Thermal degradation of the Fusarium mycotoxin deoxynivalenol

Article abstract of DOI:10.1021/jf061008g

Deoxynivalenol (DON) is a toxic secondary metabolite produced by molds of the Fusarium genus, which are able to infect cereal crops in the field. Concerning its rate of occurrence and mean concentration, DON is one of the most important mycotoxins in cereal commodities. Its toxic effects range from causing diarrhea, vomiting, and gastro-intestinal inflammation to noncompetitive inhibition of the biosynthesis of proteins in eukaryotic cells. To study the stability of DON under food-processing conditions such as cooking or baking, we performed model heating experiments and screened the residue for degradation products. Heating of DON and 3-acetyldeoxynivalenol (3-AcDON), especially under alkaline conditions, gave a mixture of compounds, which were isolated and structurally elucidated by NMR and MS experiments. Three of these compounds were already known (norDON A, norDON B, and norDON C), while four were new and named 9-hydroxymethyl DON lactone, norDON D, norDON E, and norDON F. The significance of the DON degradation products was checked by analyzing commercially available food samples. norDON A, B, and C were detected in 29-66% of the samples in mean concentrations ranging from 3 to 15 μg/kg. Furthermore, cell culture experiments using IHKE cells showed that the compounds that were detected in food samples are less cytotoxic in the formazan dye cytotoxicity assay compared to DON. Whereas DON revealed a median effective concentration (EC₅₀) at 1.1 μmol/L, all other compounds did not show any significant effect up to 100 μmol/L. These findings indicate that the degradation of DON under thermal treatment might reduce the toxicity of DON contaminated food.

Full text of DOI:10.1021/jf061008g

J. Agric. Food Chem. 2006, 54, 6445−6451 6445
Thermal Degradation of the Fusarium Mycotoxin
Deoxynivalenol
MICHAEL BRETZ, MARITA BEYER, BENEDIKT CRAMER, ANJA KNECHT, AND
HANS-ULRICH HUMPF*
Institut fu¨r Lebensmittelchemie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstrasse 45,
48149 Mu¨nster, Germany
Deoxynivalenol (DON) is a toxic secondary metabolite produced by molds of the Fusarium genus,
which are able to infect cereal crops in the field. Concerning its rate of occurrence and mean
concentration, DON is one of the most important mycotoxins in cereal commodities. Its toxic effects
range from causing diarrhea, vomiting, and gastro-intestinal inflammation to noncompetitive inhibition
of the biosynthesis of proteins in eukaryotic cells. To study the stability of DON under food-processing
conditions such as cooking or baking, we performed model heating experiments and screened the
residue for degradation products. Heating of DON and 3-acetyldeoxynivalenol (3-AcDON), especially
under alkaline conditions, gave a mixture of compounds, which were isolated and structurally elucidated
by NMR and MS experiments. Three of these compounds were already known (norDON A, norDON
B, and norDON C), while four were new and named 9-hydroxymethyl DON lactone, norDON D,
norDON E, and norDON F. The significance of the DON degradation products was checked by
analyzing commercially available food samples. norDON A, B, and C were detected in 29-66% of
the samples in mean concentrations ranging from 3 to 15 µg/kg. Furthermore, cell culture experiments
using IHKE cells showed that the compounds that were detected in food samples are less cytotoxic
in the formazan dye cytotoxicity assay compared to DON. Whereas DON revealed a median effective
concentration (EC50) at 1.1 µmol/L, all other compounds did not show any significant effect up to 100
µmol/L. These findings indicate that the degradation of DON under thermal treatment might reduce
the toxicity of DON contaminated food.
KEYWORDS: Deoxynivalenol; mycotoxin; thermal degradation; alkaline degradation; heating; stability;
GC-MS; HPLC-MS/MS; thermal treated food; food safety; toxicity
INTRODUCTION
and Fusarium crookwellense, appears to be one of the most
important mycotoxins in cereal commodities (2). Its toxicity is
attributed to the general ability of trichothecenes to noncom-
petitively inhibit the biosynthesis of proteins in eukaryotic cells
likely by affecting the ribosomal function (3). The oral LD50 of
DON was determined to be 78 mg/kg bw in B6C3F1 mice (4),
while it was 46 mg/kg bw in DDY mice (5). Pigs are especially
sensitive to DON: acute intoxications result in vomiting, feed
refusal, reduced weight gain, and diarrhea at levels as low as
1-2 mg/kg feed (1). Furthermore, inflammation and necrosis
of various tissues, mainly the gastrointestinal tract, the bone
marrow, and lymphoid tissues, are observed (3). So far, DON
is not considered to be genotoxic or carcinogenic (6). Thus, a
tolerable daily intake (TDI) value for DON of 1 µg/kg bw was
established in 1999 (1). It was derived from the “no observed
adverse effect level” (NOAEL) of 0.1 mg/kg bw of a chronic
dietary study with mice (6). Various summaries on the toxicity
of DON can be found in the literature (1, 3, 7).
Fusarium molds are a large group of phytopathogenic fungi
which are able to infect cereal crops in the field during their
growth period. They cause plant diseases, resulting in reduced
yields, deterioration of quality, and contamination of the crops
with mycotoxins. The Fusarium genus consists of more than
100 different species such as Fusarium culmorum or Fusarium
graminearum. These particular species are common on soil and
the most important in causing head blight and producing
trichothecene mycotoxins in various cereal crops. Trichothecenes
are a large group of sesquiterpenes sharing a 12,13-epoxytri-
chothec-9-ene ring system as the basic chemical structure (1).
The group is subdivided in type A trichothecenes such as T-2
toxin and type B trichothecenes such as deoxynivalenol 1 (DON,
Figure 1), the difference being a keto substitution at C-8 in the
latter case.
Considering its rate of occurrence and mean concentration,
DON, which is produced by F. graminearum, F. culmorum,
In recent years, legislation has adopted the problem of DON
in food. Maximum limits were established in the European
Union in July 2006. They range from 200 µg/kg for cereal
* Corresponding author. Tel.: +49 251 83 333 91. Fax: +49 251 83
333 96. E-mail: humpf@uni-muenster.de.
10.1021/jf061008g CCC: $33.50
© 2006 American Chemical Society
Published on Web 07/27/2006

6446 J. Agric. Food Chem., Vol. 54, No. 17, 2006
Bretz et al.
