Fumonisin production and toxic capacity in airborne black Aspergilli

Accepted Manuscript Fumonisin production and toxic capacity in airborne black Aspergilli

Daniela Jakšić, Sándor Kocsubé, Ottó Bencsik, Anita Kecskeméti, András Szekeres, Dubravko Jelić, Nevenka Kopjar, Csaba Vágvölgyi, János Varga, Maja Šegvić Klarić PII: DOI: Reference:

S0887-2333(18)30443-0 doi:10.1016/j.tiv.2018.08.006 TIV 4346

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

1 January 2018 11 June 2018 10 August 2018

Please cite this article as: Daniela Jakšić, Sándor Kocsubé, Ottó Bencsik, Anita Kecskeméti, András Szekeres, Dubravko Jelić, Nevenka Kopjar, Csaba Vágvölgyi, János Varga, Maja Šegvić Klarić , Fumonisin production and toxic capacity in airborne black Aspergilli. Tiv (2018), doi:10.1016/j.tiv.2018.08.006

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ACCEPTED MANUSCRIPT Fumonisin production and toxic capacity in airborne black Aspergilli Daniela Jakšić1, Sándor Kocsubé2, Ottó Bencsik2, Anita Kecskeméti2, András Szekeres2, Dubravko Jelić3, Nevenka Kopjar4, Csaba Vágvölgyi2, János Varga2, Maja Šegvić Klarić1*

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Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb,

Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-

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Schrottova 39, 10000 Zagreb, Croatia

6726 Szeged, Közép fasor 52, Hungary

Fidelta Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia

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Mutagenesis Unit, Institute for Medical Research and Occupational Health, Ksaverska cesta

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2, 10000 Zagreb, Croatia

*Corresponding author:

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Maja Šegvić Klarić

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Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Schrottova 39, 10000 Zagreb, Croatia

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E-mail: [email protected]; tel/fax: +385 1 6394 493 / 6394 494 During the execution of the experimental work for this paper, we were struck by the

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unexpected demise of Professor János Varga. We dedicate this paper to the memory of our beloved friend and colleague.

ACCEPTED MANUSCRIPT Abstract This study presents the distribution of fumonisin (FB)-producing and non-producing airborne Aspergilli (Nigri) in apartments (AP), basements (BS) and a grain mill (GM) in Croatia, and their cytotoxic, immunomodulation and genotoxic potency in comparison with FB1 and FB2. Concentration of black Aspergilli was 260-fold higher in GM than in living environment with

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domination of A. tubingensis and A. welwitschiae. FB2- but not FB1- was confirmed via

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HPLC-MS and detection of fum1 and fum8 genes for one isolate of A. niger (0.015 µg/mL)

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and 8/15 isolates of A. welwitschiae (0.128-13.467 µg/mL). After 24 h, both FB1 and FB2 were weakly cytotoxic (MTT assay) to human lung A549 cells and THP-1 macrophage-like

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cells. In THP-1 cells FB1 but not FB2 provoked a higher increase of IL-8, TNF- and IL-1 (ELISA). In A549 cells DNA damaging effect of FB1 was slightly higher than that of FB2

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(Comet assay). In THP-1 macrophage-like cells A. tubingensis and A. piperis evoked immunomodulatory effects which corresponded to their cytotoxicity (IC50 = 0.228-0.323

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mg/mL); A. welwitschiae, A. tubingensis and A. piperis exerted cytotoxic (IC50 = 0.214-0.460

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mg/mL) and genotoxic effects in A549 cells. Presumably, secondary metabolites other than

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FB2 may have contributed to the toxicity of black Aspergilli.

damage

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Keywords airborne fungi, A. welwitschiae, A. tubingensis, fumonisins, cytotoxicity, DNA

ACCEPTED MANUSCRIPT 1. Introduction The Aspergillus section Nigri comprises several species widely distributed in the environment. These species deserve special attention regarding their influence on human and animal health: 1) from the agricultural point of view, members of the section Nigri occur as plant pathogens and cause spoilage of various foodstuffs including grains, fruits, vegetables,

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cotton, nuts, meat and dairy products (Perrone et al., 2007; Varga et al., 2011); 2) in

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biotechnology some species are used for the production of organic acids, hydrolytic enzymes,

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and fermented foods (Hong et al., 2013; Samson et al., 2007); 3) in medical mycology increasing numbers of infections caused by black Aspergilli are reported, particularly

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otomycosis and keratomycosis in tropical and semitropical regions (Kredics et al., 2008; Szigeti et al., 2012a, 2012b); 4) from the toxicological point of view black Aspergilli are able

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to produce mycotoxins including fumonisin isomers and ochratoxin A (OTA), which are mainly introduced into organisms by contaminated food (Samson et al., 2007, 2004; Varga et

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al., 2011), but can also be taken in via inhalation.

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Since their discovery, fumonisins have been considered mycotoxins mainly produced by Fusarium species (Logrieco et al., 2003). The B group of fumonisins (FB1, FB2, and FB3) is

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frequently detected in maize and maize-based foods. The toxicological properties of FB1 as the major group of fumonisins in Fusaria have been extensively studied. The consumption of

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FB1 causes species-specific toxicological effects in animals including leukoencephalomalacia in horses and pulmonary oedema in swine. It is a hepatotoxic, nephrotoxic, carcinogenic and immunosuppressive agent in laboratory animals that has been associated with human oesophageal carcinoma in southern Africa and China (JECFA, 2001). Based on the structural similarities of FB1-3, a group provisional maximum tolerable daily intake (PMTDI) of 2 µg/kg b.w. per day for FB1-3 was established (EFSA, 2014) In Fusarium species, fum genes encode enzymes involved in the biosynthetic pathway of FBs.

ACCEPTED MANUSCRIPT Cluster genes fum1 and fum8 encode a polyketide synthase and an R-oxoamine synthase, respectively, which are responsible for the synthesis of the FB1-3 carbon backbone (Alexander et al., 2009a; Proctor et al., 2006; J. Seo et al., 2001). Other fum genes (e.g., fum2, fum3, fum6, fum10, fum13, fum14) encode enzymes that catalyse the addition or reduction of functional groups along the carbon backbone and their inactivation results in the production

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of FB2 or FB3 only (Alexander et al., 2009b).

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Frisvad et al. (2007) first reported on FB2 production in black Aspergilli and since then

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research on fumonisin production and the gene cluster required for fumonisin isomer biosynthesis in black Aspergilli has been increasing. So far fumonisin production has been

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confirmed in only two species, A. niger and A. welwitschiae (formerly A. awamori) (Palumbo et al., 2013; Perrone et al., 2011a; Varga et al., 2011, 2010). The research of fum genes

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orthologues in A. niger genome revealed the presence of the polyketide synthase (fum1), hydroxylase (fum3, fum6, fum15), dehydrogenase (fum7), aminotransferase (fum8), acyl-CoA

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synthase (fum10), carbonyl reductase (fum13), condensation-domain protein (fum14), ABC

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transporter (fum19), and transcription factor (fum21) genes and absence of fum2 gene (Pel et al., 2007; Susca et al., 2014). The absence of fum2 in A. niger, which encodes for

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monooxygenase that catalyses the hydroxylation of the fumonisin backbone at C-10, results in the production of FB2, FB4 and FB6, but the absence of FB1 and FB3 synthesis (Månsson et

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al., 2010; Proctor et al., 2006). The presence of fum1 and fum8 genes was already been previously shown essential for the production of FB2 in black Aspergilli (Susca et al., 2014, 2010). However, one study revealed the production of FB1 in black Aspergilli recovered from dried vine fruits which was confirmed by HPLC-MS analysis of the extracts (Varga et al., 2010). Although black Aspergilli are common airborne fungi, the distribution and species diversity of this group in indoor air is poorly investigated and is usually referred as to merely A. niger.

ACCEPTED MANUSCRIPT Recently, we were the first to report on the diversity of black Aspergillus species isolated from living (apartments, basements) and occupational (grain mill) air in Croatia and five species were determined by morphological and molecular methods (calmodulin gene sequence, CaM), including A. luchuensis, A. niger, A. piperis, A. tubingensis, and A. welwitschiae (Varga et al., 2014). The aim of this study was to check their ability to produce

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the FB1 and FB2 and confirm that by the production capacity accompanied by the presence of

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fum1 and fum8 genes. Since black Aspergilli were common in the mixture of airborne fungi in

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the occupational and living environment, we believed it was justified to explore the toxic properties of FB-producing and FB-non-producing Aspergilli microextracts in comparison

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with single FB1 and FB2. This was carried out using human lung adenocarcinoma A549 cells and macrophages derived from human leukemic monocyte THP-1 cells as experimental

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models. 2. Materials and methods Chemicals

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2.1.

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FB1 and FB2 standards, methanol (MeOH) and formic acid were purchased from SigmaAldrich Ltd. (Budapest, Hungary). Acetonitrile (MeCN) was obtained from VWR

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International Ltd. (Debrecen, Hungary). HPLC grade water with a resistivity of 18 MΩ was produced by ultrafiltration with a Millipore Milli-Q Gradient A10 water purification system

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(Merck Ltd., Budapest, Hungary).

2.2.

Sample collection

Airborne fungi were collected over a one-year period (2012) in two-month intervals at a grain mill (GM) situated near Zagreb, Croatia, and in residential locations which included two apartments (AP) and two basements (BS) as well as outdoor air (ODA). Locations, dynamic and conditions of sampling were described in detail in Jakšić Despot and Šegvić Klarić

ACCEPTED MANUSCRIPT (2014). Briefly, each two-month interval 20 samples were taken at each indoor location and 10 samples outdoors, the total number of samples was 420. Airborne fungi were sampled using an Airsampler Mas-100 Eco and dichloran 18% glycerol agar (DG-18) plates (Samson et al., 2010). The plates were incubated at 25°C ± 0.2°C, followed by subculturing of black Aspergilli on Czapek Yeast Agar (CYA) and Malt Extract Agar (MEA) at 25°C in the dark

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for seven days (Samson et al., 2010). . Morphological identifications were carried out

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according to the literature (Samson et al., 2007, 2010), while species identification was

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performed by sequencing a part of the calmodulin gene published previously in Varga et al.

