Discrimination between Synthetically Administered and Endogenous

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Discrimination between synthetically administered and endogenous thiouracil based on monitoring of urine, muscle and thyroid tissue. An in vivo study in young and adult bovines Jella Wauters, Lieven Van Meulebroek, Eric Fichant, Philippe Delahaut, and Lynn Vanhaecke J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01920 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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Journal of Agricultural and Food Chemistry

Discrimination between synthetically administered and endogenous thiouracil based on monitoring of urine, muscle and thyroid tissue. An in vivo study in young and adult bovines.

J. Wauters*1, L. Van Meulebroek1, E. Fichant2, P. Delahaut2, L. Vanhaecke1

1

Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety,

Laboratory of Chemical Analysis, Merelbeke, Belgium 2

CER Groupe, Health Department, Marloie, Belgium

*Corresponding

author,

(Tel:

+3292647452;

Fax:

+3292647492;

ACS Paragon Plus Environment

E-mail:

[email protected]);

1


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Abstract

2 3

Thiouracil (TU), synthesized for its thyroid-regulating capacities and alternatively misused in

4

livestock for its weight-gaining effects, is acknowledged to have an endogenous origin.

5

Discrimination between low-level abuse and endogenous occurrence is challenging and

6

unexplored in an experimental setting.

7

Therefore, cows (n = 16) and calves (n = 18) were subjected to a rapeseed-supplemented diet

8

or treated with synthetic TU.

9

Significant higher urinary TU levels were recorded after TU administration (< CCα – 15642

10

µg L-1) compared to rapeseed supplementation (< CCα – 65.8 µg L-1), however, with

11

overlapping values. TU was not detected in the edible meat, however, concentrations between

12

the CCα and 10 µg kg-1 were noted in thyroid tissue of calves and cows following rapeseed

13

supplementation. The latter concentrations were significantly higher in thyroid tissue of

14

calves (22.9-41.8 µg kg-1) and cows (16.9-36.7 µg kg-1) after synthetic TU administration.

15

These results strongly point towards thyroid analysis as a discriminatory tool.

16 17

Keywords

18

Thyreostats – natural formation – rapeseed – experimental design

19 20


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21


Introduction

22 23

Recent new insights into the endogenous formation of some substances, prohibited in food-

24

producing animals, have been compromising the zero-tolerance regulation for their use which

25

has been implemented by the European Community legislation since 19811-3. For these

26

substances, which include amongst others certain hormones and thyreostats, all European

27

member states have established targeted national residue monitoring programs to ensure the

28

absence of harmful residues and contaminants in animal-derived food and feed products 4. In

29

this context, adequate harmonized monitoring is conducted by ISO 17025 accredited

30

laboratories, whereby methods are applied that have been validated based on the criteria of

31

Commission Decision 2002/657/EC5. This also involves the definition of minimum required

32

performance limits (MRPLs) for analytical methods, which are generally largely overruled by

33

the extremely low detection capacities (CCβ) that are obtained by state-of-the-art analytical

34

technology 6, 7.

35

These low detection capabilities led to more non-compliant results for various compounds

36

(e.g. thiouracil (TU)2), for which farmers were penalized and thus imposed to a broad range of

37

consequences; i.e. an increased number of charged residue analyses for their cattle, a loss of

38

governmental grants, cancellation of their farming license, and a possible personal

39

prosecution 8. For several of these substances, including steroidal compounds such as

40

nortestosterone, boldenone, prednisolone and prednisone, an endogenous origin was rather

41

easily demonstrated 9-12. However, a similar explanation was far less evident for the suspected

42

endogenous origin in case of low-level TU levels, observed in urine from small livestock,

43

cattle and pigs. A decade ago, the endogenous origin for this substance was for the first time

44

linked to the administration of various Brassicaceae spp. to ruminants as described by Pinel et

45

al. (2006)13. The formation of TU from these Brassicaceae spp. was subsequently examined


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46

by Vanden Bussche et al. (2011)14 and Kiebooms et al. (2014)15, demonstrating the

47

involvement of enzymatic reactions with a plant and/or intestinal microbial origin.

