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Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
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Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed Toine F.H. Bovee ∗ , Gerrit Bor, Henri H. Heskamp, Johan J.P. Lasaroms, Marieke B. Sanders, Michel W.F. Nielen RIKILT Institute of Food Safety, Wageningen University and Research Centre, P.O. Box 230, 6700 AE Wageningen, The Netherlands
a r t i c l e
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a b s t r a c t
Article history:
Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening.
Received 24 April 2008
Previously we described the construction and properties of a rapid and robust yeast andro-
Received in revised form
gen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced
19 June 2008
green fluorescent protein (yEGFP), the latter in response to androgens. In the present
Accepted 23 June 2008
study this yeast androgen bioassay was validated as a qualitative screening method for
Published on line 5 July 2008
the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with
Keywords:
testosterone, 17␣-methyltestosterone, 19-nortestosterone, 17-trenbolone, 17-boldenone
Androgens
or 17␣-methylboldenone at 2 or 15 ng mL−1 in urine and 50 or 100 ng g−1 in feed. All blank
Bioassay
and spiked samples fulfilled the CC␣ and CC criterions, meaning that all 20 blank samples
Hormone abuse
gave signals below the determined decision limits CC␣ and were thus classified as compliant
Steroids
(˛ = 1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the
Validation
CC␣ and were thus classified as suspect (ˇ = 5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17␣-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography–tandem mass spectrometry. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
The illegal use of androgens in cattle breeding conflicts with principles of fair trade and may impose a risk to the animal and the consumer. In addition, hormones may be present in pharmaceutical waste illegally mixed into the feed. The use of growth promoters for fattening purposes in cattle has been banned in the European Union since 1988 [1]. This EU ban prohibits all substances having hormonal action and does
∗
Corresponding author. Tel.: +31 317480391. E-mail address: [email protected] (T.F.H. Bovee). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.06.047
not provide a list of forbidden substances. Hormone abuse or incidents may be discovered by residue analysis in calf urine or animal feed. However, in practice residue analysis is carried out on specific target compounds and can enforce the ban only to a limited extent [2]. Alternatively, receptor-based assays can be used to detect all compounds having affinity for a given receptor [3,4]. In contrast to receptor binding assays, reporter gene bioassays also mimic the transactivation step and can distinguish between receptor agonists and receptor
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a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
antagonists [5,6]. This feature is very helpful in detecting known and unknown compounds, as receptor stimulation plays a key role in the mechanism of action of growth promoters. To the best of our knowledge there are no known plant compounds with a direct androgenic mode of action, but there are plant-derived compounds, e.g. diindolylmethane (DIM), flavone, guggulsterone and equol, with an antiandrogenic mode of action [7,8]. Both yeast-based assays as well as the more established in vitro reporter assays based on mammalian cells are suitable tests to determine the androgenic potency of a given compound. In general, transcription activation assays based on mammalian cell lines have been shown to be more sensitive than yeast-based assays [6,9,10]. However, yeast-based assays have several other advantages. These include low costs and easier handling, lack of known endogenous receptors that may compete with the activity under investigation, use of media that are devoid of steroids, and last but not least, yeast cells assays are extremely robust and survive extracts from dirty sample matrices such as urine and feed [11,12]. Yeast-based assays for the screening of estrogenic activities have been shown to be sensitive, specific, robust and especially bioassays using yEGFP as a marker protein are user-friendly [12–15]. Recently, we developed a novel yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP) in response to androgens. This assay is completely performed in a 96-well plate and fluorescence is measured directly in intact yeast cells. This assay is relatively simple and sensitive, as shown by an EC50 value for testosterone of 50 nM [16]. A large number of chemically different compounds with known androgenic, but also other hormonal activities were tested in our in-house developed yeast androgen bioassay to investigate its specificity. All androgenic compounds caused a dose-related increase in the production of green fluorescent protein, whereas the glucocorticoid dexamethasone showed no response [17]. Especially with androgens, the lack of known endogenous receptors in yeast is a great advantage compared with mammalian cell lines, as androgen responsive elements (AREs) can also be activated by the progesterone and glucocorticoid receptor (PR and GR). Lots of effort were made to construct an ARE that is specific and no longer inducible by the progesterone or glucocorticoid receptor in order to avoid the potential crosstalk in mammalian cells. However, up till now such an ARE does not exist and it is doubtful whether it will be found, as the consensus progesterone and glucocorticoid responsive elements (PRE/GRE) are equal to the consensus ARE [18]. Moreover, the GR is normally expressed in all mammalian cell types. So far this resulted in cell lines that are not specific for androgens and also respond to progesterone or glucocorticoids. Other yeast androgen bioassays are about equally sensitive and specific, but our new yeast androgen bioassay is the only one with the yEGFP marker, making it not only more convenient, but also faster and cheaper [16,17]. Moreover, not one of the other androgen bioassays based on either yeast or mammalian cells has been fully validated according to the international standards as prescribed in EC 2002/657 [19]. The present study describes the validation conform EC 2002/657 of this new yeast androgen bioassay for the determination of androgenic activity in calf urine
and animal feed and demonstrates its applicability to real samples.
2.
Experimental
2.1.
Chemicals
Acetonitrile and methanol were from Biosolve (Valkenswaard, The Netherlands). Ammonium sulphate, dimethyl sulfoxide, sodium acetate and sodium carbonate were obtained from Merck (Darmstadt, Germany), dexamethasone, 19-nortestosterone, and l-leucine from Sigma and 17boldenone, 17␣-ethynylestradiol, 17␣-methylboldenone, 17␣-methyltestosterone, testosterone, and 17-trenbolone from Steraloids (Newport, RI, USA). Isolute NH2 extraction columns (500 mg) were obtained from IST (Hengoed, U.K.) and Bond Elut C18 solid phase extraction columns (1000 mg) from Varian (Harbor City, CA, USA). Dextrose and yeast nitrogen base without amino acids and without ammonium sulphate were obtained from Difco (Detroit, MI, USA). The minimal medium with l-leucine (MM/L) consisted of yeast nitrogen base without amino acids and without ammonium sulphate (1.7 g L−1 ), dextrose (20 g L−1 ) and ammonium sulphate (5 g L−1 ) and was supplemented with l-leucine (60 mg L−1 ).
2.2.
Samples and sample treatment
Aliquots of 10 mL blank calf urine or calf urine samples spiked with testosterone, 19-nortestosterone and 17-trenbolone at 2 ng mL−1 , 17-boldenone and 17␣-methylboldenone at 15 ng mL−1 , were adjusted to pH 4.8 and 20 L glucuronidase/arylsulfatase (3 U mL−1 ) was added. Enzymatic deconjugation was carried out overnight in a water bath at 37 ◦ C. Next, 10 mL of 0.25 M sodium acetate buffer pH 4.8 was added and the hydrolysed sample was subjected to solid phase extraction (SPE) on a 1000 mg C18 column, previously conditioned with 4 mL methanol and 4 mL sodium acetate buffer. Subsequently, this column was washed with 3 mL sodium acetate buffer, 6 mL water, 3 mL 10% (w/v) sodium carbonate solution, 6 mL water and finally with 6 mL methanol/water (50/50, v/v). The column was air-dried and eluted with 4 mL acetonitrile. The eluate was applied to a 500 mg NH2-column, previously conditioned with 4 mL acetonitrile. The acetonitrile eluate thus obtained was evaporated to dryness under a stream of nitrogen gas and dissolved in 1 mL acetonitrile. A 200 L part of this extract (equivalent to 2 mL urine) was transferred to a 96-well plate in triplicate and 50 L of a 4% DMSO solution was added to each well. The plate was dried overnight in a fume cupboard in order to remove the acetonitrile, and was then ready to be screened on androgenic activities with the yeast androgen bioassay. In the same way and in each series a reagent blank was prepared, using 10 mL of the 0.25 M sodium acetate buffer pH 4.8 instead of 10 mL urine. As representatives for 20 animal feed samples, 10 wet pulp feed samples which are normally used to feed pigs, and 10 regular milk replacers which are normally used to feed calves were used. Aliquots of 3 g blank feed or feed samples spiked with testosterone, 19-nortestosterone
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and 17␣-methyltestosterone at 50 ng g−1 , 17-trenbolone at 100 ng g−1 , and 17␣-methylboldenone at 500 ng g−1 were weighted. Feed samples were mixed with 6 mL methanol and 6 mL sodium acetate pH 4.8. Samples were incubated for 10 min in an ultrasonic bath and subsequently mixed for 15 min head over head. Samples were centrifuged at 3500 × g and 6 mL of the upper liquid phase was brought in a polypropylene tube. Next, the pH was adjusted to 4.8 using 4 M acetic acid and the extract was subjected to solid phase extraction (SPE) on a 1000 mg C18 column, previously conditioned with 4 mL methanol and 4 mL methanol/sodium acetate (50/50, v/v). Subsequently, this column was washed with 3 mL methanol/sodium acetate (50/50, v/v), 6 mL water, 3 mL 10% (w/v) sodium carbonate solution, three times 6 mL water and two times with 4 mL methanol/water (50/50, v/v). The column was air-dried and eluted two times with 4 mL acetonitrile. The eluate was applied to a 500 mg NH2-column, previously conditioned with 4 mL acetonitrile. The acetonitrile eluate thus obtained was evaporated to dryness under a stream of nitrogen gas and dissolved in 1.5 mL acetonitrile. A 200 L part of this extract (equivalent to 0.2 g feed) was transferred to a 96-well plate in triplicate and 50 L of a 4% DMSO solution was added to each well. The plate was dried overnight in a fume cupboard in order to remove the acetonitrile, and was then ready to be screened on androgenic activities with the yeast androgen bioassay. In the same way and in each series a reagent blank was prepared.
