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Jul 12, 2018 - The lack of information on HT-2 toxin leads to inaccurate hazard evaluations. In the present study, toxicokinetic studies of HT-2 toxin...
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Article Cite This: J. Agric. Food Chem. 2018, 66, 8160−8168

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Toxicokinetics of HT‑2 Toxin in Rats and Its Metabolic Profile in Livestock and Human Liver Microsomes Shupeng Yang,†,‡,§ Huiyan Zhang,‡,§ Marthe De Boevre,‡ Jinzhen Zhang,‡ Yanshen Li,⊥ Suxia Zhang,§ Sarah De Saeger,‡ Jinhui Zhou,† Yi Li,† and Feifei Sun*,†,§

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†

Bee Product Quality Supervision and Testing Centre, Ministry of Agriculture; Institute of Apicultural Research, Key Laboratory of Bee Products for Quality and Safety Control, Chinese Academy of Agricultural Sciences, Beijing 100093, People’s Republic of China ‡ Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium § College of Veterinary Medicine, China Agricultural University, Beijing 100193, People’s Republic of China ⊥ College of Life Science, Yantai University, Yantai, Shandong 264005, People’s Republic of China S Supporting Information *

ABSTRACT: The lack of information on HT-2 toxin leads to inaccurate hazard evaluations. In the present study, toxicokinetic studies of HT-2 toxin were investigated following intravenous (iv) and oral administration to rats at dosages of 1.0 mg per kilogram of body weight. After oral administration, HT-2 toxin was not detected in plasma, whereas its hydroxylated metabolite, 3′-OH HT-2 was identified. Following iv administration, HT-2 toxin; its 3′-hydroxylated product; and its glucuronide derivative, 3-GlcA HT-2, were observed in plasma, and the glucuronide conjugate was the predominant metabolite. To explore the missing HT-2 toxin in plasma, metabolic studies of HT-2 toxin in liver microsomes were conducted. Consequently, eight phase I and three phase II metabolites were identified. Hydroxylation, hydrolysis, and glucuronidation were the main metabolic pathways, among which hydroxylation was the predominant one, mediated by 3A4, a cytochrome P450 enzyme. Additionally, significant interspecies metabolic differences were observed. KEYWORDS: bioavailability, metabolism, phenotype, mycotoxin, risk assessment

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INTRODUCTION T-2 toxin and HT-2 toxin are type A trichothecene mycotoxins produced by various Fusarium species.1,2 Much research has been done pertaining to the toxic effects of T-2 toxin and HT-2 toxin at low concentrations, including the inhibition of protein, RNA, and DNA synthesis.3−8 T-2 toxin and HT-2 toxin were reported to be responsible for alteration of cell-membrane functions and peroxidation.9 T-2 toxin could also induce hematotoxicity and myelotoxicity.10 Additionally, alimentary toxic aleukia, a typical disease for humans, was associated with the ingestion of moldy cereal contaminated with T-2 toxin.11 HT-2 toxin is the predominant metabolite of T-2 toxin, and they differ in the functional group at the C-4 position with T-2 toxin being acetylated.4,12,13 HT-2 toxin can also be naturally produced by Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichiodes.14 T-2 toxin and HT-2 toxin are often detected and frequently observed in grains and its derived products, especially in oats and oat products.14 Considering the risk of T-2 toxin and HT-2 toxin to humans, a tolerable daily intake (TDI) of 100 ng per kilogram of body weight for the sum of these two toxins has been established by the European Food Safety Authority (EFSA).15 The chemical structures of T-2 toxin, 1, and HT-2 toxin, 2, are shown in Figure 1. Normally, the toxicity of a mycotoxin is closely associated with its exposure time and level in plasma, which would be highly affected by absorption, distribution, metabolism, and excretion.16 The mycotoxicosis caused by T-2 toxin and HT-2 toxin could lead to serious toxic effects in human and farm © 2018 American Chemical Society

Figure 1. Chemical structures of HT-2 toxin and T-2 toxin, which differ at the C-4 position.

animals. The European Food Safety Authority (EFSA) stated that it is of great need to study the efficacy of detoxifiers on the basis of toxicokinetic studies.15 The toxicokinetic characteristics of T-2 toxin was conducted in dogs. Results showed that the half-life of T-2 toxin was shorter than that of HT-2 toxin, whereas the highest concentration of T-2 toxin was much higher than that of HT-2 toxin.17 Until now, information on the toxicokinetic profile of HT-2 toxin, as a natural entity, is still scarce. However, to precisely define the toxicokinetic profile of HT-2 toxin, it is of great importance to better understand the toxicokinetic profile of HT-2 toxin by individual administration. To a great extent, the biotransformation of mycotoxins could significantly influence their toxicity.18−20 It has been reported that T-2 toxin was rapidly metabolized into a large number of Received: Revised: Accepted: Published: 8160

