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.

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.

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.

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.

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.

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%).

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.

Post-Flood Impacts on Occurrence and Distribution of Mycotoxin-Producing Aspergilli from the Sections Circumdati, Flavi, and Nigri in Indoor Environment.

Mycotoxin-producing Aspergilli (Circumdati, Flavi, and Nigri), usually associated with contaminated food, may also cause respiratory disorders and are insufficiently studied in water-damaged indoor environments. Airborne (N = 71) and dust borne (N = 76) Aspergilli collected at post-flood and control locations in Croatia resulted in eleven different species based on their calmodulin marker: A. ochraceus, A. ostianus, A. pallidofulvus, A. sclerotiorum, and A. westerdijkiae (Circumdati); A. flavus (Flavi); and A. tubingensis, A. welwitschiae, A. niger, A. piperis, and A. uvarum (Nigri). Most of the airborne (73%) and dust borne (54%) isolates were found at post-flood locations, and the highest concentrations measured in indoor air (5720 colony-forming units (CFU)/m3) and dust (2.5 × 105 CFU/g) were up to twenty times higher than in the control locations. A. flavus dominated among airborne isolates (25%) at the unrepaired locations, while 56% of the dust borne Aspergilli were identified as A. tubingensis and A. welwitschiae. The ability of identified isolates to produce mycotoxins aflatoxin B1 (AFB1), fumonisin B2 (FB2), and ochratoxin A were assessed by LC-MS analysis. All ochratoxin A (OTA)-producing Circumdati belonged to A. westerdijkiae (13.7 ± 15.81 µg/mL); in the section, FlaviA. flavus produced AFB1 (2.51 ± 5.31 µg/mL), while A. welwitschiae and A. niger (section Nigri) produced FB2 (6.76 ± 13.51 µg/mL and 11.24 ± 18.30 µg/mL, respectively). Water damage dominantly supported the occurrence of aflatoxigenic A. flavus in indoor environments. Yet unresolved, the causal relationship of exposure to indoor Aspergilli and adverse health effects may support the significance of this research.

Environment Changes, Aflatoxins, and Health Issues, a Review.

Crops contaminated by aflatoxins (AFs), the toxic and carcinogenic mycotoxins produced namely by Aspergillus flavus and Aspergillus parasiticus, have severe impacts on human health. Changes in temperature and water availability related to actual climate changes (increased temperature, heavy rainfalls, and droughts) are modulating factors of mould growth and production of mycotoxins. To protect human and animal health from the harmful effects caused by AFs, the development of a safe and effective multifaceted approach in combating food and feed contamination with AFs is necessary. This review aims to collect and analyze the available information regarding AF presence in food and feed to reinforce AF management and to prevent health issues related to the AF exposure in the light of actual climate changes.

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

Ochratoxin A (OTA) and citrinin (CIT) are mycotoxins known to co-contaminate human/animal food/feed. Their prominent nephrotoxic effects pose a threat to human and animal health. Studies have shown synergistic or additive effects of these two mycotoxins, but a clear consensus on this phenomenon does not exist. In vitro/vivo studies on OTA and CIT effects showed they elevate oxidative stress parameters. Some in vitro studies tested resveratrol (RSV) as a potential antioxidant to counteract these OTA and CIT effects. However, data on the combined effects of OTA + CIT mycotoxins and RSV on their in vivo toxicity is lacking. We used immunofluorescence microscopy and Western blotting to study the subchronic effects of individual/combined OTA (0.125 and 0.250 mg kg-1 b.w.) and CIT (20 mg kg-1 b.w.) on the localization/expression of rat renal organic anion transporters (rOats) (rOat1/Slc22a6, rOat2/Slc22a7, rOat3/Slc22a8, rOat5/Slc22a19) that mediate the secretion/reabsorption of organic anions in kidney proximal tubules. We investigated if RSV (20 mg kg-1 b.w.) can counteract the effects of both mycotoxins on the localization/expression of studied transporters. Results revealed Oat- and dose-dependent changes in protein expression of rOats. When combined with both mycotoxins, RSV decreased the protein expression of all of the studied rOats. Its effect was additive on Oat1/2/5. Thus, RSV failed to ameliorate OTA- and/or CIT-related nephrotoxic effects on the expression of studied rOats in rat kidneys.

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.

Involvement of Ahr Pathway in Toxicity of Aflatoxins and Other Mycotoxins.

The purpose of this review is to present information about the role of activation of aflatoxins and other mycotoxins, of the aryl hydrocarbon receptor (AhR) pathway. Aflatoxins and other mycotoxins are a diverse group of secondary metabolites that can be contaminants in a broad range of agricultural products and feeds. Some species of Aspergillus, Alternaria, Penicilium, and Fusarium are major producers of mycotoxins, some of which are toxic and carcinogenic. Several aflatoxins are planar molecules that can activate the AhR. AhR participates in the detoxification of several xenobiotic substances and activates phase I and phase II detoxification pathways. But it is important to recognize that AhR activation also affects differentiation, cell adhesion, proliferation, and immune response among others. Any examination of the effects of aflatoxins and other toxins that act as activators to AhR must consider the potential of the disruption of several cellular functions in order to extend the perception thus far about the toxic and carcinogenic effects of these toxins. There have been no Reviews of existing data between the relation of AhR and aflatoxins and this one attempts to give information precisely about this dichotomy.

