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  • Bioactivity Screening:

    The added value in veterinary control

    11/09/2012, J.R. Helsdingen & T.F.H. Bovee

  • Content

    [1] Background

    [2] Relevance of using Bioassays

    [3] Screening and identification using Bioassays and chemical methods

    �Gynaecomastia

    � Dietary supplements

    [4] Overall conclusion

  • [1] Background

    Steroids and other hormone active substances

    � Natural steroids and their metabolites and conjugates, e.g. OH-metabolites and glucuronidated or sulphated conjugates

    � Synthetic steroids, e.g. EE2, mestranol, hexestrol, boldenone, trenbolone, dexamethasone

    � Hormoonesters, e.g. manmade esters from both natural and synthetic steroids

    � Phytoestrogens (isoflavonoids)

    � Chemicals, e.g. BPA, PCBs, pesticides, surfactants, plastics etc.

    � Etc.

  • [1] Background

    EU regulations I

    � Directive 96/22/EC: Prohibits all substances having hormonal action

    � Regulations EC 178/2002 and EC 882/2004: oblige the member states to identify emerging risks and use validated and accredited methods for control analysis

  • [1] Background

    EU regulations II

    � Directive 96/23/EC: banns the use of Group A substances

    ● Stilbenes, derivatives, salts and esters

    ● Antithyreogene compounds

    ● Steroids

    ● Resorcyclic Acid Lactones (including zeranol)

    ● ß-agonists

    ● Others, as mentioned in the Annex of Regulation EC 37/2010

  • [1] Background

    How to obey to all these laws ?

    � The only way is bioactivity screening combined with chemical analytical confirmation and identification using validated and accredited methods for both

    � Or perhaps we should get rid of the laws. Would that be safe?

  • [1] Background

    Transcriptional Activation (TA) bioassays (yeast or mammalian cell based)

    � Detect all compounds (structures) that are able to activate the receptor, e.g. the estrogen, androgen, progesterone,

    glucocorticoid or thyroid receptor. As the main mode of action

    of all active hormones is by activating their receptor, they fulfil

    Directive 96/22/EC that prohibits all substances having

    hormonal action

    � Moreover, they are:

    ● Sensitive and specific

    ● Quick, simple and robust

    ● Applicable to urine, feed and preparations

  • [1] Background

    yeast estrogen bioassay (REA)

    ERE yEGFP

    17β- estradiol

    ER

    ER

  • [1] Background

    Dosis – response curves REA

    Bovee et al., Gene 325 (2004) 187-200

    Bovee et al., JSBMB 91 (2004) 99-109

    0.001 0.01 0.1 1 10 100 1000 10000 100000 1000000

    Concentration [ nM]

    200

    500

    800

    1100

    1400

    1700

    2000 F

    lu or

    es ce

    nc e

    E2b

    E2-benz oate

    zeara lenone

    genistein

    E1

    DES

    EE2

    estriol

  • [1] Background

    Dosis – response RAA (androgen) and RCA (corticoid)

    0.1 1 10 100 1000 10000 100000 1000000

    Concentration [nM]

    -100

    100

    300

    500

    700

    900

    1100

    F lu

    or es

    ce nc

    e

    17ß-T DHT Prog Dex 17ß-E2 Bold

    0.1 1 10 100 1000 10000

    Concentration [æM]

    -1

    0

    1

    2

    3

    4

    5

    6

    7

    8

    9

    10

    11

    12

    (T ho

    us an

    ds )

    F lu

    or es

    ce nc

    e

    dexamethasone

    budesonide

    betamethasone

    hydrocortisone

    prednisolone

    corticosterone

    Bovee et al., ABC 389 (2007) 1549-1558 Bovee et al., ABC 401 (2011) 873-882

  • Content

    [1] Background

    [2] Relevance of using Bioassays

    [3] Screening and identification using Bioassays and chemical methods

    �Gynaecomastia

    � Dietary supplements

    [4] Overall conclusion

  • [2] Relevance of using Bioassays

    � SERMs and SARMs show their specific responses in these yeast hormone bioassays too

    � Both the yeast estrogen and androgen bioassay were fully validated for both the screening of feed and calf urine samples (according to Directive 2002/657/EC and accredited ISO 17025)

    � The yeast estrogen bioassay performed well in an inter- laboratory ring test with calf urine samples