2.3.
Yeast androgen bioassay
An agar plate containing the selective MM/L medium was inoculated with the yeast hAR cytosensor from a frozen −80 ◦ C stock (20% (v/v) glycerol). The plate was incubated at 30 ◦ C for 24–48 h and then stored at 4 ◦ C. The day before running the assay, a single colony of the yeast cytosensor was used to inoculate 10 mL of selective MM/L medium. This culture was grown overnight at 30 ◦ C with vigorous orbital shaking at 225 rpm. At the late log phase, the yeast hAR cytosensor was diluted in MM/L, giving an OD at 630 nm in the range of 0.04–0.06, using a Helios Epsilon (Thermo Electron Corporation, USA). For exposure in 96-well plates, aliquots of 200 L of this diluted yeast culture were pipetted into each well, already containing the extracts of the calf urine and feed samples (see Section 2.2). A testosterone dose–response curve was included in each exposure experiment through the addition of 2 L of testosterone stock solutions in DMSO. Each feed sample extract and each testosterone stock was assayed in triplicate. Exposure was performed for 0 and 24 h. Fluorescence and OD at 630 nm at these time intervals were measured directly in a SynergyTM HT Multi Detection Microplate Reader (Bio-Tek Instruments Inc., USA) using excitation at 485 nm and measuring emission at 530 nm.
2.4.
Assay validation and data analysis
2.4.1.
Decision limit CC˛ and detection capability CCˇ
Extracts of the 20 blank calf urine and animal feed samples were analysed in the bioassay in order to determine the decision limits CC␣. Spiked calf urine and animal feed samples were analysed in order to determine whether the spiked sam-
227
ples fulfil the CC criterion. In each series an extract of a reagent blank was made as well and was also analysed in the bioassay. Each sample extract was assayed in triplicate. Fluorescence signals of the 20 blank samples and the 20 spiked samples obtained after 24 h of exposure were corrected for the signals obtained at 0 h (t24–t0) and were also corrected for the signal (t24–t0) obtained with a reagent blank. All these signals are the mean of a triplicate. In the context of EC Decision 2002/657 we define the mean signal of 20 blank samples plus three times the corresponding standard deviation as the decision limit CC␣ (˛ = 1%) [19]. Samples with a signal below this CC␣ are classified as compliant and samples with a signal above this CC␣ are classified as suspect. The criterion for the detection capability CC is that at least 19 out of the 20 spiked samples give a signal above this CC␣ and are thus classified as suspect (ˇ = 5%).
2.4.2.
Specificity
To determine the specificity of the yeast androgen bioassay for screening androgenic activity in animal feed potentially contaminated with synthetic glucocortisteroids or estrogens from pharmaceutical waste, two blank feed samples, one wet and one milk replacer, were spiked with a high dose of dexamethasone or 17␣-ethynylestradiol (both at 1000 ng g−1 ) and 200 L extracts were analysed in the yeast androgen bioassay. To check for interference, the two blank feed samples were spiked with the high dose of either dexamethasone or 17␣-ethynylestradiol in combination with a low dose of androgens: testosterone, 19-nortestosterone and 17␣methyltestosterone at 50 ng g−1 , 17-trenbolone at 100 ng g−1 , and 17␣-methylboldenone at 500 ng g−1 .
2.4.3.
Stability
For the determination of the stability of androgens in feed samples, aliquots of 3 g of blank and spiked samples were stored at their specific conditions. For this study we used one wet pulp feed sample and two milk replacers. The wet pulp samples were stored at −20 ◦ C and the milk replacers were stored in the dark at room temperature. At certain times, 0, 7, 35 and 91 days, blank and spiked feed samples were taken, extracts were made and 200 L aliquots were analysed in the yeast androgen bioassay.
3.
Results and discussion
The performance characteristics decision limit CC␣, detection capability CC, specificity and stability of the yeast androgen bioassay for screening androgenic activity in calf urine and animal feed were determined in order to validate the bioassay as a qualitative screening method according to EC 2002/657. There is no permitted limit for androgens, but for the routine screening an action level of 1–2 ng mL−1 urine is used. Similar is valid for feed and 50 ng testosterone per gram feed was selected as validation level, keeping in mind that higher levels would be expected in cases of illegal use or in feed contamination incidents. Testosterone, 17␣-methyltestosterone, 19-nortestosterone, 17-trenbolone, 17-boldenone and 17␣-methylboldenone were chosen as model compounds. Theoretically, the 200 L extract, equiva-
206b 99 78 64 49 55
38b 537 598 505 464 527 716 582 494 479 516 612 466 485 468 488 606 472 515 439 530 705 454 470 520 421
b
a
Mean, S.D. and CC␣ (CC␣ = mean of the blank + 3.0 S.D. of the blank) are determined from the 20 blank calf urine samples. Blank urine.
703 491 577 560 546 850 534 562 510 595 656 532 612 520 504 628 474 531 541 469 648 430 486 451 488 646 563 495 442 533 654 479 579 514 507 588 271 637 495 482 909 472 579 478 481 618 412 617 397 401 556 382 459 385 429 546 375 604 397 363 588 504 420 450 478 558 489 442 413 450 781 558 594 456 547 Testosterone 2 ng ml−1 19-Nortestosterone 2 ng ml−1 Trenbolone 2 ng ml−1 Boldenone 15 ng ml−1 Methylboldenone 15 ng ml−1
13 64b
14 89b
15 175b
16 80b
17 34b
18 97b
19 108b
20 60b
655 477 533 469 488
91b
S.D. (S)a Mean (X)a
12 86b 11 120b 10 110b 9 98b 8 109b 7 82b 6 3b 5 44b 4 119b 3 96b
Table 1 shows the results of the yeast androgen bioassay for 20 blank and 20 spiked calf urine samples. The signals are the responses obtained after 24 h of exposure and are corrected for the responses obtained at 0 h (t24–t0) and for the response (t24–t0) obtained with the reagent blank. All these responses are the mean of a triplicate and in general the %CV of these triplicates is less than 5% (data not shown). After 24 h of exposure there were no differences in the OD at 630 nm, meaning that no toxic effects on the yeast could be observed (data not shown). The mean response value of the 20 blank urine samples is 91 with a corresponding standard deviation of 38. The calculated decision limit CC␣, being the mean plus three times the standard deviation, for the corrected fluorescence response is therefore 206. The use of the assay can be seen as a qualitative ON/OFF method. Samples giving a signal lower than the CC␣ are compliant or negative. Samples giving a response higher than the determined CC␣ are suspect. The results in Table 1 demonstrate that the blank urine samples fulfil the CC␣ criterion, meaning that all blank urine samples give a response that is lower than the CC␣ (˛ = 1%). All blank samples are thus compliant. As all spiked urine samples give a response that is higher than the CC␣, they fulfil the CC criterion, meaning that at least 19 out of the 20 spiked samples give a signal above the CC␣ and are thus classified as suspect (ˇ = 5%). Table 2 shows the results of the yeast androgen bioassay for 20 blank and 20 spiked feed samples. Most blank feed samples have corrected responses with a negative value. These negative values are due to the relatively low responses of these samples when compared to the response of the reagent blank for which they are corrected. Moreover, these negative values are very low compared to the none reagent blank corrected
2 137b
Decision limit CC˛ and detection capability CCˇ
1 115b
3.1.
