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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
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
[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) 85-92Bovee 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
[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) 2003-2009Becue et al., ABC 339 (2011) 1031-1039Divari 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
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
[3] Screening and identification using Bioassays and
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
[3] Screening and identification using Bioassays
and chemical methods
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)
[3] Screening and identification using Bioassays and
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
[3] Screening and identification using Bioassays and
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
[3] Screening and identification using Bioassays
and chemical methods
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
[3] Screening and identification using Bioassays
and chemical methods
Unknown compound in Herb
� 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
[3] Screening and identification using Bioassays
and chemical methods
Unknown compound in Herb
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
[3] Screening and identification using Bioassays
and chemical methods
Unknown compound in Herb
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
[3] Screening and identification using Bioassays
and chemical methods
Unknown compound in Herb
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
[3] Screening and identification using Bioassays
and chemical methods
Unknown compound in Herb
[3] Screening and identification using Bioassays
and chemical methods
Unknown compound in Herb
-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
[2] Relevance of using Bioassays
[3] Screening and identification using Bioassays and chemical methods
�Gynaecomastia
� Dietary supplements
[4] Overall conclusion
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?