Cyclodextrin-Based Displacement Strategy of Sterigmatocystin from Serum Albumin as a Novel Approach for Acute Poisoning Detoxification.

This study demonstrates that sterigmatocystin (STC) interacts non-covalently with various cyclodextrins (CDs), showing the highest binding affinity for sugammadex (a γ-CD derivative) and γ-CD, and an almost order of magnitude lower affinity for ß-CD. This difference in affinity was studied using molecular modelling and fluorescence spectroscopy, which demonstrated a better insertion of STC into larger CDs. In parallel, we showed that STC binds to human serum albumin (HSA) (a blood protein known for its role as a transporter of small molecules) with an almost two order of magnitude lower affinity compared to sugammadex and γ-CD. Competitive fluorescence experiments clearly demonstrated an efficient displacement of STC from the STC-HSA complex by cyclodextrins. These results are a proof-of-concept that CDs can be used to complex STC and related mycotoxins. Similarly, as sugammadex extracts neuromuscular relaxants (e.g., rocuronium and vecuronium) from blood and blocks their bioactivity, it could also be used as first aid upon acute intoxication to encapsulate a larger part of the STC mycotoxin from serum albumin.

Fungi and their secondary metabolites in water-damaged indoors after a major flood event in eastern Croatia.

In winter and summer of 2016 and 2017, airborne fungi and house dust were collected in indoors of the village Gunja, which had been flooded, and the control village Gornji Stupnik (Croatia) in order to explore variations of fungal indoor levels, particularly Aspergilli section Nidulantes series Versicolores, as well as fungal metabolites in dust. Levels of airborne Aspergilli (Versicolores) were three times as high in winter and summer in Gunja than in the control village, while dustborne isolates were equally present in both locations. Sequencing of the calmodulin gene region revealed that among Aspergilli (Versicolores), A. jensenii and A. creber were dominant and together with A. puulaauensis, A. tennesseensis and A. venenatus produced sterigmatocystin and 5-methoxysterigmatocystin (HPLC coupled with mass spectrometry); A. amoenus, A. fructus, A. griseoaurantiacus, A. pepii, and A. protuberus produced sterigmatocystin but not 5-methoxysterigmatocystin; A. sydowii did not produce any of these toxins. A total of 75 metabolites related to Penicillium (29), Aspergillus (22), Fusarium (10), Alternaria (5), Stachybotrys (2), and other fungi (7) were detected in dust by liquid chromatography-tandem mass spectrometry. The majority of metabolites including sterigmatocystin and 5-methoxysterigmatocystin exhibited a higher prevalence in winter in Gunja.

Unique Aggregation of Sterigmatocystin in Water Yields Strong and Specific Circular Dichroism Response Allowing Highly Sensitive and Selective Monitoring of Bio-Relevant Interactions.

We demonstrated the hitherto unknown property of the mycotoxin sterigmatocystin (STC) to provide homogeneous solutions in aqueous medium by forming a unique aggregate type (not formed by analogous aflatoxins), characterized by exceptionally strong circular dichroism (CD) bands in the 300-400 nm range. Results showed that these CD bands do not originate from intrinsic STC chirality but are a specific property of a peculiar aggregation process similar to psi-DNA CD response. Transmission electron microscopy (TEM) experiments revealed a fine fiber network resembling a supramolecular gel structure with helical fibers. Thermodynamic studies of aggregates by differential scanning calorimetry (DSC) revealed high reversibility of the dominant aggregation process. We demonstrated that the novel STC psi-CD band at 345 nm could be applied at biorelevant conditions (100 nanomolar concentration) and even in marine-salt content conditions for specific and quantitative monitoring of STC. Also, we showed that STC strongly non-covalently interacts with ds-DNA with likely toxic effects, thus contrary to the previous belief requiring prior enzyme epoxidation.

Combined genotyping strategy reveals structural differences between Aspergillus flavus lineages from different habitats impacting human health.