48

More recently, a retrospective screening of urinary TU analyses (performed in the light of the

49

national monitoring programs of The Netherlands, Belgium, Poland, France, UK and

50

Norway) was performed and revealed 99% percentiles of 18.2 µg L-1 endogenous TU (95%

51

confidence interval (CI) between 15.5 and 20.0 µg L-1) for the entire bovine population and

52

35.9 µg L-1 (CI between 22.3 and 64.5 µg L-1) for the young male bovine population (6-12

53

months) 2.

54

Despite these insights, the recommended concentration (RC) of 10 µg L-1 for urinary TU,

55

suggested by the European Union of Reference Laboratories (EURLs) in 2007, has not yet

56

been revised

57

discriminate endogenous from exogenous TU, could still result in false accusation or may

58

legalize possible low-level abuse. Furthermore, it is today unclear how these low levels of

59

urinary TU may correlate with levels in other matrices, such as meat or thyroid gland,

60

matrices for which the occurrence of TU may lead to concrete health issues in both animals

61

and humans, as thyreostats are recognized to be potentially carcinogenic and teratogenic18.

62

Therefore, in this study, TU levels in urine, meat and the thyroid gland of young (n = 18) and

63

mature (n = 16) cattle, subjected to either a daily low-level treatment with synthetic TU per os

64

or either an ad libitum diet of Brassicaceae-enriched feed, were compared and evaluated for

65

their discriminatory potential towards endogenous vs. exogenous origin.

16, 17

. However, even application of a population-based threshold as cut-off to

66 67

Material and methods

68 69

Experimental Animals

70


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Sixteen non-lactating cows with an age varying between 2 and 7 years, 6 purebred, but non-

72

active, dairy cows (Holstein-Fresian), 5 purebred beef cows (Belgian Blue) and 5 dual-

73

purpose cows, were included in this study.

74

Eighteen dual-purpose calves of 3 months old were included as well.

75

The animals were housed in adequate cow pens with interior foraging capacities, but with

76

possibilities for individual feeding of concentrates, conform to the ethical guidelines for

77

housing of experimental farm animals (calves separated from cows in time and space).

78

The standard diet was fine-tuned conform to the animals’ need in terms of physiological

79

development, taking into account the crude protein content (24% versus 16% for calves and

80

cows, respectively) and the overall energy needs (administered quantity correlated with the

81

animals’ individual weights). An absolute absence of plant-sources derived from

82

Brassicaceae species was mandatory for each feed, with exception of the experimental diet

83

purposely enriched with (30%) rapeseed. The standard concentrates for calves consisted of a

84

starter grain including barley, oats, spelt and flax, while the standard concentrates for cows

85

were pelleted and contained besides the latter ingredients also wheat (and bran), palm kernel

86

oil waste products, cacao nut shells, malt roots, corn and beet pulp. These standard

87

concentrates were administered daily in the morning (1 kg/100 kg body weight for calves and

88

1.25 kg/100 kg body weight for cows). The rapeseed-enriched diet was specifically

89

formulated for our study, provided in pellet-form and containing, besides 30% rapeseed

90

(waste product from rapeseed oil production and derived from the rapeseed 00 variety,

91

containing low erucic acid and glucosinolates), similar ingredients in comparison with the

92

standard feed (palm kernel oil waste products, beet pulp, wheat (and bran), corn, barley, malt

93

and soy). The rapeseed-enriched concentrates were administered ad libitum. To complete the

94

diet, all animals were supplemented with ad libitum hay and water.


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95

The calves’ and cows’ general health status was daily monitored by recording their appetite,

96

body temperature, heart rate and general wellbeing. Their weight was monitored as well, at

97

fixed time points.

98

The experimental set-up was approved by the local Ethical Committee (CER’s Ethical

99

Committee; CE/Sante/ET/004).

100 101

Study design and sampling

102 103

Each test animal was assigned to a single experimental treatment group, i.e. the group that

104

was treated with synthetic TU per os (TU-group) or the group in which test animals were

105

subjected to a rapeseed-enriched diet (RS-group) (Supplementary Fig. 1 and 2).

