The Toxicokinetics of HT-2 Toxin in Rats and Its ... - ACS Publications

Subscriber access provided by UNIV OF DURHAM

Food Safety and Toxicology

The 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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02893 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

Journal of Agricultural and Food Chemistry

1

The Toxicokinetics of HT-2 Toxin in Rats and Its

2

Metabolic Profile in Livestock and Human Liver

3

Microsomes

4

Shupeng Yang†,‡,§, Huiyan Zhang‡,§, Marthe De Boevre‡, Jinzhen Zhang‡, Yanshen Li

5

⊥

, Suxia Zhang§, Sarah De Saeger‡, Jinhui Zhou†, Yi Li†, Feifei Sun†,§,*

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Key

6

†

7

Laboratory of Bee Products for Quality and Safety Control; Bee Product Quality

8

Supervision and Testing Centre, Ministry of Agriculture, Beijing 100093, People’s

9

Republic of China Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University,

10

‡

11

Ottergemsesteenweg 460, 9000 Ghent, Belgium

12

§

13

People’s Republic of China

14

College of Veterinary Medicine, China Agricultural University, Beijing 100193,

⊥

College of Life Science, Yantai University, Yantai, Shandong, 264005, P. R. China

15

*

16

Tel: +86-10-6259-4943; Fax: +86-10-6259-6429;

17

E-mail: [email protected]

Author to whom correspondence should be addressed:

1

ACS Paragon Plus Environment


18

ABSTRACT: The lack of information on HT-2 toxin leads to the inaccurate hazard

19

evaluation. In the present study, the toxicokinetic studies of HT-2 toxin were

20

investigated following intravenous (i.v) and oral administration to rats at a dosage of

21

1.0 mg/kg body weight. After oral administration, HT-2 toxin was not detected in

22

plasma, whereas its hydroxylated metabolite, 3'-OH HT-2 was identified. For i.v.

23

administration, HT-2 toxin, its 3'-hydroxylated product and glucuronide derivative,

24

3-GlcA HT-2, were observed in plasma, among which the glucuronide conjugate was

25

the predominant metabolite. To explore the missing HT-2 toxin in plasma, metabolic

26

studies of HT-2 toxin in liver microsomes were conducted. Consequently, eight phase

27

I and three phase II metabolites were identified. Hydroxylation, hydrolysis and

28

glucuronidation were the main metabolic pathways, among which hydroxylation was

29

the predominant one, mediated by 3A4, a cytochrome P450 enzyme. Additionally,

30

significantly inter-species metabolic differences were observed.

31

KEYWORDS: bioavailability; metabolism; phenotype; mycotoxin; risk assessment

2


Page 2 of 35

Page 3 of 35


32

INTRODUCTION

33

T-2 toxin and HT-2 toxin are type A thrichothecene mycotoxins, produced by various

34

Fusarium species, belonging to type A trichothecene.1, 2 Much research has been done

35

pertaining to the toxic effects of T-2 toxin and HT-2 toxin at a low concentration,

36

including the inhibition of protein, RNA and DNA synthesis.3-8 T-2 toxin and HT-2

37

toxin were reported to be responsible for the alteration of cell membrane functions

38

and peroxidation.9 T-2 toxin could also induce haematotoxicity and myelotoxicity.10

39

Additionally, alimentary toxic aleukia, a typical disease for humans, was associated

40

with the ingestion of moldy cereal contaminated with T-2 toxin.11 HT-2 toxin is the

41

predominant metabolite of T-2 toxin while they differ in the functional group at the

42

C-4 position, T-2 toxin being acetylated.4,12,13 HT-2 toxin can also be naturally

43

produced by Fusarium langsethiae, F. poae, and F. sporotrichiodes.14 T-2 toxin and

44

HT-2 toxin are often detected and frequently observed in grains and its derived

45

products, especially in oats and oat products.14 Considering the risk of T-2 toxin and

46

HT-2 toxin to humans, a tolerable daily intake (TDI) of 100 ng/kg b.w. for the sum of

47

these two toxins has been established by the European Food Safety Authority

48

(EFSA).15 The chemical structures of T-2 toxin, 1, and HT-2 toxin, 2, are shown in

49

Figure 1.

50

Normally, the toxicity of mycotoxins is closely associated with its exposure time and

51

level in plasma, which would be highly affected by absorption, distribution,

52

metabolism and excretion.16 The mycotoxicosis caused by T-2 toxin and HT-2 toxin

53

could lead to serious toxic effects in human and farm animals. The European Food 3



54

Safety Authority (EFSA) stated that it is of great need to study the efficacy of

55

detoxifiers on the basis of toxicokinetic studies.15 The toxicokinetic characteristics of

56

T-2 toxin was conducted in dogs. Results showed that the half-life of T-2 toxin was

57

shorter than that of HT-2 toxin whereas the highest concentration of T-2 toxin was

58

much higher than HT-2 toxin.17 Until now, information on the toxicokinetic profile of

59

HT-2 toxin, as a natural entity, is still scarce. However, to precisely define the

60

toxicokinetic profile of HT-2 toxin, it is of great importance to better understand the

61

toxicokinetic profile of HT-2 toxin by individual administration.

62

To a great extent, the biotransformation of mycotoxins could significantly influence

63

their toxicity.18-20 It has been reported that T-2 toxin was rapidly metabolized into a

64

large number of products, and HT-2 toxin was the major metabolite. Considering the

65

rapid transformation of T-2 toxin to HT-2 toxin, it is assumed that the toxicity of T-2

66

toxin should be attributed to HT-2 toxin.10 It has been proposed that the metabolic

67

routes of T-2 toxin were hydrolysis (C-4, 8, and 15), hydroxylation (C-3, 4', and 7),

68

glucuronidation (C-3) and de-epoxidation (C-11,12), in which de-epoxidation was

69

regarded as a detoxification pathway.9,

70

metabolites of T-2 toxin maintain the epoxy moiety, which seems to account for

71

typical toxic effects of trichothecenes. It has been reported that the toxicity of T-2

72

toxin was approximately 1.5 times higher than that of HT-2 toxin.10 HT-2 toxin

73

exerted deleterious effects on animals, including the failure of porcine oocyte

74

maturation and influence on porcine embryo development.7,23 Johnsen et al.24 found

75

that metabolic transformation of T-2 toxin to HT-2 toxin and neosolaniol in white

13, 21, 22

In animal tissues, most of the

4


Page 4 of 35

Page 5 of 35


76

blood cells were mediated by carboxyesterases. The toxicokinetic profile of T-2 toxin

77

showed that T-2 toxin was rapidly eliminated from plasma following intramuscular

78

injection of HT-2 toxin whereas neither T-2 toxin nor HT-2 toxin was detected after

79

oral administration of T-2 toxin.25 However, the toxicokinetic picture of HT-2 toxin,

80

as an independent entity, has not been clarified. Our team conducted the

81

toxicokinetics study of HT-2 toxin by intravenous (i.v) and oral administration (p.o)

82

of HT-2 toxin and results showed that only trace level of HT-2 toxin was detected

83

after i.v. administration and no HT-2 toxin was identified after oral administration. To

84

explore the missing HT-2 toxin, the metabolism of HT-2 toxin was investigated.