June 2, 2018 July 9, 2018 July 12, 2018 July 12, 2018 DOI: 10.1021/acs.jafc.8b02893 J. Agric. Food Chem. 2018, 66, 8160−8168

Article

Journal of Agricultural and Food Chemistry

according to the principles of the ethical committee of China Agricultural University (Beijing, China; 2016-SYXK-0124). Three rats (one female and two males) were given a single intravenous (iv) administration of HT-2 toxin, and the other three rats (two females and one male) were given HT-2 toxin orally (po) by needle lavage at the dosage of 1.0 mg per kilogram of body weight. Considering the solubility of HT-2 toxin, 1 mL of 20% ethanol was used to dissolve HT2 toxin. After a 7 day washout period, the rats were administrated (iv or po) HT-2 toxin, abiding by a crossover design (the rats given HT-2 toxin intravenously would be given the toxin orally, whereas the rats given the toxin orally would be given HT-2 toxin intravenously). Thus, a total of six rats were used for each administration route. The blood samples were collected from jugular vein prior to and 5, 10, 15, 30, 45, 60, 90, 120, 240, and 360 min after administration. About 300 μL of blood was collected at each above-mentioned time point. The collected blood samples were centrifuged at 2000g for 20 min at 4 °C. The supernatant was gathered and stored at −20 °C before analysis. Analytical Method for the Determination of HT-2 Toxin in Plasma Samples. The plasma samples were determined according to a previously reported analytical method with modifications.26 By adding 300 μL of acetonitrile/acetate (1:3, v/v) into 150 μL of centrifuged plasma, proteins were precipitated; then, they were centrifuged at 10 141g for 10 min. The supernatant was evaporated to dryness and reconstituted in the initial mobile phase. The samples were filtered through a 0.22 μm micropore membrane filter, which was purchased from Pall Corporation (New York, NY). This modified analytical method to determine HT-2 toxin in plasma was optimized and validated. The limit of detection and the limit of quantification of HT-2 toxin were 1.0 and 4.0 ng/mL. An Acquity ultrahigh-performance liquid chromatography (UPLC) system (Waters Company, Milford, MA) equipped with a mass spectrometer (Waters, Manchester, U.K.) was used to determine HT-2 toxin and its major metabolites. A BEH RP 18 column (50 × 2.1 mm i.d., 1.7 μm particle size; Waters, New Brunswick, NJ) was used to achieve chromatographic separation with a flow rate of 0.30 mL/min. The mobile phase and gradient-elution program were the same as those in the literature.26 Collision-induced dissociation was performed in positive-electrospray-ionization mode. The critical parameters were the capillary voltage, 3.2 kV; the source temperature, 120 °C; and the desolvation temperature, 350 °C. Low energy (2 eV) and high energy (10−25 eV) data were acquired via MSE mode. In Vitro Metabolism and Sample Treatment. The preparation of liver microsomes, investigation of the in vitro metabolism of HT-2 toxin, and sample treatment of microsomal incubation were performed substantially as described previously.27 Briefly, liver microsomes of rats, chickens, swine, goats, cows, and humans were incubated with HT-2 toxin. The incubation system was in 500 μL of 0.1 M phosphate buffer that contained the liver microsomes, HT-2 toxin, and NADPH. Then, the mixture was processed according to the procedure previously described.27 Phenotyping for HT-2. Recombinant human cytochrome enzymes (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and carboxylesterases (CES I and CES II) were incubated with HT-2 toxin to investigate the roles of these enzymes in metabolism. Moreover, CES I and CES II were also incubated with T-2 toxin. The incubation system contained phosphate buffer (100 mM, pH 7.4), enzymes (30 pmol), and NADPH (2 mM) along with 3 μM HT-2 toxin. After incubation for 1 h at 37 °C, 100 μL of ice-cold acetonitrile was added to terminate the reaction, and the samples were pretreated according to the procedure for in vitro metabolic samples. The in vitro metabolism assays were performed in triplicate and an additional sample without NADPH served as the negative control. UPLC-Q/TOF Conditions. Chromatographic separation was achieved through an Acquity HSS T3 (100 × 2.1 mm, 1.7 μm; Waters, Milford, MA), and the column temperature was set as room temperature. Mobile phase A was an aqueous solution with 0.05 mM aqueous ammonia, and mobile phase B was acetonitrile. The gradient program was performed as follows with a flow rate of 0.30 mL/min: 0− 1.0 min, 5−5% B; 1.0−10.0 min, 5−30% B; 10.0−13.0 min, 30−50% B; 13.0−14.0 min, 50−100% B; and 14.0−15.0 min, 5% B. The injection

