Insights into in vivo absolute oral bioavailability, biotransformation and

Subscriber access provided by WEBSTER UNIV

Food Safety and Toxicology

Insights into in vivo absolute oral bioavailability, biotransformation and toxicokinetics of zearalenone, #-zearalenol, #-zearalenol, zearalenone-14-glucoside and zearalenone-14-sulfate in pigs Amelie Catteuw, Nathan Broekaert, Siegrid De Baere, Marrianne Lauwers, Elke Gasthuys, Bart Huybrechts, Alfons Callebaut, Lada Ivanova, Silvio Uhlig, Marthe De Boevre, Sarah De Saeger, Ronette Gehring, Mathias Devreese, and Siska Croubels J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05838 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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 37

Journal of Agricultural and Food Chemistry

Insights into in vivo absolute oral bioavailability, biotransformation and toxicokinetics of zearalenone, α-zearalenol, β-zearalenol, zearalenone-14glucoside and zearalenone-14-sulfate in pigs Amelie Catteuw1°, Nathan Broekaert1°, Siegrid De Baere1, Marianne Lauwers1, Elke Gasthuys1, Bart Huybrechts2, Alfons Callebaut2†, Lada Ivanova3, Silvio Uhlig3, Marthe De Boevre4, Sarah De Saeger4, Ronette Gehring5,6, Mathias Devreese1,5 and Siska Croubels1* 1

Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine,

Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 2

Sciensano, Juliette Wytsmanstraat 14, 1050 Elsene, Belgium

3

Chemistry Section, Norwegian Veterinary Institute, Ullevålsveien 68, 0454 Oslo, Norway

4

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

Ottergemsesteenweg 460, 9000 Ghent, Belgium 5

Institute of Computational Comparative Medicine, College of Veterinary Medicine, Kansas

State University, 1700 Denison Ave, Manhattan, USA 6

Institute of Risk Assessment Sciences, Division of Toxicology/Pharmacology, Utrecht

University, Yalelaan 1, 3584 CL Utrecht, The Netherlands °

Authors contributed equally, shared first authorship

†

Deceased on June 3, 2017

* Corresponding author: Siska Croubels Tel: +32 9 264 73 45; Fax: +32 9 264 74 97; Email: [email protected]

1 ACS Paragon Plus Environment


Page 2 of 37

1

Abstract

2

its modified forms, α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalenone-14-glucoside

3

(ZEN14G) and zearalenone-14-sulfate (ZEN14S), including (pre)systemic hydrolysis in pigs.

4

Cross-over pig trials were performed by means of intravenous and oral administration of ZEN

5

and its modified forms. Systemic plasma concentrations of the administered toxins and their

6

metabolites were quantified and further processed via tailor-made compartmental toxicokinetic

7

models. Furthermore, portal plasma was analysed to unravel the site of hydrolysis, and urine

8

samples were analysed to determine urinary excretion. Results demonstrate ZEN14G and

9

ZEN14S to be (completely) presystemically hydrolysed to ZEN and demonstrate high oral

10

bioavailability for all administered compounds, with further extensive first pass

11

glucuronidation. Conclusively, the modified ZEN forms α-ZEL, β-ZEL, ZEN14G and ZEN14S

12

contribute to overall ZEN systemic toxicity in pigs and should be taken in account for the risk

13

assessment.

14

Keywords zearalenone – toxicokinetics – pig – zearalenone-14-glucoside – zearalenone-14-

15

sulfate

The aim of this study was to determine the toxicokinetic characteristics of ZEN and


Page 3 of 37


16

INTRODUCTION

17

Mycotoxins are known to be among the most hazardous of all food and feed contaminants

18

concerning chronic toxicity. Recent publications suggest that about 70% of cereal-based feeds

19

are contaminated with one or more mycotoxins 1.