Figure 1. Structures of deoxynivalenol (1) and its known degradation products isoDON (2), norDON A (3), norDON B (4), norDON C (5), and DON
lactone (6) and the new compounds 9-hydroxymethyl DON lactone (7), norDON D (8), norDON E (9), and norDON F (10).
products for young children to 1750 µg/kg for unprocessed
durum wheat and oats (8). Germany has implemented more
stringent maximum limits for DON in 2004 ranging from 100
µg/kg for infant cereal products to 500 µg/kg for most other
cereal products (9).
tography-electrospray ionization-tandem mass spectrometry
(HPLC-ESI-MS/MS).
MATERIALS AND METHODS
Reagents. DON and 3-acetyldeoxynivalenol (3-AcDON) were
prepared according to the procedure described by Altpeter et al. (26).
Water for HPLC separation was purified with a MilliQ Gradient A10
system (Millipore, Schwalbach, Germany); the other solvents for HPLC
and HPLC-MS as well as all other chemicals were purchased from
VWR (Darmstadt, Germany) or Sigma-Aldrich (Deisenhofen, Germany)
in gradient or reagent grade quality.
Model Experiments. The following compounds were used as food
models: R-D-glucose as sugar model, methyl-R-D-glucopyranoside as
starch model, and the amino acid derivatives N-R-acetyl-L-lysine methyl
ester and BOC-L-cysteine methyl ester as protein models (24). The
experiments were performed by heating 100 µg of deoxynivalenol with
1 mg of model compound without solvent. To get a homogeneous
mixture, aliquots of stock solutions of the reactants (1-20 mg/mL in
methanol) were vortexed in a 1.5 mL glass vial and the solvent was
removed under a stream of nitrogen. The mixture was heated in a
heating block for various periods (5-20 min) at different temperatures
(150-200 °C). The residue was further analyzed by gas chromatog-
raphy-mass spectrometry (GC-MS). As a control, pure DON was heated
under the conditions described above.
In general, DON is considered to be a relatively stable
compound. Most of the studies on its stability are focused on
model experiments, observing the degradation depending on
time, temperature, or pH in aqueous solutions (10-16). They
showed that DON was especially unstable under alkaline
conditions and at high temperatures. In total, five degradation
products were isolated after rather harsh thermal or alkaline
treatment of DON 1 (Figure 1) (11-13).
The results of studies on the stability of DON in food matrixes
under processing conditions such as cooking or baking seem to
be contradictory. While some authors report no degradation at
all (17, 18), most observe a moderate reduction (5-85%)
depending on the respective conditions (10, 15, 19-22). Abbas
et al. (23) studied the degradation of DON during the production
of tortillas from contaminated corn, observing a reduction of
up to 82%. This is possibly due to the nixtamalization process,
the traditional alkaline treatment of corn kernels in Latin
America.
GC-MS. Gas chromatography electron impact mass spectrometry
(GC-EI-MS) data were acquired on a HP6890 series gas chromatograph
and HP5973 mass spectrometer (Hewlett-Packard/Agilent, Bo¨blingen,
Germany) after derivatization of the compounds with 200 µL of
trimethylsilylimidazole (TMSI, 70 °C, 30 min) and addition of 300 µL
of tert-butyl methyl ether (t-BME). Data acquisition was performed
with the Chemstation software (Agilent). Chromatographic separation
was performed on a 60 m × 0.25 mm i.d. fused silica, 0.25 µm methyl
silicone J&W Scientific DB-1 column (Agilent) using 1 mL/min helium
as carrier gas. The injector temperature was set at 250 °C, and the
injection volume was 1 µL with split injection (1:9). The column
temperature was held initially at 120 °C for 1 min and then programmed
at 4 °C/min to 260 °C and then with 15 °C/min to 320 °C, which was
held constant for 10 min. The transfer line was heated at 320 °C. The
mass spectrometer was operated in the electron impact mode (EI, 70
eV electron energy) with a source temperature of 230 °C and the
quadrupole heated at 150 °C. Mass spectra were acquired in the full
scan mode ranging from m/z 40-800 with a scan rate of 2.0 scans/s.
In summary, the available data clearly show that the amount
of reduction of DON during thermal food processing is highly
variable and depends on the respective processing conditions.
However, except for isoDON 2 (Figure 1) (11), which was
detected in bread, little is known about specific degradation
products and nothing about their toxicological properties.
In an attempt to investigate the stability of DON under food
processing conditions, we used a model heating system, which
we have already applied in the investigation of the stability of
fumonisins and nivalenol (24, 25). Briefly, the mycotoxins were
heated with several model compounds simulating typical food
constituents: R-D-glucose was used as sugar model, methyl-
R-D-glucopyranoside as starch model, and the amino acid
derivatives N-R-acetyl-L-lysine methyl ester and BOC-L-cysteine
methyl ester as protein models (24, 25). The degradation
products formed were analyzed by gas chromatography-mass
spectrometry (GC-MS) and high-performance liquid chroma-
Preparation and Isolation of norDON A, norDON B, and
norDON C. The compounds were obtained by alkaline degradation of

Thermal Degradation Products of DON
J. Agric. Food Chem., Vol. 54, No. 17, 2006 6447
3-AcDON. Fifty milligrams of 3-AcDON was heated in 2 mL of 0.1
M aqueous NaOH at 75 °C for 60 min (13). The mixture was neutralized
with 200 µL of 1 M aqueous HCl and 200 µL of MeOH was added to
adjust the solution to the HPLC starting conditions. The compounds
were separated on an analytical 250 × 4.6 mm i.d., 5 µm Eurospher
100 column (Knauer, Berlin, Germany) using a linear binary gradient
delivered by a Knauer Wellchrom Maxi-Star K-1000 HPLC pump with
water as solvent A and methanol as solvent B. The HPLC was
programmed as follows: isocratic step at 20% solvent B for 1 min
followed by a linear gradient to 50% solvent B in 30 min. The flow
rate was 700 µL/ min. For injection, a Knauer A-0258 six-port valve
with a 250 µL sample loop was used. Three fractions (norDON A,
norDON B, and norDON C; yield 1.4-3.3 mg) were collected after
peak detection using a Knauer A-0293 single-wavelength detector set
at 220 nm. Furthermore, fractions between the UV-detected peaks were
collected to isolate compounds without significant UV absorbance.