Urine sample #
lent to 2 mL urine, of the 2 ng mL−1 testosterone spike that is added to a well in a final well volume of about 200 L, results in a final concentration of 69 nM in the well. The 200 L extract, equivalent to 0.2 g feed, of the 50 ng g−1 testosterone spike would theoretically result in a final concentration of 173 nM. We have shown previously that the concentration where half-maximal response is reached (EC50 ) in the yeast androgen bioassay, is about 50 nM for testosterone [16] and in general ranged between 30 and 90 nM, so theoretically the 2 ng mL−1 testosterone spike in urine and the 50 ng g−1 testosterone spike in feed should easily be detected. The relative androgenic potency (RAP), defined as the ratio between the EC50 of testosterone and the EC50 of a compound, is 1.0, 1.2, 1.7, 1.5, 0.15 and 0.13 for testosterone, 17␣-methyltestosterone, 19-nortestosterone, 17-trenbolone, 17-boldenone and 17␣-methylboldenone respectively [18]. This means that 17␣-methyltestosterone, 19-nortestosterone and 17-trenbolone are at least as potent as testosterone and that 17-boldenone and 17␣-methylboldenone are about seven times less potent than testosterone. The latter two compounds were therefore spiked to the urine and feed samples in about 7–10 times higher amounts, thereby still maintaining biological relevance, but compromising versus the practice in chemical residue analysis.
CC␣a
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Table 1 – Fluorescence response in the yeast androgen bioassay of 20 blank and 20 spiked calf urine samples and the determined decision limit CC␣
228
396 292 255 130 321
44b 28b
Mean, S.D. and CC␣ are determined from 20 feed samples. In grey is the sample that was screened as false negative. Blank feed. b
a
Testosterone 50 ng g−1 19-Nortestosterone 50 ng g−1 Methyltestosterone 50 ng g−1 Trenbolone 100 ng g−1 Methylboldenone 500 ng g−1
931 813 508 415 708
1788 981 744 399 1497
1116 962 1137 1302 859 516 597 498 1261 968
1025 1286 671 426 996
538 1057 362 458 643
481 651 258 263 439
584 652 454 372 525
68 415 −41 134 151
334 504 393 242 366
546 696 497 377 612
1356 1480 1064 543 940
910 964 747 473 900
847 1121 826 614 772
414 820 346 400 504
373 723 335 314 433
591 677 444 344 645
756 817 712 476 883
490 548 402 267 514
941 1131 780 621 969
753 889 544 412 736
40b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 −38b −72b −57b −74b −72b −54b −71b −72b −54b −68b −10b 17b −44b 4b −22b −29b −31b −19b −19b −12b
Mean (X)a S.D. (S)a CC␣a Feed sample #
Table 2 – Fluorescence response in the yeast androgen bioassay of 20 blank and 20 spiked calf urine samples and the determined decision limit CC␣
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229
signals (data not shown). However, although there is no toxic effect visible, the extracts probably contain substances that give a little inhibitory effect in the bioassay. The latter became clear when higher amounts of the feed extracts were used and showed cell toxicity. Due to this effect we were not able to validate the assay with these low amounts of androgens for dry feed as it was demonstrated before that dry feed and milk replacer samples are more complex samples compared to wet pulp feed and urine samples and dry feed samples can be considered as a worst case [12]. This is also demonstrated by the results in Table 2, as spiked milk replacer, samples #1–10, already give lower signals compared to spiked wet pulp feed, samples #11–20, corrected mean response of respectively −63 and −18. The mean response value of all 20 blank feed samples is −40 with a corresponding standard deviation of 28. The calculated decision limit CC␣ for the corrected fluorescence response is therefore 44. The results presented in Table 2 demonstrate that the blank feed samples fulfil the CC␣ criterion, meaning that all blank feed samples give a response that is lower than the CC␣ (˛ = 1%). All blank samples are thus compliant. Only sample #9, a milk replacer, spiked with methyltestosterone was screened as a false compliant or false negative. As all but one of the spiked samples give a response that is higher than the CC␣ and are classified as suspect, the spiked feed samples thus fulfil the CC criterion (ˇ = 5%).
3.2.
Specificity and interference
The specificity of the yeast androgen bioassay was determined with two blank animal feed samples, one milk replacer and one wet pulp feed sample, spiked with a high dose of dexamethasone or 17␣-ethynylestradiol (1000 ng g−1 ). Interference was checked with these two blank samples by spiking them with a low dose of testosterone, 19-nortestosterone and 17␣-methyltestosterone at 50 ng g−1 , 17-trenbolone at 100 ng g−1 , and 17␣-methylboldenone at 500 ng g−1 , in combination with the high dose of either dexamethasone or 17␣-ethynylestradiol. Extracts were made and 200 L aliquots were analysed in the bioassay. Results of corrected fluorescence signals are shown in Table 3. The data in Table 3 show that neither the glucocorticoid dexamethasone nor the estrogen 17␣-ethynylestradiol give a response in the bioassay, as the blank samples give about the same response as the corresponding dexamethasone and 17␣-ethynylestradiol spiked samples. The results in Table 3 also show that dexamethasone has no effect on the signals of the samples spiked with androgens, but 17␣-ethynylestradiol inhibits the signal of the samples spiked with androgens. Moreover, 17␣ethynylestradiol interferes with the screening result of the milk replacer spiked with testosterone, methyltestosteron and trenbolone, as these spiked samples now give a signal below the determined CC␣ and are thus falsely classified as compliant. This is not completely surprising as it was demonstrated before that 17␣-ethynylestradiol binds to the hAR without being able to show an agonistic response [17] and 17␣-ethynylestradiol has been shown to be able to displace radiolabeled dihydrotestosterone from the androgen receptor (AR), showing a relative binding affinity of about 0.8% when compared with testosterone [20]. Also other estrogenic compounds like 17-estradiol, diethylstilbestrol (DES),
230
The specificity of the yeast androgen bioassay was determined with two blank feed samples that were spiked with a high dose of dexamethasone or 17␣-ethynylestradiol, both at a level of 1000 ng g−1 . Interference was checked with the same feed samples by spiking them with the low dose of androgens in combination with the high dose of either dexamethasone or 17␣-ethynylestradiol. In grey the samples that not fulfil the criterion.
Table 3 – Specificity and interference checked with feed samples spiked with a high dose of dexamethasone or 17␣-ethynylestradiol
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
Fig. 1 – Responses of 17-testosterone (T-17) and 17␣-ethynylestradiol (EE2) in the yeast androgen bioassay and the responses of 17␣-ethynylestradiol by co-exposure with 70 and 1000 nM 17-testosterone (open symbols).
estrone, coumestrol and genistein have been shown to be able to displace radiolabeled R 1881 from the AR [21]. It is thus possible that 17␣-ethynylestradiol acts as an AR antagonist. Fig. 1 shows the response of testosterone and 17␣-ethynylestradiol in the yeast androgen assay. The figure shows that testosterone gives a full dose–response curve with an EC50 of 31 nM and that 17␣-ethynylestradiol does not give a response. To test the antiandrogenic properties of 17␣-ethynylestradiol, stock solutions of 17␣-ethynylestradiol were co-exposed with testosterone solutions giving about a half-maximal and a near maximal response, i.e. levels of 70 and 1000 nM testosterone, respectively. Fig. 1 shows that 17␣-ethynylestradiol is an AR antagonist and is able to completely inhibit the response caused by 70 and 1000 nM testosterone when its concurrent dose is high enough. Specificity and interference were not checked with urine samples as these characteristics are mainly determined by the compounds and the bioassay and not by the matrix. Moreover, as mentioned before, urine is a less complex matrix than feed.
3.3.
Stability of androgens in feed
The stability of the androgenic compounds in animal feed samples was determined with three animal feed samples, two milk replacers and one wet pulp feed sample. Again, the feed samples are tested as a worst case. Blank and spiked wet pulp feed samples were stored at −20 ◦ C and blank and spiked milk replacers were stored at room temperature in the dark. At certain times, 0, 7, 35 and 91 days, blank and spiked feed samples were taken, extracts were made and 200 L aliquots were analysed in the yeast androgen bioassay. Fluorescence signals at 24 h were corrected for the signals obtained at 0 h and the response of a reagent blank. Results are shown in Table 4. Blank samples were always screened as compliant and spiked samples were all screened as suspect. The data in Table 4 thus demonstrate that the feed samples can be stored at their specific conditions for up to 91 days, without disrupting the screening result. Urine stability tests at −20 ◦ C are ongoing, but not expected to deviate from the feed results, based on previous validation experiences with estrogens [12].
425 472 323 163 478 422 646 302 319 450 438 493 337 264 367 548 846 407 476 420
35 (7)a 7 (−26)a
3.4.
399 574 278 202 385
Days stored and (response of blank feed). a
Milk replacers were stored at room temperature in the dark and the wet pulp feed samples were stored at −20 ◦ C.
