Secondary Metabolites Of Filamentous Fungi Research Articles

Exploring the Subcellular Localization of Monascus Pigments Biosynthases: Preliminary Unraveling of the Compartmentalization Mechanism

Monascus pigments (MPs), a class of secondary metabolites produced by Monascus spp., can be classified into yellow, orange, and red MPs according to their differences in the wavelength of the maximum absorption. However, the biosynthetic sequence and cellular biosynthesis mechanism of different MPs components are still not yet completely clear in Monascus spp. In this study, the subcellular localization of five MPs synthases was investigated using fluorescent protein fusion expression. The results revealed that the proteins encoded by the MPs biosynthetic gene cluster were compartmentalized in various subcellular locations, including the mitochondrial polyketide synthase MrPigA, cytosolic enzymes consisting of the ketoreductase MrPigC, the oxidoreductase MrPigE, and the monooxygenase MrPigN, and the cell-wall-bound oxidoreductase MrPigF. Moreover, the correct localization of MrPigF to the cell wall was crucial for the synthesis of orange MPs. Lastly, we discussed the compartmentalized biosynthetic pathway of MPs. This study will not only be helpful in clarifying the biosynthetic sequence and biosynthesis mechanism of different MPs but also provides new insights into the cellular biosynthesis of secondary metabolites in filamentous fungi.

Storage mycotoxin producing fungi in Hungarian sorghum (Sorghum bicolor L. Moench) samples—molecular approach of Fusarium spp.

AbstractThe last few years have been a huge challenge for every farmer in Europe and Hungary because of the increase in hot days and the decrease in precipitation. These facts induced the farmer’s interest in sorghum because it has better stress tolerance than many other cereales, but little is known about mycotoxin-producing fungi, which can infect this crop. Mycotoxins are secondary metabolites of filamentous fungi, and they are not only phytotoxic but also harmful to humans and animals. This study aimed to determine the internal infection caused by Alternaria spp., Fusarium spp., and Aspergillus spp., which are known as mycotoxin-producing fungi in food raw material. In our study in the case of sorghum, the presence of various mycotoxin-producing fungi was detected on Fusarium selective media (Nash and Snyder media), and the results indicate that these genera are present in sorghum grains as potential mycotoxin producers. To determine which Fusarium species is occurring in our sorghum grain samples, a molecular genetic study was performed on isolated fungi using the TEF region primer pairs to identify the occurring Fusarium species. We mainly identified Fusarium proliferatum in our conventional sorghum samples. The results show that the mentioned mycotoxin-producing fungi are in the sorghum grains and may pose a risk to the safety of feed and food because they may produce mycotoxins in the field or while being stored.

Competitive, Inhibitory and Mutually Existing Interaction Mapping of Secondary Metabolites in Filamentous Fungi

Natural products are compounds that are produced by living organisms, such as bacteria and fungi. These compounds are essential for advancements in the medical field, such as the discovery of new antibiotics. When living organisms like bacteria and fungi interact with one another, they can be forced to compete for resources to survive (antagonistic) or cooperate (synergistic). It is through these relationships that new metabolites can be produced and subsequently observed. The current research on co-culturing between bacterial and fungal species is limited, thus there are many possible undiscovered metabolites. Therefore, co-culturing of this nature can be analyzed using mass spectrometry for new natural product discovery. While typical methods for microorganism culturing can be applied to co-culturing, alternatively forcing species to interact in an artificially constructed growth chamber can offer interesting insights and changes to the metabolite profile. Herein, a 3D-printed artificial chamber was created to observe the growth and interaction of each species in diverse locations, thus allowing a spatiotemporal map of natural product growth to be generated through spectral analysis. In this work, spectral analysis is conducted using the Liquid Micro-Junction Surface Sampling Probe (LMJ-SSP), which allows for non-destructive sampling of the microorganism interactions. As a result, sampling can be conducted repeatedly at different times in growth development, as well as at diverse locations throughout the chamber. Ultimately, the further exploitation of both the competitive and synergistic relationship of microorganism growth within this chamber will offer novel insights into natural product discovery.

