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STC is precursor of aflatoxin and also the end product in biosynthesis of Aspergilli from the section Versicolores. Mechanism of STC toxicity is transformation by CYP monooxygenases into epoxide, which forms DNA-adducts leading to hepatotoxicity and carcinogenicity. STC-producing Aspergilli are frequently airborne in damp dwellings and could be expected in dust contributing to STC intake by inhalation. The purpose of the study was to employ HPLC/DAD/MS method for STC analysis in STC-producing Aspergilli extracts in order to estimate health risk based on their STC-producing capacity and frequency in dust taken in winter and summer of the year 2016 from flooded and unflooded houses in Croatia. STC analysis was performed by HPLC-DAD-ESI/MS/MS gradient method: Symmetry C18 4.6x150 mm, 3.5 μm column ; mobile phase (methanol/water), flow rate 1.0 ml/min ; DAD 254 nm ; positive ionization mode. Aspergilli (Versicolores) were of similar frequency in control and flooded village (40–100 %) but the concentrations in dust were more than 10 times higher in flooded houses (4.5 x 105 CFU/g). STC production was confirmed for 6/6 control village isolates (6–30.2 mg/mg) in winter and 3/5 isolates in summer period (25.1–42.2 mg/mg). Isolates from the flooded area showed similar STCproducing abilities (3/3 isolates, 2.4–22.1 mg/mg in winter ; 9/13 isolates, 1.9–39.9 mg/mg in summer). Significantly higher concentrations of dustborne Aspergilli STC-producers in flooded houses suggest increased health risk due to expected STC content in inhalable dust. This work has been fully supported by Croatian Scientific Foundation under the project MycotoxA (IP-09-2014- 5982).

Kuća je bolesnik, a plijesan je uzrok bolesti: je li to moguće i što je lijek? Nakon poplave nastupa invazija plijesni na vodom natopljene zidove. Hranu ne trebaju, dovoljna im je slobodna voda (aktivitet vode). Pri aktivitetu vode većem od 0.9 caruju tercijalni kolonizatori, a sekundarni i primarni kolonizatori zadovoljni su i s manjim aktivitetom vode (0.6-0.8). Rastući na zdovima plijesni u zrak otpuštaju dijelove micelija i konidija koji mogu sadržavati mikotoksine, opasne za zdravlje ljudi i životinja. Koji su simptomi bolesti kuće, koje se plijesni pojavljauju kao uzročnici, kako liječiti takvu kuću, a spriječiti bolest kod ukućana, doznat ćete na prezenatciji. Tim se temama bavimo u sklopu znanstvenog projekta (IP-09-2014-5982, MycotoxA) kojeg financira Hrvatska zaklada za znanost.

Backgrounds: Sterigmatocystin (STC) is a mycotoxin produced by many fungal species, ubiquitous in working and living environment. STC exhibits hepatotoxic, nephrotoxic, mutagenic and carcinogenic effects representing a significant risk to human and animal health. LC-MS is the most commonly used technique for the STC detection. Possible complications like matrix effects on ionisation efficiency make use of isotopically labelled STC internal standard inevitable, in addition to laborious sample preparation. Circular dichroism (CD) spectroscopy is extensively used to study structural, kinetic and thermodynamic features of chiral molecules. Since STC is chiral molecule CD spectroscopy for its determination in various substrates is worth of exploring. Objectives: The purpose of this study was to demonstrate CD spectroscopy application in the determination of STC in various aqueous matrices. Methods: CD spectra of STC aqueous solutions were recorded by standard Jasco J810 setup in 1 cm quartz cuvette at various physicochemical conditions (temperature, organic solvents and salts addition). Conclusions: STC forms aggregate in aqueous solutions, yielding strong CD signal in 300-400 nm range specific to STC, with the intensity up to 1000:1 compared to the baseline. The CD signal intensity is proportional to STC concentration within 10-7 M to 10-5 M range, and none of relevant species from food or environment (including structurally similar aflatoxin B1) does not interfere with this CD- signal. These facts strongly encourage an innovative approach to STC monitoring, which could result in a completely new analytical method for the specific determination of STC, as well as studying non-covalent interactions of STC with biomacromolecules.

