T-2 Toxin-3Î±-glucoside in Broiler Chickens: Toxicokinetics, Absolute

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T-2 TOXIN-3#-GLUCOSIDE IN BROILER CHICKENS: TOXICOKINETICS, ABSOLUTE ORAL BIOAVAILABILITY AND IN VIVO HYDROLYSIS Nathan Broekaert, Mathias Devreese, Marthe De Boevre, Sarah De Saeger, and Siska Croubels J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 21, 2017

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Journal of Agricultural and Food Chemistry

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T-2 TOXIN-3α α-GLUCOSIDE IN BROILER CHICKENS:

2

TOXICOKINETICS, ABSOLUTE ORAL BIOAVAILABILITY

3

AND IN VIVO HYDROLYSIS

4 5

NATHAN BROEKAERT1, MATHIAS DEVREESE1*, MARTHE DE BOEVRE2, SARAH

6

DE SAEGER2, SISKA CROUBELS1*

7 8

1

9

and Biochemistry, Salisburylaan 133, 9820 Merelbeke, Belgium

Ghent University, Faculty of Veterinary Medicine, Department of Pharmacology, Toxicology

10

2

11

Ottergemsesteenweg 460, 9000 Ghent, Belgium

Ghent University, Faculty of Pharmaceutical Sciences, Department of Bioanalysis,

12 13

*

14

264 74 97 (corresponding authors)

[email protected]; [email protected], phone: +32 9 264 73 47, fax: +32 9

ACS Paragon Plus Environment


15

Keywords

16

Modified – Masked – Mycotoxin – Toxicokinetics

17 18

Abstract

19

Due to the lack of information on bioavailability and toxicity of modified mycotoxins,

20

current risk assessment on these modified forms assumes an identical toxicity of the

21

modified form to their respective unmodified counterparts.

22

Cross-over animal trials were performed with intravenous and oral administration of

23

T-2 toxin (T-2) and T-2 toxin-3α-glucoside (T2-G) to broiler chickens. Plasma concentrations

24

of T2-G, T-2 and main phase I metabolites were quantified using a validated liquid

25

chromatography-tandem mass spectrometry method with a limit of quantitation for all

26

compounds of 0.1 ng/mL. Resulting plasma concentration-time profiles were processed via

27

two-compartmental toxicokinetic models.

28

No T-2 triol and only traces of HT-2 were detected in the plasma samples after both

29

intravenous and oral administration. The results indicate that T-2 has a low absolute oral

30

bioavailability of 2.17±1.80%. For T2-G, an absorbed fraction of the dose and absolute oral

31

bioavailability of 10.4±8.7% and 10.1±8.5% were observed, respectively. This slight

32

difference is caused by a minimal (and neglectable) presystemic hydrolysis of T2-G to T-2, i.e.

33

3.49±1.19%. Although low, the absorbed fraction of T2-G is 5 times higher than that of T-2.

34

These differences in toxicokinetics parameters between T-2 and T2-G clearly indicate the

35

flaw in assuming equal bioavailability and/or toxicity of modified and free mycotoxins in

36

current risk assessments.

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1. Introduction

40

Fusarium is regarded as one of the most important mycotoxigenic fungi genera. It is

41

known to produce several classes of mycotoxins of which the trichothecenes are the most

42

important based on occurrence. From this class of mycotoxins, type A trichothecenes are the

43

more toxic subclass. T-2 toxin (T-2) is the most acute toxic mycotoxin of the type A

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trichothecenes [33,34]. Although in general T-2 occurs less often than the type B

45

trichothecene deoxynivalenol (DON), several European surveys have reported T-2 incidences

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over 80% for certain cereals, and maximum concentrations up to 2,406 µg/kg have been

47

observed in naturally contaminated oats [1-4].

48

Poultry is considered to have a high exposure to T-2 as their diet is mainly cereal based.

49

Toxic effects of T-2 in poultry consist of inhibition of DNA, RNA and protein synthesis;

50

immunomodulation; gastrointestinal (GI) epithelial cell lesions and neural disturbances [5].

51

Moreover, subclinical contamination levels have been found responsible for a decreased

52

animal performance with impaired feed conversion ratios and reduced feed intake [6]. HT-2

53

toxin (HT-2) is the main metabolite of T-2 and has a comparable toxicological profile and an

54

acute toxicity in the same range as T-2 [7,8].

55

For broiler chickens, first adverse effects like mucosal damage in the oral cavity occur at

56

a dose of 40 µg/kg body weight (bw) per day. For laying hens, a reduction of the egg

57

production and hatchability were observed at doses of 120 µg/kg bw per day [6]. Based on

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these data, the European Union issued indicative levels for the sum of T-2 and HT-2 in food

59

and feed commodities, from above which investigations should be performed, certainly in

60

case of repetitive findings [9,10]. For poultry compound feed, the indicative level is set at

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250 µg T-2 + HT-2/kg feed.



62

These indicative levels suggested by legislative authorities only take into account the free

63

or parent mycotoxins, i.e. T-2 and HT-2. However, based on recent research this appears to

64

be an incomplete approach, as additionally to free mycotoxins, food and feed are often co-

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contaminated with modified mycotoxins. For T-2 specifically, in planta glycosylation of the 3-

66

hydroxyl group to T-2 toxin-3-glucoside (T2-G) is an important contributor to these modified

67

forms. The occurrence of T2-G in naturally infected wheat and oats has been demonstrated

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by Busman et al. (2011) and further researched and quantified by Lattanzio et al.