Isolation of the New Compounds 9-Hydroxymethyl DON Lac-
tone, norDON D, norDON E, and norDON F. The fractions without
UV absorbance were lyophilized, redissolved in 2 mL of water-
methanol (9:1), and further purified on an analytical 250 × 4.6 mm
i.d., 5 µm MicroSorb-MV 100-5 C18 column (Varian Inc., Darmstadt,
Germany) by isocratic HPLC. The solvent (water-methanol, 9:1) was
delivered by a Knauer Wellchrom Maxi-Star K-1000 HPLC pump with
a flow rate set at 700 µL/min. Four new compounds were isolated after
peak detection using a Type 51.78 Knauer differential refractive index
detector. After solvent evaporation under nitrogen streams, all com-
pounds were further analyzed. Yield: 9-hydroxymethyl DON lactone,
1 mg; norDON D, 0.3 mg; norDON E, 0.9 mg; norDON F, 0.8 mg.
Exact Mass Measurements. The exact masses of the compounds
were measured on a Bruker Micro-TOF (Bruker Daltronics, Bremen,
Germany) mass spectrometer with flow injection and referenced on
sodium formiate. The compounds were dissolved in 1 mL of MeOH
and 10 µL of a saturated solution of NaBF4 in MeOH was added to
measure the exact mass of the sodium adducts. The resolution of the
mass spectrometer was Rfwhm ) 10000 (full width at half maximum).
ESI-MS/MS. Electrospray ionization (ESI) mass and product ion
spectra were acquired on an API 4000 QTRAP mass spectrometer
(Applied Biosystems, Darmstadt, Germany) with direct flow infusion.
For electrospray ionization, the ion spray voltage was set at -4500 V
in the negative mode and at 5500 V in the positive mode. Nitrogen
served as curtain gas (20 psi); the declustering potential, being the
accelerating current from atmospheric pressure into high vacuum, was
set at -50 V in the negative mode and 50 V in the positive mode. The
MS/MS parameters were dependent on the substances, detecting the
fragmentation of the [M - H]- or [M + H]+ molecular ions into
specific product ions after collision with nitrogen (4.5 × 10-5 Torr).
The collision energies are given at the respective compounds.
NMR Spectroscopy. 1H and 2D NMR data were acquired on a
Bruker DPX-400 (Bruker BioSpin, Rheinstetten, Germany), 13C NMR
on a Unity plus (Varian, Palo Alto, CA) NMR spectrometer. Signals
are reported in parts per million relative to d4-MeOH. For structural
elucidation and NMR signal assignment 2D NMR experiments such
as gradient-selected correlated spectroscopy (gs-COSY), heteronuclear
multiple quantum correlation (HMQC), heteronuclear multiple bond
correlation (HMBC), and 2D nuclear Overhauser enhancement spec-
troscopy (NOESY) were performed. Pulse programs for the experiments
were taken from the Bruker software library.
217 (25), 235 (17), 201 (16), 175 (14), 265 (10); positive mode: m/z
(%) 267 (100) [M + H]+, MS/MS (35 V): m/z (%) 175 (100), 213
(42), 189 (34), 161 (31), 198 (29), 231 (25), 159 (24), 145 (22).
9-Hydroxymethyl DON Lactone. Found: m/z 349.1262. Calculated
for C16H22O7 + Na+: 349.1263.
ESI-MS: negative mode: m/z (%) 325 (100) [M - H]-, MS/MS
(-30 V): m/z (%) 325 (100), 247 (73), 235 (39), 123 (34), 295 (31),
219 (29), 207 (28), 165 (28); positive mode: m/z (%) 327 (100) [M +
H]+, MS/MS (30 V): m/z (%) 309 (100), 299 (46), 269 (28), 251 (13),
327 (9), 263 (6), 225 (6), 239 (5).
1H NMR (400 MHz, d4-MeOH): δ 1.11 (3H, s, H-14), 1.35 (3H, s,
H-16), 1.62 (1H, dd, J4A,3 ) 4.2, JAB ) 14.0, H-4A), 1.72 (1H, d, JAB
) 14.8, H-10A), 2.15 (1H, dd, J4B,3 ) 10.5, JAB ) 14.0, H-4B), 2.34
(1H, dd, J10B,11 ) 4.2, JAB)14.8, H-10B), 3.26 (1H, d, JAB ) 10.6,
H-17A), 3.43 (1H, d, JAB ) 10.6, H-17B), 3.62 (1H, d, J2,3 ) 4.6,
H-2), 3.88 (1H, d, JAB ) 12.2, H-13 A), 3.96 (1H, d, JAB ) 10.2,
H-15A), 4.30 (1H, d, JAB ) 10.2, H-15B), 4.36 (1H, d, J11,10B ) 4.2,
H-11), 4.53 (1H, d, JAB ) 12.2, H-13B), 4.59 (1H, m, J3,2 ) 4.6, J3,4A
) 4.2, J3,4B ) 10.5, H-3).
13C NMR (150 MHz, d4-MeOH): δ 15.2 (C-14), 22.6 (C-16), 41.1
(C-10), 43.9 (C-4), 46.0 (C-5), 55.2 (C-9), 62.2 (C-6), 68.7 (C-17),
69.4 (C-15), 71.5 (C-3), 72.8 (C-13), 78.0 (C-11), 78.4 (C-12), 84.2
(C-2), 87.6 (C-7), 179.8 (C-8).
norDON D. Found: m/z 303.1202. Calculated for C15H20O5 + Na+:
303.1208.
ESI-MS: negative mode: m/z (%) 279 (100) [M - H]-, MS/MS
(-30 V): m/z (%) 279 (100), 179 (90), 177 (86), 249 (72), 187 (45),
205 (44), 191 (43), 207 (42); positive mode: m/z (%) 281 (100) [M +
H]+, MS/MS (25 V): m/z (%) 281 (100), 173 (79), 199 (49), 215 (48),
263 (48), 245 (46), 227 (39), 219 (39).
1H NMR (400 MHz, d4-MeOH): δ 1.07 (3H, s, H-14), 1.80 (3H, d,
J16,10 ) 1.5, H-16), 1.99 (1H,dd, J4A,3 ) 10.7, JAB ) 14.5, H-4A), 2.11
(1H, dd, J7A,6 ) 7.3, JAB ) 13.5, H-7A), 2.16 (1H, dd, J ) 12.4, JAB
) 13.5, H-7B), 2.18 (1H, dd, J4B,3 ) 5.2, JAB ) 14.5, 4B), 2.63 (1H,
m, J6,11 ) 1.4, J6,7A ) 7.3, J6,7B ) 12.4, H-6), 3.47 (1H, d, JAB ) 11.9,
H-15A), 3.53 (1H, d, J2,3 ) 4.3, H-2), 3.57 (1H, d, JAB ) 11.9, H-15B),
4.49 (1H, m, J3,2 ) 4.3, J3,4A ) 10.7, J3,4B ) 5.2, H-3), 4.69 (1H, d,
J11,10 ) 6.4, J11,6 ) 1.4, H-11), 6.93 (1H, m, J10,16 ) 1.5, J10,11 ) 6.4,
H-10).