Interaction of calcium responsive proteins and transcriptional factors with the PHO regulon in yeasts and fungi.

Phosphate and calcium ions are nutrients that play key roles in growth, differentiation and the production of bioactive secondary metabolites in filamentous fungi. Phosphate concentration regulates the biosynthesis of hundreds of fungal metabolites. The central mechanisms of phosphate transport and regulation, mediated by the master Pho4 transcriptional factor are known, but many aspects of the control of gene expression need further research. High ATP concentration in the cells leads to inositol pyrophosphate molecules formation, such as IP3 and IP7, that act as phosphorylation status reporters. Calcium ions are intracellular messengers in eukaryotic organisms and calcium homeostasis follows elaborated patterns in response to different nutritional and environmental factors, including cross-talking with phosphate concentrations. A large part of the intracellular calcium is stored in vacuoles and other organelles forming complexes with polyphosphate. The free cytosolic calcium concentration is maintained by transport from the external medium or by release from the store organelles through calcium permeable transient receptor potential (TRP) ion channels. Calcium ions, particularly the free cytosolic calcium levels, control the biosynthesis of fungal metabolites by two mechanisms, 1) direct interaction of calcium-bound calmodulin with antibiotic synthesizing enzymes, and 2) by the calmodulin-calcineurin signaling cascade. Control of very different secondary metabolites, including pathogenicity determinants, are mediated by calcium through the Crz1 factor. Several interactions between calcium homeostasis and phosphate have been demonstrated in the last decade: 1) The inositol pyrophosphate IP3 triggers the release of calcium ions from internal stores into the cytosol, 2) Expression of the high affinity phosphate transporter Pho89, a Na+/phosphate symporter, is controlled by Crz1. Also, mutants defective in the calcium permeable TRPCa7-like of Saccharomyces cerevisiae shown impaired expression of Pho89. This information suggests that CrzA and Pho89 play key roles in the interaction of phosphate and calcium regulatory pathways, 3) Finally, acidocalcisomes organelles have been found in mycorrhiza and in some melanin producing fungi that show similar characteristics as protozoa calcisomes. In these organelles there is a close interaction between orthophosphate, pyrophosphate and polyphosphate and calcium ions that are absorbed in the polyanionic polyphosphate matrix. These advances open new perspectives for the control of fungal metabolism.

Overexpression of llm1 Affects the Synthesis of Secondary Metabolites of Aspergillus cristatus.

Putative methyltransferases are thought to be involved in the regulation of secondary metabolites in filamentous fungi. Here, we report the effects of overexpression of a predicted LaeA-like methyltransferase gene llm1 on the synthesis of secondary metabolites in Aspergillus cristatus. Our results revealed that overexpression of the gene llm1 in A. cristatus significantly hindered the production of conidia and enhanced sexual development, and reduced oxidative tolerance to hydrogen peroxide. Compared with the wild-type, the metabolic profile of the overexpression transformant was distinct, and the contents of multiple secondary metabolites were markedly increased, mainly including terpenoids and flavonoids, such as (S)-olEuropeic acid, gibberellin A62, gibberellin A95, ovalitenone, PD 98059, and 1-isomangostin. A total of 600 significantly differentially expressed genes (DEGs) were identified utilizing transcriptome sequencing, and the DEGs were predominantly enriched in transmembrane transport and secondary metabolism-related biological processes. In summary, the strategy of overexpressing global secondary metabolite regulators successfully activated the expression of secondary metabolite gene clusters, and the numerous secondary metabolites were greatly strengthened in A. cristatus. This study provides new insights into the in-depth exploitation and utilization of novel secondary metabolites of A. cristatus.

The BcLAE1 is involved in the regulation of ABA biosynthesis in Botrytis cinerea TB-31.