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(2014).

2.3. Preparation of extracts for fumonisin analysis

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A. welwitschiae (N=15), A. tubingensis (N=10), A. luchuensis (N=4), A. piperis (N=1) and A. niger (N=1) isolates were cultivated on Czapek yeast autolysate (CYA) agar in darkness at

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25°C ± 0.2°C for 7 days. The extraction procedure included extracting 4 CYA agar plugs (in a

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diameter of 6 mm) with 1 mL of 75% methanol (Frisvad et al., 2007). The extracts were centrifuged at 10,000 g for 10 min, supernatants were collected and transferred to clean vials.

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Prior to LC/MS analysis, the prepared extracts were filtered through 0.45 µm filters and

2.4.

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transferred to microvials.

HPLC and MS parameters

The extracts were analyzed by HPLC-MS technique as described by Bartók et al. (2010), with minor modifications. The applied modular HPLC system (Shimadzu, Japan) was equipped with a DGU-14A vacuum degasser, LC-10AD pump, Merck Hitachi L-7200 autosampler, Shimadzu CTO-10AS column thermostat, and SCL-10A system controller and coupled with the 2010A MS (Shimadzu, Japan). The whole HPLC-MS system was controlled by

ACCEPTED MANUSCRIPT LCMSsolution (ver. 3.60.361) software. Separations were performed on a YMC-Pack J'sphere ODS H80 (YMC Europe GmbH, Dinslaken, Germany) (50mm×2.1 mm, 4 μm) HPLC column thermostated at 35°C. The mobile phase consisted of H2O (A) and MeCN (B) supplemented each with 0.1% (v/v) formic acid with a flow rate of 0.2 mL/min. The gradient elution was started with 15% B, which was increased linearly after one minute to 40% for 4 min and

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maintained at this value for 10 min; then, the B ratio was raised up to 95% for 2 min and this

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value was maintained for 6 min; then it was dropped down to the initial 15% in two minutes

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and remained constant until pressure stabilized. The run time was 38 min. The MS instrument was equipped with an ESI source and operated in positive ion mode using nitrogen as both the

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nebulizing gas (1.5 L/min) and the drying gas (0.2 bar). Positive ions were acquired in selected ion monitoring mode (event time, 0.2 s; detector voltage, 1.5 kV), while the voltages

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of the interface, curved desolvatation line (CDL) and Q-array were 4.5 kV, 25 V and 25 V, respectively, and the CDL and heat block temperatures 250 °C and 200 °C, respectively.

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During the measurements, the protonated molecular ions ([M+H]+) of FB1 (m/z 722) and FB2

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(m/z 706) were recorded, and their amount found in the samples was calculated by an external standard method using six calibration levels within the concentration range of 15.688 ng/mL

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to 502 ng/mL (FB1) and 15.625 ng/mL to 500 ng/mL (FB2) dissolved in MeCN/H2O 50/50 (v/v). The equations of the resulted calibration curves were y=14.82537x + 18.98403;

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R2=0,997 (FB1) and y=14.03072x + 156.9180; R2=0,998 (FB2).

2.5.

PCR detection of fum1 and fum8 genes

The isolated genomic DNA (Varga et al., 2014) was diluted to < 1.000 ng and used as template DNA for fumonisin biosynthetic gene specific PCR reactions. Part of polyketide synthase and R-oxoamine synthase genes (fum1 and fum8, respectively; Alexander et al.,

ACCEPTED MANUSCRIPT 2009; Proctor et al., 2006; Seo et al., 2001) were amplified in 20 μl volume containing 10X DreamTaq Buffer (with 1.5 mM MgCl2) (Thermo Scientific, USA), 0.2 mM dNTPs, 0.2µmol of primers (Table 1), 1 U DreamTaq DNA Polymerase (Thermo Scientific), nuclease free water and template DNA. Amplifications were performed in Termocycler T-100 (BioRad, Budapest, Hungary), while the PCR cycling protocol consisted of 35 cycles and included an

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initial denaturation at 95 °C for 2 min in the first and 20 s in the following runs, annealing at

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56 °C for 20 s; elongation at 72 °C for 40 s and 2 mins for the final elongation. The

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amplification products were analysed by electrophoresis on 2 % agarose gels using

Cytotoxicity, immunomodulation and genotoxicity of Aspergilli extracts and

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2.6.

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fluorescent dye GR Green (Excellgen, Budapest, Hungary).

single FB1 and FB2

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2.6.1. Cell culture and treatment

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Human lung adenocarcinoma cells A549 and human leukaemia monocytes THP-1 (European Collection of Cell Cultures, UK) were grown in 75 cm2 flasks in RPMI supplemented with 2

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mM glutamine, 10% heat-inactivated FBS, penicillin (100 IU/mL; 1 IUE 67.7 μg/mL) and streptomycin (100 μg/mL) at 37 °C in 5% CO2. Stock solutions of FB2 and FB1 (1 mg/mL)

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were prepared in sterile water and dried fungal extracts were prepared in 100% DMSO. The final concentrations of FBs and fungal extracts as well as DMSO were obtained by dilution with the culture medium. 2.6.2. MTT proliferation assay MTT proliferation test was used to test the cell viability of A549 cells and macrophage-like THP-1 cells as described previously (Jakšić Despot et al, 2016). Briefly, the differentiation of THP-1 cells into macrophages was performed using phorbol 12-myristate 13-acetate (PMA)

ACCEPTED MANUSCRIPT (20 nm/well). Prior to the experiment, Aspergilli extracts dissolved in DMSO were diluted with culture medium and the final mass concentrations ranged from 0.1 to 0.8 mg/mL.. DMSO (up to 1%) was used as the control that did not alter cell viability. The A549 (104 cells/well) and macrophage-like THP-1 cells (5x104 cells/well) were grown in a 96-well flatbottom microplate in RPMI 1640 medium supplemented with 10% of FBS. Upon 24 h

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treatment with FBs (10 to 150 µM) or with Aspergilli extracts (0.1 to 0.8 mg/mL) of FB2-

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producing and FB2-non-producing strains, the concentration that inhibits growth in 50% of

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cells (IC50) was determined. Following treatment, the medium was removed and 100 μL of MTT-tetrazolium salt reagent diluted in RPMI without FBS (0.5 mg/mL) was added into each

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well. After 3 h of incubation, the medium was replaced with 100 μL of DMSO to dissolve formazan (product of metabolised MTT reagent), and cells were incubated at room

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temperature on a rotary shaker for 15 min. The absorbance was measured using a microplate reader (Labsystem iEMS, type 1404) at a wavelength of 540 nm. All tests were performed in

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five replicates and results were expressed as percentage of control. 2.6.3. Measurement of cytokine concentration THP-1 cells were seeded on 24-well cell culture plates (1x106 cells per well) and

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differentiated for 24 h with 40 nM PMA. Cells were treated with FB1 and FB2 (10 and 100

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µM each) as well as black Aspergilli extracts (0.1 mg/mL) for 24 h. Cell medium was harvested and frozen at -80° C until analysis. Concentrations of IL-1β, IL-6, IL-8 and TNF-α were determined in harvested cell medium by DuoSet ELISA kits (R&D systems, USA) according to instructions provided by the manufacturer and as described elsewhere (Hulina et al., 2017). Cytokines concentrations were calculated from measured optical densities determined at 450 nm, using a microplate reader (En Vision® Multilabel Plate Reader, Perkin Elmer) and by standard calibration curves.

ACCEPTED MANUSCRIPT 2.6.4. Alkaline comet assay A549 cells were seeded in 6-well plates (3x105 cells per well). After 24 h, the cell medium was replaced with the treatment: FB1 and FB2 (10 and 100 µM each), black Aspergilli extracts (0.1 mg/mL) and control medium (with and without 0.1 % DMSO) for 24 h. The comet assay was carried out according to Singh et al. (1988) with minor modifications. After 24 h of

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treatment, the cells were washed, scraped with rubber, and resuspended in 300 μL of

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phosphate buffer (pH 7.4). Aliquots of 20 μL of this suspension were mixed with 100 μL

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0.5% low melting point agarose-LMP (in Ca-and Mg-free PBS) and 100 μL of agarose-cell suspension was spread onto a fully frosted slide pre-coated with 1% normal melting point

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tagarose- NMP (in sterile destiled water). After 1 h of lysis at 4 °C, denaturation for 20 min and electrophoresis for 20 min at 25 V and 300 Microgels were neutralized and then stained

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with ethidium bromide (20 μg/mL) per slide for 10 min. Slides were scored using an image analysis system (Comet assay IV, Perceptive instruments Ltd., UK) connected to a

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fluorescence microscope (Leitz, Germany). All of the experiments were performed in

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duplicate, and in each experiment images of 200 randomly selected cells (100 cells from each of the two replicate slides) were measured. Only comets with a defined head were scored. As

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a reliable measure of genotoxicity, we used tail length and the percentage of DNA in the

2.7.

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comet tail (or tail intensity) (Olive, 1999; Collins, 2004).

Statistics

The results of the MTT test, comet assay (tail length- μm; tail intensity- % of tail DNA) and concentration of cytokines (% of control) were presented as mean ± standard error of mean (SEM). Normality of the data distribution was tested by Kolmogorov-Smirnov test; One-wayANOVA was applied for normally distributed data followed by Sidak post-test, while for nonnormally distributed data Kruskal- Wallis test was applied followed by Dunn's multiple

ACCEPTED MANUSCRIPT comparison test. P<0.05 was considered statistically significant for all calculations. To obtain the IC50 from the results of MTT assay, non-linear dose-response fitting was applied using y = A1 + (A2-A1)/(1 + 10^((LOGx0-x)*p)) equation. Two-way ANOVA followed by Sidak's multiple comparison test were used for testing the significance of difference in the toxic

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response of two cell lines to black Aspergilli extracts.

