Accepted Manuscript
Toxicokinetic profile of fusarenon-X and its metabolite nivalenol in the goat (Capra hircus)
Wanchalerm Phruksawan, Saranya Poapolathep, Mario Giorgi, Kanjana Imsilp, Chainarong Sakulthaew, Helen Owen, Amnart Poapolathep
PII: S0041-0101(18)30361-1 DOI: 10.1016/j.toxicon.2018.08.015 Reference: TOXCON 5974
To appear in: Toxicon
Received Date: 16 May 2018 Revised Date: 21 August 2018 Accepted Date: 27 August 2018
Please cite this article as: Phruksawan, W., Poapolathep, S., Giorgi, M., Imsilp, K., Sakulthaew, C., Owen, H., Poapolathep, A., Toxicokinetic profile of fusarenon-X and its metabolite nivalenol in the goat (Capra hircus), Toxicon (2018), doi: 10.1016/j.toxicon.2018.08.015.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 ACCEPTED MANUSCRIPT 1 Title: Toxicokinetic profile of fusarenon-X and its metabolite nivalenol in the goat (Capra
2 hircus)
3
4 Authors: Wanchalerm Phruksawan a, Saranya Poapolathep a, Mario Giorgi b, Kanjana Imsilp a,
5 Chainarong Sakulthaew c, Helen Owen d, Amnart Poapolathep a*
6
7 Affiliations:aDepartment of Pharmacology, Faculty of Veterinary Medicine, Kasetsart
8 University, Bangkok 10900, Thailand
9 bDepartment of Veterinary Sciences, University of Pisa, Via Livornese (lato
10 monte), San Piero a Grado, 56122, Pisa, Italy
11 cFaculty of Veterinary Technology, Kasetsart University, Bangkok 10900,
12 Thailand
13 dDepartment of Veterinary Sciences, Gatton, Brisbane, University of Queensland, 14 Australia MANUSCRIPT 15 Corresponding author: Associate Prof. Dr. Amnart Poapolathep
16 Department of Pharmacology, 17 Faculty of Veterinary Medicine, 18 Kasetsart University, 19 Chatuchak, 20 Bangkok 10900, Thailand 21 Tel/Fax: +66-2-5797537 22 E-mail: [email protected]; [email protected] 23 ACCEPTED 24
25
26 2 ACCEPTED MANUSCRIPT 27 Abstract
28
29 The main aim of this research was to evaluate the toxicokinetic characteristics of
30 fusarenon-X (FX) and its metabolite, nivalenol (NIV), in goats. The amounts of FX and NIV
31 in post-mitochondrial (S-9), microsomal and cytosolic fractions of diverse tissues of the goat
32 were also investigated. FX was intravenously (iv) or orally (po) administered to goats at
33 dosages of 0.25 and 1 mg/kg bw, respectively. The concentrations of FX and NIV in plasma,
34 feces and urine were quantified by liquid chromatography tandem-mass spectrometry (LC-
35 ESI-MS/MS). The concentrations of FX in plasma were quantified up to 8 h with both routes
36 of administration. A large amount of NIV (metabolite) was quantifiable in plasma, urine and
37 feces after both administrations. The C max value of FX was 413.39 ± 206.84 ng/ml after po
38 administration. The elimination half-life values were 1.64 ± 0.32 h and 4.69 ± 1.25 h after iv
39 and po administration, respectively. In vitro experiments showed that the conversion FX-to- MANUSCRIPT 40 NIV mainly occurs in the liver microsomal fraction. This is the first study that evaluates the 41 fate and metabolism of FX in ruminant species.
42
43 KEYWORDS: fusarenon-X; nivalenol; toxicokinetic; goat
44
45
46
47 ACCEPTED 48
49
50
51 3 ACCEPTED MANUSCRIPT 52 1. Introduction
53
54 Among the trichothecenes discovered to date, deoxynivalenol (DON), T-2 toxin (T-2),
55 diacetoxyscirpenol (DAS) and Fusarenon-X (FX) are known to contaminate feed globally
56 (Pittet, 1998). Trichothecenes have been classified into A, B, C, and D types. Type A and B
57 trichothecene mycotoxins have a wide range of toxic effects on farm animals and humans
58 (WHO, 1990). In farm animals, they can cause diarrhea, feed refusal, weight loss, decreased
59 production performance, immune suppression and residues in animal food products (Eriksen
60 and Pettersson, 2004).
61 FX, a type B trichothecene mycotoxin, is mainly produced by the Fusarium fungi,
62 which naturally occurs in agricultural commodities such as cereal and cereal products,
63 including wheat, barley, corn, rye, oats, maize and multigrain (Broakaert, et al., 2015;
64 Cavaliere et al., 2005; Juan et al., 2013; Placinta et al., 1999) . FX was first isolated MANUSCRIPT 65 from Fusarium nivale strain Fn-2B, its production depends on many factors, including the 66 substrate, temperature and humidity (Cavaliere et al., 2005; Juan et al., 2013). Although
67 deoxynivalenol (DON) is the most commonly found globally, the derived products,
68 particularly breakfast cereals and bread, are susceptible to FX contamination, and are very
69 important in the human diet including in baby foods (Placinta et al., 1999; Juan et al., 2013;
70 Yzar and Omurtag, 2008). There are some reports that FX has been frequently detected along
71 with DON and nivalenol (NIV) (IARC, 1993). 72 FX predominantlyACCEPTED occurs in temperate regions of Europe and Asia, because these 73 geographical zones are suitable for Fusarium growth and FX production.
74 FX has been characterized as more potently toxic than the other members of type B
75 trichothecene mycotoxins (IARC, 1993). FX can evoke a ribotoxic stress response, which
76 inhibits protein and DNA synthesis in eukaryotic cells (Aupanun et al., 2017). FX has been 4 ACCEPTED MANUSCRIPT 77 reported to induce adverse health effects, particularly apoptosis, in organs containing actively
78 dividing cells such as the small intestine, thymus, spleen, bone marrow, testes, and in cells
79 such as reticulocytes and mitogen-stimulated human lymphocytes; these effects have also
80 been observed after exposure to other tricothecenes (Forsell and Pestka, 1985; Miura et al.,
81 1998; Poapolathep et al., 2001; 2002). Furthermore, FX dose-dependently encourages DNA
82 strand breakage of both dividing cells and differentiated Caco-2 cells (Alassane-Kpembi et
83 al., 2013). FX has been reported to induce apoptosis in developing mouse brain, thymus,
84 Peyer’s patches, spleen and human jurkat T cells (Aupanun et al., 2015, 2016; Sutjarit et al.,
85 2014).