Aspergillus flavus is a filamentous fungus which is widespread on agricultural products and also able to cause various human diseases. This species is frequently isolated from indoor air as well, furthermore, it is known as a common causal agent of keratomycosis, particularly in subtropical and tropical areas. It is also able to produce aflatoxins, one of the most carcinogenic mycotoxins which are harmful to animals and humans. In this study, 59 A. flavus isolates from four different habitats and 1 A. minisclerotigenes isolate were investigated. The isolates were identified and confirmed at the species level by the sequence analysis of a part of their calmodulin gene. Applying a combined analysis of UP-PCR, microsatellite, and calmodulin sequence data, the four group of isolates formed separate clusters on the phylogenetic tree. Examining the distribution of mating type genes MAT1-1 and MAT1-2, a ratio of approximately 3:1 was determined, and no correlation was found between the carried mating type gene and the aflatoxin production capability. HPLC analysis revealed that none of the examined isolates collected from indoor air or maize in Central Europe were able to produce aflatoxins, while about half of the isolates from India produced these mycotoxins under the test conditions.

Cytotoxic and genotoxic potencies of single and combined spore extracts of airborne OTA-producing and OTA-non-producing Aspergilli in Human lung A549 cells.

Aspergillus sclerotiorum (AS) is a well-known producer of ochratoxin A (OTA) while Aspergillus pseudoglaucus (AP) produces a wide range of extrolites with poorly investigated toxicity. These species are frequently co-occur in grain mill aeromycota. The aim of this study was to determine OTA levels in spore extracts using HPLC and immunoaffinity columns, and to examine the cytotoxicity of pure OTA, OTA-positive (AS-OTA(+)) and OTA-negative (AS-OTA(-)) spore extracts, as well as of AP spore extract, on human lung adenocarcinoma cells A549, individually and in combination, using a colorimetric MTT test (540nm). To establish which type of cell death predominated after treatments, a quantitative fluorescent assay with ethidium bromide and acridine orange was used, and the level of primary DNA damage in A549 cells was evaluated using the alkaline comet assay. OTA was detected in spore extracts (0.3-28µg/mL) of 3/6 of the AS strains, while none of the tested AP strains were able to produce OTA. Taking into account the maximum detected concentration of OTA in the spores, the daily intake of OTA by inhalation was calculated to be 1ng/kg body weight (b.w.), which is below the tolerable daily intake for OTA (17ng/kg b.w.). Using the MTT test, the following IC50 values were obtained: single OTA (53µg/mL); AS-OTA(+) (mass concentration 934µg/mL corresponds to 10.5µg/mL of OTA in spore extract); and 2126µg/mL for AP. The highest applied concentration of AS-OTA(-) spore extract (4940µg/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Single OTA and AS-OTA(+) and combinations (AP+AS-OTA(+) and AP+AS-OTA(-)) in subtoxic concentrations provoked significant primary DNA damage, apoptosis, and to a lesser extent, necrosis in A549 cells. Mixture of AP+AS-OTA(+) and AP+AS-OTA(-) in subtoxic concentrations showed dominant additive interactions. Despite the low calculated daily intake of OTA by inhalation, our results suggest that chronic exposure to high levels of OTA-producing airborne fungi in combination with other more or less toxic moulds pose a significant threat to human health due to their possible additive and/or synergistic interactions.

Identification of Aspergillus species in Central Europe able to produce G-type aflatoxins.

The occurrence of potential aflatoxin producing fungi was examined in various agricultural products and indoor air in Central European countries including Hungary, Serbia and Croatia. For species identification, both morphological and sequence based methods were applied. Aspergillus flavus was detected in several samples including maize, cheese, nuts, spices and indoor air, and several isolates were able to produce aflatoxins. Besides, three other species of Aspergillus section Flavi, A. nomius, A. pseudonomius and A. parasiticus were also isolated from cheese, maize and indoor air, respectively. This is the first report on the occurrence of A. nomius and A. pseudonomius in Central Europe. All A. nomius, A. pseudonomius and A. parasiticus isolates were able to produce aflatoxins B1, B2, G1 and G2. The A. nomius isolate came from cheese produced very high amounts of aflatoxins (above 1 mg ml⁻¹). All A. nomius, A. pseudonomius and A. parasiticus isolates produced much higher amounts of aflatoxin G1 then aflatoxin B1. Further studies are in progress to examine the occurrence of producers of these highly carcinogenic mycotoxins in agricultural products and indoor air in Central Europe.

