The Fusarium metabolite culmorin suppresses the in vitro glucuronidation of deoxynivalenol

Glucuronidation is a major phase II conjugation pathway in mammals, playing an important role in the detoxification and biotransformation of xenobiotics including mycotoxins such as deoxynivalenol (DON). Culmorin (CUL), a potentially co-occurring Fusarium metabolite, was recently found to inhibit the corresponding detoxification reaction in plants, namely DON-glucoside formation, raising the question whether CUL might affect also the mammalian counterpart. Using cell-free conditions, CUL when present equimolar (67 µM) or in fivefold excess, suppressed DON glucuronidation by human liver microsomes, reducing the formation of DON-15-glucuronide by 15 and 50%, and DON-3-glucuronide by 30 and 50%, respectively. Substantial inhibitory effects on DON glucuronidation up to 100% were found using the human recombinant uridine 5′-diphospho-glucuronosyltransferases (UGT) 2B4 and 2B7, applying a tenfold excess of CUL (100 µM). In addition, we observed the formation of a novel metabolite of CUL, CUL-11-glucuronide, identified for the first time in vitro as well as in vivo in piglet and human urine samples. Despite the observed potency of CUL to inhibit glucuronidation, no significant synergistic toxicity on cell viability was observed in combinations of CUL (0.1–100 µM) and DON (0.01–10 µM) in HT-29 and HepG2 cells, presumably reflecting the limited capacity of the tested cell lines for DON glucuronidation. However, in humans, glucuronidation is known to represent the main detoxification pathway for DON. The present results, including the identification of CUL-11-glucuronide in urine samples of piglets and humans, underline the necessity of further studies on the relevance of CUL as a potentially co-occurring modulator of DON toxicokinetics in vivo.

Introduction
Mycotoxins are toxic compounds formed as secondary metabolites by different fungal genera such as Fusarium, Aspergillus, Penicillium and Alternaria. As toxigenic molds frequently contaminate agricultural crops pre-or post-harvest, mycotoxins may enter the food and feed chains, posing a potential risk to both human and animal health. Recent multi-mycotoxin surveys as well as human biomonitoring studies point at co-occurrence of various mycotoxins (Kovalsky et al. 2016;Marin et al. 2018;Warth et al. 2013b). It is rather the rule than the exception to be exposed simultaneously to a mixture of several mycotoxins through the diet. Nonetheless, mycotoxin risk assessment is still predominantly based on single-compound toxicity studies (EFSA et al. 2018(EFSA et al. , 2017a, and combinatory interactions, in particular with co-occurring fungal metabolites considered themselves to be of low toxicological relevance, are yet rarely taken into account. Glucuronidation is a major phase II conjugation pathway for xenobiotics in most mammalian species. Glucuronide-conjugation of mycotoxins, including deoxynivalenol (DON), has been investigated in several studies (Maul et al. 2012;Schwartz-Zimmermann et al. 2017). Uridine 5′-diphospho-glucuronosyltransferases (UDP-glucuronosyltransferases, UGTs), integral membrane proteins localized in the endoplasmic reticulum, catalyze this transfer of glucuronic acid to the substrate (Dong et al. 2012). In humans, 22 UGT isoforms exist, which were classified into four gene families: UGT 1, UGT 2, UGT 3 and UGT 8 (Rowland et al. 2013). Regarding UGT tissue localization, numerous studies reported that in humans, the liver shows the highest abundance of UGT enzymes (Court et al. 2012;Izukawa et al. 2009;Ohno and Nakajin 2009), while extra-hepatic drug metabolism is considered to occur predominantly in kidneys and the gastrointestinal tract (Tourancheau et al. 2018;Tukey and Strassburg 2000). DON (Fig. 1a), a type-B trichothecene, is one of the most abundant Fusarium mycotoxins in temperate climate regions (EFSA et al. 2017b;Kovalsky et al. 2016;Streit et al. 2013). Due to its strong emetic effect, it is also referred to as vomitoxin. The C12-C13 epoxide moiety was shown to be crucial for its main mechanism of action, the interaction with the ribosomal 60S subunit, resulting in the inhibition of protein bio-synthesis (Garreau de Loubresse et al. 2014;Ueno 1977) and the induction of ribotoxic stress (Iordanov et al. 1997;Laskin et al. 2002;Pestka et al. 2004). Further mechanisms associated with DON exposure comprise, among others, proinflammatory processes (Pestka 2008(Pestka , 2010a and the activation of autophagic reactions (Del Favero et al. 2018). In several human biomonitoring studies, between 66 and 91% of ingested DON was excreted into urine as two different glucuronide conjugates: DON-3-glucuronide (DON-3-GlcA) and , the latter identified as the dominating isomer in humans (EFSA et al. 2017b;Vidal et al. 2018;Warth et al. 2013a). Maul et al. (2015) tested twelve commercially available human recombinant UGTs, whereof two isoforms led to the formation of the DON-glucuronides. UGT 2B4 predominantly formed DON-15-GlcA, whereas UGT 2B7 mainly catalyzed the formation of DON-3-GlcA.
The sesquiterpene diol culmorin (CUL, Fig. 1b), firstly isolated by Ashley et al. (1937), is another secondary metabolite produced by various Fusarium species, such as F. culmorum, F. graminearum, F. venenatum and F. cerealis (syn. crookwellense) (Greenhalgh et al. 1984;Lauren et al. 1987;Miller and MacKenzie 2000;Pedersen and Miller 1999). Occurrence levels of this fungal metabolite typically correlate with DON contamination levels, resulting in CUL concentrations about 2-3 times higher than those found for DON (Ghebremeskel and Langseth 2001;Uhlig et al. 2013). CUL was detected in 63% of 82 analyzed feed samples sourced in Europe, America and Australia at median and maximum concentrations of 195 and 44,616 µg/kg, respectively (Streit et al. 2013). Further studies identified CUL in 95-100% of Norwegian barley, oats and wheat samples, reaching median and maximum levels of 2000 and 31,500 µg/kg, respectively . CUL is often referred to as 'fungal secondary metabolite'. However, it might also be considered as an 'emerging mycotoxin', defined by Vaclavikova et al. (2013) as mycotoxins which are 'neither routinely determined, nor legislatively regulated; however, the evidence of their incidence is rapidly increasing'. Information on the toxicological relevance of CUL is limited. In the Ames test, mutagenic effects could be excluded (Pedersen and Miller 1999). CUL was shown to mediate weak antifungal activity (Strongman et al. 1987) and to be phytotoxic to wheat coleoptile tissue at concentrations between 0.1 and 1 µM (Wang and Miller 1988). Administration of CUL-contaminated feed to caterpillars and swine for 7 days (25 mg/kg) and 21 days (2 mg/kg) did not affect negatively the weight and performance of the animals (Dowd et al. 1989;Rotter et al. 1992). Nevertheless, in the same studies, potential combinatory interactions of CUL (10 mg/kg diet) with DON (25 mg/kg diet) were reported, leading to a significant weight reduction and an increased mortality of Helio zea larvae, in comparison to the effects caused by DON alone (Dowd et al. 1989). In pigs such interactions of CUL and DON could not be confirmed (Rotter et al. 1992). However, recently Michlmayr et al. (unpublished) identified CUL as an inhibitor of the UDP-glucosyltransferase Os79, resulting in a decreased DON-3-glucoside formation. Whereas mammalian enzymes use UDP-glucuronic acid as a substrate for conjugation reactions, the equivalent in plants is UDP-glucose, catalyzing, e.g., the transfer to the hydroxyl group at the C-3 position of DON (Poppenberger et al. 2003).
Based on the recent finding by Michlmayr et al. (unpublished) in planta, we aimed to address the question whether co-occurrence and presence of CUL affects also the major DON-detoxifying pathway in humans, namely glucuronidation. Therefore, we performed glucuronidation assays, using commercially available rat and human liver microscomes, as well as UGT Supersomes™. In these experiments, a new glucuronide metabolite of CUL could be identified, CUL-11-glucuronide Fig. 1c). Furthermore, the presence of CUL-11-GlcA was confirmed in urine samples of CUL-fed pigs and in a human urine sample. In addition, we determined the transcription levels of the UGTs relevant for DON glucuronidation (UGT 2B4, UGT 2B7) in human cell lines originating from intestine (HT-29, Caco-2) and liver (HepG2), and investigated whether inhibitory effects of co-occurring CUL might result in synergistic toxicity of DON and CUL.

Chemicals and reagents
CUL was produced and purified as described in Weber et al. (2018). CUL-11-GlcA was produced from rat liver microsomes according to a similar protocol as used by Schwartz-Zimmermann et al. (2017). In total, 1.5-mg CUL was treated with pooled liver microsomes from male Sprague-Dawley rats (BioIVT, Brussels, Belgium), yielding one main substance with a glucuronidation yield of > 95%. The product was isolated by preparative HPLC and consecutive NMR measurements revealed the compound to be CUL-11-GlcA. Details on NMR analysis and structure confirmation are presented in the Online Resource (Figs. S1, S2 and Table S1). DON was purchased from Romer Labs (Tulln, Austria). CUL was dissolved in dimethyl sulfoxide (DMSO) at 50 mM and DON in water (LC-MS grade) to obtain a stock solution of 10 mM. Recombinant human UGT 2B4 and UGT 2B7 were purchased from Corning Life Sciences (Amsterdam, Netherlands). Human liver microsomes (pooled from 25 donors of mixed gender, 20 mg/mL) were purchased from BioIVT (Brussels, Belgium). Alamethicin, magnesium chloride, uridine-diphosphoglucuronic acid (UDPGA) and uridine-diphospho-N-acetylglucosamine were obtained from Sigma Aldrich (Vienna, Austria). For mass spectrometric measurements, methanol (MeOH), acetonitrile (ACN), acetic acid and water of LC-MS grade were purchased from Sigma (Fluka; Vienna, Austria). A multi-component standard including DON and its metabolites (DON-3-sulfate, DON-15-sulfate, DON-3-GlcA and DOM-1) was prepared according to Warth et al. (2016). In addition, a second standard mix containing CUL and CUL-11-GlcA was prepared. For the animal experiment, polyethylene glycol 300 (PEG 300, Merck, Germany) was mixed with purified water (50/50, v/v). Thereafter, appropriate amounts of CUL were dissolved in 50% PEG 300 to obtain a stock solution of 300 µg/mL.