products, and HT-2 toxin was the major metabolite. Considering the rapid transformation of T-2 toxin to HT-2 toxin, it is assumed that the toxicity of T-2 toxin should be attributed to HT-2 toxin.10 It has been proposed that the metabolic routes of T-2 toxin were hydrolysis (C-4, C-8, and C15), hydroxylation (C-3, C-4′, and C-7), glucuronidation (C-3), and de-epoxidation (C-11 and C-12), in which de-epoxidation was regarded as a detoxification pathway.9,13,21,22 In animal tissues, most of the metabolites of T-2 toxin maintain the epoxy moiety, which seems to account for the typical toxic effects of trichothecenes. It has been reported that the toxicity of T-2 toxin was approximately 1.5 times higher than that of HT-2 toxin.10 HT-2 toxin exerted deleterious effects on animals, resulting in the failure of porcine-oocyte maturation and influencing porcine-embryo development.7,23 Johnsen et al.24 found that the metabolic transformation of T-2 toxin to HT-2 toxin and neosolaniol in white blood cells was mediated by carboxyesterases. The toxicokinetic profile of T-2 toxin showed that T-2 toxin was rapidly eliminated from plasma following intramuscular injection of HT-2 toxin, whereas neither T-2 toxin nor HT-2 toxin was detected after oral administration of T-2 toxin.25 However, the toxicokinetic picture of HT-2 toxin, as an independent entity, has not been clarified. Our team conducted a toxicokinetic study of HT-2 toxin by intravenous (iv) and oral administration (po) of HT-2 toxin, and the results showed that only trace levels of HT-2 toxin were detected after iv administration, and no HT-2 toxin was identified after oral administration. To explore the missing HT-2 toxin, the metabolism of HT-2 toxin was investigated. According to previously published literature, we know that hydroxylation of T-2 toxin is mediated via cytochrome P450 esterases of intestinal and hepatic microsomes in mice, rats, guinea-pigs, and cows but not in rabbits.9,13 However, the interspecies metabolic differences of HT-2 toxin and the specific enzymes that mediate the biotransformation of HT-2 toxin have not been comprehensively investigated. Hence, the intention of the present study was to determine the toxicokinetic profile, clarify the metabolic behaviors, and interpret the interspecies metabolic differences, which would provide a foundation for the risk assessment, detoxification, and counteraction of poisoning caused by HT-2 toxin.

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MATERIALS AND METHODS

Chemicals and Reagents. The standards of T-2 toxin (99.0%), HT-2 toxin (99.0%), and neosolaniol (98%) were purchased from Fermentek, Ltd. (Jerusalem, Israel). Pooled rat, swine, and human liver microsomes; recombinant human cytochrome P 450 enzymes (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4); and carboxylesterases (human CES1 and human CES2) were obtained from the Research Institute for Liver Diseases Company Ltd. (Shanghai, China). βNicotinamide adenine dinucleotide phosphate (NADPH) and uridine5′-diphosphoglucuronic acid (UDPGA) were acquired from SigmaAldrich Company (Hamburg, Germany). Water used in the incubation system and the aqueous solutions was purified through a Milli-Q system (Millipore, Bedford, MA). Acetonitrile and methanol in this study are LC-MS grade (Fisher Chemical, Harnover Park, IL). All the other chemicals and reagents were analytical grade. Phoenix WinNonlin 6.3 from Pharsight (Sunnyvale, CA) was used to analyze the toxicokinetic data. Animals Used for Toxicokinetic Studies of HT-2 Toxin. Six Sprague−Dawley rats (weighing about 180−220 g, 6 weeks old, half male and half female) were bought from Vital River Laboratory Animal Technology Company Ltd. (Beijing, China). The rats were housed and acclimated for 7 days under standardized conditions before dosing. Water was available ad libitum. Animal experiments were conducted 8161

DOI: 10.1021/acs.jafc.8b02893 J. Agric. Food Chem. 2018, 66, 8160−8168

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Journal of Agricultural and Food Chemistry volume was 10.0 μL. Quadrupole time-of-flight (Q-TOF), with an electrospray-ionization source, was utilized in positive mode in the present study to identify potential metabolites. The major parameters were the capillary voltage, 3.0 kV; the cone voltage, 20 V; the ion-source temperature, 100 °C; the desolvent temperature, 300 °C; and the desolvent flow rate, 600 L/h. Data was acquired in MSE mode with high and low energy at 2 eV and 10−25 eV. For MS/MS mode, the collision energy was set as 7−20 eV. Data Analysis. The obtained data was analyzed with MetabolynxXS software (Waters, Milford, MA). This software is commonly used for the identification of metabolites on the basis of a mass-defect filter (MDF) and background subtraction (BS) as well as accurate extractedion chromatography (EIC). By comparing the test samples with those of the control group, this software efficiently determines potential metabolites. To ensure the accuracy, the mass window was set within 5 ppm during the identification of metabolites.

