Role of Polyphenols from the Aqueous Extract of Aloysia citrodora in the Inhibition of Aflatoxin B1 Synthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a mycotoxin considered a potent carcinogen for humans that contaminates a wide range of crops. Various strategies have been established to reduce or block the synthesis of AFB1 in food and feed. The use of aqueous extracts derived from plants with high antioxidant activity has been a subject of study in recent years due to their efficacy in inhibiting AFB1. In this study, we assessed the effect of Aloysia citrodora aqueous extract on Aspergillus flavus growth and on AFB1 production. A bio-guided fractionation followed by High Performance Liquid Chromatography (HPLC) and Mass spectrometry analysis of the active fraction were applied to identify the candidate molecules responsible for the dose-effect inhibition of AFB1 synthesis. Our results revealed that polyphenols are the molecules implicated in AFB1 inhibition, achieving almost a total inhibition of the toxin production (99%). We identified luteolin-7-diglucuronide as one of the main constituents in A. citrodora extract, and demonstrated that it is able to inhibit, by itself, AFB1 production by 57%. This is the first study demonstrating the anti-Aflatoxin B1 effect of this molecule, while other polyphenols surely intervene in A. citrodora anti-AFB1 activity.

Effect of Popcorn (Zea mays var. everta) Popping Mode (Microwave, Hot Oil, and Hot Air) on Fumonisins and Deoxynivalenol Contamination Levels.

Mycotoxins are secondary metabolites that are produced by molds during their development. According to fungal physiological particularities, mycotoxins can contaminate crops before harvest or during storage. Among toxins that represent a real public health issue, those produced by Fusarium genus in cereals before harvest are of great importance since they are the most frequent in European productions. Among them, deoxynivalenol (DON) and fumonisins (FUM) frequently contaminate maize. In recent years, numerous studies have investigated whether food processing techniques can be exploited to reduce the levels of these two mycotoxins, which would allow the identification and quantification of parameters affecting mycotoxin stability. The particularity of the popcorn process is that it associates heat treatment with a particular physical phenomenon (i.e., expansion). Three methods exist to implement the popcorn transformation process: hot air, hot oil, and microwaves, all of which are tested in this study. The results show that all popping modes significantly reduce FUM contents in both Mushroom and Butterfly types of popcorn. The mean initial contamination of 1351 µg/kg was reduced by 91% on average after popping. For DON, the reduction was less important despite a lower initial contamination than for FUM (560 µg/kg). Only the hot oil popping for the Mushroom type significantly reduced the contamination up to 78% compared to unpopped controls. Hot oil popping appears to provide the most important reduction for the two considered mycotoxins for both types of popcorn (-98% and -58% average reduction for FUM and DON, respectively).

Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark.

Antifungal and anti-aflatoxigenic properties of organs of Cannabis sativa L.: relation to phenolic content and antioxidant capacities.

Aflatoxin B1 is a carcinogenic mycotoxin that frequently contaminates crops worldwide. Current research indicates that the use of natural extracts to combat mycotoxin contamination may represent an eco-friendly, sustainable strategy to ensure food safety. Although Cannabis sativa L. has long been known for its psychoactive cannabinoids, it is also rich in many other bioactive molecules. This study examines extracts from various organs of Cannabis sativa L. to determine their ability to limit aflatoxin production and growth of Aspergillus flavus. The results indicate that flower extract is most effective for limiting the synthesis of aflatoxin B1, leading to an almost-complete inhibition of toxin production at a concentration of 0.225 mg dry matter per gram of culture medium. Since flower extract is rich in phenolic compounds, its total antioxidant ability and radical-scavenging capacity are determined. Compared with other anti-aflatoxigenic extracts, the anti-oxidative potential of Cannabis sativa L. flower extract appears moderate, suggesting that its anti-mycotoxin effect may be related to other bioactive compounds.

Aflatoxin Biosynthesis and Genetic Regulation: A Review.