Glucuronidation assay
The potential inhibitory effect of CUL on DON glucuronidation was analyzed using liver microsomes from rats or humans. In principle, the assays were performed as described previously for DON alone (Schwartz-Zimmermann et al. 2017). Three groups were set up: (1) DON (as positive control, n = 5), (2) CUL/DON (1:1 molar ratio, n = 5), (3) CUL/DON (1:5 molar ratio, n = 5). The concentration of DON in all cases was 20 mg/L (67 µM), while the molar excess of the co-substrate UDPGA was at least 20-fold. After stopping the reactions, samples were diluted 1:10 with 20% aqueous methanol and the formed glucuronides were determined by LC-MS/MS. Furthermore, the effect of CUL on the glucuronidation of DON was tested using two different UGT isoforms (UGT 2B4, UGT 2B7) according to Maul et al. (2015). Human UGT Supersomes™ were used at a concentration of 1 mg/ mL. Mixtures of the recombinant UGT isoform containing 100 mM potassium phosphate buffer (pH 7.4), 5 mM MgCl 2 and 25 µg/mL alamethicin were placed on ice for 15 min to allow alamethicin pore formation. 100 µM CUL or the respective solvent control was added to the incubation solution and pre-incubated at 37 °C for 30 min. Afterwards, DON (10 µM) or CUL (100 µM) was added and the reaction was initiated by the addition of UDPGA (2.5 mM). After further incubation for 1 h at 37 °C, reactions were terminated by addition of 200 µL ACN. Samples were then chilled at − 20 °C for 10 min and centrifuged at 14,000×g for 5 min. Then, 200 µL of the supernatant were evaporated to dryness using a Labconco Centrivap Benchtop Vacuum Concentrator (Labconco, USA) and reconstituted in 100 µL water/ACN (9:1, v/v) for LC-MS/MS analysis. Glucuronidation experiments with the recombinant UGTs were carried out in four independent experiments.

Animals and study design
All procedures related to the animal experiment were performed according to Austrian law and following the European Guidelines for the Care and Use of Animals for Research Purpose (European Commission 2010). The experiment was approved by the office of the Lower Austrian Region Government, Group of Agriculture and Forestry, Department of Agricultural Law (approval code LF1-TVG-39/050-2017) and carried out at the Center of Applied Animal Nutrition (Biomin Holding GmbH, Tulln, Austria).
Six crossbred piglets (sow: Landrace x Large White, boar: Pietrain; approximately 5 weeks old, 8.7 ± 1.0 kg, mixed sex) were obtained from a local swine producer. Piglets were housed individually in metabolic cages, had free access to water and were allowed to acclimatize for 5 days. Feed was provided twice daily and withdrawn after 30 min to monitor feed intake. Prior to the start of the experiment, the feed was tested for mycotoxin contamination by LC-MS/MS (Malachova et al. 2014). Low levels of CUL (20 µg/kg), 5-hydroxyculmorin (107 µg/kg), 15-hydroxyculmorin (67 µg/kg) and DON (79 µg/kg) were present in the diet, whereas other relevant mycotoxins (e.g., aflatoxin B 1 , fumonisin B 1 , ochratoxin A, etc.) were not detected.
Piglets received 50% PEG 300 (solvent control) and CUL (150 µg/kg body weight) by gavage on day 1 and 7. To this end, respective solutions were orally administered via polyvinyl chloride catheters. Urine and feces of individual piglets were collected 0-8 h, 8-24 h and 24-28 h after each of the treatments. Samples were stored at − 20 °C until analysis.

LC-MS measurements
Pig urine samples were diluted to 0.2 mM creatinine with MeOH/water (50/50, v/v) and centrifuged at 14,350×g for 10 min. Measurement of liver microsome samples and pig urine samples was carried out on a 1290 Infinity series UHPLC system (Agilent Technologies, Waldbronn, Germany) coupled to a 6500+ QTrap mass spectrometer equipped with an IonDrive TurboV source (SCIEX, Foster City, CA, USA). Analytes were separated in gradient elution mode on a Kinetex C18 column (150 × 2.1 mm, 2.6 µm, Phenomenex, Aschaffenburg, Germany). Mobile phase A was water/acetic acid (99.9/0.1, v/v), mobile phase B was composed of ACN/acetic acid (99.9/0.1, v/v). The following linear gradient was used: 0.0-0.5 min: 5% B, 0.5-7.0 min: linear increase to 15% B, 7.0-14.0 min: linear increase to 90% B, 14.1-16.0 min: 100% B, 16.1-19.0 min: re-equilibration at 5% B. The flow rate was 250 µL/min, the column temperature was 30 °C and the injection volume was 3 µL. Mass spectrometric detection was performed in negative mode after electrospray ionization. The source parameters were: source temperature 400 °C, ion spray voltage − 4500 V, curtain gas 35 psi, ion source gas 1 60 psi and ion source gas 2 40 psi. Selected reaction monitoring mode was chosen as scan type and the following transitions were used: CUL-11-GlcAc quantifier m/z . Analyst software version 1.6.3 (SCIEX) was used for instrument control and data analysis. CUL-11-GlcAc eluted at 11.40 min, CUL at 12.89 min. In urine diluted to 0.2 mM creatinine, matrix effects of CUL-11-GlcAc and CUL were 96 and 95%, respectively. The limits of detection (LODs, signal-noise ratio 3:1) for CUL-11-GlcAc and CUL in pure solvent standard solution were 0.1 ng/mL and 2 ng/mL, respectively. In urine diluted to 0.2 mM creatinine, the LOD of CUL-11-Gl-cAc was 0.8 ng/mL and the LOD of CUL was 2.5 ng/mL. Limits of quantification (signal to noise ratio 10:1) were by the factor 3.3 higher than LODs.
All samples obtained from the glucuronidation assay using the recombinant enzymes UGT 2B4 and UGT 2B7 and also the human urine sample were measured by LC-HRMS applying the following method: after reconstitution, the samples were analyzed by an LC-HRMS method using a UHPLC Vanquish system hyphenated with a Q Exactive HF high-resolution mass spectrometer equipped with a heated electrospray ionization (hESI) source (all from Thermo Fisher Scientific, Waltham, USA). Chromatographic separation was achieved on a reversed-phase Acquity UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm, Waters, Milford, USA) protected by a HSS T3 VanGuard pre-column at 40 °C. At a constant flow rate of 0.4 mL/min, a linear gradient was employed with water (A) and ACN (B), both containing 0.1% acetic acid. After an initial hold time of 2 min at 10% B, the gradient was raised to 50% B at minute 6 and 100% B at minute 7. After 2 min at 100% B, the column was re-equilibrated at 10% B for 1 min. A sample volume of 2 μL was injected by the autosampler which was maintained at 4 °C. The following settings were used: negative ionization mode, capillary temperature, 325 °C; vaporizer temperature, 400 °C; sheath gas, 60 arbitrary units (a.u.); auxiliary gas, 20 a.u.; sweep gas, 3 a.u.; capillary voltage, 3 kV. The instrument was operated in profile mode (scan range, m/z 60-900) with a resolving power of 120.000 full width at half maximum and automatic gain control setting of 3 × 10 6 with a maximum injection time of 100 ms. The LOD for DON-3-GlcA and DON-15-GlcA (peak area 5000 a.u.) was estimated based on the smallest peak detected within the respective measurement sequence. For data evaluation, Xcalibur™ (version 3.0, Thermo Scientific) and Trace-Finder™ (version 3.3, Thermo Scientific) software were used. The general status of the instrument was regularly checked by the measurement of blank and quality control samples before and after the sequence.
The diluted human urine sample, obtained from pregnant women from Croatia (Sarkanj et al. 2013;Warth et al. 2016), was analyzed by the LC-HRMS full scan method to evaluate if CUL-11-GlcA may be found in human specimen. To verify the identity of the conjugate, additional MS/MS spectra of the urine sample and the reference standard were performed.

Cell culture and treatment
The human colorectal adenocarcinoma cell line HT-29 was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), the human liver hepatocellular carcinoma cells HepG2 and the human colorectal adenocarcinoma cell line Caco-2 from the American Type Culture Collection (ATCC, Manassas, USA). For HT-29 and Caco-2 cell cultivation Dulbecco's Modified Eagle's Medium and for HepG2 cells RPMI 1640 medium was used. All three basal media formulations were supplemented with 10% (v/v) heat-inactivated fetal calf serum and 1% (v/v) penicillin/streptomycin (50 U/mL). Caco-2 culture medium was additionally supplemented with 1% potassium pyruvate and 0.01 mg/mL insulin-transferrin-selenium. Culture media and supplements were purchased from GIBCO Invitrogen (Karlsruhe, Germany). For cell cultivation and treatments, humidified incubators at 37 °C and 5% CO 2 were used. Cells were routinely tested for the absence of mycoplasma contamination and used for experiments at passages 9-25. CUL, DON and their combinations were added to the incubation solutions, resulting in final solvent concentrations of 1% (v/v) LC-MS grade water and 1% (v/v) DMSO. Measurements of combinatory effects were always performed in parallel to the single substance on the same 96-well plate, to maximize comparability of the data and to allow an accurate calculation of the combinatory effects.
StepOnePlus ® software (version 2.3, Applied Biosystems, USA) was used for fluorescence signal quantification and further data analysis. Of each tested sample, at least three independent experiments were performed. Presented transcript data were normalized to the mean of transcript levels of endogenous control genes (ACTB1, GAPDH) applying if possible and necessary the ΔΔCt-method (Schmittgen and Livak 2008). Respective 2 −ΔCt data are shown as Online Resource (Fig. S5a-c).

Combinatory effects on cell viabilitysulforhodamine B assay
To determine combinatory effects of CUL with DON on cell viability, the sulforhodamine B (SRB) assay according to Skehan et al. (1990) was applied. For this purpose, reactions containing different concentrations of CUL and the respective DON concentration at a constant substance ratio of 10:1 were incubated. In both cell lines, 24-h incubations were conducted; whereas in HT-29 combinations, additionally experiments applying 48-h toxin treatment were assessed. HT-29 and HepG2 cells were seeded into 96-well plates and allowed to grow for 72 h. Cells were incubated for 24 h or 48 h with CUL and DON (CUL 0.01-100 µM; DON 0.01-10 µM) and the respective combinations. Subsequently, the cells were rinsed with pre-warmed phosphate-buffered saline, fixed with 5% (v/v) trichloroacetic acid incubated at 4 °C for 30 min. Afterwards, cells were washed four times with water, plates were dried overnight at room temperature and then stained for 1 h using a solution of 0.4% (w/v) SRB in 1% (v/v) acetic acid. To remove the remaining staining solution, cells were washed twice with water and 1% (v/v) acetic acid solution. Then, plates were dried at room temperature in the dark. Finally, 10 mM Tris buffer (pH 10; 100 µL per well) was used to dissolve the dye and single wavelength absorbance was read at 570 nm using a Cytation 3 Imaging Multi Mode Reader (BioTek, Bad Friedrichshall, Germany). A solution of 1% (v/v) triton X-100 served as positive control. Cell-free blank values were subtracted and measured data were referred to the respective solvent control. Combinatory cell viability data were compared to single treatments and to a mathematically determined expected value (EV; see "Data visualization and statistical analysis of combinatory effects").

