost inactive. No differences were found between the enantiomers of each regioisomer. Thus, results suggest that PPB(1) represent stress signals that improve plants capacity to cope better with a variety of stresses.     

Failure of plant tissues to metabolize aflatoxin B1?

After exposure of peas and wheat kernels to aflatoxin B₁ solutions the following aflatoxins could not be detected in seed extracts: aflatoxins M₁, B_2a, Q₁, aflatoxicol and tetrahydrodeoxyaflatoxin B₁.     

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/l) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

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.     

Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): screening for aflatoxin B1 and ochratoxin A detoxification

Aims: To study the interaction between Bacillus spp. and contaminating Aspergillus flavus isolated strains from Thai fermented soybean in order to limit aflatoxin production. To study the detoxification of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) by Bacillus spp. in order to find an efficient strain to remove these toxins. Methods and Results: One A. flavus aflatoxin-producing strain and 23 isolates of Bacillus spp. were isolated from soybean and fresh Thua-nao collected from the north of Thailand. Inhibition studies of A. flavus and A. westerdijkiae NRRL 3174 (reference strain) growth by all isolates of Bacillus spp. were conducted by dual culture technique on agar plates. These isolates were also tested for AFB₁ and OTA detoxification ability on both solid and liquid media. Most of the strains were able to detoxify aflatoxin but only some of them could detoxify OTA. Conclusions: One Bacillus strain was able to inhibit growth of both Aspergillus strains and to remove both mycotoxins (decrease of 74% of AFB₁ and 92·5% of OTA). It was identified by ITS sequencing as Bacillus licheniformis. The OTA decrease was due to degradation in OTα. Another Bacillus strain inhibiting both Aspergillus growth and detoxifying 85% of AFB₁ was identified as B. subtilis. AFB₁ decrease has not been correlated to appearance of a degradation product. Significance and Impact of the Study: The possibility to reduce AFB₁ level by a strain from the natural flora is of great interest for the control of the quality of fermented soybean. Moreover, the same strain could be a source of efficient enzyme for OTA degradation in other food or feeds.     

Detoxification of aflatoxin B1 by acidogenous yoghurt

Serial concentrations of aflatoxin B₁ ranged from 200 to 1000 p.p.b. were assayed for detoxification by acidogenous yoghurt. Thin-layer chromatography analysis revealed a complete transformation of 800 p.p.b. of aflatoxin B₁ to a new fluorescing compound corresponding to aflatoxin B_2a which is referred as hydroxydihydroaflatoxin B₁. Partial conversion was present in yoghurt sample containing 1000 p.p.b. Toxicity test on chickens, confirmed Ciegler findings.     

食用菌对黄曲霉毒素B1的脱毒作用研究

目的 研究平菇、金针菇、黑木耳等七种食用菌对黄曲霉毒素B1（AFB1）的脱毒效果。方法 首先通过降解圈直径与菌落直径之比（Da/Dm）筛选出具有脱毒作用的株菌;然后通过AFB1残留量筛选出脱毒能力最强的菌株;最后将该菌株接种于含AFB1的玉米粉和大米中,考查其对粮食中AFB1的脱毒效果。结果 初筛发现平菇、黑木耳、金针菇对AFB1有较强脱毒作用,其Da/Dm分别为1.6±0.02、1.5±0.01、1.4±0.02;复筛发现黑木耳的脱毒能力最强,与AFB1共培养10 d后能清除88.16%的AFB1;进一步发现黑木耳对玉米粉和大米中AFB1有一定去除作用,清除率分别为62.4%和15.73%。结论 黑木耳对受AFB1污染的粮食作物有较好的脱毒作用,可用其控制食品与饲料中的AFB1。     

Influence of gamma irradiation of aflatoxin B1 production by Aspergillus flavus growing on some Nigerian foodstuffs

Spores of Aspergillus flavus (UI, 81) inoculated into some local food materials were irradiated at 62.5, 125.0, 250.0 and 500.0 krad, and the effect on aflatoxin B1 production on subsequent incubation was measured. The results show that aflatoxin B1 production decreased with increasing gamma irradiation dose in soya bean, groundnut, palm juice, while paw paw mash showed a relatively high yield of aflatoxin at 125.0 krad as compared to other irradiation levels tested except for the control. Irradiation of soya bean and groundnut inoculated with spores of Aspergillus flavus at 500.0 krad (pre-irradiation incubation period of 2 h) inhibited aflatoxin B1 production. Analysis of variance showed that media, pre-irradiation incubation periods and irradiation levels affected the total amounts of aflatoxin produced.     

Reduction of aflatoxin B1 levels by sheep saliva

This study determined the decrease of aflatoxin B₁ by sheep saliva at concentrations of 150 and 300 μg aflatoxin B-₁/L saliva. Analyses for aflatoxins B₁, M₁, and aflatoxicol (R₀) were performed after 2, 4, 6, 24, and 48 hours of incubation. Aflatoxin M₁ and R₀ were not detected and only residues of aflatoxin B₁ were found. 4 to 13% of aflatoxin B₁ were decomposed by sheep’s saliva within 2 hrs and 33 to 43% of aflatoxin B₁ after 24 hrs. Decomposition was affected by the aflatoxin concentration. Decrease of aflatoxin B₁ at 2, 4, 6 hrs was nearly three times higher at the low concentration (150 ppb) compared to the high concentration (300 ppb). After 48 hrs incubation more than 80% of the initial aflatoxin B₁ had been decomposed by the saliva.     

Characterization of monosomes produced by aflatoxin B1.

A single injection of 1.5 mg aflatoxin B1 per kg body weight produced approx. 70% disaggregation of rat liver polysomes into monosomes within 18 h. Isolated monosomes dissociated into 40 S subunits during centrifugation in linear sucrose gradients containing 0.3 M KCI. The 4 S to 5 S molar RNA ratio of the monosomes was calculated to be 0.6, indicating 0.6 tRNA and/or aminoacyl tRNA molecule per ribosome; no peptidyl tRNA was present. These results suggest that a single injection of affatoxin B1 produces monosomes which resemble runoff ribosomes.     

真菌对黄曲霉毒素B_1污染的防治研究

黄曲霉毒素是由黄曲霉、寄生曲霉等曲霉属菌株所分泌的毒性次级代谢产物,其中,尤以黄曲霉毒素B1(Aflatoxin B1,AFB1)的毒性、致突变性、致癌性最强,而且其在农作物的栽培、收获、贮藏和加工过程中污染严重,受到全世界的广泛关注。因此,为保证食品的安全性,各国研究人员一直都在寻求安全、高效、经济、环保的方法来控制食品和饲料中AFB1的污染。近年来真菌在AFB1的生物防治方面已取得很大进展,并有部分菌株已应用于生产中。本研究就真菌对AFB1的防治机制及其前景展望进行综述。     

Thermal stability of aflatoxin B1 and ochratoxin A

Within this research project, the LCI (Lebensmittelchemisches Institut des Bundesverbandes der Deutschen Süßwarenindustrie e. V.) conducted systematic studies to determine the thermal stability of the mycotoxins ochratoxin A (OTA) and aflatoxin B₁, as the available literature provides contradictory data. Firstly, the said mycotoxins in pure form were subjected to thermal treament. Secondly, tests were conducted to determine the influence of certain matrix substances, including carbohydrates and proteins, on the thermal decomposition behaviour of the mycotoxins. As a result it can be said that OTA seems to be stable up to 180 °C; however aflatoxin B₁ was almost completely degraded at heating temperatures of 160 °C and above. In several model assays it could further be shown that the degradation of mycotoxins is improved by the existence of certain matrix compounds.