The study of fungal species evolved radically with the development of molecular techniques and produced new evidence to understand specific fungal mechanisms such as the production of toxic secondary metabolites. Taking advantage of these technologies to improve food safety, the molecular study of toxinogenic species can help elucidate the mechanisms underlying toxin production and enable the development of new effective strategies to control fungal toxicity. Numerous studies have been made on genes involved in aflatoxin B1 (AFB1) production, one of the most hazardous carcinogenic toxins for humans and animals. The current review presents the roles of these different genes and their possible impact on AFB1 production. We focus on the toxinogenic strains Aspergillus flavus and A. parasiticus, primary contaminants and major producers of AFB1 in crops. However, genetic reports on A. nidulans are also included because of the capacity of this fungus to produce sterigmatocystin, the penultimate stable metabolite during AFB1 production. The aim of this review is to provide a general overview of the AFB1 enzymatic biosynthesis pathway and its link with the genes belonging to the AFB1 cluster. It also aims to illustrate the role of global environmental factors on aflatoxin production and the recent data that demonstrate an interconnection between genes regulated by these environmental signals and aflatoxin biosynthetic pathway.

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response.

Aspergillus flavus, a soil-borne pathogen, represents a danger for humans and animals since it produces the carcinogenic mycotoxin Aflatoxin B1 (AFB1). Approaches aiming the reduction of this fungal contaminant mainly involve chemicals that may also be toxic. Therefore, identification and characterization of natural anti-aflatoxigenic products represents a sustainable alternative strategy. Piperine, a major component of black and long peppers, has been previously demonstrated asan AFB1-inhibitor; nevertheless its mechanism of action was yet to be elucidated. The aim of the present study was to evaluate piperine's molecular mechanism of action in A. flavus with a special focus on oxidative stress response. For that, the entire AFB1 gene cluster as well asa targeted gene-network coding for fungal stress response factors and cellular receptors were analyzed. In addition to this, fungal enzymatic activities were also characterized. We demonstrated that piperine inhibits aflatoxin production and fungal growth in a dose-dependent manner. Analysis of the gene cluster demonstrated that almost all genes participating in aflatoxin's biosynthetic pathway were down regulated. Exposure to piperine also resulted in decreased transcript levels of the global regulator veA together with an over-expression of genes coding for several basic leucine zipper (bZIP) transcription factors such as atfA, atfB and ap-1 and genes belonging to superoxide dismutase and catalase's families. Furthermore, this gene response was accompanied by a significant enhancement of catalase enzymatic activity. In conclusion, these data demonstrated that piperine inhibits AFB1 production while positively modulating fungal antioxidant status in A. flavus.

Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach.

Produced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation.

New insights into the organ-specific adverse effects of fumonisin B1: comparison between lung and liver.

Fumonisin B1 (FB1) is a well-known inhibitor of de novo sphingolipid biosynthesis, due to its ability to inhibit ceramide synthases (CerS) activity. In mammals, this toxin triggers broad clinical symptoms with multi-organ dysfunction such as hepatotoxicity or pulmonary edema. The molecular mechanism of CerS inhibition by FB1 remains unknown. Due to the existence of six mammalian CerS isoforms with a tissue-specific expression pattern, we postulated that the organ-specific adverse effects of FB1 might be due to different CerS isoforms. The sphingolipid contents of lung and liver were compared in normal and FB1-exposed piglets (gavage with 1.5 mg FB1/kg body weight daily for 9 days). The effect of the toxin on each CerS was deduced from the analysis of its effects on individual ceramide (Cer) and sphingomyelin (SM) species. As expected, the total Cer content decreased by half in the lungs of FB1-exposed piglets, while in contrast, total Cer increased 3.5-fold in the livers of FB1-exposed animals. Our data also indicated that FB1 is more prone to bind to CerS4 and CerS2 to deplete lung and to enrich liver in d18:1/C20:0 and d18:1/C22:0 ceramides. It also interact with CerS1 to enrich liver in d18:1/C18:0 ceramides. Cer levels were counterbalanced by those of SM. In conclusion, these results demonstrate that the specificity of the effects of FB1 on tissues and organs is due to the effects of the toxin on CerS4, CerS2, and CerS1.