86 The European Commission (EC) has regulated acceptable levels of trichothecene
87 mycotoxins in cereal grains, flours, and cereal-based products intended for human and animal
88 consumption (Gareis et al., 2003; EC, 2006) but the limited available information prevents
89 establishing a regulatory limit for FX (Gareis et al., 2003). Nowadays, the European Food 90 Safety Authority Panel on Contaminants in the MANUSCRIPT Food Chain (CONTAM) establishes the 91 tolerable daily intake of NIV as 1.2 mg/kg bw/day based on a lowest-observed-adverse-effect
92 of 0.7 mg/kg bw/day found in long-term dietary studies in mice (EFSA, 2013).
93 Toxicokinetic profiles of FX and its metabolite NIV have been reported in mice,
94 broiler chickens, ducks and piglets (Poapolathep et al., 2003, 2004, 2008; Saengtienchai et
95 al., 2014) but to the best of the authors’ knowledge no data exist on its toxicokinetics in
96 ruminants. Tremendous variations in the species specific toxicokinetic profiles of FX have 97 been observed. ACCEPTEDFurther information on the toxicokinetics of FX is also needed to evaluate 98 possible differences in toxicity between monogastric animals and ruminants. Therefore, this
99 study aimed to evaluate the toxicokinetic characteristics of FX and its metabolite NIV in
100 goats after intravenous (iv) and oral (po) administrations. The amounts of FX and NIV in 5 ACCEPTED MANUSCRIPT 101 post-mitochondrial (S-9), microsomal and cytosolic fractions of diverse tissues of the goat
102 were also investigated.
103
104 2.Materials and Methods
105
106 2.1 Animals
107
108 Eleven 9-week-old male goats (average weight 7.74 ± 0.44 kg) were purchased from a
109 commercial goat farm (Saraburi Thailand). The experimental animals were housed at the
110 Laboratory Animal Facility, Bureau of Veterinary Biologics, Department of Livestock,
111 Nakhon Ratchasima province, Thailand. Animals were acclimatized to the environment for 1
112 week prior to the commencement of the study. The animals were fed with dried pangola grass 113 and water ad libitum. All experimental proceduresMANUSCRIPT on animals were ethically approved by the 114 Animal Ethics Research Committee of the Faculty of Veterinary Medicine, Kasetsart 115 University.
116
117 2.2 Chemicals and reagents
118
119 Standard FX and NIV were purchased from Wako Pure Chemical Industries Ltd.
120 (Kyoto, Japan). Other analytical grade reagents and chemicals were purchased from Sigma 121 Chemical Co. (St.ACCEPTED Louis, MO, USA). Purified water was produced using the Milli-Q water 122 purification system from Millipore, Inc (Bedford, MA, USA). The solutions for iv and po
123 administrations were prepared in one batch at a concentration of 2 mg/ml by dissolving
124 standard FX in 0.01 M phosphate buffer saline pH 7.4 containing 10% (v/v) dimethyl
125 sulfoxide (DMSO). 6 ACCEPTED MANUSCRIPT 126
127 2.3 Toxicokinetic study
128
129 Ten goats were randomly divided into two groups (n= 5). After overnight fasting, each
130 group was administered FX iv (into the right jugular vein) or po at a dosage of 0.25 or 1.0
131 mg/kg bw, respectively. Blood samples (2.5-3.0 ml) were collected from the left jugular vein
132 of each goat with heparinized syringes at 0, 5, 15 and 30 min and 1, 2, 4, 6, 8, 12, 24, 48, and
133 72 h, whereas the urine and feces were collected at 0-2 h, 2-4 h, 4-8 h, 8-12 h, 12-24h and 24-
134 48h after FX administration. The urine and faecal samples were immediately collected from
135 the individual animals. The plasma was separated by centrifugation at 1,968 g for 15 min and
136 immediately frozen at -20 oC until analyzed. The urine and faecal samples were also stored at
137 -20 oC until analyzed.
138 MANUSCRIPT 139 2.4 Extraction and clean up 140
141 The extraction method of FX and NIV in plasma, urine and feces was based on a
142 previously published method (Poapolathep et al., 2008). Briefly, 1 ml of goat plasma or urine,
143 or 5 g of feces were extracted with 3 ml of acetonitrile (ACN)-water (3/1, v/v). Two g of
144 ammonium sulfate were added, and shaken for 30 sec by a vortex mixer. The ACN fraction
145 was separated by centrifugation at 1,968 g for 15 min. The supernatant was collected in the 146 glass tube. TheseACCEPTED extraction steps were then repeated for 2 additional cycles. The supernatant 147 fractions were combined and purified using the solid phase extraction cartridge (C18 Sep-pak
148 silica cartridge) (Waters Corp., Milford, MS, USA) as described previously (Poapolathep et
149 al., 2008). The eluate was completely evaporated under a nitrogen stream at 40 oC on a
150 heating block. The residue was reconstituted with 500 µl of methanol-water (1:4) v/v with 5 7 ACCEPTED MANUSCRIPT 151 mM ammonium acetate, and injected onto a 0.22 m syringed filter (Sartorius AG,
152 Goettingen, Germany) before being analyzed by liquid chromatography tandem-mass
153 spectrometry (LC-MS/MS).