85

According to previously published literature, we know that hydroxylation of T-2 toxin

86

is mediated via cytochrome P450 esterases of intestinal and hepatic microsomes in

87

mice, rats, guinea-pigs, cows, but not in rabbits.9,13 However, the interspecies

88

metabolic differences of HT-2 toxin and the specific enzymes that mediated the

89

biotransformation of HT-2 toxin have not been comprehensively investigated.

90

Hence, the intention of present study was to determine the toxicokinetic profile,

91

clarify the metabolic behaviors as well as to interpret the inter-species metabolic

92

differences, which would provide a foundation for the risk assessment, detoxification

93

and counteraction of poisoning caused by HT-2 toxin.

94

MATERIALS AND METHODS

95

Chemicals and reagents. The standards of T-2 toxin (99.0%), HT-2 toxin (99.0%)

96

and neosolaniol (98%) were purchased from Fermentek, Ltd. (Jerusalem, Israel). 5



Page 6 of 35

97

Pooled rats, swine and humans liver microsomes, recombinant humans cytochrome P

98

450 enzymes (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) and carboxylesterases

99

(humans CES1 and humans CES2) were obtained from the Research Institute for

100

Liver Diseases Co. Ltd (Shanghai, China). β-Nicotinamide adenine dinucleotide

101

phosphate (NADPH) and uridine-5’-diphosphoglucuronic acid (UDPGA) were

102

acquired from Sigma-Aldrich Co. (Hamburg, Germany). Water used in the incubation

103

system and the aqueous solution was purified through a Milli-Q system (Millipore,

104

Bedford, MA). Acetonitrile and methanol in this study are LC-MS grade (Fisher

105

Chemical, Harnover Park, IL). All the other chemicals and reagents were analytical

106

grade. Phoenix WinNonlin 6.3 from Pharsight (Sunnyvale, CA, United states) was

107

used to analyze the toxicokinetic data.

108

Animals used for toxicokinetic studies of HT-2 toxin. Six Sprague Dawley rats

109

(weighing about 180-220 g, 6-week, half male and half female) were bought from

110

Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The rats were

111

housed and acclimated for 7 d under standardized condition before dosing. Water was

112

available ad libitum. Animal experiment was conducted according to the principle of

113

ethical

114

(2016-SYXK-0124). Three rats (one female and two males) were given a single HT-2

115

toxin intravenous (i.v) administration and the other three rats (two females and one

116

male) were administered orally (p.o) by needle lavage at the dosage of 1.0 mg/ kg

117

body weight. Considering the solubility of HT-2 toxin, 1 mL of 20% ethanol was used

118

to dissolve HT-2 toxin. After a 7-day washout period, the rats were administrated i.v

committee

of

China

Agricultural

University

6


(Beijing,

China)

Page 7 of 35


119

or p.o abiding by a crossover design (the rats intravenously administered with HT-2

120

toxin would be administered orally whereas the rats administered orally would be

121

given HT-2 toxin intravenously. Thus, total of 6 rats were used for each

122

administration routes). The blood samples were collected from jugular vein prior to

123

and 5, 10, 15, 30, 45, 60, 90, 120, 240, 360 min after administration. About 300 µL of

124

blood was collected at each time point above-mentioned. The collected blood samples

125

were centrifuged at 2 000 g for 20 min at 4 oC. The supernatant was gathered and

126

stored at -20 oC before analysis.

127

Analytical method for determination of HT-2 toxin in plasma samples. The

128

plasma samples were determined according to a previously reported analytical method

129

with modifications.26 By adding 300 µL of acetonitrile/acetate (1:3, v/v) into 150 µL

130

of centrifuged plasma, proteins were precipitated and centrifuged at 10 141 g for 10

131

min. The supernatant was evaporated to dryness and reconstituted in initial mobile

132

phase. The samples were filtered through a 0.22 µm micropore membrane filter was

133

purchased from Pall Corporation (New York, NY). This modified analytical method

134

to determine HT-2 toxin in plasma was optimized and validated. The limit of

135

detection and the limit of quantification of HT-2 toxin were 1.0 ng/mL and 4.0 ng/mL.

136

An ACQUITY ultrahigh performance liquid chromatography (UPLC system) (Waters

137

Co., Milford, MA) equipped with mass spectrometer (Waters, Manchester, UK) was

138

used to determine HT-2 toxin and its major metabolites. A BEH RP 18 column (50

139

mm × 2.1 mm i.d., 1.7 µm particle size) (waters, New Brunswick, NJ) was used to

140

achieve chromatographic separation with a flow rate of 0.30 mL/min. Mobile phase 7



141

and gradient elution program was the same as that in the literature.26 The collision

142

induced association was performed in positive electrospray ionization mode. The

143

critical parameters were capillary voltage, 3.2 kV; source temperature, 120 oC;

144

desolvation temperature 350 oC. Low energy (2 eV) and high energy (10-25 eV) data

145

were acquired via MSE mode.

146

In vitro metabolism and sample treatment. The preparation of liver microsomes,

147

investigation of in vitro metabolism of HT-2 toxin and sample treatment of

148

microsomal incubation were performed substantially as described previously.27

149

Briefly, liver microsomes of rats, chickens, swine, goats, cows and humans were

150

incubated with HT-2 toxin, respectively. The incubation system was in 500 µL of 0.1

151

M phosphate buffer which contained liver microsomes, HT-2 toxin, and NADPH.

152

Then the mixture was processed according to the procedure previously described.27

153

Phenotyping for HT-2. Recombinant humans cytochrome enzymes (CYP 1A2, 2B6,

154

2C8, 2C9, 2C19, 2D6, 3A4) and carboxylesterases (CES I and CES II) were

155

incubated with HT-2 toxin to investigate the roles of these enzymes in the metabolism.

