Bioactivity Screening: The added value in veterinary …Dietary supplements Dietary supplements...

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

2000F

luor

esce

nce

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

Flu

ores

cenc

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

(Tho

usan

ds)

Flu

ores

cenc

e

dexamethasone

budesonide

betamethasone

hydrocortisone

prednisolone

corticosterone

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

Content

[1] Background



�Gynaecomastia




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


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


� 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



�Gynaecomastia



[3] Screening and identification using Bioassays

and chemical methods

Gynaecomastia



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)



Gynaecomastia

LC-TOFMS analysis



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



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



Dietary supplements

Negative supplement

-100

100

300

500

700

900

1100

1x 10x 100x 1000x 10000x

Flu

ores

cenc

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

Flu

ores

cenc

e

Supplement Supplement + spike after Supplement + spike before


chemical methods

Dietary supplements

Sample pretreatmentand clean-up

Gradient Liquid Chromatography

Bioassay plate

Collection plate

LC-TOFMSIdentification

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)

Flu

ores

cenc

e

ARE yEGFPARE yEGFP

Androgen yeast biosensor

ARE yEGFPARE yEGFPARE yEGFPARE yEGFP

Androgen yeast biosensor



Dietary supplements

LC-fractiones dietary supplement

-100

400

900

1400

1900

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)


chemical methods

Dietary supplements

1-testosteron

m /z80 100 120 140 160 180 200 220 240 260 280 300

%

0

100

Androstaned ione

m /z80 100 120 140 160 180 200 220 240 260 280 300

%

0

100

S19 1* ; F ractie 31+32

m /z100 120 140 160 180 200 220 240 260 280 300

%

0

100

17ß-testosteron

m /z80 100 120 140 160 180 200 220 240 260 280 300

%

0

100(a)

(d)(c)

(b)187.15

109.07131.09

145.10

159.12 205.16 289.22

253.20

188.15

109.06

97.06

123.08

253.19271.20

289.21

289.21

213.16187.14

187.14

105.07 131.08

145.10

159.11

188.15205.15 253.18

271.19

289.20

246.17289.21

271.20

253.19

213.16

197.13

171.11

161.13

143.04

97.07119.08

182.90231.17

Sample 17ß-Testosterone

1-Testosterone Androstanedione

The other one contained 4-androstene-3β,17β-diol and 5-androstane-3β,17β-diol

Rijk et al., ACA 637 (2009) 305-314


chemical methods

Dietary supplements

� Nicely validated bioassays for the detection of estrogens and androgens in calf urine and animal feed that can easily be introduced at any laboratory

� These assays have an added value compared to analytical screening alone

⇒However, bioassays can not operate on their own. Suspected samples need confirmation

⇒⇒⇒⇒ Bioassays and analytical methods are in this case complementary



Unknown compound in Herb

� I REA and RAA responses

� II MCF-7 gene profiling

� III Fractionations in the 96 and 384 well format and REA and RAA activity

� IV Identification of the responsible compounds




� REA Herb #1 response 151 (CCα animal feed 123)

� REA response Herb #2 203

� RAA response Herb #2 147 (CCα animal feed 44)

� Repeated over three times, every time Herb #1 REA positive and Herb #2 REA and RAA positive




Why MCF7 cells?

Connectivity Map (http://www.broadinstitute.org/cmap/ ):

Microarray results for 1309 compounds on MCF7 cells

2log ratio vs. appropriate control; ≥3 arrays ≥|0.8|: 1898 spots. (1.74 numerical), Treeview 1.0| 0.2

ChemCTRl ChemCTRl_E2 Yucca ChemCTRl ChemCTRl_E2 Yucca ChemCTRl ChemCTRl_E2 Yucca ChemCTRl Yuc_Old_MeOH

24h_StrippedFCS24h_StrippedFCS, StrX 40h_StrippedFCS, StrX FCS_PhRe d, StrX

Down by E2 and Herb #1:Most clearly Herb #1_old_MeOH.

Very little effect in FCS_PhRed.

Up by E2 and Herb #1 in stripped FCS but not in normal FCS.

E2 has more effect than Herb #1. For Herb #1: StrataX looks a little better than old MeOH extract but not very convincing.

Very little to no effect in FCS_PhRed.

Variable

Variable, some might be up by E2, in 24h stripped FCS.

Remarkably, no genes appear to be specifically affected by Herb #1.

Red: upregGreen: downreg

Herb #1Herb #1 Herb #1




UPLC high resolution fractionation system

using 384-well plates

15 µL per well (3 seconds)

REA results of an estrogen mixture

containing:

-17β-estradiol 50 ng/mL (E2)

-Ethinylestradiol 50 ng/mL (EE2)

-Estron 300 ng/mL (E1)

-DES 100 ng/mL

All in 2-3 wells !

Gerssen et al., (2012) in preparation




Gerssen et al., (2012) in preparation

UPLC high resolution fractionation system

using 384-well plates

REA and RAA results of an estrogen-androgen

mixture containing:

-17β-estradiol 50 ng/mL (E2)

-Ethinylestradiol 50 ng/mL (EE2)

-Estron 300 ng/mL (E1)

-DES 100 ng/mL

-Boldenone 100 µg/mL

-17β-testosterone 100 µg/mL (T)

-4-chloro-AD 100 µg/mL (CLAD)

-NanoTile-QToF-MS identification




-200

0

200

400

600

800

1000

1200

1400

a1 o2 a3 o4 a5 o6 a7 o8 a9 o10

a11

o12

a13

o14

a15

o16

a17

o18

a19

o20

a21

o22

a23

o24

RESPONS

WELL_code

BRONCHIX + TOEVOEGING LC399 RAA WELLPLATE 1

n6 Pongamone C ?

a5 E1

Compound X ?

Herb #2

Content

[1] Background



�Gynaecomastia



Overall Conclusion

� In the case of “present / not present” screening a Bioassay can be an alternative for existing chemical methods

� But, to obey 96/23/EC and 96/22/EC Bioassays and the conventional chemical methods have to be combined

� Bioassays clearly have an added value to help EU member states obey the metioned EC directives

Questions?

Bioactivity Screening: The added value in veterinary …Dietary supplements Dietary supplements...

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