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(2012)[11,12]. Based on high-resolution mass spectrometry experiments, peak area ratios

70

between glycosylated and free T2 and HT-2 in naturally contaminated wheat and oats

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indicated amounts of glycosylated T-2 and HT-2 as high as 27% of their deglycosylated

72

counterparts [12]. Furthermore, Busman et al. (2011) and Lattanzio et al. (2013) observed

73

that glycosylation is not only part of a detoxification strategy by plants, but can also be a

74

relevant route of fungal metabolism [11, 13].

75

The European Food Safety Authority (EFSA) recently published a scientific opinion on

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modified mycotoxins. In this opinion, the relative contribution of modified forms of T-2 to

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the total occurrence of T-2 was calculated to be 10%. The risk of the T2-G contribution was

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assessed for humans and several animal species (pigs, ruminants, horses, poultry and dogs)

79

where possible. The combined risk of T-2, HT-2 and their modified forms was found not to

80

be of concern, except for the highest upperbound exposure for toddlers, with levels slightly

81

exceeding the tolerable daily intake (TDI). Furthermore, the risk assessment assumed that all

82

modified forms have the same toxicity as their parent compounds, and this due to the lack

83

of information on bioavailability and toxicity of these modified forms [1]. These knowledge

84

gaps thus pose an additional uncertainty on the derived conclusions of this scientific opinion.

85

Indeed, as shown by several studies, some modified mycotoxins may have a significantly


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86

altered intrinsic toxicity and oral bioavailability; and in addition they may be subject to in

87

vivo hydrolysis as well [14-16]. Furthermore, in vivo hydrolysis of modified mycotoxins can

88

also be species dependent, for example broiler chickens do not hydrolyse DON-3-β-D-

89

glucoside (DON3G) to DON to a substantial extent, in contrast to the complete presystemic

90

hydrolysis in pigs [17].

91

Regarding T2-G, first steps have been made by McCormick et al. (2015) with an in vitro

92

digestive study performed to simulate the human GI process of T2-G. This study consisted of

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an artificial saliva digestion assay and an in vitro human colonic fermentation assay. These

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authors found that the T-2 toxin glucosides were unaltered after incubation with artificial

95

human saliva and that after a 30 min fermentation step, there was practically no hydrolysis

96

of T2-G to T-2. However, after 24 h fermentation, less than 30% of the initially added T2-G

97

was still present [24]. In vitro tests, like the ones performed by McCormick et al., provide a

98

valuable tool to make predictions on the metabolism of a compound. However,

99

comprehensive in vivo studies for metabolite profiling and identification are complementary

100

and are often required by regulatory authorities to confirm the (proposed) metabolism [18].

101

At present, no in vivo toxicokinetic data is available for T2-G in humans or animals to the

102

authors’ knowledge. It was therefore the aim of the present study to determine the

103

absolute oral bioavailability, in vivo hydrolysis and toxicokinetic characteristics of T2-G in

104

broiler chickens.

105 106 107

2. Experimental 2.1

Standards and solutions

108

T-2, HT-2 and T-2 triol were purchased from Fermentek (Jerusalem, Israel). T-2 toxin-3α-

109

glucoside (Figure 1) was synthesized by the United States Department of Agriculture (USDA)



110

as described [18,19]. All compounds were dissolved in ethanol to yield a stock solution of 10

111

mg/mL. The internal standard (IS), 25 µg/mL

112

purchased from Sigma Aldrich (Bornem, Belgium). All stock solutions were stored at ≤−15 °C.

113

Individual working standard solutions of 100 µg/mL were prepared by diluting the above

114

stock solutions with HPLC-grade acetonitrile. All working standard solutions were stored at

115

≤−15 °C. Standard mixture working solutions (containing T-2, HT-2, T-2 triol and T2-G) of

116

5,000, 500, 50 and 5 ng/mL were prepared by mixing and diluting the above individual

117

working standard solutions in HPLC-grade acetonitrile and were stored at 2-8 °C. Acetonitrile

118

solutions of T-2 and HT-2 have been reported stable for 24 months at 25 °C [20]. For T-2 triol

119

no data is published on stability/shelf life. However, suppliers claim a stock solution shelf-life

120

of at least one month at ≤−15 °C. Regarding synthesised T2-G, no data is published on

121

storage and stability neither. However, during this study no degradation of the T2-G

122

solutions into T-2, HT-2 and T-2 triol could be detected by liquid chromatography-tandem

123

mass spectrometry (LC-MS/MS) analysis.

13

C24-T-2 toxin solution in acetonitrile, was

124 125

2.2

Chemicals, materials and instrumentation

126

Water, methanol, acetic acid and ammonium acetate (all UHPLC grade) were purchased

127

from Biosolve (Valkenswaard, The Netherlands). Acetonitrile (HPLC grade) was obtained

128

from VWR (Leuven, Belgium). Microfilters Durapore PVDF (0.22 µm) were obtained from

129

Millipore (Overijse, Belgium). Separation of analytes was achieved on an Acquity UPLC® BEH

130

C18 column (150 x 2.1 mm i.d., 1.7 μm) with an Acquity UPLC® BEH C18 VanGuard pre-

131

column (5 x 2.1 mm i.d., 1.7 µm). An Acquity UPLC system coupled to a Xevo® TQ-S mass

132

spectrometer was used, operating in positive electrospray ionization (ESI+) mode, all from

133

Waters (Zellik, Belgium).


