Sara Divari
email: sara.divari@unito.it

Raffaella De Maria

Francesca Tiziana Cannizzo

Francesca Spada

Chiara Mulasso

Toine Frank

Henk Bovee

Piergiorgio Capra

Marta Leporati

Bartolomeo Biolatti

T F H Bovee

Capra

A RIKILT yeast Estrogen bioAssay (REA

Keywords: Yeast bioassay, calves, urine, estrogens, GC/MS

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated 27 animals (group B) showed as being positive up to seven days after administration of the highest 28 dosage of 17β-estradiol, while the other three groups showed as being negative. The identity of 29 estrogenic molecules in the urine of group B (17β-estradiol, 17α-estradiol, and estrone) was 30 confirmed by GS/MS. This is the first time the REA test has been applied to detect 17β-estradiol in 31 the urine of calves treated with the hormone in vivo. The technique may offer an advantageous 32 laboratory method for the veterinary surveillance of illegal steroid use. 33 34 Introduction 35

European Union (EU) regulations ban the use of growth promoters in meat-producing animals 36 (European Commission 1996a), however their illegal use raises cause for concern. By acting on 37 lipid and protein metabolism, anabolic steroids increase growth rate, stimulate muscle growth, and 38 reduce fat tissue in calves [START_REF] Groot | Combinations of growth 403 promoters in veal calves: consequences for screening and confirmation methods[END_REF]. One of the most efficient of these anabolics is 39 17β-estradiol. Histologically, it induces hyperplasia and metaplasia of prostate epithelial cells and 40 urethral epithelium [START_REF] Ruitenberg | Evaluation of the "prostate test" in checking the 449 administration of oestrogens in the calf[END_REF]Kroes et al., 1971;[START_REF] Groot | Combinations of growth 403 promoters in veal calves: consequences for screening and confirmation methods[END_REF]. Squamous 41 metaplasia is a specific response to a variety of estrogenic substances such as estradiol, 42 diethylstilbestrol (DES) and zeranol, and it is considered as a reliable histological marker to assess 43 their estrogenic action in cattle. 44 45 But for surveillance and monitoring of the ban to be effective, validated analytic methods that can Various techniques that can detect estrogenic substances include gas chromatography-mass 60 spectrometry (GC-MS) [START_REF] Daeseleire | Multiresidue analysis of anabolic 370 agents in muscle tissues and urines of cattle by GC-MS[END_REF][START_REF] Fotsis | The multicomponent analysis of estrogens in urine by ion exchange 391 chromatography and GC-MS--I. Quantitation of estrogens after initial hydrolysis of conjugates[END_REF], high-performance 61 liquid chromatography-mass spectrometry (HPLC-MS) [START_REF] Sorensen | Determination of mycotoxins in bovine milk by liquid 451 chromatography tandem mass spectrometry[END_REF] and 62 immunoassay [START_REF] Dixon | Radioimmunoassay of the anabolic agent zeranol. II. Zeranol 376 concentrations in urine of sheep and cattle implanted with zeranol (Ralgro)[END_REF][START_REF] Jansen | A 413 chemiluminescent immunoassay for zeranol and its metabolites[END_REF][START_REF] Chen | Measurement of urinary estrogen metabolites 361 using a monoclonal enzyme-linked immunoassay kit: assay performance and feasibility for 362 epidemiological studies[END_REF]. 63 64 Despite the limited number of positives found in controls, findings and analysis of illegal 65 preparations show that steroids, natural hormones and β agonists are still being used (Courtheyn et 66 al., 2002;[START_REF] Nielen | Identification of an unknown beta-agonist in feed by liquid 436 chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass 437 measurement[END_REF]. The analytical methods for measuring residues of steroids and β-67 agonists as used in control programs are unable to detect very new or outdated compounds and 68 might be one of the possible explanations for the limited positives found so far. Screening and 69 confirmatory GC-MS, and even the multi-residue liquid chromatography tandem-mass spectrometry 70 (LC/MS/MS) methods, are limited to a short