154
155 2.5 Preparation of postmitochondrial (S-9), microsomal and cytosolic fractions
156
157 In order to study the metabolites of FX, one 9-week-old male goat was sacrificed with
158 thiopentone sodium at a dosage of 20 mg/kg of bw by iv administration. Various tissues
159 including the liver, kidney, heart, lung, muscle, rumen, reticulum, omasum, abomasum,
160 duodenum, jejunum, ilium, colon and rectum were immediately collected and frozen in liquid
161 nitrogen and stored at -80 ᴏC until assayed. The postmitochondrial (S-9), microsomal and
162 cytosolic fractions were prepared as described (Esaki and Kumagai, 2002, Poapolathep et al.,
163 2008). Briefly, a portion of the thawed tissue sample was cut into small pieces using scissors 164 and homogenized in a motor driven glass homogenizer MANUSCRIPT. The homogenates were centrifuged at 165 10,000 rpm for 10 min to separate the supernatant designated as the S-9 fraction. S-9 fraction
166 was then centrifuged at 105,000 x g for 1 h to collect the cytosolic fraction. The pellet was
167 resuspended and recentrifuged at 105,000 x g. Supernatant was discarded and the microsomal
168 fraction was resuspended in 20 mM Tris buffer pH 7.4 containing 1 mM ethylene diamine
169 tetraacetic acid (EDTA) and 0.25 M sucrose. All preparation procedures were performed on
170 ice and all sample fractions were kept at -80 ᴏC until analyzed. 171 ACCEPTED 172 2.6 Protein assay
173
174 Protein concentrations in S-9, microsomal, and cytosolic fractions were determined as
175 described by Esaki and Kumagais (Esaki and Kumagai, 2002). In brief, 2 ml of S-9, 8 ACCEPTED MANUSCRIPT 176 microsomal, or cytosolic fractions from each tissue were added to 5 ml of the dye reagent:
177 water, 1:4 v/v, mixture. The samples were then incubated at room temperature for 5 min. and
178 measured spectrophotometrically at 595 nm using a GENESYS TM 20 spectrophotometer
179 (Thermo Fisher Scientific, Washington, USA). Bovine serum albumin (Bio-Rad Protein
180 Assay, Bio-Rad Lab, CA ) was used as the standard for the protein assay.
181
182 2.7 Determination of S-9, microsomal and cytosolic activity to form NIV
183
184 Conversion of FX by S-9, microsomal and cytosolic activities was performed
185 according to a published method (Esaki and Kumagai, 2002). In brief, 1 mg protein of S-9,
186 microsomal and cytosolic fractions was pre-incubated at 37 ᴏC in an incubation buffer
187 consisting of potassium phosphate buffer (100 mM, pH 7.4), G-6-P (5.6 mM), NADPH
188 (2mM) and G-6-P dehydrogenase (0.5 unit). After pre-incubation for 5 min, FX (5 mg/10 ml 189 of methanol) 5 µg of FX in 10 µl of methanol was MANUSCRIPT added and the mixtures were continuously 190 incubated whilst being shaken for 0, 15, 30, 45 and 60 min. The reaction was terminated by
191 adding an equal volume of ice-cold chloroform, followed by vigorous mixing and
192 centrifugation at 5,000 rpm for 10 min at 4 ᴏC. The incubation mixtures were extracted with
193 the mixture of ACN and water solution described above. The residue was reconstituted with
194 the mobile phase solution, and injected onto a 0.22 m syringed filter (Sartorius AG,
195 Goettingen, Germany) before being analyzed by LC-MS/MS. 196 ACCEPTED 197 2.8 LC parameters
198
199 LC analysis was performed using an Agilent 1260 Infinity (Agilent Technologies,
200 Waldbronn, Germany) consisting of a binary pump, a vacuum degasser, a column oven and 9 ACCEPTED MANUSCRIPT 201 an auto sampler. The chromatographic separation was performed on a ZORBAX Eclipse Plus
202 Rapid Resolution HT (RRHT) C18 column (4.6 x 50 mm, 1.8 m particle size, Agilent
203 Technologies) with guard column (4.6 x 5 mm, 1.8 m particle size, Agilent Technologies). 204 The column was maintained at 40 °C. The mobile phase consisted of 5 mM ammonium
205 acetate solution (mobile phase A) and methanol (mobile phase B). The gradient program of
206 the mobile phase was as follows: 0-1.0 min, 90% mobile phase A; 1.0-5.0 min, from 90 to
207 5% mobile phase A; 5-10 min, 5% mobile phase A; 10-11 min, from 5 to 90% mobile phase
208 A; 11-15 min, 90% mobile phase A. The mobile phase solution was filtered through a 0.22
209 m membrane and ultrasonically degassed prior to application. The flow rate was 0.4
210 mL/min, while the injection volume was 10 l. The temperature of the autosampler was set at
211 4 °C.
212
213 2.9 MS parameters 214 MANUSCRIPT 215 A triple quadrupole mass spectrometer was used (6460 triple, Agilent Technologies),
216 this was equipped with an electrospray ionization source operated in positive ion modes
217 (ESI+) under the multiple reaction monitoring mode (MRM). The ionization source
218 parameters were optimized as follows: capillary voltage: 3,500 V; gas temperature: 320 °C;
+ 219 gas flow: 8 l/min, and nebulizer: 50 psi. Under these conditions, FX formed [M + CH 3COO]
220 ion at m/z 413. The molecular ions and fragments employed for FX were as follows: Q1: m/z
221 413-263.4 (quantifier), CE 8 eV and Q3: m/z 413-59 (quantitation), CE 18 eV. For NIV, it
+ 222 formed [M + CHACCEPTED3COO] ion at m/z 371.1. The molecular ions and fragments employed for
223 NIV were as follows: Q1: m/z 371.1-281 (quantitation), CE 4 eV and Q3: m/z 371.1-59.1
224 (qualifier), CE 10 eV. The retention time of FX and NIV was 5.49 min and 4.45 min,
225 respectively.
226 10 ACCEPTED MANUSCRIPT 227 2.10 Method validation
228
229 Validation of the LC-MS/MS method for FX and NIV was performed to assess the
230 efficiency of this analytical method by investigating the recovery, repeatability, linear
231 working range, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision
232 and matrix effects in accordance with the guidelines on bioanalytical method validation
233 EMEA/CHMP/EWP/2012 . The linearity of an analytical procedure is its ability (within a
234 given range) to produce test results that are directly proportional to the concentration
235 (amount) of analyte in the sample. Linear regression analysis was conducted for the FX and
236 NIV standards under the optimized LC-MS/MS conditions. Linearity of the regression curve
237 was assessed on the basis of the residual plot, the fit test and the back-calculation. Recovery,
238 accuracy (%RE) and precision (repeatability, expressed as relative standard deviation (RSD)
239 in %) were determined within-day by analyzing seven replicates containing FX and NIV at 240 three different quality control (QC) levels (5, 50MANUSCRIPT and 250 ng/ml). The inter-day precisions 241 were determined by analyzing QC samples on five different days (one batch per day). The
242 calibration standard concentrations were prepared in three replicates by spiking the working
243 standard solution into blank samples to yield final concentrations of 2.5 to 1,000 ng/ml. The
244 matrix effects were determined for tested matrices by spiking blank plasma, urine and feces.
