Cellular Cytotoxicity and Oxidative Potential of Recurrent Molds of the Genus Aspergillus Series Versicolores

Molds are ubiquitous biological pollutants in bioaerosols. Among these molds, the genus Aspergillus is found in the majority of indoor air samples, and includes several species with pathogenic and toxigenic properties. Aspergillus species in the series Versicolores remain little known despite recurrence in bioaerosols. In order to investigate their toxicity, we studied 22 isolates of clinical and environmental origin, corresponding to seven different species of the series Versicolores. Spore suspensions and ethyl acetate extracts prepared from fungal isolates were subjected to oxidative potential measurement using the dithiothreitol (DTT) test and cell survival measurement. The DTT tests showed that all species of the series Versicolores had an oxidative potential, either by their spores (especially for Aspergillus jensenii) or by the extracts (especially from Aspergillus amoenus). Measurements of cell survival of A549 and HaCaT cell lines showed that only the spore suspension containing 105 spores/mL of Aspergillus jensenii caused a significant decrease in survival after 72 h of exposure. The same tests performed with mixtures of 105 spores/mL showed a potentiation of the cytotoxic effect, with a significant decrease in cell survival for mixtures containing spores of two species (on A549 cells, p = 0.05 and HaCaT cells, p = 0.001) or three different species (on HaCaT cells, p = 0.05). Cell survival assays after 72 h of exposure to the fungal extracts showed that Aspergillus puulaauensis extract was the most cytotoxic (IC50 < 25 µg/mL), while Aspergillus fructus caused no significant decrease in cell survival.


Introduction
Air pollution is a complex and dynamic phenomenon involving the exposure of living organisms to harmful airborne substances. The health effects of indoor and outdoor air pollutants are a major public health issue due to the duration of exposure, even to relatively low concentrations of air pollutants [1][2][3]. According to the World Health Organization, three million deaths per year worldwide are due to stroke (36%), ischemic heart disease (36%), lung cancer (14%), chronic obstructive pulmonary disease (8%), or acute lower respiratory disease (6%) caused by exposure to airborne particles [4]. Airborne physical, chemical, and biological particles form a heterogeneous mixture whose composition varies continuously in space and time [2,3,5]. One of the mechanisms explaining part of the health effects of airborne particles is their ability to synthesize or catalyze the formation of reactive oxygen species (ROS) when they reach the lung cells. These molecules then cause oxidative stress and inflammation of the airways. This ability to synthesize and/or catalyze the formation of ROS is determined by the particles' oxidative potential (OP), which can be easily measured by biochemical and cell-free assays. Although the physical and chemical

Molecular Identification
Isolates belonging to the genus Aspergillus series Versicolores are phenotypically similar, which makes identification by culture and microscopy difficult, even from selective media. We therefore used molecular biology to identify the 21 isolates [15,17].
DNA extraction was performed as described in our previous study [20] using a modified protocol of the Nucleospin™ Plant II kit (Macherey-Nagel, Duren, Germany). Fungal material was transferred to a 2 mL microcentrifuge tube with glass beads. The microcentrifuge tube was incubated twice 15 min at 80 • C and then 15 min at −80 • C. It was then placed into a swing-mill with 400 µL of lysis buffer PL1 for 15 min at 20 Hz, and incubated with 10 µL of RNAse and 20 µL of proteinase K (Promega, Madison, WI, USA) at 10 mg/mL at 65 • C for 15 min. Then, 400 µL of chloroform (MP Biomedicals-Thermo Fisher Scientific, Waltham, MA, USA) was added. The clear supernatant was recovered and extracted as described by the supplier.
DNA was purified using the NucleoSpin gDNA Clean-up kit (Macherey-Nagel, Duren, Germany) following the instructions of the manufacturer. Quantification and quality of the fungal DNA were performed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [39].
All Aspergillus isolates belonging to the series Versicolores (n = 22) were grown on MEA+ and stored on slant agar at 4 • C and in a cryoprotective agent composed of sterile water and 10% glycerol (Carlo Erba, Val-de-Reuil, France) at −80 • C before any further testing. We also included a reference strain (CBS 245.65) to validate our identification technique by amplification and sequencing of the BenA gene.