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3. Results

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3.1. Occurrence of airborne black Aspergilli

The abundance of airborne Aspergillus species from the section Nigri in occupational (GM),

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and living (AP and BS) environments as well as outdoor air (ODA) in the six periods of

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sampling is presented in Table 2. The proportion of black Aspergilli in the total number of viable airborne fungi regardless of location and period of sampling was low (0.05-4.4%), with mean absolute concentrations ranging from 1 at AP/BS to 800 CFU/m3 at GM and mean

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estimated concentration ranging from 22.5 to 1550 CFU/m3 at GM. The mean absolute concentrations of black Aspergilli were approximately 10 times higher in the GM compared to the levels detected in the living indoor environment (AP and BS) and outdoor air. Black

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Aspergilli peaked in May (GM), when the estimated concentration (probable viable number)

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rise up to 5x103 CFU/m3, while in the remainder of the sampling periods, the estimated levels were below 500 CFU/m3. The recovery of aspergilli (Nigri) from samples taken both indoors (AP and BS) and outdoors was poor; the maximum absolute concentrations were below 100 CFU/m3, except in the AP in March (140 CFU/m3). Additionally, black Aspergilli were detected outdoors only in warmer months (July and September). 3.2. The ability of black Aspergilli to produce fumonisins Among the identified isolates of black Aspergilli, which include A. welwitschiae (N=15), A. tubingensis (N=10), A. luchuensis (N=4), A. piperis (N=1) and A. niger (N=1), FB2 was

ACCEPTED MANUSCRIPT quantified in 8 out of 15 isolates of A. welwitschiae and the A. niger isolate. FB1 was not detected in any sample (Table 3.; Fig.1). Among A. welwitschiae isolates that produced FB2, seven isolates were recovered from the GM and one from the AP samples, while the A. niger isolate was originated from the living area BS. Molecular screening for the presence of fum1 and fum8 genes revealed that the majority of the isolates that produced FB2 in the agar plug

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test also present these genes, except one strain of A. welwitschiae that yielded an amplicon

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from fum8, but not from fum1. Two strains of A. welwitschiae produced approximately 10

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µg/mL of FB2; three isolates produced FB2 in amounts between 3 and 8 µg/mL; and three isolates produced FB2 at concentrations below 1 µg/mL. The weakest producer was A. niger

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(0.015 g/mL) with an FB2 level near to the lowest level of calibration. FB2 was not found at a detectable level in the isolates belonging to the A. tubingensis, A. luchuensis and A. piperis

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species and the absence of amplification of fum1 and fum8 support that these isolates are not

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3.3. Cytotoxic effects

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FB2 producing fungi.

The MTT test revealed that both FB1 and FB2 were weakly cytotoxic to A549 cells and

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macrophages derived from THP-1 cells; although all of the applied concentrations of single FBs (10-150 M) significantly affected cell viability compared to control at 24 h of exposure.

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Nevertheless none of them was sufficient to induce a reduction of more than 50% in the cell metabolic activity (IC50) of A549 or THP1 cell lines (Fig.2). THP-1 macrophage-like cells were more sensitive to FBs than A549 cells, particularly to FB1; the viability of THP-1 macrophage-like cells dropped to 60% after exposure to the highest concentration of FB1. Unlike FBs, black Aspergilli extracts of FB2-producers and non-producers were more cytotoxic for both cell lines (Table 4.; Fig. 3). FB2 content in A. welwitschiae (0.09-0.76 µM of FB2) and A. niger (0.000088-0.0007 µM of FB2) extract dilutions (0.1-0.8 mg/mL) was

ACCEPTED MANUSCRIPT between one to five orders of magnitude lower that the lowest concentration of single FB2 (10 M) used in the MTT test. Considering the calculated IC50 of extract mass concentrations, A. welwitschiae was the most cytotoxic to A549 cells, followed by A. tubingensis and A. piperis. According to IC50 values, the last two exerted similar cytotoxic potency in both cell lines (Table 4). The dose-response for A. niger and A. luchuensis extracts in both cell lines was

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similar and the calculated IC50 was > 0.8 mg/mL.

3.4. Immunomodulatory effects

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FB1 application resulted in the increase of all of the measured cytokines (IL-1β, IL-6, IL-8

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and TNF-α), while the most pronounced effect was on IL-8 (Fig. 4). An interesting pattern could be observed comparing the effect on IL-1β and IL-6. Applied at a concentration of 10

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μM, FB1 caused the increase of IL-6, IL-1β was not different than the control, while a ten times higher concentration of FB1 caused a reversed effect: the increase of IL-1β and the

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decrease of IL-6. In both cases, the concentration of TNF-α remained increased. In contrast to

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FB1, FB2 provoked a significant decrease of IL-1β and did not have an effect on IL-8. Both FBs applied in 10-fold lower concentration significantly increased IL-6. Among the extracted

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black Aspergilli, A. piperis and A. tubingensis had the strongest effect causing a significant increase of IL-1β, TNF-α and IL-8 while the concentration of IL-6 was not different than the

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control. Among FB2 producers, A. niger provoked a significant increase of TNF- only, while the other tested cytokines did not differ significantly from the control regardless of whether or not they were treated with A. niger or A. welwitschiae.

3.5. Genotoxic effects Genotoxic effects were evaluated in A549 cells using alkaline comet assay. Generally, the DNA damaging effect of FB1 (in terms of increased tail length and tail intensity) was slightly

ACCEPTED MANUSCRIPT higher than that of FB2. While no significant changes were observed in comet tail length, except slightly when FB2 was applied at 100 μM, a significant increase of tail intensity was observed when FB1 or FB2 was applied at 10 μM. Application of 100 μM FB1/FB2 resulted in a tail intensity similar to the control sample. Application of black Aspergilli extracts caused a significant increase of comet tail length in

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the range: A. tubingensis > A. piperis > A. luchuensis > A. welwitschiae > A. niger. However,

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only A. welwitschiae, A. piperis and A. tubingensis extracts caused a significant increase of

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tail intensity, although the effect was more than twice less pronounced than that of FB1/FB2.

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(Fig. 5).

4. Discussion

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4.1. Indoor occurrence, species diversity and FB2 production in airborne black Aspergilli This study is the continuation of our recently published ones on species diversity of airborne

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black Aspergilli in Croatia (Varga et al., 2014) as well as Aspergillus species from the section

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Versicolores in the occupational (GM) and living (AP and BS) indoor environment over a one year sampling period (Jakšić Despot et al., 2016). Unlike Aspergillus from the section

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Versicolores (0.7-55%), black Aspergilli comprised a significantly lower proportion (0.054.4%) of the total airborne fungi detected in the studied indoor environment. Mean

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concentrations of viable black Aspergilli in the GM (20-240 CFU/m3), as a working environment, are in line with reports on these species in the food industry (220.6 CFU/m3), poultry farms (101.2 CFU/m3), and a bakery (29.5 CFU/m3) (Sharma et al., 2010). Taking into account the statistically estimated concentration of black Aspergilli (5256 CFU/m3) in the GM, the levels of these species are 50-260 times higher compared to those obtained in the AP and BS. Seasonal variations of the proportion of airborne black Aspergilli in the AP (0.084.4%) in Zagreb are comparable to the levels of these species (2.86-6.37%) in urban homes in

ACCEPTED MANUSCRIPT Tekirdag city (Turkey), although Tekirdag has a borderline Mediterranean/humid subtropical climate and Zagreb is situated near the boundary of the humid continental climate (Sen and Asan, 2009). A study conducted in a school in Perth (Australia) during winter and summer revealed that black Aspergilli were common indoor species present at similar levels in both seasons at mean concentrations between 4 to 40 times higher than in the Zagreb AP (Zhang et

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al., 2013). This comes as no surprise because Perth has a hot-summer Mediterranean climate,

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favourable for the growth of xerophilic fungi such as black Aspergilli. A limited number of

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studies on the diversity and occurrence of indoor black Aspergilli have been published so far. The diversity of indoor black Aspergilli in Croatia from our preliminary study was similar to

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other European countries including Hungary, the Netherlands, Serbia and Turkey; A. luchuensis, A. niger, A. welwitschiae and A. tubingensis were detected in all of the listed

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countries, except for A. niger which was not found in samples from Turkey (Varga et al., 2014, 2011). Greater species diversity was obtained from Thailand indoor air samples; among

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the 22 isolates assigned to 7 different species (including A. fijiensis, A. japonicus, A. neoniger,

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A. trinidadensis, and one new species related to A. saccharolyticus), only A. niger and A. tubingensis matched our isolates. Also, a different composition of indoor black Aspergilli was

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reported in the samples taken in 17 US states, Bermuda, Martinique and Trinidad & Tobago (Jurjević et al., 2012), as compared to Croatia; among 56 isolates 6 species were identified

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including A. aculeatus, A. fijiensis, A. floridensis, A. japonicas, A. trinidadensis and A. uvarum. These studies, in addition to previously discussed results on levels of indoor black Aspergilli, confirm the significant influence of climatic conditions on the distribution and occurrence of these fungi in indoor environments. Among the 31 isolates of black Aspergilli obtained in the present study, only that identified as Aspergillus niger (1/1) and some A. welwitschiae isolates (8/15) were shown to have the ability to produce FB2. None of the tested isolates produced FB1, which is in accordance with

ACCEPTED MANUSCRIPT Nielsen et al. (2015). The majority of the FB2-producer isolates (7/9) originated from the GM, showing that workers in occupational indoor environments of this type may be more exposed to FB2 via inhalation than people in living environments. The concentration of FB2 detected in isolates of A. welwitschiae following agar plug extraction was 600 times higher than that of the toxin produced by an A. niger strain. The production of FB2 in these isolates correlated

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with the detection of fum1 and fum8 genes involved in the biosynthesis of fumonisins. The

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results of our study provide additional evidence that the dominant species and significant

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producer of FB2 among black Apergilli is A. welwitschiae. This species was also predominantly isolated from onions (Gherbawy et al., 2015) and grapes in the Mediterranean

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Basin and California, but together with A. niger (Palumbo et al., 2013; Susca et al., 2014). These two species show many similarities in the secondary metabolism, sharing biosynthetic

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pathways for ochratoxin A (OTA), pyranonigrin A, tensidol B, funalenone, malformins naphtho-γ-pyrones as well as FB2 (Jens Christian Frisvad et al., 2007; Perrone et al., 2011a;

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Susca et al., 2010). In this study, we analysed only the production of FB2 but the biosynthesis

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of other secondary metabolites should not be excluded, which was supported by the cytotoxic

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assays.