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RESULTS AND DISCUSSION Toxicokinetic Trials. Plasma concentrations of HT-2 toxin were determined using a developed and validated ultrahighperformance-liquid-chromatography−tandem-mass-spectrometry (UPLC-MS/MS) method.26 The limit of quantitation was 4.0 ng/mL. Toxicokinetic studies of HT-2 toxin in rats show that the plasma concentrations, after oral administration, are all below the limit of quantitation (LOQ). For iv administration, HT-2 toxin is detected in plasma, with a maximum concentration of 80.55 ng/mL. 3-GlcA HT-2 and 3′-OH HT2 are the major compounds of HT-2 toxin in plasma, and the corresponding plasma-concentration−time curves are presented in Figure 2. Figure 2A is the plotted mean-plasma-concentration−time curve of HT-2 toxin after iv administration. Figure 2B shows the mean-concentration−time curves of 3′-OH HT-2 after iv and po administration. Figure 2C illustrates the concentration−time curve of 3-GlcA HT-2 after iv administration. The typical pharmacokinetic parameters of HT-2 toxin following iv administration are as follows: the elimination halflife (t1/2λz) is 10.5 ± 0.6 min, the area under the plasmaconcentration−time curve from 0 to infinity is 1077.2 ± 195.1 min·ng/mL, the total body clearance is 943.8 ± 141.0 mL/min/ kg, and the mean residence time is 15.4 ± 0.5 min. After oral administration, HT-2 toxin is not detected in plasma; a hydroxylated metabolite of HT-2 toxin, 3′-OH HT-2 is identified from the first-time point and then decreases gradually for the remaining time. MS/MS-Spectrum Interpretation of HT-2 Toxin. HT-2 toxin, the most important metabolite of T-2 toxin in mammals, also naturally exists in Fusarium-contaminated grains and often coexists with T-2 toxin.28 HT-2 toxin and T-2 toxin have the similar chemical structures; they differ only at the C-4 position, with T-2 toxin being acetylated. Thus, the MS behavior for the metabolic study of HT-2 toxin should be much like that of T-2 toxin. To form the stable molecular ion [M + NH4]+, m/z 442.2411, 0.05 mM aqueous ammonia is added in aqueous solution. Then, the MS/MS spectrum of HT-2 toxin is acquired under collision-induced dissociation (CID) and fragment ions are generated, such as m/z 425, 323, 263, 245, 233, 215, 197, and 169. The MS/MS pattern is shown in detail in Figure 3. Seen from Figure 3, the precursor ion could form m/z 425 by loss of NH3. Then, the product ion could generate m/z 323 by the cleavage of the side chains at the C-8 position (C5H11O, amyl acyl) and the C-3 or -4 position (H2O). The product ion, m/z 263, is formed from m/z 323 by loss of the acetyl group at the C15 position. After that, the ions m/z 233 and 245 are generated from m/z 263 by the loss of CH2O and H2O, respectively, and

Figure 2. Plotted mean-plasma-concentration−time curves of HT-2 toxin. The highest concentration was 80.0 ng/mL. (A) Plotted meanplasma-concentration−time curve of HT-2 toxin after iv administration to rats at doses of 1.0 mg per kilogram of body weight (n = 6). (B) Mean-peak-area−time curves of 3′-OH HT-2 after iv and po administration. (C) Peak-area−time curve of 3-GlcA HT-2 after iv administration (n = 6).

finally, product ion m/z 215 is formed. Fragment ion m/z 215 generated ions m/z 197 and 169 by the subsequent loss of H2O and CO. Phase I Metabolism of HT-2 Toxin in Vitro. After incubation, all the positive and control samples were simply pretreated and determined using UPLC-Q/TOF. As a result, a total of eight metabolites were identified. The elemental composition of each metabolite and measured values, theoretical values, mass error, and double-bond-equivalent (DBE) values of each product ion are summarized in Table 1, and the corresponding spectra are illustrated in Figure 3. Metabolic annotation of potential metabolites of HT-2 toxin is depicted in the following sections. Structural Interpretation of Potential Metabolites of HT-2 Toxin. Hydrolytic Metabolites, MHT-1 and MHT-2. The 8162

DOI: 10.1021/acs.jafc.8b02893 J. Agric. Food Chem. 2018, 66, 8160−8168

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Journal of Agricultural and Food Chemistry