156

Moreover, CES I and CES II were also incubated with T-2 toxin. The incubation

157

system contained phosphate buffer (100 mM, pH 7.4), enzymes (30 pmol) and

158

NADPH (2 mM) along with 3 µM of HT-2 toxin. After incubation for 1 h at 37 °C,

159

100 µL of ice-cold acetonitrile was added to terminate the reaction and pretreated

160

according to the procedure for in vitro metabolic samples. The in vitro metabolism

161

assays were performed in triplicate and an additional sample without NADPH served

162

as the negative control. 8


Page 8 of 35

Page 9 of 35


163

UPLC-Q/TOF conditions. The chromatographic separation was achieved through an

164

Acquity HSS T3 (100 mm×2.1 mm, 1.7 µm) (Waters, Milford, MA) while the column

165

temperature was set as room temperature. Mobile phase A was aqueous solution with

166

0.05 mM aqueous ammonia and mobile phase B was acetonitrile. Gradient program

167

was performed as follows with a flow rate of 0.30 mL/min: 0-1.0 min, 5-5%B;

168

1.0-10.0 min, 5-30%B; 10.0-13.0 min, 30-50%B; 13.0-14.0 min, 50-100%B;

169

14.0-15.0 min, 5%B. The injection volume was 10.0 µL. Quadrupole-Time- of- Flight

170

(Q-TOF), with an electrospray source ionization was utilized in positive mode in the

171

present study to identify potential metabolites. The major parameters were: capillary

172

voltage, 3.0 kV; cone voltage, 20 V; ion source temperature, 100 oC; de-solvent

173

temperature, 300 oC; de-solvent flow rate, 600 L/h. Data was acquired in MSE mode

174

with high and low energy at 2 eV and 10-25 eV. For MS/MS mode, the collision

175

energy was set as 7-20 eV.

176

Data analysis. Obtained data was analyzed through the software, MetabolynxXS

177

(waters). This software is commonly used for the identification of metabolites based

178

on mass defect filter (MDF), background subtraction (BS) as well as the accurate

179

extracted ion chromatograph (EIC). In comparison with the samples of control group,

180

it is of great efficiency to determine potential metabolites. To ensure the accuracy,

181

mass window was set within 5 ppm during identification of metabolites.

182

RESULTS AND DISCUSSION

9



183

Toxicokinetic trials. Plasma concentrations of HT-2 toxin were determined using a

184

developed and validated ultrahigh performance liquid chromatography tandem mass

185

spectrometry (UPLC-MS/MS) method.26 The limit of quantitation was 4.0 ng/mL.

186

Toxicokinetic studies of HT-2 toxin in rats show that the plasma concentrations, after

187

oral administration, are all below limit of quantitation (LOQ). For i.v administration,

188

HT-2 toxin is detected in plasma with the maximum concentration of 80.55 ng/mL.

189

3-GlcA HT-2 and 3'-OH HT-2 are the major compounds of HT-2 toxin in plasma and

190

the corresponding plasma concentration-time curves are presented in Figure 2. Figure

191

2A is the plotted mean plasma time vs concentration curve of HT-2 toxin after i.v

192

administration. Figure 2B shows the mean concentration vs time curves of 3'-OH

193

HT-2 after i.v and po administration. Figure 2C illustrates the concentration-time

194

curve of HT-2-3-GlcA after i.v administration. The typical pharmacokinetic

195

parameters of HT-2 toxin following i.v administration are as follows: elimination

196

half-life (t1/2λz), 10.5 ± 0.6 min; area under the plasma concentration-time curve from

197

0 to infinite, 1077.2 ± 195.1 min·ng/mL; total body clearance, 943.8 ± 141.0 mL

198

/min/kg; mean residence time, 15.4 ± 0.5 min. After oral administration, HT-2 toxin is

199

not detected in plasma, a hydroxylated metabolite of HT-2 toxin, 3'-OH HT-2 is

200

identified from the first-time point and then decreases gradually for the remaining

201

time.

202

MS/MS spectrum interpretation of HT-2 toxin. HT-2 toxin, the most important

203

metabolite of T-2 toxin in mammals, also naturally exists in Fusarium contaminated

204

grains and often co-exists with T-2 toxin.28 HT-2 toxin and T-2 toxin have the similar 10


Page 10 of 35

Page 11 of 35


205

chemical structure and they differ at the C-4 position, with T-2 toxin being acetylated.

206

Thus, the MS behavior for metabolic study of HT-2 toxin should be much like that of

207

T-2 toxin. To form the stable molecular ion [M+NH4]+, m/z 442.2411, 0.05 mM

208

aqueous ammonia is added in aqueous solution. Then the MS/MS spectrum of HT-2

209

toxin is acquired under collision induced dissociation (CID) and fragment ions are

210

generated, such as m/z 425, 323, 263, 245, 233, 215, 197 and m/z 169. The MS/MS

211

pattern is shown in detail in Figure 3. Seen from Figure 3, the precursor ion could

212

form m/z 425 by loss of NH3. Then the product ion could generate m/z 323 by the

213

cleavage of side chain at the C-8 position (C5H11O, amyl acyl) and the C-3 or the C-4

214

position (H2O). The product ion, m/z 263, is formed from m/z 323 by loss of acetyl

215

group at the C-15 position. After that, the ions, m/z 233 and m/z 245 are generated

216

from m/z 263 by loss of CH2O and H2O, respectively, and finally product ion, m/z 215

217

is formed. Fragment ion, m/z 215 generated ions, m/z 197 and m/z 169 by the

218

subsequent loss of H2O and CO.

219

Phase I metabolism of HT-2 toxin in vitro. After incubation, all the positive and

220

control samples were simply pretreated and determined using UPLC-Q/TOF. As a

221

result, a total of eight metabolites were identified. The elemental composition of each

222

metabolite, measured values, theoretical values, mass error and double bond

223

equivalent values (DBE) of each product ion are summarized in Table 1 and the

224

corresponding spectra illustrated in Figure 3. Metabolic annotation of potential

225

metabolites of HT-2 toxin is depicted in the following sections.

