Preparation and Characterization of Cysteine Adducts of

May 27, 2016 - Norwegian Veterinary Institute, P.O. Box 750, Sentrum, NO-0106 Oslo, Norway. ‡ Department of Chemistry, University of Oslo, P.O. Box ...
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Preparation and Characterization of Cysteine Adducts of Deoxynivalenol Ana Stanic,*,†,‡ Silvio Uhlig,† Anita Solhaug,† Frode Rise,‡ Alistair L. Wilkins,†,§ and Christopher O. Miles† †

Norwegian Veterinary Institute, P.O. Box 750, Sentrum, NO-0106 Oslo, Norway Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, NO-0315 Oslo, Norway § Chemistry Department, University of Waikato, Private Bag 3105, 3240 Hamilton, New Zealand ‡

S Supporting Information *

ABSTRACT: Conjugation with the biologically relevant thiol glutathione is one of the metabolic pathways for the mycotoxin deoxynivalenol (DON) in wheat. The occurrence of putative DON−cysteine conjugates has also been shown in wheat, likely in part as a result of degradation of the glutathione conjugates. It was reported that thiols react in vitro with DON at two positions: reversibly at C-10 of the α,β-unsaturated ketone and irreversibly at C-13 of the epoxy group. We synthesized pure DON− cysteine adducts and made analytical standards using quantitative NMR experiments. Compounds were characterized using NMR and LC−HRMS/MS and tested in vitro for toxicity. Cysteine conjugates were much less toxic than DON at the same concentration, and LC−HRMS analysis demonstrated that there was no detectable metabolism of the conjugates in human monocytes or human macrophages. KEYWORDS: DON, thiol, Michael adduct, HRMS, NMR, qNMR, ERETIC, LC−MS

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INTRODUCTION Deoxynivalenol (DON), 1 (Figure 1), is one of the most widely distributed mycotoxins, mainly produced by Fusarium species and contaminating grains and cereal-based food and feed.1 DON belongs to the type-B group of trichothecenes, characterized by a ketone group at the C-8 position. Trichothecenes inhibit protein synthesis,2 and ingestion of DON by animals can lead to acute gastrointestinal symptoms such as emesis and feed refusal.3 The immune system of several species, including humans, is sensitive to trichothecenes, and low concentrations of DON induce the expression of early response and proinflammatory genes at the mRNA and protein levels, while high concentrations promote leukocyte apoptosis.4 Several detoxification pathways for DON are known in plants, including glycosylation,5 sulfation,6 and glutathione conjugation.7 Kluger et al.8 reported formation of DON−glutathione (DON−GSH), as well as the presence of the degradation products DON−cysteinylglycine and DON−cysteine (DON− Cys) in DON-treated wheat lines.8 The glutathione pathway is one of the most common types of xenobiotic modifications during phase II metabolism, where the conjugation reaction occurs between the thiol group of the amino acid cysteine in GSH and an electrophilic feature in xenobiotic.9 Studies with model thiols showed that thiol addition to DON can follow two reaction mechanisms: reversible Michael addition to C-10 of the α,β-unsaturated ketone, and irreversible addition to C-13 of the epoxide ring.10 These adducts were shown to exist as equilibrium mixtures of the hemiketal and ketone forms of the DON−thiol model compounds.11,10 The reported DON− GSH, DON−Cys, and DON−CysGly conjugates8 have so far only been tentatively identified by LC−HRMS/MS, and their exact chemical structures are yet to be determined. © 2016 American Chemical Society

DON−Cys is expected to be one of the major breakdown products of DON−GSH in vivo.7 Here we report synthesis of the major products from the reaction of DON with L-cysteine, identification of their structures, and determination of their toxicities in vitro.

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MATERIALS AND METHODS

General. Deoxynivalenol (DON) (≥98%), L-cysteine (≥98%), 5.0 mm Norell Standard Series NMR tubes, D2O (99.9 atom % D), lipopolysaccharide (LPS) from Escherichia coli, and sodium carbonate (pro analysis) were from Sigma-Aldrich (Steinheim, Germany), HPLC grade water and acetonitrile were from Thermo Fisher Scientific (Waltham, MA), and ammonium formate (puriss p.a. for HPLC) and dioxane were from Fluka (Steinheim, Germany). Sodium bicarbonate (pro analysis) (Merck, Darmstadt, Germany) and sodium carbonate were used to prepare 0.2 M carbonate buffer (pH 10.7). The buffer pH was measured at ambient temperature with a Mettler Delta 320 pH meter. Acetic acid-d4 (99.5 atom % D) was from Cambridge Isotope Laboratories (Tewksbury, MA). C18-E, C18-U, and Strata-X SPE columns (500 mg) were from Phenomenex (Torrance, CA). C18-U and Strata-X solid-phase extraction (SPE) columns were conditioned with 5 mL of MeOH and 5 mL of water. The deoxynivalenol standard used for quantitation in cells was from Romer Laboratories (Tulln, Austria). Alamar Blue and human TNF-α Cytoset were from Invitrogen (Life Technologies, Carlsbad, CA), and RPMI 1640 culture medium, penicillin/streptomycin, fetal bovine serum (FBS; EU standard), and PBS were from Lonza (Verviers, Belgium). Phorbol 12-myristate 13-acetate (PMA) was from Calbiochem (La Jolla, CA). Celltox Green was from Promega (Fitchburg, WI). Methanol was from Received: Revised: Accepted: Published: 4777

March 13, 2016 May 26, 2016 May 27, 2016 May 27, 2016 DOI: 10.1021/acs.jafc.6b01158 J. Agric. Food Chem. 2016, 64, 4777−4785