106

Within each group (TU or RS group), two categories of experimental animals were

107

considered: those subjected to the treatment (Test; n = 5 for cows (2 beef, 1 dual-purpose, 2

108

dairy cow(s)), n = 6 for calves) and those who served as controls (Control; n = 3 for cows (1

109

beef, 1 dual-purpose, 1 dairy cow in the RS-group and 2 dual-purpose and 1 dairy cow(s) in

110

the TU-group), n = 3 for calves). Animals were assigned to a particular treatment group

111

aiming for an equal distribution of animals with peculiar characteristics such as breed

112

(purpose) and age (only relevant in case of cows).

113

Several cow pens were available for each treatment (sub)group, to prevent uptake of

114

rapeseed-enriched feed by control animals or animals belonging to the TU group. Cows and

115

calves were subjected to the same study designs (TU-group and RS-group, Supplementary

116

Fig. 1 and 2), but the study in cows was performed during spring, while for calves the study

117

was undertaken during early summer.

118

For each animal, the experimental treatment period was preceded by two weeks of

119

acclimatization. During this period, the animals were given time to adapt to their new housing


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120

facilities, the (standard) diet, the water supplies, their care-takers and the technicians involved

121

in treatment and sampling.

122

The standard diet was also maintained during treatment (1 week) and washout (1 week) in the

123

TU group, for both test and control animals. During TU treatment test animals were

124

supplemented per os, with 2 mg synthetic 2-TU per kg body weight. This was effectuated by

125

flushing (using water) two capsules through the mouth and subsequently esophagus into the

126

stomach. At the end of the experiment, euthanasia of the animals was performed by captive

127

bolt quickly followed by exsanguination (Supplementary Fig. 1 and 2).

128

Animals belonging to the RS group received an alternative treatment procedure without a

129

washout period. A three-week-treatment period with an ad libitum diet of 30% rapeseed-

130

enriched concentrate (and ad libitum straw instead of hay) was followed by euthanasia of the

131

animal. The control animals were administered the standard diet formulation.

132

Urine and serum samples were taken daily. Samples from the muscle tissue (diaphragm, hind

133

leg and loin) and the thyroid gland were taken post mortem. All samples were immediately

134

stored at -20°C, however, prior to storage, every batch of urine was treated with a TU

135

stabilizing step by adding 0.25 M ethylenediaminetetraacetic acid (EDTA) and 37% HCl 19.

136

The sampling frequency of urine, serum, muscle (diaphragm, hind leg, loin) and thyroid is

137

illustrated in Table 1.

138 139

Analytical methods

140 141

Reagents and chemicals

142 143

The chemical standard 2-TU (TU) and deuterated internal standard 6-propyl-2-TU-D5 (PTU-

144

D5) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Toronto Research


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145

Chemicals, Inc. (Toronto, ON, Canada), respectively. Stock solutions were prepared in

146

methanol at a concentration of 200 ng µL-1. Working solutions were prepared by 200x and

147

2000x dilutions in methanol (1.0 and 0.1 ng µL-1, respectively). When necessary, sonication

148

was applied to ensure the complete dissolution of the substances. Solutions were stored in

149

dark glass bottles at 7°C.

150

Reagents were of analytical grade when used for extraction, and of Optima® LC-MS grade

151

when used for U-HPLC-MS/MS analysis. They were obtained from VWR International

152

(Merck, Darmstadt, Germany) and Fisher Scientific (Loughborough, UK), respectively.

153

Phosphate buffer, dissolved in deionized water, was made up from 0.5M Na2HPO4.2H2O and

154

0.5 M KH2PO4, adjusted to pH 7. For extraction purposes the required amount of phosphate

155

buffer, pH 7, was saturated with 1% of DL-dithiothreitol (DTT, purity 99%, Sigma-Aldrich,

156

St. Louis, MO, USA).

157 158

Urine

159 160

Extraction and detection of TU in urine was performed according to Vanden Bussche et al.