13C NMR (150 MHz, d4-MeOH): δ 14.4 (C-14), 15.6 (C-16), 37.0
(C-7), 41.8 (C-4), 47.9 (C-6), 54.6/55.1 (C-13/C-5), 63.7 (C-15), 69.1
(C-11), 72.7 (C-3), 85.8 (C-2), 87.6 (C-12), 139.0 (C-9), 143.7 (C-
10), 203.1 (C-8).
norDON E. Found: m/z 305.1341. Calculated for C15H22O5 + Na+:
305.1365.
ESI-MS: negative mode: m/z (%) 281 (100) [M - H]-, MS/MS
(-25 V): m/z (%) 129 (100), 281(57), 251 (54), 121 (32), 179 (12),
233 (11), 151 (10), 205 (8); positive mode: m/z (%) 283 (100) [M +
H]+, MS/MS (25 V): m/z (%) 265 (100), 247 (82), 187 (67), 229 (65),
217 (53), 201 (51), 159 (50), 175 (46).
1H NMR (400 MHz, d4-MeOH): δ 1.01 (3H, d, J16,9 ) 6.7, H-16),
1.01 (3H,s, H-14), 1.87 (1H, m, JAB ) 14.1, J10A,11 ) 2.0, J10A,9
)
12.3, H-10A), 1.90 (1H, dd, JAB ) 13.7, J7A,6 ) 12.7, H-7A), 1.95
(1H, dd, J4A,3 ) 10.7, JAB ) 14.4, H-4A), 2.11 (1H, dd, J4B,3 ) 5.4,
JAB ) 14.4, H-4B), 2.18 (1H, dd, JAB ) 13.7, J7B,6 ) 7.3, H-7B), 2.21
(1H, m, JAB ) 14.1, J10B,11 ) 3.6, J10B,9 ) 6.5, H-10B), 2.63 (1H, m,
J6,11 ) 1.7, J6,7A ) 12.7, J6,7B ) 7.3, H-6), 3.10 (1H, m, J9,16 ) 6.7,
J9,10B ) 6.5, J9,10A ) 12.3, H-9), 3.39 (1H, d, JAB ) 12.0, H-15A),
3.52 (1H, d, JAB ) 12.0, H-15B), 3.65 (1H, d, J2,3 ) 4.4,H-2), 4.40
(1H,m, J11,10A ) 2.0, J11,10B ) 3.6, J11,6 ) 1.7, H-11), 4.51 (1H, m, J3,2
) 4.4, J3,4A ) 10.7, J3,4B ) 5.4, H-3).
Spectroscopy Data. For known compounds only the MS data are
given, while the NMR data are included for the new ones.
norDON A. ESI-MS: negative mode: m/z (%) 265 (100) [M - H]-,
MS/MS (-35 V): m/z (%) 163 (100), 217 (39), 189 (31), 229 (27),
214 (24), 199 (23), 265 (22), 171 (16); positive mode: m/z (%) 267
(100) [M + H]+, MS/MS (10 V): m/z (%) 203 (100), 213 (35), 151
(33), 185 (26), 231 (25), 125 (17), 201 (16), 175 (16).
13C NMR (150 MHz, d4-MeOH): δ 14.3 (C-14/C-16), 35.2 (C-7),
35.6 (C-9), 37.0 (C-10), 42.0 (C-4), 52.7 (C-6), 54.7 (C-5), 57.9 (C-
13), 63.6 (C-15), 72.0 (C-11), 73.1 (C-3), 86.2 (C-2), 88.3 (C-12), 217.3
(C-8).
norDON F. Found: m/z 319.1152. Calculated for C15H20O6 + Na+:
319.1158.
ESI-MS: negative mode: m/z (%) 295 (100) [M - H]-, MS/MS
(-30 V): m/z (%) 121 (100), 295 (72), 175 (46), 127 (45), 111 (42),
177 (37), 293 (34), 135 (29); positive mode: m/z (%) 297 (100) [M +
norDON B. ESI-MS: negative mode: m/z (%) 265 (100) [M - H]-,
MS/MS (-35 V): m/z (%) 163 (100), 214 (48), 229 (44), 123 (19),
217 (17), 247 (16), 202 (15), 235 (11), 265 (7); positive mode: m/z
(%) 267 (100) [M + H]+, MS/MS (35 V): m/z (%) 175 (100), 137
(26), 157 (24), 219 (24), 173 (23), 161 (22), 159 (22), 142 (21).
norDON C. ESI-MS: negative mode: m/z (%) 265 (100) [M - H]-,
MS/MS (-35 V): m/z (%) 163 (100), 229 (87), 214 (44), 247 (33),

6448 J. Agric. Food Chem., Vol. 54, No. 17, 2006
Bretz et al.
H]+, MS/MS (30 V): m/z (%) 187 (100), 159 (43), 251 (38), 145 (34),
175 (34), 161 (31), 297 (29), 233 (25).
1H NMR (400 MHz, d4-MeOH): δ 0.96 (3H, s, H-14), 1.31 (3H, d,
J16,9 ) 7.5, H-16), 1.72 (1H, dd, JAB ) 14.1, J4A,3 ) 5.6, H-4A), 1.82
(1H, d, JAB ) 14.2, H-13A), 1.92 (1H, dd, JAB ) 14.1, J4B,3 ) 10.5,
Cell Culture. Immortalized human kidney epithelial cells (IHKE
cells, passage 164-167) were kindly provided by M. Gekle (Wu¨rzburg,
Germany) (IHKE cells are originally from S. Mollerup, National
Institute of Occupational Health, Norway). They were cultured as
described by Tveito (28) in DMEM (Dulbecco’s modified Eagle’s
medium)/Ham’s-F12 medium (100 µL/cm2) enriched with 13 mmol/L
NaHCO3, 15 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic
acid (HEPES), 36 µg/L hydrocortisone, 5 mg/L human apotransferrin,
5 mg/L bovine insulin, 10 µg/L mouse epidermal growth factor, 5 µg/L
Na selenite, 10% fetal calf serum, and in addition 1% penicillin/
streptomycin under standard cell culture conditions (37 °C, 5% CO2).