Article

Journal of Agricultural and Food Chemistry

operated with a spray voltage of 3.9 kV in negative and 3 kV in positive mode, with a temperature of 300 °C. The temperature of the transfer capillary was set to 280 °C, and the sheath gas and auxiliary gas flow rates were set to 35 and 10 units (approximately 35 and 10 L/min, respectively). For targeted HRMS/MS, the precursor m/z was set to 418.2 for [M + H]+ in positive mode and 416.1 for [M − H]− in negative mode with an isolation window (i.e., scan range of the quadrupole) of 4.0 m/z. The normalized collision energy for higherenergy collisional dissociation (HCD) was set to 35%, and the resolution for the product ions was set to 17,500. This method was used for obtaining high-resolution mass spectrometric data for DON− Cys adducts and their fragments. Method 2B. A Dionex UltiMate 3000 UPLC (Thermo Fisher Scientific) was used. Method 2B was otherwise identical to method 2A, except that the Q-Exactive was run in both positive and negative modes in the mass range m/z 150−900, with fast polarity switching, and the spray voltage was set to 4 kV and the transfer capillary temperature at 250 °C. This method was used for the UPLC−HRMS analysis of the cell cultures treated with 1, 2, and 3. Adduct Stability. L-Cysteine (360 μg, 3 μmol) in the carbonate buffer (1 mL) was added to 1 mg of DON (3 μmol) and left at ambient temperature. After 20 d, the reaction mixture was applied to a C18-E SPE column that had been conditioned with 5 mL of MeCN and 5 mL of water. The column was washed with 2 × 1 mL of water and eluted with 2 mL each of 1%, 2%, 5%, 7%, 10%, and 100% MeCN in water (v/v). The 1% MeCN contained 2, 3, and 4. Aliquots of this fraction were regularly analyzed using LC−MS method 1B. After 4 d, acetic acid (5 μL) was added to an aliquot of the 1% MeCN fraction (150 μL), and the acidified aliquot and the original fraction were followed for another week (Figure 2).

Figure 1. Structures of deoxynivalenol (DON), 1, and purified DON− cysteine conjugates 2 and 3. 1−3 were present as equilibrating ketone and hemiketal isomers (designated a and b, respectively). Compounds 4−6 were tentatively identified as additional stereoisomers (at C-9 and C-10) of the Michael adduct, and 7−10 as DON−(Cys)2 double adducts. Romil (Cambridge, United Kingdom). Dimethyl sulfoxide (DMSO) (pro analysis) was from Merck (Darmstadt, Germany). LC−MS. Chromatographic separations were carried out on a 150 × 2.1 mm, i.d. 3 μm, Atlantis T3 column (Waters, Milford, MA). The flow rate was 300 μL/min, and the injection volume was 1 μL. The mobile phase consisted of 5 mM aqueous ammonium formate (A) and 5 mM ammonium formate in acetonitrile/water (95:5, v/v) (B). Gradient 1 was a linear gradient from 0.5−15% B over 15 min, then to 100% B over 5 min, with a 3 min hold, followed by a return to 0.5% B and 3 min hold for equilibration. Gradient 2 was identical to gradient 1, except that the gradient started and ended with 2% A. Method 1A. A Finnigan Surveyor HPLC system was coupled with an LTQ linear ion trap mass spectrometer (both Thermo Fischer Scientific) operated in full scan mode (m/z 100−1000) and equipped with an electrospray ionization (ESI) interface, operated in positive or negative mode. The source voltage was set to 5 kV, the capillary voltage was 35 V, the capillary temperature was 275 °C, the tube lens offset was 80 V, the sheath gas flow rate was 58 units (approximately 58 L/min), and the auxiliary gas flow rate was 11 units (approximately 11 L/min). Elution was with gradient 1. This method was used to monitor the reaction of DON and cysteine and for recording the MSn fragmentation spectra of 2 and 3 (Figure 1), in both positive and negative modes. Multistage fragmentation (MS2−4) of 2 and 3 was studied under the following parameters: collision energy of 35%, activation Q 0.25, isolation width of 2 m/z, and product ion mass range m/z 110−550 for ionization in positive mode and m/z 105−500 for ionization in negative mode. Method 1B. was identical to method 1A, except that elution was with gradient 2, and was used to analyze SPE fractions during Michael adduct stability tests. Method 2A. An Acquity UPLC (Waters) was coupled to a QExactive Fourier-transform high-resolution mass spectrometer (Thermo Fischer Scientific). Elution was with gradient 1, and the mass spectrometer was run in either positive or negative full-scan mode in the mass range m/z 100−1000 with a mass resolution set to 70,000. The mass spectrometer was equipped with a heated ESI interface, and

Figure 2. Ratio of concentrations of 3:2 (circles) and 4:2 (triangles) with (hollow symbols) and without (filled symbols) addition of acetic acid, as determined by LC−MS (method 1B). Curves show the data fitted to 3-parameter exponential decay curves (no acid; solid lines) while straight lines were fitted to data after addition of acetic acid (dashed lines). The formulas for the fitted curves and lines are shown in the figure. Calculated t1/2 was ca. 3 d for 3 and ca. 9 d for 4.

DON−Cys Epoxide Adduct, 2, and DON−Cys Michael Adduct, 3. Two parallel reactions were set up, where DON (1 mg, 3 μmol) was dissolved in freshly prepared solutions of L-cysteine (8.5 mg, 70 μmol) in carbonate buffer (1 mL, pH 10.7), and left at room temperature. One reaction was set up with the aim of producing epoxide adduct 2 and was followed for 5 d using LC−MS (method 1A), after which an aliquot of the reaction mixture (600 μL) was diluted with water to 1 mL and applied to a Strata-X SPE column. The column was eluted with water (4 × 1 mL), 5% MeOH (4 × 1 mL), and 7% MeOH (1 mL). The fractions were analyzed using LC−MS method 1A. The fourth water fraction, which contained most of the 4778

