Toxicology Letters 284 (2018) 205–212

Contents lists available at ScienceDirect

Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

Less-toxic rearrangement products of NX-toxins are formed during storage T

and food processing
Elisabeth Vargaa,1, Gerlinde Wiesenbergerb,1, Lydia Woelflingsederb,c, Krisztian Twaruschekb,
Christian Hametnerd, Marta Vaclavikováa, Alexandra Malachováa, Doris Markoc,
⁎
Franz Berthillera, , Gerhard Adamb
a
Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural
Resources and Life Sciences, Vienna (BOKU), Tulln, Austria
b
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Tulln, Austria
c
Department of Food Chemistry and Toxicology, University of Vienna, Vienna, Austria
d
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria

G RA P H I C A L AB S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: A new type A trichothecene mycotoxin, NX-2, was previously reported to be produced by North American
Trichothecene isolates of the cereal pathogen Fusarium graminearum. Here we describe the isolation and structural character-
Detoxification ization of a rearrangement product, called NX2-M1, and related compounds with different acetylation patterns
Fusarium graminearum (NX3-M1 and NX4-M1). In the NX-M1 derivatives, the epoxide ring is opened, and a covalent bridge between C-
Alamar Blue assay
10 and C-12 of the trichothecene backbone is formed. In vitro translation assays showed that NX3-M1 is less toxic
Sulforhodamine B assay
for eukaryotic ribosomes than NX-3. NX3-M1 also has a greatly reduced cytotoxic potential on two tested human
colon cell lines. Formation of NX3-M1 can therefore be regarded as a detoxification reaction. The related F.

Abbreviations: DAS, diacetoxyscirpenol; DMEM, Dulbecco’s Modified Eagle’s Medium; DON, deoxynivalenol; ELSD, evaporative light scattering detector; HRMS, high resolution mass
spectrometry; MeOH, methanol; MS/MS, tandem mass spectrometry; NOEL, no observed effect level; (U)HPLC, (ultra) high performance liquid chromatography; PBS, phosphate buffered
saline; PCR-RFLP, polymerase chain reaction restriction fragment length polymorphism; QTOF, quadrupole time-of-flight mass spectrometer; SEM, standard error of the mean; SRB,
sulforhodamine B; TCA, trichloroacetic acid
⁎
Corresponding author at: Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of
Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 20, 3430 Tulln, Austria.
E-mail addresses: elisabeth.varga@boku.ac.at (E. Varga), gerlinde.wiesenberger@boku.ac.at (G. Wiesenberger), lydia.woelflingseder@univie.ac.at (L. Woelflingseder),
krisztian.twaruschek@boku.ac.at (K. Twaruschek), christian.hametner@tuwien.ac.at (C. Hametner), marta.vaclavikova@boku.ac.at (M. Vaclaviková),
alexandra.malachova@boku.ac.at (A. Malachová), doris.marko@univie.ac.at (D. Marko), franz.berthiller@boku.ac.at (F. Berthiller), gerhard.adam@boku.ac.at (G. Adam).
1
Both authors contributed equally to this article.

https://doi.org/10.1016/j.toxlet.2017.12.016
Received 8 November 2017; Received in revised form 15 December 2017; Accepted 20 December 2017
Available online 23 December 2017
0378-4274/ © 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
E. Varga et al.
graminearum mycotoxin deoxynivalenol (DON), which is frequently occurring worldwide, is very stable during
food processing. Testing NX-3 at different pH-values and temperature conditions, as well as under conditions
that simulate the storage of infected grains and bread-making process, revealed a strongly reduced stability of
NX-3 and concurrent formation of NX3-M1. Although the NX-3 formed in planta is as toxic as DON, the extensive
formation of the non-toxic rearrangement product should be taken into account for risk assessment of this
emerging food contaminant.
1. Introduction
Mycotoxins are secondary metabolites produced by filamentous
fungi that can cause acute toxicity or chronic health effects in humans
and animals (Bennett and Klich, 2003). Fusarium graminearum is the
causative agent of important plant diseases, such as head blight of small
grain cereals and ear rot in maize, and contaminates food and feed with
trichothecene mycotoxins (Logrieco et al., 2003; Goswami and Kistler,
2004). More than 200 of these polycyclic sesquiterpenoids have been
identified from natural sources (Grove, 2007) and they are grouped into
four classes: type A, B, C and D trichothecenes (McCormick et al.,
2011). In most wheat-producing areas, deoxynivalenol (DON) produ-
cing F. graminearum strains are dominant. They are subdivided into 3-
acetyl-DON and 15-acetyl-DON chemotype strains based on the loca-
tion-specific deacetylation of the biosynthetic precursor 3,15-diacetyl-
DON (Alexander et al., 2011).
Gale et al. (2010) discovered a new population of F. graminearum
(sensu stricto) isolated from wheat in Minnesota, which was genotyped
as a 3-acetyl-DON producer, but produced none of the known
trichothecenes. In Varga et al. (2015), we described the isolation,
characterization, and structure elucidation of novel type A trichothecenes,
named NX-toxins, which are formed by these strains. The initially isolated
NX-2 is similar in structure to 3-acetyl-DON, but lacks the keto group at C-
8, defining type B trichothecenes (see Fig. 1). In infected wheat, NX-2 is
deacetylated to NX-3 in the same manner as the acetylated precursors of
DON. NX-3 shows a similar inhibitory effect on protein synthesis as DON,
as has been verified by in vitro translation assays. By introducing the TRI1
allele of the NX-2 producing strain into a tri1Δ mutant of a 15-acetyl-DON
producing strain, a strain producing NX-4 was obtained. NX-4 differs from
NX-2 by acetylation at C-15 instead of C-3. This experiment proved that the
TRI1 allele is responsible for the different hydroxylation at C-8 between
DON- and NX-chemotypes (Varga et al., 2015).