Preparation of Calibrated Spore Suspensions
Spores suspensions were made from isolates grown for 10 days on MEA+. The surface of the colonies was covered with collection liquid. The resulting crude suspensions were filtered through sterile cotton to remove hyphae and fungal debris and then through sterile polytetrafluoroethylene (PTFE) filters of 5 µm porosity (Sartorius-Thermo Fisher Scientific, Waltham, MA, USA) to remove clusters of spores stuck together. The spores were then counted on a KOVA®Glasstic slide (KOVA-Thermo Fisher Scientific, Waltham, MA, USA) before performing decimal dilutions ranging from 10 5 to 10 spores/mL, which are the most commonly encountered concentrations in indoor environments [13]. Final concentrations and quality of spore suspensions were confirmed by flow cytometry on a Cytoflex S hemocytometer (Beckman Coulter, Brea, CA, USA).

Preparation of Fungal Extracts
Fungal extracts were made from isolates grown for 21 days on MEA+. For all isolates, 12 agar plugs were taken and introduced into 5 mL glass tubes. To each of these tubes, 2 mL of ethyl acetate (Sigma-Aldrich-Merck, Darmstadt, Germany) acidified with 1% acetic acid (Sigma-Aldrich-Merck, Darmstadt, Germany) was added. After vortexing each tube for 30 s, the tubes were centrifuged at 1500 rpm for 15 min. The supernatant (1.5 mL) was collected and filtered through a 0.22 µm pore size syringe tip filter (Thermo Fisher Scientific, Waltham, MA, USA) to remove spores. The extracts were then evaporated in a SpeedVac Plus concentrator (Savant-Thermo Fisher Scientific, Waltham, MA, USA) at room temperature. The dry extracts were stored in the dark at room temperature until use. The dry extracts were then solubilized in a mixture of culture medium and dimethyl sulfoxide (DMSO) (Pan Biotech-Dominique Dutscher, Tourgéville, France) (5%). Each extract was then diluted to obtain four concentrations: 250 µg/mL, 125 µg/mL, 50 µg/mL, and 25 µg/mL.

Oxydative Potential Measurement
Measurement of the oxidative potential of spore suspensions and fungal extracts was performed by the dithiothreitol (DTT) assay. This test is a commonly used cell-free method for assessing the oxidative potential of redox-active chemicals in air. The consumption of DTT (in excess) upon contact with the tested suspensions or solutions is monitored and the depletion of DTT is proportional to the concentration of ROS in the reaction mixture. Existing protocols [8,40] were adapted to suit our tests. The reaction mixture consisted of 550 µL of Dulbecco's Phosphate Buffer Solution (DPBS) (Gibco-Thermo Fisher Scientific, Waltham, MA, USA) incubated in 2 mL microtubes at 37 • C, to which 50 µL of 0.5 mM DTT (Thermo Fisher Scientific, Waltham, MA, USA) and 50 µL of spore suspension or fungal extract were added. Then, 100 µL of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (Thermo Fisher Scientific, Waltham, MA, USA) was added after 0, 15, and 30 min of contact staining the reaction medium a yellowish color with an optical density (OD) proportional to the amount of DTT remaining. For each incubation time, 150 µL of mixture was transferred in triplicates to a 96-well microplate. OD was then measured at 412 nm by spectrophotometry (BioTek-Agilent Technologies, Santa Clara, CA, USA). For each assay, a blank (DPBS) was performed in triplicates. For the measurement of the oxidative potential of the extracts, a solvent control (DMSO) was added.