list of a priori known hormone residues (Cai et al., 71 1997;[START_REF] Hewitt | Screening and confirmatory 410 strategies for the surveillance of anabolic steroid abuse within Northern Ireland[END_REF][START_REF] Van Poucke | Development and validation of a multi-analyte method for 456 the detection of anabolic steroids in bovine urine with liquid chromatography-tandem mass 457 spectrometry[END_REF]. The multi-analyte screening 72 ability of radio-and enzyme immunoassays depends on the limited degree of cross-reactivity of the 73 antibody used (Vanoosthuyze et al., 1997;[START_REF] Haasnoot | Immunofiltration as sample cleanup for the 408 immunochemical detection of beta-agonists in urine[END_REF]. Alternatively, receptor-based 74 assays can be used to detect compounds having affinity for a given receptor [START_REF] Mueller | Overview of in vitro tools to assess the estrogenic and antiestrogenic activity of 426 phytoestrogens[END_REF]; 75 specifically, yeast-based assays feature several advantages, including robustness, low cost, lack of 76 known endogenous receptors, and the use of media devoid of steroids [START_REF] Breithofer | Quantitative multi-residue determination of beta-agonists in bovine urine 358 using on-line immunoaffinity extraction-coupled column packed capillary liquid chromatography-359 tandem mass spectrometry[END_REF]77 Graumann et al., 1999;[START_REF] Witters | Detection of estrogenic activity in Flemish 462 surface waters using an in vitro recombinant assay with yeast cells[END_REF]. 78 79 Recently, Bovee et al. (2004aBovee et al. ( , 2004b) developed a bioassay called RIKILT yeast Estrogen 80 bioAssay (REA), which is fully validated according to the guidelines as prescribed in EC Decision 81 2002/657 and accredited ISO 17025. The assay consists of a recombinant yeast cell that stably 82 expresses the human estrogen receptor α (ERα) and a yeast-enhanced green fluorescent protein 83 (yEGFP) as a reporter protein in response to estrogens. The GFP is a protein that exhibits green much more easily than an extra-chromosomal reporter construct with β galactosidase as a substrate-86 based reporter protein, a system frequently used by older yeast-based assays (Routledge and 87 Sumpter, 1997;[START_REF] Gaido | Evaluation 394 of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene 395 transcription assay[END_REF][START_REF] Rehmann | Applicability of a yeast oestrogen screen for the 445 detection of oestrogen-like activities in environmental samples[END_REF][START_REF] Morito | Interaction of phytoestrogens with estrogen receptor α and β[END_REF]Le Guevel and 88 Pakdel, 2001). 89 90The EC50 revealed by the REA test was 0.5 nM for 17β-estradiol and was comparable with 91 reporter EC50 values for yeast estrogen bioassays that contain β galactosidase as a reporter (Bovee 92 et al., 2004c). The yEGFP assay can be performed completely in 96-well plates within 24 h and 93 does not need cell wall disruption nor the addition of a substrate. Finally, the REA test is extremely 94 robust and survives extracts from dirty sample matrices such as urine and feed and it is able to 95 recognize estrogen molecules (natural and artificial hormones, aflatoxin and phytoestroges) (Bovee 96 et al., 2004b) 97 98 This bioassay is routinely applied as a qualitative screening method in both calf urine and feed 99 sample matrices in Dutch laboratories: only sample extracts showing a fluorescence response 100 beyond the decision limit CCα will be declared suspect for estrogen activity. Subsequently, the 101 identity of substances causing estrogenicity must be determined by conventional residue analytical 102 methods such as GC-MS or by using bioassay-directed quadrupole time-of-flight MS (QTOFMS) 103 identification approaches [START_REF] Nielen | Bioassay-directed Identification of estrogen residues in urine by liquid chromatography 429 electrospray quadrupole time-of-flight mass spectrometry[END_REF](Nielen et al., , 2006)). Moreover, the bioassay could be a good tool 104 alongside instrumental analysis for determining estradiol residues in bovine urine. 105 106