Toxicokinetics of fumonisin B1 in turkey poults and tissue persistence after exposure to a diet containing the maximum European tolerance for fumonisins in avian feeds.

The kinetic of fumonisin B1 (FB1) after a single IV and oral dose, and FB1 persistence in tissue were investigated in turkey poults by HPLC after purification of samples on columns. After IV administration (single-dose: 10mg FB1/kg bw), serum concentration-time curves were best described by a three-compartment open model. Elimination half-life and mean residence time of FB1 were 85 and 52min, respectively. After oral administration (single-dose: 100mg FB1/kg bw) bioavailability was 3.2%; elimination half-life and mean residence time were 214 and 408min, respectively. Clearance of FB1 was 7.6 and 7.5ml/min/kg for IV and oral administration, respectively. Twenty-four hours after the administration of FB1 by the intravenous route, liver and kidney contained the highest levels of FB1 in tissues, level in muscle was low or below the limit of detection (LD, 13microg/kg). The persistence of FB1 in tissue was also studied after administration for 9 weeks of a feed that contained 5, 10 and 20mg FB1+FB2/kg diet. Eight hours after the last intake of 20mg FB1+FB2/kg feed (maximum recommended concentration of fumonisins established by the EU for avian feed), hepatic and renal FB1 concentrations were 119 and 22microg/kg, level in muscles was below the LD.

Sex-related differences in the immune response of weanling piglets exposed to low doses of fumonisin extract.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides, a fungus that commonly contaminates maize. Sex-related effects of FB1 have been observed with respect to carcinogenicity in rodents, to performances in pigs and immunosuppression in mice. In the present study the sex-related effect of FB1 on the pig immune response was determined. Female and castrated male piglets received for 28 d either control feed or feed contaminated with 8 mg FB1/kg feed in the form of F. verticillioides culture material. At day 7 and day 21, animals were immunised subcutaneously with a Mycoplasma agalactiae vaccine. Ingestion of FB1-contaminated feed significantly decreased weight gain in males but had no effect in females. No sex-related difference was observed in biochemical parameters, but a higher level of creatinine was noted in toxin-treated animals. FB1 also altered the pig immune response in a sex-specific manner. In males, ingestion of FB1-contaminated feed significantly decreased specific antibody levels after vaccination as well as the mRNA expression level of IL-10. In females, the toxin has no effect on specific antibodies or on cytokine mRNA levels. The results of the present study indicate that FB1 is immunosuppressive in pigs. The magnitude of this FB1-induced immunosuppression is highly dependent on sex, with males being more susceptible than females.

Mycotoxin fumonisin B1 alters the cytokine profile and decreases the vaccinal antibody titer in pigs.

Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, may contaminate feed and food. In the present study, we investigated the effect of FB1 on the modulation of the cytokine profile and on the establishment of a vaccinal antibody response. In vitro investigations on pig peripheral blood mononuclear cells (PBMC) indicate that FB1 decreased interleukin-4 (IL-4) and increased interferon-gamma (IFN-gamma) synthesis at both the protein and mRNA levels. A short in vivo exposure (7 days) of weanling piglets to 1.5 mg/kg body weight of purified FB1 altered the cytokine balance in mesenteric lymph nodes and spleen similarly to the in vitro PBMC results. We also investigated the effect of FB1 on the antibody response during a vaccination process. A prolonged in vivo exposure (28 days) of weanling piglets to feed contaminated with 8 mg FB1/kg significantly decreased the expression of IL-4 mRNA by porcine whole blood cells and diminished the specific antibody titer after vaccination against Mycoplasma agalactiae. By contrast, ingestion of the contaminated feed had no effect on the serum concentration of the immunoglobulin subset (IgG, IgA, and IgM). Taken together, our data suggest that FB1 alters the cytokine profile and decreases the specific antibody response built during a vaccination protocol. These results may have implications for humans or animals eating contaminated food or feed.