Liang et al. (2014) performed a study involving 463 F. graminearum
strains from Minnesota, North Dakota and South Dakota in three sampling
periods (1999–2000, 2006–2007 and 2011–2013) to determine the spatial
and temporal dynamics of the NX-strain. A polymerase chain reaction
restriction fragment length polymorphism (PCR-RFLP) assay targeting
specifically the TRI1 gene for the identification of NX-2 alleles was used.
The overall frequency within the F. graminearum population was low
(2.8%), but NX-strains were detected in all sampling periods. In a follow
up study, Kelly et al. (2016) investigated 2515 F. graminearum strains from
all over the world for the presence of the NX-2 TRI1 allele. NX-2 isolates
were only identified in southern Canada and northern United States at a
low frequency (1.8%). They concluded that the NX-2 chemotype might be
indigenous and endemic in this area. Furthermore, there are indications
that they all have a single evolutionary origin and evolved recently from a
type B trichothecene producing ancestor (Kelly et al., 2016). An important
remaining question is, whether NX producers may have an ecological
advantage in certain environments and may consequently be increasing to
levels relevant for food safety. It has been argued (Varga et al., 2015) that
lack of the conjugated C-8 keto group prevents formation of a Michael
adduct with glutathione to the double bond of DON, which still remains a
barely understood detoxification reaction in plants (Gardiner et al., 2010;
Stanic et al., 2016). Very recently, Lofgren et al. (in press) reported that
the NX-genotype has a much higher frequency (up to 20% of the total F.
graminearum population) in an area of upstate New York (Willsboro, Lake
Champlain). Furthermore, for the first time, NX-2 genotypes were also
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Toxicology Letters 284 (2018) 205–212
reported in maize ears, stubbles and air samples (Lofgren et al., in press).
During the initial screening for novel metabolites in the NX-isolates of
F. graminearum, we found two compounds, NX-2 and NX-1, the latter one
turned out to be a rearrangement product of NX-2 and was therefore re-
named to NX2-M1. This led us to investigate the stability of NX-toxins, and
to determine the toxicological properties of the formed rearrangement
products, which are presented here. We furthermore examined the stabi-
lity of NX-3, that major NX-metabolite in planta during storage and in a
bread baking simulation experiment. If NX-strains become more abundant,
NX-3 might also become a contaminant in cereals and should no longer be
regarded as negligible. The formation of derivatives during food proces-
sing, as well as the elucidation of their toxicity, is crucial for proper risk
assessment, and therefore, for food safety.
2. Materials and methods
2.1. Chemicals and reagents
Methanol (MeOH, LC-grade) and glacial acetic acid were obtained
from VWR International GmbH (Vienna, Austria). Technical grade ethyl
acetate was purchased at Wagner&Munz GmbH (München, Germany)
and petroleum ether (40–60 °C) was obtained from Lactan GmbH & Co
KG (Graz, Austria). Water was purified using a Purelab Ultra system
(ELGA LabWater, Celle, Germany). Ammonium acetate and formic acid
(both LC–MS grade) were purchased from Sigma Aldrich (Vienna,
Austria) and ammonium formate was obtained as 5 mM aqueous solu-
tion from Agilent Technologies (Waldbronn, Germany). Deuterated
solvents were purchased from Eurisotop (Gif sur Yvette Cedex, Paris,
France) and standards of NX-2, NX-3 and NX-4 were previously purified
as described in Varga et al. (2015).
Cell culture media and supplements were obtained from Fisher
Scientific (Austria) GmbH (Vienna, Austria), Invitrogen™ Life
Technologies (Karlsruhe, Germany), Lonza Group (Basel, Switzerland),
Sarstedt AG & Co (Nümbrecht, Germany), Sigma-Aldrich (Darmstadt,
Germany), Szabo-Scandic HandelsgmbH & Co KG (Vienna, Austria) and
VWR International (Vienna, Austria).
2.2. Purification of NX2-M1, NX3-M1 and NX4-M1
Cultivation of the rice cultures and the first steps of the purification of
NX2-M1 were the same as previously described for NX-2 (Varga et al.,
2015). Briefly, the NX-isolate 06-156 (NRRL 66038) of F. graminearum was
routinely grown on Fusarium minimal medium (Leslie and Summerell,
2006). Sporulation of the strain was performed on mung bean broth. In
total, 105 spores were used to inoculate 10 g of autoclaved rice. Incubation
Fig. 1. Structures of the three previously reported NX-metabolites (A: NX-2, B: NX-3 and
C: NX-4) and the corresponding newly described NX-M1 derivatives (D: NX2-M1, E: NX3-
M1 and F: NX4-M1).