4.2. Toxic potency of FBs vs. FB2-producing and non-producing black Aspergilli –

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possible health risk 4.2.1. Cytotoxicity

The MTT assay revealed that both FB1 and FB2 were weakly toxic to both human lung A549 cells and THP-1 macrophage-like cells. The A549 cells were equally sensitive to the effects of both FB1 and FB2, while the THP-1 macrophage-like cells were more sensitive to FB1. These results are consistent with literature data on the cytotoxicity of fumonisins in various cell lines, including human bronchoalveolar cells BEAS-2B, in which the IC50 concentrations

ACCEPTED MANUSCRIPT for both FB1 and FB2 were above 150 µM after 24 h of exposure (Gutleb et al., 2002; McKean et al., 2006; Shier et al., 1991). The A. welwitschiae extract was the most toxic among the extracts tested in A549 cells, but it was not cytotoxic to THP-1 macrophage-like cells. Taking into account that the A. welwitschiae extract mass concentration that significantly decreased cell viability contained FB2 at a 10-fold lower concentration than those used in FB2 individual

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treatment, it can be concluded that other metabolites in the extract but not FB2 contributed to

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the cytotoxicity of A. welwitschiae. Since we did not analyse the presence of OTA or other

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secondary metabolites (e.g. pyranonigrin A, tensidol B, funalenone, malformins, naphtho-γpyrones) we can only speculate that some of them contributed to the cytotoxicity. Among

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them, malformins and naphto-y-pyrones exerted cytotoxic properties at nanomolar (IC50 = 130 and 90 nM) and micromolar concentrations (IC50<100 µM) in human cancer cell lines

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(Huang et al., 2011; Liu et al., 2016). Apart from that, elastase production, an important virulence factor in A. fumigatus during respiratory system infection, was also described in

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black Aspergilli (Kothary et al., 1984; Varga et al., 2011). Blanco et al. (2002) showed that A.

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welwitschiae (ex A. awamori) isolates possessed significantly more intensive elastase activity in elastin lyses compared to A. niger. This may also be one of the reasons for the stronger

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cytotoxic effect of A. welwitschiae than A. niger, which was particularly expressed in lung A549 cells. Bünger et al. (2004) showed that the extract of A. niger at a concentration of 800

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mg/mL was cytotoxic to A549 cells. In our study, applied at the concentration of 0.8 mg/mL, A. niger extract reduced the cell viability of both A549 cells and THP-1 macrophages to approximately 54% of the control. It is possible that the A. niger used in the study of Bünger et al. (2004) was misidentified since more accurate tools of identification have been introduced since (Perrone et al., 2011b; Samson et al., 2007). Therefore, it is questionable whether the described toxic effect belongs to the extract of A. niger or to some other species from the section Nigri.

ACCEPTED MANUSCRIPT THP-1 macrophage-like cells were more susceptible to extracts of A. tubingensis and A. piperis than A549 cells, but the concentration of 0.8 mg/mL almost completely reduced the cell viability of both cell lines. This observation is particularly important from the aspect of respiratory toxicity and/or infection in susceptible individuals. Recently, Lamboni et al. (2016) reported on the prevalence of A. tubingensis in nuts from Benin, which produced

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naphto-y-pyrones (auraspenones, fonsecinones, flavasperones), asperazine and malformins at

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high rates. In addition to cytotoxic naphto-y-pyrones and malformins, asperazine showed

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selective cytotoxicity against human leukaemia cells (Bugni and Ireland, 2004). Recently, A. tubingensis was detected as the most common black Aspergilli after A. niger in clinical

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isolates of patients with chronic respiratory diseases, including cystic fibrosis and chronic obstructive pulmonary disease (Gautier et al., 2016). Additionally, A. tubingensis was

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presented in patients with cutaneous infections and otomycoses (Balajee et al., 2009; Szigeti et al., 2012a, 2012b) as well as corneal infections in tropical regions (Kredics et al., 2008). A.

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piperis was reported to produce pyranonigrins, naphto-y-pyrones and aflavinins.

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Pyranonigrins and aflavinins showed insecticidal properties, but pyranonigrins did not exert cytotoxicity in human cancer cell lines (Hiort et al., 2004; Wang et al., 1995), while, to the

lines.

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best of our knowledge, there are no data on cytotoxic properties of aflavinins in human cell

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4.2.2. Immunomodulation When macrophages are exposed to inflammatory stimuli they produce cytokines including TNF-, IL-1, IL-6, IL-8 and IL-12. Thus, we explored the differences in secretion of proinflammatory cytokines (TNF-, IL-1, IL-6, IL-8) by THP-1 macrophage like cells upon exposure to pure FBs or Aspergilli extracts. Endogenous pyrogen TNF- has control of the cytokines network; it is responsible for the production of IL-1, IL-6 and IL-8. Deficiency of TNF-α in experimental animals promoted cancer (Suganuma et al., 1999). Similarly to TNF,

ACCEPTED MANUSCRIPT IL-1 is also produced at the early stage of inflammation and macrophages are the main source of this cytokine. IL-1 is synthesized as a leaderless precursor, cleaved by inflammasome-activated caspase-1 and released by autophagy from macrophages. This cytokine has a beneficial role of mediating an immune response against the pathogenic infiltration, but it can also promote the pathogenesis of tissue damage that leads to chronic

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inflammation (Eder, 2009). IL-6 is a pleiotropic cytokine showing both proinflammatory and

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antiinflammytory activities but in chronic inflammation it is rather proinflammatory. The

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synthesis of this cytokine is induced by IL-1 and TNF-α (Duque and Descoteaux, 2014). IL-8 is a monocyte- and macrophage-derived cytokine that serves as chemoatractant of neutrophils

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to the site of infection or injury and its secretion from macrophages can be stimulated with TNF-, IL-1 or a lipopolysaccharide (Carré et al., 1991). Our results showed that FBs

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exerted a different immunomodulatory pattern in THP-1 marcophage-like cells; FB1 but not FB2 provoked higher increase of cytokines IL-8, TNF- and IL-1; FB2 effectively decreased

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the levels of IL-1β and did not have an effect on TNF- and IL-8. IL-6 was significantly increased only when both FBs were applied at a 10-fold lower concentration. Several studies on different in vivo and in vitro models showed contradictory results regarding FB1 effect on

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cytokine release, in some cases FB1 stimulated production of inflammatory cytokines, while in some cases it exerted an inhibitory effect (Taranu et al., 2005). In the human cell lines of

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the stomach and colon, FB1 caused an increase in the production of TNF-α and IL-1β (Mahmoodi et al., 2012)., which is in agreement with our findings. In the same study, a decrease in the production of IL-8 was observed. Also, in the human colon cell line HT-29, FB1 induced a decrease in IL-8 synthesis but only after exposure to LPS stimulation (Minervini et al., 2014). In lymphocytes and neutrophils obtained from the peripheral blood of healthy people and patients with breast and gastrointestinal cancers, FB1 inhibited the production of TNF-α and G-CSF cytokines demonstrating immune suppression particularly in

ACCEPTED MANUSCRIPT cancer patients (Odhav et al., 2008). Chiba et al. (2007) suspected that FB1-induced an increase of TNF-α and IL-6 in mice mast cells as a result of FB1-inhibited synthesis of ceramide. Inhibition of ceramide synthesis and disruption of membrane phospholipids have been indicated as the key mechanisms of FB1 toxicity and our obtained immunomodulatory effects of FB1 may be the result of changes in the expression of cell surface markers important

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in immune cell communication (Odhav et al., 2008). To the best of our knowledge, no data on

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the immunomodulatory effect of FB2 has yet been published. Based on the chemical structure

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similarities between FB1 and FB2, as well as the known inhibition of ceramide synthesis, we expected a similar immunomodulatory effect in THP-1 like macrophages. Surprisingly, FB2

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significantly increased only the production of IL-6, unlike FB1 significantly decreased IL-1, and exerted a much weaker effect on TNF- and IL-8 than FB1. We can only surmise that

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these events are probably linked to the weaker cytotoxicity of FB2 for THP-1 like macrophages. Among black Aspergilli, only A. piperis and A. tubingensis evoked a significant

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increase in the production of IL-1, IL-8 and TNF-, but not IL-6. The inflammatory effect of

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A. piperis and A. tubingensis extracts corresponded to their cytotoxic potential in THP-1-like macrophages. The effect on cytokine signalling is best studied in Aspergillus fumigatus as the

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most common fungal pathogen in humans, while data on different black Aspergilli are scarce.