245 The recovery, LOD, LOQ, accuracy and precision were assessed. The LOD and LOQ of the
246 method were evaluated as the signal versus noise value (S/N) of 3:1 and 10:1, respectively. 247 ACCEPTED 248 2.11 Toxicokinetic parameters calculations
249
250 The concentrations of FX and NIV in experimental goats vs time were described by a
0 251 non-compartmental model using WinNonlin software (version 5.3.1). Cp was the peak 11 ACCEPTED MANUSCRIPT
252 concentration at the initial time, Cmax was peak plasma concentration, Tmax was time at peak
253 plasma concentration, AUC was the area under the curve, Vd was the volume of distribution,
254 t1/2 λ was the elimination half-life, Cl was the plasma clearance, MRT was the mean residence
255 time, Vd ss was the volume of distribution at steady state. The oral bioavailability ( F) was
256 calculated using the following equation:
257 (%)F (po) = [(AUC po) x dose iv/(AUC iv) × dose po] 100
258
259 2.12 Statistical analysis
260
261 Toxicokinetic variables were evaluated using the student’s t-test to determine
262 statistically significant differences between the treatment groups (iv vs po). Both
263 toxicokinetic parameters and toxin (FX and NIV) plasma concentrations are presented as
264 means ± SD (normality tested by Shapiro-Wilk test). All analyses were conducted using 265 GraphPad InStat (GraphPad Software, La Jolla, MANUSCRIPT CA, USA). In all experiments, differences 266 were considered significant with a p < 0.05.
267
268 3.Results
269
270 3.1 Method validation and quality assurance
271 272 RegardingACCEPTED FX and NIV detection, the LC-MS/MS method used in this study showed 273 linearity range, intra-day and inter-day precision, and accuracy for quantification of FX and
274 NIV within the limits requested by the EMA (2012). Linearity of the calibration curves for
275 plasma, urine and feces matrices, expressed as the determination coefficients (R 2), gave
276 values that were all above 0.998. The method validation parameters are shown in Table 1. 12 ACCEPTED MANUSCRIPT 277
278 3.2 Toxicokinetic study
279
280 Both FX and NIV were detectable in the plasma of goats following single iv or po
281 administration. The semilogarithmic plots of the mean (±SD) plasma concentration-time
282 curve of FX and NIV at a dosage of 0.25 and 1 mg/kg bw in goats following iv and po
283 administrations are shown in Figs. 1a and 1b. FX was quantifiable from 5 min to 8 h while
284 NIV was quantifiable from 5 min to 48 h, after iv administration of FX. After oral
285 administration of FX, FX was quantifiable from 5 min to 8 h whereas, NIV was quantifiable
286 from 5 min to 24 h. The mean plasma vs time curve profiles showed a lower concentration of
287 NIV after po as compared to iv administration. This is also reflected by the AUC values for iv
288 vs po, reporting a F% value of FX of about 15.81%. The toxicokinetic parameters are shown 289 in Table 2. The toxicokinetic parameters althoughMANUSCRIPT variable among the animals, were however 290 normally distributed (Shapiro-Wilk test) and reported as mean ± SD. This variability is 291 common in farm animals when not inbreed subjects are used (Lee et al., 2017; De Vito et al.,
292 2018; Kim et al., in press). Following iv administration, the value for the t1/2 λ of FX (1.64 ±
293 0.32 h) was shorter than that obtained after po administration (4.69 ± 1.25 h). The Cl value of
294 FX was low (310.98 ± 90.60 ml/h/kg) while the Vd was reasonably wide (730.10 ± 257.40
295 ml/kg). The AUC of NIV was higher than FX both in iv and po administrations. The
296 pharmacokinetic parameters are shown in Table 2. 297 ACCEPTED 298 3.3 Urine and faeces excretion
299
300 In urine, FX and NIV were quantified in urine after iv administration whereas, after po
301 administration only NIV was quantifiable. In feces, a large amount of NIV was quantifiable 13 ACCEPTED MANUSCRIPT 302 after po administration of FX in goats (Table 3).
303
304 3.4 Metabolic conversion of FX in S-9, microsomal and cytosolic fractions
305
306 To investigate the tissues that contribute to the metabolism of FX into NIV in goats, FX
307 was incubated with S-9, microsomal and cytosolic fractions of different tissues (liver, kidney,
308 heart, lung, muscle, rumen, reticulum, omasum, abomasum, duodenum, jejunum, ilium, colon
309 and rectum) for 15, 30, 45 and 60 min. The results showed that the biotransformation of FX
310 into NIV mainly occurred in the microsomal fraction of liver. The highest activity was 80.8
311 % at 60 min after incubation (Fig 2a).The biotransformation of FX to NIV also occurred in
312 the microsomal fraction of the kidney and lung although at lower rate, but no conversion was
313 found in the S-9 and cytosolic fractions (Fig 2a). Fig 2b shows the biotransformation of FX to
314 NIV in the stomach, including rumen, reticulum, omasum and abomasum of goats. The 315 biotransformation was detected in the S-9, microsom MANUSCRIPTal and cytosolic fractions of goat 316 stomach, and the highest activity was shown in the microsomal fractions. Among intestinal
317 tissues, including duodenum, jejunum, ileum, colon, and rectum (Fig 2c), the highest activity
318 was shown to be 24.5% at 60 min after incubation in the microsomal fraction of the jejunum.