Citrinin Provoke DNA Damage and Cell-Cycle Arrest Related to Chk2 and FANCD2 Checkpoint Proteins in Hepatocellular and Adenocarcinoma Cell Lines.

Citrinin (CIT), a polyketide mycotoxin produced by Penicillium, Aspergillus, and Monascus species, is a contaminant that has been found in various food commodities and was also detected in house dust. Several studies showed that CIT can impair the kidney, liver, heart, immune, and reproductive systems in animals by mechanisms so far not completely elucidated. In this study, we investigated the CIT mode of action on two human tumor cell lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma). Cytotoxic concentrations were determined using an MTT proliferation assay. The genotoxic effect of sub-IC50 concentrations was investigated using the alkaline comet assay and the impact on the cell cycle using flow cytometry. Additionally, the CIT effect on the total amount and phosphorylation of two cell-cycle-checkpoint proteins, the serine/threonine kinase Chk2 and Fanconi anemia (FA) group D2 (FANCD2), was determined by the cell-based ELISA. The data were analyzed using GraphPad Prism statistical software. The CIT IC50 for HepG2 was 107.3 µM, and for A549, it was >250 µM. The results showed that sensitivity to CIT is cell-type dependent and that CIT in sub-IC50 and near IC50 induces significant DNA damage and cell-cycle arrest in the G2/M phase, which is related to the increase in total and phosphorylated Chk2 and FANCD2 checkpoint proteins in HepG2 and A549 cells.

Respiratory health and breath condensate acidity in sawmill workers.

PURPOSE: The aim of the study was to evaluate exhaled breath condensate acidity (EBC pH) as a biomarker of airway response to occupational respiratory hazards present in sawmill. METHODS: Sixty-one sawmill workers in total (26 from Sawmill 1 and 35 from Sawmill 2) provided EBC samples at the beginning and at the end of the working week. Respiratory symptoms, lung function, bronchodilator test and atopy status were assessed. Occupational environment was checked for the levels of respiratory hazards. RESULTS: Airborne dust concentrations were below threshold limit value. Endotoxin in Sawmill 1 and Sawmill 2, and moulds in Sawmill 1 were at the levels able to induce inflammatory response in the airways. Mould levels were 2.5 times higher in Sawmill 1 than in Sawmill 2. Compared to Sawmill 2 workers, lower spirometry values, higher prevalence of dry cough and positive bronchodilator test were found in Sawmill 1 workers. Monday EBC pH values did not differ between sawmills, but declined after one working week in Sawmill 1 workers (from 7.88 to 7.49, P = 0.012) and not in Sawmill 2 workers. Similar results were obtained when only respiratory healthy non-smokers were analysed. Monday-to-Friday change of other respiratory parameters was not observed. CONCLUSION: The results suggest EBC pH as a biomarker of acute respiratory effects related to occupational exposure to respiratory hazards in sawmills, presumably increased mould levels. The effect was present even at subclinical level, namely in respiratory healthy subjects. The long-term health implications remain unclear and should be evaluated in a follow-up study.

Combined Toxicity of the Most Common Indoor Aspergilli.

The most common Aspergilli isolated from indoor air samples from occupied buildings and a grain mill were extracted and analyzed for their combined (Flavi + Nigri, Versicolores + Nigri) cytotoxic, genotoxic and pro-inflammatory properties on human adenocarcinoma cells (A549) and monocytic leukemia cells induced in macrophages (THP-1 macrophages). Metabolite mixtures from the Aspergilli series Nigri increase the cytotoxic and genotoxic potency of Flavi extracts in A549 cells suggesting additive and/or synergistic effects, while antagonizing the cytotoxic potency of Versicolores extracts in THP-1 macrophages and genotoxicity in A549 cells. All tested combinations significantly decreased IL-5 and IL-17, while IL-1ß, TNF-α and IL-6 relative concentrations were increased. Exploring the toxicity of extracted Aspergilli deepens the understanding of intersections and interspecies differences in events of chronic exposure to their inhalable mycoparticles.

Azithromycin-loaded liposomal hydrogel: a step forward for enhanced treatment of MRSA-related skin infections.