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IL-1 and TNF are induced in alveolar macrophages in response to A. fumigatus antigens and in monocytes in response to Aspergilli conidia and hyphae (Park and Mehrad, 2009). It was shown that only live but not heat-inactivated A. niger could evoke the secretion of IL-1 and IL-6 in MPI macrophages (Chacko, 2016), which is in agreement with our results of no significant cytokine release induced by A. niger extract, that could also be linked to its low cytotoxic potential in THP-1-like macrophages. The difference in the immunomodulatory pattern of black Aspergilli extracts is hard to explain but it cannot be linked to the production

ACCEPTED MANUSCRIPT of FB2; it is more likely that the previously discussed metabolites contribute to both the cytotoxicity and immunomodulation in THP-1-like macrophages. 4.2.3. Genotoxicity Considering a similar pattern of FBs-induced DNA damage in A549 cells obtained by the

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alkaline comet assay, we can assume that these toxins share the same mechanism of genotoxicity. However, in the interpretation of the alkaline comet assay results, one has to

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take into account that often there is no simple association between the amount of primary

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DNA damage caused by exposure to a particular chemical and the biological effects of that damage. Based on the knowledge gathered in several in vitro and in vivo studies, FB1 induces

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a dose-dependent increase of genotoxicity expressed as increased comet’s tail intensity or

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increase in micronuclei formation, and oxidative stress has been implicated as one of the mechanisms involved in DNA damage (Domijan et al., 2006; Galvano et al., 2002a, 2002b; Peraica et al., 2008). We found that both FBs evoked a significant increase in the tail intensity

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of A549 cells when applied at a lower concentration, but without significant changes in tail length values, which is in accordance with previous studies. In a previous comet assay study, Ehrlich et al. (2002) observed a dose-dependent genotoxic effects of FB1 in HepG2 cells at

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concentrations ≥25 µg/mL. Since FB1 has clastogenic potential per se, and could lead to the

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disturbance of the cellular oxidative-antioxidative equilibrium, DNA instability observed at the lower FBs concentrations tested might be associated with the formation of simple types of DNA lesions as single strand breaks and alkali labile sites, which could be sensitively detected by the alkaline comet assay. Furthermore, the observed difference in the response of comet parameters is not surprising because as the level of damage increases so does the relative intensity of staining of DNA in the tail, rather than tail length which consists of an increasing number of relaxed DNA loops (Collins et al., 2008). However, it is worth to mention that when FBs were applied at a higher concentration (100 µM) in A549 cells, we

ACCEPTED MANUSCRIPT observed a more than 3-fold lower tail intensity than after treatment with 10 µM. Based on these findings, and the fact that both FBs were weakly cytotoxic to A549 cells and decrease in cell viability after treatment with 10 and 100 µM was not significantly different, we conclude that at least a part of the genotoxic effects at the higher tested concentrations could be related to the formation of more complex DNA lesions as cross-links (both inter- and intrastrand) and

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possibly DNA-protein adducts.

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Cross-linking (DNA–DNA and DNA–protein) is the only-known mechanism which

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contributes to an actual decrease in DNA migration in the comet assay (Tice et al., 1997). Cross-linking obstructs the DNA uncoiling during alkaline denaturation, which retards the

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migration of DNA fragments during electrophoresis. Interstrand cross-links especially can lead to a tighter binding of the DNA core (Fairbairn et al., 1995). As known, (Miyamae et al.,

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1997), cells with DNA cross-linking lesions frequently show no increased or even decreased DNA migration in the alkaline comet assay. Our results agree well with those observations

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and suggest cross-linking as a possible mechanism of genotoxicity. Based on the available

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literature, FBs alone did not contribute to the formation of cross-links, but at increasing concentrations at least FB1 led to extensive lipid peroxidation (Abado-Becognee et al., 1998;

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Kouadio et al., 2005), which ended with the formation of malondialdehyde, known as a potent cross-linking agent (Abel and Gelderblom, 1998). Considering that in the present study we

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did not apply special modifications of the comet assay, our assumption regarding the involvement of cross-links in overall primary DNA damage measured after exposure to FBs has to be proven in future studies, and to obtain an unambiguous answer regarding the possible cross-linking potential of FBs and/or their metabolites, further investigations are needed. All extracts of black Aspergilli significantly increased tail length, but only A. welwitschiae, A. piperis and A. tubingensis provoked a marked increase of tail intensity, which corresponded

ACCEPTED MANUSCRIPT to the cytotoxic potential of the extracts in A549 cells. To our knowledge, this is the first report on the toxic potential of different Aspergillius species from the section Nigri to reveal significant species-related differences in toxic effects in A549 cells and THP-1-like macrophages.

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5. Conclusions

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The statistically estimated concentration of black Aspergilli (up to 5x103 CFU/m3) in the

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grain mill, dominated by A. welwitschiae and A. tubingensis, showed a potential health risk for workers, because these concentrations were up to 260-folds higher than in residential

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environments.

Among black Aspergilli, only A. welwitschiae and A. niger produced FB2, which was also

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confirmed with the presence of fum1 and fum8 genes involved in FB production. FB2 did not exert cytotoxic properties in human lung adenocarcinoma A549 cells and THP-1 macrophage-

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like cells, which means that FB2 alone did not contribute to the cytotoxic potential of the

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black Aspergilli. Extracts of A. welwitschiae, A. tubingensis and A. piperis exerted a more pronounced cytotoxic and genotoxic effects in A549 cells than A. niger and A. luchuensis. In

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THP-1 macrophage-like cells, only A. tubingensis and A. piperis evoked cytotoxic and immunmodulatory effects. Secondary metabolites including naphto-y-pyrones malformins

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and asperazine as well as elastase activity might contribute to the toxicity of the extracts, but the combined effects of these metabolites should be explored in future studies. In future studies, it would be extremely useful to explore the combined effects of black Aspergilli secondary metabolites and enzymes to clarify their interactions in toxic action and possible involvement in chronic respiratory diseases.

Acknowledgements

ACCEPTED MANUSCRIPT This work was financially supported by the University of Zagreb (Grant No. 1126). This study forms part of the project GINOP-2.3.2-15-2016-00012, supported by the European Social Fund. This work was also supported by OTKA grant Nos. K115690 and K8407 as well as through András Szekeres, who received support through the new national excellence

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program of the Hungarian Ministry of Human Capacities.

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References

Abado-Becognee, K.T.A., Fleurat-Lessard, R.E. á F., W. T. Shier á F. Badria Ennamany, R.,

SC

Creppy, Â., 1998. Cytotoxicity of fumonisin B 1 : implication of lipid peroxidation and

NU

inhibition of protein and DNA syntheses. Arch. Toxicol. 72, 233–236. Abel, S., Gelderblom, W.C.A., 1998. Oxidative damage and fumonisin B 1 -induced toxicity

MA

in primary rat hepatocytes and rat liver in vivo 131, 121–131. Alexander, N.J., Proctor, R.H., McCormick, S.P., 2009a. Genes, gene clusters, and

D

biosynthesis of trichothecenes and fumonisins in Fusarium. Toxin Rev. 28, 198–215.

PT E

doi:10.1080/15569540903092142

Alexander, N.J., Proctor, R.H., McCormick, S.P., 2009b. Genes, gene clusters, and

CE

biosynthesis of trichothecenes and fumonisins in Fusarium. Toxin Rev. 28, 198–215. doi:10.1080/15569540903092142

AC

Balajee, S.A., Kano, R., Baddley, J.W., Moser, S.A., Marr, K.A., Alexander, B.D., Andes, D., Kontoyiannis, D.P., Perrone, G., Peterson, S., Brandt, M.E., Pappas, P.G., Chiller, T., 2009. Molecular Identification of Aspergillus Species Collected for the TransplantAssociated Infection Surveillance Network. J. Clin. Microbiol. 47, 3138–3141. doi:Doi 10.1128/Jcm.01070-09 Blanco, J.L., Hontecillas, R., Bouza, E., Blanco, I., Pelaez, T., Muñoz, P., Perez Molina, J., Garcia, M.E., 2002. Correlation between the elastase activity index and invasiveness of

ACCEPTED MANUSCRIPT clinical isolates of Aspergillus fumigatus. J. Clin. Microbiol. 40, 1811–1813. doi:10.1128/JCM.40.5.1811-1813.2002 Bugni, T.S., Ireland, C.M., 2004. Marine-derived fungi: a chemically and biologically diverse group of microorganisms. Nat. Prod. Rep. 21, 143–163. doi:10.1039/b301926h Bünger, J., Westphal, G., Mönnich, A., Hinnendahl, B., Hallier, E., Müller, M., 2004.

PT

Cytotoxicity of occupationally and environmentally relevant mycotoxins. Toxicology

RI

202, 199–211. doi:10.1016/j.tox.2004.05.007

SC

Carré, P.C., Mortenson, R.L., King, T.E., Noble, P.W., Sable, C.L., Riches, D.W., 1991. Increased expression of the interleukin-8 gene by alveolar macrophages in idiopathic

NU

pulmonary fibrosis. A potential mechanism for the recruitment and activation of neutrophils in lung fibrosis. J. Clin. Invest. 88, 1802–10. doi:10.1172/JCI115501

MA

Chacko, S., 2016. Comparison of pro-inflammatory cytokines ( IL-6 , IL- 1α and IL - 1β ) released by MPI and MARCO ( - / - ) knockout cells when stimulated by heat killed

D

fungi- Candida albicans and Aspergillus niger. Plymouth Student Sci. 9, 4–23.

PT E

Collins, A.R., Oscoz, A.A., Giovannelli, L., Brunborg, G., Gaiva, I., Kruszewski, M., Smith, C.C., 2008. The comet assay : topical issues 23, 143–151. doi:10.1093/mutage/gem051

CE

Domijan, A.-M., Zeljezić, D., Kopjar, N., Peraica, M., 2006. Standard and Fpg-modified comet assay in kidney cells of ochratoxin A- and fumonisin B(1)-treated rats.