Cell Culture
In order to study the activity of spores and fungal extracts on the skin and at the respiratory level, we chose two cell lines: the A549 (adenocarcinomic human alveolar basal epithelial cells) (ATCC®CRM-CCL-185TM, USA) cell line and the HaCaT (aneuploid immortal keratinocytes) (AddexBio T0020001, USA) cell line. Cells were grown in 25 mL flasks at 37 • C in an environment containing 5% CO 2 with 5 mL of culture medium containing Dulbecco's Modified Eagle Medium (DMEM) (Gibco-Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS) (Pan Biotech-Dominique Dutscher, Tourgéville, France), 1% penicillin-streptomycin 10,000 U/mL (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), and 0.01% gentamicin 10 mg/mL (Sigma-Aldrich-Merck, Darmstadt, Germany). Cells were resuspended by adding 1 mL of trypsin 0.05% (Gibco-Thermo Fisher Scientific, Waltham, MA, USA) in the case of A549 cells or 0.25% (Pan Biotech GmbH, Aidenbach, Germany) in the case of HaCaT cells. Cells were counted using a KOVA™ Glasstic™ slide (KOVA-Thermo Fisher Scientific, Waltham, MA, USA) and then suspended in DMEM at a cell concentration of 75,000 cells/mL. In each well of a 96-well microplate, 200 µL of the cell suspension was introduced. The microplate was placed in the incubator for 24 h. The culture medium was then replaced with the medium containing the spore suspensions or fungal extracts before being incubated again for 24 to 72 h. For each line and for each condition, six replicates were performed.
After exposure, cells were fixed with 50 µL of 50% cold (4 • C) trichloroacetic acid (Sigma-Aldrich-Merck, Darmstadt, Germany) for one hour. The plate was rinsed five times with running water and then air-dried. Then, 50 µL of 0.4% sulforhodamine B (SRB) (Sigma-Aldrich-Merck, Darmstadt, Germany) in 1% acetic acid was deposited for 30 min at room temperature in each well to stain the proteins. The plate was then washed four times with 1% acetic acid and air-dried. The dye was solubilized with 100 µL of 10 mM tris(hydroxymethyl)aminomethane (TRIS) base buffer pH 10.5 (Sigma-Aldrich-Merck, Darmstadt, Germany) with 10 min of agitation at room temperature. OD was then measured at 570 nm using a microplate reader by subtracting the reference OD read at 655 nm to eliminate interference. The result obtained for each condition was compared to a culture control (DMEM + 10% FBS) consisting of eight replicates for each experimental condition, expressing the result as percentage of cell survival for each condition compared to the control. For the extracts, a culture control with 5% DMSO was performed in eight replicates in order to estimate the toxicity of the fungal extracts independently of the solvent.

Statistical Analyses
Descriptive statistics were calculated to provide information on oxidative potential. Rates of DTT consumption by spore suspensions and fungal extracts were subjected to the Mann-Whitney test. The percentages of cell survival were subjected to the Kruskal-Wallis test. Only results at p < 0.05 were considered statistically significant. Statistical analyses were performed using SAS system v.9.4 (SAS Institute Inc. Cary, NC, USA) and XLSTAT v.2021.2.1.1120 (Addinsoft, Paris, France).

Isolates Identification
Identification of isolates by amplification and sequencing of the BenA gene is presented in Table 1. All sequences are presented in Supplementary Table S1. The reference strain CBS 245.65 was well identified as belonging to the species Aspergillus amoenus. The other 21 isolates could be identified as belonging to seven different species of the series Versicolores: Aspergillus amoenus (n = 1), A. creber (n = 7), A. fructus (n = 1), A. jensenii (n = 3), A. protuberus (n = 1), A. puulaauensis (n = 1), and A. sydowii (n = 7). Aspergillus creber and A. sydowii were the most represented species (n = 7 isolates for each of the two species). Aspergillus creber was only found in bioaerosols, while Aspergillus sydowii was only found in clinical samples.

Oxidative Potential of Spores
The DTT consumption rate obtained for the blank (natural oxidation in the reaction mixture in open air) was 0.040 nmol/min. We were able to measure an oxidative potential for each of the 22 isolates of the seven species tested as well as a decrease in DTT consumption rate proportional to the concentrations of the spore suspensions. However, we observed a very heterogeneous oxidative potential even between isolates of the same species. Indeed, the lowest concentration causing a significant increase in DTT oxidation compared to blank for Aspergillus amoenus was 10 5 and 10 spores/mL for strain CBS 245.65 and isolate HAB06, respectively. The same observation was made for Aspergillus creber isolates recovered only from the environment (10 5 spores/mL for 08FM2_A49, 10 3 spores/mL for HAB07 and HOSP150313_5_98, 10 2 spores/mL for HAB02 and HAB32, and 10 spores/mL for HAB64 and HOSP050413_5_135), and Aspergillus sydowii recovered only from clinical samples (10 4 spores/mL for 8051266672_C3 and 0062415698_C7, 10 3 spores/mL for 9071870945_C5 and 0062445522_C8, 10 2 spores/mL for 4040348777_C2, and 10 spores/mL for 0062445523_C9 and 0112723999_C11). For Aspergillus jensenii, a species for which we identified both clinical and environmental isolates, the finding was the same: the lowest concentration significantly increasing DTT consumption compared to blank was 10 3 spores/mL for 9041799386_C4 and 10 spores/mL for 4070377575_C6 and HAB01. Of the seven species used for the DTT test, Aspergillus protuberus was the species with the lowest oxidative potential, while Aspergillus jensenii was the species with the highest oxidative potential.

