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5-metoksisterigmatocistin (5-MET) je slabo istraženi mikotoksin za kojeg je zabilježeno da ima citotoksična i genotoksična svojstva. Strukturno je srodan sterigmatocistinu (STC), zajedno s kojim ga mogu proizvoditi plijesni roda Aspergillus serije Versicolores. Te su plijesni često prisutne u zatvorenim vlažnim i prašnjavim prostorima. Takvi prostori mogu biti skladišta i proizvodni pogoni u pivovarama. S obzirom na to da je pivo u Hrvatskoj konzumirano u velikim količinama, prisutnost ovih aspergila i biosinteza mikotoksina predstavljaju javnozdravstveni rizik ako je pivo kao krajnji proizvod kontaminirano 5-MET. Za analizu je skupljeno 58 različitih uzoraka piva s hrvatskog tržišta. Ekstrakcija je provedena korištenjem acetonitrila kao otapala, isoljavanjem i propuštanjem uzorka kroz SPE kolonu. Pripremljeni ekstrakti analizirani su tankoslojnom kromatografijom. Stacionarna faza bio je silikagel 60, a mobilna faza smjesa toluena, etil-acetata i mravlje kiseline (90% v/v) u omjeru 5:4:1. Za pojačanje fluorescencije ploče su sušene, grijane i prskane etanolnom otopinom aluminijeva klorida. Limit detekcije za 5-MET iznosio je 3.2 μg/mL. 5-MET je detektiran u 27 od 58 (46,55%) uzoraka. Njegova prisutnost potvrđena je UV-Vis spektroskopijom nakon ekstrakcije strugotine s TLC ploče. Rezultati ovog istraživanja predstavljaju prvi nalaz 5-MET u pivu. Također, potvrđuju prikladnost primijenjene metode te predstavljaju signal za potencijalnu javnozdravstvenu opasnost, imajući na umu ogromnu potrošnju piva. Istraživanja koja uključuju veći broj uzoraka te primjenjuju sofisticiranije kromatografske tehnike (poput LC/MS) omogućila bi precizniju interpretaciju ovih rezultata i kvantitativnu analizu 5-MET. Uz to, omogućila bi osjetljiviju detekciju (detekciju 5-MET u manjim koncentracijama) i selektivniju detekciju (s većom razinom sigurnosti da je u pitanju 5-MET a ne neki strukturno srodan spoj). 5-methoxysterigmatocystin (5-MET) is unsufficiently investigated cytotoxic and genotoxic mycotoxin. It is structurally related to sterigmatocystin (STC) and produced namely by molds from the genus Aspergillus, series Versicolores. These molds are frequently found in damp and dusty indoor environments. In beer production, storage and production spaces fit this description. Given the high consumption of beer in Croatia, presence of these Aspergillus molds amd their production of mycotoxins constitutes a public health risk if beer in its end product state is contaminated with 5-MET. Fifty eight different samples of beer marketed in Croatia were collected for the purposes of this analysis. The extraction was performed by acetonitrile as a solvent, salt-induced precipitation and phase extraction with SPE columns. The prepared extracts were analysed by thin-layer chromatography. Silica gel 60 was used as stationary phase and a 5:4:1 mixture of toluene, ethyl-acetate and formic acid (90% v/v) as mobile phase. To enhance fluorescence, TLC plates were dried, heated and sprayed with an ethanol solution of aluminium chloride. Limit of detection (LOD) for 5-MET was 3.2 μg/mL. 5-MET was detected in 27 of 58 (46,55%) samples. Its presence was confirmed by UV-Vis spectroscopy following the extraction of the spot of interest from the TLC plate. This is the first time 5-MET has been detected in beer. Additionally, this study confirms the suitability of the applied method and serves as a signal for a potential public health risk, considering the high levels of beer consumption. Research that includes a larger number of samples and uses more sophisticated analytic techniques (such as LC/MS) would allow for a more accurate interpretation of these results and enable quantitative analysis. Additionally, it would increase sensitivity (making detection of smaller quantities of 5-MET possible) and selectivity (the likelihood that 5-MET is detected, as opposed to structurally related compounds).