161

(2010)

162

supplemented with 1 mL phosphate buffer, containing 1% DDT. Next, a two-fold liquid-

163

liquid extraction with 5 mL ethyl acetate was executed after which the obtained extract was

164

evaporated to dryness. Finally, the dried extract was dissolved in a solution of acidified ultra

165

pure water and methanol (both acidified with 0.1% formic acid) (90/10, v/v). Analysis of

166

extracts was effectuated by ultra-high performance liquid-chromatography (UHPLC),

167

followed by tandem mass spectrometry (MS/MS). Chromatographic separation of the target

168

analytes was thereby achieved on an Accela UHPLC system (Thermo Fisher Scientific, San

169

José, USA), equipped with a HSS T3 column (1.8 µm, 100 x 2.1 mm) (Waters, Milford, MA,

19

. Briefly, 1 mL urine was enriched with 50 ng PTU-d5 internal standard and


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170

USA), applying a binary solvent gradient program19. Mass spectrometric detection was

171

realized on a TSQ VantageTM triple-quadrupole instrument (Thermo Fisher Scientific, San

172

José, USA) using a HESI II ionization source in positive ion mode19.

173 174

Thyroid and muscle tissue

175 176

Analysis of TU in thyroid and muscle tissue was performed according to Pinel et al. (2005) 20.

177

The protocol started with 250 mg of lyophilized tissue, which was extracted with 5 mL

178

methanol. After centrifugation, the obtained supernatant was filtered through a 0.45 µm

179

membrane (Whatman, Springfield Mill, UK) and enriched with 100 ng of 5-dihydroxyboryl-

180

6-propyl-2-TU (BPTU) internal standard. Subsequently, the mixture was evaporated to

181

dryness, dissolved in phosphate buffer (pH 8), and supplemented with 3-iodobenzylbromide.

182

The extract was then purified by multiple diethyl ether extractions and consecutive solid-

183

phase extractions, using silica gel (1 g) and reversed-phase C18 (2 g) cartridges (UCT,

184

Bristol, PA, USA). Chromatographic analysis of the extract was achieved on a Waters

185

Acquity System (Manchester, UK), equipped with a PurospherTM STAR C18 column (5 µm,

186

125 x 2.0 mm) (Merck Millipore, Darmstadt, Germany) and LiChroCART cartridge. Mass

187

spectrometric detection was performed on a Quattro Premier tandem mass spectrometer

188

(Waters, Manchester, UK), operating in the positive ion electrospray mode20.

189 190

Data analysis

191 192

Due to the obvious lack of homoscedacity of the data in any (sub)dataset, TU levels were

193

statistically assessed based on non-parametric testing whereby the Kruskal-Wallis test with

194

post-hoc Dunn’s comparisons was applied in case that at least three groups were compared,


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195

while the analysis between two groups was based on the Mann-Whitney U test. Differences

196

were considered to be significant if p < 0.05.

197

TU levels were compared in several subsets of data, which were created with the split file

198

function in SPSS 22.0 (IBM, Brussels, Belgium) after all experimental data derived from the

199

several study-set ups (Supplementary Fig. 1 and 2) were combined in one data file. The

200

grouping variables were defined as the experimental animal group (cows vs. calves), the

201

treatment group (synthetic TU treatment vs. the rapeseed-enriched diet), the treatment status

202

(control vs. treated animals), the experimental period (acclimatization, treatment (RS vs. TU),

203

washout), the refined experimental period (in which the RS treatment was further subdivided

204

in the first, the second and the third week of treatment, whereby the first and second week ran

205

parallel with the TU treatment and washout period of the TU group, respectively), and the

206

time of day (AM versus PM). Defining these grouping variables allowed to split the file and

207

thus compare (sub)groups based on 4 of these factors, depending on the analysis of interest,

208

while the fifth grouping variable served as the final variable and basis on which TU levels

209

were compared.

210 211

Results and discussion

212 213

Low-level urinary TU samples have been frequently observed in several European countries

214

whilst performing routine monitoring in the frame of the National Monitoring Programs 1, 2, 21.