827 1311 731 378 1115 609 633 447 639 876 415 417 203 188 489 688 968 256 846 738 Testosterone 50 ng g−1 19-Nortestosterone 50 ng g−1 Methyltestosterone 50 ng g−1 Trenbolone 100 ng g−1 Methylboldenone 500 ng g−1
0
594 798 355 631 611
7
305 358 162 142 309
35
(−9)a (−54)a
1 Milk replacer
(−12)a Feed sample #
Table 4 – Stability of low amounts of androgens in animal feed
91
(−50)a
0
(−58)a
7
296 346 206 169 344
35
(−56)a (−85)a
4 Milk replacer
91 (−44)
0
(−4)a
14 Wet pulp feed
91 (−22)a
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231
Application to real samples
Two hundred and thirty seven calf urine samples from a national control program were send to RIKILT and screened for androgenic activity using the validated yeast androgen bioassay procedure described here. 19 samples were screened as suspect and 218 were screened as compliant. These 19 suspect samples and 20 randomly picked compliant samples were analysed with GC/MS/MS as described before [22]. Results are shown in Table 5. The 20 negative screened samples (#1–20) contained no norethandrolone (NE), 17␣ethyl-5-estrane-3␣,17-diol (EED), 17-nortestosterone (NT17), 17␣-nortestosterone (NT-17␣) or 17␣-methyltestosterone (MT-17␣) and 16 of these 20 bioassay negative screened samples contained no 17-testosterone (T-17), while four contained less than 1 ng mL−1 . All samples (#1–39) contained 17␣-testosterone (T-17␣), but this compound is known as a very weak androgen receptor agonist and is almost inactive in the yeast androgen bioassay [17]. It was demonstrated before that NT-17 is a little more potent and that MT-17␣ is just as potent as 17testosterone [17,23,24]. Norethandrolone (NE), 17␣-ethyl-5estrane-3␣,17-diol (EED) and 17␣-nortestosterone (NT-17␣) were not tested before in the bioassay and their dose–response curves are shown in Fig. 2. Results were as expected, as based on its structure EED, a metabolite of the synthetic androgen norethandrolone (NE) [25], will be less active than NE and 17testosterone, because of its H-group at the 5-position and its OH-group at the 3␣-position [17]. Also 17␣-nortestosterone (NT-17␣) was expected to be less active as it was demonstrated before that changing the position of the OH-group at position 17 from 17 to 17␣ strongly decreases the potency of the compound [17,23]. These differences in potencies between 17-testosterone and 17-nortestosterone on one hand and 17␣-testosterone and 17␣-nortestosterone on the other, show that the 17-OH configuration is very important for androgenic activity [26]. NE is expected to be a little more potent than 17testosterone, because compared to -testosterone the absence of the methyl group at position 19 increases the potency (just as with NT-17), while the ethyl group at the 17␣-position hardly influences the potency (just as with the methyl group of MT-17␣) [17]. However, the EC50 value for NE is about the same as that of T-17. Moreover, we cannot explain the higher maximal response of NE, as all potent compounds tested so far reach the same maximal response as T-17, while less potent compounds reach either the same or a lower maximal value. Six of the 19 bioassay suspect screened samples (#21–39) contained 17-testosterone levels above 1 ng mL−1 (range 1.2–14.9 ng mL−1 , mean 4.5 ng mL−1 ). From the other 13 bioassay suspect screened samples there were five containing less than 1 ng 17-testosterone per mL and another one contained a small amount of 17␣-ethyl-5-estrane-3␣,17-diol (EED). When these 13 bioassay suspect screened samples are regarded as false positives, the percentage of false positives is 5%, which is acceptable for a qualitative screening method. However, it cannot be excluded that there are other metabolites of endogenous androgens, e.g. 5␣-androstane-3,17-diol, 5␣-androstane-3␣,17-diol, and 5␣-androstane-17-ol-3-one (5␣-DHT) and its isomers, that are not included in the GC/MS/MS analysis yet and are active in
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Table 5 – Levels (ng mL−1 ) of natural and synthetic steroids and their metabolites determined in calf urine samples from a national control program using GC/MS/MS. Bioassay result
NE
EEDa
NT-17
NT-17␣
T-17
T-17␣a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N <1 N <1 N <1 N N N <1 N N N N
1.2 <1 9.6 62 1.1 <1 4.8 26 3.7 100 21 72 2.9 1.2 2.2 114 32 7.3 8.9 1.7
N N N N N N N N N N N N N N N N N N N N
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
S S S S S S S S S S S S S S S S S S S
N N N N N N N N N N N N N N N N N N N
N <1 N N N N N N N N N N N N N N N N N
N N N N N N <1 N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N
1.2 N N 3.8 N N 1.2 <1 N <1 2.4 N 14.9 3.4 N <1 <1 N <1
54.6 23 5.2 225 <1 26 85 13 29 60 135 2.4 160 168 3.0 4.0 80 20 22
N N N N N N N N N N N N N N N N N N N
Sample #
MT-17␣
N = negative, S = suspect, NE = norethandrolone (NE), EED = 17␣-ethyl-5-estrane-3␣,17-diol, NT-17 = 17-nortestosterone, NT-17␣ = 17␣nortestosterone, T-17 = 17-testosterone, T-17␣ = 17␣-testosterone and MT-17␣ = 17␣-methyltestosterone. a
Not validated for urine
the bioassay, that could account for these 13 suspect bioassay results [17,27,28].
4.
Fig. 2 – Responses of 17-testosterone (T-17), norethandrolone (NE), 17␣-nortestosterone (NT-17␣), and 17␣-ethyl-5-estrane-3␣,17-diol (EED) in the yeast androgen bioassay.
Conclusions
The data presented and the determined performance characteristics prove that the yeast androgen bioassay can detect low levels of androgens in calf urine and animal feed samples. All blank samples gave responses lower than the determined decision limits CC␣ and were classified as compliant. Thus there were no false suspect results. Signals of all spiked calf urine samples were higher than the determined decision limit CC␣, classified as suspect and thus fulfilled the CC criterion. However, the minimum required performance limit for 17-boldenone glucuronide in urine is 1 ng mL−1 , but because this compound is about
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
seven times less potent than testosterone, it was spiked to urine at a level of 15 ng mL−1 . Although the sensitivity of this screening method is too low for compounds with a relative low androgenic potency compared to testosterone, such as 17-boldenone and 17␣-methylboldenone, the assay has several advantages. It is robust, easy, quick and inexpensive, it obeys to the principles of the EU ban, which prohibits all substances having hormonal action. Moreover, the assay is able to detect new designer steroids as already proven for the designer steroid tetrahydrogestrinone in human urine samples [29]. Regarding the feed samples, the spiked animal feed samples fulfilled the CC criterion as well. High levels of the glucocorticoid dexamethasone or the estrogen 17␣-ethynylestradiol did not give a response in the bioassay. Dexamethasone did not interfere with the screening result of spiked animal feed samples, but high levels of 17␣-ethynylestradiol can interfere and lead to false negative screening results. Here we show that 17␣-ethynylestradiol acts as an androgen receptor antagonist in a yeast androgen bioassay expressing yEGFP in response to androgens. However, illegal cocktails in general contain more androgens than estrogens and thus this inhibiting effect is not relevant in veterinary practice [30]. Moreover, the presence of compounds with an antagonistic mode of action can easily be determined by spiking the samples or extracts with a dose of an agonist that gives a half-maximal or near maximal response [31]. The observation that feed samples could be stored at their specific conditions for up to 91 days without influencing the screening result, showed that androgens in feed are stable. The stability study also demonstrates that the outcome of the assay, compliant/suspect, is reproducible and that the procedure is robust. As all the performance characteristics met the criteria that were put forward in EC Decision 2002/657 for validation of a qualitative screening method [19], the above described clean-up/yeast androgen bioassay procedure is proven to be valid for the determination of androgenic activity in calf urine and animal feed. Indeed the assay could successfully be applied to real urine samples from a control program and GC/MS/MS identification and quantification showed that negative screened samples contained no or low levels of androgens. As a result 218 of 237 samples were screened negative in the bioassay and can be regarded androgen free, demonstrating the applicability and strength of this yeast androgen bioassay as a screening test. The percentage of false positives was only 5%, but this percentage might drop if GC/MS/MS analysis would include more metabolites like 5␣-DHT and its isomers. The latter will be part of our future research efforts. Moreover, the GC/MS/MS results are in agreement with earlier findings, showing that 17␣testosterone is the most abundant natural hormone residue in urine of both male and female calves and is associated with an increased 17-testosterone level [22].
Acknowledgements This project was financially supported by the Dutch Ministry of Agriculture, Nature and Food Quality. Authors would like to thank Ron L.A.P. Hoogenboom for his adjustments to the manuscript.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed Toine F.H. Bovee ∗ , Gerrit Bor, Henri H. Heskamp, Johan J.P. Lasaroms, Marieke B. Sanders, Michel W.F. Nielen RIKILT Institute of Food Safety, Wageningen University and Research Centre, P.O. Box 230, 6700 AE Wageningen, The Netherlands
a r t i c l e
i n f o
a b s t r a c t
Article history:
Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening.