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2.3

Plasma sample preparation

136

Blank plasma samples.

Heparinized blood from 12 h fasted broiler chickens

137

(Ross 308, Institute for Agricultural and Fisheries Research (ILVO) animal unit, Merelbeke,

138

Belgium) was centrifuged (2,851 x g, 10 min, 4 °C) to obtain blank plasma. This plasma was

139

pooled, homogenized, aliquoted and stored at ≤ – 15 °C for later use as matrix-matched

140

calibrators and quality control samples.

141

Calibrator and quality control samples.

To 200 µL of blank plasma, 20 µL of a 100

142

ng/mL IS working solution and appropriate volumes of the standard mixture working

143

solutions (5,000, 500, 50 and 5 ng/mL) were added to obtain calibrator samples with

144

mycotoxin concentrations of 0.1, 1, 5, 10, 50, 100, 500 and 1,000 ng/mL. Acetonitrile was

145

added up to a volume of 1.0 mL to precipitate plasma proteins. After vortex mixing and

146

centrifugation (8,517 x g, 10 min, 4 °C), the supernatant was isolated and evaporated to

147

dryness under nitrogen at 40 °C. The sample was then redissolved in 200 µL of a UHPLC-

148

grade water/methanol mixture (85/15; v/v), filtered (0.22 µm) and analyzed by LC-MS/MS.

149

Incurred samples from animal trials (2.6).

To 200 µL of plasma, 20 µL of a 100

150

ng/mL IS working solution were added and the samples were subjected to the same sample

151

preparation procedure as the calibrator samples.

152 153

2.4

LC-MS/MS analysis

154

Analytes were eluted at a flow rate of 300 µL/min with mobile phase A consisting of

155

water, methanol and acetic acid (97/2/1; v/v/v) + 5 mM aqueous ammonium acetate, and

156

mobile phase B consisting of water, methanol and acetic acid (2/97/1; v/v/v) + 5 mM

157

ammonium acetate. A gradient elution program was applied: 0-1 min (70% A, 30% B), 1.0-1.1



158

min (linear gradient to 50% B), 1.1-5.0 min (50% A, 50% B), 5.0-5.1 min (linear gradient to

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95% B), 5.1-8.0 min (5% A, 95% B), 8.0-8.1 min (linear gradient 30% B), 8.1-10.0 min (70% A,

160

30% B). Column and autosampler temperatures were set to respectively 40 and 8 °C, the

161

injection volume (partial loop) was fixed at 10 µL.

162

The mass spectrometer was operated in the multiple reaction monitoring (MRM)

163

mode with two ion transitions for each analyte. Instrumental and compound specific

164

parameters were optimized by the direct infusion of 10 ng/mL standard solutions in a 50/50

165

(v/v) mixture of mobile phase A and B at a flow rate of 50 µL/min. Capillary voltage was set

166

at 2.5 kV, cone at 15 V, cone gas flow at 150 L/h, desolvation temperature at 600 °C,

167

desolvation gas flow at 1000 L/h and collision gas flow at 150 µL/min. Compound specific

168

instrumental parameters, together with precursor and product ions used for quantification

169

and qualification, are given in Table 1.

170 171

2.5

Analytical method validation

172

Validation was performed on spiked blank plasma samples. The recommendations

173

issued by the European Community [21, 22] and the Veterinary International Conference on

174

Harmonisation (VICH) [23] were used as validation guidelines.

175

Linearity of the analytes’ response was assessed by means of three matrix-matched

176

calibration curves consisting of at least seven calibration points in the range of 0.1-100

177

ng/mL for HT-2, T-2 triol and T2-G, and in the range of 0.1-1,000 ng/mL for T-2. The criteria

178

and corresponding acceptability ranges for linearity were a correlation coefficient (r) of

179

≥0.99 and a goodness-of-fit coefficient (gof) of ≤20%.

180

Within-day accuracy & precision were determined by analyzing six samples spiked at

181

a low concentration level (limit of quantitation or LOQ) and a high concentration level (linear


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range maximum). Values for the relative standard deviation (RSD) should not exceed 2/3 of

183

the RSDmax, calculated according to the Horwitz equation [24]. The acceptance criteria for

184

accuracy were: −50% to +20%, −30% to +10% and −20% to +10% for concentraƟons ≤ 0.1

185

ng/mL, between 1 and 10 ng/mL, and ≥10 ng/mL, respectively.

186

Between-day accuracy & precision were assessed by analyzing three samples spiked

187

at a low concentration level (LOQ) and a high concentration level (linear range maximum) on

188

three consecutive days (n=3x3). The acceptance criteria for accuracy were identical to the

189

values given above and RSD values should not exceed the RSDmax [24].

190

The LOQ was determined as the lowest concentration for which the method had

191

acceptable results with regards to accuracy and precision. It was assessed by spiking six

192

plasma samples at 0.1, 0.2, 0.5, 1 or 2 ng/mL. The LOQ was also established as the lowest

193

point of the calibration curve. The limit of detection (LOD) was calculated using the signal-to-

194

noise (S/N) ratio of the samples spiked at the LOQ level (n=6), corresponding to the

195

concentration with a S/N ratio of 3.