The aim of this study was to evaluate the ability of the REA to detect estradiol residues in the urine 107 of experimentally treated veal calves, and to compare the results with those from official analytical 108 methods (GC/MS). 109 Twenty-four cross-bred Friesian male calves six months old were randomly assigned to four 112 experimental groups: group A (n=6) treated daily with 1ml/animal of gliburide (0.2 mg/L) and 113 androsterone (8 mg/L) cocktail i.m. [START_REF] Krentz | Oral antidiabetic agents: current role in type 2 diabetes mellitus[END_REF]; group B (n=6) treated weekly with 17β-114 estradiol (diluted in 10 ml of benzylic alcohol and 1 ml of ethyl oleate ) i.m. six times until one 115 week before slaughter, for a total of 190 mg/animal (Table 1); group C (n=6) treated weekly with 116 testosterone propionate (diluted in sesame oil) i.m. , for a total of 1.050 g/animal (Table 1). Group 117 D (n=6) was the control. 118

The entire treatment phase lasted for a total of 44 days. 119 120

The average body weight of the animals at the start of the animal housed was approximately 140 121 kg; the calves were tethered and fed with liquid milk replacer twice a day (providing per kg: dry 122 matter (DM) 950 g, crude protein (CP) 230 g, ether extract (EE) 210 g, ash 60 g, cellulose 1 g, 123 retinol 75 mg, ascorbic acid 50 mg, Cu 5 mg, cholecalciferol 0.125 mg, alphatocopherol 80 mg). 124

The amount of the fodder was increased gradually up to 8 l/calf/day, then increased gradually up to 125 16 l/calf/day; after 1 month, a quantity of 0•5 kg of barley straw (per kg: DM 900 g, CP 20 g, EE 10 126 g, ash 60 g, crude fiber 410 g) was added to the diet according to the indications suggested by the 127 European Commission ( (97/182/EC). Aliquots of 2 ml of calf urine of each experimental group were adjusted to pH 4.8 and 6 µl β-139 glucuronidase/arylsulfatase (300 and 22.5 Uml -1 , respectively) were added. Enzymatic 140 deconjugation was carried out overnight in a water bath at 37°C. Next, 2 ml of 0.25 M sodium 141 acetate buffer, pH 4.8, was added and the hydrolysed sample was subjected to solid-phase 142 extraction (SPE) on a C18 column fitted on a vacuum manifold (Supelco). The column was 143 previously conditioned with 2.5 ml methanol and 2.5 ml sodium acetate buffer. The column was 144 then washed with 1.5 ml 10% (w/v) sodium carbonate solution, 3.0 ml water, 1.5 ml sodium acetate 145 buffer, pH 4.8, 3.0 ml water, and finally with 2 ml methanol/water (50:50, v/v). The column was 146 air-dried and eluted with 4 ml acetonitrile, which was applied to an NH2-column (Supelco) 147 previously conditioned with 3.0 ml acetonitrile. The acetonitrile eluate was evaporated to 2 ml 148 under a stream of nitrogen gas. A 100-µl aliquot of this extract (equivalent to 100 µl urine) was 149 transferred to a 96-well plate in triplicate and 50 µl water and 2 µl DMSO were added to each well. 150

To remove the acetonitrile from this mixture, the plate was dried overnight in a fume cupboard and 151 was then ready to be screened for estrogenic activities with the yeast estrogen bioassay. 152