215

Currently, a threshold of 10 µg L-1 is applied to differentiate between synthetic and

216

endogenously formed TU, detected in urine16. However, based on recent studies, a threshold

217

of 30 µg L-1 would be more justified 2. Nevertheless, defining a threshold is an artificial

218

measure exposed to several shortcomings, potentially leading to facultative false (positive or

219

negative) conclusions. A major issue regarding the validation of this threshold value is the


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220

lack of studies reporting recovered urinary TU concentrations following low-level synthetic

221

TU abuse, and a comparison with (endogenous) levels resulting from supplementation with a

222

Brassicaceae-enriched diet.

223

In this study, groups of mature cattle (n = 5) and calves (n=6) were treated with synthetic TU

224

or with rapeseed meal allowing a comparison of TU levels in urine, thyroid, and meat tissue

225

achieved under similar experimental conditions (Tables 2-4), with cows and calves

226

undergoing the same experimental workflow (Supplementary Fig. 1 and 2) being handled by

227

the same persons, but with experiments performed during (early) spring (February-April) and

228

(early) summer (May-July), for cows and calves respectively.

229 230

Urinary TU levels

231 232

Administration of synthetic 2-TU

233 234

A significant increase in urinary TU was observed after onset of synthetic TU administration

235

in both calves and cows (Mann-Whitney U test; control versus test animals; p < 0.05), with

236

significantly higher concentration levels during treatment and washout as compared to the

237

acclimatization period (Fig. 1 and 2; Kruskal-Wallis met post hoc Dunn’s test; p < 0.05). TU

238

levels were significantly higher in the afternoon (PM) compared to the morning (AM) urine,

239

with the latter samples taken prior to daily treatment (Mann-Whitney U test; p < 0.05). Values

240

were comparable for cows (median

241

(median AM = 263 µg L-1; median PM = 5454 µg L-1). Peaking urinary values in the afternoon-

242

samples can be explained by TU administration during the morning and a consequent and fast

243

elimination from then on. A rapid absorption of TU, or other thyreostats, from the gastro-

244

intestinal tract, with a short half-life in serum (T1/2=1.65 h in humans) and an additional fast

AM

= 302 µg L-1; median

PM


= 4989 µg L-1) and calves

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245

elimination from the body, mainly through renal excretion, has been documented in literature

246

with maximum levels in serum, urine and milk between 4 to 8h after administration 22-25. This

247

is particularly true for single-dose administration, whereas daily repeated doses could be

248

responsible for prolonged half-lives in urine (and serum) with postponed elimination,

249

particularly if (high-dose) treatment was maintained for at least two weeks 25, 26. In this study,

250

however, the animals were treated one week with a dose that is at least 5 times lower as

251

compared to the listed studies. As such, it may be explained that very low levels of TU were

252

sometimes observed in individual cows (minimum values of 29.7 µg L-1) and calves

253

(minimum below CCα) despite synthetic TU treatment. Moreover, elimination of TU from the

254

circulation will likely be completed by morning, particularly after single (first) dose

255

treatment. These synthetically induced low TU levels are a first indication for possible

256

shortcomings of a revised RC.

257

An exponential decrease in TU was noticed once the washout was initiated with a faster

258

elimination profile in calves vs. cows, returning to acclimatization levels on the third day after

259

arrest of the treatment (Fig. 1 and 2) (Mann-Whitney U test; calves versus cows; p < 0.05).

260

The latter data are largely in line with the elimination profile of methyl-TU (MTU) and TU

261

previously described by other authors 24, 26.

262 263

Supplementation of a rapeseed-enriched diet

264 265

The effects of supplementing a rapeseed-enriched diet to cows or calves were less pronounced

266

than those of artificial TU administration. It should hereby be noted that relatively high levels

267

of urinary TU were observed during acclimatization of test animals (table 3). The detection of

268

TU in urine of control animals after acclimatization resulted in an unexpected profile, with

269

significant higher TU levels in the afternoon compared to the morning urine sample (for both


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calves and cows) (Mann-Whitney U test; p < 0.05). Since the animals were fed with

271

concentrates only once a day (in the morning, after urine sampling), an obvious metabolic link

272

with this feed, even if containing no rapeseed, is presumed.