E. Varga et al.
was performed at 20 °C, 55% humidity and constant light. The 24-day old
rice cultures were homogenized with 80 mL ethyl acetate and an Ultra-
Turrax T25 (IKA-Werke, Staufen, Germany), and extracted for 90 min at
room temperature on a GFL rotary shaker (Burgwedel, Germany). The
extracts were evaporated to dryness and normal phase chromatography
was carried out on a 40 × 800 mm self-packed silica gel (63–200 μm,
Sigma Aldrich) column. Packing and washing of the column was per-
formed with petroleum ether:ethyl acetate (3:1, v/v). In contrast to NX-2,
the more polar NX2-M1 eluted with ethyl acetate:MeOH (3:1, v/v). The
NX2-M1 fraction was dried down, reconstituted in MeOH:H2O (20:80, v/
v) and further purified with an 1100 series preparative high performance
liquid chromatography (HPLC) system (Agilent Technologies, Waldbronn,
Germany) equipped with an evaporative light scattering detector (ELSD;
Sedex 85, Sedere LT-ELSD, Altfortville Cedex, France). A reversed phase
Gemini NX-C18 column (150 × 21.2 mm, 5 μm, Phenomenex, Aschaffen-
burg, Germany) equipped with a guard column of the same material was
used and the following gradient at a flow rate of 16 mL/min was applied:
an initial 1 min holding time of 20% MeOH was followed by a linear in-
crease to 60% MeOH within the next 7 min. Thereafter, a fast switch to
100% MeOH for a 2 min column flushing period was performed before the
column was equilibrated at the starting conditions. NX2-M1 was collected
by peak-based fractionation between 4.5 and 6.5 min. Approximately
20 mg of a white powder was obtained from 42 rice cultures after eva-
poration of MeOH and freeze-drying.
NX3-M1 was obtained by incubating previously purified NX-3 at
37 °C in a 50 mM sodium tetraborate buffer (pH = 10.3) for 90 min.
This solution was purified using the preparative HPLC system, a SunFire
C18 OBD (19 × 100 mm, 5 μm, Waters, Eschborn, Germany) column
and the following gradient: after an initial 1 min holding period at 10%
MeOH, a linear gradient to 40% MeOH within the next 5 min was ap-
plied. The column was flushed with 100% MeOH for 2 min before
equilibration at the starting conditions started. Again the MeOH from
the combined fraction was evaporated and the aqueous solution was
lyophilized, yielding approximately 4.5 mg NX3-M1 from 5.3 mg NX-3.
NX4-M1 was isolated from 14 day old rice cultures of strain IAWP48
as a by-product of NX-4 purification. IAWP48 is a transformant of a
tri1Δ F. graminearum PH-1 (NRRL 31084, FGSC 9075) containing the
NX-version of the TRI1-allele (Varga et al., 2015). Inoculation of rice
cultures and growth conditions were the same as reported above for
NX-isolate 06-156. Following the above extraction procedure, the
pooled extract was evaporated to dryness. Thereafter, the solution was
re-dissolved in acetonitrile, defatted with 2.5 vol of hexane and purified
via preparative HPLC using a SunFire C18 OBD (19 × 100 mm, 5 μm)
column. In the first step, NX-4 was separated from NX-3, NX3-M1, and
NX4-M1 using the following gradient: 0–1 min: 20% MeOH, 2–6 min:
linear gradient from 20 to 50% MeOH, 6.1–10 min 100% MeOH,
10.1 min 20% MeOH. In the second step, NX4-M1 was separated from
the other compounds applying the same gradient as described above for
the purification of NX3-M1. Approximately 3 mg NX4-M1 could be
obtained from 21 glasses of 14 day old IAWP48 cultures on rice.
2.3. Structural elucidation of the NX-M1 derivatives
Tandem mass spectrometric measurements using high resolution
mass spectrometry (MS/HRMS) were performed on a 1290 ultra high
performance liquid chromatography (UHPLC) system equipped with a
Zorbax SB C18 column (150 × 2.1 mm, 1.8 μm) and a 6550 iFunnel
quadrupole time-of-flight (QTOF) LC–MS instrument (all Agilent
Technologies, Waldbronn, Germany). The method parameters were the
same as described in Varga et al. (2015), with the exception that in MS/
MS-mode, the lower mass range was decreased to m/z 25. Data were
acquired with MassHunter Workstation Software B.06.01 and were
evaluated with MassHunter Qualitative Analysis Version B.07.00.
NMR spectra were obtained on a Bruker Avance DRX–400 MHz
spectrometer (NX2-M1; operating at 400 MHz for 1H and 100 MHz for
13
C) or a Bruker Avance III HD 600 FT-NMR spectrometer (NX3-M1 and
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Toxicology Letters 284 (2018) 205–212
NX4-M1; operating at 600 MHz for 1H and 150 MHz for 13C). The
samples were dissolved in methanol-d4 and measured at ambient tem-
perature. All chemical shifts are provided in ppm relative to tetra-
methylsilane, and calibration was done on the basis of residual solvent
resonances (3.31 ppm for 1H, 49.15 ppm for 13C). All pulse programs
were obtained from the Bruker software library. The NMR data were
evaluated using TOPSPIN 3.5 (Bruker BioSpin GmbH).