AC

Toxicology 222, 53–9. doi:10.1016/j.tox.2006.01.024 Duque, G.A., Descoteaux, A., 2014. Macrophage cytokines : involvement in immunity and infectious diseases 5, 1–12. doi:10.3389/fimmu.2014.00491 Eder, C., 2009. Mechanisms of interleukin-1 b release. Immunobiology 214, 543–553. doi:10.1016/j.imbio.2008.11.007 EFSA, 2014. Evaluation of the increase of risk for public health related to a possible temporary derogation from the maximum level of deoxynivalenol, zearalenone and

ACCEPTED MANUSCRIPT fumonisins for maize and maize products European Food Safety Authority. EFSA J. 12123699, 1–61. doi:10.2903/j.efsa.2014.3699 Ehrlich, V., Darroudi, F., Uhl, M., Steinkellner, H., Zsivkovits, M., 2002. Fumonisin B 1 is genotoxic in human derived hepatoma ( HepG2 ) cells. Mutagenesis 17, 257–260. Fairbairn, D.W., Olive, P.L., Neill, K.L.O., 1995. The c o m e t assay : a comprehensive

PT

review. Mutat. Res. 339, 37–59.

RI

Frisvad, J.C., Smedsgaard, J. orn, Samson, R.A., Larsen, T. homas O., Thrane, U. lf, 2007.

SC

Fumonisin B 2 Production by Aspergillus niger. J. Agric. Food Chem. 55, 9727–9732. Frisvad, J.C., Smedsgaard, J., Samson, R.A., Larsen, T.O., Thrane, U., 2007. Fumonisin B2

NU

production by Aspergillus niger. J. Agric. Food Chem. 55, 9727–9732. doi:10.1021/jf0718906

MA

Galvano, F., Campisi, A., Russo, A., Galvano, G., Palumbo, M., Renis, M., Barcellona, M.L., Perez-Polo, J.R., Vanella, A., 2002a. DNA damage in astrocytes exposed to fumonisin

D

B1. Neurochem. Res. 27, 345–351. doi:10.1023/A:1014971515377

PT E

Galvano, F., Russo, A., Cardile, V., Galvano, G., Vanella, A., Renis, M., 2002b. DNA damage in human fibroblasts exposed to fumonisin B1. Food Chem. Toxicol. 40, 25–31.

CE

doi:10.1016/S0278-6915(01)00083-7 Gautier, M., Ollivier, C.L., Cassagne, C., Reynaud-gaubert, M., Dubus, J., Hendrickx, M.,

AC

Gomez, C., Piarroux, R., 2016. Aspergillus tubingensis : a major filamentous fungus found in the airways of patients with lung disease. Med. Mycol. 1–12. doi:10.1093/mmy/myv118 Gherbawy, Y., Elhariry, H., Kocsube, S., Bahobial, A., Deeb, B. El, Altalhi, A., Varga, J., Vagvolgyi, C., 2015. Molecular characterization of black Aspergillus species from onion and their potential for ochratoxin a and fumonisin b2 production. Foodborne Pathog. Dis. 12, 414–423. doi:10.1089/fpd.2014.1870

ACCEPTED MANUSCRIPT Gutleb, A.C., Morrison, E., Murk, A.J., 2002. Cytotoxicity assays for mycotoxins produced by Fusarium strains: a review. Environ. Toxicol. Pharmacol. 11, 309–20. Hiort, J., Maksimenka, K., Reichert, M., Perović-Ottstadt, S., Lin, W.H., Wray, V., Steube, K., Schaumann, K., Weber, H., Proksch, P., Ebel, R., Müller, W.E.G., Bringmann, G., 2004. New natural products from the sponge-derived fungus Aspergillus niger. J. Nat.

PT

Prod. 67, 1532–1543. doi:10.1021/np030551d

RI

Hong, S.-B., Lee, M., Kim, D.-H., Varga, J., Frisvad, J.C., Perrone, G., Gomi, K., Yamada,

SC

O., Machida, M., Houbraken, J., Samson, R.A., 2013. Aspergillus luchuensis, an industrially important black Aspergillus in East Asia. PLoS One 8, e63769.

NU

doi:10.1371/journal.pone.0063769

Huang, H.-B., Xiao, Z., Feng, X., Huang, C., Zhu, X., Ju, J., Li, M., Lin, Y., Liu, L., She, Z.,

MA

2011. Cytotoxic naphtho- g -pyrones from the mangrove endophytic fungus Aspergillus tubingensis ( GX1-5E ). Helv. Chim. Acta 94, 1732–1740.

D

Hulina, A., Grdić Rajković, M., Jakšić Despot, D., Jelić, D., Dojder, A., Čepelak, I., Rumora,

PT E

L., 2017. Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells. Cell Stress Chaperones 1–12.

CE

doi:10.1007/s12192-017-0847-0

Jakšić, D., Puel, O., Canlet, C., Kopjar, N., Kosalec, I., Šegvić Klarić, M., 2012. Cytotoxicity

AC

and genotoxicity of versicolorins and 5-methoxysterigmatocystin in A549 cells. Arch. Toxicol. 86, 1583–91. doi:10.1007/s00204-012-0871-x Jakšić Despot, D., Kocsubé, S., Bencsik, O., Kecskeméti, A., Szekeres, A., Vágvölgyi, C., Varga, J., Šegvić Klarić, M., 2016. Species diversity and cytotoxic potency of airborne sterigmatocystin-producing Aspergilli from the section Versicolores. Sci. Total Environ. 562, 296–304. doi:10.1016/j.scitotenv.2016.03.183 Jakšić Despot, D., Šegvić Klarić, M., 2014. A year-round investigation of indoor airborne

ACCEPTED MANUSCRIPT fungi in Croatia. Arh Hig Rada Toksikol 209–218. doi:10.2478/10004-1254-65-20142483 Joint FAO/WHO Expert Committee on Food Additives., 2001. Safety evaluation of certain mycotoxins in food, FAO food and nutrition paper. Jurjević, Z., Peterson, S.W., Stea, G., Solfrizzo, M., Varga, J., Hubka, V., Perrone, G., 2012.

PT

Two novel species of Aspergillus section Nigri from indoor air. IMA Fungus 3, 159–73.

RI

doi:10.5598/imafungus.2012.03.02.08

SC

Kothary, M.H., Chase, T., MacMillan, J.D., 1984. Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in

NU

mice. Infect. Immun. 43, 320–325.

Kouadio, J.H., Mobio, A., Baudrimont, I., Moukha, S., 2005. Comparative study of

MA

cytotoxicity and oxidative stress induced by deoxynivalenol , zearalenone or fumonisin B1 in human intestinal cell line Caco-2. Toxicology 213, 56–65.

D

doi:10.1016/j.tox.2005.05.010

PT E

Kredics, L., Varga, J., Antala, Z., Samson, R.A., Sandor, K., Narendranc, V., Bhaskard, M., Manoharane, C., Csaba, V., Manikandanc, P., 2008. Black aspergilli in tropical

CE

infections Black aspergilli in tropical infections. Rev. Med. Microbiol. 19, 65–78. doi:10.1097/MRM.0b013e3283280391

AC

Lamboni, Y., Nielsen, K.F., Linnemann, A.R., Gezgin, Y., Hell, K., Nout, M.J.R., Smid, E.J., Tamo, M., van Boekel, M.A.J.S., Hoof, J.B., Frisvad, J.C., 2016. Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa. PLoS One 11, e0164310. doi:10.1371/journal.pone.0164310 Liu, Y., Wang, M., Wang, D., Li, X., Wang, W., Lou, H., Yuan, H., 2016. Malformin A 1 promotes cell death through induction of apoptosis , necrosis and autophagy in prostate

ACCEPTED MANUSCRIPT cancer cells. Cancer Chemother. Pharmacol. 77, 63–75. doi:10.1007/s00280-015-2915-4 Logrieco, A., Bottalico, A., Mulé, G., Moretti, A., Perrone, G., 2003. Epidemiology of toxigenic fungi and their associated mycotoxins for some Mediterranean crops. Eur. J. Plant Pathol. doi:10.1023/A:1026033021542 Mahmoodi, M., Alizadeh, A.M., Sohanaki, H., Rezaei, N., Amini-najafi, F., 2012. Impact of

PT

fumonisin B1 on the production of inflammatory cytokines by gastric and colon cell

RI

lines. Iran J Allergy Asthma Immunol 11, 165–173.

SC

Månsson, M., Klejnstrup, M.L., Phipps, R.K., Nielsen, K.F., Frisvad, J.C., Gotfredsen, C.H., Larsen, T.O., 2010. Isolation and NMR characterization of fumonisin b2 and a new

NU

fumonisin B6 from aspergillus niger. J. Agric. Food Chem. 58, 949–953. doi:10.1021/jf902834g

MA

McKean, C., Tang, L., Billam, M., Tang, M., Theodorakis, C.W., Kendall, R.J., Wang, J.S., 2006. Comparative acute and combinative toxicity of aflatoxin B1 and T-2 toxin in

PT E

doi:10.1002/jat.1117

D

animals and immortalized human cell lines. J. Appl. Toxicol. 26, 139–147.

Minervini, F., Garbetta, A., Antuono, I.D., Cardinali, A., Antonio, N., Lucantonio, M.,

CE

Visconti, A., 2014. Toxic Mechanisms Induced by Fumonisin B 1 Mycotoxin on Human Intestinal Cell Line 115–123. doi:10.1007/s00244-014-0004-z

AC

Miyamae, Y., Zaizen, K., Ohara, K., Mine, Y., 1997. Detection of DNA lesions induced by chemical mutagens by the single cell gel electrophoresis (Comet) assay. 1. Relationship between the onset of DNA damage and the characteristics of mutagens. Mutat. Res. 393, 99–106. Nielsen, K.F., Frisvad, J.C., Logrieco, A., 2015. Letter to the Editor: “‘Analyses of Black Aspergillus Species of Peanut and Maize for Ochratoxins and Fumonisins,’” A Comment on: J. Food Prot. 77(5):805–813 (2014). J. Food Prot. 78, 6–12. doi:10.4315/0362-

ACCEPTED MANUSCRIPT 028X.78.1.6 Odhav, B., Adam, J.K., Bhoola, K.D., 2008. Modulating effects of fumonisin B1 and ochratoxin A on leukocytes and messenger cytokines of the human immune system. Int. Immunopharmacol. 8, 799–809. doi:10.1016/j.intimp.2008.01.030 Palumbo, J.D., O’Keeffe, T.L., Gorski, L., 2013. Multiplex PCR analysis of fumonisin

PT

biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains.