Data visualization and statistical analysis of combinatory effects
Combinatorial interactions of two or more substances are present if the experimentally determined effect differs from the "additive effect" of the two or more single compounds (Chou 2006). To assess interactions that do not follow a linear dose-response relationship, mathematical models must be applied to calculate this "additive effect". Chou's "multiple drug effect equation" (Chou 2006) is currently recognized as the most accurate model existing to describe these effects (Aichinger et al. 2018;Alassane-Kpembi et al. 2017;Vejdovszky et al. 2017). However, the determination of an IC 50 value and, therefore, a measurable effect of at least 50% is a prerequisite for the application of this model. Since in some of our experimental setups this criterion was not fulfilled, the model of independent joint action (IJA) was chosen for the analysis of combinatory effects of CUL and its co-contaminant DON (Webb 1963). Applying this model, an EV is calculated for each combination taking into consideration the determined single effects via the formula: with f ab being the EV, and f a and f b being the measured effects of the single substances. Then, the EV was compared to the measured combinatory data set applying two-tailed Student's t test. In the glucuronidation assay, statistical differences between the determined single effects of DON and the combination with CUL in glucuronide formation were calculated applying two-tailed Student's t test using for the combinatory data 50% relative standard deviation (RSD), defined as LOD. For statistical analysis and data visualization, Origin 2018 software (Northampton, USA) was used.

Inhibitory effect of CUL on in vitro glucuronidation of DON
The ability of CUL to interfere with DON glucuronidation was assessed using two commercially available liver microsome preparations from human and rats. As expected, the main metabolite of DON incubated with liver microsomes from rats was DON-3-GlcA (data not shown). No significant concentration differences of DON-3-GlcA in the DON, CUL/ DON (1:1) and CUL/DON (1:5) groups were seen. Thus, it seems that CUL has no modulatory effect on the glucuronidation activity of rat liver microsomes. Interestingly, the picture was quite different using human liver microsomes (Fig. 2a, b). Only a small part of DON was glucuronidated, of which the major metabolite was DON-15-GlcA (Fig. 2a), formed about 3.5 times more than DON-3-GlcA (Fig. 2b). However, using the human microsomes, combinatory incubations resulted in a significant decrease in the concentration of both formed DON-glucuronides with increasing CUL concentrations. In the equimolar combinations, only 85% DON-15-glucuronide and 70% DON-3-glucuronide of the initial glucuronide formation were found. Applying fivefold molar excess of CUL resulted in about 50% inhibition.
In case of human recombinant UGTs, both UGT 2B isoforms tested (2B4 and 2B7), accepted DON as a substrate, leading to the formation of one glucuronide each (Fig. 2c, d), even though the activities of both recombinant enzymes for the respective glucuronide formation were low. UGT 2B4 primarily formed DON-15-GlcA (Fig. 2c), whereas UGT 2B7 solely catalyzed the formation of DON-3-GlcA (Fig. 2d).
Combinatory incubations of 100 µM CUL together with 10 µM DON resulted in reduced glucuronidation activity in the enzyme preparations. In the UGT 2B4 incubations, the DON-15-GlcA signal was significantly reduced to 3% of the initial glucuronide formation, whereby signal detection was possible in only one of the four replicates very close to the method's detection limit. In UGT 2B7, which showed lower glucuronidation activity with DON alone, the combination with CUL decreased glucuronidation to a level not detectable by the applied method.
When incubated with 100 µM CUL, both UGT 2B isoforms also led to the formation of a CUL-11-GlcA (Fig. 1c), firstly identified in in vitro enzyme incubations. CUL-11-GlcA formation was not affected by the presence of DON.

Differences in UGT gene transcription in HepG2, HT-29 and Caco-2 cells
Encouraged by these results, we set out to test whether an interaction between DON and CUL is also evident at the level of cell lines in toxicity assays. We first tested whether the relevant UGTs are expressed in the cell lines used, and whether the presence of DON or CUL had an effect on expression of the respective genes. Messenger RNA (mRNA) levels of the two UGT isoforms responsible for DON glucuronidation in three different cancer cell lines were investigated by qRT-PCR and are depicted relative to UGT 2B4 mRNA expression in HepG2 cells (Fig. 3). Transcription levels of UGT 2B4 in HepG2 and Caco-2 were found to be similar. In Caco-2, mRNA levels of UGT 2B4 were only 2.60 ± 2.67-fold higher than in HepG2. In contrast, in HT-29, mRNA of UGT 2B4 was found only at low levels in comparison to the other two cell lines, as a Transcript levels of UGT 2B7 were much higher in the analyzed cell lines: about 62-fold in HepG2, only 1.8-fold in HT-29 and around 32-fold in Caco-2. Comparing the different UGT transcription levels of each cell line, in HepG2 cells, UGT 2B7 levels were comparably high. In HT-29, UGT 2B4 was determined to be the least transcribed isoform under the tested conditions. In Caco-2, a similar transcription pattern as in HepG2 cells was found.

Impact of sub-toxic concentrations on UGT gene transcription
The human colon cancer cell line HT-29, known to effectively glucuronidate other mycotoxins such as alternariol and alternariol methyl ether (Pfeiffer et al. 2007), was selected exemplarily for further analysis of the UGT transcription levels and cytotoxicity studies (see "Combinatory cytotoxic effects"). qRT-PCR was used to investigate the modulatory effect of sub-toxic concentrations of CUL (100 µM) and DON (0.1 µM) after 3-h, 24-h and long-term exposure on UGT mRNA levels (Fig. 4a, b). Only marginal impact of short-term CUL exposure (3 h) on UGT transcription levels could be determined, resulting in a slightly increased mRNA concentration of UGT 2B7. However, 24-h incubation with 100 µM CUL resulted in a decrease in the transcription levels of both UGT isoforms tested. Due to limited availability (in-house purification of CUL), long-term cell exposure for 7 days with CUL was not tested.
Incubations with DON for 3 h caused a tentative reduction of the UGT 2B7 transcription level (Fig. 4b). While after 24-h cell exposure, UGT 2B4 transcript levels were increased to 1.3 ± 0.2, UGT 2B7 mRNA levels remained below solvent control level. Long-term treatment of HT-29 cells for 7 days caused a significant reduction of the transcription level in case of UGT 2B7 in comparison to the solvent control, resulting in the following relative transcript level: 0.5 ± 0.1 (Fig. 4b).

Combinatory cytotoxic effects
In both cell lines, HT-29 and HepG2, CUL (0.1-100 µM) did not cause any statistically significant cytotoxic effect in the SRB assay after 24-h incubation (Fig. 5a, b). Since a constant 10:1 ratio has already been used in the glucuronidation assay (see "Inhibitory effect of CUL on in vitro glucuronidation of DON") and as DON concentrations ≥ 10 µM are known to cause strong cytotoxic effects after 24-h incubations, combinations with CUL were performed in the concentration ratio 10:1 (CUL:DON; Fig. 5a-c). In both cell lines, DON mediated similar cytotoxic effects, resulting in a statistically significant reduction of the cell viability at DON concentrations ≥ 1 µM. Combinations of CUL and DON caused similar cytotoxic effects to the ones mediated by DON alone and none of the tested combinations differed significantly from the mathematically calculated EVs.
To assess combinatory effects of CUL on DON-induced cytotoxicity after longer incubation times, HT-29 cells were treated for 48 h. Nevertheless, even cell treatments for 48 h did not cause significant cytotoxic effects of CUL, nor combinatory effects of CUL and DON (Fig. 5c). Thus, with respect to cytotoxicity, no significant effects of the binary mixtures in comparison to the single treatments with DON or the calculated EV were observed. Fig. 4 Impact of CUL and DON exposure on gene transcription levels of (a) UGT 2B4 and (b) UGT 2B7 in HT-29 cells after 3-h, 24-h and 7-day incubation measured by qPCR. Transcription data are normalized to the respective solvent controls (solv. ctr., solid line). Due to a lack of substance availability of CUL 7-day treatments have not been assessed in this case (n. a.). As sub-toxic substance concentrations were applied in this experiment, transcript levels are not affected by cytotoxicity. Data are expressed as mean values ± SD of at least three independent experiments performed in technical duplicates. Data were tested by Kruskal-Wallis ANOVA to compare different time-points and one-sample t test to assess differences to the solvent control. # indicates significant differences in comparison to the solvent control level (p < 0.05)

Fig. 5 Combinatory effects of CUL with DON on cell viability of HT-29 (24-h incubation: a;
48-h incubation: c) and HepG2 cells (24-h incubation: b) in the sulforhodamine B assay. Combinations of CUL and DON were combined 10:1. 1% water (LC-MS grade) + 0.5% DMSO served as solvent controls (solid line). Data are expressed as mean values ± SD of at least five independent experiments performed in triplicates normalized to the respective solvent control. 1% triton-X 100 was used as positive control (TX-100). Significant differences to the respective lowest tested concentration are indicated in the graphs with (b) for DON and (c) for the combination (exponents represent: (1) p < 0.05 and (2) p < 0.01)

Formation of CUL-11-GlcA in vivo
Urine samples of the piglet trial were diluted to the same concentration of creatinine (0.2 mM) and concentrations of CUL and CUL-11-GlcA were determined by LC-MS/MS. CUL-11-GlcA eluted at 11.40 min, CUL at 12.89 min and a potential further CUL-GlcA at 4.27 min. In urine diluted to 0.2 mM creatinine, matrix effects of CUL-11-GlcA and CUL were 96 and 95%, respectively. The LODs (signal to noise ratio 3:1) for CUL-11-GlcA and CUL in pure solvent standard solution were 0.1 ng/mL and 2 ng/mL, respectively. In urine diluted to 0.2 mM creatinine, the LOD of CUL-11-GlcA was 0.8 ng/mL and the LOD of CUL was 2.5 ng/mL. Limits of quantification (signal-noise ratio 10:1) were by the factor 3.3 higher than LODs. Figure 6 shows chromatograms of CUL-11-GlcA and CUL in pure solvents (Fig. 6a) and in a pig urine sample (Fig. 6b).
As with the recombinant UGT enzymes and (human and rat) liver microsomes, CUL was also readily glucuronidated in vivo to CUL-11-GlcA by pigs. In measured urine samples from four animals, the concentration of CUL-11-GlcA was higher than of CUL (maximum CUL-11-GlcA/CUL ratio: 8.7, average ratio 6.3). In urine samples of two animals, no CUL-11-GlcA was detected. One likely explanation for the latter is that insufficient separation of urine and feces in the metabolic cages led to hydrolysis of the formed glucuronides by gut microbes during the sampling period.