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.

Aflatoxin production and in vitro toxicity of Aspergilli section Flavi isolated from air samples collected from different environments.

Aspergilli section Flavi, originally isolated from air samples collected from inhabited apartments (AP), unoccupied basements (BS), and processing facilities of a grain mill (GM), were analyzed for their potential to produce aflatoxin B1 (AFB1) on solid media. The isolates were further characterized with regard to their cytotoxic, genotoxic, and pro-inflammatory properties in vitro. Aspergilli were identified based on partial calmodulin (CaM) gene sequencing; the producing capacities of isolates were analyzed by HPLC/FLD and confirmed by genes in biosynthesis (aflR, norA, omtA). In the grain mill, the Aspergilli section Flavi (up to 1.3 × 106 cfu/m3) dominated by AFB1-producing Aspergillus flavus (71%, 4.5-5254 ng/ml) which showed a serious health risk for workers. Living environments were not relevant sources of exposure. After 24 h, AFB1 (1-100 µmol/l) reduced cell viability (MTT test) in both A549 cells and THP-1 macrophage-like cells without reaching IC50. In A549 cells, the extract of the AFB1-producing A. flavus significantly decreased cell viability but not below 50%. THP-1 macrophage-like cells were more sensitive to both extracts, but IC50 was obtained only for the AFB1-producing strain (0.37 mg/ml; AFB1 2.78 µmol/l). AFB1 (1 and 10 µmol/l) induced significant DNA damage (tail intensity, alkaline comet assay) in A549 cells in contrast to Aspergilli extracts. AFB1 elevated IL-6 and IL-8, while Aspergilli extracts increased IL-1ß, TNF-α, and IL-17 release in THP-1 macrophages (ELISA). Chronic exposure to AFB1 and/or other metabolites in airborne A. flavus from occupational environments may stimulate epithelial damage of airways accompanied by lowered macrophage viability.

Azithromycin-loaded liposomes for enhanced topical treatment of methicillin-resistant Staphyloccocus aureus (MRSA) infections.

Antibiotic delivery via liposomal encapsulation represents a promising approach for the efficient topical treatment of skin infections. The present study aimed to investigate the potential of using different types of azithromycin (AZT)-loaded liposomes to locally treat skin infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains. Conventional liposomes (CLs), deformable liposomes (DLs), propylene glycol-containing liposomes (PGLs) and cationic liposomes (CATLs) encapsulating AZT were prepared, and their physical characteristics, drug release profiles, ex vivo skin penetration/deposition abilities, in vitro anti-MRSA activities (planktonic bacteria and biofilm) and cell biocompatibilities were assessed. The (phospho)lipid composition and presence of surfactant or propylene glycol affected the physical characteristics of the liposomes, the release profile of AZT, its deposition inside the skin, as well as in vitro antibacterial efficacy and tolerability with the skin cells. All the liposomes retained AZT inside the skin more efficiently than did the control and were biocompatible with keratinocytes and fibroblasts. CATLs, DLs and PGLs efficiently inhibited MRSA strain growth and were superior to free AZT in preventing biofilm formation, exhibiting minimal inhibitory concentrations and minimal biofilm inhibitory concentrations up to 32-fold lower than those of AZT solution, thus confirming their potential for improved topical treatment of MRSA-caused skin infections.

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.

Differential activation of MAPKs by individual and combined ochratoxin A and citrinin treatments in porcine kidney PK15 cells.

The aim of this study was to investigate the underlying mechanisms of OTA and CTN individual and combined toxicity in porcine kidney PK15 cells of proximal tubule origin. Activation and expression of mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 were determined by Western blot analysis. MAPKs were differentially activated by single or dual OTA and CTN treatments. Single OTA and CTN stimulated transient ERK and prolonged JNK activation, while phospho-p38 signal was more persistent after OTA treatment. Mycotoxin mixture provoked significant down-regulation of ERK activation, more prolonged phospho-p38 signal, and two-stage JNK phosphorylation pattern. In order to define the role of particular MAPKs in mycotoxin(s) cytotoxicity, we performed MTT assay with specific MAPKs inhibitors. In both individual and combined treatments JNK and p38 inhibition significantly induced cell survival. When cells were exposed to toxin mixture, inhibition of ERK also promoted cell survival, although to a lesser extent that JNK and p38 inhibition. Next we investigated the association between calcium (Ca(2+)) and MAPKs after OTA and/or CTN treatments, and we employed Ca(2+) chelator BAPTA-AM. We demonstrated that p38 activation was significantly down-regulated in cells treated with CTN alone or OTA + CTN suggesting the role of Ca(2+) in mycotoxin-induced cell death.