Received 24 April 2008
Previously we described the construction and properties of a rapid and robust yeast andro-
Received in revised form
gen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced
19 June 2008
green fluorescent protein (yEGFP), the latter in response to androgens. In the present
Accepted 23 June 2008
study this yeast androgen bioassay was validated as a qualitative screening method for
Published on line 5 July 2008
the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with
Keywords:
testosterone, 17␣-methyltestosterone, 19-nortestosterone, 17-trenbolone, 17-boldenone
Androgens
or 17␣-methylboldenone at 2 or 15 ng mL−1 in urine and 50 or 100 ng g−1 in feed. All blank
Bioassay
and spiked samples fulfilled the CC␣ and CC criterions, meaning that all 20 blank samples
Hormone abuse
gave signals below the determined decision limits CC␣ and were thus classified as compliant
Steroids
(˛ = 1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the
Validation
CC␣ and were thus classified as suspect (ˇ = 5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17␣-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography–tandem mass spectrometry. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
The illegal use of androgens in cattle breeding conflicts with principles of fair trade and may impose a risk to the animal and the consumer. In addition, hormones may be present in pharmaceutical waste illegally mixed into the feed. The use of growth promoters for fattening purposes in cattle has been banned in the European Union since 1988 [1]. This EU ban prohibits all substances having hormonal action and does
∗
Corresponding author. Tel.: +31 317480391. E-mail address: [email protected] (T.F.H. Bovee). 0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2008.06.047
not provide a list of forbidden substances. Hormone abuse or incidents may be discovered by residue analysis in calf urine or animal feed. However, in practice residue analysis is carried out on specific target compounds and can enforce the ban only to a limited extent [2]. Alternatively, receptor-based assays can be used to detect all compounds having affinity for a given receptor [3,4]. In contrast to receptor binding assays, reporter gene bioassays also mimic the transactivation step and can distinguish between receptor agonists and receptor
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antagonists [5,6]. This feature is very helpful in detecting known and unknown compounds, as receptor stimulation plays a key role in the mechanism of action of growth promoters. To the best of our knowledge there are no known plant compounds with a direct androgenic mode of action, but there are plant-derived compounds, e.g. diindolylmethane (DIM), flavone, guggulsterone and equol, with an antiandrogenic mode of action [7,8]. Both yeast-based assays as well as the more established in vitro reporter assays based on mammalian cells are suitable tests to determine the androgenic potency of a given compound. In general, transcription activation assays based on mammalian cell lines have been shown to be more sensitive than yeast-based assays [6,9,10]. However, yeast-based assays have several other advantages. These include low costs and easier handling, lack of known endogenous receptors that may compete with the activity under investigation, use of media that are devoid of steroids, and last but not least, yeast cells assays are extremely robust and survive extracts from dirty sample matrices such as urine and feed [11,12]. Yeast-based assays for the screening of estrogenic activities have been shown to be sensitive, specific, robust and especially bioassays using yEGFP as a marker protein are user-friendly [12–15]. Recently, we developed a novel yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP) in response to androgens. This assay is completely performed in a 96-well plate and fluorescence is measured directly in intact yeast cells. This assay is relatively simple and sensitive, as shown by an EC50 value for testosterone of 50 nM [16]. A large number of chemically different compounds with known androgenic, but also other hormonal activities were tested in our in-house developed yeast androgen bioassay to investigate its specificity. All androgenic compounds caused a dose-related increase in the production of green fluorescent protein, whereas the glucocorticoid dexamethasone showed no response [17]. Especially with androgens, the lack of known endogenous receptors in yeast is a great advantage compared with mammalian cell lines, as androgen responsive elements (AREs) can also be activated by the progesterone and glucocorticoid receptor (PR and GR). Lots of effort were made to construct an ARE that is specific and no longer inducible by the progesterone or glucocorticoid receptor in order to avoid the potential crosstalk in mammalian cells. However, up till now such an ARE does not exist and it is doubtful whether it will be found, as the consensus progesterone and glucocorticoid responsive elements (PRE/GRE) are equal to the consensus ARE [18]. Moreover, the GR is normally expressed in all mammalian cell types. So far this resulted in cell lines that are not specific for androgens and also respond to progesterone or glucocorticoids. Other yeast androgen bioassays are about equally sensitive and specific, but our new yeast androgen bioassay is the only one with the yEGFP marker, making it not only more convenient, but also faster and cheaper [16,17]. Moreover, not one of the other androgen bioassays based on either yeast or mammalian cells has been fully validated according to the international standards as prescribed in EC 2002/657 [19]. The present study describes the validation conform EC 2002/657 of this new yeast androgen bioassay for the determination of androgenic activity in calf urine
and animal feed and demonstrates its applicability to real samples.
2.
Experimental
2.1.
Chemicals
Acetonitrile and methanol were from Biosolve (Valkenswaard, The Netherlands). Ammonium sulphate, dimethyl sulfoxide, sodium acetate and sodium carbonate were obtained from Merck (Darmstadt, Germany), dexamethasone, 19-nortestosterone, and l-leucine from Sigma and 17boldenone, 17␣-ethynylestradiol, 17␣-methylboldenone, 17␣-methyltestosterone, testosterone, and 17-trenbolone from Steraloids (Newport, RI, USA). Isolute NH2 extraction columns (500 mg) were obtained from IST (Hengoed, U.K.) and Bond Elut C18 solid phase extraction columns (1000 mg) from Varian (Harbor City, CA, USA). Dextrose and yeast nitrogen base without amino acids and without ammonium sulphate were obtained from Difco (Detroit, MI, USA). The minimal medium with l-leucine (MM/L) consisted of yeast nitrogen base without amino acids and without ammonium sulphate (1.7 g L−1 ), dextrose (20 g L−1 ) and ammonium sulphate (5 g L−1 ) and was supplemented with l-leucine (60 mg L−1 ).
2.2.
Samples and sample treatment
Aliquots of 10 mL blank calf urine or calf urine samples spiked with testosterone, 19-nortestosterone and 17-trenbolone at 2 ng mL−1 , 17-boldenone and 17␣-methylboldenone at 15 ng mL−1 , were adjusted to pH 4.8 and 20 L glucuronidase/arylsulfatase (3 U mL−1 ) was added. Enzymatic deconjugation was carried out overnight in a water bath at 37 ◦ C. Next, 10 mL of 0.25 M sodium acetate buffer pH 4.8 was added and the hydrolysed sample was subjected to solid phase extraction (SPE) on a 1000 mg C18 column, previously conditioned with 4 mL methanol and 4 mL sodium acetate buffer. Subsequently, this column was washed with 3 mL sodium acetate buffer, 6 mL water, 3 mL 10% (w/v) sodium carbonate solution, 6 mL water and finally with 6 mL methanol/water (50/50, v/v). The column was air-dried and eluted with 4 mL acetonitrile. The eluate was applied to a 500 mg NH2-column, previously conditioned with 4 mL acetonitrile. The acetonitrile eluate thus obtained was evaporated to dryness under a stream of nitrogen gas and dissolved in 1 mL acetonitrile. A 200 L part of this extract (equivalent to 2 mL urine) was transferred to a 96-well plate in triplicate and 50 L of a 4% DMSO solution was added to each well. The plate was dried overnight in a fume cupboard in order to remove the acetonitrile, and was then ready to be screened on androgenic activities with the yeast androgen bioassay. In the same way and in each series a reagent blank was prepared, using 10 mL of the 0.25 M sodium acetate buffer pH 4.8 instead of 10 mL urine. As representatives for 20 animal feed samples, 10 wet pulp feed samples which are normally used to feed pigs, and 10 regular milk replacers which are normally used to feed calves were used. Aliquots of 3 g blank feed or feed samples spiked with testosterone, 19-nortestosterone
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and 17␣-methyltestosterone at 50 ng g−1 , 17-trenbolone at 100 ng g−1 , and 17␣-methylboldenone at 500 ng g−1 were weighted. Feed samples were mixed with 6 mL methanol and 6 mL sodium acetate pH 4.8. Samples were incubated for 10 min in an ultrasonic bath and subsequently mixed for 15 min head over head. Samples were centrifuged at 3500 × g and 6 mL of the upper liquid phase was brought in a polypropylene tube. Next, the pH was adjusted to 4.8 using 4 M acetic acid and the extract was subjected to solid phase extraction (SPE) on a 1000 mg C18 column, previously conditioned with 4 mL methanol and 4 mL methanol/sodium acetate (50/50, v/v). Subsequently, this column was washed with 3 mL methanol/sodium acetate (50/50, v/v), 6 mL water, 3 mL 10% (w/v) sodium carbonate solution, three times 6 mL water and two times with 4 mL methanol/water (50/50, v/v). The column was air-dried and eluted two times with 4 mL acetonitrile. The eluate was applied to a 500 mg NH2-column, previously conditioned with 4 mL acetonitrile. The acetonitrile eluate thus obtained was evaporated to dryness under a stream of nitrogen gas and dissolved in 1.5 mL acetonitrile. A 200 L part of this extract (equivalent to 0.2 g feed) was transferred to a 96-well plate in triplicate and 50 L of a 4% DMSO solution was added to each well. The plate was dried overnight in a fume cupboard in order to remove the acetonitrile, and was then ready to be screened on androgenic activities with the yeast androgen bioassay. In the same way and in each series a reagent blank was prepared.