DOI: 10.1021/acs.jafc.6b01158 J. Agric. Food Chem. 2016, 64, 4777−4785

Article

Journal of Agricultural and Food Chemistry epoxide adduct, was evaporated to dryness at 30 °C under a gentle stream of nitrogen. The second reaction was set up with the aim of preparing Michael adduct 3, and was followed for 4 d using LC−MS (method 1A), after which the mixture was applied to a C18-U SPE column. The column was eluted with water (2 × 1 mL) followed by 2 mL each of 2%, 5%, 7%, 10%, and 15% MeOH in water. The fractions were analyzed using LC−MS (method 1A), and those containing predominantly 3 (2% and 5% MeOH) were combined and evaporated at 30 °C under a stream of nitrogen. Oxidation of DON Cysteine Conjugates. Hydrogen peroxide (30%, 50 μL) was added to diluted aliquots (450 μL) of purified 2 and 3. The reaction was followed for approximately 5 h using LC−MS (method 2A). NMR Spectroscopy. One-dimensional (1H, SELTOCSY, 13C, JMOD, and DEPT135) and two-dimensional (COSY, TOCSY, HSQC, HMBC, NOESY, and ROESY) NMR experiments were conducted at 304.6 K using an Avance AVII 600 MHz NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a 5 mm CP-TCI (1H/13C, 15N−2H) triple-resonance inverse cryoprobe with Z-gradient coils. DON (1) and purified 2 and 3 were dissolved in ca. 550 μL of D2O to which 5 μL of acetic acid-d4 had been added. Data were recorded and processed using Bruker TopSpin (version 3.0), and chemical shifts are reported relative to internal CD2HCOOD, 2.08 ppm, and CD3COOD, 21.03 ppm.12 Quantitative NMR (qNMR), ERETIC2. Solutions of dioxane in D2O (25.3 ± 0.4, 158 ± 3, 527 ± 7 μM) were used as external standards to generate reference electronic signals. Uncertainties for the calibrant concentrations were calculated in accordance with NMKL procedure No. 5.13 Samples for structural analysis by NMR were freeze-dried and redissolved in 550 μL of D2O containing 25 μM DMSO, as an internal standard for the validation of the ERETIC2. NMR was based on the ERETIC14 and PULCON15 (pulse length based concentration determination) methodologies, where a reference signal from an external standard of known concentration is electronically inserted into the NMR spectrum of the test sample, and its integrated peak area is used to calculate the concentration of the test compound. Experiments were conducted on an Avance AVIII 400 MHz NMR spectrometer, equipped with a 5 mm BBO probe with temperature set to 294.7 K. Data were recorded and processed using Bruker TopSpin 3.2. For each compound, a set of 10 consecutive 1H NMR experiments was performed, using a 90° flip angle, 64 scans, a relaxation delay (d1) of 10 s, an acquisition time of 3.412 s, a pulse width (p1) of 9.04 μs, and a receiver gain of 203.0, with low-power continuous wave presaturation on the water peak. The areas of the methyl resonances (H-14 and H-16) and the H-15b doublet for compounds 2a and 2b, and 3a and 3b, were manually integrated, and concentrations were calculated based on the three dioxane standards. The calculated concentrations were obtained from the average of 30 independent measurements of H-14 (for 2a, 2b, and 3a) or H-16 (for 3b). Cell Culture and Treatments. The human acute monocyte leukemia cell line (THP-1) from the European Collection of Cell Cultures (ECACC) was grown in RPMI 1640 supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 and kept in logarithmic growth phase at 5−15 × 105 cells/mL through routine subculturing, according to standard ECACC protocol. The passage number was kept below 20. DON and its derivatives in D2O−DMSO (from qNMR) were applied to the cells such that the final concentration of deuterated solvent in the medium was below 1.5%. Appropriate controls containing the same amount of toxin-free deuterated solvent were included in each experiment. Proliferation. THP-1 cells (monocytes) were seeded at 150,000 cells/cm2. Following exposure to DON and the conjugates, the metabolic activity of the cells was measured using the Alamar Blue assay according to the manufacturer’s protocol. The dark blue oxidized form of Alamar Blue is reduced to a highly fluorescent form in functional mitochondria, and the measured fluorescence intensity is thus proportional to the number of viable cells.16 The fluorescence (λex

555 nm, λem 585 nm) was quantitated using a Spectramax i3x (Molecular Devices, Sunnyvale, CA) plate reader. Cytokine Measurement. THP-1 cells were seeded at 125,000/ cm2 and differentiated into macrophages by treatment with PMA (50 ng/mL) for 24 h. The medium was then replaced, and the cells were rested in complete growth medium for 24 h. The cells were subsequently treated with LPS (0.05 ng/mL) for 3 h, followed by exposure to the test compounds for an additional 24 h. The medium was harvested and centrifuged (500g, 4 °C, 10 min) to remove cell debris. Levels of secreted TNF-α in supernatants were measured by ELISA, using human TNF-α Cytoset, according to the manufacturers’ guidelines. The absorbance was measured using a plate reader (Spectramax i3x) and the data analyzed using the SoftMax Pro 6.5 software. Cell Toxicity. The CellTox Green Dye is a nontoxic and proprietary asymmetric cyanine dye that stains the DNA of cells with impaired membrane integrity (necrotic cells) where the binding interaction with DNA produces a fluorescence signal that is proportional to cytotoxicity.17 CellTox Green Dye mixed with PBS (1:10) was added (1:100) to the cells in the 96-well plate, and the fluorescence (λex 485 nm, λem 520 nm) was quantitated with a Spectramax i3x platereader after 15 min incubation at room temperature in the dark. DON (20 μM) served as a positive control for cell death. Cellular Effects and LC−HRMS Analysis of Cell Culture Medium and Cell. Upon completion of the cell culture experiments, the medium (with and without LPS) was removed from the macrophages, and cells and medium were frozen separately until further processing, whereas monocytes were frozen together with the cell culture medium containing Alamar Blue. All samples were allowed to thaw at room temperature prior to LC−MS analyses. Macrophage medium and monocytes with the medium were then transferred to 1.5 mL Eppendorf tubes, and MeOH containing 0.1% acetic acid was added (300 μL). Samples were then vortex-mixed and centrifuged (10 min, 15000g) on an Eppendorf 5424 microcentrifuge (Hamburg, Germany). Macrophages were extracted directly from the cell culture plate with MeOH/H2O (3:1, v/v) (300 μL), containing 0.1% acetic acid, shaken on an IKA MTS4 plate shaker for 10 min (ca. 10 Hz), and then centrifuged as above. Supernatants were analyzed by LC−MS (method 2B).