319 The data for the metabolism of FX into NIV in various tissues of goats are shown in Fig 2.
320
321 4. Discussion 322 ACCEPTED 323 The results of this study revealed that FX is rapidly absorbed into the systemic
324 circulation after po administration. A large proportion of NIV was rapidly quantified in the
325 goat, indeed the plasma concentration ratio FX/NIV was already less than one 5 min after iv
326 and po FX administration. NIV was quantified in the plasma from 5 min to 24 h after oral 14 ACCEPTED MANUSCRIPT 327 administration of FX, indicating FX was absorbed and metabolized rapidly. These results are
328 also consistent with previous research, showing that FX was rapidly transformed into NIV in
329 liver of mice, broiler chickens, ducks and piglets (Poapolathep et al., 2003, 2008;
330 Saengtienchai et al., 2014). Indeed FX was rapidly absorbed after po administration (Tmax =
331 0.08 h) and the oral bioavailability was 15.81%. The oral bioavailability of FX in goats was
332 lower than in piglets (74.4%) similar to ducks (19.4%) (Poapolathep et al., 2008;
333 Saengtienchai et al., 2014), but higher than in broilers (9.8%) (Poapolathep et al., 2008). The
334 present study demonstrated that the elimination half-life value ( t1/2 λ) was longer in the po
335 (4.69 ± 1.25 h) group than in the iv group (1.64 ± 0.32 h). This might be due to a flip flop
336 effect already described for NIV in broilers (Kongkapan et al., 2016). On the other hand, the
337 t1/2 λ of NIV obtained in the po group (7.96 ± 2.99 h) was shorter than in the iv group (22.85 ±
338 4.62 h) after FX administration in goats. This might be due to inhibition of the metabolic
339 enzyme as a result of the sudden high FX plasma concentration that takes place in the IV 340 group (Wen et al., 2016). This parameter was shorterMANUSCRIPT in goats than that reported in piglets 341 (1.71 h; Saengtienchai et al., 2014), ducks (2.1 h; Poapolathep et al., 2008) but was longer
342 than that in broilers (1.2 h; Poapolathep et al., 2008) after iv administration of FX. The Cl
343 value was 310.98 ± 90.60 ml.h/kg after iv administration of FX in goats. This value was
344 greater than that reported in broiler chickens (113.57 ± 18.95 ml.h/kg) but similar to that
345 reported in ducks (331.98 ± 66.38 ml.h/kg). This might indicate that FX is more efficiently
346 cleared in ducks and goats compared to broiler chickens. Following po administration, the 347 maximum plasmaACCEPTED concentration (Cmax) of FX (413.39 ± 206.84 ng/ml) was reached at the 348 time of maximum concentration (Tmax), 0.08 h. The rather low plasma concentration of FX
349 after po administration is indicative off all of the phenomena that might lower the amount of
350 FX that reaches the systemic circulation. These include GI tract microbial activity absorption
351 process from the gut lumen into the intestinal tissue, and metabolism by all of the tissues 15 ACCEPTED MANUSCRIPT 352 through which FX must pass to get to the systemic circulation: intestinal tissue, blood
353 capillaries, and the liver (first-pass effect). All together, these likely processes produce a low
354 oral bioavailability (15.8%) of FX. However, a large proportion of NIV was detectable in
355 goat plasma after po administration of FX. These findings might indicate that FX was in fact,
356 efficiently absorbed in the gastrointestinal tract, and it was metabolized to NIV in liver of
357 goats. In urine and feces, a large proportion of NIV was detectable after iv and po
358 administration of FX in goats. These findings suggest that FX is excreted largely as NIV in
359 the urine and feces of goats. The results also are consistent with previous investigations of FX
360 in monogastric animals including, mice, broilers, ducks and piglets, which showed that a
361 large proportion of FX was changed into NIV following FX administration (Poapolathep et
362 al., 2003, 2008; Saengtienchai et al., 2014). However, NIV has been reported to be
363 metabolized to a de-epoxidated form (de-epoxynivalenol) by microorganisms in
364 gastrointestinal tract (Wu et al., 2010) but, a de-epoxyidated form was not investigated in this 365 study. MANUSCRIPT 366
367 Using the assumption that trichothecenes undergo a variety of different metabolic
368 reactions including hydrolysis to split off side groups, hydroxylation and de-epoxidation, the
369 data from the present study suggest a higher efficiency of microsomal enzymes in the liver,
370 compared to other tissues of goats. This also indicated the liver as the likely major site of the
371 FX-to-NIV metabolism in goats. The intestinal microflora is important for the 372 biotransformationACCEPTED of trichothecenes. The presence or absence of particular intestinal 373 microflora species can influence the extent to which an animal is sensitive to NIV, because
374 the epoxidated products were shown to be less toxic than parental molecules. In previous
375 studies, its metabolite NIV was observed in in vitro liver microsomes and S-9 fractions from
376 broilers, ducks, and piglets (Poapolathep et al., 2003, 2008, 2014). The current study 16 ACCEPTED MANUSCRIPT 377 produced consistent results for FX-to-NIV metabolism. It is possible that liver microsomal
378 fractions of goats have a high capacity for FX biotransformation. The more active
379 metabolism of goats compared other animals is well known (Toutain et al., 2010). This is
380 linked to their respective feeding behaviour where goats are natural browsers. When eating
381 plants, they preferentially eat the most nutritious part available but will also eat the portions
382 containing many toxic alkaloids that need to be metabolised by a hepatic first pass effect. The
383 highest capacity of FX-to-NIV biotransformation in microsomal fractions was found in the
384 jejunum (25%, among intestinal part) and rumen (3%, among stomach part) of goats.
385 However, it has been reported that ruminants have a high ruminal capacity for
386 biotransforming some mycotoxins to less toxic metabolites (Muller et al., 1998), microbes
387 transform mycotoxins in the intestinal tract of animals prior to absorption (Schatzmayr et al.,
388 2006; Escriva, et al., 2015) and in vivo biotransformation might be larger.
389 390 5. Conclusions MANUSCRIPT 391
392 In conclusion, FX is absorbed from the gastrointestinal tract with a relatively low
393 bioavailability in goats. The liver is the organ responsible for the FX-to-NIV
394 biotransformation. FX can be excreted mainly as NIV in the feces of goats after p.o.
395 administration of FX, whereas it can be excreted mainly in urine after iv administration. The
396 results obtained in this study contribute to the knowledge of toxicokinetic behaviour of FX in 397 the goat. However,ACCEPTED a depoxidated form as de-epoxynivalenol and the other modified forms 398 should be further investigated.