196 197

Carry-over was evaluated by analyzing a mixture of mobile phase A and B (50/50; v/v) directly after the highest calibrator sample.

198

The specificity, the capability of the method to distinguish between the analytes and

199

other substances or interferences, was determined on six blank plasma samples. For an

200

acceptable specificity, the S/N ratio of possible interfering peaks with similar retention times

201

in these samples should not exceed the S/N ratio of the analyte(s)’ LOD.

202

Recovery and matrix effects. Two types of matrix-matched calibration curves were

203

prepared. One curve was prepared by spiking blank calibrator samples before extraction and

204

one curve was prepared by spiking blank calibrator samples after extraction. A third

205

calibration curve was prepared in standard solution. All curves consisted of five calibration



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points in the range of 1-100 ng/mL. The slopes of these calibration curves (x-axis: theoretical

207

concentration; y-axis: MS signal intensity uncorrected by isotopically labeled IS) were

208

compared

209

extraction/slope standard solution).

210

suppression/enhancement (SSE = 100 × slope spiked after extraction/slope standard

211

solution), and the extraction recovery RE was calculated as follows: RE = 100 × slope spiked

212

before extraction/slope spiked after extraction. Regarding SSE, values ≤ 100% indicate ion

213

suppression due to matrix effects, values ≥ 100% are caused by ion enhancement [25].

to

calculate

the

apparent

recovery

(RA = 100 × slope

spiked

The matrix effect was denoted as

before signal

214 215

2.6

Toxicokinetic animal trials

216

Six broilers chickens (Ross 308, mixed sex, 18 days of age) were housed at the Faculty

217

of Veterinary Medicine of Ghent University. Water and feed were present ad libitum. The

218

birds were allowed to acclimatize for 1 week in a climate-controlled stable with

219

temperatures between 18 and 25 °C, a relative humidity between 40 and 80% and a 18 h/6 h

220

light/dark cycle. Three broilers were given a single T2-G intravenous injection (IV) and three

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broilers a single per os bolus (PO, by means of gavage directly into the crop). After a one-day

222

washout period, the same animals were administered T-2 by IV or PO route in a two-way

223

cross-over design, with a one-day washout period (Figure 1). Twelve hours before

224

administration of T-2 or T2-G, the animals were fasted. In order to obtain detectable plasma

225

concentrations after PO administration, it was opted to use a dose of 1 mg/kg bw T2-G and

226

an equimolar dose of T-2, i.e. 0.74 mg/kg bw. Mycotoxin solutions (10 mg/mL in ethanol)

227

were diluted with saline (0.9% NaCl, VWR, Leuven, Belgium) to obtain a volume of about one

228

mL, that was appropriate for administration. Before the administration of the toxins (0 min),

229

and at 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min afterwards, blood (0.5 – 1 mL) was


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collected in heparinized tubes from the vena metatarsea plantaris. Blood samples were

231

centrifuged (2851 x g, 10 min, 4 °C) within 2 h after collection and plasma was stored at -

232

20°C. The animal trial was approved by the ethical committee of the Faculty of Veterinary

233

Medicine and the Faculty of Bioscience Engineering (EC2013/139).

234 235

2.7

Toxicokinetic modeling and statistical analysis

236

Toxicokinetic analysis was performed using WinNonlin Professional version 5.2.1.

237

(Pharsight, St-Louis, MO, USA). Plasma concentrations below the LOQ were excluded from

238

the analysis. A two-compartmental model (central compartment (Vc) and peripheral

239

compartment (Vp)) with first-order elimination kinetics was applied for the analysis of both

240

T-2 and T2-G.

241

The following primary and secondary toxicokinetic parameters for both T-2 and T2-G

242

were calculated from the IV data: total body clearance (CL), central volume of distribution

243

(Vc), peripheral volume of distribution (Vp), disposition rate constant (β) and disposition

244

half-life (t1/2β). Maximal plasma concentration (Cmax) and time to maximal plasma

245

concentration (tmax) were calculated from the PO data. A 1/ŷ weighing was applied for all

246

calculations.

247

The absolute oral bioavailability F, i.e. the fraction absorbed in the systemic circulation

248

in its unaltered form, was calculated for T-2 and T2-G from the area under the plasma

249

concentration-time curves from time 0 to infinity (AUC0-inf) as follows:

250 2 =

251



252 253

The fraction of the dose absorbed in either form, FRAC, was calculated for T-2 and T2-G by correcting for molecular weight (MW) and CL.

254 ∗ CL ∗ CL + ! 2 = ∗ CL ! 255 256 257

The presystemic hydrolysis (Pres. Hydr.) of T2-G was determined as the percentage of

258

the dose that is hydrolysed presystemically to T-2 and absorbed as T-2 (%), and was

259

calculated by correcting for molecular weight (MW) and CL.

260

#$%&. ()*$. =

∗ CL

∗ CL ∗ CL + !

261 262

Statistical analyses were performed to compare the results for FRAC, F, Cmax and tmax

263

after T2-G administration to the results obtained after T-2 administration. A one-way ANOVA

264

with post-hoc Scheffé tests was conducted (p < 0.05) (SPSS 20.0, IBM, Chicago, IL, USA). All

265

variables were log transformed in order to fulfill the equality of variances criterion as

266

determined by the Levene’s test for homogeneity of variances (p value > 0.01).