H-4B), 2.19 (1H, m, J9,16 ) 7.5, J9,10 ) 2.1, H-9), 2.38 (1H, d, JAB
)
14.2, H-13B), 3.63 (1H, d, J2,3 ) 4.1, H-2), 3.84 (1H, d, J10,9 ) 2.1,
H-10), 4.06 (1H, s, H-11), 4.51 (1H, m, J3,2 ) 4.1, J3,4A ) 5.6, J3,4B
10.5, H-3).
)
13C NMR (150 MHz, d4-MeOH): δ 15.4 (C-14), 17.6 (C-16), 33.1
(C-13), 41.9 (C-4), 53.3 (C-5), 53.5 (C-9), 59.2 (C-7), 67.6 (C-15),
70.4 (C-6), 73.2 (C-3), 83.9 (C-11), 86.1 (C-10), 86.3 (C-2), 89.6 (C-
12), 184.5 (C-8).
Cytotoxicity Assay. Cytotoxicity was evaluated colorimetrically with
the Cell Counting Kit-8 (CCK-8) from Dojindo Laboratories (Tokyo,
Japan) similar to the manufacturer’s instruction. Briefly, cells were
grown on a 96-well microplate. One hundred microliters of a cell
suspension, containing 3 × 103 cells, was added to each well. After 48
h of growth, culture medium was replaced by serum free medium for
24 h. Test compounds were dissolved in methanol and added to serum
free medium (the final MeOH concentration was <1%). After 24 h of
incubation, the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-
5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] solution was
added and the cells were incubated for 1 h. WST-8 produces a water-
soluble formazan dye upon reduction in the presence of an electron
carrier. The absorbance of each well was measured with a FLUOstar
Optima microplate reader (BMG Labtechnologies, Jena, Germany) at
450 nm. The amount of the formazan generated by the activity of
dehydrogenases in cells is directly proportional to the number of viable
cells per well. The absorbance of the treated wells was compared with
a solvent-treated control.
Sample Preparation Method 1. Sixty grams of commercially
available food samples (n ) 43) were finely ground in a laboratory
blender. Twenty-five grams was weighed in an Erlenmeyer flask and
2 µg of zearalanone was added as internal standard. The sample was
extracted three times with 100 mL of methanol-water (4:1), and the
supernatant was filtered, evaporated to dryness, and redissolved in 10
mL of acetonitrile. For analysis by HPLC-MS/MS, 500 µL of the extract
was evaporated to dryness and redissolved in 500 µL of methanol-
water (1:9). For quantification, standard solutions of the analytes and
the internal standard were mixed to cover concentration ranges equaling
2.5-480 µg/kg for DON, 1-300 µg/kg for norDON A, B, and C, and
80 µg/kg for zearalanone (ZAN). The limits of detection were
determined to be 2.5 µg/kg for DON and norDON A and 1 µg/kg for
norDON B and C (S/N 10:1).
Sample Preparation Method 2. Twenty-five grams of commercially
available food samples (n ) 18, tortilla chips, bread, corn flakes, and
pretzels) was weighed in an Erlenmeyer flask and 2.5 µg of 15-d1-
DON (29) was added as internal standard. Acetonitrile-water (4:1)
(100 mL) was added and the samples were homogenized with an Ultra
Turrax T25B (Janke & Kunkel IKA, Staufen, Germany) for 3 min at
20000 rpm. After filtration through a Schleicher & Schuell filter (5951/
2, 320 mm), the extract was cleaned up using the method of Klo¨tzel et
al. (27): 4 mL of the extract was passed through a Varian Bond Elut
Mycotoxin cartridge. Exactly 2 mL of the eluate was evaporated to
dryness under a nitrogen stream (42 °C), and the residue was redissolved
in 500 µL of water-methanol (9:1) and further analyzed by HPLC-
MS/MS. For quantification, five standard solutions containing DON
(10-500 ng/mL), norDON A, B, and C (1-75 ng/mL), and the internal
standard 15-d1-DON (100 ng/ mL) were prepared and analyzed by
HPLC-MS/MS in the MRM mode. For the calculation of calibration
curves, the peak area ratios of the analytes to the internal standard were
plotted against the concentration ratios.
HPLC-ESI-MS/MS Analysis. For HPLC-ESI-MS/MS analysis, an
Agilent 1100 series HPLC was linked to the API 4000 QTRAP mass
spectrometer. Data acquisition was performed with the Analyst 1.4
software (Applied Biosystems). Chromatographic separation was
performed on a 150 × 2.1 mm i.d., 4 µm Phenomenex SynergiFusion
column (Phenomenex, Aschaffenburg, Germany) using a linear binary
gradient. The injection volume was 5 µL, and the flow rate was 200
µL/ min. Solvent A was methanol and solvent B was water. The HPLC
was programmed as follows: isocratic step at 10% solvent A for 1
min followed by a linear gradient to 80% solvent A in 26 min. After
each HPLC run, the column was washed with 100% solvent A and
equilibrated for 15 min at the starting conditions. For HPLC-MS/MS
the mass spectrometer was operated in the multiple reaction monitoring
mode (MRM), detecting negative ions. Zero-grade air served as
nebulizer gas (35 psi) and, heated at 300 °C, as turbo gas for solvent
drying (50 psi). The following transition reactions were monitored for
a duration of 150 ms each. Declustering potential (DP), collision energy
(CE) and collision cell exit potential (CXP) are given in brackets:
DON: m/z 295-265 (DP -80 V, CE -16 V, CXP -7 V); norDON
A, B, and C: m/z 265-163 (DP -95 V, CE -28 V, CXP -11 V);
9-hydroxymethyl DON lactone: m/z 325-295 (DP -70 V, CE -25
V, CXP -8 V); norDON D: m/z 279-249 (DP -70 V, CE -25 V,
CXP -8 V); norDON E: m/z 281-251 (DP -70 V, CE -25 V, CXP
-8 V); norDON F: m/z 295-177 (DP -65 V, CE -30 V, CXP -11
V), d1-DON: m/z 296-265 (DP -80 V, CE -16 V, CXP -7 V);
ZAN: m/z 319-275 (DP -90 V, CE -30 V, CXP -15 V).