20

Fusarium fungi frequently infect cereals, i.e. wheat, barley, rye, maize and oats, in temperate

21

regions including Western Europe. The mycotoxins produced by these Fusarium species cause

22

significant economic losses in animal production, and are a hazard to public health and animal

23

welfare. With an incidence of up to 80% in unprocessed cereals and up to nearly 100% in

24

compound feed, zearalenone (ZEN) is one of the most critical Fusarium mycotoxins, due to its

25

universal occurrence and toxic potential 2,3. ZEN is a non-steroidal mycoestrogen, which binds

26

to the 17β-estradiol receptor (ER) in target cells, leading to hyperestrogenism and reproductive

27

disorders in both humans and pigs. Additionally, hemotoxic and genotoxic properties are

28

reported 4.

29

Aside from ZEN, the modified ZEN forms frequently (co-)contaminate cereal-based foods

30

and feedstuff 5–7. They are formed by plants and rival fungi by altering the chemical structure

31

mediated by biotransformation reactions, as a natural protective strategy against xenobiotics.

32

Important examples of these modified forms of ZEN are α-zearalenol (α-ZEL), β-zearalenol (β-

33

ZEL), zearalenone-14-glucoside (ZEN14G) and zearalenone-14-sulfate (ZEN14S)

34

Although in lower concentrations than for the free (or unconjugated) forms, relative high

35

incidence rates have been noted for modified mycotoxins in cereal-based food and feed

36

matrices, with values of 53%, 63%, 30% and 20% for α-ZEL, β-ZEL, ZEN14G and ZEN14S

37

respectively 5. The same study suggests that approximately 60% of the available ZEN was

38

found as modified form 6.

5,7,8

.

39

The susceptibility of humans and different animal species to a certain xenobiotic strongly

40

depends on the oral bioavailability and biotransformation processes of that xenobiotic. For 3 ACS Paragon Plus Environment


Page 4 of 37

41

example, ZEN is efficiently absorbed after oral (PO) administration in pigs, with an absorbed

42

fraction of 85% 9. After oral absorption, its fate is determined by biotransformation, biliary

43

excretion and enterohepatic recycling (EHC) 9. As for the biotransformation, phase I reactions

44

consist of reduction of ZEN to α-ZEL and β-ZEL, catalysed by 3α- and 3β-hydroxysteroid

45

dehydrogenases. Phase II biotransformation consists of glucuronidation of ZEN, α-ZEL and β-

46

ZEL, catalysed by uridine diphosphate glucuronyl transferase (UDPGT)

47

biotransformation reactions mainly occur in the liver, however in vitro experiments

48

demonstrated the intestinal mucosa to be a possible extrahepatic biotransformation site

49

Species that mainly metabolise ZEN to α-ZEL, such as humans and pigs, are relatively sensitive

50

due to the higher affinity of α-ZEL for the ER, resulting in more potent toxic properties 11,16–21.

51

Curiously, Warth et al. did not detect α-ZEL in urine of one human volunteer after ZEN

52

administration 21. In contrast, species that predominantly form β-ZEL, such as broiler chickens

53

and rats, tend to be less sensitive due to the lower affinity of this metabolite for the ER 11. More

54

specifically, α-ZEL has a 73 times higher binding affinity to the ER than ZEN, whereas β-ZEL

55

exhibits about half of the affinity of ZEN 22. Phase II metabolites have lost their affinity for the

56

ER 23,24. Nevertheless, cleavage (hydrolysis) during mammalian digestion of phase II modified

57

forms produced by plants, can release the free ZEN 25. Consequently, the total exposure to ZEN

58

and its risk assessment, based only on the free ZEN, might be underestimated.

59

10,11

. Both

9,12–15

.

Hydrolysis of the phase II modified ZEN forms mainly occurs in the gastrointestinal tract by 25

60

both enzymes of the bacterial microbiota and of the mammalian intestinal epithelium

61

Hydrolysis of ZEN14G during digestion in the pig, was first demonstrated by Gareis et al. 26.