267 268 269

3. Results and discussion

270

3.1 Method validation


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The results for linearity (r, gof), sensitivity (LOD, LOQ), SSE, RE and RA are given in Table

272

2. The results for linearity were all in accordance with the acceptance criteria, with r ≥ 0.99

273

and gof ≤ 20%. The LOD varied from 0.01 ng/mL (T-2 triol), 0.02 ng/mL (T-2 and T2-G) to 0.04

274

ng/mL (HT-2). The LOQ was set at 0.1 ng/mL for all mycotoxins. For RE unusually high values

275

were obtained, however, as the values obtained for RA were situated between 55 and 82%,

276

we do not believe this compromised the obtained results. Moreover, since an isotopically

277

labeled internal standard was used, the results were compensated for extraction recovery.

278 279

Table 3 displays the results for accuracy (%) and precision (RSD, %). All results fell within the specified acceptability ranges.

280

The method complied with specificity requirements as no interfering peaks were

281

observed in the chromatographic elution zone of the analytes with a S/N ratio ≥ 3 (results

282

not shown). Furthermore, carry-over was evaluated, and for none of the analytes carry-over

283

signals were detected that could interfere with the response/area of the analytes at their

284

given retention time (results not shown).

285 286

3.2 Toxicokinetic trials

287

In a previous toxicokinetic study with T-2 in broiler chickens, an oral dose of 0.02 mg/kg

288

bw was administered and was extrapolated from EU maximum guidance values in feed. This

289

dose however did not give rise to plasma concentrations above the LOQ [26]. Consequently,

290

this dose did not allow calculation of the absolute oral bioavailability of T-2. Sun et al. orally

291

dosed broilers at 2.0 mg T-2/kg bw, resulting in detectable plasma profiles [27].

292

Consequently, the dose administered in this study was chosen at 1 mg T2-G/kg bw and at an

293

equimolar dose of T-2, i.e. 0.74 mg/kg bw.



294

During the animal trials, none of the broiler chickens showed any adverse effects after

295

acute IV or PO administration of T-2G. On the other hand, within 24 h after the conduct of

296

the T-2 cross-over study, one broiler died. However, this could not be directly related to the

297

administration of the mycotoxin. The other broilers showed no clinical symptoms after the T-

298

2 IV and PO administration.

299

Plasma concentration-time profiles for PO and IV administration of T-2 (n=6) and T2-G

300

(n=3) to broiler chickens are presented in Figure 2. Each profile is represented as the mean +

301

standard deviation (SD).

302

A first observation from the plasma analyses is that after both PO and IV administration

303

of T-2 and T2-G, no T-2 triol and only traces of HT-2 were detected in plasma. In contrast,

304

Sun et al. detected high amounts of HT-2 and T-2 triol after IV administration of T-2 (0.5

305

mg/kg bw), as well as high concentrations of T-2 triol after PO T-2 administration (2 mg/kg

306

bw) [27].

307

The toxicokinetic parameters are shown in Table 4. The F of T-2 in broilers is limited to

308

2.17±1.80%. Sun et al. calculated a notably higher F of T-2, i.e. 17.1% [27]. Osselaere et al.

309

administered T-2 PO (20 µg/kg bw) to chickens and no plasma concentrations above the LOQ

310

could be detected for T-2 and its metabolite HT-2. Consequently, no determination of F was

311

possible. Given the LOQ of 2.5 ng/mL of the latter method, it does indicate a likely low F, in

312

correspondence with the findings of this study.

313

The values for CL (191 mL/min/kg), β (0.0346/min), t1/2β (21.4 min) and Vss (sum of Vc

314

and Vp; 3240 mL/kg) correspond well with the values obtained by Sun et al., i.e. 120

315

mL/min/kg, 0.04/min, 17.3 min and 3170 mL/kg, respectively [27]. In contrast, the values for

316

these toxicokinetics parameters described by Osselaere et al. differ significantly, this

317

discrepancy is probably caused by the application of a non-compartmental model [26].


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318

For T2-G a FRAC and F of 10.4±8.7% and 10.1±8.5% were determined, respectively. This

319

slight difference is caused by a partial presystemic hydrolysis of T2-G to T-2, i.e. 3.49±1. 19%.

320

McCormick et al. performed an in vitro digestive study on T2-G, developed to simulate the

321

human GI process. They concluded that after a 30 min incubation there was practically no

322

hydrolysis of T2-G to T-2. However, after an incubation period of 24 h, less than 30% of the

323

initially added T2-G remained [18]. These results are in accordance with the low degree of

324

hydrolysis found in this study, given the short GI transit time of max. 3-4 h, in broilers [28].

325

Similar results have been obtained for DON3G, another glycosylated trichothecene, where

326

after IV and PO administration a complete absence of in vivo hydrolysis was observed in

327

broilers [29].

328

Table 4 also shows a FRAC of T2-G which is five times higher than that of T-2. In contrast,

329

the FRAC of DON3G is about three times lower than that of DON in broiler chickens. This

330

discrepancy can possibly be explained by the higher log P of T-2 (not described so far)

331

compared to that of DON (log P -1.0) [30]. Glycosylation in general will increase polarity and

332

thus lower the log P value. For T2-G this glycosylation may result in a more favourable log P

333

for transport of the molecule across cell membranes and for intestinal absorption, as stated

334

by Lipinski’s rule of 5 or certain variants thereof, with satisfactory log P values situated

335

between -0.4 and +5.6.