Oxidative Potential of Fungal Extracts
Kinetics of DTT consumption by fungal extracts grouped by species are presented in Figure 1.
Because the majority of the fungal extracts were yellowish, the final concentration chosen for the DTT assays in the reaction mixture was 25 µg/mL, a concentration for which the OD measurement at 412 nm was not different from the blank (DPBS) in the absence of DTT and DTNB. In microtubes containing only DPBS (blank), we measured an average of 23.87 nmol of DTT after 30 min of incubation (DTT consumption rate of 0.038 nmol/min). Our solvent control (DMSO) showed no oxidative potential (no significant difference from the blank (DPBS)). All extracts showed oxidative potential at a concentration of 25 µg/mL with significantly higher DTT consumption (p < 0.05) than those measured for the blank and solvent control. Among the fungal extracts (n = 22) of the seven species tested, the extracts (n = 2) of Aspergillus amoenus showed a significantly higher oxidative potential than the other extracts. However, similarly to the spore suspensions, a significant intraspecific variability in DTT consumption was observed. Because the majority of the fungal extracts were yellowish, the final concentration chosen for the DTT assays in the reaction mixture was 25 µg/mL, a concentration for which the OD measurement at 412 nm was not different from the blank (DPBS) in the absence of DTT and DTNB. In microtubes containing only DPBS (blank), we measured an average of 23.87 nmol of DTT after 30 min of incubation (DTT consumption rate of 0.038 nmol/min). Our solvent control (DMSO) showed no oxidative potential (no significant difference from the blank (DPBS)). All extracts showed oxidative potential at a concentration of 25 µg/mL with significantly higher DTT consumption (p < 0.05) than those measured for the blank and solvent control. Among the fungal extracts (n = 22) of the seven species tested, the extracts (n = 2) of Aspergillus amoenus showed a significantly higher oxidative potential than the other extracts. However, similarly to the spore suspensions, a significant intraspecific variability in DTT consumption was observed.
In order to evaluate the impact of the presence of spores of different species, we prepared spore suspensions containing spores of the two, three, four, or five most recurrent species with a final concentration of 10 5 spores/mL, to which our cells were exposed for 72 h, considering the results already obtained. The mixtures were made from the four species found in the environment: Aspergillus amoenus, A. creber, A. jensenii, and A. protuberus, plus Aspergillus sydowii, as it was the most frequent species in our clinical samples. As shown in Figure 2, all combinations containing spores belonging to two different species showed a significant decrease in cell survival (p < 0.05) for the A549 cell line, with the lowest value for the Aspergillus amoenus/A. creber mixture (92.04% ± 0.93), and the highest value for the Aspergillus protuberus/A. sydowii mixture (96.21 ± 1.66). HaCaT cells showed a higher sensitivity than A549 cells at equivalent exposure time ( Figure 2B). Indeed, all mixtures containing spores of two or three different species showed a significant decrease in cell survival (p < 0.001 and p < 0.05, respectively). The lowest percentage of cell survival was observed for the Aspergillus creber/A. jensenii mixture (95.43% ± 0.57), while the highest percentage of cell survival, significantly lower than the negative control, was observed for the Aspergillus amoenus/A. creber/A. sydowii mixture (97.12% ± 1.22). In the absence of 50% or less cell survival for any of these experimental conditions, no IC50 (50% cell survival inhibiting concentration) could be calculated. 72 h, considering the results already obtained. The mixtures were made from the four species found in the environment: Aspergillus amoenus, A. creber, A. jensenii, and A. protuberus, plus Aspergillus sydowii, as it was the most frequent species in our clinical samples. As shown in Figure 2, all combinations containing spores belonging to two different species showed a significant decrease in cell survival (p < 0.05) for the A549 cell line, with the lowest value for the Aspergillus amoenus/A. creber mixture (92.04% ± 0.93), and the highest value for the Aspergillus protuberus/A. sydowii mixture (96.21 ± 1.66). HaCaT cells showed a higher sensitivity than A549 cells at equivalent exposure time ( Figure 2B). Indeed, all mixtures containing spores of two or three different species showed a significant decrease in cell survival (p < 0.001 and p < 0.05, respectively). The lowest percentage of cell survival was observed for the Aspergillus creber/A. jensenii mixture (95.43% ± 0.57), while the highest percentage of cell survival, significantly lower than the negative control, was observed for the Aspergillus amoenus/A. creber/A. sydowii mixture (97.12% ± 1.22). In the absence of 50% or less cell survival for any of these experimental conditions, no IC50 (50% cell survival inhibiting concentration) could be calculated.