REA 154

The yeast cytosensor expressing the human estrogen receptor α (hERα) and yeast-enhanced green 155 fluorescent protein (yEGFP) in response to estrogens has been described previously (Bovee et al., 156 2004a, b). The yeast growth was performed according to published data (Bovee et al. 2004a). For 157 exposure in 96-well plates, 200-µl aliquots of this yeast culture were pipetted into each well 158 already containing the extracts of the urine samples. A 17β-estradiol dose-response curve in 159 1). Each urine sample extract and each 160 17β-estradiol standard was assayed in triplicate. Exposure of the yeast to the sample was 161 performed 24 h and the yeast fluorescence at 0 h and 24 h was measured directly in a Victor 162 31420 Multilabel Counter (PerkinElmer) using excitation at 485 nm and emission measurement at 163 530 nm. Differences of the fluorescence measurement at 24 h and 0 h of yeast exposed to samples 164 (t24-t0) were calculated and corrected for the reagent blank data, thus providing the final estrogenic 165 activity data for each sample. The samples are reported 'suspect' when the t 24-t 0 fluorescence 166 measurement exceeds the CCα (fluorescence of 4409) obtained in the ring test in our laboratory at 167 the University of Turin, Italy [START_REF] Bovee | Inter-laboratory comparison of a yeast 352 estrogen bioassay for screening calf urine[END_REF]; otherwise, the samples are reported 'compliant'. 168 The bioassay is a qualitative screening method, i.e. no concentrations are reported. We have 169 analyzed a total of 114 samples. 170

GC/MS analysis 172

The deconjugated estrogens were extracted from 2 mL of aqueous samples, derivatized and 173 analyzed with an Agilent Technologies 6890 N GC connected to an Agilent 5975 MSD (Akre et 174 al., 2004;[START_REF] Mouatassim-Souali | Validation of a 423 quantitative assay using GC/MS for trace determination of free and conjugate estrogens in 424 environmental water samples[END_REF][START_REF] Daeseleire | Validation of multi-residue methods for the 373 detection of anabolic steroids by GC/MS in muscle tissues and urine samples from cattle[END_REF]. The internal standard was 175 methyl testosterone at a final concentration of 75 ng/mL. In order to hydrolyze the glucurono-176 conjugate of 17α-and 17β-estradiol, the pH was adjusted between 6÷ to 7 with HCl 1 M; 50 µL 177 of enzyme β-glucuronidase (441 I.U.) were added and hydrolyzed for 1 h at 42°C. We extracted 178 17α-and 17β-estradiol at pH 9 for NaOH 1M by liquid/liquid extraction with 10 mL of tert-butyl-179 methyl-ether. The organic phase was dried under nitrogen stream at 50°C. The analytes were 180 derivatized with 50 µL of tetramethylsilane and heated to 70°C for 30 min in thermoblock. 181