RESULTS AND DISCUSSION
To study the formation of degradation products of deoxyni-
valenol under food-processing conditions such as baking, model
heating experiments with compounds mimicking typical food
constituents were performed. DON was heated at various
temperatures (150-200 °C) for different time periods (5-20
min) and with R-D-glucose (sugar model), methyl-R-D-glucopy-
ranoside (starch model), and the amino acid derivatives N-R-
acetyl-L-lysine methyl ester and BOC-L-cysteine methyl ester
(protein models). The resulting residues were screened using
gas chromatography-mass spectrometry (GC-MS) after trim-
ethylsilylation. Degradation of DON was observed under all
conditions, generally accelerating with rising temperatures. As
previously reported for nivalenol (25), heating with N-R-acetyl-
L-lysine methyl ester resulted in the fastest degradation: after
10 min at 150 °C, only 20% of DON was left, while it was
50-90% with the other model compounds. Although significant
losses of DON were observed in all experiments, only in the
heating experiment with the lysine derivative appreciable
quantities of three degradation products (up to 40% of the total
DON used) were detected. The losses that cannot be ascribed
to the formation of degradation products are most likely caused
by pyrolysis or polymerization reactions, as heating DON alone
results in a similar rate of decay. The formation of the
degradation products in the course of heating of DON with the
lysine derivative is most likely due to alkaline catalysis by the
ꢀ-amino group of the model compound. DON has previously
been reported to be unstable under alkaline conditions and
degrade into at least four products, namely, norDON A 3,
norDON B 4, norDON C 5, and DON lactone 6 (Figure 1)
(13). For the preparation of these compounds, 3-AcDON, which
was available and rapidly hydrolyzes to DON under alkaline
conditions, was heated with 0.1 M NaOH as described in the
literature (13). The solution was neutralized and separated by
HPLC, collecting fractions after UV detection at 220 nm as
well as the fractions between the UV peaks. The compounds

Thermal Degradation Products of DON
J. Agric. Food Chem., Vol. 54, No. 17, 2006 6449
1
with UV absorption were analyzed by HPLC-MS/MS and H
Table 1. Semiquantitative Results of the Analysis of DON and
NMR and identified as norDON A, norDON B, and norDON
C.
norDON A, B, and C by HPLC-MS/MS in 34 Food Samples
positive samples
The degradation products without or with only weak UV
absorption were separated by isocratic HPLC and fractions were
collected after RI detection. For structural elucidation of the
isolated compounds, the exact masses were measured and 1D
and 2D NMR spectra such as 1H, 13C, H,H-COSY, H,C-HMQC,
H,C-HMBC, and H,H-NOESY were recorded. Due to the
limited amount of available material (<1 mg), structural
elucidation was achieved only for the four compounds 7, 8, 9,
and 10 shown in Figure 1. DON lactone was not detected;
however, substances 7 and 10 are structurally similar to it: 7
has an additional CH2O moiety at position 9 of the DON lactone
skeleton; therefore, it was called 9-hydroxymethyl DON lactone
7 (9-HM DON lactone). The formation of this product can
probably be explained by the addition of formaldehyde, which
is released in the process of alkaline degradation of DON to
norDON A, B, or C.
compound
(>LOQ)
LOQ
−
10
µ
g/kg
10
−
50
µ
g/kg
>50 µg/kg
DON
32 (94%)
21 (62%)
12 (35%)
10 (29%)
4
9
10
7
10
11
2
18
1
norDON A
norDON B
norDON C
n.d.a
2
1
a n.d.: not detectable.
Table 2. Concentration of DON and norDON A, B, and C in
Representative Food Samples ( g/kg, Mean of Duplicate Analyses)
µ
concentration, µg/kg
sample
DON
norDON A
norDON B
norDON C
wholemeal cookies
wholemeal crackers
pretzels 1
301
134
64
10
81
78
208
24
39
26
148
45
216
10
24
62
31
36
12
<LOQ
15
10
18
<LOQ
<LOQ
<LOQ
13
<LOQ
3
1
<LOQ
3
1
<LOQ
n.d.a
n.d.a
<LOQ
1
<LOQ
4
2
<LOQ
5
3
<LOQ
n.d.a
n.d.a
<LOQ
2
pretzels 2
pretzel sticks 1
pretzel sticks 2
tortilla chips 1
tortilla chips 2
tortilla chips 3
tortilla chips 4
tortilla shells
mini-wheats
corn flakes 1
corn flakes 2
oat flakes
bread
maize snack
maize (nixtamalized)
Compound 10 was named norDON F and is a pentacyclic
isomer of DON. In comparison to DON lactone, it is hydroxyl-
ated at position 10 of the skeleton and the ring between C-7
and C-13 is linked by a C-C bond instead of an ether bridge.
The compounds 8 and 9 were named norDON D and norDON
E, respectively, and differ only regarding the type of bond
between C-9 and C-10 of the skeleton: while norDON D has
a double bond in this position, norDON E is saturated. Due to
the complex changes in the molecular structure, in which
released formaldehyde was most likely involved, no mechanisms
for the formation of the new degradation products are proposed.
7
16
n.d.a
< LOQ
n.d.a
n.d.a
<LOQ
n.d.a
6
n.d.a
< LOQ
n.d.a
n.d.a
<LOQ
n.d.a
12
n.d.a
<LOQ
6
77
107
5
14
To study the relevance of the DON degradation products in
food, thermally processed cereal products mainly from the
German market were analyzed using a fast screening method
without sample cleanup. As the structures of the new compounds
had not yet been elucidated, the analysis was focused only on
norDON A, B, and C. The samples were homogenized, spiked
with zearalanone as internal standard, and extracted with
methanol-water (3:1). The extract was filtered, evaporated to
dryness, and redissolved in water-methanol (9:1) to adjust the
solution to the HPLC starting conditions. No further cleanup
was performed prior to analysis by HPLC-MS/MS. The analytes
were ionized by electrospray in the negative mode and the mass
spectrometer was operated in the multiple reaction monitoring
mode (MRM). This method was developed for screening
purposes and thus the results discussed below are only semi-
quantitative.
positive samples (>LOQ)
mean (positive samples)
median
100%
92
70
66%
15
14
33%
3
2
33%
5
3
a n.d.: not detectable
A total of 43 thermally processed samples were analyzed,
three of which were from Mexico. These particular samples had
been nixtamalized, that is, the traditional cooking of corn kernels
with alkaline calcium hydroxide solution in Central America
prior to the preparation of tortillas. Due to strong signal
suppression of the internal standard, 9 of the 43 samples were
not analyzable. DON was detected in 32 of the samples,
norDON A in 21, norDON B in 12, and norDON C in 10. The
results of the analyses, which are summarized in Table 1, clearly
demonstrate the occurrence of DON degradation products in
thermally processed food.
Figure 2. HPLC-ESI-MS/MS analysis of a nixtamalized maize sample
(DON, 107 g/kg; norDON A, 14 g/kg; norDON B, 6 g/kg; norDON C,
12 g/kg).