399
400 Conflict of interest
401 17 ACCEPTED MANUSCRIPT 402 The authors declare that there are no conflicts of interest in publishing this work.
403
404 Acknowledgments
405
406 The authors gratefully acknowledge the Graduate School, Kasetsart University for
407 providing research funds and we would like to thank our colleagues at the Department of
408 Pharmacology in Faculty of Veterinary Medicine, Kasetsart University for their helpful
409 recommendations for this work.
410
411 References
412
413 Alassane-Kpembi, Kolf-Clauw, M., Gauthier, T., Abrami, R., Abiola, F.A., Oswald, I.P.,
414 Puel. O. 2013. New insights into mycotoxin mixtures: the toxicity of low doses of Type MANUSCRIPT 415 B trichothecenes on intestinal epithelial cells is synergistic. Toxicol. Appl. Pharmacol. 416 272, 191-198.
417 Aupanan, S., Poapolathep, S., Imsilp, P., Prapong, T., Poapolathep, A. 2015. Oral exposure
418 of fusarenon-X induced apoptosis in Peyer’s patches, thymus, and spleen of mice.
419 Res.Vet. Sci. 102, 217-222.
420 Aupanan, S., Phuektes, P., Poapolathep, S., Sutjarit, S., Giorgi, M., Poapolathep, A. 2016.
421 Apoptosis and gene expression in Jurkat human T cells and lymphoid tissues of 422 fusarenon-X-treatedACCEPTED mice. Toxicon 123, 15-24. 423 Aupanan, S., Poapolathep, S., Giorgi, M., Imsilp, K., Poapolathep, A. 2017. An overview of
424 the toxicology and toxicokinetics of fusarenon-X, a type B trichothecene mycotoxin.
425 J.Vet. Med. Sci. 79, 6-13.
426 Broekaert, N., Devreese, M., De Baere, S., Croubels, S. 2015. Modified Fusarium 18 ACCEPTED MANUSCRIPT 427 mycotoxins unmasked: From occurrence in cereals to animal and human excretion. Food
428 Chem. Toxicol.80, 17-31.
429 Cavalier, C., Ascenzo, G.D., Foglia, P., Pastorini, E. 2005. Determination of type B
430 trichothecenes and macrocyclic lactone mycotoxins in field contaminated maize. Food
431 Chem. 92, 559-568.
432 De Vito, V., Łebkowska-Wieruszewska, B., Lavy, E., Lisowski, A., Owen, H., Giorgi, M.,
433 2018. Pharmacokinetics of meloxicam in lactating goats ( Capra hircus ) and its
434 quantification in milk after a single intravenous and intramuscular injection. Small Rum.
435 Res. 160, 38–43.
436 Eriksen, G.S., Pettersson, H., 2004. Toxicological evaluation of trichothecenesin animal feed.
437 Anim. Feed Sci. Technol. 114, 205-239.
438 Esaki, H., Kumagai, S. 2002. Glutathione-S-transferase activity toward aflatoxin epoxide in
439 livers of mastomys and other rodents. Toxicon 40, 941-945. 440 Escriva, L., Font, G., Manyes, L. 2015. In vivo tox MANUSCRIPTicity studies of fusarium mycotoxins in the 441 last decade: A review. Food Chem. Toxicol. 78, 185-206.
442 443 European Commission, 2006. Commission Regulation (EC) No. 1881/2006 of 19 December
444 2006. Setting maximum levels for certain contaminants in foodstuffs. Off. J. Eur. U. L
445 364, 5-24
446 European Food Safety Authority (EFSA), 2013. Scientific opinion on risks for animal and
447 public health related to the presence of nivalenol in food and feed. EFSA J. 11, 3262. 448 European MedicinesACCEPTED Agency, 2012. EMEA/CHMP/EWP/ 192217 of 1 February 2012. 449 Guideline on bioanalytical method validation.
450 Forsell, J.H., Pestka, J.J. 1985. Relation of 8-ketotrichothecene and zearalenone analog
451 structure to inhibition of mitogen-induced human lymphocyte blastogenesis. Appl.
452 Environ. Microbiol. 50, 1304-1307. 19 ACCEPTED MANUSCRIPT 453 Gareis, M., Zimmermann, C., Schothorst, R., Paulsch, W., Vidnes, A., Bergsten, C., Paulsen,
454 B., Brera, C., Miraglia, M. 2003. Collection of occurrence data of Fusarium toxins in food
455 and assessment of dietary intake by population of EU member states. Report of SCOOP
456 task 3.2.10. April 2013. http://ec.europa.eu/food/fs/scoop/task3210.pdf
457 IARC, 1993. Toxins derived from Fusarium graminearum, F. culmorum and F.
458 crookwellense; zearalenone , deoxynivalenol, nivalenol and fusarenon-X. IARC
459 Monographs on the Evaluation of Carcinogenic Risks to Humans 56, 397-444.
460 Juan, C., Ritieni, A., Manes, J. 2013. Occurrence of Fusarium mycotoxins in Italian cereal
461 and cereal products from organic farming. Food Chem. 141, 1747-1755.
462 Kim, T.W., Łebkowska-Wieruszewska, B., Sitovs, A., Poapolathep, A., Owen, H., Lisowski,
463 A., Abilova, Z., Giorgi, M. (in press). Pharmacokinetic profiles of metamizole (dipyrone)
464 active metabolites in goats and its residues in milk. J. Vet. Pharmacol. Ther.
465 Kongkapan, J., Giorgi, M., Poapolathep, S., Isariyodom, S., Poapolathep, A. 2016. 466 Toxicokinetics and tissue distribution of niva lenolMANUSCRIPT in broiler chickens. Toxicon 111, 31- 467 46.
468 Lee, H.K., DeVito, V., Vercelli, C., Tramuta, C., Nebbia, P., Re, G., Kovalenko, K., Giorgi,
469 M., 2017. Ex vivo antibacterial activity of levofloxacin against Escherichia coli and its
470 pharmacokinetic profile following intravenous and oral administrations in broilers. Res.
471 Vet. Sci. 112, 26-33.
472 Miura, K., Nakajima, Y., Yamanaka, N., Terao, K., Shibato, T., Ishino, S.1998. Induction of 473 apoptosis withACCEPTED fusarenon-X in mouse thymocytes. Toxicology 127, 195-206. 474 475 Muller, H.M., Lerch, C., Muller, K., Eggert, W. 1998. Kinetic profiles of ochratoxin A and
476 ochratoxin α during in vitro incubation in buffered forestomach and abomasal content
477 from cows. Nat. Tox. 6:,251-258.