226

Structural interpretation of potential metabolites of HT-2 toxin. 11



227

Hydrolytic metabolites MHT-1 and MHT-2. The retention time of MHT-1 and

228

MHT-2 are 3.29 min and 5.85 min, respectively, but they had the same mass value of

229

m/z 358.1866 ([C17H28NO7]+), which represents the scission at amyl acyl group of the

230

C-8 position to HT-2 toxin, suggesting that these two metabolites are hydrolytic

231

products. For MHT-1, its fragment ions m/z 263, m/z 245, m/z 215, m/z 197 and m/z

232

169, are similar to those of HT-2 toxin, inferring the core structure of MHT-1

233

maintains unchanged. Besides, typical neutral loss of 102 Da (amyl acyl group) at the

234

C-8 position is not observed in MS/MS spectrum of MHT-1, which further

235

demonstrates that hydrolysis occurs at the C-8 position. Thus, MHT-1 is identified as

236

4-de-Ac neosolaniol. For MHT-2, the acetyl group at the C-15 is hydrolyzed, forming

237

T-2 triol. However, T-2 triol is extremely polar and not easily ionized. It is likely to

238

subsequently be acetylated at the C-4 and cleaved at the C-8. Thus, MHT-2 is

239

previously identified as a metabolite of T-2 toxin and designated as 4-acetoxy T-2

240

tetraol.

241

Hydroxylated metabolites MHT-3 to MHT-8. For MHT-3 to MHT-8, they yields

242

the same ammonia adduct, [M+NH4]+ with the mass value of m/z 358.1866

243

([C17H28NO7]+), with different retention times (MHT-3, 8.44; MHT-4, 8.65; MHT-5,

244

8.91; MHT-6, 10.21; MHT-7, 10.43; MHT-8, 12.63). These metabolites are

245

considered as mono-hydroxylated or oxidative products of HT-2 toxin considering the

246

addition of a hydroxy group within 5 ppm mass error with calculated exact mass

247

value.

12


Page 12 of 35

Page 13 of 35


248

In MS/MS spectra of MHT-3, MHT-4 and MHT-5, the neutral loss of 118 Da is 16

249

Da higher than the neutral loss of 102 Da in HT-2 toxin, implying that these

250

metabolites are hydroxylated products at side chain of the C-8 side chain. In

251

comparison with our previously reported literature regarding metabolism of T-2 toxin,

252

MHT 3-5 share the same MS/MS spectra as MT-4, a metabolite of T-2 toxin, but

253

only MHT-3 has the same retention time as MT-4, showing that MHT-3 and MT-4

254

are the same compound.4, 13 MHT-3, thus, is interpreted as 3'-OH HT-2. It has been

255

reported that for T-2 toxin, the hydroxylation could occur at the C-3' (ω-1) position as

256

well as at the C-4' (ω) position. Hence, MHT-4 and MHT-5 are considered as

257

isomers of 4'-OH HT-2.

258

MHT-6 and MHT-7 have almost the same MS/MS spectra, showing they are likely to

259

be the stereoisomers. Additionally, a typical neutral loss of 102 Da (amyl acyl group)

260

at the C-8 position is observed, which indicates that the side chain remains

261

unchanged. Furthermore, the product ions (m/z 339, 279, 261, 249 and m/z 231) are

262

16 Da higher than product ions (m/z 263, 245, 233, 245, and m/z 215) of HT-2 toxin,

263

which further verifies the introduction of a hydroxy group on the core structure.

264

Therefore, MHT-6 and -7 are hydroxylated derivatives on the core structure.

265

Compared with the metabolism results of T-2 toxin in rats reported by our group13,

266

MHT-6 and -7 have the same m/z values and MS/MS pattern as that of 7-OH HT-2, a

267

metabolite identified in rats’ urine. Therefore, MHT-6 and -7 are tentatively

268

identified as stereoisomers of 7-OH HT-2.

13



269

For MHT-8, similar to MHT-6 and -7, loss of 102 Da (amyl acyl group) is observed,

270

implying that the C-8 position maintains unchanged. However, the MS/MS spectrum

271

of MHT-8 is significantly different from those of MHT-6 and -7, which could be

272

attributed to hydroxylated position. Interestingly, the neutral loss of 28 Da (CO) from

273

m/z 243 to m/z 215 is observed, which means that the hydroxylation occurs at a

274

carbon double-bond. Therefore, MHT-8 is tentatively considered to be 10-OH HT-2.

275

Metabolic differences of HT-2 toxin in various species and major metabolic

276

pathway in phase I incubation system. After 2 h incubation, more than half of the

277

amount of HT-2 toxin remains un-metabolized. The predominant metabolic routes of

278

HT-2 toxin are hydrolysis and hydroxylation in liver microsomes. Moreover, there is

279

a significant inter-species metabolic difference for HT-2 toxin. Metabolites and

280

corresponding amount of these metabolites are illustrated in Figure 4A. It is obvious

281

that MHT-3 (3'-OH HT-2) is the major metabolite of HT-2 toxin in all animal and

282

humans microsomes. In addition, in rat microsomes, MHT-7 (7-OH HT-2) also has a

283

high yield, indicating that the critical hydroxylation sites of HT-2 toxin in rats are at

284

the C-3' and the C-7 positions. Additionally, a small amount of MHT-7 is found in

285

liver microsomes of swine, goats, cows and humans, but not in chickens. However, in

286

chickens, MHT-8 (10-OH HT-2) has a higher amount and even more than MHT-3.

287

Thus, hydroxylation at the C-10 position is a predominant metabolic pathway of HT-2

288

toxin in chickens. MHT-4 and MHT-5, the hydroxylated products at the C-4' position,

289

are isomers, which are merely detected in swine. MHT-6 is not detected in chickens,

290

but identified in liver microsomes of other species at trace level concentrations. 14


Page 14 of 35

Page 15 of 35


291

MHT-1, the acetyl-hydrolyzed derivative of HT-2 toxin at the C-15 position, is

292

detected in liver microsomes of rats, swine, goats, cows and humans. MHT-1 is

293

identified in swine in trace level, but undetectable in chickens. For MHT-2, it is

294

higher in liver microsomes of chickens and swine and smaller in rats. Due to the

295

potential difference of the ionization efficiency concerning HT-2 toxin metabolites, it

296

is better to synthesize the metabolites to elucidate the derivatives, not to rely solely on

297

the high-resolution mass spectrometry. In conclusion, hydroxylation at the C-3', C-7

298

and C-10 positions are the primary metabolic pathways of HT-2 toxin.