µ
µ
µ
µ
cartridge. After a further concentration step, the sample was
analyzed by HPLC-MS/MS.
For the determination of the recovery rates, a blank corn
flakes sample was spiked with norDON A, B, and C at three
different levels (5, 25, and 50 µg/kg), prepared in duplicate,
and analyzed by HPLC-MS/MS. The recovery rates ((SD) were
determined to be 113.1 ( 5.6% for norDON A, 42.0 ( 3.7%
for norDON B, and 63.5 ( 4.5% for norDON C (n ) 6). The
low recovery rates for norDON B and C can be explained by
losses in the cleanup procedure compared to the internal
standard.
To confirm the semiquantitative results, 18 other samples
were analyzed in duplicate for degradation products, including
the new ones, with a different method based on the determi-
nation of DON by isotope dilution mass spectrometry (ID-MS)
(29). The samples were homogenized, spiked with 15-d1-DON
as internal standard, and extracted with ACN-water (4:1). The
extract was filtered and purified using a Bond Elut Mycotoxin
The results of the analyzed food samples, which were
calculated without correction by the respective recovery rates,
are shown in Table 2. They confirm the concentration range of

6450 J. Agric. Food Chem., Vol. 54, No. 17, 2006
Bretz et al.
Figure 3. Cytotoxicity of (A) DON and (B) the norDON compounds in IHKE cells. The cells were incubated with the shown concentrations for 24 h, and
then the viable cells were determined colorimetrically.
LITERATURE CITED
norDON A, B, and C determined with the first method. norDON
A is more abundant than norDON B and C and its concentration
is higher in most samples. The new degradation products
9-hydroxymethyl DON lactone and norDON D, E, and F were
not detected in any of the samples. As a representative example
Figure 2 shows the HPLC-MS/MS chromatogram of the
nixtamalized maize sample (DON, 107 µg/kg; norDON A, 14
µg/kg; norDON B, 6 µg/kg; norDON C, 12 µg/kg). Due to
higher ionization efficiency, the signal intensity of norDON B
and C is higher than that of DON and norDON A.
To determine the cytotoxicity of the deoxynivalenol degrada-
tion products that were detected in food, cell culture experiments
were performed using immortalized human kidney epithelial
cells (IHKE). As shown in Figure 3, DON revealed a median
effective concentration (EC50) at approximately 1.1 ( 0.09
µmol/L, whereas norDON A, B, and C did not exert any
significant effect up to 100 µmol. These findings are in
agreement with the fact that the epoxy group plays an important
role in the toxicity of trichothecenes (3, 30). Cell culture
experiments using the 5-bromo-2′-deoxyuridine incorporation
assay accessing DNA synthesis showed that the de-epoxides
of deoxynivalenol were 24 times less toxic compared to the
toxin with an intact epoxide ring (30). It was also shown in the
brine shrimp bioassay that the de-epoxides are much less acutely
toxic than the corresponding trichothecenes (31). Only recently,
Cetin and Bullerman (32) showed that the extrusion of cereal-
based products at temperatures from 150 to 200 °C reduced
not only DON but also the toxicity of an extract of the extrudate
in an MTT bioassay. Furthermore, our own studies showed that
the structurally analogous compounds derived from the alkaline
degradation of nivalenol are considerably less cytotoxic than
nivalenol (25).
(1) European Commission, Scientific Committee On Food, Opinion
of the Scientific Committee On Food on Fusarium toxins, Part
6: Group evaluation of T-2 toxin HT-2 toxin, nivalenol and
deoxynivalenol, 2002 (http://europa.eu.int/comm/food/fs/sc/scf/
out123_en.pdf, accessed date: 02/01/2006).
(2) Directorate-General Health and Consumer Protection, Reports
on tasks for scientific cooperation, report of experts participating
in Task 3.2.10: Collection of occurrence data of Fusarium toxins
in food and assessment of dietary intake by the population of
EU member states, 2003 (http://europa.eu.int/comm/food/fs/
scoop/task3210.pdf, accessed date: 02/01/2006).
(3) Rotter, B. A.; Prelusky, D. B.; Pestka, J. J. Toxicology of
deoxynivalenol (vomitoxin). J. Toxicol. EnViron. Health 1996,
48, 1-34.
(4) Forsell, J. H.; Jensen, R.; Tai, J.-H.; Witt, M.; Lin, W. S.: Pestka,
J. J. Comparison of acute toxicities of deoxynivalenol (vomi-
toxin) and 15-acetyldeoxynivalenol in the B6C3F1 mouse. Food
Chem. Toxicol. 1987, 25, 155-162.
(5) Yoshizawa, T.; Morooka, N. Studies on the toxic substances in
infected cereals: acute toxicities of new trichothecene mycotox-
ins deoxynivalenol and its monoacetate. J. Food Hyg. Soc. Jpn.
1974, 15, 261-269.
(6) Iverson, F.; Armstrong, C.; Nea, E.; Truelove, J.; Fernie, S.; Scott,
P. M.; Stapley, R.; Hayward, S.; Gunner, S. Chronic feeding
study of deoxynivalenol in B6C3F1 male and female mice.
Teratogenesis Carcinog. Mutagen. 1995, 15, 283-306.
(7) Pieters, M. N.; Fiolet, D. C. M.; Baars, A. J. Deoxynivalenol:
Derivation of concentration limits in wheat and wheat containing
food products. RIVM report 388802018, 1999 (http://www-
.mykotoxin.de/Dokumente/don_rivm.pdf, accessed date: 02/01/
2006).
(8) Commission Regulation (EC) No 856/2005. Off. J. Eur. Union
2005, L 143, 3-8.
(9) Verordnung zur A¨ nderung der Mykotoxin-Ho¨chstmengenver-
ordnung und der Dia¨tverordnung. Bundesgesetzblatt 2004, 151-
152.
In summary, heating experiments with DON were performed
to study its degradation under thermal food processing condi-
tions. The degradation products were isolated and structural
elucidation was achieved by NMR and MS experiments.
Subsequently, various cereal-based food samples were analyzed
for these compounds. norDON A, B, and C were detected in
29-66% of the samples in mean concentrations ranging from
3 to 15 µg/kg. Since these degradation products are less
cytotoxic compared to DON in the cell culture assay used, we
conclude that the formation of the degradation products during
heating processes might reduce the toxicity of DON-contami-
nated samples.
(10) Kamimura, H.; Nishijima, M.; Saito, K.; Yasuda, K.; Ibe, A.;
Nagayama, T.; Ushiyama, H.; Naoi, Y. Studies on mycotoxins
in foods. XII. The decomposition of trichothecene mycotoxins
during food processing. Shokuhin Eiseigaku Zasshi 1979, 20,
352-357.