2.4. LC–MS/MS method for the quantitation of NX-2, NX-3, NX2-M1 and
NX3-M1
Chromatographic separation was achieved on a Gemini C18 column
(150 × 4.6 mm, 5 μm; Phenomenex, Aschaffenburg, Germany) at 25 °C
using a 1290 UHPLC system from Agilent Technologies (Waldbronn,
Germany). The eluents were composed of H2O and MeOH (A: 80:20, v/
v; B: 3:97, v/v) and contained both 5 mM ammonium acetate. The
applied gradient was as follows: 0–2 min: 0% B; 2–9 min: linear gra-
dient from 0% B to 70% B; 9–11 min: flushing of the column with 100%
B; 11–13 min column equilibration at 0% B. Between 3.5 and 10 min
the LC flow of 800 μL/min was directed to the mass spectrometer,
otherwise to the waste. Target routine analysis was performed on a low
resolution mass spectrometer, namley a QTrap 4000 (Sciex, Foster City,
CA, USA), equipped with a TurboV electrospray ionization source. The
following source parameters were applied: curtain gas 35 psi (240 kPa),
ion spray voltage (4 kV), temperature 550 °C, ion source gas 1 and 2
both 50 psi (344 kPa) and the interface heater on. The following se-
lected reaction monitoring (SRM) transitions with a dwell time of 25 ms
were used: NX3-M1 (retention time 3.8 min) m/z 318.2 (declustering
potential, DP, 46 V), product ions m/z 301.1 (collision energy, CE,
15 V) and m/z 283.1 (CE, 23 V); NX2-M1 (6.1 min) m/z 360.1 (DP,
41 V), product ions m/z 229.0 (CE, 31 V) and m/z 307.0 (CE, 25 V); NX-
3 (6.5 min) m/z 300.2 (DP, 36 V), product ions m/z 217.0 (CE, 13 V)
and m/z 247.0 (CE, 9 V) and NX-2 (9.1 min) m/z 342.1 (DP, 41 V),
product ions m/z 171.1 (CE, 27 V) and m/z 307.1 (CE, 9 V). Data were
acquired and analyzed using Analyst 1.6.3 software from Sciex and
further data processing was performed using Microsoft Excel 2010. The
same method was applied on an 1100 HPLC system (Agilent
Technologies) coupled to a 3500 triple quadrupole MS/MS system
(Sciex). On that system the retention times were as follows: NX3-M1
(4.8 min), NX2-M1 (8.0 min), NX-3 (8.2 min) and NX-2 (10.7 min).
2.5. In vitro translation inhibition and human cell culture assay
In vitro translation experiments were performed as described in
Varga et al. (2015). Three experiments were performed with three in-
dependent dilutions of NX3-M1. NX-3 curves were taken from Varga
et al. (2015), while DON controls are shown as mean of four in-
dependent experiments.
HT-29 human colorectal adenocarcinoma cells were purchased from
the German Collection of Microorganisms and Cell Cultures (DSMZ,
Braunschweig, Germany). Cells were cultured with Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat in-
activated fetal calf serum and 50 U/mL penicillin and 50 μg/mL strep-
tomycin. Non-tumorigenic human colon epithelial cells HCEC-1CT
(Roig and Shay, 2010) were kindly provided by Prof. Jerry W. Shay (UT
Southwestern Medical Center, Dallas, TX, USA) and cultivated with
DMEM high glucose supplemented with 2% (v/v) Medium 199 (10X),
2% (v/v) cosmic calf serum, 20 mM 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid (HEPES), 50 μg/mL gentamicin, insulin-
transferrin-selenium-G (10 μg/mL; 5.5 μg/mL; 6.7 ng/mL), recombinant
human epidermal growth factor (18.66 ng/mL) and hydrocortisone
(1 μg/mL). Both cell lines were cultivated in a humidified incubator at
37 °C and 5% CO2 and routinely tested for the absence of mycoplasm
contamination.
E. Varga et al.
2.5.1. Coupled cytotoxicity assays (alamarBlue® and sulforhodamine B
assay)
Per well, 7500 or 8500 HT-29 and HCEC-1CT cells were seeded in
200 μL culture medium in black 96-well plates with clear bottom and
allowed to grow for 72 or 48 h. Then culture medium was withdrawn
and cells were treated for 24 h with different concentrations of DON,
NX-3 and NX3-M1 in the respective cell culture medium. As a solvent
control 1% water (LC–MS grade) was incubated and 1% (v/v) Triton X-
100 served as a positive control. After the treatment period the medium
was withdrawn, cells were rinsed once with phosphate buffered saline
(PBS; 100 μL/well) and incubated with DMEM containing 10% (v/v)
alamarBlue® reagent (Invitrogen™ Life Technologies, Karlsruhe,
Germany) for 75 min. Subsequently, fluorescence intensity was mea-
sured (excitation: 530 nm; emission: 600 nm) on a Cytation 3 Imaging
Multi Mode Reader (BioTek, Bad Friedrichshall, Germany). For quan-
tification, blank-values were subtracted and measured data were re-
ferred to solvent control.
Cells were rinsed twice with PBS (100 μL/well) and fixed with 20 μL
of 50% (w/v) trichloroacetic acid (TCA) per well for 30 min at 4 °C. To
remove TCA, cells were washed three times with water. The plate was
allowed to dry overnight at room temperature, before 50 μL of a sul-
forhodamine B (SRB; 0.4% (w/v) in 1% (v/v) acetic acid) solution were
added to the cells and incubated for 1 h at room temperature protected
from light. After rinsing the stained cells twice with water and another
two times with 1% (v/v) acetic acid, the plate was dried at room
temperature in the dark. The staining was eluted with 100 μL 10 mM
Tris buffer (pH 10) per well and single wavelength absorbance at
570 nm was read on a Cytation 3 Imaging Multi Mode Reader (BioTek,
Bad Friedrichshall, Germany). Blank values were subtracted and final
results were referred to solvent control. Data are presented as a mean of
at least five independent experiments each performed in technical
quadruplicate ± standard error of mean (SEM) and analyzed with
OriginPro 9.1 v G (Origin Lab, Massachusetts). All data were tested for
normality by Shapiro-Wilk test. Significant differences of all measure-
ments, compared to the no observed effect level (NOEL), were eval-
uated via one-way analysis of variance (ANOVA) and Bonferroni post
hoc test. For statistical comparison of all measurements to the re-
spective DON concentration and between the different cell lines two-
tailed Student’s t tests were applied. Significance levels were set to 5%
(“1” = p < .05; “2” = p < .01; “3” = p < .001).