Discussion
Mycotoxins not only cause substantial economic losses due to contaminated food, feed and loss of animal productivity, but also pose a serious health threat to both humans and livestock. Numerous studies tried to characterize the toxic potential and mechanistic activities of various Fusarium mycotoxins (Alshannaq and Yu 2017;Fraeyman et al. 2017;Pestka 2010b). Nevertheless, so far this study is, to the best of our knowledge, the first to address combinatory effects of the two co-occurring Fusarium metabolites DON and CUL in human cell models. CUL, previously reported to have limited toxic potential in mammals (Dowd et al. 1989;Miller and MacKenzie 2000;Pedersen and Miller 1999;Prelusky et al. 1989;Rotter et al. 1992), was found to inhibit the metabolic activity of purified plant UDP-glucosyltransferases (Michlmayr et al., unpublished). To investigate interactions of CUL and DON on DON-detoxifying, mammalian enzymes, glucuronidation activities of human liver microsomes and two human UGTs were determined. Using human liver microsomes, significantly lower levels of DON-glucuronides were found in the presence of CUL in a concentration-dependent manner (Fig. 2a, b). Regarding the two tested UGT isoforms, UGT 2B4 and 2B7, combinations with CUL led in both cases to conjugate concentrations near or even below the method's detection limit (Fig. 2c, d). Furthermore, we confirmed that DON-15-GlcA is predominantly formed by UGT 2B4 and that DON-3-GlcA formation is mainly catalyzed by UGT 2B7 (Maul et al. 2015). In accordance with Maul et al. (2015), only low glucuronidation activities were observed by both UGTs.
In the course of the in vitro enzyme experiments, a new CUL metabolite, CUL-11-GlcA, was identified and characterized. This finding argues presumably for a competitive inhibition of the UGTs, as the terpenoid CUL and DON might compete for the same binding site on the enzyme. However, CUL-11-GlcA was not only detected after incubation with UGT 2B4 and 2B7 in vitro (Fig. 2), but also identified as a novel in vivo metabolite in pig urine samples and in a human urine sample obtained from pregnant women in Croatia ( Fig. 6; Online Resource: Fig. S4) (Sarkanj et al. 2013;Warth et al. 2016). This sample has been demonstrated before to contain high concentrations of DON (275 µg/L) and of its metabolites DON-3-GlcA (298 µg/L), DON-15-GlcA (1238 µg/L), and DON-3-sulfate (58 µg/L) (Sarkanj et al. 2013;Warth et al. 2016). Furthermore, a second CUL-GlcA was detected in the pig urine sample, which due to a lack of reference standard could be only tentatively identified as CUL-8-GlcA (Fig. 6).
In cell culture experiments, we aimed to further evaluate potential synergistic toxic effects of DON and CUL probably resulting from the UGT inhibition. To select in this respect appropriate cell lines, expressing the two UGT isoforms of interest, three well-known cancer cell lines (HepG2, HT-29 and Caco-2) were tested for their UGT transcription levels (Fig. 3). Even though mRNAs of both isoforms were present in all the addressed cell lines, expression levels differed substantially when comparing the three cell models, but also among the different isoforms. So far, UGT mRNA expression levels of these cell lines have not been compared, even though numerous studies investigated already UGT expression levels in human tissues, focusing mainly on the liver and the gastrointestinal tract (Court et al. 2012;Izukawa et al. 2009;Ohno and Nakajin 2009;Strassburg et al. 2000;Tukey and Strassburg 2000;Wu et al. 2011). As all three selected cell lines are commonly used for various toxicological studies and also to investigate xenobiotic and drug metabolism in vitro (Akbari et al. 2017;Bohets et al. 2001;Weaver et al. 2017), this characterization of UGT transcription levels is of crucial importance for the scientific community. A study, using real-time PCR to generate a quantitative expression level profile of the UGTs in 26 adult and 3 fetal tissues, confirmed that liver is the organ containing the highest levels of UGT mRNAs, among which UGT 2B4 was the most abundant isoform (40% of total UGT mRNA content; up to 90% in fetal liver) (Court et al. 2012). Ohno and Nakajin (2009) analyzed beyond liver tissue extrahepatic mRNA levels of various UGT isoforms. Intestinal tissue expressed predominantly UGT 1A1, 1A10, 2B7, 2B15 and 2B17. Relatively low levels of various UGTs were reported in esophageal tissue and even in steroidogenic tissues, such as breast, prostate, heart and adrenal gland (Ohno and Nakajin 2009). However, most transcript profiles of UGTs in human tissue-derived cell lines are not consistent with those in corresponding tissue samples (Hart et al. 2010;Nakamura et al. 2008). Using semiquantitative reversetranscription PCR Nakamura et al. (2008) determined UGT mRNA levels from various cell lines including HepG2 and Caco-2. In HepG2, all UGT 2B isoforms tested were all highly expressed, whereas comparably low mRNA levels of most 1A isoforms were determined. For Caco-2, similar UGT expression levels for UGT 1A1, 2B7 and 2B15 were elucidated; however, UGT 2B isoforms were less present in comparison to the levels in HepG2 cells (Nakamura et al. 2008). Studies comparing transcript levels or enzyme activities of HepG2 cells with those of human tissue samples, of primary human hepatocytes or of other liver cell lines, such as HepaRG, mainly reported in HepG2 cells lower UGT transcript levels and enzyme activities (Hart et al. 2010;Westerink and Schoonen 2007;Yokoyama et al. 2018).
Performing a similar data analysis as Ohno and Nakajin (2009), normalizing the Ct-values obtained in our qRT-PCR experiments to the mean of transcript levels of endogenous control genes, multiplying by a factor 10 4 , relative values for UGT 2B4 of 5375 and 5026 and for UGT 2B7 of 6695 and 5781 can be obtained for HepG2 and HT-29, respectively. As Ohno and Nakajin (2009) reported in liver tissue samples values above 37,900 for UGT 2B4 expression, our results clearly show that the transcript levels of this UGT isoform in HepG2 cells are much lower than in the liver tissue. However, with respect to UGT 2B7, similar relative expression values to the ones calculated and reported above have been described by Ohno and Nakajin (2009).
Various studies in animal and in vitro models have demonstrated that the consumption of certain xenobiotics (e.g., aflatoxin B 1 , β-naphthoflavone, ethanol,etc.) may increase UGT levels and respective glucuronidation activities via different induction pathways (Court 2010;Hanioka et al. 2012Hanioka et al. , 2006Kardon et al. 2000;Li et al. 2000). Since mRNA levels of the selected UGT isoforms were partly expressed at relatively low levels, exemplarily HT-29 cells were exposed to sub-toxic concentrations of DON (0.1 µM) and CUL (100 µM). We speculated that these short-and long-term incubations using respective UGT enzyme substrates might have modulatory or even inducing effects on UGT transcript levels (Fig. 4a, b). Incubations for 3 h only marginally affected UGT mRNA levels and after 24-h, UGT 2B4 was the only one slightly induced after DON incubation. Up to 2.5-fold inductions, such as reported by Hanioka et al. (2012) in HepG2 cells after 48-h aflatoxin B 1 treatment, could not be corroborated in this study. To date, similar studies assessing modulatory effects of DON or CUL treatment on UGT mRNA levels have not been described in literature so far.
Despite the fact that UGT transcription levels in all three cell lines were relatively low and even an extended exposure to sub-toxic substrate concentrations could not induce UGT mRNA expression, potential synergistic effects of the two Fusarium secondary metabolites on cell viability were assessed (Fig. 5). In HT-29 and HepG2 cells after 24 h and 48 h, no significant interactions between CUL and DON could be determined. Hence, interactions between DON and CUL observed in planta (Michlmayr et al., unpublished) and by Dowd et al. (1989) in caterpillars, could not be confirmed in our cell models. While humans excrete in urine up to 90% of total DON as glucuronide conjugates, LC-MS/MS analysis of cell extracts could not detect DON-glucuronides in HT-29 and HepG2 cells (data not shown). However, in the same experiments, high glucuronide formation by both cell lines was observed for the Alternaria mycotoxin alternariol, known to be predominantly conjugated by UGT 1A1 and UGT 1A9 (data not shown). Thus, as also transcription levels of UGT 2B4 were about tenfold lower than levels reported in liver tissue (Ohno and Nakajin 2009), potential inhibitory effects of CUL might be of minor importance for the overall toxicity in cell culture and might not be measurable at the determined endpoints. However, negative evidence from our cell culture experiments does not exclude a synergistic effect in the mammalian organism. Due to a lack in exposure and toxicokinetic data, one can only speculate, if relevant systemic concentrations of CUL and DON, at which competitive inhibition of UGTs might take place, can be reached by dietary toxin exposure. As technical issues rendered the findings of the above-mentioned in vivo study inconclusive, a respective animal study needs to be repeated to gain further insights into these CUL-DON interactions.
In conclusion, the present study reported for the first time CUL to partially inhibit the glucuronidation activity of human liver microsomes and recombinant UGTs. Furthermore, we were able to detect two novel CUL-glucuronides both in vitro, as well as, in vivo in pig and human urine samples. Even though in HT-29 and HepG2 cells this presumably competitive effect of CUL on DON glucuronidation did not induce considerably higher cytotoxicity in mixtures, which may be due to the limited glucuronide formation in the tested cell lines, in the human organism DON-glucuronide formation is the main detoxification reaction. Hence, to elucidate if this inhibitory mechanism and resulting combinatory effects are of importance in the mammalian organism and to gain further insight into systemic CUL concentrations and respective toxicokinetics, a more detailed characterization in vivo is required. This may contribute substantially to the discussion about classifying CUL not only as a secondary fungal metabolite but also as an "emerging mycotoxin". Furthermore, this study shows that we are still at the very beginning in the toxicological profiling of chemical mixtures, and underlines also that the assessment of drug-exposome interactions and their further understanding are of crucial importance.


extfieldsofstudy: Medicine Chemistry

text:
Screening of Cellular Stress Responses Induced by Ambient Aerosol Ultrafine Particle Fraction PM0.5 in A549 Cells

Effects of airborne particles on the expression status of markers of cellular toxic stress and on the release of eicosanoids, linked with inflammation and oxidative damage, remain poorly characterized. Therefore, we proposed a set of various methodological approaches in order to address complexity of PM0.5-induced toxicity. For this purpose, we used a well-characterized model of A549 pulmonary epithelial cells exposed to a non-cytotoxic concentration of ambient aerosol particle fraction PM0.5 for 24 h. Electron microscopy confirmed accumulation of PM0.5 within A549 cells, yet, autophagy was not induced. Expression profiles of various cellular stress response genes that have been previously shown to be involved in early stress responses, namely unfolded protein response, DNA damage response, and in aryl hydrocarbon receptor (AhR) and p53 signaling, were analyzed. This analysis revealed induction of GREM1, EGR1, CYP1A1, CDK1A, PUMA, NOXA and GDF15 and suppression of SOX9 in response to PM0.5 exposure. Analysis of eicosanoids showed no oxidative damage and only a weak anti-inflammatory response. In conclusion, this study helps to identify novel gene markers, GREM1, EGR1, GDF15 and SOX9, that may represent a valuable tool for routine testing of PM0.5-induced in vitro toxicity in lung epithelial cells.