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.

Sterigmatocistin (STC) je poliketidni mikotoksin kojeg proizvode neke vrste plijesni iz roda Aspergillus. Strukturno je sličan aflatoksinu B1 (AFB1) te se unsen hranom u jetri, kao i AFB1, pomoću enzima iz porodice citokroma P450 metabolizira u epoksid koji stvara adukte s DNA djelujući hepatotoksično i hepatokancerogeno. Međutim, na temelju istraživanja na mužjacima štakora zaključeno je da STC zbog slabije bioraspoloživosti ima za tri reda veličine manji hepatokancerogeni potencijal od AFB1. Plijesni iz roda Aspergillus sekcije Versicolores primarni su kolonizatori vlažnih zidova gdje mogu proizvoditi STC i njegove derivate, primjerice 5-metoksisterigmatocistin (5-MET-STC) te se u organizam mogu unijeti inhalacijom. Istraživanja na poksunim životinjama pokazuju da mikotoksini uneseni inahlacijom imaju do deset puta jači toksični učinak nego pri unosu ingestijom. Naša istraživanja u poplavljenom području općine Gunja pokazuju da je dvije godine nakon poplave (2014.) u obnovljenim kućama vidljiva kolonizacija zidova plijesnima, a u zraku i kućnoj prašini dominirale su aspergile iz sekcije Versicolores. U kućnoj prašini Gunje i u kontrolnom selu Gornji Stupnik dominirali su STC i 5-MET-STC ; STC je u detektiran u 70% uzoraka prašine u Gunji i 50% uzoraka prašine u Gornjem Stupniku, dok je 5-MET-STC bio podjednako zastupljen (50%) na obje lokacije. Maksimalne koncentracije ovih toksina su bile 1, 3 do 8 puta veće u Gunji negoli u Gornjem Stupniku (maksimum STC 0.59 μg/g, 5-METSTC, 7, 70 μg/g). Prema podacima iz literature dnevno inhaliramo 10-12 m3 zraka u kojem je prosječna maksimalna koncentracija respirabilnih čestica 54 μg/m3 Uzimajući u obzri maksimalne koncentracije STC i 5-MET-STC detektirane u kućnoj prašini u Gunji možemo pretpostaviti da čovjek dnevno udisanjem može unijeti 0.4 μg STC, odnosno 5 μg 5-METSTC. Na temelju spomenute procjene izloženosti i naših dosadašnjih istraživanja mehanizma djelovanja STC in vitro postavili smo hipotezu: a) STC i 5-MET-STC uzrokuju genotoksičan učinak u plućima štakora nakon jednokratnog tretiranja intartrahealnom instilacijom u dozama koje su detektirane u kućnoj prašini vlažnih zatvorenih prostora ; b) STC ima jači genotoksični potencijal od 5-metoksi-STC ; c) uzimajući u obzir sličnost u kemijskoj strukturi istodobno aplicirani toksini imat će antagonistički učinak. U pokusu intratrahealne instilacije korišteni su 3 mjeseca stari mužjaci štakora soja Wistar. Životinje raspoređene u 4 skupine (po 6 životinja) jednokratno su tretirane s STC (0, 4 ug/mL), 24 5-MET-STC (5 ug/mL), STC+5-MET-STC (0, 4+5 ug/mL), 0, 3% dimetilsulfoksidom (DMSO) (kontrola) te su 24 sata nakon tretmana žrtvovane. Genotoksično djelovanje u homogenatu pluća ispitano je primjenom alkalnog i neutralnog kometnog testa. Kao parametri kometnog testa koji pokazuju oštećenje DNA uzeti su duljina repa (TL) i intenzitet repa (TI). Statistička analiza mjerenih parametara je napravljena primjenom programskog jezika R ; razlike između svih parova tretmana testirane su Tukeyevim testom za razinu značajnosti p

Predavanje je održano u sklopu Godišnje Skupštine Hravatskog toksikološkog društva na kojem su se predstavljale teme koje su zastupljene u časopisu Arhiv za higijenu rada i tokikologiju, a mikotoksini su jedna od zastupljenih tema. Predstavljeni su razultati prikazani u sklopu pozvanog predavanja održanog na Balkan Fungus 2018. u Temišvaru (Rumunjska) čiji je sažetak pod brojem 966716.