2.6. Stability of NX-3 in solution and ground wheat
The stability of NX-3 was tested in different buffer solutions: pH 3
(sodium-acetate buffer), pH 7 and pH 8 (sodium/potassium dihydrogen
phosphate buffer), pH 9 and pH 10 (sodium tetraborate buffer), pH 11
(sodium-bicarbonate buffer). Buffers were prepared according to the
directions at De Lloyd (2000) and checked afterwards, the measured pH
values were: pH 3.0, pH 7.0, pH 8.2, pH 9.0, pH 10.3 and pH 11.5.
The reactions were prepared in 2 mL screw-cap vials (Sarstedt,
Nümbrecht, Germany) containing 60 μL of NX-3 (1000 mg/L in water),
300 μL buffer (100 mM) and 240 μL water. The first sample was taken
immediately after mixing of the samples (t = 0, several minutes) and
was frozen in dry ice/ethanol. The reactions were incubated side-by-
side at 37 °C and 65 °C, and aliquots of each sample were removed after
6, 24, 48 h and 15 days. The aliquots were transferred to room tem-
perature and after 10 min centrifuged for 1 min at 10,000 rpm (ca.
9,390 g). One hundred μL of the centrifuged supernatants were trans-
ferred to Eppendorf tubes, frozen immediately and kept at −80 °C until
analysis. Since the reactions were not chemically stopped, the time
between thawing and measurement was kept as short as feasible. For
measurement, a subsample was diluted 1:10 with water after thawing
and the remaining samples were immediately frozen again.
Measurements were performed with a UHPLC–HRMS method using the
same settings as in the HRMS-characterization described above. The
only exception was that data acquisition was performed solely in HRMS
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Toxicology Letters 284 (2018) 205–212
mode (m/z 50–1,000, 3 scans per second).
To investigate storage stability, 30 wheat heads of the cultivar
“Apogee” were spray inoculated during flowering with a spore sus-
pension of the NX-2 producing F. graminearum strain 02-264 (WG-9,
NRRL 66037) and grown at 20 °C, 18 h light and 6 h dark at 60% hu-
midity. After ripening, the infected ears were harvested and im-
mediately frozen at −80 °C. Grinding of the frozen wheat heads (ap-
prox. 4–5 ears/beaker) was performed with a ball mill (MM400, Retsch,
Haan, Germany) at maximum speed for 30 s. Afterwards they were
pooled and stored again at −80 °C until further processing. The pooled
fractions were thawed in a desiccator and 100-mg samples were either
transferred into 15-mL Greiner tubes (for storage) or into glass tubes
(for determination of dry weight). Four samples were immediately
transferred to −80 °C and served as control. All other samples were
stored at 20 or 37 °C and were transferred to −80 °C after 1, 2, 4 or 8
weeks (six replicates per condition and time point). For determination
of the dry weight, the glass tubes were incubated at 105 °C for several
days until the weight was constant (about 105 h).
Samples were thawed at room temperature for ca. 10 min, before
adding 2 mL extraction buffer (MeOH:H2O 50:50, v/v) to each sample.
They were incubated for 5 min at room temperature, properly re-sus-
pended using a vortex and/or pipetted up and down and extracted for
1 h at 20 °C with vigorous shaking (200 rpm). After extraction, the tubes
were centrifuged for 5 min at 20 °C (2,934g) and 1.4 mL of the super-
natants were transferred to Eppendorf tubes. The samples were cen-
trifuged again at a maximum speed (20,238g) for 10 min. Aliquots of
the supernatants were transferred to HPLC vials and stored at −20 °C
until HPLC–MS/MS analysis, which was performed using the 3500
LC–MS/MS system. When necessary, samples were further diluted with
MeOH:H2O (50:50, v/v).
2.7. Stability of NX-3 during simulated bread baking
The dough was prepared by incubating 500 mg wheat flour and 400 μL
water for 15–30 min at room temperature and then “kneading” it with a
spatula until the dough did not stick to the tube and spatula any more. For
the pilot experiment, each dough was spiked with either NX-3 or NX3-M1
at one concentration in triplicate and rested for 20 h at room temperature
to allow complete diffusion of the analytes. Thereafter, the samples were
frozen and stored at −20 °C for at least 1 h. The samples were thawed and
the three-fold volume of extraction solvent (2.7 mL; MeOH:H2O 50:50, v/
v) was added. The dough was then cut into small pieces with a spatula and
homogenized with an Ultra-Turrax. After 1 h extraction at 20 °C, the
samples were transferred to Eppendorf tubes and centrifuged at 20,000g
for 10 min. The supernatants were then transferred to HPLC vials and
measured with the QTrap 4000 system. Furthermore, blank doughs were
prepared following the same procedure and the final extract was spiked
with either NX-3 or NX3-M1. This procedure allowed the assessment of
matrix effects and extraction recovery of the analytes.
In the main experiment, the dough was prepared as described
above, but after the “kneading” process, the dough was rested at 20 °C
for 24 h and then heated at two different temperatures (65 °C and 95 °C)
in a water bath (for better control of temperature) for two different time
spans (30 min and 1 h). For each temperature/duration combination,
six samples of simulated bread were baked. Of those, five were pre-
pared with water containing NX-3, while one served as control and only
water was used. Further sample preparation and extraction procedure
were the same as in the pre-experiment described above. The control
sample was spiked after extraction in five replicates and served as a
matrix matched control to correct for matrix effects, which might occur
during the ionization in the mass spectrometer. For NX3-M1, only the
harshest conditions (1 h baking at 95 °C) were tested, using the same
process as described above for NX-3. Additionally, a pure standard
solution in water was likewise processed (24 h at 20 °C and 1 h at 95 °C).