Abscisic acid (ABA), as a classic plant hormone, is a key factor in balancing the metabolism of endogenous plant hormones, and plays an important role in regulating the activation of mammalian innate immune cells and glucose homeostasis. Currently, Botrytis cinerea has been used for fermentation to produce ABA. However, the mechanism of the regulation of ABA biosynthesis in B. cinerea is still not fully understood. The putative methyltransferase LaeA/LAE1 is a global regulator involved in the biosynthesis of a variety of secondary metabolites in filamentous fungi. In this study, we demonstrated that BcLAE1 plays an important role in the regulation of ABA biosynthesis in B. cinerea TB-31 by knockout experiment. The deletion of Bclae1 caused a 95% reduction in ABA yields, accompanied by a decrease of the transcriptional level of the ABA synthesis gene cluster Bcaba1-4. Further RNA-seq analysis indicated that deletion of Bclae1 also affected the expression level of key enzymes of BOA and BOT in secondary metabolism, and accompanied by clustering regulatory features. Meanwhile, we found that BcLAE1 is involved in epigenetic regulation as a methyltransferase, with enhanced H3K9me3 modification and attenuated H3K4me2 modification in ΔBclae1 mutant, and this may be a strategy for BcLAE1 to regulate ABA synthesis.

Risk Assessment of Mycotoxins Intake through the Consumption of Maize, Peanuts, Rice and Cassava in Côte D’Ivoire

Mycotoxins are secondary metabolites of filamentous fungi that colonize a wide range of crops, including cereals and oilseeds, both in the field and after harvest, especially during storage. Several studies carried out on the occurrence of mycotoxins in crops and their derived products such as maize, peanuts, rice and attieke (cassava product), reported substantial levels of Aflatoxins (AFs), Fumonisins (FBs), Ochratoxin A (OTA) and Zearalenone (ZEA). The aim of the present study was to analyze findings available on Aflatoxins, Ochratoxin A and Fusarium toxins occurrence in maize, peanuts, rice and attieke in order to assess the exposure level and cancers risk in the Ivorian population. References and publications related to OTA in C?te d’Ivoire were searched and selected. Medline/PubMed, Elsevier Bibliographic Databases, BioInfoBank Library, DOAJ (Directory of Open Acess Journal), Australian Journal of Basic and Applied Sciences and https://medwelljournals.com/home.php were used as databases. Data available showed Estimated Daily Intake (EDI) of AFs and OTA were above of their Tolerable Daily Intake (TDI) as recommended by the Joint FAO/WHO Experts Committee on Food Additives through rice, maize, peanut and attieke consumption in C?te d’Ivoire. In addition, there is a veritable incidence of cancers with the abundant and frequent consumption of foods maize, rice, peanuts and attieke. However, maize and rice seemed to be sources of FBs

Polyamines Upregulate Cephalosporin C Production and Expression of β-Lactam Biosynthetic Genes in High-Yielding Acremonium chrysogenum Strain.

The high-yielding production of pharmaceutically significant secondary metabolites in filamentous fungi is obtained by random mutagenesis; such changes may be associated with shifts in the metabolism of polyamines. We have previously shown that, in the Acremonium chrysogenum cephalosporin C high-yielding strain (HY), the content of endogenous polyamines increased by four- to five-fold. Other studies have shown that the addition of exogenous polyamines can increase the production of target secondary metabolites in highly active fungal producers, in particular, increase the biosynthesis of β-lactams in the Penicillium chrysogenum Wis 54–1255 strain, an improved producer of penicillin G. In the current study, we demonstrate that the introduction of exogenous polyamines, such as spermidine or 1,3-diaminopropane, to A. chrysogenum wild-type (WT) and HY strains, leads to an increase in colony germination and morphological changes in a complete agar medium. The addition of 5 mM polyamines during fermentation increases the production of cephalosporin C in the A. chrysogenum HY strain by 15–20% and upregulates genes belonging to the beta-lactam biosynthetic cluster. The data obtained indicate the intersection of the metabolisms of polyamines and beta-lactams in A. chrysogenum and are important for the construction of improved producers of secondary metabolites in filamentous fungi.