336

Mean T2-G volumes of distribution, CL and Cmax values are significantly higher than

337

those observed for T-2. Mean values for β (0.0285/min) and t1/2β (24.3 min) of T2-G do not

338

significantly differ from those obtained for T-2.

339

These differences in toxicokinetics parameters between T-2 and T2-G, specifically the five

340

times higher FRAC for T2-G, clearly indicate the flaw in assuming equal toxicity of modified

341

and parent compounds in risk assessment [1]. For every amount of T2-G in feed, compared



342

to T-2, the systemic exposure to the glucoside conjugate will be five times higher.

343

Consequently, the relative contribution of modified forms of T-2 to the occurrence of T-2 in

344

feed, estimated at 10% would result in an in vivo contribution of 50%. Assuming equal

345

intrinsic toxicity is similarly flawed. Although no T2-G toxicity data is available, a clearly

346

decreased toxicity has been demonstrated for DON3G compared to DON [17,29,31,32].

347

Given the high degree of structural similarity between DON and T-2, a similar decrease in

348

toxicity may reasonably be expected for T2-G. Furthermore, as seen with DON3G, T2-G was

349

not hydrolysed in broiler chickens to its respective free mycotoxin to a significant extent.

350

These results may thus contribute to a more accurate risk assessment of T2-G in broiler

351

chickens.

352 353

Acknowledgements

354

The financial support by Ghent University (Special Research Fund grant no.

355

I/00105/01) as well as from the Federal Public Service of Health, Food Chain Safety and

356

Environment (FOD) ZENDONCONVERT RT14/09 project is gratefully acknowledged.

357

358

1.

EFSA Panel on Contaminants in the Food Chain (CONTAM) (2014). Scientific Opinion

359

on the risks for human and animal health related to the presence of modified forms

360

of certain mycotoxins in food and feed. EFSA J, 12(12), 3916.

361

2.

Mankevičienė, A., Butkutė, B., Gaurilčikienė, I., Dabkevičius, Z., & Supronienė, S.

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(2011). Risk assessment of Fusarium mycotoxins in Lithuanian small cereal grains.

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Food Control, 22(6), 970-976.


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3.

Scudamore, K. A., Baillie, H., Patel, S., & Edwards, S. G. (2007). Occurrence and fate of

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Fusarium mycotoxins during commercial processing of oats in the UK. Food Additives

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and Contaminants, 24(12), 1374-1385.

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4.

oats. Food Additives and Contaminants, 26(7), 1063-1069.

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5.

Escrivá, L., Font, G., & Manyes, L. (2015). In vivo toxicity studies of fusarium mycotoxins in the last decade: A review. Food and Chemical Toxicology, 78, 185-206.

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Edwards, S. G. (2009). Fusarium mycotoxin content of UK organic and conventional

6.

EFSA Panel on Contaminants in the Food Chain (CONTAM) (2011). Scientific opinion

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on the risks for animal and public health related to the presence of T-2 and HT-2

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7.

Current Drug Metabolism, 9(1), 77-82.

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Kuca, K., Dohnal, V., Jezkova, A., & Jun, D. (2008). Metabolic pathways of T-2 toxin.

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Schuhmacher-Wolz, U., Heine, K., & Schneider, K. (2010). Report on toxicity data on

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trichothecene mycotoxins HT-2 and T-2 toxins. EFSA Supporting Publications, 7(7).9.

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European Commission. (2006). Commission Recommendation of 17 August 2006 on

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the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and

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fumonisins in products intended for animal feeding. Off. J. Eur. Union, 229, 7-9.

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European Commission. (2013). Commission Recommendation 2013/165/EU of 27

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March 2013 on the presence of T-2 and HT-2 toxin in cereals and cereal products. Off.

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Busman, M., Poling, S. M., & Maragos, C. M. (2011). Observation of T-2 toxin and HT-

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2 toxin glucosides from Fusarium sporotrichioides by liquid chromatography coupled

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to tandem mass spectrometry (LC-MS/MS). Toxins, 3(12), 1554-1568.

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12.

Lattanzio, V. M., Visconti, A., Haidukowski, M., & Pascale, M. (2012). Identification

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and characterization of new Fusarium masked mycotoxins, T2 and HT2 glycosyl

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derivatives, in naturally contaminated wheat and oats by liquid chromatography–

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high-resolution mass spectrometry. Journal of Mass Spectrometry, 47(4), 466-475.

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Lattanzio, V. M., Ciasca, B., Haidukowski, M., Infantino, A., Visconti, A., & Pascale, M.

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(2013). Mycotoxin profile of Fusarium langsethiae isolated from wheat in Italy:

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production of type-A trichothecenes and relevant glucosyl derivatives. Journal of

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Mass Spectrometry, 48(12), 1291-1298.

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Broekaert, N., Devreese, M., De Mil, T., Fraeyman, S., Antonissen, G., De Baere, S., De

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Backer, P., Vermeulen, A., & Croubels, S. (2015). Oral Bioavailability, Hydrolysis, and

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Comparative Toxicokinetics of 3-Acetyldeoxynivalenol and 15-Acetyldeoxynivalenol

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in Broiler Chickens and Pigs. Journal of Agricultural and Food Chemistry, 63(39), 8734-

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8742.