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RESULTS AND DISCUSSION Reaction of DON with Cysteine. The conditions for the reaction of DON with thiols had previously been optimized with 2-mercaptoethanol and other model thiols.10 When we applied these protocols to the reaction of DON with L-cysteine, LC−MS showed that two major products, 5 and 6, were rapidly formed (Figure 3) due to addition of a single molecule of cysteine to DON (m/z 418 and 416 in positive and negative ionization modes, respectively) (Figure 4). Initially, 5 and 6 dominated the chromatograms, but, over time, two other products (2 and 3) affording ions of identical mass came to dominate the chromatograms (Figure 3). Compounds 4−6 were likely transient Michael adducts, based on the mass spectrometric data, which isomerized to 3 as the thermodynamically favored Michael adduct. The chemical structures of 3 and epoxide adduct 2 were thus subsequently identified (Figure 1) by NMR spectroscopy. LC−MS also showed the presence of four early eluting products 7−10, with m/z 537 and 539 in negative and positive modes, respectively, detectable within 30 min (Figure 4), corresponding to addition of two cysteine molecules to DON. Addition of two thiol molecules to DON has previously been reported using model thiols, and their structures were characterized as Michael and epoxide double adducts.10 All DON−Cys conjugates, including the double adducts, afforded singly charged ions upon electrospray ionization. 4779

DOI: 10.1021/acs.jafc.6b01158 J. Agric. Food Chem. 2016, 64, 4777−4785

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Journal of Agricultural and Food Chemistry

fraction containing 3 also contained residual DON (ca. 10% measured by NMR and LC−HRMS, on a molar basis). LC− HRMS (method 2A) revealed only single peaks for purified 2 and 3, consistent with C18H28O8NS+ ([M + H]+, m/z 418.1532 for 2 and m/z 418.1529 for 3) in positive mode and C18H26O8NS− ([M − H]−, m/z 416.1388 for 2 and m/z 416.1389 for 3) in negative mode (Figure 4). However, subsequent NMR experiments showed that 1, 2, and 3 were equilibrating mixtures of the ketone and hemiketal forms (Figure 1). Structural Elucidation. The chemical structures of DON− Cys epoxide conjugate (2a/2b) and the DON−Cys Michael conjugate (3a/3b) (Figure 1) were unambiguously identified using NMR spectroscopy. All samples were dissolved in D2O/ CD3COOD because acid was necessary to stabilize the Michael adduct, 3. In this solvent, 1−3 were present as equilibrating pairs of ketone−hemiketal isomers, although the ketone:hemiketal ratios, as measured from the areas under the methyl peaks (H-16 and H-14), varied (1a:1b, 89:11; 2a:2b, 8:92; and 3a:3b, 29:71). This resulted in exchange peaks in the TOCSY and ROESY spectra attributable to fast isomerization of the hemiketal and ketone during the mixing time of the experiments due to the presence of the acid in the solvent. Exchange peaks in the ROESY NMR spectrum were easily distinguished from through-space correlations by their opposite phase. These exchange peaks did not hinder the assignment of NMR resonances and are not discussed further. COSY and TOCSY NMR spectra of DON (1a/1b) were used to identify neighboring protons and spin systems. HSQC NMR spectra identified the corresponding 13C resonances, HMBC NMR spectra were used to connect the spin systems and identify quaternary carbons, and the stereochemistry was confirmed with ROESY NMR spectra. The H-16 methyl protons of major isomer 1a (1.85 ppm) (Table 1) showed HMBC correlations to signals at 138.6 ppm (C-10), 138.7 ppm (C-9), and 203.5 ppm (C-8), while the H-16 methyl protons of

Figure 3. Extracted ion chromatograms (m/z 418, [M + H]+ for DON−Cys) from LC−MS (method 1A) in positive ionization mode, for the reaction of DON and cysteine used to prepare epoxide adduct 2. Compounds 2 and 3 were subsequently purified and characterized as DON−Cys epoxide and Michael adduct, respectively, whereas 4−6 were tentatively identified as diastereoisomeric DON−Cys Michael adducts.

Purification of 2 and 3. Preparative reactions of DON with cysteine were followed by LC−MS (method 1A), and reaction mixtures were extracted when the desired product reached optimum intensity in the chromatogram. Due to their high polarity, compounds 2 and 7−10 (Figure 4) eluted shortly after excess cysteine when the SPE columns were washed with water. However, the separation was sufficient to obtain pure 2 for NMR analyses. Separation of Michael adduct 3 using SPE was also successful, but LC−MS (method 1A) revealed that the

Figure 4. Extracted ion chromatograms (LC−HRMS, 5 ppm mass tolerance) corresponding to the protonated ([M + H]+) and deprotonated ([M − H]−) ions of DON−Cys (A and C, respectively) and DON−(Cys)2 (B and D, respectively). The extracted values of m/z are A, 418.1530; B, 416.1385; C, 539.1728; and D, 537.1582. 4780

DOI: 10.1021/acs.jafc.6b01158 J. Agric. Food Chem. 2016, 64, 4777−4785

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Journal of Agricultural and Food Chemistry

Table 1. 1H and 13C NMR Assignments (δ in ppm) for DON (1a, 1b), DON−Cys Epoxide Adduct (2b), and DON−Cys Michael Adduct (3a and 3b) in D2O with CD3COOD (1% v/v)a 1a 1

1b 13

2 3

3.73 (d, 4.5) 4.50 (dt, 11.4, 4.5)

81.6 69.3

3.53 (d, 4.6) 4.45 (dt, 11.2, 4.6)

81.5 69.05

4

2.30 (dd, 15.1, 4.5) 2.10 (dd, 15.1, 11.4)

43.6

2.34 (dd, 15.0, 4.6) 2.09 (dd, 15.0,11.2)b

43.8

5 6 7 8 9 10 11 12 13 14 15 16 1′

2′ 3′

4.87 (bs)