62

An in vitro digestion study by Dall’Erta et al. showed that ZEN14G and ZEN14S are

63

completely hydrolysed to ZEN by the human colonic microbiota after an incubation time of

64

only 30 minutes

65

Glucosides seem to be more resistant to acid conditions in comparison with sulfate conjugates

27

.

. Besides enzymatic hydrolysis, also acidic hydrolysis is described.


Page 5 of 37


66

28

67

Unfortunately, experiments studying the exact site of hydrolysis, are still lacking for ZEN

68

conjugates. Sampling of portal plasma facilitates possible differentiation between presystemic

69

(gastrointestinal) and systemic (hepatic, blood enzymes, …) hydrolysis of ZEN14G and

70

ZEN14S, as previously described for deoxynivalenol-3-glucoside (DON3G) 30.

. In contrast, an in vivo study in rats revealed hydrolysis of ZEN14G in the stomach

29

.

71

Following a request from the European Commission (EC), the European Food Safety

72

Authority Panel on Contaminants in the Food Chain (EFSA CONTAM Panel) assessed whether

73

it is appropriate and feasible to set a group health-based guidance value for ZEN and its

74

modified forms related to their presence in food and feed. The EFSA CONTAM panel found it

75

appropriate to set a group total daily intake (TDI) of 0.25 µg/kg bodyweight (BW) per day,

76

based on a no-observed-effect level (NOEL) in gilts of 10 µg/kg BW, expressed as ZEN

77

equivalents for ZEN and its modified forms. To take in account differences in in vivo estrogenic

78

potency, each phase I metabolite was attributed a potency factor, relative to ZEN, to use for

79

exposure estimates of the respective ZEN metabolites. Phase II metabolites of ZEN and of its

80

phase I metabolites, which do not have an estrogenic activity, were assumed to be cleaved,

81

releasing ZEN and its phase I metabolites. These conjugates were attributed the same relative

82

potency factors as the unconjugated forms. As such, relative potency factors of 60, 0.2, 1 and 1

83

were attributed to α-ZEL, β-ZEL, ZEN14G and ZEN14S respectively 8.

84

Current potency factors were derived from rat, and until today scientific data concerning the

85

estrogenicity and ADME processes (absorption, distribution, metabolisation and excretion) of

86

modified ZEN in humans and pigs are lacking

87

associated with the present risk assessment. The panel recommends that more data on

88

toxicokinetics of modified forms of ZEN is needed, particularly on their oral bioavailability and

89

in vivo hydrolysis 25,31,32.

31

. Various assumptions and uncertainties are



Page 6 of 37

90

Therefore, the goal of this study was to determine the absolute oral bioavailability and the

91

main toxicokinetic parameters of α-ZEL, β-ZEL, ZEN14G and ZEN14S, as well as the degree

92

of (pre)systemic hydrolysis of ZEN14G and ZEN14S in pigs, a species which in addition to its

93

susceptibility to the effects of ZEN, also provides a reliable model to extrapolate to humans 33.

94

In vivo models are preferred to in vitro models, as these do not account for important

95

physiological and anatomical factors, including intestinal mucosa and luminal content

96

composition (enzymes, microbiota), internal organ blood flow and enterohepatic recirculation

97

34

98

MATERIALS AND METHODS

99

Standards, reagents and solutions

.

100

ZEN, α-ZEL and β-ZEL (>99% purity) were obtained from Fermentek (Jerusalem, Israel). α-

101

zearalanol (α-ZAL), β-zearalanol (β-ZAL) and zearalanone (ZAN) were purchaised from

102

Sigma-Aldrich (Bornem, Belgium).