RI

Mycologia 105, 277–284. doi:10.3852/11-418

22, 535–551. doi:10.1128/CMR.00014-09

SC

Park, S.J., Mehrad, B., 2009. Innate Immunity to Aspergillus Species. Clin. Microbiol. Rev.

NU

Pel, H.J., de Winde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap, P.J., Turner, G., de Vries, R.P., Albang, R., Albermann, K., Andersen, M.R., Bendtsen, J.D., Benen, J.A.E.,

MA

van den Berg, M., Breestraat, S., Caddick, M.X., Contreras, R., Cornell, M., Coutinho, P.M., Danchin, E.G.J., Debets, A.J.M., Dekker, P., van Dijck, P.W.M., van Dijk, A.,

D

Dijkhuizen, L., Driessen, A.J.M., d’Enfert, C., Geysens, S., Goosen, C., Groot, G.S.P.,

PT E

de Groot, P.W.J., Guillemette, T., Henrissat, B., Herweijer, M., van den Hombergh, J.P.T.W., van den Hondel, C.A.M.J.J., van der Heijden, R.T.J.M., van der Kaaij, R.M.,

CE

Klis, F.M., Kools, H.J., Kubicek, C.P., van Kuyk, P.A., Lauber, J., Lu, X., van der Maarel, M.J.E.C., Meulenberg, R., Menke, H., Mortimer, M.A., Nielsen, J., Oliver, S.G.,

AC

Olsthoorn, M., Pal, K., van Peij, N.N.M.E., Ram, A.F.J., Rinas, U., Roubos, J.A., Sagt, C.M.J., Schmoll, M., Sun, J., Ussery, D., Varga, J., Vervecken, W., van de Vondervoort, P.J.J., Wedler, H., Wösten, H.A.B., Zeng, A.-P., van Ooyen, A.J.J., Visser, J., Stam, H., 2007. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat. Biotechnol. 25, 221–31. doi:10.1038/nbt1282 Peraica, M., Ljubanovi, D., Domijan, A., 2008. The effect of a single dose of fumonisin B 1 on rat kidney. Croat. Chem. acta 81, 119–124.

ACCEPTED MANUSCRIPT Perrone, G., Stea, G., Epifani, F., Varga, J., Frisvad, J.C., Samson, R.A., 2011a. Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biol. 115, 1138– 1150. doi:10.1016/j.funbio.2011.07.008 Perrone, G., Stea, G., Epifani, F., Varga, J., Frisvad, J.C., Samson, R.A., 2011b. Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biol. 115, 1138–

PT

1150. doi:10.1016/j.funbio.2011.07.008

RI

Perrone, G., Susca, A., Cozzi, G., Ehrlich, K., Varga, J., Frisvad, J.C., Meijer, M., Noonim,

SC

P., Mahakamchanakul, W., Samson, R.A., 2007. Biodiversity of Aspergillus species in some important agricultural products. Stud. Mycol. 59, 53–66.

NU

doi:10.3114/sim.2007.59.07

Proctor, R.H., Plattner, R.D., Desjardins, A.E., Busman, M., Butchko, R.A.E., 2006.

MA

Fumonisin production in the maize pathogen Fusarium verticillioides: Genetic basis of naturally occurring chemical variation. J. Agric. Food Chem. 54, 2424–2430.

D

doi:10.1021/jf0527706

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Samson, R.A., Houbraken, J.A.M.P., Kuijpers, A.F.A., Frank, J.M., Jens, C., Lyngby, D.-K., 2004. New ochratoxin A or sclerotium producing species in Aspergillus section Nigri.

CE

Stud. Mycol. 50, 45–61.

Samson, R.A., Noonim, P., Meijer, M., Houbraken, J., Frisvad, J.C., Varga, J., 2007.

AC

Diagnostic tools to identify black Aspergilli. Stud. Mycol. 59, 129–45. doi:10.3114/sim.2007.59.13 Samson, R. a., Houbraken, J., Thrane, U., Frisvad, J.C., Andersen, B., 2010. Food and Indoor Fungi, CBS Laboratory Manual Series. Sen, B., Asan, A., 2009. Fungal flora in indoor and outdoor air of different residential houses in Tekirdag City (Turkey): Seasonal distribution and relationship with climatic factors. Environ. Monit. Assess. 151, 209–219. doi:10.1007/s10661-008-0262-1

ACCEPTED MANUSCRIPT Seo, J. a, Proctor, R.H., Plattner, R.D., 2001. Characterization of four clustered and coregulated genes associated with fumonisin biosynthesis in Fusarium verticillioides. Fungal Genet. Biol. 34, 155–165. doi:10.1006/fgbi.2001.1299 Seo, J., Proctor, R.H., Plattner, R.D., 2001. Characterization of four clustered and coregulated genes associated with fumonisin biosynthesis in Fusarium verticillioides. Fungal Genet.

PT

Biol. 34, 155–165. doi:10.1006/fgbi.2001.1299

RI

Sharma, D., Dutta, B.K., Singh, A.B., 2010. Exposure to indoor fungi in different working

SC

environments: A comparative study. Aerobiologia (Bologna). 26, 327–337. doi:10.1007/s10453-010-9168-9

NU

Shier, W.T., Abbas, H.K., Mirocha, C.J., 1991. Toxicity of the mycotoxins fumonisins B1 and

Mycopathologia 116, 97–104.

MA

B2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells.

Suganuma, M., Okabe, S., Marino, M.W., Sakai, A., Sueoka, E., Fujiki, H., 1999. Advances

D

in Brief Essential Role of Tumor Necrosis Factor ␣ ( TNF- ␣ ) in Tumor Promotion as

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Revealed by TNF- ␣ -deficient Mice 1 4516–4518. Susca, A., Proctor, R.H., Butchko, R.A.E., Haidukowski, M., Stea, G., Logrieco, A., Moretti,

CE

A., 2014. Variation in the fumonisin biosynthetic gene cluster in fumonisin-producing

AC

and nonproducing black aspergilli. Fungal Genet. Biol. 73, 39–52. doi:10.1016/j.fgb.2014.09.009 Susca, A., Proctor, R.H., Mulè, G., Stea, G., Ritieni, A., Logrieco, A., Moretti, A., 2010. Correlation of mycotoxin fumonisin B2 production and presence of the fumonisin biosynthetic gene fum8 in Aspergillus niger from grape. J. Agric. Food Chem. 58, 9266– 9272. doi:10.1021/jf101591x Szigeti, G., Kocsubé, S., Dóczi, I., Bereczki, L., Vágvölgyi, C., Varga, J., 2012a. Molecular identification and antifungal susceptibilities of black Aspergillus isolates from

ACCEPTED MANUSCRIPT otomycosis cases in Hungary. Mycopathologia 174, 143–147. doi:10.1007/s11046-0129529-8 Szigeti, G., Sedaghati, E., Mahmoudabadi, A.Z., Naseri, A., Kocsubé, S., Vágvölgyi, C., Varga, J., 2012b. Species assignment and antifungal susceptibilities of black aspergilli recovered from otomycosis cases in Iran. Mycoses 55, 333–338. doi:10.1111/j.1439-

PT

0507.2011.02103.x

RI

Šegvić Klarić, M., Jakšić Despot, D., Kopjar, N., Rašić, D., Kocsubé, S., Varga, J., Peraica,

SC

M., 2015. Cytotoxic and genotoxic potencies of single and combined spore extracts of airborne OTA-producing and OTA-non-producing Aspergilli in Human lung A549 cells.

NU

Ecotoxicol. Environ. Saf. 120, 206–214. doi:10.1016/j.ecoenv.2015.06.002 Taranu, I., Marin, D.E., Bouhet, S., Pascale, F., Bailly, J., Miller, J.D., Pinton, P., Oswald,

MA

I.P., 2005. Mycotoxin fumonisin B1 alters the cytokine profile and decreases the vaccinal antibody titer in pigs. Toxicol. Sci. 84, 301–307. doi:10.1093/toxsci/kfi086

D

Tice, R.R., Yager, J.W., Andrews, P., Crecelius, E., 1997. Effect of hepatic methyl donor

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status on urinary excretion and DNA damage in B6C3Fl mice treated with sodium arsenite. Mutat. Res. 386, 315–334.

CE

Varga, J., Frisvad, J.C., Kocsubé, S., Brankovics, B., Tóth, B., Szigeti, G., Samson, R.A., Varga, A., Frisvad, A., 2011. New and revisited species in Aspergillus section Nigri

AC

Extrolite analysis. Stud. Mycol. 69, 1–17. doi:10.3114/sim.2011.69.01 Varga, J., Kocsubé, S., Suri, K., Szigeti, G., Szekeres, a., Varga, M., Tóth, B., Bartók, T., 2010. Fumonisin contamination and fumonisin producing black Aspergilli in dried vine fruits of different origin. Int. J. Food Microbiol. 143, 143–149. doi:10.1016/j.ijfoodmicro.2010.08.008 Varga, J., Kocsube, S., Szigeti, G., Baranyi, N., Vagvlogyi, C., Despot, D.J., Magyar, D., Meijer, M., Samson, R.A., Klaric, M.S., 2014. Occurrence of black Aspergilli in indoor

ACCEPTED MANUSCRIPT environments of six countries. Arh. Hig. Rada Toksikol. 65, 219–223. doi:Doi 10.2478/10004-1254-65-2014-2450 Wang, H., Gloer, J.B., Wicklow, D.T., Dowd, P.F., 1995. Aflavinines and other antiinsectan metabolites from the ascostromata of Eupenicillium crustaceum and related species. Appl. Environ. Microbiol. 61, 4429–4435.