273

Despite the relatively high urinary TU values in control samples (both acclimatization

274

samples as samples derived from control animals), significant higher TU levels were observed

275

in the urine samples of the RS treated cows (Mann-Whitney U test; acclimatization versus

276

treatment; p < 0.05) (Fig. 3).

277

Such an increase was however not observed for calves. The discrepant results between cows

278

and calves may be attributed to their respective inherent gastro-intestinal physiologies. It is

279

well known that the digestive tract of bovines is very particular and unique, whereby the

280

stomach is represented by 4 entities, with each its proper metabolic function. The maturation

281

of the rumen, including bacterial colonization, which is supposed to be still in progress in

282

calves with the age of three months, is hypothesized as responsible for the different results. It

283

is indeed very well described that the development, colonization and final homeostasis of the

284

rumen is strongly depending on several parameters such as age 27, 28 and diet 29, 30. From fig. 3

285

and based on the control calves, it might be concluded that no complete acclimatization (to

286

the concentrates) was achieved until the end of the first treatment week, hereby masking a

287

potential significant effect of the rapeseed- enriched diet in the test animals. This phenomenon

288

was statistically assessed by splitting the treatment period for calves belonging to the RS

289

group into three different weeks, whereby TU levels were significantly lower in control

290

animals during the last two treatment weeks compared to the acclimatization period and the

291

first week of treatment (Kruskal-Wallis with post hoc Dunn’s test; p < 0.05). This decrease

292

was not observed for the test animals which may indirectly point towards a significant

293

inducing effect of the rapeseed-enriched diet on TU formation. In addition, significantly


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294

different urinary TU levels were observed between control and test animals during the second

295

and third week of treatment (Mann-Whitney U-test; control versus test, p < 0.05).

296

An important finding relates to the persistent occurrence of relatively high urinary TU levels

297

in the control samples. Although endogenous TU production is currently only linked to the

298

inclusion of Brassicaceae spp. in animals feed

299

bacterial strains (in monogastric animals)

300

needed for this conversion, e.g. the glucosinolates, are also present in other plant materials 32.

301

Generally, glucosinolates, abundantly present in Brassicaceae, are metabolized after chewing

302

and ingesting whereby myrosinases from plant- or bacterial origin are responsible for the

303

hydrolysis. During this process, there is a release of glucose and an unstable intermediate,

304

which can be further converted to a wide range of metabolites such as oxazolidine-thiones,

305

nitriles, epithionitriles, thiocyanates, isothiocyanates and thiourea

306

products have been suggested as candidate-precursors for TU, but the exact metabolic

307

pathway is currently unknown. Nevertheless, in animal feed production, rapeseed side chain

308

products are wanted due to their high protein content. At the same time they are disliked

309

because of their high glucosinolate content. Glucosinolates are – depending on the specific

310

metabolite – demonstrated to be goitrogenic, mutagenic, hepatogenic or nephrotoxic 35. These

311

growth-inhibiting properties have led to the cultivation of low-glucosinolate lines of Brassica

312

spp.

313

these low-glucosinolate- varieties, but still resulted in increased TU formation. However,

314

since low-level urinary TU concentrations were also present in control samples one can only

315

speculate that also other common animal feed ingredients derived from plants may also

316

contain glucosinolate-like or chemical related compounds, enabling the TU formation.

317

Control animals were fed with concentrates and hay in this study. The supplementation of ad

318

libitum hay, in contrast to the administration of wheat straw for (RS) test animals in the

13, 14

and requires the intervention of specific

15, 31

, it is suggested that the molecular substrates

15, 33-35

. These breakdown

36, 37

. The rapeseed meal added to the feed pellets in this study was also derived from


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319

treatment phase, may be a first explanation for the relatively high basal levels of urinary TU

320

during acclimatization and in other control samples. A pasture may be colonized by a variety

321

of herbs mixed within the grass. Some of these herbs (e.g. Eruca sativa (rocket salad),

322

Sinapsis arvensis (wild mustard), Sisymbrium irio (London rocket), Capsella bursapastoris

323

(shepherd’s purse) and Raphanus raphanistrum (wild radish)) belong to the Brassicacae spp.