6.70 (dq, 5.9, 1.4) 4.90 (d, 5.9) 3.28 3.29 1.09 3.79 3.86 1.85

(d, 3.7) (d, 3.7) (s) (d, 12.6) (d,12.6) (s)

1

2b

H

atom

C

47.0 54.0 75.3 203.5 138.7 138.6 72.4 67.9 49.1 15.0 61.5 15.8

H

4.01 (bs)

5.58 (br s) 4.76 (m) 3.02 2.93 1.13 4.22 3.48 1.81

(d, 3.8) (d, 3.8) (d, 9.1) (d, 9.1) (s)

13

44.6 54.6 75.7 105.7 143.4 122.0 77.1 68.6 50.85 15.33 67.42 16.3

3a

1

C

13

H

C

1

H

3b 13

1

C

H

13

C

4.18 (d, 4.6) 4.72 (dq, 10.5, 4.6) 1.65 (dd, 14.5, 4.6)c 2.21 (dd, 14.5, 10.5)

79.0 68.7

3.77 (d, 4.5) 4.52 (m)

81.5 69.49d

3.74 (d, 4.5) 4.52 (dt, 10.9, 4.5)

82.4 69.53d

43.3

2.25 (dd, 14.8, 4.3)

44.0

1.89 (dd, 4.5, 14.5)

44.9

4.23 (bs)

50.2 50.6 75.5 104.2 141.4

2.09 (dd, 14.8, 11.5)b

5.65 (dq, 5.4, 1.5) 4.96 (d, 5.4)b 3.32 3.61 1.40 4.37 3.57 1.90 3.30

(d, 13.5) (d, 13.5) (s) (d, 9.3) (d, 9.3) (br s) (2H, m)

4.09 (br t, 5.0)

121.3 76.1 81.8 34.7 14.8 68.9 15.1 33.6

53.5 172.5

4.80 (d, 1.1) 2.83 (dq,12.8, 6.5) 3.48 (dd, 12.8, 3.4) 4.77 (d, 5.3) 3.32 (d, 3.8) 3.23 (d, 3.8) 1.03 (s) 3.70 (d, 12.4) 3.86 (d, 12.4) 1.25 (d, 6.5) 3.43 (dd, 14.8, 8.0)c 3.20 (dd, 14.8, 4.1) 4.15 (dd, 8.0, 4.1)

2.07 (dd, 10.9, 14.5)c 46.9 56.4 75.9 213.6 44.1 52.5 76.2 67.9 49.5 14.3 60.2 13.0 33.0

54.4 173.6

3.99 (s) 1.86 (dq, 11.9, 6.5)e 3.05 (dd, 11.9, 5.8) 4.59 (d, 5.8) 3.35 (d, 3.8) 3.27 (d, 3.8) 1.08 (s) 4.23 (d, 8.5) 3.70 (d, 8.5) 1.16 (d, 6.5) 3.46 (dd, 14.8, 6.3)c 3.15 (dd, 14.8, 4.5) 4.00 (dd, 6.3, 4.5)

44.4 56.6 77.0 107.3 43.0 50.2 75.5 68.4 49.2 15.2 68.5 13.1 32.0

54.9 173.6

a In the format: chemical shift in ppm (multiplicity, coupling constants in Hz). With the exception of H-15 of 1a and 3a, chemical shifts for methylene protons are in the order Hpro‑R, Hpro‑S. Full assignment of chemical shifts for 2a was not possible, however, the following partial assignments were established from various experiments: 2.08 (H-4), 4.93 (H-7), 6.68 (H-10), 5.07 (H-11), 3.42, 3.50 (H-13), 1.36 (H-14), 3.83, 4.11 (H-15), 1.95 (H-16), 73.3 (C-7), 201.4 (C-8), 135.6 (C-9), 137.4 (C-10), 71.8 (C-11), 63.2 (C-15). bCoupling constant measured from TOCSY spectrum. cCoupling constant measured from HSQC spectrum. dCarbon chemical shifts assigned to two decimal places due to very close, but distinguishable, resonances. eCoupling constants measured from SELTOCSY spectrum.

(C-13), and 53.5 ppm (C-2′) and 172.5 ppm (C-3′), established the attachment of the cysteinyl thiol to the epoxide group in 2 as shown in Figure 1. Full assignment of the resonances of minor isomer 2a was not possible due to signal overlap and the small amount of 2a present. However, 2a was obviously a DON−Cys epoxide adduct containing a C-8 ketone because its H-16 methyl signal (1.95 ppm) showed an HMBC correlation to 135.6 ppm (C-9), 137.4 ppm (C-10), and 201.4 ppm (C-8), as well as having an olefinic proton (H-10, 6.68 ppm) at a higher chemical shift than for corresponding C-8 hemiketal (5.65 ppm). The same type of NMR experiments were conducted for 3a/ 3b, as for 2, revealing a similar set of spin systems (H-16/H-9/ H-10/H-11, H-2/H-3/H-4α/H-4β/H-14, H-7, H-13a/H-13b, H-15α/H-15β, and H-1′/H-2′) and correlations for most of their structures. The HMBC NMR spectrum confirmed the minor isomer, 3a, as a ketone, and the major isomer, 3b, as a hemiketal, based on correlations from H-16 (1.25/1.16 ppm) to C-9 (44.1/43.0 ppm), C-10 (52.5/50.2 ppm), and C-8 (213.6/107.3 ppm) for 3a/3b. HMBC correlations were observed from the cysteine side-chain methylene protons, H1′a and H-1′b (3.20 and 3.43/3.15 and 3.46 ppm for 3a/3b, respectively), to C-10 (52.5/50.2 ppm), C-2′ (54.4/54.9), and C-3′ (173.6/173.6 ppm), as were correlations from H-10 (3.48/3.05 ppm) to C-1′ (33.0/32.0 ppm) and C-9 (44.1/43.0 ppm). Thus, the HMBC NMR spectra confirmed attachment