Compounds of interest were separated on a HP-5 column (17 m x 0.20 mm x 0.33 µm film 182 thickness). The temperature program was 90°C for 1.50 min, ramped at 25°C/min to 260°C and 183 held for 3 min, ramped at 25°C/min to 320 °C and held for 1.30 min. A 2-µl aliquot was injected 184 into the GC-MS system in splitless mode. The injector port temperature was 280°C, the transfer 185 line from the GC system to the mass-selective detector was held at 280°C. Helium was the carrier 186 gas at 2.1 mL/min initial flow. The pressure was constant. The mass-selective detector was operated 187 in electronic impact mode using selected ion monitoring (SIM). The validity of the REA test was evaluated by comparing the test results with those of the GC-MS 203 method which represents the "golden test" or "gold standard" [START_REF] Petrie | Statistics for veterinary and animal science[END_REF]. Validity 204 criteria were sensitivity, specificity and concordance index or efficiency , which is the concordance 205 degree of the test. Evaluation was easily done using a 2x2 contingency table and the sensitivity (%) 206 was calculated as 100 x the number of true positives /(number of true positives + number of false 207 negatives), the specificity (%) as 100 x the number of true negatives /(number of false positives + 208 number of true negatives), and the concordance index (%) as 100 x (number of true positives+ 209 number of true negatives)/total number of samples. 210 after the first dose and four out of six samples collected 48 h after the third dose showed 225 fluorescence over 4409 (Fig. 2a and2b). After one week, all urine samples from animals that had 226 received the first and third dose showed a fluorescence lower than the CCα. Instead, at the sixth 227 dose all the 17β-estradiol-treated animals (Fig. 2c) had fluorescence data higher than 4409 also 228 after 168 h. 229 230 Table 2 compares the data from the REA test and the GC-MS analysis. The GC-MS results of all 231 urine samples after administration of dose I at T0 demonstrate that endogen 17α-estradiol and 232 17β-estradiol were absent (sensitivity of method was 1 ppb). At 5 h after the first dose a small 233 quantity of 17β-estradiol and a significant increase in its metabolite (17α-estradiol) was detectable 234 in the animals. After 24 h, the 17β-estradiol level began to decrease, whereas the 17α-estradiol 235 Estrogens are among the most important growth promoters used illegally in animal production and 252 their use is prohibited in the European Union (Council Directive 1996). An alternative approach to 253 chemical analysis [START_REF] Courtheyn | Recent developments in the use and abuse of growth promoters[END_REF] for the identification of treated animals might be the use 254 of a bioassay. Bovee et al. (2004aBovee et al. ( , 2004b) ) In this study we evaluated the ability of REA to detect estradiol residues in the urine of 266 experimentally treated veal calves, and compared the results with an official analytical method 267 (GC/MS). 268 269In hormone treatment of cattle, the urinary profile usually contains a mixture of metabolites of the 270 anabolic drug that was used. Since the bioassay provides an additive measurement of all estrogenic 271 residues and metabolites (having different relative estrogenic potencies), the chance of overlooking 272 illegal treatment is relatively low, as compared with other screening assays such as highly specific 273 immunoassays. In a previous study (Bovee et al., 2004b), it was shown that 17β-estradiol 274 metabolites (17α-estradiol, estrone, estriol and hydroxylated estrogens) give a response in the 275

bioassay. 276 277

We demonstrated that GC/MS analysis of urine samples confirmed the results obtained with the 278 REA method, except for five samples of group B. Three real false negatives were: the sample 279 taken at 48 h after injection III in animal 290403, the sample taken at 72 h after injection VI in 280 animal 252732 and the sample taken at 0 h after injection III in animal 300408 resulting in a 281 percentage of false negatives that is below the accepted level of 5%. 282

283

The other two samples corresponded to animal 301095 collected at 48 and 168 h after the first 284 treatment.; they did not contain 17β-estradiol and contain small quantities of 17α-estradiol (6.29 285 and 2.52 ng/ml respectively) and resulted negative at REA. However, the 17α-estradiol metabolite 286 In conclusion, the animals can metabolize the first two treatments in a week, while subsequent 312 doses, stronger, leaving significant residues of 17α-estradiol and less 17β-estradiol after one week 313 to treatment. The 17α-estradiol is systematically present in concentration 10-50 times higher than 314 the 17β-estradiol, index of quick conversion of one form in the other. 315 316 Statistical analysis confirmed that the bioassay is a valid method that can discriminate between 317 estrogen-treated and non-estrogen-treated calves when compared against the results of an official 318 chemical test like the GC-MS. However, screening with this bioassay is limited to calf urine 319 because older bovines produce large amounts of endogenous 17α-estradiol and estrone, which 320 would cause too many false-suspect urine values. 321 322 Sample preparation is quite similar in both methods, but the cost of this test and the easier 323 interpretation of the REA results procedure make it a more practical method for large scale 324 screening to support the chromatography method. Currently, it is not possible to discriminate 325 natural estrogen molecules from synthetic estrogenic hormones illegally injected into animals. 326 Furthermore, the REA method can be applied only to samples from prepuberal veal calves fed 327 regular milk replacer. Nonetheless, the REA method is a fast and simple assay for routine 328 surveillance and monitoring and it can significantly reduce the GC-MS sample workload. 329

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  i.m. (group B), androsterone and gliburide (group A), and testosterone (group C) 25 molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to 26