103

IS, 25.4 µg/mL) stock solution in acetonitrile (ACN) was purchased from Biopure (Tulln,

104

Austria). ZEN14G and ZEN14S were enzymatically synthesised, purified and verified using

105

nuclear magnetic resonance (NMR) and liquid chromatography-tandem mass spectrometry

106

(LC-MS/MS)

107

ZEN14S. Following characterised glucuronides (GlcAs) were available from earlier work:

108

ZEN-14-O-glucuronide (ZEN-14-GlcA), α-ZEL-14-O-glucuronide (α-ZEL-14-GlcA), α-ZEL-

109

7-O-glucuronide (α-ZEL-7-GlcA), β-ZEL-14-O-glucuronide (β-ZEL-14-GlcA) and β-ZEL-16-

110

O-glucuronide (β-ZEL-16-GlcA)

111

dimethylsulfoxide (DMSO) (Sigma-Aldrich, Diegem, Belgium), providing a stock solution of

112

10 mg/mL, to use for animal trials. ZEN, ZEN14G, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN

113

were dissolved in methanol (MeOH), providing a stock solution of 1 mg/mL, to use for

114

analytical experiments. ZEN14S was dissolved in water, providing a stock solution of 0.821

35,36

13

C18-ZEN (stable isotopically labelled internal standard,

. No remaining ZEN (ZEN). Secondary parameters were: AUC0-∞, ke and t1/2el. Estimated

294

secondary parameters of ZEN after IV ZEN14G administration were ke ZEN and t1/2el ZEN. The

295

percentage of systemic hydrolysis (Syst. Hydr.) of ZEN14G to ZEN, was calculated according

296

to the following formula:

297

(3)

298

After IV ZEN14S administration, no LC-MS/MS plasma concentrations could be detected, and

299

consequently no further toxicokinetic modeling could be performed.

300

Although no toxicokinetic modeling could be performed on LC-MS/MS data obtained after PO

301

administration of the toxins, some estimates could be calculated based on the LC-MS/MS and

302

LC-HRMS data. These are only estimates because results are only based on data from three

303

pigs. An equimolar correction was applied on all values. Absolute oral bioavailability (F), i.e.

304

the fraction of the administered toxin which is absorbed in the systemic circulation in its

305

unchanged forms, could be estimated as follows:

306

(4)

307

For phase II modified mycotoxins, the absorbed fraction (FRAC), i.e. the fraction of the

308

administered toxin which is absorbed in the systemic circulation in its unchanged and in its

309

hydrolysed form, could be estimated as follows:

310

(5)

311

RESULTS AND DISCUSSION

Syst. Hydr. (%) = AUC

AUC𝑍𝐸𝑁 ∗CL𝑍𝐸𝑁 ∗100

𝑍𝐸𝑁14𝐺 ∗(CL𝑍𝐸𝑁14𝐺 +CL𝑍𝐸𝑁14𝐺→𝑍𝐸𝑁 )+ AUC𝑍𝐸𝑁 ∗CL𝑍𝐸𝑁

F ( %) ≈

AUC𝑃𝑂 (𝑎𝑑𝑚𝑖𝑛𝑖𝑠𝑡𝑒𝑟𝑒𝑑 𝑡𝑜𝑥𝑖𝑛+𝑚𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑡𝑒𝑠) ∗100 AUC𝐼𝑉 (𝑎𝑑𝑚𝑖𝑛𝑖𝑠𝑡𝑒𝑟𝑒𝑑 𝑡𝑜𝑥𝑖𝑛+𝑚𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑡𝑒𝑠)

FRAC (%) ≈

AUC𝑃𝑂 (𝑎𝑑𝑚𝑖𝑛𝑖𝑠𝑡𝑒𝑟𝑒𝑑 𝑡𝑜𝑥𝑖𝑛+ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑒𝑑 𝑡𝑜𝑥𝑖𝑛+𝑚𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑡𝑒𝑠) ∗100 AUC𝐼𝑉 (𝑎𝑑𝑚𝑖𝑛𝑖𝑠𝑡𝑒𝑟𝑒𝑑 𝑡𝑜𝑥𝑖𝑛+ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑒𝑑 𝑡𝑜𝑥𝑖𝑛+𝑚𝑒𝑡𝑎𝑏𝑜𝑙𝑖𝑡𝑒𝑠)