Critical Assessment of Mycotoxins in Beverages and Their Control Measures.

Mycotoxins are secondary metabolites of filamentous fungi that contaminate food products such as fruits, vegetables, cereals, beverages, and other agricultural commodities. Their occurrence in the food chain, especially in beverages, can pose a serious risk to human health, due to their toxicity, even at low concentrations. Mycotoxins, such as aflatoxins (AFs), ochratoxin A (OTA), patulin (PAT), fumonisins (FBs), trichothecenes (TCs), zearalenone (ZEN), and the alternaria toxins including alternariol, altenuene, and alternariol methyl ether have largely been identified in fruits and their derived products, such as beverages and drinks. The presence of mycotoxins in beverages is of high concern in some cases due to their levels being higher than the limits set by regulations. This review aims to summarize the toxicity of the major mycotoxins that occur in beverages, the methods available for their detection and quantification, and the strategies for their control. In addition, some novel techniques for controlling mycotoxins in the postharvest stage are highlighted.

Recent Advances in Mycotoxin Analysis and Detection of Mycotoxigenic Fungi in Grapes and Derived Products

Mycotoxins are secondary metabolites of filamentous fungi that can cause toxic effects in human and animal health. Most of the filamentous fungi that produce these mycotoxins belong to four genera, namely, Aspergillus, Penicillium, Fusarium, and Alternaria. Mycotoxigenic fungi, along with mycotoxins, create a constant and serious economic threat for agriculture in many terms, counting product losses due to crop contamination and food spoilage, as well malnutrition when considering nutritional quality degradation. Given the importance of robust and precise diagnostics of mycotoxins and the related producing fungi in the grape food chain, one of the most important agricultural sectors worldwide, the present review initially delivers a comprehensive presentation of mycotoxin reports on grape and derived products, including a wide range of commodities such as fresh grapes, raisins, wine, juices, and other processed products. Next, based on worldwide regulations’ requirements for mycotoxins, and referring to the relative literature, this work presents methodological approaches for mycotoxin determination, and stresses major methods for the detection of fungal species responsible for mycotoxin production. The principle of function and basic technical background on the available analytical and molecular biology techniques developed—including chromatography, mass spectrometry, immunochemical-based assays, biosensors, and molecular assays—is briefly given, and references for their application to grape and derived product testing are highlighted.

Applications of CRISPR/Cas9 in the Synthesis of Secondary Metabolites in Filamentous Fungi.

Filamentous fungi possess the capacity to produce a wide array of secondary metabolites with diverse biological activities and structures, such as lovastatin and swainsonine. With the advent of the post-genomic era, increasing amounts of cryptic or uncharacterized secondary metabolite biosynthetic gene clusters are continually being discovered. However, owing to the longstanding lack of versatile, comparatively simple, and highly efficient genetic manipulation techniques, the broader exploration of industrially important secondary metabolites has been hampered thus far. With the emergence of CRISPR/Cas9-based genome editing technology, this dilemma may be alleviated, as this advanced technique has revolutionized genetic research and enabled the exploitation and discovery of new bioactive compounds from filamentous fungi. In this review, we introduce the CRISPR/Cas9 system in detail and summarize the latest applications of CRISPR/Cas9-mediated genome editing in filamentous fungi. We also briefly introduce the specific applications of the CRISPR/Cas9 system and CRISPRa in the improvement of secondary metabolite contents and discovery of novel biologically active compounds in filamentous fungi, with specific examples noted. Additionally, we highlight and discuss some of the challenges and deficiencies of using the CRISPR/Cas9-based genome editing technology in research on the biosynthesis of secondary metabolites as well as future application of CRISPR/Cas9 strategy in filamentous fungi are highlighted and discussed.