Figure 3. Spectra and proposed fragmentation pathway of HT-2 toxin and its metabolites. The mass tolerance of each metabolite was within 5 ppm.

and cleaved at C-8. Thus, MHT-2 was identified as a metabolite of T-2 toxin and designated as 4-acetoxy T-2 tetraol. Hydroxylated Metabolites, MHT-3 to MHT-8. MHT-3−8 yielded the same ammonia adduct, [M + NH4]+, with the mass value of m/z 358.1866 ([C17H28NO7]+), but they had different retention times (MHT-3, 8.44; MHT-4, 8.65; MHT-5, 8.91; MHT-6, 10.21; MHT-7, 10.43; and MHT-8, 12.63). These metabolites are considered as monohydroxylated or oxidative products of HT-2 toxin, considering the addition of a hydroxy group within a 5 ppm mass error with calculated exact mass values. In the MS/MS spectra of MHT-3, MHT-4, and MHT-5, the neutral loss of 118 Da is 16 Da higher than the neutral loss of 102 Da in HT-2 toxin, implying that these metabolites are hydroxylated products at the side chain of the C-8 side chain. In comparison with the previously reported literature regarding

retention times of MHT-1 and MHT-2 are 3.29 and 5.85 min, respectively, but they had the same mass value of m/z 358.1866 ([C17H28NO7]+), which represents the scission at the amyl acyl group of the C-8 position of HT-2 toxin, suggesting that these two metabolites are hydrolytic products. For MHT-1, its fragment ions, m/z 263, 245, 215, 197, and 169, are similar to those of HT-2 toxin, allowing us to infer that the core structure of MHT-1 remains unchanged. Moreover, the typical neutral loss of 102 Da (amyl acyl group) at the C-8 position is not observed in the MS/MS spectrum of MHT-1, which further demonstrates that hydrolysis occurs at the C-8 position. Thus, MHT-1 is identified as 4-de-Ac neosolaniol. For MHT-2, the acetyl group at the C-15 is hydrolyzed, forming T-2 triol. However, T-2 triol is extremely polar and not easily ionized. It is likely to have been subsequently acetylated at the C-4 position 8163

DOI: 10.1021/acs.jafc.8b02893 J. Agric. Food Chem. 2018, 66, 8160−8168

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Journal of Agricultural and Food Chemistry

Table 1. Summary of Proposed Phase I and Phase II Metabolites (MHT-1−11) of HT-2 Toxin Observed in Liver Microsomes from Humans and Different Species of Animalsa liver microsomesb

no.

metabolites

c

retention time (min)

rat

chicken

swine

goat

cow

human

major fragmentsd 425, 323, 263*, 245, 215, 197, 169 323, 275, 263*, 245, 233, 215, 197, 169, 145 323, 293, 263, 245, 233, 215*, 203, 197, 157, 145 323, 263, 245, 215, 197*, 185, 157 441, 323, 263, 215, 197*, 185, 157 441, 323, 263, 215, 197* 441, 339, 321, 279*, 261, 149, 243, 231, 213, 189, 185 441, 339, 321, 279*, 261, 149, 243, 231, 213, 189, 185 441, 423, 339, 321, 279, 261, 243*, 215, 213, 189, 157 601, 499, 425, 323, 263*, 245, 215, 197, 187, 169 601, 499, 439, 425, 323, 275, 263, 245, 215*, 197, 185 421, 323, 305, 263*, 245, 215, 197, 185, 169

composition

[M + NH4]+ (m/z) 442.2441

2.4

5.38

√

√

√

√

√

√

error (ppm)

HT-2

HT-2 toxin

C22H36NO8+

MHT-1

C17H28NO7+

358.1866

−1.7

3.92

√

ND

√

√

√

√

C17H28NO7+

358.1866

3.4

5.86

√

√

√

ND

ND

ND

MHT-3

4-de-Ac neosolaniol 4-acetoxy T-2 tetraol 3′-OH HT-2

C22H36NO9+

458.2390

2.8

8.44

√

√

√

√

√

√

MHT-4

4′-OH HT-2

C22H36NO8+

458.2390

−1.0

8.65

ND

ND

√

√

ND

ND

MHT-5 MHT-6

4′-OH HT-2 7-OH HT-2

C22H36NO8+ C22H36NO8+

458.2390 458.2390

−1.7 1.3

8.91 10.23

ND √

ND ND

√ √

ND √

√ √

ND √

MHT-7

7-OH HT-2

C22H36NO8+

458.2390

0.9

10.43

√

ND

√

√

√

√

MHT-8

10-OH HT-2

C22H36NO8+

458.2390

3.1

12.63

√

√

√

√

√

√

MHT-9

3-GlcA HT-2

C28H44NO14+

618.2762

−3.2

3.41

√

√

√

√

√

√

MHT-10

4-GlcA HT-2

C28H44NO14+

618.2762

2.4

3.20

√

ND

√

√

√

√

MHT-11

3-GlcA-4-de-Ac neosolaniol

C23H36NO13+

534.2187

−1.9

3.46

√

ND

ND

ND

ND

√

MHT-2

a

The [M + NH4]+ (m/z) values were calculated from the proposed structural formulas. The error (ppm) is the difference between the calculated and observed m/z values. These are tentative identifications based on LC-HRMS. b√, detected; ND, not detected. cAc, acetylation; GlcA, glucuronic acid. dAsterisks (*) indicate the base peaks in the MS/MS spectra.