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Nagl, V., Schwartz, H., Krska, R., Moll, W. D., Knasmüller, S., Ritzmann, M., Adam, G.,

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& Berthiller, F. (2012). Metabolism of the masked mycotoxin deoxynivalenol-3-

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glucoside in rats. Toxicology Letters, 213(3), 367-373.

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Nagl, V., Woechtl, B., Schwartz-Zimmermann, H. E., Hennig-Pauka, I., Moll, W. D.,

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Adam, G., & Berthiller, F. (2014). Metabolism of the masked mycotoxin

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deoxynivalenol-3-glucoside in pigs. Toxicology Letters, 229(1), 190-197.


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Broekaert, N., Devreese, M., van Bergen, T., Schauvliege, S., De Boevre, M., De

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Saeger, S., Vanhaecke, L., Berthiller, F., Michlmayr, H., Malachová, A., Adam, G.,

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Vermeulen, A., & Croubels, S. (2017). In vivo contribution of deoxynivalenol-3-β-D-

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glucoside to deoxynivalenol exposure in broiler chickens and pigs: Oral bioavailability,

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hydrolysis and toxicokinetics. Archives of Toxicology, 91, 699-712.

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18.

McCormick, S. P., Price, N. P., & Kurtzman, C. P. (2012). Glucosylation and other

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biotransformations of T-2 toxin by yeasts of the Trichomonascus clade. Applied and

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Environmental Microbiology, 78(24), 8694-8702.

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McCormick, S. P., Kato, T., Maragos, C. M., Busman, M., Lattanzio, V. M., Galaverna,

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G., Dall-Asta, C., Crich, D., Price, N.P.J., & Kurtzman, C. P. (2015). Anomericity of T-2

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toxin-glucoside: masked mycotoxin in cereal crops. Journal of Agricultural and Food

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Chemistry, 63(2), 731-738.

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Widestrand, J., & Pettersson, H. (2001). Effect of time, temperature and solvent on

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the stability of T-2 toxin, HT-2 toxin, deoxynivalenol and nivalenol calibrants. Food

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Additives & Contaminants, 18(11), 987-992.

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European Communities. Commission Decision 2002/657/EC of 12 August 2002

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implementing Council Directive 96/23/EC concerning the performance of analytical

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methods and the interpretation of results. Official Journal of the European

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Communities L 221, 8-36

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Heitzman, R. J. (1994). Veterinary drug residues. Residues in food producing animals and their products: Reference materials and methods. EUR 15127 EN.



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Matuszewski, B. K., Constanzer, M. L., & Chavez-Eng, C. M. (2003). Strategies for the

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assessment of matrix effect in quantitative bioanalytical methods based on HPLC−

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MS/MS. Analytical chemistry, 75(13), 3019-3030.

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of deoxynivalenol, T-2 toxin and zearalenone in broiler chickens. Food and Chemical

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Toxicology, 51, 350-355.

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Sun, Y. X., Yao, X., Shi, S. N., Zhang, G. J., Xu, L. X., Liu, Y. J., & Fang, B. H. (2015).

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

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intravenous and oral administration. Journal of Veterinary Pharmacology and

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Golian, A., & Maurice, D. V. (1992). Dietary poultry fat and gastrointestinal transit time of feed and fat utilization in broiler chickens. Poultry Science, 71(8), 1357-1363.

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Pierron, A., Mimoun, S., Murate, L. S., Loiseau, N., Lippi, Y., Bracarense, A. P. F., Liaubet, L., Schatzmayr, G., Berthiller, F., Moll, W.D., & Oswald, I. P. (2015). Intestinal


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toxicity of the masked mycotoxin deoxynivalenol-3-β-Dd-glucoside. Archives of

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Toxicology, 1-10.

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30.

Maresca, M. (2013). From the gut to the brain: Journey and pathophysiological

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effects of the food-associated trichothecene mycotoxin deoxynivalenol. Toxins, 5(4),

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784-820.

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Poppenberger, B., Berthiller, F., Lucyshyn, D., Sieberer, T., Schuhmacher, R., Krska, R.,

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Kuchler, K., Glössl, J., Luschnig, C., & Adam, G. (2003). Detoxification of the Fusarium

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mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana.

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Journal of Biological Chemistry, 278(48), 47905-47914.

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Wu, W., He, K., Zhou, H. R., Berthiller, F., Adam, G., Sugita-Konishi, Y., Watanabe, M.,

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Krantis, A., Durst, T., Zhang, H., & Pestka, J. J. (2014). Effects of oral exposure to

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naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory

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Pharmacology, 278(2), 107-115.

466 467

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from simple to complex mycotoxins. Toxins, 3(7), 802-814.

468 469 470

McCormick, S., Stanley, A.M., Stover, N.A., Alexander, A.J. (2011). Trichothecenes:

34.

Desjardins A.E. (2006). Fusarium Mycotoxins Chemistry, Genetics and Biology. APS Press; Eagan, MN, USA, 1–260.

471



472


Page 22 of 28

Page 23 of 28


473

Figures

474

Figure 1. Chemical structure of T-2 toxin-3-glucoside and overview of the two-way cross-

475

over study design for intravenous (IV) and oral (PO) T-2 toxin (T2) administration to six

476

broiler chickens with a one-day wash-out period (blue). T-2 toxin-glucoside (T2G) was

477

administered IV and PO to each 3 broiler chickens (green).