Zearalenone and Its Metabolites-General Overview, Occurrence, and Toxicity.

Mycotoxins are secondary metabolites of filamentous fungi and represent one of the most common groups of food contaminants with low molecular weight. These toxins are considered common and can affect the food chain at various stages of production, harvesting, storage and processing. Zearalenone is one of over 400 detected mycotoxins and produced by fungi of the genus Fusarium; it mainly has estrogenic effects on various organisms. Contaminated products can lead to huge economic losses and pose risks to animals and humans. In this review, we systemize information on zearalenone and its major metabolites.

High-Yielding Lovastatin Producer Aspergillus terreus Shows Increased Resistance to Inhibitors of Polyamine Biosynthesis

The biosynthesis of pharmaceutically significant secondary metabolites in filamentous fungi is a multistep process that depends on a wide range of various factors, one of which is the intracellular content of polyamines. We have previously shown that in Aspergillus terreus lovastatin high-yielding strain (HY) exogenous introduction of polyamines during fermentation can lead to an increase in the production of lovastatin by 20–45%. However, the molecular mechanisms of this phenomenon have not been elucidated. In this regard, we carried out an inhibitory analysis at the key stage of polyamine biosynthesis, the conversion of L-ornithine to putrescine by the enzyme ornithine decarboxylase (ODC). A. terreus HY strain showed upregulation of genes for biosynthesis of polyamines, 3–10-fold, and increased resistance compared to the original wild-type strain upon inhibition of ODC on synthetic medium with 5 mM α-difluoromethylornithine (DFMO), by 20–25%, and 5 mM 1-aminooxy-3-aminopropane (APA), by 40–45%. The data obtained indicate changes in the metabolism of polyamines in A. terreus HY strain. The observed phenomenon may have a universal character among fungal producers of secondary metabolites improved by classical methods, since previously the increased resistance to ODC inhibitors was also shown for Acremonium chrysogenum, a high-yielding producer of cephalosporin C.

Mycotoxins in food and the risk to consumer health

Mycotoxins are secondary metabolites of filamentous fungi mainly produced by Fusarium, Alternaria, Aspergillus and Penicillium species. Presence of mycotoxins in food and feed may have numerous health consequences, for both human and animals. Furthermore, occurrence of fungi and mycotoxins in food and feed can cause great economic losses. Therefore, in the recent decades mycotoxins are recognized as significant worldwide concern. Although several hundred different mycotoxins are known, obligatory control as well as maximum levels are defined only for the following mycotoxins: aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, fumonisins and patulin. However, in the recent years, scientific attention is increasingly focused on many new mycotoxins and their involvement in different adverse effects on human and animals health. Furthermore, climate changes are recognized as important factors that seriously affect recent increases in fungi and mycotoxins occurrence worldwide. Based on everything stated above it could be noticed that mycotoxins, as one of the most toxic and the most frequent chemical contaminants of food chain, represent serious concerns and a growing global problem. However, due to non-implementation of continuous control and permanent monitoring of mycotoxins, there is still lack of data related to the occurrence of mycotoxins in the Republic of Srpska. There is a need for Republic of Srpska to enhance the control strategy of mycotoxins which would begreatly contribute to the increase of available data and further improvement of food and feed quality and safety, as well as decrease of economic losses. The aim of this chapter is to present an overview of the most significant and most recent researches related to the occurrence of mycotoxins in food.

The Putative Methyltransferase TlLAE1 Is Involved in the Regulation of Peptaibols Production in the Biocontrol Fungus Trichoderma longibrachiatum SMF2.