Figure 4. (A) Phase I metabolism of HT-2 toxin in the liver microsomes of humans and different species. (B) Phase II metabolism of HT-2 toxin in human and various animal liver microsomes. (C) Attribution of CES-I and CES-II enzymes to T-2 toxin and HT-2 toxin. The x-axis is the relative percentage. (D) CYP-reaction phenotyping for HT-2 toxin. The x-axis is the relative percentage.

the metabolism of T-2 toxin, MHT-3−5 share the same MS/MS spectra as MT-4, a metabolite of T-2 toxin, but only MHT-3 has the same retention time as MT-4, showing that MHT-3 and MT-4 are the same compound.4,13 MHT-3, thus, is interpreted

as 3′-OH HT-2. It has been reported that for T-2 toxin, the hydroxylation could occur at the C-3′ (ω-1) position as well as at the C-4′ (ω) position. Hence, MHT-4 and MHT-5 are considered as isomers of 4′-OH HT-2. 8164

DOI: 10.1021/acs.jafc.8b02893 J. Agric. Food Chem. 2018, 66, 8160−8168

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Journal of Agricultural and Food Chemistry

in microsomes of livestock and humans. By comparing the spectra with those of T-2 toxin, two phase II metabolites of HT2 toxin were identified, 3-GlcA HT-2 and 4-GlcA HT-2. The precursor of MHT-11 is m/z 534.2187 ([M + NH4]+, [C23H36NO13]+), which is 176 Da higher than that of MHT-1 (4-deacetyl neosolaniol), indicating that MHT-11 is likely to be the GlcA-binding product of MHT-1. Additionally, in the MS/ MS spectrum of MHT-9, the typical neutral loss of GlcA (176 Da, m/z 439 to 263) is observed, and the fragment ions of MHT-11, m/z 263, 245, 215, and 197, are identical to those of MHT-1 (4-deacetyl neosolaniol).13 Therefore, MHT-11 is considered as 3-GlcA- 4-deacetyl neosolaniol. Phase II In Vitro Metabolic Routes of HT-2 Toxin and Their Interspecies Metabolic Differences. Hydroxylation (C-3′, C-4′, and C-7), hydrolysis (C-15), and glucuronidation (C-3 and C-4) are the major routes for HT-2 toxin. The metabolic pathways of HT-2 toxin and T-2 toxin have much in common, but there are still significant metabolic differences between HT-2 toxin and T-2 toxin. Hydroxylation is the major metabolic pathway for HT-2 toxin, especially at C-3′. Interestingly, for chickens, the dominant product of HT-2 toxin is 10-OH HT-2. The relative amount of each metabolite of HT-2 toxin is illustrated in Figure 4B. It is clear that the most important conjugation site of HT-2 toxin in swine liver microsomes is C-4, followed by the C-3 position. Metabolic Phenotype of HT-2 Toxin. Wu et al.29,30 put forward that both CYP 450 and carboxylesterase enzymes played a role in the metabolism of T-2 toxin. In the present study, Figure 4C shows that T-2 toxin is mainly hydrolyzed by CES-I and CES-II, and the potency of CES-II is approximately 4fold that of CES-I. However, the hydrolysis of HT-2 toxin mediated by CES-I and CES-II is weak. CYP 450 isoenzyme 3A4 plays vital roles in the metabolism of HT-2 toxin, which could be observed in Figure 4D. Isoenzymes 2C19 and 2C8 also play a small role in the transformation of HT-2 toxin. The cytochrome P450 superfamily has been reported to be the major phase I metabolic enzymes, accounting for 75% of in vivo drug metabolism. Metabolic Differences between T-2 Toxin and HT-2 Toxin. Ohta et al.31 investigated the metabolism of the T-2 toxin in rats, mice, and rabbits and reported the hydrolyzed product at the acetyl group (HT-2 toxin). They also determined the nonspecific enzyme (carboxylesterase) in the liver that mediated the hydrolysis by chemical enzyme inhibition. Subsequently, Yoshizawa et al.32 proposed that the predominant metabolite of T-2 toxin was HT-2 toxin, a hydrolyzed product at the C-4 position. However, it was then observed that the major metabolite of T-2 toxin was HT-2 toxin in terrestrial animals and neosolaniol in aquatic animals.9 Swanson et al.33 reported that the de-epoxide reaction was a vital pathway for the T-2 toxin for detoxification, and de-epoxidation was mediated by intestinal microbial action, not by the liver. Until now, the metabolic profile of HT-2 toxin was still limited. The importance of HT-2 toxin was emphasized and evaluated by the Joint Expert Committee on Food Additives (JECFA), which noted that the toxicity of T-2 toxin resulted from T-2 toxin and its major metabolite, HT-2 toxin, simultaneously. Despite the similar chemical structures, the metabolic profile of HT-2 toxin varied from that of T-2 toxin. It was revealed in our study that hydroxylation, especially hydroxylation at the C-3′ position, is the dominant metabolic route of HT-2 toxin. Compared with those of the unmetabolized parent toxin, the metabolic rate and extent of HT-2 toxin are