Mycotoxins are secondary metabolites produced by moulds abundant in the different environments. Among these, aflatoxin B1 (AFB1) is considered one of the most toxic and is human cancerogen [1]. Structurally related furofurane derivatives (Fig. 1) sterigmatocystin (STC) and 5- methoxysterigmatocystin (5-MET-STC) are direct biosynthetic precursors in AFB1 producing Aspergilli, but also the final products of biosynthesis occurring in the different types of moulds [2]. Despite similarities in their chemical structures, their toxic properties differ and thus suggesting the differences in their physicochemical properties in water solutions. In this study we showed CD spectra of AFB1, STC and 5-MET-STC in organic and water based solvents for the first time. While the CD signal in organic solvent is of low intensity and quite similar for all of the three compounds (Fig. 2A), STC showed distinctive CD signal of unusually high intensity in water (Fig. 2B). In contrast to its structural analogues we suspect such signal of STC is due to its ability to aggregate in water as was confirmed with other methods we applied that will be presented and discussed. The significance of these results is yet to be explored, but they made the basis of a new approach in the analysis of STC in water based solutions, in addition to understanding its behavior in biological systems.

Plijesni uglavnom povezujemo sa ružnim, štetnim i trošnim, a cilj ove radionice je prikazati ih u mnogima nepoznatom svjetlu- raskošne, simpatične i korisne. Suze štite naše oči od živih i neživih opasnosti, a na sličan način i plijesni duguju svoj opstanak u nemilosrdnoj konkurenciji živoga svijeta brojnim sastavnicama svojih “suza”. Pred kojim plijesnima bakterije strepe? Kakve veze imaju plijesni s “eliksirom mladosti”? Kako plijesni pomažu da smo manje masni? Odgovore na ova i brojna druga pitanja saznati ćete ako nas posjetite na našoj radionici.

Black Aspergilli (section Nigri) are ubiquitous moulds which contaminate foodstuffs and occur in indoor and outdoor environments. These species may represent a health hazard due to their irritant, allergic and toxic effects. Recent studies show that black Aspergilli can produce several fumonisin isomers including FB2 which toxic effects are poorly explored. The purpose of this study was: 1) to determine airborne black Aspergilli based on calmodulin gene sequence (CaM) ; 2) to explore FB2 production capacity in culture microextracts by LC/MS accompanied with the presence of fum1 and fum8 genes involved in fumonisins biosynthesis ; 3) to assess the cytotoxic and genotoxic potential of FB2 compared to FB1 as well as to FB2-positive and -negative Aspergilli microextracts in human lung adenocarcinoma cells (A549). In A. welwitschiae (7/15) and A. niger (1/1) isolates presence of both fum1 and fum8 genes correlated with FB2 presence in culture microextracts, in concentrations from 127.79 ng/mL to 13467.74 ng/mL (A. welwitschiae) and 15.16 ng/ml (A. niger). Upon 24 h treatment with 100 µM both FB1 and FB2 slightly altered A549 cells viability (83.40% and 71.25%, respectively). Microextracts (mg of microextract per mL of culture media) prepared from A. tubingensis and A. piperis were the most cytotoxic (IC20= 0.400 ± 0.002 mg/mL and 0.62 ± 0.06 mg/mL, respectively) followed by A. welwitschiae (IC50= 0.246 ± 0.007 mg/mL). A. luchensis and A. niger extracts (0.8 mg/mL) decreased cell viability to 58.88 % and 88.80 %, respectively. Tail intensity (% of DNA in tail) was the comet assay parameter selected to evaluate DNA damage compared to control treatment (0.1% DMSO). After 24 h treatment both FB1 and FB2 (10 µM) caused significant DNA damage (0.35 % and 0.33 %, respectively) as compared to control (P