Azithromycin (AZT) encapsulated into various types of liposomes (AZT-liposomes) displayed pronounced in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) (1). The present study represents a follow-up to this previous work, attempting to further explore the anti-MRSA potential of AZT-liposomes when incorporated into chitosan hydrogel (CHG). Incorporation of AZT-liposomes into CHG (liposomal CHGs) was intended to ensure proper viscosity and texture properties of the formulation, modification of antibiotic release, and enhanced antibacterial activity, aiming to upgrade the therapeutical potential of AZT-liposomes in localized treatment of MRSA-related skin infections. Four different liposomal CHGs were evaluated and compared on the grounds of antibacterial activity against MRSA, AZT release profiles, cytotoxicity, as well as texture, and rheological properties. To our knowledge, this study is the first to investigate the potential of liposomal CHGs for the topical localized treatment of MRSA-related skin infections. CHG ensured proper viscoelastic and texture properties to achieve prolonged retention and prolonged release of AZT at the application site, which resulted in a boosted anti-MRSA effect of the entrapped AZT-liposomes. With respect to anti-MRSA activity and biocompatibility, formulation CATL-CHG (cationic liposomes in CHG) is considered to be the most promising formulation for the treatment of MRSA-related skin infections.

A Novel Approach for the Treatment of Aerobic Vaginitis: Azithromycin Liposomes-in-Chitosan Hydrogel.

Biocompatible mucoadhesive formulations that enable a sustained drug delivery at the site of action, while exhibiting inherent antimicrobial activity, are of great importance for improved local therapy of vaginal infections. The aim of this research was to prepare and evaluate the potential of the several types of azithromycin (AZM)-liposomes (180-250 nm) incorporated into chitosan hydrogel (AZM-liposomal hydrogels) for the treatment of aerobic vaginitis. AZM-liposomal hydrogels were characterized for in vitro release, and rheological, texture, and mucoadhesive properties under conditions simulating the vaginal site of application. The role of chitosan as a hydrogel-forming polymer with intrinsic antimicrobial properties was explored against several bacterial strains typical for aerobic vaginitis as well as its potential effect on the anti-staphylococcal activity of AZM-liposomes. Chitosan hydrogel prolonged the release of the liposomal drug and exhibited inherent antimicrobial activity. Additionally, it boosted the antibacterial effect of all tested AZM-liposomes. All AZM-liposomal hydrogels were biocompatible with the HeLa cells and demonstrated mechanical properties suitable for vaginal application, thus confirming their potential for enhanced local therapy of aerobic vaginitis.

A potential role of calcium in apoptosis and aberrant chromatin forms in porcine kidney PK15 cells induced by individual and combined ochratoxin A and citrinin.

The aim of this study was to establish the involvement of calcium signalling in genotoxicity, apoptosis and necrosis evoked by ochratoxin A (OTA) and citrinin (CTN) alone or in combination in porcine kidney PK15 cells. Cell proliferation test (MTT) and trypan blue assays (24 h) demonstrated that CTN (IC(50) = 73.5 ± 1.0, 75.4 ± 1.4 µM, respectively) was less toxic than OTA (IC(50) = 14.0 ± 2.4, 20.5 ± 1.0 µM, respectively). To test their cytotoxic interactions, two doses of single OTA (6 and 10 µM) and CTN (30 and 50 µM) and their combinations were applied. Combined treatment showed additive cytotoxic effects. OTA and CTN induced dose-dependent increase in cytosolic calcium level (assessed with Fura-2 AM). However, combined treatment did not provoke additional increase in calcium signal. The rate of apoptosis and necrosis (DAPI-antifade staining) was significantly higher after 12 h than 24 h, while the frequencies of micronuclei (MNs) and nuclear buds (NBs) were higher after 24 h than 12 h treatment. Combined exposure resulted in apoptotic and necrotic synergism, while genotoxic effects of OTA + CTN were noted as antagonistic or additive. Co-exposure of cells to calcium chelator BAPTA-AM significantly reduced CTN and OTA + CTN-evoked apoptosis. Twenty-four hour after co-exposure to BAPTA-AM and a single OTA and CTN, MNs significantly decreased while NBs dropped significantly after co-treatment with BAPTA-AM and OTA + CTN. In conclusion, disturbance of Ca(2+) homeostasis caused by OTA and CTN plays a significant role in cell genotoxicity and death.