299

Phase II metabolism of HT-2 toxin in microsomes of rats, chickens, swine, goats,

300

cows and humans. Three phase II metabolites and two hydrolytic metabolites

301

(4-de-Ac neosolaniol and 4-acetoxy T-2 tetraol), are identified. Figure 3 illustrates

302

MS/MS spectra of the three Phase II metabolites in microsomes of livestock and

303

humans. By comparing the spectra with those of T-2 toxin, two phase II metabolites

304

of HT-2 toxin are identified, which are 3-GlcA HT-2 and 4-GlcA HT-2. The

305

precursor of MHT-11 is m/z 534.2187, ([M+NH4]+, [C23H36NO13]+), which is 176 Da

306

higher than that of MHT-1 (4-deacetyl neosolaniol), indicating MHT-11 is likely to

307

be the GlcA binding product of MHT-1. Additionally, in the MS/MS spectrum of

308

MHT-9, typical neutral loss of GlcA, 176 Da (m/z 439 to m/z 263) is observed, and

309

the fragment ions of MHT-11, m/z 263, 245, 215 and m/z 197, are identical to those

310

of MHT-1 (4-deacetyl neosolaniol).13 Therefore, MHT-11 is considered as 3-GlcA-

311

4-deacetyl neosolaniol.

15



312

Phase II metabolic routes in vitro of HT-2 toxin as well as their interspecies

313

metabolic differences. Hydroxylation (C-3', 4', 7), hydrolysis (C-15) and

314

glucuronidation (C-3 and C-4) are the major routes for HT-2 toxin. The metabolic

315

pathways of HT-2 toxin and T-2 toxin have much in common but there are still

316

significant metabolic differences between HT-2 toxin and T-2 toxin. Hydroxylation is

317

the major metabolic pathway for HT-2 toxin, especially at the C-3'. Interestingly, for

318

chickens, the dominant product of HT-2 toxin is 10-OH HT-2. The relative amount of

319

each metabolites of HT-2 toxin is illustrated in Figure 4B. It is clear that the most

320

important conjugative site of HT-2 toxin in swine liver microsomes is at the C-4,

321

followed by the C-3 position.

322

Metabolic phenotype of HT-2 toxin. Wu et al.29,30 put forward that both CYP 450

323

and carboxylesterase enzymes played a role in the metabolism of T-2 toxin. In the

324

present study, Figure 4C shows that T-2 toxin is mainly hydrolyzed by CES-I and

325

CES-II while the potency of CES-II is approximately 4-fold that of CES-I. However,

326

the hydrolysis of HT-2 toxin mediated by CES-I and CES-II is weak. CYP 450

327

isoenzyme, 3A4 plays vital roles in the metabolism of HT-2 toxin, which could be

328

observed in Figure 4D. Isoenzyme 2C19 and 2C8 also plays a little role in

329

transformation of HT-2 toxin. The cytochrome P450 superfamily has been reported as

330

the major phase I metabolic enzymes, accounting for 75% of in vivo drug metabolism.

331

Metabolic differences between T-2 toxin and HT-2 toxin. Ohta et al.31 investigated

332

the metabolism of T-2 toxin in rats, mice and rabbits and reported the hydrolyzed

333

product at acetyl group (HT-2 toxin). They also determined the non-specific enzyme 16


Page 16 of 35

Page 17 of 35


334

(carboxylesterase) in liver that mediated the hydrolysis by chemical enzyme inhibition.

335

Subsequently, Yoshizawa et al.32 proposed that the predominant metabolite of T-2

336

toxin was HT-2 toxin, a hydrolyzed product at the C-4 position. However, it was then

337

observed that the major metabolite of T-2 toxin was HT-2 toxin in terrestrial animals,

338

but neosolaniol in aquatic animals.9 Swanson et al.33 reported that de-epoxide reaction

339

was a vital pathway of T-2 toxin for detoxification and the de-epoxidation was

340

mediated by intestinal microbial action, not by liver.

341

Until now, the metabolic profile of HT-2 toxin is still limited. The importance of

342

HT-2 toxin was emphasized and evaluated by Joint Expert Committee on Food

343

Additives (JECFA) and noted that the toxicity of T-2 toxin resulted from T-2 toxin

344

and its major metabolite, HT-2 toxin, simultaneously. Despite the similar chemical

345

structure, the metabolic profile of HT-2 toxin varied from that of T-2 toxin. It is

346

revealed, in our study, that hydroxylation, especially hydroxylation at the C-3'

347

position, is the dominant metabolic route of HT-2 toxin. Compared with the

348

un-metabolized parent toxin, the metabolic rate and extent of HT-2 toxin are both

349

lower than that of T-2 toxin in liver microsomes. In addition to the hydroxylation at

350

the C-3' position, the same as that of T-2 toxin, HT-2 toxin has several specific

351

hydroxylated sites, including the C-4', C-7 and C-10 positions. A novel hydroxylated

352

metabolite was observed by Yoshizawa et al.34 through in vivo metabolism of T-2

353

toxin, but its chemical structure was not elucidated. Subsequently, this hydroxylated

354

product was elucidated into 3'-OH-7-OH HT-2 by Pawlosk and Mirocha35using MS

355

techniques. In the present study, hydroxylation, hydrolysis and glucuronidation are the 17



356

major metabolic routes for HT-2 toxin. Lin et al.36 conducted the species metabolic

357

difference of T-2 toxin in liver microsomes. In the current study, the metabolic

358

differences among species are observed as well. MHT-1 is identified in the liver

359

microsomes of rats, goats, cows and humans, but not in chickens. Nevertheless,

360

MHT-2, the isomer of MHT-1, is identified in liver microsomes of chickens, and also

361

trace level in liver microsomes of rats and swine. Similar to the phase II metabolism

362

of T-2 toxin, GlcA binding sites of HT-2 toxin is primarily at the C-3 hydroxy group,

363

followed by the C-4 hydroxy group. For phase II metabolism in the liver microsomes

364

of chickens, no GlcA conjugate of T-2 toxin is observed, but GlcA conjugate of HT-2

365

toxin is detected in trace level.

366

The metabolic pathways of HT-2 toxin and T-2 toxin have much in common but there

367

are still significant metabolic differences between HT-2 toxin and T-2 toxin.