Introduction
Particulate matter (PM) in the ambient air is known to play an important role in the development of cardiovascular and respiratory diseases and is classified as carcinogenic to humans (Group 1; [1]). Especially ultrafine particles with an aerodynamic diameter (d ae ) <100 nm could easily enter and transfer within organisms and interact with cells and subcellular components [2]. At the ultrastructural cellular level, aerosol particles were observed either enclosed in membrane vesicles and autophagosomes, or associated with mitochondrial membranes and lamellar bodies in A549 pulmonary cells [3]. In order to reveal the mechanism of PM-induced toxicity, in vitro studies in human airway cells have been performed using size segregated aerosol particles. Water suspension of fine particles PM2.5 were shown to induced oxidative stress, aryl hydrocarbon receptor (AhR)-mediated gene expression, inflammatory responses, DNA damage, changes in the cell cycle and autophagy in human bronchial and lung cells [4][5][6][7]. The ultrafine fraction was found to cause inflammatory, cytotoxic and genotoxic responses [8][9][10][11][12]. However, in addition to well-known effects, such as oxidative stress and inflammation, other mechanisms of nanoparticle toxicity were suggested and encompass endoplasmic reticulum

Results
In order to determine whether the aerosol particles smaller than 0.5 µm are able to induce cellular stress responses, pulmonary A549 cells were exposed to PM0.5 in a concentration of 25 µg/cm 2 for 24 h, which was chosen according to the study Gualtieri et al. [3] and shown to be non-cytotoxic in the time frame of 72 h (WST-1 assay; data not shown).

Intracellular Localization
To ensure that aerosol particles were taken into the cells, ultrathin sections of A549 cells exposed to PM0.5 were examined by transmission electron microscopy. After 3 h of exposure, particles were not found on the ultrathin sections, but after 6, 12 and 24 h exposure, particles occurring in intracellular aggregates of various sizes were observed. They were engulfed by plasmatic membrane in some cases, while in other cases the membrane was not clear. Cellular membrane structures often occurred inside the vesicles with larger aggregates of particles, suggesting that the particles were deposited in multivesicular bodies or autophagosomes (Figure 1).

Expression Analysis of a New Set of Genes Involved in Various Signaling Pathways Including AhR and p53
Next, we examined the expression status of model genes associated with AhR (cytochrome P450 1A1; CYP1A1) and p53 (CDK1A, PUMA, NOXA) activation. Furthermore, gremlin 1 (GREM1) and SOX9 have been shown recently to have a similar 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like expression fingerprint during short-term adaptive response of A549 cells to both toxic exogenous and endogenous ligands, and may thus serve as markers of AhR-dependent transcriptional activity in lung epithelial cells [19]. Herein, we therefore included GREM1 and SOX9 expression analysis into testing of PM0.5-induced toxicity. PM0.5 exposure at non-cytotoxic concentration changed mRNA levels of AhR model target gene CYP1A1, as well as expression of proposed AhR lung-specific targets GREM1 (increased expression) and SOX9 (decreased expression, as shown in Figure 2A).
DNA damage responses were identified in A549 cells on both mRNA and protein levels as previously described [31]. Weak genotoxic effects of PM0.5 were suggested by slightly but significantly induced expression of p53 transcriptional target genes, cyclin dependent kinase inhibitor 1A (CDKN1A, the gene encoding p21 protein), PUMA and NOXA, as determined by realtime quantitative qRT-PCR ( Figure 2B). Moreover, the levels of phosphorylated histone H2AX protein (γH2AX), which is involved in cellular responses to DNA double-strand breaks and DNA replication stress [32], as well as levels of phosphorylated p53 (Ser15) and p21 proteins, were also elevated. Despite increased mRNA level of pro-apoptotic genes PUMA and NOXA, apoptosis was not massively induced by PM0.5 as confirmed by the absence of signal specific for cleaved PARP in western blots (Figure 3).

Expression Analysis of a New Set of Genes Involved in Various Signaling Pathways Including AhR and p53
Next, we examined the expression status of model genes associated with AhR (cytochrome P450 1A1; CYP1A1) and p53 (CDK1A, PUMA, NOXA) activation. Furthermore, gremlin 1 (GREM1) and SOX9 have been shown recently to have a similar 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like expression fingerprint during short-term adaptive response of A549 cells to both toxic exogenous and endogenous ligands, and may thus serve as markers of AhR-dependent transcriptional activity in lung epithelial cells [19]. Herein, we therefore included GREM1 and SOX9 expression analysis into testing of PM0.5-induced toxicity. PM0.5 exposure at non-cytotoxic concentration changed mRNA levels of AhR model target gene CYP1A1, as well as expression of proposed AhR lung-specific targets GREM1 (increased expression) and SOX9 (decreased expression, as shown in Figure 2A).
DNA damage responses were identified in A549 cells on both mRNA and protein levels as previously described [31]. Weak genotoxic effects of PM0.5 were suggested by slightly but significantly induced expression of p53 transcriptional target genes, cyclin dependent kinase inhibitor 1A (CDKN1A, the gene encoding p21 protein), PUMA and NOXA, as determined by real-time quantitative qRT-PCR ( Figure 2B). Moreover, the levels of phosphorylated histone H2AX protein (γH2AX), which is involved in cellular responses to DNA double-strand breaks and DNA replication stress [32], as well as levels of phosphorylated p53 (Ser15) and p21 proteins, were also elevated. Despite increased mRNA level of pro-apoptotic genes PUMA and NOXA, apoptosis was not massively induced by PM0.5 as confirmed by the absence of signal specific for cleaved PARP in western blots ( Figure 3). Relative mRNA levels of CYP1A1, GREM, SOX9, CDKN1A, NOXA, and PUMA were measured by qRT-PCR in A549 cells exposed to PM0.5 (in concentration 25 μg/cm 2 ) for 24 h. A total of 10 nM TCDD and 1 μM benzo[a]pyrene (BaP) were used as positive controls for activation of AhR signaling and DNA damage response, respectively. The results are expressed as means ± SD of three independent experiments. The means are significantly different from the negative control (ctrl) at * p < 0.05 and ** p < 0.01. Relative mRNA levels of CYP1A1, GREM, SOX9, CDKN1A, NOXA, and PUMA were measured by qRT-PCR in A549 cells exposed to PM0.5 (in concentration 25 µg/cm 2 ) for 24 h. A total of 10 nM TCDD and 1 µM benzo[a]pyrene (BaP) were used as positive controls for activation of AhR signaling and DNA damage response, respectively. The results are expressed as means ± SD of three independent experiments. The means are significantly different from the negative control (ctrl) at * p < 0.05 and ** p < 0.01. Figure 3. Western blot detection of proteins involved in DNA damage response and apoptosis. A549 cells were exposed to PM0.5 for 24 h and protein levels of phosphorylated H2AX (γH2AX), p53 phosphorylated on Ser15, p21, and cleaved PARP were detected. A total of 10 μM BaP and UV-C irradiation were used as positive controls. Ctrl, negative control. Detection of β-actin was used to confirm the equal loading.

Induction of Novel Toxicity Markers-Early Stress Response Genes and Unfolded Protein Response
Cellular response to various environmental stress conditions includes an activation of unspecific genes, mainly transcription factors that modulate expression of genes involved in maintenance of cellular homeostasis [22][23][24]. Our results show that 24h exposure of A549 cells to PM0.5 significantly induced the expression of early response genes EGR1 and GDF15 but not that of ATF3 ( Figure 4).
ER stress leads to activation of genes involved in compensatory response, the UPR. We measured changes in HSPA5 (also known as binding immunoglobulin protein, BiP), DDIT3 (CHOP) and XBP1s mRNA and protein levels, representing activation of all three UPR pathways [33]. We found that, in contrast to commonly used ER stress inducers thapsigargin and tunicamycin, PM0.5 did not change mRNA levels of tested UPR-associated genes ( Figure 5). A549 cells were exposed to PM0.5 for 24 h and protein levels of phosphorylated H2AX (γH2AX), p53 phosphorylated on Ser15, p21, and cleaved PARP were detected. A total of 10 µM BaP and UV-C irradiation were used as positive controls. Ctrl, negative control. Detection of β-actin was used to confirm the equal loading.

Induction of Novel Toxicity Markers-Early Stress Response Genes and Unfolded Protein Response
Cellular response to various environmental stress conditions includes an activation of unspecific genes, mainly transcription factors that modulate expression of genes involved in maintenance of cellular homeostasis [22][23][24]. Our results show that 24 h exposure of A549 cells to PM0.5 significantly induced the expression of early response genes EGR1 and GDF15 but not that of ATF3 ( Figure 4).  ER stress leads to activation of genes involved in compensatory response, the UPR. We measured changes in HSPA5 (also known as binding immunoglobulin protein, BiP), DDIT3 (CHOP) and XBP1s mRNA and protein levels, representing activation of all three UPR pathways [33]. We found that, in contrast to commonly used ER stress inducers thapsigargin and tunicamycin, PM0.5 did not change mRNA levels of tested UPR-associated genes ( Figure 5).  were determined by qRT-PCR and western blotting, respectively. A total of 1 µM BaP, 10 nM TCDD, 100 nM thapsigargin (Tha) and 4 µg/mL tunicamycin (Tun) were used as positive controls. The results are expressed as means ± SD of three independent experiments. The means are significantly different from the negative control (ctrl) at * p < 0.05 and ** p < 0.01. Detection of β-actin was used to confirm equal loading.

Autophagy
To determine whether autophagy is increased after the exposure to aerosol particles, flow cytometry using a CYTO-ID ® Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) and western blot detection of proteins LC3B (microtubule associated protein 1 light chain 3 beta) and sequestosome (SQSTM1/p62) were used. Starvation and exposure to chloroquine were used as positive controls. Flow cytometry did not reveal any increase in the percentage of autophagic cells after 12 or 24 h exposure as shown in Figure 6A. Similarly, levels of protein LC3B-II, a marker of autophagosomes, and SQSTM1/p62, which is a selective substrate for autophagy, were not elevated during 24 h exposure, as shown in Figure 6B. No autophagy was identified in A549 cells exposed to PM0.5. (A) Flow cytometric detection of autophagy using CYTO-ID ® staining in A549 cells. The percentage of autophagic cells was related to the negative control (100%). Starvation (Starv.) and 10 μM chloroquine (CQ) were used as positive controls. Neither 12 h nor 24 h exposure to PM0.5 increased autophagy in exposed cells. (B) Western blot detection of LC3B and SQSTM1/p62 protein levels in A549 cells exposed to PM0.5, CQ or starved for 24 h. Detection of β-actin was used to confirm equal loading. The means are significantly different from the negative control at * p <0.05.