minor isomer 1b (1.81 ppm) correlated to signals at 143.4 ppm (C-9), 122.0 ppm (C-10), and 105.7 ppm (C-8). The chemical shifts of the C-8, C-10, and the H-10 (1a, 6.70 ppm, and 1b, 5.58 ppm) resonances were the most diagnostic for differentiating the ketone and hemiketal. Additionally, the observed coupling constant between H-15α and H-15β was 12.6 Hz in hemiketal 1b and 9.1 Hz in ketone 1a, consistent with previously reported data.10 The proton and carbon chemical shifts, as well as the coupling constants, of the ketone form of DON (1a) in D2O/CD3COOD were similar to those previously reported in CD3CN.10 The hemiketal−ketone equilibrium of 1 has already been reported by Jarvis et al.,18 who analyzed DON in different solvents using NMR. 1 H−1H connectivities for 2a/2b were established via COSY, TOCSY, and SELTOCSY experiments, revealing six rather short spin systems: H-16/H-10/H-11; H-2/H-3/H-4α/H-4β/ H-14; H-7; H-13a/H-13b; H-15α/H-15β; and H-1′/H-2′. Correlations observed in the HSQC and HMBC NMR spectra led to a full assignment of the resonances of the major isomer (2b). Correlations observed in the HMBC spectrum of 2b from H-16 (1.90 ppm) to 141.4 ppm (C-9), 121.3 ppm (C-10), and 104.2 ppm (C-8) identified it as the hemiketal isomer. 2b also showed HMBC correlations from the epoxide protons at 3.32 and 3.61 ppm (H-13a/H-13b) to 33.6 ppm (C-1′), 50.2 ppm (C-5), 79.0 ppm (C-2), and 81.8 ppm (C-12). This, together with HMBC correlations from 3.30 ppm (H-1′) to 34.7 ppm 4781

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Journal of Agricultural and Food Chemistry of cysteine at C-10 via a sulfide linkage, consistent with the observed absence of olefinic resonances in 3. In ROESY NMR experiments, 3a/3b showed NOE correlations between H-9 (2.83/1.86 ppm) and H-7 (4.80/ 3.99 ppm), and between H-10 (3.48/3.05 ppm) and both H-11 (4.77/4.59 ppm) and H-16 (1.25/1.16 ppm), with an additional correlation from H-10 of 3b (3.05 ppm) to H-15α (3.70 ppm). Thus, H-16 and H-10 were on the α-face, and H-9 on the β-face of the molecule (Figures 1 and 5).

these compounds were obtained in microgram amounts, weighing was not feasible. Quantitative NMR was selected for quantitation because of the direct proportionality of 1H signal intensity to the number of nuclei,19 and the availability of the ERETIC2 method to measure analytes based on the signal of an external reference compound without the necessity of additional hardware equipment. NMR samples from structure elucidation were freeze-dried and redissolved in D2O containing DMSO (as an internal standard) for quantitative NMR. This resulted in minor changes to the chemical shifts of some signals, and in the hemiketal:ketone ratios. As their H-14 peaks coincided, 2a and 2b were quantitated together, while the concentration of the 3 was measured as the sum of the major and minor methyl signals (for 3b and 3a), using the ERETIC2 NMR experiment. Calculated masses for both 2 and 3 were 70.2 μg and 71.4 μg, respectively, with relative standard deviations of 2.8% and 1.5%, respectively. LC−HRMS and LC−MSn. High-resolution mass spectrometry and targeted MS/MS with positive and negative ESI were used to study the MS fragmentation patterns of DON−Cys derivatives. Compounds 2 and 3 afforded [M − H]− ions with m/z 416.1388 and 416.1389, respectively, during negative ESI, consistent with C18H26O8NS− (Δ 0.8 and 1.1 ppm) and [M + H]+ ions with m/z 418.1532 and 418.1529, respectively, in positive ESI mode, consistent with C18H28O8NS+ (Δ 0.4 and −0.3 ppm). In extracted ion chromatograms, the signal intensity for 2 relative to 3 was lower in positive than in negative ionization mode (Figure 4). This was in part attributable to their propensity to form sodium adducts ([M + Na]+, m/z 440.1350) in positive ionization mode, as these constituted ca. 50% of the signal intensity of [M + H]+ for 2, but only about 25% of the intensity of [M + H]+ for 3. The fragmentation patterns for 2 and 3 were different in both positive and negative ionization modes (Figure 6), making it possible to differentiate the two types of adducts by LC−MS/ MS. The HRMS/MS of [M + H]+, 2 (m/z 418.2) of the

Figure 5. 3D model of the hemiketal form of the DON−Cys Michael adduct, 3b, showing selected, structurally significant, correlations observed in the ROESY NMR spectrum that define the stereochemistry of 3 at C-9 and C-10.

ERETIC2. In order to prepare analytical standards of 2 and 3, it was necessary to determine their exact concentrations. As

Figure 6. Product ion mass spectra from LC−HRMS/MS of DON−Cys conjugates in positive (A) and negative (B) ion modes. Compound 2 is the epoxide adduct from addition of cysteine to C-13, and 3 and 4 are from Michael addition of cysteine to C-10. 4782