368

Additionally, It is revealed that the metabolism of HT-2 toxin is primarily mediated

369

by isoenzyme 3A4. It is first proposed that the hydrolysis of T-2 toxin is mediated by

370

CES-II, followed by CES-I. It has been reported that the double bond formed by the

371

C-9 and C-10 as well as the epoxy ring formed by the C-12 and C-13, were the toxic

372

groups.37 In addition, the acetyl amyl group at the C-8 position and acetyl group at the

373

C-4 and C-5 motif could enhance the toxicity.37 Other hydroxylated products were

374

prone to be excreted due to increased polarity. GlcA binding reaction is a critical

375

mechanism for detoxification and the GlcA conjugate often had no activity and easily

376

eliminated. The detailed metabolic pattern of HT-2 toxin is shown in Figure 5.

18


Page 18 of 35

Page 19 of 35


377

The cleavage of the acetyl group at the C-4 position does not distinctly attenuate the

378

toxicity whereas the loss of the amyl acyl group strikingly decreases the

379

cytotoxicity.37 Hydroxylation at the C-3' position is a theoretical activation pathway

380

for HT-2 toxin and formation from HT-2 toxin to T-2 tetraol or to T-2 triol is an

381

inactivation pathway. Namely, the toxicity of MHT-1, MHT-2, MHT-10 and three

382

GlcA conjugates would sharply decrease whereas the toxicity of MHT-3-5 would

383

maintain or even increase.37 Overall, the obtained toxicokinetics, metabolic and

384

phenotyping information would facilitate means for detoxification and risk

385

assessment.

386

ABBREVIATIONS USED

387

GlcA, glucuronic acid; i.v, intravenous; p.o, oral administration; MDF, mass defect

388

filtering; BS, background subtraction; EIC, extracted ion chromatograms; NADPH,

389

β-nicotinamide

390

uridine-5’-diphosphoglucuronic acid; CYP 450, cytochrome P450 enzymes;

391

carboxylesterases, CES; CID, collision-induced dissociation; LOQ, limit of

392

quantitation;

393

ultrahigh-performance

394

tolerable daily intake.

395

ACKNOWLEDGMENTS

adenine

DBE,

double liquid

dinucleotide

bond

equivalent

phosphates;

values;

UDPGA,

UPLC-Q/TOF,

chromatography−quadrupole/time-of-flight;

19


TDI,


396

This work is financially supported by the National Natural Science Foundation of

397

China (No. 31702296).

398

CONFICT OF INTEREST

399

Authors declare that there are no conflicts of interest.

400

SUPPORTING INFORMATION

401

The accurate MS/MS spectra and proposed fragmentation pathway of HT-2 toxin

402

(Figure S1), extracted ion chromatograms of HT-2 toxin phase I (Figure S2), phase II

403

(Figure S3) metabolites, chromatogram of HT-2 toxin at the limit of quantitation

404

(Figure S4), chromatogram of blank sample (Figure S5), Analytical method validation

405

(Table S1) and toxicokinetic parameters for HT-2 toxin (Table S2) were supplied as

406

Supporting Information, which was available free of charge via the internet at

407

http://pubs.acs.org.

408

REFERENCES

409

1.

Scott, P. M., Trichothecenes in grains. Cereal Food World 1990, 35, 661-666.

410

2.

Ueno, Y., Mode of action of trichothecenes. Ann. Nutr. Aliment. 1977, 31,

411

885-900.

412

3.

413

T-2 toxin, HT-2 toxin and emetine correspond to plasma elevations of peptide

414

YY3-36 and 5-hydroxytryptamine. Arch. Toxicol. 2016, 90, 997-1007.

Wu, W.; Zhou, H. R.; Bursian, S. J.; Link, J. E.; Pestka, J. J., Emetic responses to

20


Page 20 of 35

Page 21 of 35


415

4.

Yang, L.; Tu, D.; Zhao, Z.; Cui, J., Cytotoxicity and apoptosis induced by mixed

416

mycotoxins (T-2 and HT-2 toxin) on primary hepatocytes of broilers in vitro. Toxicon.

417

2017, 129, 1-10.

418

5.

419

oxidative stress induced by T-2 toxin and HT-2 toxin in broilers and broiler

420

hepatocytes. Food Chem. Toxicol. 2016, 87, 128-137.

421

6.

422

Exposure to HT-2 toxin causes oxidative stress induced apoptosis/autophagy in

423

porcine oocytes. Sci. Rep. 2016, 6, 33904.

424

7.

425

on mouse oocytes and its possible mechanisms. Arch. Toxicol. 2016, 90, 1495-1505.

426

8.

427

induces thymic apoptosis in vivo in mice. Toxicol Appl. Pharmacol. 1998, 148,

428

205-214.

429

9.

430

Curr. Drug Metab. 2008, 9, 77-82.

431

10. Lautraite, S.; Parent-Massin, D.; Rio, B.; Hoellinger, H., Comparison of toxicity

432

induced by HT-2 toxin on human and rat granulo-monocytic progenitors with an in

433

vitro model. Hum. Exp. Toxicol. 1996, 15, 208-213.

434

11. Choi, S. U.; Choi, E. J.; Kim, K. H.; Kim, N. Y.; Kwon, B. M.; Kim, S. U.; Bok,

435

S. H.; Lee, S. Y.; Lee, C. O., Cytotoxicity of trichothecenes to human solid tumor

436

cells in vitro. Arch. Pharm. Res. 1996, 19, 6-11.

Yang, L.; Yu, Z.; Hou, J.; Deng, Y.; Zhou, Z.; Zhao, Z.; Cui, J., Toxicity and

Zhang, Y.; Han, J.; Zhu, C. C.; Tang, F.; Cui, X. S.; Kim, N. H.; Sun, S. C.,

Zhu, C. C.; Zhang, Y.; Duan, X.; Han, J.; Sun, S. C., Toxic effects of HT-2 toxin

Islam, Z.; Nagase, M.; Yoshizawa, T.; Yamauchi, K.; Sakato, N., T-2 toxin

Dohnal, V.; Jezkova, A.; Jun, D.; Kuca, K., Metabolic pathways of T-2 toxin.

21



437

12. Pace, J. G.; Matson, C. F., Stability of T-2, HT-2, and T-2 tetraol in biological

438

fluids. J. Anal. Toxicol. 1988, 12, 48-50.

439

13. Yang, S. P.; Li, Y. S.; Cao, X. P.; Hu, D. F.; Wang, Z. H.; Wang, Y.; Shen, J. Z.;

440

Zhang, S. X., Metabolic pathways of T-2 toxin in in vivo and in vitro systems of

441

wistar rats. J. Agric. Food Chem. 2013, 61, 9734-9743.