Arachidonic and Linoleic Acid Metabolites
The concentrations of AA and linoleic acid metabolites were measured in cell culture medium harvested after 24 h exposure to PM0.5, namely prostaglandins (PGE2, PGA2, PGD2, PGF2α, PGF2β, 6keto-PGF1α, i.e., a non-enzymatic product of PGI2, 8-iso-PGF2α, 15-keto-PGE2, 13,14-dihydro-15-keto-PGE2), hydroxyeicosatetranoic acids (5-HETE, 8-HETE, 11-HETE, 12-HETE, 15-HETE), and hydroxyoctadecadienoic acids (9-HODE, 13-HODE), thromboxane TxB2, and leukotriene LxA4. No significant differences in concentrations of the measured metabolites were found in a medium of control and exposed cells. The only exception was the significant change in the levels of PGD2 and 13,14-dihydro-15-keto-PGE2, the final metabolite of PGE2. Concentrations of several metabolites were under the detection limit (PGJ2, 9-HETE, 20-HETE, and the epoxyeicosatrienoic acids-8,9-EET, 11,12-EET, 14,15-EET). Concentrations of selected eicosanoids are shown in Table 1, the other data are not shown. Table 1. Concentrations of selected eicosanoids. The cell growth medium was harvested after 24 h exposure of A549 cells to PM0.5. The concentrations of eicosanoids in the medium, measured by LC/MS-MS, are expressed as mean in pg/mL ± standard deviation. The experiment was done independently three times in technical duplicates. The data were analyzed by Student's t-test. The mean of PGD2 and 13,14-dihydro-15-keto-PGE2 were significantly different from the negative control (Ctrl) at * p < 0.05. Abbreviations: PG, prostaglandin; Tx, thromboxane. No autophagy was identified in A549 cells exposed to PM0.5. (A) Flow cytometric detection of autophagy using CYTO-ID ® staining in A549 cells. The percentage of autophagic cells was related to the negative control (100%). Starvation (Starv.) and 10 µM chloroquine (CQ) were used as positive controls. Neither 12 h nor 24 h exposure to PM0.5 increased autophagy in exposed cells. (B) Western blot detection of LC3B and SQSTM1/p62 protein levels in A549 cells exposed to PM0.5, CQ or starved for 24 h. Detection of β-actin was used to confirm equal loading. The means are significantly different from the negative control at * p <0.05.

Discussion
Cellular stress responses are adaptive mechanisms that are activated to maintain homeostasis and survive adverse conditions [16]. Cellular stress responses are not as specific as, e.g., AhR-dependent CYP1A1 expression and are known to be induced by various stimuli; they represent integral parameters affected by different pathways linked to toxicity [34]. Modulation of cellular stress markers stands somewhere between determination of cytotoxicity/cell death and induction of specific biomarkers, such as CYP1A1 expression or DNA adduct formation. Therefore, they constitute an ideal first-line set of parameters suitable for identification of in vitro toxicity. Determination of stress markers in the cells exposed to xenobiotics such as drugs, environmental contaminants, or their mixtures is a novel approach in in vitro toxicity profiling [34][35][36].
In this study, we compared the expression profiles of selected cellular stress response genes (namely early response genes and UPR genes) with expression profiles of genes involved in multiple cellular processes, including AhR or p53 signaling in A549 cells exposed to non-cytotoxic concentrations of PM0.5. We observed expression changes in CYP1A1, SOX9 and GREM1 that were recently shown to be associated with activation of AhR signaling in lung epithelial cells [19] together with induction of p53-dependent transcriptional responses (monitored here by induced levels of both P-p53 and p21 protein and p53 target genes CDKN1A, NOXA and PUMA) [31]. The level of γH2AX protein, the marker of double strand breaks and/or DNA repair, was not significantly elevated, which together with the absence of PARP cleavage and unchanged mitochondrial activity even after 72 h exposure to PM0.5 (WST-1 assay, data not shown) suggests that PM0.5 at a concentration of 25 µg/cm 2 does not induce apoptosis. Parts of genes, suggested here as positive markers of PM0.5 toxicity, includes early response genes, EGR1 and GDF15, that were previously shown to participate in adaptive response of cells to various stress stimuli including, e.g., ionizing radiation or viral infection [37,38]; both of them may be linked to AhR activation [39,40]. However, other transcription factor(s), for example p53, may be also involved in EGR1 and GDF15 induction after PM0.5 exposure [28]. EGR1 has been shown to be induced by cigarette smoke in lung cells, which again documents its association with AhR activation, and with chemokine production, MAPK activation, HSP70 signaling and with inflammatory responses [41][42][43][44]. GDF15 has been reported in the airway epithelium of smokers with chronic obstructive pulmonary disease and in human airway epithelial cells exposed to cigarette smoke [45]. A possible link between mixed AhR/p53 activation and induced levels of cellular stress markers EGR1 and GDF15 is also strongly supported by OTFBS (overrepresented transcription factor binding sites) analysis of human EGR1 and GDF15 genes ( Table 2) as the binding site matrices for both transcription factors are significantly predicted in regulatory regions of GDF15 gene. In concordance with previously published data, showing EGR1 in human lung epithelial cells being a direct AhR target [39], OTFBS analysis did indeed confirm the prediction of DRE in regulatory regions of EGR1. Furthermore, the existence of AhR and p53 functional crosstalk has been suggested earlier [46][47][48]. It supports the hypothesis that various stress agents may activate simultaneously both signaling pathways and thus contribute to mixed induction of cellular stress marker expression. Besides, EGR1 may directly affect its own expression levels as well as levels of GDF15 as indicated by OTFBS analysis (Table 2). Finally, since EGR1 has been documented to bind regulatory regions of p53 promoter and to induce p53-dependent apoptosis, it is tempting to speculate that this early response gene is the key member of stress response circuit composed by AhR and p53. Surprisingly, another early response gene, ATF3, was not deregulated by PM0.5. ATF3 may be linked to genetic integrity and promotion of malignance of lung cells [24,49] and was identified as an AhR target gene in human liver HepaRG cells [40]. This is not consistent with recent finding that ATF3 is involved in PM-induced airway inflammation [21]. However, it was shown that ATF3 induction after DNA damage stimuli is only transient [50] and, moreover, it is associated with induction of apoptosis or with suppression of cell growth; these parameters were not observed as significantly changed in our study.
ER stress and autophagy are other possible mechanisms of nanoparticle-induced toxicity [7,14,15]. In our study, we did not detect any changes in mRNA or protein levels of UPR markers HSPA5 (BiP), DDIT3 (CHOP) and XBP1s [33]. Although we found PM0.5 localized in structures similar to autophagosomes or late endosomal multivesicular bodies, autophagy was not induced in the PM0.5-exposed A549 cells (as determined by LC3B and p62 protein level and CYTO-ID ® marker).
PGs, HETEs and HODEs, the oxidative products of AA and its precursor linoleic acid, are lipid mediators playing an important role in inflammation, atherosclerosis and carcinogenesis [51][52][53][54]. We detected no differences in the levels of AA, PGs, HETEs and HODEs released into the cell growth medium after the exposure of A549 cells to PM0.5. The only exception was the increase in PGD 2 and the decrease in PGE 2 metabolites, which may suggest a slight induction of anti-inflammatory response [30] at low, non-cytotoxic concentration of PM0.5. Importantly, we found no significant modulation of levels of 8-iso-PGF 2alpha , 9-and 13-HODE, the markers of oxidative stress and lipid peroxidation [29,55], although oxidative stress is generally a well-accepted mechanism of nanoparticle-induced toxicity (e.g., [56]).
In this study, various types of cellular stress responses-early stress response genes, UPR, autophagy and inflammation-related eicosanoids-were studied after the exposure of lung epithelial cells to fraction of PM0.5 in order to suggest novel readouts for testing PM0.5-induced toxicity. Moreover, the stress markers' expression was compared with known AhR-and p53-dependent effects. We conclude that (1) the most sensitive events for assessment of exposure to low PM0.5 concentration are the activation of AhR-and p53-dependent pathways together with GREM1, EGR1 and GDF15 induction and that (2) chemical components adhered to the surface of nanoparticles (such as polycyclic aromatic hydrocarbons and other AhR agonists) are responsible for the observed adverse effects. This hypothesis has been already suggested previously [8,54] and is supported by studies on the effects of extractable organic matter from PM [57][58][59].

Aerosol Collection
Ambient air aerosol particles were collected in the Prague city center in March 2015 by a high-volume cascade impactor (BGI-900; BGI Incorporated, Waltham, MA, USA) as was described in detail previously [17]. A lower accumulation fraction (0.17 < d ae < 0.5 µm) of aerosol particles was collected on polyurethane foam impaction substrates. Besides the 49% of particles of relevant size, the fraction contained 37% of ultrafine particles (<0.17 µm) [17]. Therefore, lower accumulation fraction of aerosol particles was chosen as representative PM0.5 for in vitro experiments.

Particle Separation
Aerosol particles of fraction D were separated from PUF substrates using a slightly modified procedure according to Gualtieri et al. [3]. Briefly, PUF substrates were placed into glass beakers with deionized MilliQ water and sonicated in an ultrasonic bath (Teson 10, 650 W; Tesla, Czech Republic) three times for 20 min. Each time the particle suspension was collected and pure MilliQ water was added to the substrate. The mass of the extracted particles was determined gravimetrically; particle concentration in water was 980 µg/mL. Particle suspension was aliquoted and stored in glass vials at −20 • C until use. Prior to in vitro experiments, a frozen aliquot of airborne particle suspension was thawed, vortexed and sonicated for 20 min in an ultrasonic bath, and then into the cell culture medium in concentration 25 µg/cm 2 [17].

Cell Culture
Human lung carcinoma A549 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in Dulbecco's Modified Eagle's medium (D-MEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1.0 g/L glucose and pyruvate, 10% fetal bovine serum, 200 mM glutamine and 10 mg/mL gentamycin sulfate (Sigma-Aldrich, Prague, Czech Republic). The same medium supplemented with 10% fetal bovine serum was used also for the exposures. Cells were cultivated in plastic cell culture flasks (TPP, Trasadingen, Switzerland) in humidified atmosphere of 5% CO 2 at 37 • C.

Real-Time Quantitative RT-PCR
The confluent A549 cells were exposed to the PM0.5 suspension in concentrations 25 µg/cm 2 for 24 h. A total of 1 µM BaP (benzo[a]pyrene), 10 nM TCDD, 100 nM Tha or 400 µM H 2 O 2 were used as positive controls. The cells were washed twice with PBS and harvested into the cell lysis buffer provided with the NucleoSpin RNA II Purification Kit (Macherey Nagel, Düren, Germany). Total RNA was isolated according to the manufacturer's instructions. The levels of ATF3, CDKN1A, CYP1A1, DDIT3, EGR1, GDF15, GREM1, HSPA5, NOXA, PUMA, SOX9, and XBP1s mRNAs were determined by real-time RT-PCR using QuantiTect RT-PCR kit (Qiagen GmbH, Hilden, Germany). Sequences of primers and probes used for detection of GDF15, SOX9, GREM1, CYP1A1, and XBP1s were described previously [19,60,61]. The sequences of the other primers (all designed and synthesized by Generi Biotech, Czech Republic) and numbers of Universal Probe Library probes (UPL; Roche Life Sciences, Mannheim, Germany) are listed in Table 3. Hydroxymethylbilane synthase (HMBS; NM_000190) was used as the reference gene (predesigned qPCR assay, 3032-F, Generi-Biotech, Hradec Králové, Czech Republic). The RT-PCR reactions were carried out in 10 µL reaction mixture containing: 5 µl of QuantiTect Probe RT-PCR Master Mix, 0.1 µL of QuantiTect RT mix, 1.1 µL of solution of primers and probe, 1.8 µL of water and 2 µL of sample and were run on the LightCycler ® 480 System (version 1.5.0, Roche Diagnostics, Prague, Czech Republic) using the program published previously [19]. Changes in gene expression were calculated using the comparative threshold cycle method [62].