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Journal of Agricultural and Food Chemistry DON−Cys epoxide adduct showed a fragment at m/z 388.1427, corresponding to loss of CH2O, most likely as a result of cleavage of the side chain attached at C-6. MS3 fragmentation of m/z 388 using the linear ion trap afforded fragments at m/z 370, 352, and 334, attributable to loss of up to three molecules of H2O. The even masses of the product ions showed that the ions contained a nitrogen atom, and thus cysteine was still attached to these ions. Furthermore, MS4 fragmentation of m/z 370 from 2 afforded ions at m/z 281 and 263, which were also visible in the HRMS/MS spectrum (Figure 6). The accurate masses of these two ions were m/z 281.0846 and 263.0739, consistent with elemental compositions of C14H17O4S+ and C14H15O3S+ (Δ 1.1 and Δ 0.9, respectively), showing that sulfur stays attached to the molecule during the fragmentation. In contrast, HRMS/MS of [M + H]+ of Michael adduct 3 gave a prominent product ion at m/z 122.0274 (C3H8O2NS+, Δ 2.4 ppm), corresponding to [M + H]+ for cysteine. The intensity of the m/z 122.0274 product ion was higher in the HRMS/MS spectrum of compound 3 than 2, indicating that it is more easily eliminated from the Michael adduct than from the epoxide adduct. Other notable product ions were m/z 401.1268 (C18H25O8S+, Δ 0.8 ppm), corresponding to loss of ammonia (Figure 6), and m/z 297.1332 (C15H21O6+, Δ 0.3 ppm), corresponding to [M + H]+ for DON, and 249.1122 (C14H17O4+, Δ 1.0 ppm), corresponding to a further loss of 48.9209 Da, attributable to losses of H2O and the epoxide or C-6 side chain (CH4O2, Δ 2.2 ppm) from DON. HRMS/MS data from [M − H]− of 3 (m/z 416.1) gave only one high intensity fragment at m/z 120.0113 (C3H6O2NS−, Δ − 10.0 ppm), consistent with [M − H]− for cysteine (Figure 6). In contrast, HRMS/MS fragmentation of [M − H]− of 2 afforded more product ions (Figure 6): m/z 386.1285 (loss of CH2O, Δ 2.4 ppm) and m/z 299.0964 (C14H19O5S−, Δ 1.7 ppm). Other product ions in the HRMS/MS spectrum of [M − H]− of 2 were observed at m/z 265.1086 (C14H17O5−, Δ 1.6 ppm) and 247.0978 (C14H15O4−, Δ 0.8 ppm) (Figure 6) that appeared to arise from fragmentation of DON itself, as previously reported for mercaptoethanol epoxy adducts of DON.10 Stability of Michael Adduct 3. It has been proposed that Michael addition of a thiol to the C-10 carbon of the α,βunsaturated ketone of DON (1a) is reversible,10 but no experiments were conducted in order to study the reversibility directly. During the development of the isolation procedure for 2 and 3, we observed slow deconjugation of 3 and 4, but not of 2, during storage of the fraction from the initial SPE. This result supports that 4 was a structural isomer of 3 from Michael addition of cysteine to C-10. We found that this deconjugation reaction became negligible upon acidification of an aliquot of the mixture, while the control sample continued to deconjugate at the same rate (Figure 2). This is consistent with basecatalyzed elimination of thiolate from the Michael adduct, but not from the epoxy adduct, as previously proposed based on studies with model thiols.10 For this reason, small amounts of acetic acid were added to the samples during purification, storage, and analysis. The initial formation of 5 and 6 (Figure 3) is consistent with a kinetically favored Michael addition, whereas 3 and 4 appear to be thermodynamically more favored stereoisomers from Michael-type addition of cysteine to DON. The relative peak ratios also indicated that epoxide adduct 2 was formed more

slowly than the Michael adducts (3−6), but its formation is irreversible. Oxidation of 2 and 3. When diluted aliquots of purified 2 and 3 were treated with hydrogen peroxide, LC−HRMS analysis showed two main products for each of the adducts, consistent with the elemental composition C18H28O9NS+ (m/z 434.1471−434.1474, Δ 1.1−2.2 ppm) and C18H26O9NS− (m/z 432.1324−432.1326, Δ 1.8−1.9 ppm) (in positive and negative modes, respectively), corresponding to addition of one oxygen atom to 2 and 3. These oxidation products are likely the diastereoisomeric pairs of the sulfoxides of DON−Cys. LC− HRMS analysis of the oxidation of 2 also revealed a single slowly growing peak at m/z 450.1428 (C18H28O10NS+, Δ −0.2 ppm) and two slowly growing peaks in the oxidation of 3 at m/ z 450.1423 (C18H28O10NS+, Δ 1.1 ppm), corresponding to the addition of two oxygen atoms, attributable to the sulfone derivatives of 2 and 3. Trace levels of sulfoxides were also observed in samples of 2 and 3 stored at −20 °C for 2 months. The sulfoxides of 2 and 3 showed different fragmentation patterns in both positive and negative LC−HRMS/MS. MS/ MS fragmentation of the 3-sulfoxides gave two prominent product ions in both positive and negative modes. The highermass product ions (m/z 138.0217, C3H8O3NS+, Δ 1.7 ppm, and m/z 136.0058, C3H6O3NS−, Δ 3.6 ppm) correspond to oxidized cysteine (sulfenic acid form) that was easily eliminated from 3-sulfoxide, in contrast to 2-sulfoxide. Oxidation of the sulfide to a sulfoxide followed by LC−MS/MS could be used to help to confirm the presence of thiol adducts in samples, in a manner similar to that reported for identifying the presence of methionine residues in cyclic peptides.20 Autoxidation of sulfides to their sulfoxides is a common process occurring in nature,21 and such compounds could potentially also occur via metabolism. Cellular Effects and LC−HRMS Analysis of Cell Culture Medium and Cell. DON is known to induce proinflammatory effects, as well as cytotoxicity in macrophages.22 In our previous study,10 we used the THP-1 cell line as a model system to investigate cellular effects of DON and DON−mercaptoethanol derivatives. Here, the same model was used to compare the effects of DON and cysteine derivatives 2 and 3. DON (1) severely inhibited proliferation of THP-1 monocytes, whereas 3 showed only slight inhibition and 2 gave no detectable effect (Figure 7A). The weak effect observed for 3 could in part be attributable to its contamination with ca. 10 mol % of 1. We also treated THP-1 monocytes with different concentrations of the test compounds (2, 4, and 6 μM) and measured the proliferation at different time points (24, 48, and 72 h). There was a pronounced decrease in proliferation of the cells treated with DON at all time points and concentrations, but no significant effect on proliferation when cells were exposed to 2 or 3. This is consistent with the results from Fruhman et al.,11 who found that 2.1 μM DON reduced in vitro translation to 50%, but that 22.5 μM of the methanethiol Michael adduct of DON was needed in order to get the same effect. Subsequent semiquantitative LC−HRMS analysis of the treated monocytes showed that the concentrations of 1, 2, and 3 were unchanged after 48 h, indicating that there had been no detectable metabolism or oxidation of 1 or its cysteine derivatives. Furthermore, the monocytes and medium treated with Michael adduct 3 (which contained ca. 10 mol % 1) showed the same ratio between 1 and 3 at 0, 24, and 48 h. The cytokine TNF-α assay was used to test whether 2 and 3 induce proinflammatory responses in macrophages similar to 4783