Cell Survival after Exposure to Fungal Extracts
The percentages of cell survival for the A549 and HaCaT lines after 72 h of exposure to the fungal extracts are shown in Figure 3. DMEM with 5% DMSO used to solubilize the fungal dry extracts did not show a significant decrease in cell survival on the A549 and HaCaT lines (98.65% and 98.68%, respectively). Cell survival of the A549 and HaCaT lines after exposure to fungal extracts showed a dose effect for all species. An overall comparison of cell survival percentages obtained for all concentrations and extracts, regardless of species, revealed a greater sensitivity of HaCaT cells to fungal extracts than A549 cells (p < 0.0001). For both cell lines, among the fungal extracts tested, Aspergillus puulaauensis was the most cytotoxic (IC50 < 25.0 µg/mL for both cell lines), followed by Aspergillus creber (IC50 = 116.9 µg/mL for cell line A549 and < 50.0 µg/mL for cell line HaCaT) and Aspergillus jensenii (IC50 = 148.9 µg/mL and 209.3 µg/mL for A549 and HaCaT cell lines, respectively). In decreasing order of cytotoxicity: Aspergillus amoenus and Aspergillus protuberus (for which the comparison of their cytotoxic activity showed no significant difference), Aspergillus sydowii, and finally Aspergillus fructus, for which no significant decrease in cell survival was observed.
son of cell survival percentages obtained for all concentrations and extracts, regardless of species, revealed a greater sensitivity of HaCaT cells to fungal extracts than A549 cells (p < 0.0001). For both cell lines, among the fungal extracts tested, Aspergillus puulaauensis was the most cytotoxic (IC50 < 25.0 µg/mL for both cell lines), followed by Aspergillus creber (IC50 = 116.9 µg/mL for cell line A549 and < 50.0 µg/mL for cell line HaCaT) and Aspergillus jensenii (IC50 = 148.9 µg/mL and 209.3 µg/mL for A549 and HaCaT cell lines, respectively). In decreasing order of cytotoxicity: Aspergillus amoenus and Aspergillus protuberus (for which the comparison of their cytotoxic activity showed no significant difference), Aspergillus sydowii, and finally Aspergillus fructus, for which no significant decrease in cell survival was observed.

Isolates Identification
Amplification and sequencing of the BenA gene allowed the identification of the 21 isolates collected as belonging to seven different species of the series Versicolores: Aspergillus amoenus, A. creber, A. fructus, A. jensenii, A. protuberus, A. puulaauensis, and A. sydowii.
Aspergillus creber was the most common species in the bioaerosols (7 out of 11 isolates), which was consistent with data on fungal diversity in bioaerosols from the USA [17], Croatia [16], Italy [41], and our previous study [20] (France). Aspergillus amoenus, A. jensenii, and A. protuberus have also been found in bioaerosols in other studies [16,20]. However, the specific diversity here was quite low. Indeed, Aspergillus cvjetkovicii, A. fructus, A. griseoaurantiacus, A. pepii, A. tabacinus, A. sydowii, A. tennesseensis, or A. venenatus have also been isolated from bioaerosols [16,17,26], which was not the case in our study.
Regarding the species found in clinical samples, our results were only partially consistent with available clinical data [18,42]: Aspergillus sydowii seemed to be the most frequent species found in clinical samples, but we did not find A. creber or A. amoenus, which were the two most frequent species cited after A. sydowii.