Page 15 of 37


312

Systemic plasma analysis and toxicokinetic analysis

313

The aim of this study was to determine the absolute oral bioavailability and biotransformation

314

of ZEN, α-ZEL, β-ZEL, ZEN14G and ZEN14S, the degree of in vivo hydrolysis of ZEN14G

315

and ZEN14S to ZEN (differentiating between presystemic and systemic hydrolysis) and the

316

toxicokinetic parameters of different ZEN forms in pigs. Tailor-made compartmental

317

toxicokinetic models were developed, which offer the advantage, compared with non-

318

compartmental analysis, to take into account metabolites as well as to make predictions about

319

concentrations at unsampled time points 46.

320

Aside from oedematous swelling of the vulva, no adverse effects were detected during the

321

animal trials following all bolus administration.

322

Plasma concentration-time profiles, obtained after LC-MS/MS and LC-HRMS analysis, for all

323

bolus administrations are presented in Figure 3. Each profile represents the mean + standard

324

deviation (SD) of n animals for systemic plasma concentrations (n can deviate because of

325

problems during blood sampling due to catheter occlusions). Values below the LOQ were not

326

included. No ZAN, α-ZAL or β-ZAL were detected after IV and PO administration of ZEN, α-

327

ZEL, β-ZEL, ZEN14G or ZEN14S, and is in accordance with previously published data 42,47.

328

After oral administration, plasma concentrations of ZEN, its modified forms as well as its

329

phase I metabolites, were too often below the LOQ to construct reliable plasma concentration-

330

time profiles. This can be attributed to the extensive first pass biotransformation of ZEN and

331

its modified forms. Pfeiffer et al. determined the in vitro glucuronidation activities for ZEN and

332

its phase I metabolites using hepatic microsomes from various farm animals, experimental

333

animals as well as humans, showing the highest activity for porcine microsomes 14. Indeed, a

334

fast and extensive glucuronidation of ZEN, α-ZEL and β-ZEL was detected after PO

335

administration.



Page 16 of 37

336

After IV ZEN administration, a partial phase I biotransformation of ZEN to α-ZEL, but not to

337

β-ZEL, was observed. This conversion was estimated to be approximately 60%, based on the

338

area under the curve (AUC0∞) corrected for differences in CL of both ZEN and α-ZEL. These

339

findings are similar to the data obtained by Fleck et al. after IV ZEN administration (0.1 mg/kg

340

BW)

341

generally considered the main phase I metabolite in the pig. Nevertheless, some studies report

342

the presence of β-ZEL and β-ZEL-GlcA in pig urine after oral administration of ZEN, with α/β-

343

ratio’s ranging from 3 up to 36.5

344

genetic variation within the different pig breeds or even individuals. After all, in vitro studies

345

have demonstrated porcine hepatic microsomal reduction of ZEN to both α- and β-ZEL 52–54.

346

The derived main toxicokinetic parameters after IV administration of ZEN, α-ZEL, β-ZEL and

347

ZEN14G are shown in Table 1.

47

. Some controversy exists concerning β-ZEL as ZEN metabolite in the pig. α-ZEL is

10,47–51

. Differences in α/β-ZEL ratio could be explained by

348

Within multiple in vivo pig studies concerning toxicokinetics of ZEN, the elimination half-

349

life of ZEN showed high variation (2.63 – 86.6 h) 9,47,50,55–57. These values are higher than the

350

elimination half-life of ZEN (t1/2el ZEN = 0.95 ± 0.45h) calculated in the current study. This

351

discrepancy can be explained by the fact that the other studies took into account the GlcAs,

352

which show a longer persistence in biological matrices such as plasma and urine. Only one in

353

vivo study was performed in which toxicokinetic parameters of the free ZEN were calculated

354

after IV administration of ZEN to pigs

355

resulted in rapid distribution from the plasma (t1/2 = 0.3-1h). This is comparable with the

356

elimination half-life of ZEN, obtained in the present study. Additionally, a similar CL rate was

357

observed in both studies, with values of 5.1 ± 1.4 L/kg*h and 6.66 ± 2.74 L/kg*h in the study

358

of Fleck et al. and in present study, respectively 47.