2.3.
Yeast androgen bioassay
An agar plate containing the selective MM/L medium was inoculated with the yeast hAR cytosensor from a frozen −80 ◦ C stock (20% (v/v) glycerol). The plate was incubated at 30 ◦ C for 24–48 h and then stored at 4 ◦ C. The day before running the assay, a single colony of the yeast cytosensor was used to inoculate 10 mL of selective MM/L medium. This culture was grown overnight at 30 ◦ C with vigorous orbital shaking at 225 rpm. At the late log phase, the yeast hAR cytosensor was diluted in MM/L, giving an OD at 630 nm in the range of 0.04–0.06, using a Helios Epsilon (Thermo Electron Corporation, USA). For exposure in 96-well plates, aliquots of 200 L of this diluted yeast culture were pipetted into each well, already containing the extracts of the calf urine and feed samples (see Section 2.2). A testosterone dose–response curve was included in each exposure experiment through the addition of 2 L of testosterone stock solutions in DMSO. Each feed sample extract and each testosterone stock was assayed in triplicate. Exposure was performed for 0 and 24 h. Fluorescence and OD at 630 nm at these time intervals were measured directly in a SynergyTM HT Multi Detection Microplate Reader (Bio-Tek Instruments Inc., USA) using excitation at 485 nm and measuring emission at 530 nm.
2.4.
Assay validation and data analysis
2.4.1.
Decision limit CC˛ and detection capability CCˇ
Extracts of the 20 blank calf urine and animal feed samples were analysed in the bioassay in order to determine the decision limits CC␣. Spiked calf urine and animal feed samples were analysed in order to determine whether the spiked sam-
227
ples fulfil the CC criterion. In each series an extract of a reagent blank was made as well and was also analysed in the bioassay. Each sample extract was assayed in triplicate. Fluorescence signals of the 20 blank samples and the 20 spiked samples obtained after 24 h of exposure were corrected for the signals obtained at 0 h (t24–t0) and were also corrected for the signal (t24–t0) obtained with a reagent blank. All these signals are the mean of a triplicate. In the context of EC Decision 2002/657 we define the mean signal of 20 blank samples plus three times the corresponding standard deviation as the decision limit CC␣ (˛ = 1%) [19]. Samples with a signal below this CC␣ are classified as compliant and samples with a signal above this CC␣ are classified as suspect. The criterion for the detection capability CC is that at least 19 out of the 20 spiked samples give a signal above this CC␣ and are thus classified as suspect (ˇ = 5%).
2.4.2.
Specificity
To determine the specificity of the yeast androgen bioassay for screening androgenic activity in animal feed potentially contaminated with synthetic glucocortisteroids or estrogens from pharmaceutical waste, two blank feed samples, one wet and one milk replacer, were spiked with a high dose of dexamethasone or 17␣-ethynylestradiol (both at 1000 ng g−1 ) and 200 L extracts were analysed in the yeast androgen bioassay. To check for interference, the two blank feed samples were spiked with the high dose of either dexamethasone or 17␣-ethynylestradiol in combination with a low dose of androgens: testosterone, 19-nortestosterone and 17␣methyltestosterone at 50 ng g−1 , 17-trenbolone at 100 ng g−1 , and 17␣-methylboldenone at 500 ng g−1 .
2.4.3.
Stability
For the determination of the stability of androgens in feed samples, aliquots of 3 g of blank and spiked samples were stored at their specific conditions. For this study we used one wet pulp feed sample and two milk replacers. The wet pulp samples were stored at −20 ◦ C and the milk replacers were stored in the dark at room temperature. At certain times, 0, 7, 35 and 91 days, blank and spiked feed samples were taken, extracts were made and 200 L aliquots were analysed in the yeast androgen bioassay.
3.
Results and discussion
The performance characteristics decision limit CC␣, detection capability CC, specificity and stability of the yeast androgen bioassay for screening androgenic activity in calf urine and animal feed were determined in order to validate the bioassay as a qualitative screening method according to EC 2002/657. There is no permitted limit for androgens, but for the routine screening an action level of 1–2 ng mL−1 urine is used. Similar is valid for feed and 50 ng testosterone per gram feed was selected as validation level, keeping in mind that higher levels would be expected in cases of illegal use or in feed contamination incidents. Testosterone, 17␣-methyltestosterone, 19-nortestosterone, 17-trenbolone, 17-boldenone and 17␣-methylboldenone were chosen as model compounds. Theoretically, the 200 L extract, equiva-
206b 99 78 64 49 55
38b 537 598 505 464 527 716 582 494 479 516 612 466 485 468 488 606 472 515 439 530 705 454 470 520 421
b
a
Mean, S.D. and CC␣ (CC␣ = mean of the blank + 3.0 S.D. of the blank) are determined from the 20 blank calf urine samples. Blank urine.
703 491 577 560 546 850 534 562 510 595 656 532 612 520 504 628 474 531 541 469 648 430 486 451 488 646 563 495 442 533 654 479 579 514 507 588 271 637 495 482 909 472 579 478 481 618 412 617 397 401 556 382 459 385 429 546 375 604 397 363 588 504 420 450 478 558 489 442 413 450 781 558 594 456 547 Testosterone 2 ng ml−1 19-Nortestosterone 2 ng ml−1 Trenbolone 2 ng ml−1 Boldenone 15 ng ml−1 Methylboldenone 15 ng ml−1
13 64b
14 89b
15 175b
16 80b
17 34b
18 97b
19 108b
20 60b
655 477 533 469 488
91b
S.D. (S)a Mean (X)a
12 86b 11 120b 10 110b 9 98b 8 109b 7 82b 6 3b 5 44b 4 119b 3 96b
Table 1 shows the results of the yeast androgen bioassay for 20 blank and 20 spiked calf urine samples. The signals are the responses obtained after 24 h of exposure and are corrected for the responses obtained at 0 h (t24–t0) and for the response (t24–t0) obtained with the reagent blank. All these responses are the mean of a triplicate and in general the %CV of these triplicates is less than 5% (data not shown). After 24 h of exposure there were no differences in the OD at 630 nm, meaning that no toxic effects on the yeast could be observed (data not shown). The mean response value of the 20 blank urine samples is 91 with a corresponding standard deviation of 38. The calculated decision limit CC␣, being the mean plus three times the standard deviation, for the corrected fluorescence response is therefore 206. The use of the assay can be seen as a qualitative ON/OFF method. Samples giving a signal lower than the CC␣ are compliant or negative. Samples giving a response higher than the determined CC␣ are suspect. The results in Table 1 demonstrate that the blank urine samples fulfil the CC␣ criterion, meaning that all blank urine samples give a response that is lower than the CC␣ (˛ = 1%). All blank samples are thus compliant. As all spiked urine samples give a response that is higher than the CC␣, they fulfil the CC criterion, meaning that at least 19 out of the 20 spiked samples give a signal above the CC␣ and are thus classified as suspect (ˇ = 5%). Table 2 shows the results of the yeast androgen bioassay for 20 blank and 20 spiked feed samples. Most blank feed samples have corrected responses with a negative value. These negative values are due to the relatively low responses of these samples when compared to the response of the reagent blank for which they are corrected. Moreover, these negative values are very low compared to the none reagent blank corrected
2 137b
Decision limit CC˛ and detection capability CCˇ
1 115b
3.1.