Oxidative Potential
To our knowledge, this study was the first to provide data on the oxidative potential of species belonging to the series Versicolores. Each isolate showed a significant oxidative potential, but for different spore concentrations. The species whose spores showed the highest oxidative potential was Aspergillus jensenii, which is considered as one of the most frequently found species in bioaerosols after A. creber. DTT consumption rates were between 0.129 and 0.085 nmol/min for suspensions with 10 5 and 10 2 spores/mL, respectively, which was lower than those known for Aspergillus fumigatus (between 0.422 and 0.185 nmol/min for spore suspensions with 10 5 and 10 2 spores/mL, respectively), but closer to those measured for Aspergillus brasiliensis (between 0.240 and 0.007 nmol/min for spore suspensions with 10 5 and 10 2 spores/mL, respectively) [8]. Due to the lack of studies on this subject, we have no comparative information regarding the intraspecific variability that we observed with our isolates.
At a concentration of 25 µg/mL, all extracts showed a higher oxidative potential than the blank. We were able to measure an oxidative potential with an average rate of DTT consumption of 0.116 nmol/min for all species of the series Versicolores combined. This value was not significantly different from that obtained for spore suspensions at 10 5 spores/mL (p = 0.16). The species for which the extracts showed the highest DTT consumption was Aspergillus amoenus, which has previously been found in air, but is more common in food [17,43]. Since this was the first study of the oxidative potential of fungal extracts, we did not have a point of comparison to determine whether or not these values were high compared to extracts of Aspergilli from the series Versicolores made with other solvents, or to extracts made with the same solvent but for other species.
Thiols (such as DTT) can interact with spores, and are able to modify their structure by giving a giant cell a shape close to that of chlamydospores [44]. This interaction could account for some of the DTT consumption; however, the incubation time required to obtain these giant cell forms is three days, which is much longer than the 30 min incubation time in our study. As mentioned in the introduction and in previous studies, the species of the series Versicolores showed a significant phenotypic polymorphism. This polymorphism was found at the microscopic level with the presence of spores of variable colors, and can be smooth, rough, or verrucous; and at the macroscopic level with a variety of textures and synthesis of pigments and exudates, even between isolates belonging to the same species [17,20,45].
Pigments and exudates contain secondary metabolites with various activities: antifungal and antibacterial agents (asperversin, averufins, aspergillomarasmine A) [46,47] or cytotoxic agents (versixanthones) [27]. One of the molecules found in the pigments of Aspergillus of the series Versicolores is melanin, which, although historically known for its antioxidant properties, in fact has a duality of functional activity through eumelanin (photoprotective and antioxidant) and pheomelanin (phototoxic and prooxidant) [48][49][50]. A variable production of pheomelanin and/or eumelanin or other unidentified compounds from one isolate to another could explain this variability in the oxidative potential measured for the extract from isolates belonging to the same species of the series Versicolores.