Vrste aspergila učestale su u zraku radnog i stambenog okolišu. U povećanim koncentracijama, ali i zbog izraženog potencijala tvorbe mikotoksina, pridonose kroničnim bolestima dišnog sustava. Izolati aspergila sekcija Flavi, Versicolores i Nigri, prikupljeni tijekom jednogodišnjeg uzorkovanja na lokacijama mlin žitarica (M) te u stambenim prostorima (podrum-P, stan-S) identificirani su do razine vrste primjenom makro- i mikroskopskih obilježja te analizom genskog slijeda CaM. Ukupne koncentracije plijesni u zraku na lokaciji M bile su do 30 puta više u odnosu na ostale lokacije, a dosezale su 40000 CFU/m3. Kao najčešće vrste u M identificirane su A. flavus u sekciji Flavi koja se uz A. parasiticus pokazala potentnim proizvođačem B i G skupina aflatoksina, na što upućuje detekcija analita u mikroekstraktima te detekcija gena aflR, norA i omtA uključenih u biosintezu aflatoksina. Na lokacijama P i S dominirale su vrste A. jensenii i A. creber unutar sekcije Versicolores, a vrste A. tubingensis i A. welwitschiae unutar crnih aspergila izoliranih na svim lokacijama tijekom cijele godine. Pored sedam identificiranih vrsta među izolatima iz sekcije Versicolores izolirana je i jedna nova vrsta a sve vrste pokazale su se dobrim proizvođačima STC koji je detektiran u njihovim ekstraktima, dok je od gena uključenih u biosintezu STC detektiran gen norA. Od pet identificiranih vrsta unutar sekcije Nigri, samo A. niger i A. welwitschiae proizvode FB2 što je potvrđeno na razini gena fum1 i fum8, a FB2 je dokazan u ekstraktima kultura. In vitro ispitivanjima toksičnosti, A549 stanice i THP-1 makrofagi bili su izražene osjetljivosti na djelovanje STC te ekstrakata vrsta proizvođača STC. Citotoksični učinci kombinacija mikotoksina AFB1+FB1/FB2 te STC+ FB1/FB2 uglavnom su se antagonizirali. Međutim, izlučivanje IL-6 u THP-1 makrofagima koreliralo je s koncentracijom AFB1 i STC čak i nakon dodatka FB1/FB2. Aditivni ili sinergistički učinci mikotoksinogenih ekstrakata vrsta sekcija Flavi+Nigri te Versicolores+Nigri odrazili su se i na izlučivanje citokina, među kojima dominantno TNF-α. Genotoksičnost AFB1 i STC u A549 stanicama korelirala je s povećanom ekspresijom p53 proteina sa i bez dodatka FB1 ili FB2. Ispitivanja interakcija s DNA i polinukleotidima u vodenom mediju ukazala su na snažno nekovalentno vezanje STC, bitno različitim mehanizmima od AFB1.

In order to investigate post-flood mycological patterns in Gunja compared to an unaffected area (Gornji Stupnik) 20 indoor airsamples (IAS), 5 outdoor airsamples (ODAS) and 10 dust samples (DS) were taken at 5 repaired (RH) and 5 unrepaired houses (UNRH) in Gunja, 5 control houses in Gornji Stupnik (SH) and 10 IAS and 2 DS in the elementary schools in Gunja (ESG) and Gornji Stupnik (ESS). Additionally, food samples (FS) were collected in both Gunja (FSG, N=10) and Stupnik (FSS, N=5). We used Dichloran 18%- Glycerol Agar (DG-18) and Malt Extract Agar (MEA) for AS, while for DS and FS Dichlorane Rose- Bengal Chloramphenicol Agar (DRBC) and DG- 18 were used. Mean concentration of aeromycota (cfu/m3) in Gunja in RH (2.1x103) was similar to ODAS (1.8x103 cfu/m3), while in UNRH concentration was significantly higher in IAS (4.6x103) but not in ODAS (3.6x103). Compared to both UNRH and RH, concentration of aeromycota in control area (SH) was significantly lower in IAS (460 cfu/m3) and ODAS (70). Similary, concentration of airborne fungi was significantly lower in ESS (212) than in ESG (1.6x103). Penicillium, Cladosporium and Aspergillus (sections Aspergillus, Circumdati, Nigri) genera were prevailing except in EES where Rhizopus sp. were frequently isolated (60%). Aspergilli (section Aspergillus) were present in all IAS (13-47%), section Nigri in RH and SH (13%), Circumdati in ESG (18%) and Versicolores in RH and SH (7 and 33%). High concentration of Aspergilli from the section Versicolores (5.7x103) indicated heavy contamination in one of the RH in Gunja. Mean concentration of fungi in DS (cfu/g) from RH (2.7x106) was higher than in UNRH (1.x105) and SH (1.3x105). Also, higher concentrations were observed in DS from ESG (1.2x105) than ESS (4.8x104). Composition of aeromycota corresponds to dustmycota, but Aspergilli (Versicolores) were more frequent in DS (40- 100%). In food samples mean concentration of fungi (cfu/g ) was higher in FSS (2x106) than in FSG (5x105) and dominated by Fusarium spp. in both FSG (75%) and FSS (80%). These results indicate that two years after major flood in Gunja concentration of airborne fungi is much higher comparing to unflooded area and significat source of aeromycota are unrepaired houses, heavily contaminated with secondary and tertiary colonizers. High concentration of airborne and dustborne fungi represent significant treath to humans due to inhalatory exposure to potential mycotoxin producers.