47

. In that study, IV injection of 0.1 mg/kg BW ZEN

359

After both IV α-ZEL and β-ZEL administration, a partial conversion to ZEN was

360

demonstrated. Using a similar calculation approach as for the conversion of ZEN to α-ZEL, the


Page 17 of 37


361

conversion of α-ZEL to ZEN was estimated to be approximately 7% after IV α-ZEL

362

administration. No conversion between α-ZEL and β-ZEL was observed.

363

After IV ZEN14G administration, ZEN14G undergoes partial hydrolysis to ZEN (19.95 ±

364

1.59%). Systemic hydrolysis can be attributed to the effects of liver enzymes and blood esterase

365

enzymes. This hydrolysis of ZEN14G is in contrast to the observations for DON3G by

366

Broekaert et al., where no systemic hydrolysis was detected of DON3G after IV administration

367

to pigs 30. However, Broekaert et al. did describe the presence of systemic hydrolysis for the 3-

368

and 15-acetylated forms of deoxynivalenol.

369

In remarkable contrast to ZEN14G, no ZEN14S, ZEN or ZEL were detected after IV ZEN14S

370

administration, as well with the LC-MS/MS as with the LC-HRMS method. This indicates that

371

ZEN14S is not systemically hydrolysed, but assumably rapidly cleared from the system, hereby

372

swiftly reducing plasma concentrations of ZEN14S below the relatively high LOQ (20 ng/mL).

373

Elimination half-life of the modified ZEN forms appeared to be shorter than that of ZEN itself,

374

caused by either a higher CL (α-ZEL) or lower volumes of distribution (β-ZEL and ZEN14G),

375

due to their higher polarity compared to ZEN. Another explanation could possibly be

376

differences in plasma protein binding.

377

In contrast to ZEN and its phase I metabolites, the GlcA metabolites of ZEN, α-ZEL, β-ZEL

378

could be detected after PO administration, demonstrating systemic exposure to all of the orally

379

administered ZEN forms and demonstrating the potential of these phase II metabolites as

380

biomarkers for ZEN exposure in pigs. This is in line with the study of Binder et al. and Fleck

381

et al., in which phase II glucuronidation of ZEN was described

382

glucuronide (ZEN-GlcA) has additionally been demonstrated to be the main urinary excretion

383

product after oral administration of ZEN to one human volunteer

384

groups, i.e. at position 14 and 16, ZEN can theoretically form two regio-isomeric mono-

385

glucuronides. Results for ZEN- and ZEL-GlcAs revealed that exclusively the 14-isomers, i.e.

47,50

21

. Besides pigs, ZEN-

. Due to its two hydroxyl



Page 18 of 37

386

ZEN-14-GlcA and α-ZEL-14-GlcA isomers, were formed after ZEN, α-ZEL, ZEN14G and

387

ZEN14S bolus administration. This is in accordance with Pfeiffer et al., who incubated ZEN

388

with hepatic microsomes from different species in the presence of uridine-5'-

389

diphosphoglucuronic acid (UDPGA). ZEN-14-GlcA was the predominant glucuronide in all

390

species 14. Results after β-ZEL bolus administration showed that both β-ZEL-14-glucuronide

391

and β-ZEL-16-glucuronide were formed. In contrast to the results of Ueberschär et al., no

392

sulfate conjugates of α-ZEL were observed 58. Curiously, Ueberschär described 62% of the total

393

dose of ZEN-metabolites, recovered in urine, to be in the sulfated form, although pigs are

394

generally known to have a low phase II sulfation activity 58–60.