Cytotoxicity and genotoxicity of versicolorins and 5-methoxysterigmatocystin in A549 cells.

Aspergillus versicolor and A. flavus are primary colonizers in damp dwellings, and they produce sterigmatocystin (ST) and aflatoxin B1 (AFB(1)), respectively. These hepatotoxic and carcinogenic mycotoxins and their precursors and derivates possess a furofuran ring, which has proven responsible for their toxicity. The aim of this study was to investigate the cytotoxicity and genotoxicity of versicolorin A (VER A) and versicolorin B (VER B), as the furofuran precursors of aflatoxins and ST, and of 5-methoxysterigmatocystin (5-MET-ST), a methoxy derivative of ST, in human adenocarcinoma lung cells A549. The IC(50) values of the tested compounds were obtained by the cell proliferation MTT test as follows: 109 ± 3.5 µM (VER A), 172 ± 4 µM (VER B) and 181 ± 2.6 µM (5-MET-ST). The comet assay and micronucleus test were used to assess their genotoxic potential after 24 h of treatment with concentrations corresponding to ½ and » IC(50) in comparison with AFB(1) and ST, applied in concentrations corresponding to ½ IC(50), as previously determined in A549 cells. DNA damage parameters assessed by the comet assay were tail length, tail intensity and tail moment, while the level of DNA damage in the micronucleus test was evaluated by the number of formed micronuclei (MN), nuclear buds (NB) and nucleoplasmic bridges (NPB) in 1,000 binucleated cells. Considering the three comet parameters, all applied toxins exerted significant DNA damage compared to the control, while ST and VER B produced the highest DNA damage. All toxins provoked a statistically significant increase in MN, and a slightly decreased formation of NB and NPB. AFB(1), ST and 20 µM VER A showed a statistically significant increase in all three micronucleus parameters compared to the control, and the highest increase in the number of MN occurred in cells treated with 50 µM VER A. The differences between results obtained by the micronucleus test and comet assay could be explained by the fact that the micronucleus detects irreversible DNA damage, which is usually correlated with the previously determined cytotoxic potential of the AFB(1) precursors.

Subchronic exposure of individual and combined ochratoxin A and citrinin selectively affects the expression of rat renal organic cation transporters.

Ochratoxin A (OTA) and citrinin (CIT) are nephrotoxins found co-occurring in various human/animal food/feed and recognized as a health threat. However, most studies investigate individual effects and neglect their combined nephrotoxic effects in mammals. Previous studies have indicated that organic anion/cation transporters (OATs/OCTs) localized in renal proximal tubules mediate the transport of OTA and CIT. Still, little is known about the in vivo effects of individual/combined OTA and CIT on protein localization/expression of OCTs, physiologically/pharmacologically important renal transporters. Here, we used Western blot and immunofluorescence microscopy to study the effects of subchronic (21-day) exposure to individual/combined OTA (0.125 and 0.250 mg kg-1 b.w.) and CIT (20 mg kg-1 b.w.) on protein localization/expression of organic cation transporters (rOct1/Slc22a1 and rOct2/Slc22a2) in kidneys of Wistar rats. Since the antioxidant resveratrol (RSV) has shown measurable protective effects against OTA- and CIT-related oxidative stress toxicity in vitro, we investigated the effects of an OTA + CIT + RSV combination on rOct1/2 localization/expression in the same model. Individual OTA induced a dose-dependent decrease of rOct1 but not rOct2 protein expression, whereas their localization pattern remained unchanged. Individual CIT did not affect the renal rOct1/2 protein localization/expression. Combined OTA + CIT exposure induced a significant decrease of rOct1 protein expression by an OTA250 dose, whereas oral co-administration of OTA + CIT + RSV resulted in a significant decrease of rOct1/2 protein expression. Thus, we revealed an OTA-related selective effect on the rOct1/2 protein expression and a non-specific adverse effect of RSV in the OTA + CIT + RSV combination on the renal organic cation transport system in rat.