The biocontrol fungus Trichoderma longibrachiatum SMF2 secretes a large quantity of peptaibols that have been shown to have a range of biological activities and therefore great application values. However, the mechanism of the regulatory expression of peptaibols is still unclear. The putative methyltransferase LaeA/LAE1 is a global regulator involved in the biosynthesis of some secondary metabolites in filamentous fungi. In this study, we demonstrated that the ortholog of LaeA/LAE1 in the biocontrol fungus T. longibrachiatum SMF2, TlLAE1, plays an important role in the regulation of peptaibols production. Deletion of Tllae1 resulted in a slight negative impact on mycelial growth, and a significant defect in conidial production. Deletion of Tllae1 also compromised the production of peptaibols to a large degree. Further analyses indicated that this defect occurred at the transcriptional level of the two synthetases-encoding genes, tlx1 and tlx2, which are responsible for peptaibols production. By contrast, constitutive expression of Tllae1 in T. longibrachiatum SMF2 led to 2-fold increased peptaibols production, suggesting that this is a strategy to improve peptaibols production in Trichoderma fungi. These results demonstrate the important role of LAE1 in the regulation of peptaibols production in T. longibrachiatum SMF2.

Restricting mycotoxins without killing the producers: a new paradigm in nano-fungal interactions.

Over the past several years, numerousstudies havedemonstrated the feasibility of using engineered nanoparticles as antifungals, especially against those fungal pathogens that produce mycotoxins and infect plants, animals, and humans. The high dosage of nanoparticles has been a concern in such antifungal applications due to the potential toxicological and ecotoxicological impacts. To address such concerns, we have recently introduced the idea of inhibitingmycotoxin biosynthesis using low doses ofengineered nanoparticles. At such low doses these particles are minimally toxic to humans and the environment. From our studies we realize that for the effective use of nanotechnology to intervene in the biology of fungal pathogens and for an accurate evaluation of the impacts of the increasingly growing nanomaterials in the environment on fungi and their interacting biotic partners, there is a pressing need for a rigorous understanding of nano-fungal interactions, which is currently far from complete. In this minireview, we build on the available evidence from nano-bio interaction research and our recent interaction studies with Aspergillus cells and engineered silver nanoparticles to introduce a potentialtheoretical model for nano-fungal interactions. The aim of the proposed model is to provide an initial insight on how nanoparticle uptake and their transformation inside fungal cells, possibly influence the production of mycotoxins and other secondary metabolites of filamentous fungi .

Comparative Transcriptome Analysis Shows Conserved Metabolic Regulation during Production of Secondary Metabolites in Filamentous Fungi.

Filamentous fungi possess great potential as sources of medicinal bioactive compounds, such as antibiotics, but efficient production is hampered by a limited understanding of how their metabolism is regulated. We investigated the metabolism of six secondary metabolite-producing fungi of the Penicillium genus during nutrient depletion in the stationary phase of batch fermentations and assessed conserved metabolic responses across species using genome-wide transcriptional profiling. A coexpression analysis revealed that expression of biosynthetic genes correlates with expression of genes associated with pathways responsible for the generation of precursor metabolites for secondary metabolism. Our results highlight the main metabolic routes for the supply of precursors for secondary metabolism and suggest that the regulation of fungal metabolism is tailored to meet the demands for secondary metabolite production. These findings can aid in identifying fungal species that are optimized for the production of specific secondary metabolites and in designing metabolic engineering strategies to develop high-yielding fungal cell factories for production of secondary metabolites. IMPORTANCE Secondary metabolites are a major source of pharmaceuticals, especially antibiotics. However, the development of efficient processes of production of secondary metabolites has proved troublesome due to a limited understanding of the metabolic regulations governing secondary metabolism. By analyzing the conservation in gene expression across secondary metabolite-producing fungal species, we identified a metabolic signature that links primary and secondary metabolism and that demonstrates that fungal metabolism is tailored for the efficient production of secondary metabolites. The insight that we provide can be used to develop high-yielding fungal cell factories that are optimized for the production of specific secondary metabolites of pharmaceutical interest.

A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici.