MHT-6 and MHT-7 have almost the same MS/MS spectra, showing that they are likely to be stereoisomers. Additionally, the typical neutral loss of 102 Da (amyl acyl group) at the C-8 position is observed, which indicates that the side chain remains unchanged. Furthermore, the product ions (m/z 339, 279, 261, 249, and 231) are 16 Da higher than the product ions of HT-2 toxin (m/z 263, 245, 233, 245, and 215), which further verifies the introduction of a hydroxy group on the core structure. Therefore, MHT-6 and MHT-7 are hydroxylated derivatives on the core structure. Compared with the metabolism results of T-2 toxin in rats reported by our group,13 MHT-6 and MHT-7 have the same m/z value and MS/MS pattern as those of 7-OH HT-2, a metabolite identified in the rats’ urine. Therefore, MHT-6 and MHT-7 are tentatively identified as stereoisomers of 7-OH HT2. For MHT-8, similar to MHT-6 and MHT-7, the loss of 102 Da (amyl acyl group) is observed, implying that the C-8 position maintains unchanged. However, the MS/MS spectrum of MHT-8 is significantly different from those of MHT-6 and MHT-7, which could be attributed to hydroxylated position. Interestingly, the neutral loss of 28 Da (CO) from m/z 243 to 215 is observed, which means that the hydroxylation occurs at a carbon double bond. Therefore, MHT-8 is tentatively considered to be 10-OH HT-2. Metabolic Differences of HT-2 Toxin in Various Species and Major Metabolic Pathways in the Phase I Incubation System. After 2 h of incubation, more than half of the amount of HT-2 toxin remains unmetabolized. The predominant metabolic routes of HT-2 toxin are hydrolysis and hydroxylation in liver microsomes. Moreover, there are significant interspecies metabolic differences for HT-2 toxin. Metabolites and the corresponding amounts of these metabolites are illustrated in Figure 4A. It is obvious that MHT-3 (3′OH HT-2) is the major metabolite of HT-2 toxin in all animal and human microsomes. In addition, in rat microsomes, MHT-7 (7-OH HT-2) also has a high yield, indicating that the critical hydroxylation sites of HT-2 toxin in rats are at the C-3′ and C-7 positions. Additionally, a small amount of MHT-7 is found in the liver microsomes of swine, goats, cows, and humans but not in chickens. However, in chickens, MHT-8 (10-OH HT-2) is present in a higher amount, which is even more than that of MHT-3. Thus, hydroxylation at the C-10 position is a predominant metabolic pathway of HT-2 toxin in chickens. MHT-4 and MHT-5, the hydroxylated products at the C-4′ position, are isomers that are barely detected in swine. MHT-6 is not detected in chickens, but it is identified in liver microsomes of other species at trace-level concentrations. MHT-1, the acetyl-hydrolyzed derivative of HT-2 toxin at the C-15 position, is detected in the liver microsomes of rats, swine, goats, cows, and humans. MHT-1 is identified in swine at trace levels but undetectable in chickens. For MHT-2, it is higher in the liver microsomes of chickens and swine and lower in rats. Because of the potential differences in ionization efficiency concerning HT2 toxin metabolites, it is better to synthesize the metabolites to elucidate the derivatives and not rely solely on high-resolution mass spectrometry. In conclusion, hydroxylation at the C-3′, C7, and C-10 positions are the primary metabolic pathways of HT-2 toxin. Phase II Metabolism of HT-2 Toxin in Microsomes of Rats, Chickens, Swine, Goats, Cows, and Humans. Three phase II metabolites and two hydrolytic metabolites (4-de-Ac neosolaniol and 4-acetoxy T-2 tetraol) were identified. Figure 3 illustrates the MS/MS spectra of the three phase II metabolites 8165