(11) Greenhalgh, R.; Gilbert, J.; King, R. R.; Blackwell, B. A.; Startin,
J. R.; Shepherd, M. J. Synthesis, characterization, and occurrence
in bread and cereal products of an isomer of 4-deoxynivalenol
(vomitoxin). J. Agric. Food Chem. 1984, 32, 1416-1420.
(12) Grove, J. F. Phytotoxic compounds produced by Fusarium
equiseti. Part 7: Reactions and rearrangement of the 7-hydroxy-
12,13-epoxytrichothec-9-en-8-one skeleton. J. Chem. Soc., Perkin
Trans. 1 1985, 1731-1736.
ACKNOWLEDGMENT
(13) Young, J. C.; Blackwell, B. A.; ApSimon, J. W. Alkaline
degradation of the mycotoxin 4-deoxynivalenol. Tetrahedron
Lett. 1986, 27, 1019-1022.
(14) Wolf, C.; Bullerman, L. Heat and pH alter the concentration of
deoxynivalenol in an aqueous environment. J. Food Prot. 1997,
61, 365-367.
We thank H. Luftmann for the exact mass measurements, K.
Bergander for setting up the 13C NMR experiments, A.
Klusmeyer-Ko¨nig for technical assistance, and M. Gekle, G.
Schwerdt, and R. Freudinger for support with the cell culture
experiments.

Thermal Degradation Products of DON
J. Agric. Food Chem., Vol. 54, No. 17, 2006 6451
(15) Lauren, D. R.; Smith, W. A. Stability of the Fusarium myc-
otoxins nivalenol, deoxynivalenol and zearalenone in ground
maize under typical cooking environments. Food Addit. Contam.
2001, 18, 1011-1016.
(16) Abramson, D.; House, J. D.; Nyachoti, C. M. Reduction of
deoxynivalenol in barley by treatment with aqueous sodium
carbonate and heat. Mycopathologia 2005, 160, 297-301.
(17) Scott, P. M.; Kanhere, S. R.; Lau, P. Y.; Dexter, J. E.;
Greenhalgh, R. Effects of experimental flour milling and
breadbaking on retention of deoxynivalenol (vomitoxin) in hard
red spring wheat. Cereal Chem. 1983, 60, 421-424.
(18) Wolf-Hall, C.; Hanna, M.; Bullerman, L. Stability of deoxyni-
valenol in heat-treated foods. J. Food Prot. 1999, 62, 962-964.
(19) Young, J. C.; Fulcher, R. G.; Hayhoe, J. H.; Scott, P. M.; Dexter,
J. E. Effect of milling and baking on deoxynivalenol (vomitoxin)
content of eastern Canadian wheats. J. Agric. Food Chem. 1984,
32, 659-664.
(20) Seitz, L. M.; Eustace, W. D.; Mohr, H. E.; Shogren, M. D.;
Yamazaki, W. T. Cleaning, milling, and baking tests with hard
red winter wheat containing deoxynivalenol. Cereal Chem. 1986,
63, 146-150.
(21) Boyacioglu, D.; Hettiarachchy, N. S.; D’Appolonia, B. L.
Additives affect deoxynivalenol (vomitoxin) flour during bread-
baking. J. Food Sci. 1993, 58, 416-418.
(22) Wolff, J. Untersuchungen der Gehaltsvera¨nderungen der Fusari-
umtoxine Deoxynivalenol und Zearalenon durch Be- und
Verarbeitungsprozesse in Getreide und Getreideprodukten. 26.
Mykotoxin-Workshop, Herrsching, Germany, 2004.
(23) Abbas, H. K.; Mirocha, C. J.; Rosiles, R.; Carvajal, M.
Decomposition of zearalenone and deoxynivalenol in the process
of making tortillas from corn. Cereal Chem. 1988, 65, 15-19.
(24) Seefelder, W.; Knecht, A.; Humpf, H.-U. Bound fumonisin B1:
Analysis of fumonisin-B1 glyco and amino acid conjugates by
liquid chromatography-electrospray ionization-tandem mass spec-
trometry. J. Agric. Food Chem. 2003, 51, 5567-5573.
(25) Bretz, M.; Knecht, A.; Goeckler, S.; Humpf, H.-U. Structural
elucidation and analysis of thermal degradation products of the
Fusarium mycotoxin nivalenol. Mol. Nutr. Food Res. 2005, 49,
309-316.
(26) Altpeter, F.; Posselt, U. K. Production of high quantities of
3-acetyldeoxynivalenol and deoxynivalenol. Appl. Microbiol.
Biotechnol. 1994, 41, 384-387.
(27) Klo¨tzel, M.; Lauber, U.; Humpf, H.-U. A new solid-phase
extraction clean-up method for the determination of 12 type A
and B trichothecenes in cereals and cereal based food by LC-
MS/MS. Mol. Nutr. Food Res. 2006, 50, 261-269.
(28) Tveito, G.; Hansteen, I.; Dalen, H.; Haugen, A. Immortalization
of normal human kidney epithelial cells by nickel II. Cancer
Res. 1989, 49, 1829-1835.
(29) Bretz, M.; Beyer, M.; Cramer, B.; Humpf, H.-U. Stable isotope
dilution analysis of the Fusarium mycotoxins deoxynivalenol
and 3-acetyldeoxynivalenol. Mol. Nutr. Food Res. 2006, 50,
251-260.
(30) Sundstol Eriksen, G.; Pettersson, H.; Lundh, T. Comparative
cytotoxicity of deoxynivalenol, nivalenol, their acetylated deriva-
tives and de-epoxy metabolites. Food Chem. Toxicol. 2004, 42,
619-624.
(31) Swanson, S. P.; Rood, H. D.; Behrens, J. C., Jr.; Sanders, P. E.
Preparation and characterization of the deepoxy trichothecenes:
deepoxy HT-2, deepoxy T-2 triol, deepoxy T-2 tetraol, deepoxy
15-monoacetoxyscirpenol, and deepoxy scirpentriol. Appl. En-
Viron. Microbiol. 1987, 53, 2821-2826.
(32) Cetin, Y.; Bullerman, L. B. Confirmation of reduced toxicity of
deoxynivalenol in extrusion-processed corn grits by the MTT
bioassay. J. Agric. Food Chem. 2006, 54, 1949-1955.
Received for review April 11, 2006. Revised manuscript received June
20, 2006. Accepted June 20, 2006.
JF061008G