Multilocus DNA sequence-based identification methods raised the number of known species assigned to the Aspergillus section Versicolores. Currently, there are 16 species accepted in the section, including A. amoenus, A. austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. griseoaurantiacus, A. hongkongensis, A. jensenii, A. protuberus, A. puulaauensis, A. subversicolor, A. sydowii, A. tabacinus, A. tennesseensis, A. venenatus, and A. versicolor. Based on morphological identifications, most of these species were identified as either A. sydowii or A. versicolor, with the latter reported to have a world-wide distribution, growing in many habitats. Aspergillus versicolor has been implicated in health hazards including sick building syndrome, human and animal mycoses, and contamination of food and feed were assigned primarily to this species. A. versicolor is still commonly isolated from indoor surveys, even though species such as A. jensenii and A. creber seem more common. From indoor air samples collected at a grain mill in Croatia, we isolated an undescribed species assigned to the Aspergillus section Versicolores. A polyphasic approach, including sequence-based methods, morphological and physiological studies, was used for species characterization and in this paper is described as Aspergillus pepii. Additionally, sterigmatocystin producing abilities have been confirmed. Based on a combined phylogenetic tree, morphological features and sterigmatocystin producing abilities, A. pepii is closely related to A. versicolor. Further studies should explore the frequency of the species in indoor environments and its medical, industrial, and environmental significance.

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and sterigmatocystin (STC) are mycotoxins produced by filamentous fungi of the genus Aspergillus which exert various toxic properties in vivo and in vitro. The superficial layer of the skin (epidermis) is the most exposed part of the human body, and it's composed mostly of keratinocytes which have an important immune role by excreting various cytokines and could be subjected to harmful chemicals, e.g. the carcinogenic effect of mycotoxins such as AFB1. The purpose of this study was to investigate the cytotoxic, proinflammatory and genotoxic effects of single AFB1, OTA and STC, and their binary combinations on human keratinocyte HaCaT cells. Cells were exposed to single and combined mycotoxins for 24 h. Cytotoxicity was determined by MTT reduction assay. Excretion of cytokines IL- 1β, IL-6, IL-8, TNFα and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA) in cell supernatant. The level of DNA damage was determined by alkaline comet assay. According to concentrations that inhibit metabolic activity of HaCaT cells by 50% (IC50), cytotoxic potencies of mycotoxins were as follows: STC (IC50= 44.42 μM ) > OTA (IC50= 98.93 μM), while AFB1 at 50 μM reduced cell viability by 40% and increase to 150 μM did not provoke further decrease of cell viability. AFB1+OTA and OTA+STC applied in subcytotoxic concentrations showed additive and synergistic effects while AFB1+STC exerted dominant antagonism. ELISA showed that untreated HaCaT cells do not produce IL-1β, TNFα and IL-10, while IL-6 and IL-8 were produced in concentrations of 507 pg/mL and 547 pg/mL, respectively. All three mycotoxins provoked the increase of IL-6 and IL-8 by 123-297% and 131- 228%, respectively. The binary mycotoxin combinations showed an antagonistic effect on cytokine excretion. The alkaline comet assay showed that all mycotoxins individually and in binary combinations provoke DNA fragmentation by significant increase of the tail intensity (1.68- 5.57%) as compared to untreated cells (0.57%). The effect of binary combinations on DNA fragmentation were additive (AFB1+OTA) ; additive or antagonistic (AFB1+STC) ; and dominantly antagonistic (OTA+STC). In conclusion individual mycotoxins AFB1, OTA and STC and their binary mixtures evoke cytotoxic, genotoxic and proinflammatory effects in HaCaT cells. Taking into account result of combined treatments, cytotoxic potency of particular binary mixtures may overcome genotoxic and proinflammatory action.

Species assigned to Aspergillus section Versicolores are commonly isolated from different indoor environments. Due to a great variation in macrophenotypic characteristics and only few in microphenotypic, DNA based phylogenetic study elucidated appropriate assignement of isolates to this section. Previously, Aspergillus versicolor was the most commonly reported species isolated from indoor air of damp indoor environment and a major contributor to sick building syndrome described as a complex of respiratory dissorders among inhabitants of damp dwellings. One of incriminating factors in such conditions is a high frequency of sterigmatocystin (STC) producers among the isolates assigned to this species. Moreover, its presence is confirmed in building materials such as wall scrapes, house dust and grain dust. Although mechanisms are not completely understood and clarified, significant toxic properties of STC are confirmed on various cell cultures and animal models. The purpose of our research was to examine the distributon of isolates assigned to section Versicolores in indoor air in Croatia and to test their capability of STC biosynthesis. We included samples isolated from indoor environments including basements, apartments and grain mill. A polyphasic approach including sequence-based methods, morphological and physiologicyal examinations and mycotoxin producing abilities was used for species identification. Accordingly, we identified following species: A. jensenii (22), A. creber (13), A. venenatus (4), A. tenneesseensis (1), A. amoenus (5), A. griseoaurantiacus (1) and Aspergillus section Versicolores spec. nov. (3). All samples examined were capable of sterigmatocystin production as confirmed in associated microextracts by HPLC/UV-Vis methods. Highest contents of STC were found in samples belonging to A. jensenii ranging from 1.192 μg/mL to 133.63 μg/mL while the lowest concentration of STC were detected in extracts of A. protuberus in range of 0.117 to 2.749 μg/mL and A. tenneseensis which was 0.871 μg/mL. STC was detected in 13/25 dust samples collected in grain mill (0.06 μg/g to 2.35 μg/g). This research confirmed variability in species assigned to section Versicolores and underlined the actual and possible presence of STC in selected indoor environments.