  The experiment was authorized by the Italian Ministry of 128 Health and the Ethical Committee of the University of Turin. The carcasses of the treated animals 129 were destroyed (2003/74/CE -DL 16 March 2006, No. 158). 130 131 Urine samples of experimental group B were collected at 5, 24, 48 and 168 h after the injection of 132 estradiol (I, III, and VI doses), analyzed by REA and then confirmed by GC-MS. Urine samples 133 from the experimental groups (A, C) were analyzed with the REA test at 15 and 44 days into the 134 urine samples from each experimental group were compared with the fluorescence data 135 obtained from the control animals. 136 137 Steroid extraction of calf urine samples 138

  in each exposure experiment (Fig.

  ions was carried out after acquisition of complete mass spectra for analyte, 190 both alone and in the working standard, under the same chromatographic conditions as for the 191 samples. The masses for 17α-and 17β-estradiol were m/z = 416, m/z = 401, m/z = 326, m/z = 285, 192 m/z = 232, and m/z = 446 and m/z = 301 for methyl testosterone. The dwell time was 80 ms. The 193 retention times were 11.80 min for 17α-estradiol, 12.20 min for 17β-estradiol, and 12.80 min for 194 internal standard. Calibration curves were constructed by extracting urine spiked with 17α-and 195 17β-estradiol at a final concentration of 5, 10, 30, 50 and 100 ng/mL. The calibration graph for each 196 analyte is described by the equation Y = mX + b, whereby Y represents the ratio between the 197 response value of the analyte (peak area) and the response value of the internal standard (peak area), 198and X is the concentration in ng/mL of analyte in the sample. We have analyzed a total of

Figure 1 Figure 2

 12 Figure1shows the dose-response-curve obtained with the REA test after 24-h exposure to 212

  rise. At 48 h after injection, 17β-estradiol had been completely metabolized and 236 converted into 17α-estradiol. 237 238 At one week (168 h) after the administration of the first dose, 17β-estradiol was absent, except in 239 animal 301095 where traces of 17α-estradiol were found. At T0 of the third dose no analytes were 240 found, but an increase in both 17α-estradiol and 17β-estradiol was detectable after treatment. 241 REA test specificity was evaluated by comparing the urine samples from calves in groups A 242 (cocktail treatment), C (testosterone treatment) and D (control animal); no animals were found 243 positive to estrogens (

Page

  developed a bioassay, (REA), fully validated according 255 to the guidelines as prescribed in EC Decision 2002/657 and accredited ISO 17025. In The 256 Netherlands, the test is routinely applied as a screening test for the detection of illegal estrogen 257 treatment in calves. 258 259 In 2006, Nielen et al. analyzed more than 120 calf urine samples randomly collected from different 260 /MS. The data for REA validation were obtained from aliquots of blank calf urine and calf 262 urine samples spiked with 17β-estradiol and other estrogen molecules. Currently, there are no data 263 on REA application in animals experimentally treated with estrogenic hormones. 264 265

  Figure. 1. Response of REA after 24-h exposure to 17β-estradiol. Exposure was started by adding 2 467

Table 3

 3

	244	).
	245

Table 4

 4 illustrates the 2x2 contingency table for evaluating the validity of the REA method as a 246 qualitative screening test versus the results obtained with GC/MS. The REA test was found to have: 247

	248	100 x (83/(83+5))= 94.3% sensitivity, 100 x (16/(0+16)=100% specificity, and a concordance index
	249	of 100 x (83+16)/104= 95.2%
	250
	251	Discussion