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.

The purpose of this study was to investigate post-flood mycological patterns in Gunja compared to an unaffected area (Gornji Stupnik) in Croatia and to compare qualitative and quantitative composition in repaired and unrepaired houses in flooded area in winter and summer sampling period. During 2016 in each sampling period twenty indoor airsamples (IAS), five outdoor airsamples (ODAS) and ten dust samples (DS) were taken at five repaired (RH) and five unrepaired houses (UNRH) in Gunja, five control houses in Gornji Stupnik (SH) and ten IAS and two 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). Dichloran 18%- Glycerol Agar (DG-18) and Malt Extract Agar (MEA) were used for AS, while for DS and FS Dichlorane Rose-Bengal Chloramphenicol Agar (DRBC) and DG-18 were used. Similar patterns of aeromycota were observed in both sampling periods at the locations specified, while the concentrations of airborne fungi were significantly higher in summer period than in winter period, as well as in flooded compared to control area. The highest concentrations of aeromycota were measured in UNRH (IAS and ODAS). Penicillium, Cladosporium and Aspergillus (sections Aspergillus, Versicolores, Circumdati, Nigri) genera were prevailing although in EES Rhizopus sp. were frequently isolated. Aspergilli from the section Versicolores were less common in UNRH compared to RH and SH. Concentration of fungi was significantly higher in DS from Gunja compared to control area. In winter period the highest concentrations were observed in RH while in summer period they were higher in UNRH. Although the composition of dustmycota corresponded to aeromycota, Aspergilli (Versicolores) were more frequent in DS. Higher concentrations of fungi were observed in FSS than in FSG in winter while they were similar in summer. Fusarium spp. were the dominating species in both sampling periods. The most frequent Aspergilli were those assigned to the section Aspergillus both in FSG and FSS followed by those from the section Flavi, Circumdati and Nigri, especially in summer period. Significantly higher concentrations of airborne and dustborne fungi in flood affected compared to control area increase the health risk for humans due to inhalatory exposure to potential mycotoxin producers. These results underline the issue of yet unrepaired houses in flood affected area as these represent the significant source of secondary and tertiary colonizers. Acknowledgements: This work has been fully supported by Croatian Scientific Foundation under the project MycotoxA (IP-09- 2014-5982)

Purpose: Samples from different environmental sources were screened for the presence of Aspergillus, and the distribution of the different species-complexes was determined in order to understand differences among that distribution in the several environmental sources and which of these species complexes are present in specific environmental settings. Methods: Four distinct environments (beaches, poultries, swineries and hospital) were studied and analyzed for which Aspergillus complexes were present in each setting. After plate incubation and colony isolation, morphological identification was done using macro- and microscopic characteristics. The universal fungal primers ITS1 and ITS4 were used to amplify DNA from all Aspergillus isolates, which was sequenced for identification to species complex level. SPSS v15.0 for Windows was used to perform the statistical analysis. Results: Thirty-nine isolates of Aspergillus were recovered from both the sand beach and poultries, 31 isolates from swineries, and 80 isolates from hospital environments, for a total 189 isolates. Eleven species complexes were found total. Isolates belonging to the Aspergillus Versicolores species-complex were the most frequently found (23.8%), followed by Flavi (18.0%), Fumigati (15.3%) and Nigri (13.2%) complexes. A significant association was found between the different environmental sources and the distribution of the several species-complexes (p