Furthermore, the spiking solutions, which were frozen immediately
after preparation, were measured in five replicates. The determined
E. Varga et al.
average concentrations in bread and the extract were compared to those
of the immediately frozen spiking solution. The percentage of the re-
maining NX-3 after the baking procedure was calculated by dividing the
concentration obtained in the bread samples by the concentration
found in the spiked extract. For the calculation of the standard devia-
tion of the NX-3 or NX3-M1 recovered from the bread, the error pro-
pagation was followed, taking into account the standard deviations of
the compounds recovered in the spiked samples before and after the
bread baking simulation.
3. Results and discussion
3.1. Initial identification, purification and characterization of NX-M1
derivatives
As described in Varga et al. (2015), the so called NX-strains did not
produce any of the then-known trichothecenes, despite causing typical
Fusarium head blight symptoms in wheat. During the LC–MS-based
screening for metabolites, we identified two peaks, which we initially
termed NX-1 and NX-2, and which were unique to the NX-strains. While
NX-1 had similarities in the MS/MS fragmentation pattern to NX-2, it was
not UV active – a first hint pointing to a reaction involving the double
bond between C-9 and C-10. Structure elucidation (see below and Sup-
plementary information) revealed that NX-1 is related to NX-2 (Varga
et al., 2015), but contains one water molecule more than NX-2. Grove
(1985) first showed the formation of acid-catalyzed rearrangement pro-
ducts of various type A trichothecenes to e.g. 10,13-cyclotrichothecane, a
reaction which is prevented with the introduction of an 8-ketone in type B
trichothecenes. Likewise, Shams et al. (2011) reported the formation of
DAS-M1 during thermal treatment of diacetoxyscirpenol (DAS) in aqueous
solution. Since we expected a similar reaction for the NX metabolites, we
changed the earlier working designation NX-1 to NX2-M1 for consistency.
Correspondingly, the proposed rearrangement products of NX-3 (the
deacetylated version of NX-2) and NX-4 (acetylation at C-15) were termed
NX3-M1 and NX4-M1, respectively (see Fig. 1).
LCeHRMS measurements confirmed the sum formulas of C17H26O7
for both NX2-M1 and NX4-M1, and C15H24O6 for NX3-M1, corre-
sponding to the addition of water compared to the respective NX-me-
tabolite. Whereas in case of NX2-M1 and NX3-M1, the signal corre-
sponding to the ammonium adduct was slightly higher than that of the
protonated ion, the protonated ion signal was more than two times
higher than that of the ammonium adduct for NX4-M1. HRMS/MS
spectra of the purified compounds are shown in Supplementary in-
formation Fig. A1. While many NX-M1 fragments occur also in the
parent compounds, m/z 283.15 (corresponding to a loss of the acetyl
group and hence the mass of NX-3) is not visible neither in NX2-M1, nor
in NX4-M1, whereas it is a fragment in NX3-M1 and prominently pre-
sent in the NX-2 and NX-4 spectra. The presence of m/z 265.14 (cor-
responding to a loss of one acetyl and two hydroxy groups) indicates
that the bonds of at least two hydroxy groups are weaker in the NX-M1
derivatives than in the NX-metabolites.
Structure elucidation and signal assignment was carried out based
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Toxicology Letters 284 (2018) 205–212
on 1D (1H, 13C) and 2D (1H1H correlation spectroscopy, 1H13C het-
eronuclear single quantum correlation, and 1H13C hetero-nuclear mul-
tiple bond correlation) NMR spectroscopy (see Supplementary in-
formation Table A1 and A2). Furthermore, Nuclear Overhauser Effect
Spectroscopy (NOESY) was performed to determine the correct ste-
reochemistry. Fig. 1 shows the structures of the (previously reported)
NX-metabolites (NX-2, NX-3 and NX-4) and the corresponding NX-M1
derivatives (NX2-M1, NX3-M1 and NX4-M1).
3.2. In vitro translation inhibition assays
Trichothecenes are well known inhibitors of eukaryotic protein
biosynthesis. The epoxy group is essential for the inhibitory effect, and
the opening of the epoxide ring was shown to alleviate the toxicity in
case of DAS (Shams et al., 2011). Thus, a similar effect was expected for
the rearrangement products of NX-metabolites. However, acetylation at
C-3 already strongly reduces translation inhibition of trichothecenes
(Kimura et al., 1998), consequently, NX-2 is not an effective transla-
tional inhibitor. The apparent inhibition of wheat ribosomes in a
translation assay was indeed due to deacetylation of NX-2 and forma-
tion of NX-3 during the assay (Varga et al., 2015). For this reason, all
following experiments were performed with NX-3 and its derivative
NX3-M1. As shown in Fig. 2, NX3-M1 has no inhibitory effect on animal
ribosomes, while in the wheat germ extract it leads to about 10%
translation inhibition only at a concentration of 200 μM. In contrast,
NX-3 and DON inhibit almost completely the translation at 20 μM in
both assays (Fig. 2 and Varga et al., 2015). Thus, we conclude that the
opening of the epoxide ring leads to detoxification of NX-toxins.