324

family

325

glucosinolate-precursors of TU and consequently be responsible for an elevated basal level in

326

the control samples. In contrast, the concentrates that were administered to the control calves

327

and cows were supposed to be free of glucosinolates. Cereal grains such as oats, barley, spelt

328

and wheat, which are the ingredients of our control study concentrates, largely consists of

329

carbohydrates (approximately 60%) (mainly starch), proteins (approximately 10%) and fiber

330

(approximately 10%)

331

other possible precursors for TU. Flax (Linum usitatissimum), however, was also part of the

332

feed. Similar to plants from the Brassicaceae spp. family, these seeds have been demonstrated

333

to produce a cyanogenic compound with comparable repellent properties towards insects,

334

herbivores and microorganisms as described for glucosinolates 42-46.

38, 39

. Hay derived from a field with the latter mixed flora may then evidently contain

40, 41

and do not contain glucosinolates nor are suspected to contain

335 336

TU residues in soft tissues

337 338

In the edible meat tissue (diaphragm muscle, loin and hind leg) no residues of TU were

339

detected, at least if synthetic TU treatment was consequently followed by a one-week washout

340

period. However, during a pilot study (data not shown), one cow was slaughtered immediately

341

after one week of synthetic TU administration (2 mg/kg body weight), without washout

342

period, resulting in TU residues in the meat above the CCα (diaphragm: 6.8 µg kg-1; loin: 5.4

343

µg kg-1; hind leg: 5.9 µg kg-1). In this particular animal, TU levels were dramatically elevated


15


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344

in the thyroid gland (580 µg kg-1 versus below CCα for control animals). This is more than ten

345

times higher compared to the maximum value for TU retrieved in the thyroid of cows after

346

one week of washout. High concentrations of thyreostats in thyroid tissue have been

347

documented in several reports, and are not attributed to a hampered elimination of these

348

compounds, but merely to the accumulation of this compound in its target tissue

349

Accumulation following low-level TU administration is further evidenced by the overall in

350

vivo study results in which TU levels were demonstrated to be significantly higher in TU

351

treated calves and cows, compared to both their control groups and the respective calves and

352

cows supplemented with a rapeseed diet (Mann-Whitney U test, test versus control; TU

353

versus RS; p < 0.05 for every comparison). Lees et al. (1972)47 describe a faster accumulation

354

in rats, if smaller doses of TU were administered, whereby increasing ratios between thyroid

355

and plasma were noticed. This dose-dependent accumulation might explain why detectable

356

but low TU levels are also monitored in rapeseed-administered cattle despite the large

357

discrepancy in urinary levels of rapeseed treated cattle versus TU treated cattle.

358

In our study, TU levels in thyroid tissue did not show any overlapping results between the RS

359

group (maximum in calves and cows respectively 10.2 and 5.0 µg kg-1) and the TU group

360

(minimum in calves and cows respectively 22.9 and 23.5 µg kg-1), endorsing the possible

361

relevance of thyroid gland analysis as a tool to discriminate between endogenous TU

362

formation and synthetic administration, even at low-level abuse and with prolonged

363

withdrawal periods of at least one week.

364

Although TU residue levels in the thyroid tissue were significantly lower for animals

365

supplemented with rapeseed compared to synthetic TU treated animals, the levels following

366

rapeseed supplementation were significantly higher compared to control animals in calves,

367

but not in cows (Mann-Whitney U test; p < 0.05). The presence of TU in the thyroid gland

368

after/during rapeseed supplementation and the parallel absence in control calves, are another


22, 24-26, 47

.

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369

indication for the significant effect of rapeseed administration on the endogenous TU

370

formation, despite the impeded interpretation based on the respective urinary levels. In

371

addition, it was demonstrated that thyroid TU residues after rapeseed supplementation were

372

significantly higher in calves compared to cows (Mann-Whitney U, cows versus calves; p

Discrimination between Synthetically Administered and Endogenous

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