Table . 1

 . . Treatment of group B and group C calves. 2

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	4	Table 2. 17α-and 17β-estradiol screening of urine samples by REA and GC-MS methods. 168 n.t. n.t. 48 61 <1 + -
	3 5	ID animal Dose VI III I 301096 VI III I 301095 I VI I 297627 I III 252732 III VI I 290403 III 300408 VI VI III *n.t.: not tested	Dose I II III IV V VI F o r P Group B 17β β β β-estradiol (mg/animal) 20 20 30 40 40 40 e e r R e Group C Testosterone (mg/animal) 150 150 150 200 200 200 v i e w O n l y Time from injection (h) 17α-α-α-α-estradiol (ng/ml) 17β-β-β-β-estradiol 0 148 5.8 + 168 <1 <1 -REA response 5 820 183 + 0 <1 <1 -(ng/ml) 0 <1 <1 -5 247.2 30.53 24 2022 112 + 5 351 39 + 48 420 20 + 24 560 15 + + 24 464.74 <1 72 72 1.8 -48 n.t. n.t. -+ 48 98.68 <1 96 100 4.6 + 168 n.t. n.t. -+ 168 <1 <1 120 n.t. n.t. + 0 73 1.4 + -0 <1 <1 -5 568 76 144 100 2.3 + 5 1618 158 + 168 67 3.8 + 24 2761 110 + + 24 609 13 0 <1 <1 -48 319 7.9 + + 48 248 4.5 5 341.62 67.74 + 72 87 1.7 + + 168 n.t* n.t 24 865.94 29.63 + 96 60 1.2 + -0 117 3.1 + 5 418 96 + 24 1356 80 + 48 582 23 + 72 239 8.7 + 96 23 <1 + 120 187.59 9.26 144 82.97 4.72 168 216.14 10.37 + 0 <1 <1 -5 255.57 5.92 + 24 294.91 22.02 + 48 20.63 <1 + 168 <1 <1 -0 233.67 28.83 -5 411. 34 24 399.65 20.84 + 48 155.85 7.36 + 168 n.t. n.t. 0 20 <1 + 5 2202 273 + 24 525 19 + 48 111 4.8 + 72 129 3.6 + 96 183 8.7 + 120 96.03 4.29 + 144 26.54 <1 + 168 n.t. n.t. + 0 <1 <1 -5 156.04 1.45 + 24 238.25 <1 + 48 4.57 <1 + 168 <1 <1 -0 <1 <1 -5 401.35 62.5 24 704.99 23.28 + 48 180.67 5.09 24 435 8.9 + + y 0 <1 <1 -5 601 108 + y + O n l 48 365.52 20.72 + 72 137 5.4 + 96 100 4.7 + 120 179.56 8.02 + 144 111.14 <1 + 168 61.15 <1 + l n O e v i e w 48 74 <1 + 168 <1 <1 0 <1 <1 -5 637 137 + 24 684 18 + 48 176 6.0 -168 n.t. n.t. 0 38.46 0 + 5 296.93 74.77 + 24 664.62 57.28 + w -e i v -e -R 0 <1 <1 -5 353 13 + 24 1601 41 + R + P e e r 5 1142.03 369.12 + 24 765.91 59.08 + 48 141.34 <1 72 58 2 + 96 36 1.1 120 70.46 <1 + 144 16.11 <1 + 168 64 2.8 + r e + e + P + 0 45.3 <1 + + 48 6.29 <1 -120 n.t n.t. + F o r 168 2.52 <1 -0 <1 <1 -5 606.53 126.04 + 24 297.43 8.16 + 48 104.19 <1 + 168 n.t. n.t. -144 44 <1 + 168 71 1.0 + F r o
			2 3 4

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	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	5

Table 3 .

 3 Fluorescence data of REA test in groups D, A and C (CCα = 4409[START_REF] Bovee | Inter-laboratory comparison of a yeast 352 estrogen bioassay for screening calf urine[END_REF]. 6

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		Treatment day 15	Treatment day 44
	Group D	1213 ± 1714	-117 ± 2569
	Group A	3348 ± 1223	1346 ± 1340
	Group C	376 ± 330	1818 ± 1860
	7
	8
	9

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Table 4 .

 4 The 2x2 contingency table for the evaluation of REA validity as a qualitative screening test 10 (GC-MS method considered the golden test). 11

	REA results	GC-MS estrogens positive	GC-MS estrogens negative
	+	83 a	0 b

fluorescence that can be measured directly[START_REF] Cormack | Yeast-enhanced 364 green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans[END_REF]. The yEGFP can be measured

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