3.3. Impact on cellular viability and protein content of two human colon cell
lines (alamarBlue® and sulforhodamine B assay)
In order to evaluate the impact of NX-3 and its rearrangement
product NX3-M1 on the viability of human cell lines, the colon carci-
noma cell line HT-29 and the non-tumorigenic colon epithelial cells
HCEC-1CT were used. Viability was assessed using two different assays,
the alamarBlue® assay to determine cellular metabolic activity and the
sulforhodamine B assay to detect changes in cellular protein levels. Due
to structural similarity between NX-3 and DON, the cytotoxic effects of
NX-3 and NX3-M1 were compared to that of DON. Incubations for 24 h
with DON and NX-3 resulted in both test systems and in both cell lines
in a concentration dependent cytotoxicity. In HT-29 cells, significant
cytotoxic effects were observed in concentrations > 10 μM (Fig. 3A and
C), whereas in HCEC-1CT cells, incubations with 1 μM of DON and NX-3
resulted already in a significant decrease of cell viability (Fig. 3B and
D). NX3-M1 did not cause any significant cytotoxic effects in both cell
lines, even though incubations with concentrations of 10 μM up to 100
μM in HT-29 cells and 100 μM in HCEC-1CT cells resulted in slightly
reduced cell viability. In Supplementary information Fig. A2 (A: DON;
B: NX-3; C: NX3-M1), the SRB data are presented as a comparison of the
two cell lines, and it can be clearly seen that HCEC-1CT cells are more
susceptible to DON and NX-3 mediated toxicity. In case of NX3-M1, no
Fig. 2. In vitro toxicity of deoxynivalenol (DON) and NX-3 and
NX3-M1 in A) wheat germ extract and B) rabbit reticulocyte lysate
based translation assays. Error bars show standard deviations of
replicates.
E. Varga et al.
significant differences between HT-29 and HCEC-1CT cells could be
determined. These data demonstrate that, while NX-3 is comparable in
its toxicity to DON, the formation of NX3-M1 is clearly a detoxification
reaction. We therefore set out to investigate to what extent this con-
version may occur during cereal storage and food processing condi-
tions.
3.4. Stability of NX-3 in solution and ground wheat
First, we investigated the stability of NX-3 in aqueous solutions at
different pH conditions during incubation at different temperatures of
37 °C or 65 °C over a period of 15 days. The pH-ranges tested were pH 3
and pH 7 to pH 11. The fastest degradation was observed at pH 9 and
pH 10 with almost no detectable NX-3 after 6 h (see Fig. 4). At 65 °C a
faster degradation was observed than at 37 °C. A second, earlier eluting
peak (2.2 min instead of 3.6 min) with the same molecular mass of NX3-
M1 was observed. At pH 3, even a third peak at 4.6 min with the same
accurate mass could be determined after 15 days at 37 °C and already
after 6 h at 65 °C. These minor compounds were not purified, but we
assume that the epoxide ring is opened, and a bridge e.g. to C-9 instead
of C-10 might be formed (similar to the DON sulfonates reported by
Schwartz et al. (2013)) or an opening of the epoxide ring without ring
formation.
A relevant question is also the fate of NX-3 during the storage of
contaminated grains. To investigate this issue, we artificially inoculated
ears of the highly susceptible wheat cultivar Apogee with the NX-2
producing F. graminearum strain 02-264 (WG-9). While the fungus only
produces NX-2 on autoclaved rice, the main trichothecene observed in
planta is NX-3 (Varga et al., 2015), which is formed by deacetylation of
NX-2. For stability tests, infected wheat heads were harvested after ri-
pening, ground, and incubated for various time periods at two different
storage temperatures. It was assessed how much NX-2 and NX-3 had
remained, and how much NX2-M1 and NX3-M1 had been formed
during storage of up to eight weeks at 20° and 37 °C.
At 20 °C, the concentration of NX3-M1 increased over time (Fig. 5),
while we found no NX2-M1, or only traces of it, at any condition. Sur-
prisingly, the amount of NX-2 increased over time, while NX-3 did not
decrease, as one would expect from the corresponding formation of NX3-
M1. A likely explanation is that F. graminearum was still metabolically
active and synthesized new NX-2, which was converted into NX-3 and
NX3-M1. The molar sum of compounds roughly doubled during the
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Toxicology Letters 284 (2018) 205–212
Fig. 3. Effects of deoxynivalenol (DON), NX-3 and NX3-M1 on the
cellular viability and protein content of the two human colon cell
lines HT-29 and HCEC-1CT determined in the alamarBlue® (HT-
29: A; HCEC‐1CT: B) and sulforhodamine B assay (HT-29: C;
HCEC‐1CT: D). Significant differences to the respective NOEL/
0.01 nM are indicated by “a” for DON and “b” for NX-3 (“1”
p < .05; “2” p < .01; “3” p < .001). Significant differences to
the respective concentration of DON are indicated by “x” (“1”
p < .05; “2” p < .01; “3” p < .001). Values are shown as
mean ± SEM of at least five independent experiments performed
in quadruplicates. In the graph, the grey line (100%) shows sol-
vent control data.
incubation time. At 37 °C, a temperature above typical F. graminearum
growth temperatures, NX3-M1 increased over time, NX-3 diminished, and
the molar sum was roughly constant. In conclusion, during extended grain
storage, probably already under conditions of grain filling, as well as at
high temperatures in the field before harvest, a significant portion of NX-3
may be converted into the non-toxic NX3-M1. Some NX3-M1
(5.3 ± 0.3 μmol/g dry weight, corresponding to 14% of NX-3) was al-
ready found in the control samples of the wheat ears.
Assuming that a frequency of 2% NX producers leads to a 2% NX-3
contamination besides DON, at a 2 mg/kg DON contamination level one
would only expect 40 μg/kg NX-3. If potentially more than 70% of that
amount is already converted to NX3-M1 in grain, the task to find NX-3
becomes analytically challenging due to relatively bad ionization
properties. It is therefore not surprising that reports of NX-3 occurrence
in naturally contaminated grain are still lacking. Yet, NX-3 may become
more relevant if NX-3 producers would indeed have a selective ad-
vantage, and if their frequency would increase, as has already been
reported for upstate New York (Lofgren et al., in press). However, our
results show that this scenario would be still preferable to contamina-
tion with DON, unless, due to unexpected differences in uptake and
metabolization of NX-3, this compound would prove to be more toxic to
humans.