442

14. EFSA, Scientific opinion on the risks for animal and public health related to the

443

presence of T-2 and HT-2 toxin in food and feed. EFSA panel on contaminants in the

444

food chian (CONTAM). EFSA J. 2011, 9, 2481.

445

15. EFSA, Appropriateness to set a group health based guidance value for T-2 and

446

HT-2 toxin and its modified forms. EFSA J. 2017, 15, 53.

447

16. Osselaere, A.; Li, S. J.; De Bock, L.; Devreese, M.; Goossens, J.;

448

Vandenbroucke, V.; Van Bocxlaer, J.; Boussery, K.; Pasmans, F.; Martel, A.; De

449

Backer, P.; Croubels, S., Toxic effects of dietary exposure to T-2 toxin on intestinal

450

and hepatic biotransformation enzymes and drug transporter systems in broiler

451

chickens. Food Chem. Toxicol. 2013, 55, 150-155.

452

17. Sintov, A.; Bialer, M.; Yagen, B., Pharmacokinetics of T-2 toxin and its

453

metabolite HT-2 toxin, after intravenous administration in dogs. Drug Metab Dispos.

454

1986, 14, 250-254.

455

18. Beeton, S.; Bull, A. T., Biotransformation and detoxification of T-2 toxin by soil

456

and freshwater bacteria. Appl. Environ. Microbiol. 1989, 55, 190-197.

457

19. Gareis, M.; Ertl, B.; Bauer, J.; Gedek, B., Biotransformation of T-2 toxin and

458

diacetoxyscirpenol in the isolated perfused rat liver. Mycotoxin Res. 1985, 1, 77-82. 22


Page 22 of 35

Page 23 of 35


459

20. Rafai, P.; Papp, Z.; Jakab, L., Biotransformation of trichothecenes alleviates the

460

negative effects of T-2 toxin in pigs. Acta. Vet. Hung. 2013, 61, 333-343.

461

21. Meng-Reiterer, J.; Bueschl, C.; Rechthaler, J.; Berthiller, F.; Lemmens, M.;

462

Schuhmacher, R., Metabolism of HT-2 toxin and T-2 toxin in oats. Toxins (Basel)

463

2016, 8, 364.

464

22. Nathanail, A. V.; Varga, E.; Meng-Reiterer, J.; Bueschl, C.; Michlmayr, H.;

465

Malachova, A.; Fruhmann, P.; Jestoi, M.; Peltonen, K.; Adam, G.; Lemmens, M.;

466

Schuhmacher, R.; Berthiller, F., Metabolism of the fusarium mycotoxins T-2 toxin

467

and HT-2 toxin in wheat. J. Agric. Food Chem. 2015, 63, 7862-7872.

468

23. Zhang, Y.; Jia, R. X.; Pan, M. H.; Lu, Y.; Cui, X. S.; Kim, N. H.; Sun, S. C.,

469

HT-2 toxin affects development of porcine parthenotes by altering DNA and histone

470

methylation in oocytes matured in vitro. Theriogenology 2017, 103, 110-116.

471

24. Johnsen, H.; Odden, E.; Lie, O.; Johnsen, B. A.; Fonnum, F., Metabolism of T-2

472

toxin by rat liver carboxylesterase. Biochem Pharmacol 1986, 35, 1469-1473.

473

25. Sun, Y. X.; Yao, X.; Shi, S. N.; Zhang, G. J.; Xu, L. X.; Liu, Y. J.; Fang, B. H.,

474

Toxicokinetics of T-2 toxin and its major metabolites in broiler chickens after

475

intravenous and oral administration. J. Vet. Pharmacol. Ther. 2015, 38, 80-85.

476

26. Yang, S.; Wang, Y.; Beier, R. C.; Zhang, H.; De Ruyck, K.; Sun, F.; Cao, X.;

477

Shen, J.; Zhang, S.; Wang, Z., Simultaneous Determination of Type A and B

478

Trichothecenes and Their Main Metabolites in Food Animal Tissues by

479

Ultraperformance Liquid Chromatography Coupled with Triple-Quadrupole Mass

480

Spectrometry. J. Agric. Food Chem. 2015, 63, 8592-8600. 23



Page 24 of 35

481

27. Sun, F.; Yang, S.; Zhang, H.; Zhou, J.; Li, Y.; Zhang, J.; Jin, Y.; Wang, Z.; Li,

482

Y.; Shen, J.; Zhang, S.; Cao, X., Comprehensive Analysis of Tiamulin Metabolites in

483

Various

484

Chromatography Coupled to Quadrupole/Time-of-Flight. J. Agric. Food Chem. 2017,

485

65, 199-207.

486

28. Yoshinari, T.; Takeuchi, H.; Aoyama, K.; Taniguchi, M.; Hashiguchi, S.; Kai, S.;

487

Ogiso, M.; Sato, T.; Akiyama, Y.; Nakajima, M.; Tabata, S.; Tanaka, T.; Ishikuro, E.;

488

Sugita-Konishi, Y., Occurrence of four Fusarium mycotoxins, deoxynivalenol,

489

zearalenone, T-2 toxin, and HT-2 toxin, in wheat, barley, and Japanese retail food. J

490

Food Prot. 2014, 77, 1940-1946.

491

29. Wu, Q.; Huang, L.; Liu, Z.; Yao, M.; Wang, Y.; Dai, M.; Yuan, Z., A comparison

492

of hepatic in vitro metabolism of T-2 toxin in rats, pigs, chickens and carp.

493

Xenobiotica. 2011, 41, 863-873.

494

30. Wu, Q.; Wang, X.; Yang, W.; Nusseler, A.; Xiong, L.; Kuca, K.; Dohnal, V.;

495

Zhang, X.; Yuan, Z., Oxidative stress-mediated cytotoxicity and metabolism of T-2

496

toxin and deoxynivalenol in animals and humans: an update. Arch Toxicol. 2014, 88,

497

1309-1326.

498

31. Ohta, M.; Ishii, K.; Ueno, Y., Metabolism of trichothecene mycotoxins. I.

499

Microsomal deacetylation of T-2 toxin in animal tissues. J. Biochem. 1977, 82,

500

1591-1598.

501

32. Yoshizawa, T.; Mirocha, C. J.; Behrens, J. C.; Swanson, S. P., Metabolic fate of

502

T-2 toxin in a lactating cow. Food Cosmet. Toxicol. 1981, 19, 31-39.