Cell Survival
For the first time, we exposed cells of the A549 and HaCaT lines directly to spore suspensions and total acidified ethyl acetate extracts of series Versicolores species.
The cell survival measured after 24, 48, and 72 h of exposure did not show any significant difference with those measured for the negative control, except for the A549 cell line exposed to spore suspensions at 10 5 spores/mL of the Aspergillus jensenii species, considered as the most frequently found species of the series Versicolores in bioaerosols after A. creber [16,20].
The tests performed with spore suspension mixtures aimed at mimicking the interactions between different species of the series Versicolores most frequently and simultaneously found in bioaerosols and their impact on cell survival. After 72 h of exposure, all mixtures containing 10 5 spores/mL of two species in equivalent amounts showed a significant decrease in cell survival of the A549 and HaCaT lines. We could make the same observation for mixtures containing 10 5 spores/mL of three different species in equivalent amounts for the HaCaT cell line. This indicated that the simultaneous presence of spores of at least two different species generated interactions leading to the stimulation of secondary metabolite biosynthetic pathways [51]. These metabolites, absent when spores of only one species were present in suspension, caused a decrease in cell survival. In other words, spore toxicity was exacerbated by the concomitant presence of spores of other species. An increase in the species richness of airborne Aspergilli of the series Versicolores is therefore a risk factor that increases their toxicity. The lack of a significant decrease in cell survival with mixtures of spores from more than three different species probably was related to a relative decrease in the number of spores of each species in the mixture. Similarly, cytotoxicity was higher for mixtures containing spores from two species than those from three different species.
Cell survival tests performed on total extracts with acidified ethyl acetate showed that the HaCaT cell line was more sensitive than the A549 cell line, and that the extract made from Aspergillus puulaauensis was the most toxic. However, this mold was not the most common species of the series Versicolores in the air [20,22,41]. On the other hand, the two other species that showed the highest decrease in cell survival (Aspergillus creber and A. jensenii) were the most frequent in the air [16,20]. These species are indeed known to synthesize sterigmatocystin, 5-methoxysterigmatocystin, and versicolorin A and B, which are cytotoxic mycotoxins that can be extracted by acidified ethyl acetate [52][53][54].

A Link between Oxidative Potential and Cell Survival?
All spore suspensions showed a significant decrease in the amount of DTT present in the reaction medium, especially for the 10 5 spores/mL Aspergillus jensenii suspensions.
Only the A549 cell line after 72 h of exposure to Aspergillus jensenii spore suspensions at a concentration of 10 5 spores/mL showed a significant decrease in cell survival. This may suggest a possible correlation between these two tests.
However, measurement of the oxidative potential revealed that extracts of Aspergillus amoenus consumed DTT most rapidly, while at an equivalent concentration (25 µg/mL), cell survival was not altered. On the contrary, the oxidative potential of A. puulaauensis was not significantly different from that measured for the other species but showed the highest cytotoxicity.
An overall comparison of the data obtained for oxidative potential and cell line survival for extracts at 25 µg/mL clearly showed that there was no relationship between the results of these two tests. This meant that the oxidation generated by the spores and extracts was not sufficient to decrease cell survival, and that the observed toxicity was due to other mechanisms than oxidation alone, such as a direct activation of caspase-3 by sterigmatocystin [55].

Are Clinical Isolates More Dangerous Than Environmental Isolates?
In our study, we observed an important intraspecific heterogeneity in terms of toxicity. Statistical tests performed did not show a higher oxidative potential or cytotoxicity for the clinical isolates than for the environmental isolates. However, it is interesting to note that the most represented species among the clinical isolates was Aspergillus sydowii, which grows more easily at 37 • C than the other species of the series Versicolores [20].

Limitations
In this first study, the measurement of the oxidative potential was only performed with the DTT test, which is a cell-free test. Similarly, the cytotoxicity tests were conducted on two cell lines (one for skin and one for the airways) using SRB staining. Moreover, other reference strains (such as Aspergillus amoenus CBS 245.65) should be tested in future studies.

Conclusions
In conclusion, DTT tests allowed us to highlight that Aspergillus jensenii spores and A. amoenus extracts had the highest oxidative potentials. Cell survival assays showed a decrease in cell survival only for Aspergillus jensenii spores at a concentration of 10 5 spores/mL after 72 h of exposure of A549 cells and for all fungal extracts, especially for A. puulaauensis. We were able to demonstrate a greater sensitivity of HaCaT cells to the fungal extracts than for A549 cells. The cell survival tests performed with the spore mixtures allowed us to observe a potentiation of the toxicity when spores of different species were present concomitantly in suspension. The comparison of the two methods showed that the measurement of an oxidizing potential alone was not predictive of cellular toxicity.
These first data on the toxicity of spores and extracts of Aspergilli species of the series Versicolores allowed us to affirm that a great intraspecific variability exists in terms of biological activity, and that these species do not all present the same hazard for human health, hence the need to identify them within bioaerosols. Identification and quantification of new metabolites must be undertaken to explain this variability in biological activity. Studies on toxicity of these metabolites are also necessary to contribute to health risk assessment of molds belonging to Aspergillus series Versicolores.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms10020228/s1, Table S1: BenA gene sequences of the isolates used in this study.