478

479 480 481

482



483

Figure 2. Plasma concentration-time profiles of T-2 toxin (T-2) and T-2-glucoside (T2-G) after

484

oral (PO) and intravenous (IV) administration of T-2 (0.76 mg/kg bodyweight, n=6) and T2-G

485

(1 mg/kg bodyweight, n=3) to broiler chickens. Values are presented as mean + SD.

486 487 488 489 490 491 492 493 494 495


Page 24 of 28

Page 25 of 28


496

Tables

497

Table 1. Compound specific MRM ion transitions and MS parameters of T-2 toxin (T-2), its metabolites HT-2

498

toxin (HT-2) and T-2 triol, T-2 glucoside (T2-G) and the isotopically labelled internal standard (13C24-T-2); Rt=

499

retention time; IS= internal standard; quantifier ion

a

T-2

Measured

Precursor ion

Product ion

form/adduct

(m/z)

(m/z)

[M+NH4]+

484.1

245.4a

Rt (min)

Collision Energy 12

5.7 305.4 HT-2

[M+Na]+

447.2

12

285.1a

20 4.2

345.1 T-2 triol

[M+Na]

+

405.0

124.9

18 a

15 3.3

303.0 T2-G

[M+Na]+

650.9

15

326.9a

40 4.5

406.9 13

C24-T-2 (IS)

[M+NH4]

+

508.2

198.0

40 a

22 5.7

228.9

500 501 502 503 504 505


22


Page 26 of 28

506

Table 2. Method validation results for analysis of T-2 toxin (T-2), HT-2 toxin (HT-2), T-2 triol and T-2 toxin-

507

glucoside (T2-G) in broiler chicken plasma: linearity (correlation coefficient r and goodness-of-fit coefficient gof,

508

minimum 7 concentration points), limit of detection and limit of quantification (LOD and LOQ; n=6), matrix

509

effects (signal suppression/enhancement (SSE)), apparent recovery (RA) and extraction recovery (RE).

510 Range

Correlation

Goodness of

LOD

LOQ

SSE

RA

RE

(ng/mL)

coefficient (r)

fit (gof, %)

(ng/mL)

(ng/mL)

(%)

(%)

(%)

T-2

0.1-1000

0.9999

6.52

0.02

0.1

96.6

77.8

80.5

HT-2

0.1-100

0.9981

17.33

0.04

0.1

31.6

81.3

257

T-2 triol

0.1-100

0.9984

10.79

0.01

0.1

19.7

55.4

282

T2-G

0.1-100

0.9910

13.36

0.02

0.1

41.8

76.7

183

511 512

Table 3. Method validation results for analysis of T-2 toxin (T-2), HT-2 toxin (HT-2), T-2 triol and T-2 toxin-

513

glucoside (T2-G) in broiler chicken plasma: within-day precision (n=6) and between-day precision (n=3x3) with

514

corresponding accuracy at low (limit of quantification (LOQ)) and high (linear range maximum) concentration

515

level

Within-day (n=6) Accuracy (%)

Between-day (n=3x3)

Precision (RSD, %)

Accuracy (%)

Precision (RSD, %)

LOQ

Range max

LOQ

Range max

LOQ

Range max

LOQ

Range max

8.3

-0.4

18.8

2.6

11.1

-0.5

7.0

1.4

HT-2

-42.0

-6.0

18.9

2.7

-40.0

-5.1

18.6

2.7

T-2 triol

-10.0

-4.5

17.6

2.1

-1.1

-3.3

19.2

2.8

T2-G

-9.-9

-3.8

19.6

2.3

-31.1

-3.2

30.2

2.5

T-2

516


Page 27 of 28


517

Table 4. Toxicokinetic parameters, absolute oral bioavailability (%) and presystemic hydrolysis (%) of T-2 toxin

518

(T-2) (n=6) and T-2 toxin-glucoside (T2-G) (n=3) after intravenous (IV) and oral (PO) administration to broiler

519

chickens.

Administration route

IV

PO

Parameter

T-2

T2-G

CL (mL/min/kg)

191±42

377±85*

Vc (mL/kg)

1173±387

2705±1281*

Vp (mL/kg)

2067±744

4568±1838*

β (1/min)

0.0346±0.0088

0.0285±0.0017

t1/2β (min)

21.4±6.6

24.3±1.4

FRAC (%)

2.17±1.80

10.4±8.7*

Pres.Hydr. (%)

-

3.49±1.19*

F (%)

2.17±1.80

10.1±8.5*

Cmax (ng/mL)

5.55±7.49

33.9±27.0*

tmax (min)

8.14±4.38

4.67±4.62

Values are presented as mean ± SD. β=disposition rate constant (1/min); t1/2β=disposition half-life (min); CL=total body clearance (mL/min/kg); Vc=volume of distribution of the central compartment (mL/kg); Vp=volume of distribution of the peripheral compartment (mL/kg); FRAC=absorbed fraction (%); Pres.Hydr.=percentage of the dose that is hydrolysed presystemically (%); F=absolute oral bioavailability (%); Cmax=maximum plasma concentration (ng/mL); tmax= time to maximum plasma concentration (min); Statistical analysis was performed by means of ANOVA,* statistically significant difference (p

T-2 Toxin-3Î±-glucoside in Broiler Chickens: Toxicokinetics, Absolute

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