3.5. Stability of NX-3 during bread baking
Since humans rarely consume grain in raw form, a further question
is the stability of NX-3 during food processing, in particular bread-
making and baking. As described in the previous section, we observed
conversion of NX-3 into the less toxic NX3-M1 at higher pH values and/
or elevated temperatures (65 °C). Therefore, we further investigated the
degree of conversion in an experiment simulating the bread baking
process. In a pilot experiment, we showed that the extraction recovery
of NX-3 and NX3-M1 was around 100%, and the determined matrix
effects were 90% for NX-3 and 130% for NX3-M1. A maximum of 4
molar% NX3-M1 was formed during the extraction procedure.
In the main experiment, we spiked the dough before the bread-making
procedure as well as the blank bread after extraction with NX-3 for each of
the four temperature/duration combinations (30 min at 65 °C, 60 min at
65 °C, 30 min at 95 °C and 60 min at 95 °C). Spiking of the extract served as
matrix-matched control to compensate for matrix effects that occurred
during the ionization of the compounds in the mass spectrometer. As
E. Varga et al.
Fig. 4. Degradation of NX-3 and formation of NX3-M1 during storage at 37 °C or 65 °C at six different pH-values. Note that at pH 9 and 10 (D and E), formation of NX3-M1 was already
observed at time point zero.
expected, we observed conversion of NX-3 to NX3-M1 during bread-
making: At 65 °C, the difference between baking for 30 min (88% ± 22%
of remaining NX-3) and 60 min (86% ± 21% NX-3) was not significant
(Fig. 6). At a baking temperature of 95 °C, however, only 60% ± 17% of
the NX-3 was recovered after 30 min and only 30% ± 15% NX-3 was
found after 60 min incubation. At the same time, the amount of generated
NX3-M1 increased gradually. For all these values, the average matrix ef-
fects for NX-3 ranged between 87 and 96% and were taken into con-
sideration. NX3-M1 was also used for spiking and was stable during the
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Toxicology Letters 284 (2018) 205–212
Fig. 5. Degradation of NX-3 in ground wheat and formation of
NX3-M1 over a two-month period at two different temperatures
(20 °C and 37 °C). “Control” indicates the results of the control
measurements which were frozen immediately after grinding. On
the far right side, the concentrations of NX-compounds in the
samples after determination of the dry weight are provided. All
data are displayed as mean values and standard deviation of six
replicates (except control only four replicates).
baking process (103% ± 15%), already taking into account signal sup-
pression and enhancement values of 74% ± 9%.
During bread baking, the crust reaches temperatures higher than
100 °C, but the crumb heats up only slowly. Bread is considered finished
when a temperature of 98 °C in the inside is reached. In the absence of
real baking experiments, the scenario involving 30 min at 95 °C seems
most realistic, indicating that most likely, approx. 50% of the NX-3
present in flour are degraded to non-toxic NX3-M1 during the process.
In the case of DON, only a small amount of degradation (5–10%) was
E. Varga et al.
Fig. 6. Bread baking simulation experiment under four different conditions showing the
formation of NX3-M1. Prior to the “baking” process the dough was incubated for 24 h at
20 °C for all conditions. The data are displayed as mean values and standard deviations of
five replicates.
observed in most studies (for review see Wu et al., 2017).
Our results, on the other hand, indicate that during baking, at least
some NX-3 is likely to remain.
NX-3 is currently confined to the northern US and Canada, and is
present only in very low concentrations. Yet, due to international grain
trade, introduction of NX-2 producing strains into countries that heavily
import North American wheat seems to be a likely scenario. Countries
such as Egypt, Philippines or Mexico were not sampled in the study of
Kelly et al. (2016). Even if NX producers are introduced there and reach
relevant abundance due to an ill understood selective advantage, this
will seemingly not cause a major toxicological threat.
Concluding, NX-3 possesses similar toxicity for human cell lines as
DON. However, the lower stability and the expected substantial con-
version into the nearly non-toxic compound NX3-M1 during food pro-
cessing should be taken into account for further risk assessment.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This research was supported by the Austrian Science Fund (FWF) via
the special research project Fusarium (F3702-B11, F3706-B11, F3718-
B11). Furthermore, the authors acknowledge the financial support of the
Austrian Federal Ministry of Science, Research and Economy, the
National Foundation for Research, Technology and Development and
BIOMIN Holding GmbH for funding the Christian Doppler Laboratory for
Mycotoxin Metabolism. The Lower Austrian Government is thanked for
partly financing the LC–MS/MS systems as well as for supporting the
Christian Doppler Laboratory. The authors would like to express their
gratitude towards Pia Spörhase for providing the inoculated Apogee
wheat heads and Giorgia Del Favero for support during cell culture ex-
periments. Furthermore, the authors would like to thank Prof. Jerry W.
Shay (UT Southwestern Medical Center, Dallas, TX, USA) for providing
HCEC-1CT cells and Prof. Christoph Gasche, MD (Division of
Gastroenterology and Hepatology, Medical University of Vienna, Austria)
for the support in the HCEC-1CT cell cultivation. The picture of the skull
in the graphical abstracts was taken from: https://commons.wikimedia.
org/wiki/File:Death_skull.svg (accessed 04.11.2017) with permission.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.toxlet.2017.12.016.
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Toxicology Letters 284 (2018) 205–212
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