Flow Cytometry
The A549 cells were grown in 24-well cell culture plates. After reaching confluency, the cell culture medium was replaced by fresh one and the cells were exposed to the PM0.5 suspension in a concentration of 25 µg/cm 2 for 12 or 24 h. Exposure to 10 µM chloroquine and starvation (cells without changing fresh medium) were used as positive controls. The increase in the percentage of autophagic cells was measured using a CYTO-ID ® Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer's instructions on a flow cytometer FACSCalibur (BD, Franklin Lakes, NJ, USA). The cells exposed to PM0.5 had high autofluorescence in FL1, FL2 and FL3 (data not shown). In order to avoid misinterpretation of the data, isocontrol fluorescence was subtracted from the fluorescence of samples with the CYTO-ID ® probe.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
After exposure of A549 cells to PM0.5, the cell culture medium was harvested and solid-phase extraction was used for extraction and purification of arachidonic acid metabolites using SELECT HLB SPE cartridges (Supelco, Sigma-Aldrich, St. Luis, MO, USA). Subsequently, the analytes were detected by LC-MS/MS using the Agilent 1200 chromatographic system (610 Triple Quad LS/MS, Agilent Technologies, Santa Clara, CA, USA) as published earlier [64]. All eicosanoid standards were purchased from the Cayman Chemical Company (Ann Arbor, MI, USA).

Statistical Analysis
The data were analyzed by Student's t-test or Mann-Whitney tests. Differences were considered significant when p < 0.05.

Conclusions
Effects of airborne particles on the expression status of markers specific for cellular toxic stress and on release of eicosanoids related to inflammation and oxidative damage are not yet characterized. Therefore, in this study, we analyzed the expression profiles of various cellular stress response genes that have been previously shown to be involved in, e.g., early stress response, endoplasmic reticulum stress, unfolded protein response and autophagy, and compared them with the expression profiles of genes dependent on aryl hydrocarbon receptor (AhR) or p53 signaling. We used a well-characterized model of pulmonary cells (A549) exposed to non-cytotoxic concentration of ambient aerosol particle fraction PM0.5 for 24 h. Effects of PM0.5 were compared with those induced by prototypical toxic compounds, including, e.g., BaP and TCDD. Our results show that only early stress response genes EGR1 and GDF15 were upregulated together with AhR-and p53-dependent genes and thus suggest the major role of both signaling pathways in PM0.5-mediated toxicity. The accumulation of PM0.5 was found in A549 cells, however, markers of ER stress, UPR or autophagy were not induced. Finally, extracellular release of prostaglandin D 2 was also slightly increased. Based on our experimental data we can conclude that PM0.5 effects strongly resemble those elicited by BaP. Therefore, the induction of AhR-dependent and p53-dependent transcriptional activity (caused probably by polycyclic aromatic hydrocarbons and other AhR agonists bound to PM0.5) together with increased expression of cellular markers EGR1 and GDF15 are the most sensitive events, which may lead to adverse effects of PM0.5.

Acknowledgments:
The authors would like to thank Jan Hovorka (Institute for Environmental Studies, Faculty of Science, Charles University, Prague) for the PM sampling.

Conflicts of Interest:
The authors declare no conflict of interest.


extfieldsofstudy: Chemistry Medicine

text:
The effects of simulated +Gz and microgravity on intervertebral disc degeneration in rabbits

The overall objective of this study was to test the hypothesis that +Gz (hypergravity/positive acceleration) and microgravity can both aggravate intervertebral disc degeneration (IVDD). Due to +Gz and microgravity, many pilots develop IVDD. However, the lack of animal models of IVDD under conditions of simulated +Gz and microgravity has hampered research on the onset and prevention of IVDD. Rabbits were randomly allotted to a control group, microgravity group, +Gz group, or mixed (+Gz + microgravity) group. A tail-suspension model was utilized to simulate a microgravity environment and an animal centrifuge to mimic +Gz conditions. After exposure to the above conditions for 4, 8, and 24 weeks, the body weights (BW) of animals in the control group gradually increased over time, while those of animals in the microgravity and mixed groups both decreased (p < 0.001). As compared with the control group, the proteoglycan content of animals in the other three groups was significantly reduced (F = 192.83, p < 0.001). The imageological, histopathological, and immunohistochemical changes to the L6–S1 intervertebral disc samples suggests that the effects of +Gz and microgravity can aggravate IVDD over time. The mixed effects of +Gz and microgravity had the greatest effect on degeneration and +Gz had a particularly greater effect than microgravity.

the impact of microgravity on skeletal muscles; however, most have focused on bone loss rather than IVDD [13][14][15] . To the best of our knowledge, no study has focused on the cumulative and interaction effects of both +Gz (positive acceleration) and microgravity, which are thought to be closely related to the degree and speed of IVDD 16 . Therefore, the present experiment simulated conditions of +Gz and microgravity in order to test the hypothesis that +Gz and microgravity can both aggravate IVDD.
To examine the impact of microgravity or hypergravity on a living body, many animal experiments have been performed aboard the International Space Station 17 . However, due to high costs and limited resources, performing experiments in actual +Gz and microgravity environments is not feasible. An improved tail-suspension model was utilized to simulate a microgravity environment 18 and an animal centrifuge to mimic +Gz conditions 19 . Using up-to-date technologies, including recording of changes in body weight (BW), imageological, biochemical, histopathological, and immunohistochemical studies were conducted to systematically explore the effects of simulated +Gz and microgravity on IVDD (Fig. 1).

Results
General conditions of animals. Throughout the experiment, only a small number of the experimental animals exhibited signs of anorexia, diarrhea, listlessness, or infection. To maintain the overall health of the experimental animals, a series of measures were employed, which included actively improving the feeding environment and regular use of prophylactic antibiotics. There were significant differences in the BWs of the rabbits among the four groups at all time periods. The BWs of animals in the control group gradually increased over time (F = 484.76, p < 0.001), while those of animals in the microgravity group (F = 66.02, p < 0.001) and mixed (+Gz + microgravity) group (F = 212.09, p < 0.001) both decreased. Meanwhile, +Gz had no effect on the BWs of the rabbits in the +Gz group (F = 0.07, p = 0.98). As shown in Table 1, with an increase in exposure time (from week 4 to 24), the effect of microgravity significantly decreased the BWs of the rabbits and the mixed effects of +Gz and microgravity had the greater effect on BW loss. The tail-suspension model was used to simulate microgravity condition. (C) A high-speed animal centrifuge was used to simulate +Gz condition. The speed of the centrifuge was set to 79.1 rpm to obtain +7 Gz. www.nature.com/scientificreports www.nature.com/scientificreports/ Imageological changes of IVD. MRI showed different degrees of signal intensities depending on the different tissues embedded in the vertebral body marrow and spinal cord, while signals of the intervertebral disks on sagittal and axial T2WIs indicated the level of lumbar IVDD according to the grading measurements of the Pfirrmann category. At weeks 4, 8, and 24, there was no indication of narrowing of the disc space, modic changes (a common observation by MRI marked by signal intensity changes in vertebral body marrow adjacent to the endplates of degenerating discs), disc herniation, or spinal stenosis among the animals in the control and microgravity groups. MRI revealed narrowing of the intervertebral spaces in seven rabbits in the +Gz group and nine in the mixed group. At week 24, modic changes and water content decreases in the discs were observed in 40% and 80% of rabbits in the +Gz group, and 40% and 100% of rabbits in the mixed group, respectively (Fig. 2). The results showed that the effect of +Gz plays an important role in IVDD over time.

Changes in PG content in IVDs at L6-L7.
To evaluate the effects of +Gz and microgravity on PG content, changes in PG content in IVDs at L6-L7 were compared among all groups. At week 24, the PG content dramatically decreased, as compared with that in the control group. Results of phloroglucinol method 20 interactively indicated that the PG content of animals in other groups was significantly reduced (F = 192.83, p < 0.001) (Fig. 3).
Pathological changes to IVDs at L7-S1. To determine the effects of +Gz and microgravity, the pathological changes of IVDs at L7-S1 at weeks 4, 8, and 24 were analyzed by HE staining. At weeks 4 and 8, observation by light microscopy indicated that most NPs were complete and had a clear boundary with the AF, while NP cells were evenly distributed and loose reticular fibers formed throughout the extracellular matrix. At week 24, a few tissue sections in the control group showed partial absence of NPs and formation of cell clusters, while the inner layer of the AF was slightly distorted. Furthermore, internal fissures were observed with the occasional appearance of rounded chondrocytes in the NPs of animals in the microgravity group. There were fewer and scattered NP cells in sections obtained from animals in the +Gz and mixed groups, accompanied with metaplasia of chondrocytes. The number of NP cells was significantly reduced and fissures existed between the inner and outer layers ( Table 2). NP cells and PG from all the groups were stained with safranin O, which exhibited a red color. Safranin O staining was significantly stronger in the samples from the control group, as compared with the other three groups. There was no significant difference in safranin O staining between the microgravity and +Gz groups. In the mixed group, safranin O staining was the lightest (Fig. 4).
Changes in collagen-1, collagen-2, TGF-β and matrix metallopeptidase (MMP) concentrations in IVDs at L7-S1. Next, the effects of +Gz and microgravity on the expression levels of collagen-1, Figure 2. Imageological results of animals in each group. As shown by the sagittal T2WIs at 24 weeks, the IVD at L7-S1 of one animal in the control group was classified as Pfirrmann grade I (red arrow). The structure of the disc is homogeneous with a bright hyperintense white signal intensity and a normal disc height. Similarly, the IVD at L7-S1 of one animal in the microgravity group was classified as grade II (red arrow). The structure of the disc is inhomogeneous with a hyperintense white signal. The distinction between the NP and AF is clear, and the disc height is normal, with or without horizontal gray bands. Grade III (red arrow) in the +Gz group: The structure of the disc is inhomogeneous with an intermediate gray signal intensity. The distinction between the NP and AF is unclear, and the disc height is normal or slightly decreased. Grade IV (red arrow) in the mixed group: The structure of the disc is inhomogeneous, with a hypointense dark gray signal intensity. The distinction between the NP and AF is lost, and the disc height is normal or moderately decreased. As shown by the axial T2WIs, spinal stenosis developed in all of the experimental groups, but not the control group.
collagen-2, and TGF-β in IVDs at L7-S1 were examined. Positive granularity of collagen-1, collagen-2, and TGF-β were all clay brown or brownish yellow and located in the AF and NP. Immunohistochemical results at 4, 8, and 24 weeks showed significant upregulation of both collagen-1 and TGF-β in the microgravity group, +Gz group, and mixed group (p < 0.001), but not the control group (collagen-1: p = 0.991 and TGF-β: p = 0.996). As compared with the control group, collagen-1 and TGF-β levels were gradually upregulated in both AF and NP over time, with significantly increases at 8 and 24 weeks. On the contrary, collagen-2 was gradually downregulated (p < 0.001) over time in the three experimental groups, but not the control group (p = 0.991). There were few positive cells in the control group at all time periods and there was no significant difference between the time points (MMP-1: p = 0.126 and MMP-3: p = 0.134). Positive expression rates in the microgravity, +Gz, and mixed groups increased gradually with prolonged exposure time, with significant differences between time points in each group (p < 0.001). The strongest expression was in the mixed group at all time points, followed by the +Gz group, and the microgravity group (Fig. 5).