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Zhang, G., Neumeister-Kemp, H., Garrett, M., Kemp, P., Stick, S., Franklin, P., 2013.

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Exposure to Airborne Mould in School Environments and Nasal Patency in Children.

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Indoor Built Environ. 22, 608–617. doi:10.1177/1420326X12447534

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Table 1. The PCR primers used for the detection of fum genes

fum1-F2

TGGCGATTGGTCGTCCAGGTCT

fum1-R2

GCCACCGATGTCCACAAGCGAA

vnF1

TTCGTTTGAGTGGTGGCA

vnR3

CAACTCCATASTTCWWGRRAGCCT

Gene

Reference

fum1

(Palumbo et al., 2013)

fum8

(Susca et al., 2014)

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Sequence

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Primers

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Table 2. Abundance of black Aspergilli in indoor and outdoor air for each sampling period

Perio CFU/m3

ing

Mean ± SD Abso lute

Estim ated*

40 ±

263 ±

ry

52

339

Marc

20 ±

h

52

131 ± 344

May

240

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±

260 July

30 ± 57

Abso lute

Estim ated*

0-

Basement

Outdoor air

(AP)

(BS)

(ODA)

CFU/m3

CFU/m3

CFU/m3

Me Ra

Me Ra

Me Ra

%

0.

an

nge

%

an

nge

%

SD

SD

1 ± 020

0.

1±

0-

0. 14

15 4.5

62

4

20

0- 0200 1314

0. 8 ± 04. 05 31 140 40

-

-

-

-

-

-

0. 1 ± 090 4 20

0.

-

-

-

-

-

-

-

1± 5

020

0.

2±

0-

0.

10

6

20

10

020

0. 47

-

-

0. 08

0- 0400 2628

0. 8 ± 035 19 80

1.

1± 73 4

020

0.

0200 0-225

0. 1 ± 032 4.5 20

0. 3 ± 45 7

020

0. 25

124

Nove mber

20 ± 52

22.5 ± 59

-

SD

0- 0200 1314

mber

-

±

197 ± 375 302 ± 809

-

±

0- 0800 5256

50 ±

%

±

1550 ± 1730

Septe

nge

an

100 0-657

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Janua

Range

D

sampl

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d of

Apartment

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Grain mill (GM)

-

08

4± 10 8 -

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* The probable number of viable particles calculated from Feller’s formula (Pr=N 1/N + 1/N1 + 1/N-2 + 1/N-r+1), given by the manufacturer (Merck KgaA, Darmstadt, Germany; Pr=probable statistical total; r=Number of cfu counted; N=total number of holes in the sampling head). % – mean concentration of black Aspergilli / mean concentration of total airborne fungi** x 100

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** – Mean concentration of total airborne fungi adopted from Jakšić Despot and Šegvić Klarić (2014)

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- – not detected

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Table 3. PCR screening of fum genes in fumonisin B2 producing and non-producing black

Isolate code

Sampling site

Detected FB1

Detected FB2

(3 agar plugs)

(3 agar plugs)

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Species

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Aspergilli

fum1

fum8

g/mL

µg/mL

MFBF AN10927B

Grain mill

nd

nd

-

-

A. tubingensis

MFBF AN10928B

Grain mill

nd

nd

-

-

A. tubingensis

MFBF AN10868A

Grain mill

nd

nd

-

-

A. tubingensis

MFBF AN11053A

Grain mill

nd

nd

-

-

A. tubingensis

MFBF AN11053B

Grain mill

nd

nd

-

-

A. tubingensis

MFBF AN11054B

Grain mill

nd

nd

-

-

A. tubingensis

MFBF AN11119A

nd

nd

-

-

A. tubingensis

MFBF AN10856A

Apartment

nd

nd

-

-

A. tubingensis

MFBF AN10862A

Apartment

nd

nd

-

-

Grain mill

A. tubingensis

MFBF AN11058B

Outdoor air

nd

nd

-

-

A. luchuensis

MFBF AN10927A

Grain mill

nd

nd

-

-

MFBF AN11054A

Grain mill

nd

nd

-

-

A. luchuensis

MFBF AN11133A

Apartment

nd

nd

+

-

A. luchuensis

MFBF AN11000B

Outdoor air

nd

nd

-

-

A. welwitschiae

MFBF AN10868B

Grain mill

nd

nd

+

+

A. welwitschiae

MFBF AN10924B

Grain mill

nd

13.467

+

+

A. welwitschiae

MFBF AN10925A

Grain mill

nd

7.991

+

+

A. welwitschiae

MFBF AN10925B

Grain mill

nd

0.128

+

+

A. welwitschiae

MFBF AN10926A

Grain mill

nd

5.762

+

+

A. welwitschiae

MFBF AN10926B

Grain mill

nd

10.476

+

+

A. luchuensis

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A. tubingensis

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Grain mill

nd

0.290

+

+

A. welwitschiae

MFBF AN10929B

Grain mill

nd

0.156

+

+

A. welwitschiae

MFBF AN11113A

Grain mill

nd

nd

+

-

A. welwitschiae

MFBF AN10545

Apartment

nd

nd

+

+

A. welwitschiae

MFBF AN11060A

Apartment

nd

3.988

-

+

A. welwitschiae

MFBF AN11061B

Apartment

nd

nd

+

-

A. welwitschiae

MFBF AN11062A

Apartment

nd

nd

+

-

A. welwitschiae

MFBF AN 11139 B

Basement

nd

nd

+

+

A. welwitschiae

MFBF AN 11140 B

Basement

nd

nd

+

-

A. niger

MFBF AN 11066 B

Basement

nd

0.015

+

+

A. piperis

MFBF AN 11119 B

Grain mill

nd

nd

+

-

SC

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A. welwitschiae

Table 4. The calculated IC50 of black Aspergilli extracts (0.1-0.8 mg/mL) in A549 and THP-1

IC50 (A549)

IC50 (THP-1)

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Treatment

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macrophage-like cell lines using non-linear curve fitting (R2=0.9767)

mg/mL (mean± SEM) > 0.8

A. welwitschiae 0.214 ± 0.004 > 0.8

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A. luchuensis

> 0.8 > 0.8

D

A. niger

> 0.8

0.329 ± 0.001 0.228 ± 0.009

A. piperis

0.460 ± 0.011 0.323 ± 0.009

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CE

A. tubingensis

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A. welwitschiae

D

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A. niger

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Fig. 1. Extracted ion chromatograms at m/z= 706 corresponding to FB2 in FB2-producing black Aspergilli

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FB1 (*c)

100

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FB2 (*c)

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80

70

SC

A549 cells viability (% of control)

90

60

40 0

30

60

90

120

150

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Treatment (M)

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50

D

A

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100

FB2 (*c)

CE

80

70

60

50

40 0

AC

THP-1 cells viability (% of control)

90

FB1 (*c)

30

60

90

120

150

Treatment (M)

B Fig. 2. Survival of A549 cells (A) and THP-1 macrophage like cells (B) after 24 h of treatment with single FB1 and FB2 measured by MTT test. Each data point represents mean ± SEM of cell viability (% of control, control = 100 % of cell viability) for FB1 and FB2

ACCEPTED MANUSCRIPT concentrations ranging from 10 to 150 µM. All treatments both in A549 and THP-1 cells were

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significantly different compared to control (*c, P<0.05).

AC

CE

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D

MA

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SC

RI

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Fig. 3. Cytotoxicity of extracts prepared from different species assigned to section Nigri in A549 and THP-1 cells measured by MTT test. Dried extracts were dissolved in DMSO and diluted in cell culture medium (RPMI 1640) for the treatment (0.1-0.8 mg/mL). Up to 1% DMSO was used as the control. All treatments both in A549 and THP-1 cells were significantly different compared to control* (P<0.05). The statistical significance of the same type of treatment in THP-1 compared to A549 cells is marked with an asterisk (*, P<0.05).

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Fig. 4. Relative concentration of cytokines measured in the supernatant of THP-1 macrophage-like cells upon the treatment specified. The statistical significance of the

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treatment compared to control is marked with an asterisk (*, P<0.05) above each histogram.

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C o n tr o l ( 0 .1 % D M S O ) FB1 10 µM FB1 1 0 0 FB 2 1 0 M FB2 1 0 0 A . n i g e r 0 .1 m g / m L ( 0 .0 0 0 0 9  M F B 2 ) A . w e l w i ts c h i a e 0 .1 m g /m L ( 0 .0 9 5  M F B 2 ) A . l u c h u e n s i s 0 .1 m g /m L A . p i p e r i s 0 .1 m g /m L

40

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A . t u b i n g e n s i s 0 .1 m g /m L

4

*

*

*

*

*

*

2

SC

20

* 3

RI

30

T a il in te n s ity (% )

T a il le n g t h (  m )

*

10

0

*

*

*

1

NU

0

Fig. 5. Genotoxicity of FB1/FB2 and black Aspergilli extracts applied in subcytotoxic

MA

concentrations determined by alkaline comet assay in A549 cells. The statistical significance of the treatment compared to control is marked with an asterisk (*, P<0.05) above each

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histogram.

ACCEPTED MANUSCRIPT Highlights  A. tubingensis and A. welwitschiae were dominant black Aspergilli in grain mill  A. welwitschiae and A. niger produced fumonisin B2 (FB2) 

A. welwitschiae, A. tubingensis and A. piperis were cyto- and genotoxic to A549 cells

 A. tubingensis and A. piperis were the most cyto- and immunotoxic to THP-1 cells

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 Other metabolites in the extracts, but not FB2 contribute to toxicity

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5