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Figure 5. Detailed metabolic pattern of HT-2 toxin.

reaction is a critical mechanism for detoxification, and the GlcA conjugate often has no activity and is easily eliminated. The detailed metabolic pattern of HT-2 toxin is shown in Figure 5. The cleavage of the acetyl group at the C-4 position does not distinctly attenuate the toxicity, whereas the loss of the amyl acyl group strikingly decreases the cytotoxicity.37 Hydroxylation at the C-3′ position is a theoretical activation pathway for HT-2 toxin, and the conversion from HT-2 toxin to T-2 tetraol or to T2 triol is an inactivation pathway. Namely, the toxicity sharply decrease with the formation of MHT-1, MHT-2, MHT-10, and the three GlcA conjugates, whereas the toxicity remains or even increases with the formation of MHT-3−5.37 Overall, the obtained toxicokinetic, metabolic, and phenotyping information facilitates means for detoxification and risk assessment.

both lower in liver microsomes. In addition to hydroxylation at the C-3′ position, the same as that of T-2 toxin, HT-2 toxin has several specific hydroxylation sites, including the C-4′, C-7, and C-10 positions. A novel hydroxylated metabolite was observed by Yoshizawa et al.34 through the in vivo metabolism of T-2 toxin, but its chemical structure was not elucidated. Subsequently, this hydroxylated product was elucidated to be 3′OH-7-OH HT-2 by Pawlosk and Mirocha35 using MS techniques. In the present study, hydroxylation, hydrolysis, and glucuronidation are the major metabolic routes for HT-2 toxin. Lin et al.36 determined species-level differences in T-2 toxin metabolism in liver microsomes. In the current study, the metabolic differences among species are observed as well. MHT1 is identified in the liver microsomes of rats, goats, cows, and humans but not in chickens. Nevertheless, MHT-2, the isomer of MHT-1, is identified in the liver microsomes of chickens and also at trace levels in the liver microsomes of rats and swine. Similar to the phase II metabolism of T-2 toxin, the GlcAbinding sites of HT-2 toxin are primarily at the C-3 hydroxy group, followed by the C-4 hydroxy group. For phase II metabolism in the liver microsomes of chickens, no GlcA conjugate of T-2 toxin is observed, but a GlcA conjugate of HT-2 toxin is detected at trace levels. The metabolic pathways of HT-2 toxin and T-2 toxin have much in common, but there are still significant metabolic differences between HT-2 toxin and T-2 toxin. Additionally, it was revealed that the metabolism of HT-2 toxin is primarily mediated by isoenzyme 3A4. It was first proposed that the hydrolysis of T-2 toxin is mediated by CES-II, followed by CESI. It has been reported that the double bond formed by C-9 and C-10 as well as the epoxy ring formed by C-12 and C-13 were the toxic groups.37 In addition, the acetyl amyl group at the C-8 position and the acetyl group at the C-4 and C-5 motifs could enhance the toxicity.37 Other hydroxylated products were prone to be excreted because of increased polarity. The GlcA-binding

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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.8b02893. Accurate MS/MS spectra and proposed fragmentation pathway of HT-2 toxin, extracted-ion chromatograms of HT-2 toxin phase I and phase II metabolites, chromatogram of HT-2 toxin at the limit of quantitation, chromatogram of a blank sample, analytical-method validation, and toxicokinetic parameters for HT-2 toxin (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-10-6259-4943. Fax: +86-10-6259-6429. E-mail: [email protected]. ORCID

Shupeng Yang: 0000-0001-7879-403X Marthe De Boevre: 0000-0002-6151-5126 8166

DOI: 10.1021/acs.jafc.8b02893 J. Agric. Food Chem. 2018, 66, 8160−8168

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Journal of Agricultural and Food Chemistry

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Sarah De Saeger: 0000-0002-2160-7253 Feifei Sun: 0000-0002-0824-0821 Funding

This work is financially supported by the National Natural Science Foundation of China (No. 31702296). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED GlcA, glucuronic acid; iv, intravenous; po, oral administration; MDF, mass-defect filtering; BS, background subtraction; EIC, extracted-ion chromatogram; NADPH, β-nicotinamide adenine dinucleotide phosphate; UDPGA, uridine-5′-diphosphoglucuronic acid; CYP 450, cytochrome P450 enzyme; CES, carboxylesterase; CID, collision-induced dissociation; LOQ, limit of quantitation; DBE, double-bond-equivalent values; UPLC-Q/TOF, ultrahigh-performance liquid chromatography−quadrupole time-of-flight; TDI, tolerable daily intake.

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