Discussion
Using a centrifuge and tail-suspension model designed for rabbit studies, the effects of +Gz and microgravity on IVDD were systematically explored. Given that acquiring and maintaining rats and rabbits is easier and more affordable than that of other animals, recent studies of IVDD have mostly used the rat 17,19 . However, the rabbit is much larger than the rat, with morphologic characteristics that are more similar to those of humans 21,22 .
Analysis of the changes in BW of the experimental animals found that weight loss was mainly caused by severe restriction of physical activities induced by tail-suspension, while the animals' resistance led to more physical compression, and the posture of the head downward and feet upward had harmful effects on digestion and absorption of food, as well as breathing and circulation 23 .
The normal NP and the medial side of the AF in the IVDs had high signal intensities on sagittal T2WIs, and with the development of IVDD, the water content of the NP was reduced, which resulted in weakening of the signal intensity 24,25 . In combination with the Pfirrmann's grading system showed no significant short-term degeneration in the lumbar IVDs. In the long-term (24 weeks), IVDD was occasionally observed. Similarly, several rabbits in the microgravity group were graded as level II at 24 weeks due to the effect of microgravity. Although +Gz alone and in combination with microgravity can accelerate degeneration in the early stage (4 weeks), with an increase in exposure time, disc degeneration becomes more obvious. It is worth mentioning that the assessment of disc degeneration in the early stage was not observed by MRI, but was only confirmed by histological examination 26 . As IVDD is initiated by histomorphological and molecular changes, only when the degeneration achieved  www.nature.com/scientificreports www.nature.com/scientificreports/ a certain degree, imageological changes can be depicted. Besides, pathological performance at the micro level is easier to detect early, while imageological examinations are macroscopic.
The healthy NP is rich in PG and collagen-2, whereas the AF is rich in collagen-1 9,26,27 . Collagen-1 and collagen-2 are the most abundant collagens in discs, accounting for about 70%-80% of all collagen. Previous studies suggest that members of the TGF-β family have pleiotropic effects on IVD cells 28 . Yang et al. 29 found that the TGF-β content was significantly increased in degenerative disc disease. Jin et al. 30 revealed that TGF-β was a protective factor of NP and AF, regulated collagen-1 and collagen-2, and inhibited or even reversed the process of IVDD 31,32 . The present experiment showed the contents of collagen-1, collagen-2, and TGF-β did not change significantly with no intervention, and there was no significant degeneration of the NP and AF in the control group, while under the conditions of +Gz and/or microgravity, the contents of collagen-1 and TGF-β gradually increased and that of collagen-2 decreased with an increase in exposure time. Meanwhile, the degeneration of NP and AF were positively correlated with exposure time.
Humans retained notochordal cells in ageing discs, the number is very low. In comparison, rabbit discs have a high number of notochordal cells 33 . It was shown that notochord cells can effectively maintain the size of the NP and prevent disc degeneration, which was often caused by the disappearance of notochord cells in animal models 34 . Our experiment showed that the cell phenotype had changed in the central region of NP, which probably resulted from the phenotype changes of notochordal cells, or caused by the growth of fibrocartilaginous tissue from cartilage endplate. These changes reduced the hydration ability of intervertebral disc and the resistance to degeneration, which can be considered as the initial changes of IVDD.
What determines the mechanisms by which +Gz and/or microgravity induces disc degeneration? The substance and energy metabolism of IVDs is maintained by osmosis because of the lack of a direct nutrient supply 5 . Many scholars have acknowledged that this nutrient delivery method is required by the vertebrae to bear loads and to maintain normal hydrostatic pressure between the vertebrae, thereby further ensuring the distribution www.nature.com/scientificreports www.nature.com/scientificreports/ of body fluids and blood, as well as normal metabolism among the discs and vertebrae [35][36][37][38] . Under conditions of +Gz or/and microgravity, IVDs and vertebrae had to bear loads that were much greater than physical hydrostatic pressure, resulting in weakened permeability of cartilage and endplates, and subsequent dramatic reductions in the nutrient supply and metabolism of NP and AF, especially oxygen and glucose supplies, thereby reducing the synthesis and concentration of PG in the NP, causing dehydration and atrophy of the IVD tissue, and hindering the repair and reconstruction of NP and AF, ultimately accelerating the progression of IVDD. Besides, it has been confirmed that with the changes of pressure and stress on the IVD, interleukin 1 (IL-1), tumor necrosis factor, MMPs, and other serum inflammatory factors can mediate and accelerate the process of IVDD, and their expression is regulated by a series of signal channels, such as the mitogen-activated protein kinase pathway, nuclear factor-κB pathway, and Wnt/β-catenin pathway 39 .
In conclusion, the conditions of simulated +Gz and microgravity can be widely applied to studies of the causes and prevention of IVDD. The results of this study suggest that the mixed effects of +Gz and microgravity were more obvious than the single impact of +Gz or microgravity alone, which could accelerate IVDD. Besides, the degree of degeneration was positively correlated with exposure time. Further neurologic function testing should include other spinal segments and consider secondary injuries.

Ethics statement. The study protocol was approved by the Ethics Committee of Peking Union Medical
College Hospital, Peking Union Medical College and Chinese Academy of Medical Science, and all animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Peking Union Medical College Hospital.

Rabbits.
A total of 120 healthy New Zealand male white rabbits, 7-8 months old (median: 7.6 months), weighing 3.0-3.5 kg (median: 3.1 kg), were purchased from the Experimental Animal Center of the First Affiliated Hospital of Chinese People's Liberation Army (Beijing, China). The rabbits were assigned to one of four groups of 30 rabbits each: a control group, microgravity group, +Gz group, or mixed group. According to the principle of statistical reliability, if an experimental animal was dead or missing, a new experimental rabbit was added.
Description of the devices for simulating microgravity and +Gz. An improved tail-suspension model, based on the mouse model proposed by Wronski and Morey-Holton 18 , was utilized to simulate microgravity, where the hind limbs were hung with no weight bearing on the fore legs to maintain the body in a horizontal Figure 5. Immunohistochemical results observed by optical microscopy (×400) of the IVDs at L7-S1 of different groups at 24 weeks. Positive granularity of collagen-1, collagen-2, and TGF-β were all clay brown or brownish yellow (red arrow). Gray values (%) of collagen-1, collagen-2, and TGF-β in the IVDs at L7-S1 of different groups. Positive cells are indicated by brown-yellow or brown particles in the cytoplasm and bluestained nuclei (black arrow). Data are presented as the mean ± SD.

Procedures.
To maintain the overall health of the experimental animals, a series of measures were employed, which included actively improving the feeding environment and regular use of prophylactic antibiotics. All rabbits were housed in a light-and temperature-controlled environment with free access to food and water, and were weighed weekly. The control group received no intervention. Rabbits in the microgravity group were suspended for 5 days per week, while the others for two days. Those in the +Gz group were exposed to +7 Gz three times for 60 s each at 10-min intervals and rest at other times, which was repeated every other day. The rabbits in the mixed group were also suspended for 5 days as with the microgravity group and exposed to +Gz following in the same manner as in the +Gz group. Ten rabbits were randomly chosen and assigned to subgroups at 4, 8, and 24 weeks after weighing. Simulation of +Gz and microgravity were performed at the Air Force Institute of Aviation Medicine (Beijing, China). Imageological, biochemical, histopathological, and immunohistochemical studies were conducted at the Key Laboratory of Peking Union Medical College Hospital.
Imageological study. At 4,8,and 24 weeks, all animals were anesthetized with ketamine at a concentration of 10 mg/kg BW and underwent magnetic resonance imaging (MRI) examinations. MRI was performed using a 3.0 T clinical magnet (Siemens AG, Munich, Germany) to observe structural changes on sagittal T2-weighted images (T2WIs) (repetition time = 3200.0 ms; echo time = 112.0 ms). The T2WIs were evaluated according to the classification of IVDD as reported by Pfirrmann et al. 41 . All images were evaluated by another orthopedist blinded to the study conditions. Selection of IVD samples for biochemical, histopathological, and immunohistochemical studies. The rabbits were immediately sacrificed upon completion of the imageological examination. IVD samples of L6-L7 and L7-S1 were obtained with a sharp scalpel, then fixed in 10% paraformaldehyde and stored at room temperature. The reason for collecting IVD samples from L6-L7 and L7-S1 was to obtain samples subjected to the largest load, as changes at these locations were the most obvious. According to a report by Phillips et al. 42 , the caudal vertebrae (L7-S1) usually sustained a greater load than the proximal vertebral segments (L7-S1 > L6-7 > L5-6 > L4-5). Therefore, IVD samples from L6-L7 and L7-S1 were selected for key observations and evaluations of the range of interest.
Biochemical study. At 24 weeks, IVD samples from L6-L7, together with the adjacent AF and surrounding soft tissue, were resected and fixed in 20% formalin for the determination of PG content. In a preliminary experiment, PG content was slightly reduced with no statistical significance at 4 and 8 weeks; however, levels were significantly attenuated at 12 weeks 22 . After degreasing, 10 mg of NP were added to 0.05 mL of 3% NaOH in a 4-mL centrifuge tube, which was incubated at 40 °C in a water bath using a constant temperature vibrator for 3 h. Then, 2 mL of glacial acetic acid and 50 μL of 0.25% trypsin was added to the tube, which was further incubated at 50 °C in a water bath for 2 h. The enzyme mixture was transferred to a flask and divided into 10-mL aliquots. One milliliter of this solution was used for PG analysis according to the phloroglucinol method 20 . Absorbance of the reaction mixture was detected using a spectrophotometer (UV-2201; Shimadzu Corporation, Tokyo, Japan) at a wavelength of 558 nm, then transformed into PG content.
Histopathological study. The IVD samples of L7-S1 collected from each subgroup were subjected to standard dehydration in baths of increasing percentages of alcohol, cleared in dimethylbenzene, embedded in paraffin, frozen for 20 min, and then serially sectioned into 5 µm-thick slices. The samples were stained with hematoxylin and eosin (HE), as well as safranin O by routine methods, and observed under a light microscope (Olympus Corporation, Tokyo, Japan). The cellularity and morphology of NP and AF were evaluated according to the Nishimura classification 43 and all images were examined by another experienced histologist in a blinded manner. Safranin O binds to PG, so a stronger red staining with indicates a higher amount of PG, from which the concentration of PG can be determined to study the degeneration of NP cells.