478 Pittet, A., 1998. Natural occurrence of mycotoxins in foods and feeds-an updated reviews. 20 ACCEPTED MANUSCRIPT 479 Rev. Med. Vet. 149, 479-492.
480 Placinta, C.M., D’Mello, J.P.F., Macdonald, A.M.C. 1999. A review of worldwide
481 contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim Feed
482 Sci. Technol. 78, 21-37.
483 Poapolathep, A., Suzuki, K., Katayama, K.I., Ohtsuka, R., Nagata, T., Uetsuka, K.,
484 Nakayama, H., Doi,K. 2001. Development of apoptosis and changes in apoptosis-related
485 genes expression in the thymus of nivalenol-treated mice. J. Toxicol. Pathol. 1, 299-304.
486 Poapolathep, A., Ohtsuka, R., Kiatipattanasakul, W., Ishigami, N., Nakayama, H., Doi, K.
487 2002. Nivalenol-induced apoptosis in thymus, spleen and Peyer’s patches of mice.
488 Exp. Toxicol. Pathol. 5, 441–446.
489 Poapolathep, A., Sugita-Konishi, Y., Doi, K., Kumagai, S. 2003. The fates of trichothecene
490 mycotoxins, nivalenol and fusarenon-X, in mice. Toxicon 41, 1047-1054.
491 Poapolathep, A., Sugita-Konishi, Y., Phitsanu, T., Doi, K., Kumagai, S. 2004. Placenta and 492 milk transmission of trichothecene mycotoxins MANUSCRIPT, nivalenol and fusarenon-X, in mice. 493 Toxicon 44, 111-113.
494 Poapolathep, A., Poapolathep, S., Sugita-Konishi, Y., Imsilk, K., Tassanawat, T., Sinthusing,
495 C., Itoh, Y., Kumagai, S. 2008. Fate of fusarenon-X in broilers and ducks. Poult. Sci. 87,
496 1510-1515.
497 Saengtienchai, T., Poapolathep, S., Isariyodom, S., Ikenaka, Y., Ishizuka, M., Poapolathep,
498 A. 2014.Toxicokinetics and tissue depletion of fusarenon-X and its metabolite nivalenol 499 in piglets. FoodACCEPTED Chem Toxicol. 66, 307-312. 500 Schatzmayr, G., Zehner, F., Taubel, M., Schatzmayr, D., Klimitsch, A., Loibner, A.P.,
501 Binder, E.M. 2006. Microbiologicals for deactivating mycotoxins. Mol. Nutrition Food
502 Res. 50, 543-551.
503 Sutjarit, S., Nakayama, S.M.M., Ikenaka, Y., Ishizuka, M., Banlunara, W., 21 ACCEPTED MANUSCRIPT 504 Rerkamnuaychoke, W., Kumagai, S., Poapolathep, A. 2014. Apoptosis and gene
505 expression in the developing mouse brain of fusarenon-X-treated pregnant mice.
506 Tox. Letters 229, 292-302.
507 Toutain, P.L., Ferran, A., Bousquet-Mélou, A. 2010. Species differences in
508 pharmacokinetics and pharmacodynamics. Handb. Exp. Pharmacol. 199, 19-48.
509 Wen, J., Mu, P., Deng, Y. 2016. Mycotoxins: Cytotoxicity and biotransformation in animal
510 cells. Toxicol. Res. 5, Issue 2, 377-387.
511 World Health Organization (WHO), 1990. Selected mycotoxins: ochratoxins, trichothecenes,
512 ergot. Environmental Health Criteria 105. Geneva: International Programme on Chemical
513 Safety, WHO. 71-164.
514 Wu, Q., Dohnal, V., Huang, L., Kuca, K., Yuan, Z. 2010. Metabolic pathways of
515 trichothecenes. Drug Metab. Rev. 42, 250-267.
516 Yzar, S., Omurtag, G.Z. 2008. Fumonisins, Trichothecenes and Zearalenone in Cereals. 517 Int. J. Mol. Sci. 9, 2062-2090. MANUSCRIPT 518
ACCEPTED ACCEPTED MANUSCRIPT
Table 1. Precision (RSD%), accuracy (%RE) and recovery (%R) of fusarenon-X (FX) and nivalenol (NIV) in biological matrices of goats .
Analyte Matrice Concentration Precision (%RSD) Accuracy (%RE) LOD LOQ of QC sample Recovery ±SD Intra-day Inter-day Intra-day Inter-day (ng/ml or (ng/ml or (ng/ml or ng/g) (n=7) (n=21) (n=7) (n=21) ng/g) ng/g) FX Plasma 5 4.35 7.6 1.24 2.62 101.24±4.58 50 3.82 3.27 1.89 1.17 101.89±3.49 1.50 5.0 250 1.05 0.76 0.34 0.40 100.34±1.06 Urine 5 4.20 4.19 1.68 4.47 101.68±4.27 50 4.07 4.17 4.54 3.62 104.54±4.26 1.46 4.87 250 0.94 1.15 -0.27 0.97 99.73±0.94 Feces 10 7.82 5.94 2.56 2.51 102.58±8.02 50 3.96 4.07 2.06 2.60 102.04±4.04 2.54 8.45 250 1.06 0.82 0.67 0.39 100.67±1.07 NIV Plasma 5 4.04 4.88 -0.72 1.23 99.28±4.37 50 3.67 3.84 -4.87 -4.87 95.32±3.69 1.73 5.78 250 0.64 0.46 -0.72 -0.57MANUSCRIPT 99.48±0.39 Urine 5 2.90 5.26 -5.76 -2.39 94.24±2.73 50 4.90 4.60 -3.38 -0.70 96.62±4.25 1.54 5.13 250 0.89 0.61 -0.32 -0.22 99.68±0.89 Feces 10 1.50 6.53 -8.44 -2.79 91.56±1.37 50 4.04 4.62 -3.58 -1.70 96.42±3.90 3.0 10.0 250 0.98 0.80 -0.69 -0.68 99.31±0.37
ACCEPTED
ACCEPTED MANUSCRIPT
Table 2. Mean ± SD value of the toxicokinetic parameters of fusarenon-X (FX) and nivalenol (NIV) following iv and po administration at a dosage of 0.25 and 1 mg/kg bw, respectively in goats (n= 5).