Pokazana su osnove mikološke analize: RAD U MIKOLOŠKOM LABORATORIJU KLASIFIKACIJA PLIJESNI UZGOJNA SVOJSTVA PLIJESNI (hranjive podloge) METODE KVANTITATIVNE MIKOLOŠKE ANALIZE - uzorci hrane - uzorci plijesni u zraku SMJERNICE (zrak)

The aim of this study was to assess qualitative and quantitative composition of aeromycota based on airsampling conducted during one year in two-month intervals in Zagreb, Croatia at the locations including grain mill (GM), apartment (AP) and basement (BS) compared to the outdoor air (ODA). In addition, the incidence of potentially harmful species of Aspergilli belonging to the sections Flavi, Nigri and Versicolores has been observed. The number of fungal spores (CFU/m3) was scored based on colony growth after five days of incubation on DG-18 agar plates (Dichloran-18% glycerol-agar) at 25 °C for each sample (420 in total). Isolation and determination of the fungi based on morphological characteristics on the appropriate culture media like CYA (Czapek Yeast Extract Agar) followed. The average concentrations of airborne fungi scored in grain mill samples were significantly higher (P

Airborne fungi have been incriminated to cause several respiratory disorders based on different immunological and toxic pathways including hypersensitivity pneumonitis, asthma, rhinitis, chronic bronchitis and organic dust toxic syndrome. Mycotoxins in fungal spores have been proposed to cause adverse health effects particularly after chronic exposure to high levels of airborne fungi in indoor environments. Recent study (January 2012-November 2012) taken in grain mill (GM), apartments, and basements (Zagreb, Croatia) showed that average concentration of airborne fungi in GM was about 30 times higher (up to 40000 cfu/m3) than in other locations (up to 1244 cfu/m3). At GM species of Aspergillus, Penicillium and Eurotium dominated over other detected airborne fungi. Among Aspergilli and Eurotia, A. ochraceus and E. herbariorum were constantly present in the samples taken at GM. Aspergillus ochraceus (AO) is well-known producer of ochratoxin A (OTA) and Eurotium herbariorum (EH) produces a wide range of metabolites with poorly investigated toxicity. In the current investigation OTA was detected (HPLC, immunoaffinity columns) in spore extracts (0.3-28 µg/mL) of 5/6 AO strains isolated in November 2012, while none of tested EH strains were able to produce OTA. Taking into account maximum detected concentration of OTA in the spores, daily intakes of OTA by inhalation were calculated to be 0.08 and 3 ng/kg b.w., which is below the tolerable daily intake of OTA (17 ng/kg b.w.). Cytotoxicity of pure OTA, OTA-positive (AO-OTA+) and OTA- negative (AO-OTA-) spore extracts, as well as spore extract of EH, was tested on human lung adenocarcinoma cells A549, individually and in combination, using colorimetric MTT test (570 nm). Concentrations that decreased the cell viability by 50% (IC50) were as it follows: pure OTA (53 μg/mL) ; AO-OTA+ (mass concentration 934 μg/mL corresponds to 10.5 μg/mL of OTA in spore extract) ; and 2126 μg/mL for EH. Highest applied concentration of AO-OTA- spore extract (4940 g/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Combinations EH + AO-OTA+ and EH + AO-OTA- applied in subtoxic concentrations showed dominant additive interactions. Also, synergistic effect was obtained for one combination of EH and AO- OTA+. Despite the low calculated daily intake of OTA by inhalation, chronic exposure to high levels of OTA-producing airborne fungi in combination with other more or less toxic moulds pose significant threat to human health due to their possible additive and/or synergistic interactions.