Urine sample #
lent to 2 mL urine, of the 2 ng mL−1 testosterone spike that is added to a well in a final well volume of about 200 L, results in a final concentration of 69 nM in the well. The 200 L extract, equivalent to 0.2 g feed, of the 50 ng g−1 testosterone spike would theoretically result in a final concentration of 173 nM. We have shown previously that the concentration where half-maximal response is reached (EC50 ) in the yeast androgen bioassay, is about 50 nM for testosterone [16] and in general ranged between 30 and 90 nM, so theoretically the 2 ng mL−1 testosterone spike in urine and the 50 ng g−1 testosterone spike in feed should easily be detected. The relative androgenic potency (RAP), defined as the ratio between the EC50 of testosterone and the EC50 of a compound, is 1.0, 1.2, 1.7, 1.5, 0.15 and 0.13 for testosterone, 17␣-methyltestosterone, 19-nortestosterone, 17-trenbolone, 17-boldenone and 17␣-methylboldenone respectively [18]. This means that 17␣-methyltestosterone, 19-nortestosterone and 17-trenbolone are at least as potent as testosterone and that 17-boldenone and 17␣-methylboldenone are about seven times less potent than testosterone. The latter two compounds were therefore spiked to the urine and feed samples in about 7–10 times higher amounts, thereby still maintaining biological relevance, but compromising versus the practice in chemical residue analysis.
CC␣a
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
Table 1 – Fluorescence response in the yeast androgen bioassay of 20 blank and 20 spiked calf urine samples and the determined decision limit CC␣
228
396 292 255 130 321
44b 28b
Mean, S.D. and CC␣ are determined from 20 feed samples. In grey is the sample that was screened as false negative. Blank feed. b
a
Testosterone 50 ng g−1 19-Nortestosterone 50 ng g−1 Methyltestosterone 50 ng g−1 Trenbolone 100 ng g−1 Methylboldenone 500 ng g−1
931 813 508 415 708
1788 981 744 399 1497
1116 962 1137 1302 859 516 597 498 1261 968
1025 1286 671 426 996
538 1057 362 458 643
481 651 258 263 439
584 652 454 372 525
68 415 −41 134 151
334 504 393 242 366
546 696 497 377 612
1356 1480 1064 543 940
910 964 747 473 900
847 1121 826 614 772
414 820 346 400 504
373 723 335 314 433
591 677 444 344 645
756 817 712 476 883
490 548 402 267 514
941 1131 780 621 969
753 889 544 412 736
40b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 −38b −72b −57b −74b −72b −54b −71b −72b −54b −68b −10b 17b −44b 4b −22b −29b −31b −19b −19b −12b
Mean (X)a S.D. (S)a CC␣a Feed sample #
Table 2 – Fluorescence response in the yeast androgen bioassay of 20 blank and 20 spiked calf urine samples and the determined decision limit CC␣
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
229
signals (data not shown). However, although there is no toxic effect visible, the extracts probably contain substances that give a little inhibitory effect in the bioassay. The latter became clear when higher amounts of the feed extracts were used and showed cell toxicity. Due to this effect we were not able to validate the assay with these low amounts of androgens for dry feed as it was demonstrated before that dry feed and milk replacer samples are more complex samples compared to wet pulp feed and urine samples and dry feed samples can be considered as a worst case [12]. This is also demonstrated by the results in Table 2, as spiked milk replacer, samples #1–10, already give lower signals compared to spiked wet pulp feed, samples #11–20, corrected mean response of respectively −63 and −18. The mean response value of all 20 blank feed samples is −40 with a corresponding standard deviation of 28. The calculated decision limit CC␣ for the corrected fluorescence response is therefore 44. The results presented in Table 2 demonstrate that the blank feed samples fulfil the CC␣ criterion, meaning that all blank feed samples give a response that is lower than the CC␣ (˛ = 1%). All blank samples are thus compliant. Only sample #9, a milk replacer, spiked with methyltestosterone was screened as a false compliant or false negative. As all but one of the spiked samples give a response that is higher than the CC␣ and are classified as suspect, the spiked feed samples thus fulfil the CC criterion (ˇ = 5%).
3.2.
Specificity and interference
The specificity of the yeast androgen bioassay was determined with two blank animal feed samples, one milk replacer and one wet pulp feed sample, spiked with a high dose of dexamethasone or 17␣-ethynylestradiol (1000 ng g−1 ). Interference was checked with these two blank samples by spiking them with a low dose of testosterone, 19-nortestosterone and 17␣-methyltestosterone at 50 ng g−1 , 17-trenbolone at 100 ng g−1 , and 17␣-methylboldenone at 500 ng g−1 , in combination with the high dose of either dexamethasone or 17␣-ethynylestradiol. Extracts were made and 200 L aliquots were analysed in the bioassay. Results of corrected fluorescence signals are shown in Table 3. The data in Table 3 show that neither the glucocorticoid dexamethasone nor the estrogen 17␣-ethynylestradiol give a response in the bioassay, as the blank samples give about the same response as the corresponding dexamethasone and 17␣-ethynylestradiol spiked samples. The results in Table 3 also show that dexamethasone has no effect on the signals of the samples spiked with androgens, but 17␣-ethynylestradiol inhibits the signal of the samples spiked with androgens. Moreover, 17␣ethynylestradiol interferes with the screening result of the milk replacer spiked with testosterone, methyltestosteron and trenbolone, as these spiked samples now give a signal below the determined CC␣ and are thus falsely classified as compliant. This is not completely surprising as it was demonstrated before that 17␣-ethynylestradiol binds to the hAR without being able to show an agonistic response [17] and 17␣-ethynylestradiol has been shown to be able to displace radiolabeled dihydrotestosterone from the androgen receptor (AR), showing a relative binding affinity of about 0.8% when compared with testosterone [20]. Also other estrogenic compounds like 17-estradiol, diethylstilbestrol (DES),
230
The specificity of the yeast androgen bioassay was determined with two blank feed samples that were spiked with a high dose of dexamethasone or 17␣-ethynylestradiol, both at a level of 1000 ng g−1 . Interference was checked with the same feed samples by spiking them with the low dose of androgens in combination with the high dose of either dexamethasone or 17␣-ethynylestradiol. In grey the samples that not fulfil the criterion.
Table 3 – Specificity and interference checked with feed samples spiked with a high dose of dexamethasone or 17␣-ethynylestradiol
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
Fig. 1 – Responses of 17-testosterone (T-17) and 17␣-ethynylestradiol (EE2) in the yeast androgen bioassay and the responses of 17␣-ethynylestradiol by co-exposure with 70 and 1000 nM 17-testosterone (open symbols).
estrone, coumestrol and genistein have been shown to be able to displace radiolabeled R 1881 from the AR [21]. It is thus possible that 17␣-ethynylestradiol acts as an AR antagonist. Fig. 1 shows the response of testosterone and 17␣-ethynylestradiol in the yeast androgen assay. The figure shows that testosterone gives a full dose–response curve with an EC50 of 31 nM and that 17␣-ethynylestradiol does not give a response. To test the antiandrogenic properties of 17␣-ethynylestradiol, stock solutions of 17␣-ethynylestradiol were co-exposed with testosterone solutions giving about a half-maximal and a near maximal response, i.e. levels of 70 and 1000 nM testosterone, respectively. Fig. 1 shows that 17␣-ethynylestradiol is an AR antagonist and is able to completely inhibit the response caused by 70 and 1000 nM testosterone when its concurrent dose is high enough. Specificity and interference were not checked with urine samples as these characteristics are mainly determined by the compounds and the bioassay and not by the matrix. Moreover, as mentioned before, urine is a less complex matrix than feed.
3.3.
Stability of androgens in feed
The stability of the androgenic compounds in animal feed samples was determined with three animal feed samples, two milk replacers and one wet pulp feed sample. Again, the feed samples are tested as a worst case. Blank and spiked wet pulp feed samples were stored at −20 ◦ C and blank and spiked milk replacers were stored at room temperature in the dark. At certain times, 0, 7, 35 and 91 days, blank and spiked feed samples were taken, extracts were made and 200 L aliquots were analysed in the yeast androgen bioassay. Fluorescence signals at 24 h were corrected for the signals obtained at 0 h and the response of a reagent blank. Results are shown in Table 4. Blank samples were always screened as compliant and spiked samples were all screened as suspect. The data in Table 4 thus demonstrate that the feed samples can be stored at their specific conditions for up to 91 days, without disrupting the screening result. Urine stability tests at −20 ◦ C are ongoing, but not expected to deviate from the feed results, based on previous validation experiences with estrogens [12].
425 472 323 163 478 422 646 302 319 450 438 493 337 264 367 548 846 407 476 420
35 (7)a 7 (−26)a
3.4.
399 574 278 202 385
Days stored and (response of blank feed). a
Milk replacers were stored at room temperature in the dark and the wet pulp feed samples were stored at −20 ◦ C.