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primed with LPS, only 1 showed a pronounced increase in the cytokine level, while 3 gave a slight increase and 2 showed no effect (Figure 7B). At the end of the experiments, the macrophages and the media were separated, extracted, and analyzed by LC−HRMS. All of the tested compounds were present in the media, but were not detected in the cellular extracts. Again, the concentrations indicated that there was no metabolism or deconjugation of 1−3 in the cells during the experiment. To ensure that we worked in the noncytotoxic concentration range with regard to the cytokine measurements, the CellTox Green cytotoxicity assay was used to test the cytotoxicity of 1, 2, and 3 in the THP-1 macrophages. None of the test compounds induced cytotoxicity at 4 μM (Figure 7C). We here present a simple method for preparation and purification of DON−Cys adducts and have shown that the addition of cysteine occurs both at the 9,10-double bond and at the epoxide ring in DON. The structures of the two final products were identified using NMR spectroscopy, and their concentrations were determined using quantitative NMR. The obtained data should facilitate identification of the type of DON−Cys adducts that occur in natural samples such as plant or mammalian tissues. Furthermore, the prepared solutions of defined concentration can be used as stock solutions for analytical standards and thereby allow quantification of DON− Cys adducts in real samples. The DON−Cys adducts were much less toxic than DON in vitro. This might open new possibilities for chemical detoxification strategies of DONcontaminated raw materials.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01158. NMR spectra (1H and 13C NMR, COSY, TOCSY, HSQC, HMBC, NOESY/ROESY), structures, accurate mass data, LC−HRMS chromatograms, possible fragmentation pathways, and proliferation of THP-1 cells following exposure to 1−3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +47 23216221. Fax: +47 23216201. E-mail: ana.stanic@ vetinst.no, [email protected]. Funding

This work was part of the project “Mycotoxins and toxigenic fungi in Norwegian pig farming: consequences for animal health and possible intervention strategies” funded by the Research Council of Norway (Project No. 225332) and cofinanced by Animalia, Lantmännen Research Foundation, and Felleskjøpet Fôrutvikling.

Figure 7. (A) Proliferation of THP-1 monocytes after exposure for 24 h to 4 μM DON (1), its epoxide (2), or Michael (3) cysteine adducts, measured using Alamar Blue. (B) TNF-α secretion measured by ELISA in THP-1 macrophages, with and without priming by LPS for 3 h, and followed by exposure to 4 μM DON (1), its epoxide (2), or Michael (3) cysteine adducts for 24 h. (C) Cell toxicity induced by 4 μM DON (1), its epoxide (2), or Michael (3) cysteine adducts in THP-1 macrophages for 24 h. Positive control = DON (20 μM). Data are the arithmetic mean ± SD of 3 independent experiments. Data significantly different from the control (1-way ANOVA with Dunnett’s post-test, p < 0.05) are marked with asterisks (*).

Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED COSY, correlation spectroscopy; DEPT, distortionless enhancement of polarization transfer; DON, deoxynivalenol; GSH, glutathione; ERETIC, electronic reference to access in vivo concentrations; ESI, electrospray ionization; FBS, fetal bovine serum; HMBC, heteronuclear multiple-bond correlation; HRMS, high-resolution mass spectrometry; HSQC, heteronuclear single quantum coherence; IL-1β, interleukin-1

those induced by DON.23,24 In the absence of LPS, all of the compounds gave an increase in TNF-α response compared to the control (Figure 7B). However, when the macrophages were 4784

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resources/protocols/technical%20manuals/101/ celltox%20green%20cytotoxicity%20assay%20protocol.pdf. (18) Jarvis, B. B.; Mazzocchi, D. B.; Ammon, H. L.; Mazzola, E. P.; Flippen-Anderson, J. L.; Gilardi, R. D.; George, C. F. Conformational effects in trichothecenes: structures of 15-hydroxy C4 and C8 ketones. J. Org. Chem. 1990, 55, 3660−3662. (19) Bharti, S. K.; Roy, R. Quantitative 1H NMR spectroscopy. TrAC, Trends Anal. Chem. 2012, 35, 5−26. (20) Miles, C. O.; Melanson, J. E.; Ballot, A. Sulfide oxidations for LC-MS Analysis of methionine-containing microcystins in Dolichospermum flos-aquae NIVA-CYA 656. Environ. Sci. Technol. 2014, 48, 13307−13315. (21) Jacob, C. A scent of therapy: pharmacological implications of natural products containing redox-active sulfur atoms. Nat. Prod. Rep. 2006, 23, 851−863. (22) Pestka, J.; Zhou, H. R. Toll-like receptor priming sensitizes macrophages to proinflammatory cytokine gene induction by deoxynivalenol and other toxicants. Toxicol. Sci. 2006, 92, 445−455. (23) Arunachalam, C.; Doohan, F. M. Trichothecene toxicity in eukaryotes: cellular and molecular mechanisms in plants and animals. Toxicol. Lett. 2013, 217, 149−158. (24) Islam, Z.; Pestka, J. J. LPS priming potentiates and prolongs proinflammatory cytokine response to the trichothecene deoxynivalenol in the mouse. Toxicol. Appl. Pharmacol. 2006, 211, 53−63.

beta; JMOD, J-modulated spin−echo; LPS, lipopolysaccharide; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser spectroscopy; PMA, phorbol 12-myristate 13-acetate; ROESY, rotating frame Overhauser spectroscopy; SELTOCSY, selective total correlation spectroscopy; SPE, solid-phase extraction; TOCSY, total correlation spectroscopy; TNF-α, tumor necrosis factor alpha

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REFERENCES

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