Species

of

Farm

Animals

Using

Ultra-High-Performance

24


Liquid

Page 25 of 35


503

33. Swanson, S. P.; Nicoletti, J.; Rood, H. D., Jr.; Buck, W. B.; Cote, L. M.;

504

Yoshizawa, T., Metabolism of three trichothecene mycotoxins, T-2 toxin,

505

diacetoxyscirpenol and deoxynivalenol, by bovine rumen microorganisms. J.

506

Chromatogr. 1987, 414, 335-342.

507

34. Yoshizawa, T.; Sakamoto, T.; Kuwamura, K., Structures of deepoxytrichothecene

508

metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats. Appl. Environ.

509

Microbiol. 1985, 50, 676-679.

510

35. Pawlosky, R. J.; Mirocha, C. J., Structure of a metabolic derivative of T-2 toxin

511

(TC-6) based on mass spectrometry. J. Agric. Food Chem. 1984, 32, 1420-1423.

512

36. Lin, N.; Guo, L.; Chen, J.; Xie, J., Species difference of T-2 toxin metabolism in

513

liver microsomes by high performance liquid chromatography-tandem mass

514

spectrometry. Chin J Pharmacol Toxicol. 2017, 31, 754-759.

515

37. Wu, Q.; Dohnal, V.; Huang, L.; Kuca, K.; Yuan, Z., Metabolic pathways of

516

trichothecenes. Drug Metabo. Rev. 2010, 42, 250-26

25



FIGURE CAPTIONS Figure 1: The chemical structures of HT-2 toxin and T-2 toxin, which differs at C-4 position. Figure 2: Plotted mean plasma Time vs Concentration curve of HT-2 toxin after i.v administration to rats at a dose of 1.0 mg/kg body weight (n=6). The highest concentration was 80.0 ng/mL. (A) Plotted mean plasma time vs concentration curve of HT-2 toxin after i.v administration to rats at a dose of 1.0 mg/kg body weight (n=6). (B) Mean peak area-time curves of 3’-OH HT-2 after i.v and po administration. (C) Peak area-time curve of 3-GlcA HT-2 after i.v administration (n=6). Figure 3: Spectra and proposed fragmentation pathway of HT-2 toxin and its metabolites. The mass tolerance of each metabolite was within 5 ppm. Figure 4: (A) Phase I metabolism of HT-2 toxin in different species and human liver microsomes. (B) Phase II metabolism of HT-2 toxin in various animals and human liver microsomes. (C) Attribution of CES-I and CES-II enzymes to T-2 toxin and HT-2 toxin, x-axis is the relative percentage. (D) CYP reaction phenotyping for HT-2 toxin, x-axis is the relative percentage. Figure 5: Detailed metabolic pattern of HT-2 toxin.

26


Page 26 of 35

Page 27 of 35


Figure 1

27



Figure 2

28


Page 28 of 35

Page 29 of 35


Figure 3

29



Figure 4

30


Page 30 of 35

Page 31 of 35


Figure 5

31



Page 32 of 35

Table 1. Summary of Proposed Phase I and Phase II metabolites (MHT-1-11) of HT-2 Toxin Observed in Liver Microsomes from Different Species of Animals and Humans [M+NH4]+ No.

Metabolites

Composition (m/z)

Error

Liver microsomes

Retention

(ppm) time (min)

Major fragments rat

chicken

swine

goat

cow

human

HT-2

HT-2 toxin

C22H36NO8+

442.2441

2.4

5.38

√

√

√

√

√

√

425, 323, 263*, 245, 215, 197, 169

MHT-1

4-de-Ac neosolaniol

C17H28NO7+

358.1866

-1.7

3.92

√

ND

√

√

√

√

323, 275, 263*, 245, 233, 215, 197, 169, 145

MHT-2

4-acetoxy T-2 tetraol

C17H28NO7+

358.1866

3.4

5.86

√

√

√

ND

ND

ND

MHT-3

3'-OH HT-2

C22H36NO9+

458.2390

2.8

8.44

√

√

√

√

√

√

323, 263, 245, 215, 197*, 185, 157

MHT-4

4'-OH HT-2

C22H36NO8+

458.2390

-1.0

8.65

ND

ND

√

√

ND

ND

441, 323, 263, 215, 197*, 185, 157

MHT-5

4'-OH HT-2

C22H36NO8+

458.2390

-1.7

8.91

ND

ND

√

ND

√

ND

441, 323, 263, 215, 197*

MHT-6

7-OH HT-2

C22H36NO8+

458.2390

1.3

10.23

√

ND

√

√

√

√

441, 339, 321, 279*, 261, 149, 243, 231, 213, 189, 185

MHT-7

7-OH HT-2

C22H36NO8+

458.2390

0.9

10.43

√

ND

√

√

√

√

441, 339, 321, 279*, 261, 149, 243, 231, 213, 189, 185

MHT-8

10-OH HT-2

C22H36NO8+

458.2390

3.1

12.63

√

√

√

√

√

√

441, 423, 339, 321, 279, 261, 243*, 215, 213, 189, 157

MHT-9

3-GlcA HT-2

C28H44NO14+

618.2762

-3.2

3.41

√

√

√

√

√

√

601, 499, 425, 323, 263*, 245, 215, 197, 187, 169

MHT-10

4-GlcA HT-2

C28H44NO14+

618.2762

2.4

3.20

√

ND

√

√

√

√

601, 499, 439, 425, 323, 275, 263, 245, 215*, 197, 185

C23H36NO13+

534.2187

-1.9

3.46

√

ND

ND

ND

ND

√

421, 323, 305, 263*, 245, 215, 197, 185, 169

MHT-11

3-GlcA-4-de-Ac neosolaniol

32


323, 293, 263, 245, 233, 215*, 203, 197, 157, 145

Page 33 of 35


The [M+NH4]+ (m/z) values were calculated from the proposed structural formulae. The Error (ppm) is the difference between the calculated and observed m/z values. * The base peak in the MS/MS spectra; √, detected; and ND, not detected. These are tentative identification based on LC-HRMS. Ac is the abbreviation of acetylation; GlcA is short of Glucuronic acid.

33



TOC:

34


Page 34 of 35

Page 35 of 35


35


The Toxicokinetics of HT-2 Toxin in Rats and Its ... - ACS Publications

Recommend Documents