Mupirocin calcium microencapsulation via spray drying: feed solvent influence on microparticle properties, stability and antimicrobial activity.

OBJECTIVES: The aim of this research was to design a controlled release, spray dried, mupirocin calcium-loaded microparticles (MP) with acrylic polymer and assess the influence of a feed solvent at preselected drug:polymer proportions (1:5 and 2:1 (w/w)) on the performance and stability of the prepared MP. METHODS: Physicochemical properties of MP were assessed using modulated differential scanning calorimetry (MDSC), and thermogravimetric analyses (TGA), Fourier transformed infrared spectroscopy (FTIR) and X-ray analyses and were correlated with drug release. Morphology and particle size were determined using low-angle laser light scattering and a scanning electron microscope. A time-kill assay was conducted on two strains of Staphylococcus aureus to evaluate the antimicrobial activity of MP. RESULTS AND DISCUSSION: The MP formed solid dispersions without apparent drug crystallization. Drug-polymer miscibility, morphology, drug release and consequently antimicrobial activity were dependent on drug loading (DL) and the used solvent. The superior control of drug release from MP was achieved for the higher DL (2:1 (w/w) drug:polymer proportion) using solvents in the following order: methanol ≈ methanol:ethanol (50:50, w/w) > isopropanol:acetone (40:60, w/w). Moreover, a time-kill assay performed on S. aureus (ATCC 29213) and methicillin-resistant S. aureus strains confirmed the prolonged release and preservation of antimicrobial activity of the microencapsulated drug. The physical aging of the solid dispersion after 10 months of storage had negligible impact on the MP performance. CONCLUSIONS: Acrylic-based MP were confirmed as suitable microcarriers for prolonged drug release using a well-established spray drying technique, while solvent influence was strongly related to the DL employed.

Spray dried microparticles for controlled delivery of mupirocin calcium: process-tailored modulation of drug release.

Spray dried microparticles containing mupirocin calcium were designed as acrylic matrix carriers with modulated drug release for efficient local drug delivery at minimum daily dose. Particle generation in spray drying and its effect on release performance were assessed by varying drug : polymer ratios with consequently altered initial saturations. Narrow-sized microparticles with mean diameters of 1.7-2.5 µm were obtained. Properties of the generated solid dispersions were examined by X-ray, thermal (thermogravimetric analysis, modulated differential scanning calorimetry) and spectroscopic (Fourier transformed infrared, Fourier transformed Raman) methods and correlated with drug loading and in vitro release. The best control over mupirocin release was achieved for 2 : 1 (w/w) drug : polymer ratio and found to be strongly process-dependent. For a particular ratio, increased feed concentration (>4%) boosted while increased inlet temperature (≥ 100 °C) reduced drug release. Antimicrobial activity testing confirmed that encapsulated drug preserved its antibacterial effectiveness. Conclusively, spray drying was proven as a suitable method for preparing structured microparticles which can control drug release even at exceptionally high drug loadings.

Sterigmatocystin, 5-Methoxysterigmatocistin, and Their Combinations Are Cytotoxic and Genotoxic to A549 and Hepg2 Cells and Provoke Phosphorylation of Chk2, but Not Fancd2 Checkpoint Proteins.

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are structurally related mycotoxins with cytotoxic and genotoxic properties. In the present study, we hypothesized that DNA damage induced by non-cytotoxic concentrations of single and combined mycotoxins could alter the phosphorylation of the checkpoint proteins Chk2 and FANCD2 (ELISA) in HepG2 and A549 cells. The cytotoxic potential (MTT test) of single and combined STC and 5-M-STC, the nature of their interaction (additivity, antagonism, or synergy) and DNA damage level (alkaline comet assay) in HepG2 and A549 cells were also investigated. All experiments were performed after 24 h of mycotoxin treatment. 5-M-STC was 10-folds more cytotoxic than STC to both HepG2 and A549 cells. Both mycotoxins are genotoxic to HepG2 and A549 cells by inducing both double and single DNA strand breaks that activate Chk2 (especially in HepG2 cells) but not the FANCD2 protein. STC exerted higher genotoxic potential than 5-M-STC in HepG2 and A549 cells when both toxins were applied individually at the same concentration. Dual combinations of non-cytotoxic mycotoxin concentrations showed additive to antagonizing cytotoxic and genotoxic effects. The absence and low activation of checkpoint proteins during prolonged exposure to non-cytotoxic concentrations of STC and 5-M-STC could support cell proliferation and carcinogenesis.