395

Based on LC-MS/MS and LC-HRMS data, estimated oral bioavailability for each of the

396

administered toxins (except for ZEN14S) could be calculated (see Table 1). For ZEN this

397

results in an estimated F of 61%, which is comparable to the finding of Fleck et al., were an F

398

of 59% was calculated for pigs of the same age 47. In a study in older pigs (10-14 weeks old) a

399

higher F of 80-85% was found 9. For α-ZEL an estimated F of 123% was calculated. Evidently,

400

this value is an overestimation as no correction for clearance could be applied, and as Table 1

401

shows a relatively high clearance of α-ZEL. For β-ZEL the estimated F was 98%. These high

402

values for α- and β-ZEL could indicate that ZELs are more easily absorbed than ZEN itself,

403

approaching complete oral absorption.

404

For ZEN14G this resulted in an estimated FRAC of 61%, suggesting complete hydrolysis of

405

ZEN14G to ZEN after PO administration, and absorption as such.

406

After PO ZEN14S administration a lag time of over 4 h p.a. can be observed, before

407

quantifiable plasma concentrations of ZEN-14-GlcA were detected. Due to this lag time, the

408

plasma concentration-time profile for ZEN14S metabolites exceeds the sampling period of 480

409

minutes, resulting in an incomplete profile. This lag time suggest hydrolysis of ZEN14S to

410

occur distally in the gastrointestinal tract, presumably in the colon by microflora, which is in


Page 19 of 37


411

accordance with a study published by Dall’Erta et al. on in vitro digestion, which showed that

412

ZEN14S is rapidly deconjugated to ZEN by colonic microbiota during the simulation of the

413

large intestines 27. This is in contrast to the results after oral ZEN14G administration, were no

414

lag time was observed, suggesting hydrolysis occurs proximally at the main site of absorption

415

(duodenum and proximal jejunum), presumable by the acidic conditions (stomach, duodenum),

416

or enzymes of the intestinal epithelium or microbiota 62. Proximal gastrointestinal hydrolysis

417

of ZEN14G is supported by equal FRAC and F for ZEN14G and ZEN respectively. Due to the

418

incomplete profile for ZEN14S, no estimates for FRAC could be made, although presystemic

419

(gastrointestinal) hydrolysis of ZEN14S is clearly present. This is in accordance with previous

420

studies, both in the pig as after exposure to human intestinal microflora 27,50.

421

After all PO administrations, the profiles of the GlcAs show a second peak in the plasma

422

concentration-time profiles during the elimination phase, at about 2 h p.a.. This phenomenon

423

can possibly be attributed to the EHC processes and has previously been described by several

424

authors 9,63.

425

Portal plasma analysis

426

Additionally, portal plasma samples were analysed after all oral bolus administrations

427

(Supplementary Figure 2). No ZEN14G or ZEN14S could be detected in portal plasma,

428

supporting the hypothesis that hydrolysis of the modified ZEN forms to ZEN must occur before

429

portal plasma is reached, i.e. in the gastrointestinal tract by both enzymes of the bacterial

430

microbiota and/or of the mammalian intestinal epithelium, as well as in acidic conditions of the

431

stomach

432

α-ZEL and β-ZEL could be detected in the portal plasma and not in the jugular plasma,

433

suggesting these phase I modified mycotoxins are intestinally absorbed as such, followed by an

434

extensive first pass hepatic glucuronidation. Unfortunately, this could not be confirmed for

25–27

. After PO α-ZEL and β-ZEL administration, low concentrations of respectively



Page 20 of 37

435

ZEN , assumably due to the lower F for ZEN in comparison with α/β-ZEL and consequently

436

the lower plasma concentrations of ZEN (

Insights into in vivo absolute oral bioavailability, biotransformation and

Recommend Documents