827 1311 731 378 1115 609 633 447 639 876 415 417 203 188 489 688 968 256 846 738 Testosterone 50 ng g−1 19-Nortestosterone 50 ng g−1 Methyltestosterone 50 ng g−1 Trenbolone 100 ng g−1 Methylboldenone 500 ng g−1
0
594 798 355 631 611
7
305 358 162 142 309
35
(−9)a (−54)a
1 Milk replacer
(−12)a Feed sample #
Table 4 – Stability of low amounts of androgens in animal feed
91
(−50)a
0
(−58)a
7
296 346 206 169 344
35
(−56)a (−85)a
4 Milk replacer
91 (−44)
0
(−4)a
14 Wet pulp feed
91 (−22)a
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
231
Application to real samples
Two hundred and thirty seven calf urine samples from a national control program were send to RIKILT and screened for androgenic activity using the validated yeast androgen bioassay procedure described here. 19 samples were screened as suspect and 218 were screened as compliant. These 19 suspect samples and 20 randomly picked compliant samples were analysed with GC/MS/MS as described before [22]. Results are shown in Table 5. The 20 negative screened samples (#1–20) contained no norethandrolone (NE), 17␣ethyl-5-estrane-3␣,17-diol (EED), 17-nortestosterone (NT17), 17␣-nortestosterone (NT-17␣) or 17␣-methyltestosterone (MT-17␣) and 16 of these 20 bioassay negative screened samples contained no 17-testosterone (T-17), while four contained less than 1 ng mL−1 . All samples (#1–39) contained 17␣-testosterone (T-17␣), but this compound is known as a very weak androgen receptor agonist and is almost inactive in the yeast androgen bioassay [17]. It was demonstrated before that NT-17 is a little more potent and that MT-17␣ is just as potent as 17testosterone [17,23,24]. Norethandrolone (NE), 17␣-ethyl-5estrane-3␣,17-diol (EED) and 17␣-nortestosterone (NT-17␣) were not tested before in the bioassay and their dose–response curves are shown in Fig. 2. Results were as expected, as based on its structure EED, a metabolite of the synthetic androgen norethandrolone (NE) [25], will be less active than NE and 17testosterone, because of its H-group at the 5-position and its OH-group at the 3␣-position [17]. Also 17␣-nortestosterone (NT-17␣) was expected to be less active as it was demonstrated before that changing the position of the OH-group at position 17 from 17 to 17␣ strongly decreases the potency of the compound [17,23]. These differences in potencies between 17-testosterone and 17-nortestosterone on one hand and 17␣-testosterone and 17␣-nortestosterone on the other, show that the 17-OH configuration is very important for androgenic activity [26]. NE is expected to be a little more potent than 17testosterone, because compared to -testosterone the absence of the methyl group at position 19 increases the potency (just as with NT-17), while the ethyl group at the 17␣-position hardly influences the potency (just as with the methyl group of MT-17␣) [17]. However, the EC50 value for NE is about the same as that of T-17. Moreover, we cannot explain the higher maximal response of NE, as all potent compounds tested so far reach the same maximal response as T-17, while less potent compounds reach either the same or a lower maximal value. Six of the 19 bioassay suspect screened samples (#21–39) contained 17-testosterone levels above 1 ng mL−1 (range 1.2–14.9 ng mL−1 , mean 4.5 ng mL−1 ). From the other 13 bioassay suspect screened samples there were five containing less than 1 ng 17-testosterone per mL and another one contained a small amount of 17␣-ethyl-5-estrane-3␣,17-diol (EED). When these 13 bioassay suspect screened samples are regarded as false positives, the percentage of false positives is 5%, which is acceptable for a qualitative screening method. However, it cannot be excluded that there are other metabolites of endogenous androgens, e.g. 5␣-androstane-3,17-diol, 5␣-androstane-3␣,17-diol, and 5␣-androstane-17-ol-3-one (5␣-DHT) and its isomers, that are not included in the GC/MS/MS analysis yet and are active in
232
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
Table 5 – Levels (ng mL−1 ) of natural and synthetic steroids and their metabolites determined in calf urine samples from a national control program using GC/MS/MS. Bioassay result
NE
EEDa
NT-17
NT-17␣
T-17
T-17␣a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N N
N N N N N N N <1 N <1 N <1 N N N <1 N N N N
1.2 <1 9.6 62 1.1 <1 4.8 26 3.7 100 21 72 2.9 1.2 2.2 114 32 7.3 8.9 1.7
N N N N N N N N N N N N N N N N N N N N
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
S S S S S S S S S S S S S S S S S S S
N N N N N N N N N N N N N N N N N N N
N <1 N N N N N N N N N N N N N N N N N
N N N N N N <1 N N N N N N N N N N N N
N N N N N N N N N N N N N N N N N N N
1.2 N N 3.8 N N 1.2 <1 N <1 2.4 N 14.9 3.4 N <1 <1 N <1
54.6 23 5.2 225 <1 26 85 13 29 60 135 2.4 160 168 3.0 4.0 80 20 22
N N N N N N N N N N N N N N N N N N N
Sample #
MT-17␣
N = negative, S = suspect, NE = norethandrolone (NE), EED = 17␣-ethyl-5-estrane-3␣,17-diol, NT-17 = 17-nortestosterone, NT-17␣ = 17␣nortestosterone, T-17 = 17-testosterone, T-17␣ = 17␣-testosterone and MT-17␣ = 17␣-methyltestosterone. a
Not validated for urine
the bioassay, that could account for these 13 suspect bioassay results [17,27,28].
4.
Fig. 2 – Responses of 17-testosterone (T-17), norethandrolone (NE), 17␣-nortestosterone (NT-17␣), and 17␣-ethyl-5-estrane-3␣,17-diol (EED) in the yeast androgen bioassay.
Conclusions
The data presented and the determined performance characteristics prove that the yeast androgen bioassay can detect low levels of androgens in calf urine and animal feed samples. All blank samples gave responses lower than the determined decision limits CC␣ and were classified as compliant. Thus there were no false suspect results. Signals of all spiked calf urine samples were higher than the determined decision limit CC␣, classified as suspect and thus fulfilled the CC criterion. However, the minimum required performance limit for 17-boldenone glucuronide in urine is 1 ng mL−1 , but because this compound is about
a n a l y t i c a c h i m i c a a c t a 6 3 7 ( 2 0 0 9 ) 225–234
seven times less potent than testosterone, it was spiked to urine at a level of 15 ng mL−1 . Although the sensitivity of this screening method is too low for compounds with a relative low androgenic potency compared to testosterone, such as 17-boldenone and 17␣-methylboldenone, the assay has several advantages. It is robust, easy, quick and inexpensive, it obeys to the principles of the EU ban, which prohibits all substances having hormonal action. Moreover, the assay is able to detect new designer steroids as already proven for the designer steroid tetrahydrogestrinone in human urine samples [29]. Regarding the feed samples, the spiked animal feed samples fulfilled the CC criterion as well. High levels of the glucocorticoid dexamethasone or the estrogen 17␣-ethynylestradiol did not give a response in the bioassay. Dexamethasone did not interfere with the screening result of spiked animal feed samples, but high levels of 17␣-ethynylestradiol can interfere and lead to false negative screening results. Here we show that 17␣-ethynylestradiol acts as an androgen receptor antagonist in a yeast androgen bioassay expressing yEGFP in response to androgens. However, illegal cocktails in general contain more androgens than estrogens and thus this inhibiting effect is not relevant in veterinary practice [30]. Moreover, the presence of compounds with an antagonistic mode of action can easily be determined by spiking the samples or extracts with a dose of an agonist that gives a half-maximal or near maximal response [31]. The observation that feed samples could be stored at their specific conditions for up to 91 days without influencing the screening result, showed that androgens in feed are stable. The stability study also demonstrates that the outcome of the assay, compliant/suspect, is reproducible and that the procedure is robust. As all the performance characteristics met the criteria that were put forward in EC Decision 2002/657 for validation of a qualitative screening method [19], the above described clean-up/yeast androgen bioassay procedure is proven to be valid for the determination of androgenic activity in calf urine and animal feed. Indeed the assay could successfully be applied to real urine samples from a control program and GC/MS/MS identification and quantification showed that negative screened samples contained no or low levels of androgens. As a result 218 of 237 samples were screened negative in the bioassay and can be regarded androgen free, demonstrating the applicability and strength of this yeast androgen bioassay as a screening test. The percentage of false positives was only 5%, but this percentage might drop if GC/MS/MS analysis would include more metabolites like 5␣-DHT and its isomers. The latter will be part of our future research efforts. Moreover, the GC/MS/MS results are in agreement with earlier findings, showing that 17␣testosterone is the most abundant natural hormone residue in urine of both male and female calves and is associated with an increased 17-testosterone level [22].
Acknowledgements This project was financially supported by the Dutch Ministry of Agriculture, Nature and Food Quality. Authors would like to thank Ron L.A.P. Hoogenboom for his adjustments to the manuscript.
233
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