Secondary metabolite (SM) production and development are correlated processes in fungi that are often coordinated by pleiotropic regulators. The eukaryotic regulators are critical players in mediating SM production related to fungal development, yet little data are available to support this hypothesis. In this study, a global regulator, RsdA (regulation of secondary metabolism and development), was identified through genome-wide analysis and deletion of the regulator gene in the endophytic fungus Pestalotiopsis fici. Here, we established that RsdA regulation of SMs is accompanied by the repression of asexual development. Deletion of rsdA significantly reduces not only asexual development, resulting in low sporulation and abnormal conidia, but also the major SM production, while remarkably increasing the melanin production. Overproduction of melanin leads to the formation of unusual, heavily pigmented hyphae. Transcriptome analysis data provide the evidence that RsdA globally regulates genes involved in secondary metabolism and asexual development. Double deletion of rsdA and the melanin polyketide synthase gene PfmaE confirm that RsdA regulation of asexual development is independent of the melanin biosynthetic pathway. Finally, our results demonstrate that RsdA can be used for the discovery of secondary metabolites in filamentous fungi.

An in vitro investigation on the cytotoxic and nuclear receptor transcriptional activity of the mycotoxins fumonisin B1 and beauvericin

Fumonisin B1 (FB1) and beauvericin (BEA) are secondary metabolites of filamentous fungi, which under appropriate temperature and humidity conditions may develop on various foods and feeds. To date few studies have been performed to evaluate the toxicological and endocrine disrupting effects of FB1 and BEA. The present study makes use of various in vitro bioassays including; oestrogen, androgen, progestagen and glucocorticoid reporter gene assays (RGAs) for the study of nuclear receptor transcriptional activity, the thiazolyl blue tetrazolium bromide (MTT) assay to monitor cytotoxicity and high content analysis (HCA) for the detection of pre-lethal toxicity in the RGA and Caco-2 human colon adenocarcinoma cells. At the receptor level, 0.001-10μM BEA or FB1 did not induce any agonist responses in the RGAs. However at non-cytotoxic concentrations, an antagonistic effect was exhibited by FB1 on the androgen nuclear receptor transcriptional activity at 10μM and BEA on the progestagen and glucocorticoid receptors at 1μM. MTT analysis showed no decrease in cell viability at any concentration of FB1, whereas BEA showed a significant decrease in viability at 10μM. HCA analysis confirmed that the reduction in the progestagen receptor transcriptional activity at 1μM BEA was not due to pre-lethal toxicity. In addition, BEA (10μM) induced significant toxicity in both the TM-Luc (progestagen responsive) and Caco-2 cells.

Interaction effects of enniatin B, deoxinivalenol and alternariol in Caco-2 cells.

Enniatin B (ENN B), deoxinivalenol (DON) and alternariol (AOH) are secondary metabolites of filamentous fungi. These mycotoxins are contaminants of vegetables and cereals. They are cytotoxic and their effects are enhanced by their mixtures. The objectives of this study were to compare the cytotoxicity of ENN B, DON and AOH alone or in combination in human adenocarcinoma (Caco-2) cells and to evaluate the type of interactions of mycotoxin mixtures by the isobologram analysis. Cells were treated with concentrations ranging from 1.85 to 90μM (AOH) and from 0.312 to 10μM (for ENN B and DON), individually and in combination of two and three mycotoxins (from 1.85 to 30μM for AOH and from 0.312 to 5μM for ENN B and DON). The relation ratios between the mixtures DON+ENN B was 1:1; AOH+DON and ENN B+AOH was 1:6, and for the tertiary combination DON, ENN B and AOH 1:1:6. The IC50 value of ENN B and DON were 3.87 and 5.54μM, respectively. No IC50 values were obtained for the AOH at any time tested in Caco-2 cells. With the isobologram the type of interaction between mycotoxin was evaluated. Synergistic, antagonistic and addictive effect was observed for the combination studied depending on the concentration affected. Mycotoxins combinations reduce cellular viability in the following increasing order: (DON+ENN B)>(ENN B+AOH)>(DON+AOH)>(DON+AOH+ENN B).