Fungi and their metabolites in grain from individual households in Croatia.

A total of 117 fungal metabolites were detected in grains collected in Gunja-G (flooded village) and Gornji Stupnik-GS (control village), located in the Zagreb County, Croatia. Major mycotoxins and derivatives (17), ergot alkaloids (14), Fusarium (23), Aspergillus (18), Penicillium (18), Alternaria (7) and other fungal and unspecific metabolites (20) were found. A higher number of metabolites co-occurred per sample in grains from G (115) than in GS (91). Regulated mycotoxins were below maximum limits except fumonisins B1,2 in 15-20% of grains and aflatoxin B1. Fusarium metabolites contaminated more than 50% of grains at both locations. Besides FB1,2, bikaverin, aurofusarin, culmorin and 15-hidroxyculmorin were detected at relatively high concentrations. Ergot alkaloids were detected at 2-18 times higher concentrations in grains from G as compared to GS. Majority of Aspergillus mycotoxins were present at a low frequency (5-15%). Penicillium metabolites recovered with higher frequency in GS (55-70%) than in G (20-55%). Alteranaria metabolites prevailed in grains from G (60-80%).

Beauvericin and ochratoxin A genotoxicity evaluated using the alkaline comet assay: single and combined genotoxic action.

This study was aimed at investigating the genotoxic potential of single beauvericin (BEA) and ochratoxin A (OTA) as well as their interaction in porcine kidney epithelial PK15 cells and human leukocytes using the alkaline comet assay. IC(50) of BEA (5.0 +/- 0.6) and OTA (15.8 +/- 1.5) estimated by MTT reduction assay shows that BEA is three times more toxic than OTA. BEA (0.1 and 0.5 microM) and OTA (1 and 5 microM) were applied alone or in combination of these concentrations for 1 and 24 h in PK15 cells and human leukocytes. Genotoxicity of these toxins to PK15 cells was time- and concentration dependent. After 1 h, significant increase in tail length, tail intensity, tail moment, and abnormal sized tails (AST) was noted upon exposure to 1 muM of OTA alone and BEA + OTA combinations. Single BEA (0.5 microM) and OTA (1 and 5 microM) and their combinations evoked significant DNA damage in PK15 cells, considering all comet tail parameters measured after 24 h of treatment. Human leukocytes were slightly concentration but not time dependent. After 1 h of exposure, there were no significant changes in the tail length. Tail intensity, tail moment, and/or incidence of AST were significantly higher in cells treated with single OTA or BEA and their combinations than in control cells. DNA damage in leukocytes was significantly higher after 24 h of exposure to single toxins and their combinations, considering all comet tail parameters, but these changes were less pronounced than in PK15 cells. Combined toxins showed additive and synergistic effects in PK15 cells, while only additive effects were observed in human leukocytes. Combined prolonged exposure to BEA and OTA in subcytotoxic concentrations through food consumption could induce DNA damage contributing to the carcinogenicity in animals and humans.

Assessing the Effect of Mycotoxin Combinations: Which Mathematical Model Is (the Most) Appropriate?

In the past decades, many studies have examined the nature of the interaction between mycotoxins in biological models classifying interaction effects as antagonisms, additive effects, or synergisms based on a comparison of the observed effect with the expected effect of combination. Among several described mathematical models, the arithmetic definition of additivity and factorial analysis of variance were the most commonly used in mycotoxicology. These models are incorrectly based on the assumption that mycotoxin dose-effect curves are linear. More appropriate mathematical models for assessing mycotoxin interactions include Bliss independence, Loewe's additivity law, combination index, and isobologram analysis, Chou-Talalays median-effect approach, response surface, code for the identification of synergism numerically efficient (CISNE) and MixLow method. However, it seems that neither model is ideal. This review discusses the advantages and disadvantages of these mathematical models.