    � Was shown a cheap alternative for real practise: estrogen bioassay screening calf urine samples vs GC-MS analysis

    Bovee et al., JSBMB 118 (2010) 85-92 Bovee et al., ACA 529 (2005) 57-64 Bovee et al., FAC 23 (2006) 556-568 Bovee et al., ACA 637 (2009) 225-234 Bovee et al., ACA 637 (2009) 265-272 Nielen et al., FAC 23 (2006) 1123-1131

  • [2] Relevance of using Bioassays

    � Was validated by Waternet/Waterproef Laboratorium in The Netherlands for screening estrogens in water samples

    � The yeast estrogen and androgen bioassay are successfully used at Ghent University for the screening of food supplements (including bio-directed identification)

    � Both bioassays validated and used for calf urine and feed at the Polish RIKILT analogue Institute: National Veterinary Research Institute, Pulaway, Poland

    � Both bioassays are being used for the screening of calf urine samples at the University of Veterinary Medicine, Turin, Italy

    Nguyen et al., TiV 25 (2011) 2003-2009 Becue et al., ABC 339 (2011) 1031-1039 Divari et al., FAC 27 (2010) 1123-1131

  • [2] Relevance of using Bioassays

    � RIKILT: Showed an added value by the identification of anabolic steroids and derivatives in supplements, using bioassay-guided fractionation, UHPLC/TOFMS analysis and accurate mass database searching

    Peters et al., ACA 664 (2010) 77-88

  • Content

    [1] Background

    [2] Relevance of using Bioassays

    [3] Screening and identification using Bioassays and chemical methods

    �Gynaecomastia

    � Dietary supplements

    [4] Overall conclusion

  • [3] Screening and identification using Bioassays

    and chemical methods

    Gynaecomastia

  • [3] Screening and identification using Bioassays

    and chemical methods

    Gynaecomastia

    �Batch #1 of the Prostasol capsules contained about 0.3 mg 17β-E2 equivalents per gram

    �Batch #2 of the Prostasol capsules contained about 3.6 mg 17β-E2 equivalents per gram

    �Batch #3 were recently released Prostasol tablets and contained no estrogenic compounds (below 5 ng 17β-E2 equivalents per gram)

  • [3] Screening and identification using Bioassays

    and chemical methods

    Gynaecomastia

    LC-TOFMS analysis

  • [3] Screening and identification using Bioassays

    and chemical methods

    Gynaecomastia

    � Male 60 years with prostate problems (elevated PSA level)

    � Supplement positive in Bioassay screening

    � High signals revealing high estrogenic activity

    � Confirmation and identification using LC/TOF-MS and NMR

    �Diethylstilbestrol (DES) Batch1: 0.9 mg DES/G and Batch 2: 4.1 mg DES/G

    Combined methods of bioassay and chemical methods revealed

    that the effects of Gynaecomastia where developed by

    exposure to DES by the intake of a Chinese herbal supplement

  • [3] Screening and identification using Bioassays

    and chemical methods

    Dietary supplements � Dietary supplements � analysed by LC-MS/MS for 49

    steroids

    ● 18 supplements - 11 positive and 7 negative

    Van Poucke et al., ACA 586 (2007) 35-42

    2 supplements show androgenic activity in the

    yeast androgen bioassay

  • [3] Screening and identification using Bioassays

    and chemical methods

    Dietary supplements

    Negative supplement

    -100

    100

    300

    500

    700

    900

    1100

    1x 10x 100x 1000x 10000x

    F lu

    or es

    ce nc

    e

    supplement supplement + spike after supplement + spike before

  • [3] Screening and identification using Bioassays and

    chemical methods

    Dietary supplements

    Positive supplement

    -100

    100

    300

    500

    700

    900

    1100

    1x 10x 100x 1000x 10000x

    F lu

    or es

    ce nc

    e

    Supplement Supplement + spike after Supplement + spike before

  • [3] Screening and identification using Bioassays and

    chemical methods

    Dietary supplements

    Sample pretreatment and clean-up

    Gradient Liquid Chromatography

    Bioassay plate

    Collection plate

    LC-TOFMS Identification

    Flow split

    Bioactivity screening

    -100

    100

    300

    500

    700

    900

    0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

    Retention time(min)

    F lu

    or es

    ce nc

    e

    ARE yEGFPARE yEGFP

    Androgen yeast biosensor

    ARE yEGFPARE yEGFPARE yEGFPARE

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