Toxicokinetic parameter Intravenous administration Oral administration (unit) FX NIV FX NIV t1/2λ (h) 1.64 ± 0.32 22.85 ± 4.62 4.69 ± 1.25 7.96 ± 2.99
Kel (h -1) 0.44 ± 0.09 0.03 ± 0.01 0.16 ± 0.05 0.10 ± 0.04
Tmax (h) - 0.38 ± 0.38 0.08 ± 0.00 6.05 ± 4.82
Cmax (ng/ml) - 393.56 ± 104.81 413.39 ± 206.84 181.88 ± 32.45 0 Cp (ng/ml) 2051.80 ± 439.31 - - -
AUC last (h ng/ml) 847.20 ± 248.70 - - -
AUC inf (h ng/ml) 867.27 ± 257.10 4716.99 ± 1523.44 243.31 ± 66.45 2777.97 ± 410.98 Vd (ml/kg) 730.10 ± 257.40 - MANUSCRIPT- - CL (ml/h/kg) 310.98 ± 90.60 - - - MRT (h) 1.17 ± 0.27 27.37 ± 7.57 4.79 ± 1.16 12.69 ± 2.55
Vd ss (ml/kg) 354.65 ± 89.53 - - - F (%) - - 15.81 -
0 Note: t1/2λ = elimination half-life; Kel = elimination rate constant; Tmax = time at maximum concentration; Cmax = the maximum concentration; Cp = plasma concentration at initial time; AUC last = area under the curve from zero to 24 h; AUC inf = area under the curve from zero to the last, Vd = volume of ACCEPTED § distribution, CL = clearance; MRT = mean residence time; Vdss = volume of distribution at steady state; F = oral bioavailability. This data is divided for bioavailability when calculated for the po administration ACCEPTED MANUSCRIPT
Table 3. Mean ± SD values of fusarenon-X (FX) and nivalenol (NIV) in urine and feces at dosages of 0.25 and 1.0 mg/kg bw after iv and po administrations of FX in goats (n = 5 per group)
Intravenous administration Oral administration Time (h) Urine (ng/ml) Feces (ng/g) Urine (ng/ml) Feces (ng/g)
FX NIV FX NIV FX NIV FX NIV
0-2 57.48 ± 29.14 330.63 ± 294.81 ND ND NS NS ND ND
2-4 46.87 ± 21.01 296.25 ± 261.66 ND 62.43 ± 10.31 ND 52.35 ± 0.01 ND ND
4-8 51.66 ± 16.07 125.31 ± 80.30 ND 83.90 ± 55 ND 172.75 ± 171.31 ND 359.23 ± 165.40
8-12 ND 50.36 ± 0.01 ND 165.94 ± 39.69 ND 51.72 ± 1.51 ND 242.13 ± 109.05 MANUSCRIPT 12-24 ND ND ND ND ND ND ND 304.01 ± 287.68
24-48 ND ND ND ND ND ND ND ND
NS = No sample, ND = not detected
ACCEPTED ACCEPTED MANUSCRIPT a)
10000
1000
100
10
Toxin concentration Toxin concentration (ng/ml) 1 0 10 20 30 40 50 Time (h) b) b) MANUSCRIPT 1000
100
10
Toxin concentration Toxin concentration (ng/ml) ACCEPTED 1 0 5 10 15 20 25 Time (h)
ACCEPTED MANUSCRIPT
Fig. 1. Mean values (±SD) of fusarenon-X ( □) and nivalenol ( ●) concentration in plasma goats at dosages of 0.25 and 1 mg/kg bw of fusarenon-X; a) intravenous administration, and b) oral administration (n= 5).
MANUSCRIPT
ACCEPTED
ACCEPTED MANUSCRIPT a) b) 30 100 15min 30min 45min 60min 15min 30min 45min 60min 25 80 20 60 15 40 10 20 5 0 0 S9 S-9 S-9 S-9 S-9 S-9 S-9 S-9 cytosolic cytosolic cytosolic cytosolic cytosolic cytosolic cytosolic cytosolic microsomal microsomal microsomal microsomal microsomal Conversion Conversion rate to Nivalenol (%) microsomal microsomal microsomal
Conversion Conversion rate to Nivalenol (%) Liver Kidney Lung Duodenum Jejunum Ileum Colon Rectum Tissues Tissues
4 c) MANUSCRIPT15 min 30 min 45 min 60 min 3 2 1 0 S-9 S-9 S-9 S-9 cytosolic cytosolic cytosolic cytosolic microsomal microsomal microsomal microsomal Conversion Conversion rate to Nivalenol (%) RumenACCEPTED Reticulum Omasum Abomasum Tissues
ACCEPTED MANUSCRIPT
Fig. 2. Metabolic conversion of fusarenon-X to nivalenol at concentration of 5 µµµg/10 µµµl in S-9 fractions, microsomal fractions and cytosolic fractions of liver, kidney and lung (a), intestine (duodenum, jejunum, ileum, colon and rectum) (b) and stomach (rumen, reticulum, omasum and abomasum) (c) from goats.
MANUSCRIPT
ACCEPTED ACCEPTED MANUSCRIPT Highlights
• This is the first study that evaluates the fate and metabolism of FX in ruminant species.
• FX is absorbed from the gastrointestinal tract with a relatively low bioavailability in goats.
• The liver is the organ responsible for the FX-to-NIV biotransformation in goats.
• FX is excreted mainly as NIV in the feces of goats after p.o. administration of FX.
MANUSCRIPT
ACCEPTED ACCEPTED MANUSCRIPT Ethical Form
Title: Toxicokinetic profile of fusarenon-X and its metabolite nivalenol in the goat (Capra hircus)
All experimental procedures were performed according to the Guideline for All Experiments, and approved by the Ethics Research Committee of the Faculty of Veterinary Medicine, Kasetsart University.
Amnart Poapolathep
Corresponding author
MANUSCRIPT
ACCEPTED