monitoring of endocrine disruption in different milieu matrices
DESCRIPTION
Monitoring of endocrine disruption in different milieu matrices. W. Dhooge , F.H. Comhaire, A. Mahmoud, F. Eertmans, J.M. Kaufman Endocrinology/Andrology, University Hospital Ghent, Belgium FlandersBio, Belgium. Introduction. A few facts. - PowerPoint PPT PresentationTRANSCRIPT
Monitoring of endocrine Monitoring of endocrine disruption in different milieu disruption in different milieu
matricesmatrices
W. DhoogeW. Dhooge, F.H. Comhaire, A. Mahmoud, F. , F.H. Comhaire, A. Mahmoud, F. Eertmans, J.M. KaufmanEertmans, J.M. Kaufman
Endocrinology/Andrology, University Hospital Endocrinology/Andrology, University Hospital Ghent, BelgiumGhent, Belgium
FlandersBio, BelgiumFlandersBio, Belgium
IntroductionIntroduction
A few factsA few facts
Man made chemicals are found everywhere on the Man made chemicals are found everywhere on the planetplanet
Many of these xeno-biotics may interfere with the Many of these xeno-biotics may interfere with the endocrine systemendocrine system
Mainly with (anti-)estrogenic actionMainly with (anti-)estrogenic action
These include PCBs, pesticides, plastics, heavy These include PCBs, pesticides, plastics, heavy metalsmetals
““Possible” effectsPossible” effects
CancersCancers
Obesity, diabetes Obesity, diabetes
Genital tract anomaliesGenital tract anomalies
Pubertal disturbancesPubertal disturbances
InfertilityInfertility
Skakkebaek et al (2001), Hum Reprod 16: 972–978
The spectrum of Testicular dysgenesis syndromeThe spectrum of Testicular dysgenesis syndrome
Problems of analytical testingProblems of analytical testing
Number of chemicals is growingNumber of chemicals is growing
It is costly & difficult to test each separatelyIt is costly & difficult to test each separately
Frequently, no standard method is availableFrequently, no standard method is available
Analytical tests do not detect mixture effectsAnalytical tests do not detect mixture effects
Biological testsBiological tests
Receptor-based assaysReceptor-based assays
Sensitive (signal amplification), detect mixture Sensitive (signal amplification), detect mixture effectseffects
Receptor activation Receptor activation Signal (color change etc) Signal (color change etc)
Cells expressing receptor (yeast, liver, ..)Cells expressing receptor (yeast, liver, ..)
Biological testsBiological tests
Cell-based assays Cell-based assays possible false negative possible false negative results (cell toxicity) in heavily polluted results (cell toxicity) in heavily polluted environmental samplesenvironmental samples
Receptor test without a cell !!!Receptor test without a cell !!!
ObjectivesObjectives
Develop screening tools: Develop screening tools: affordable, affordable, sensitive, rapid sensitive, rapid biologically relevantbiologically relevant
Allow screeningAllow screening:: environmental samples environmental samples Humans: exogenous, endogenous substancesHumans: exogenous, endogenous substances Low doses of highly active substances (natural estrogens)Low doses of highly active substances (natural estrogens)
The Yeast assay (YES)The Yeast assay (YES)
Estrogen inducible expression system in yeastEstrogen inducible expression system in yeast
Adapted from Routledge Adapted from Routledge et al.et al., 199, 19966
Nucleus
ER
Hsp Hsp
HspHsp
ERE
Lac-ZLuc
-Galactosidase
Absorbance read at 540 nm
log(Conc.) (g/L)
Abs
orba
nce
-13-13-12-12-11-11-10-10 -9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00
1.01.0
1.51.5
2.02.0
2.52.5 Estradiol Methoxychlor Benzylbutylphthalate Lindane Endosulfan Blanc
Yeast assay as developed by Routledge and Yeast assay as developed by Routledge and SumpterSumpter (1996) (1996)
11
22
33 Estradiol Bisphenol ADicofol Paraquat Chloropicrin Blanc
log(Conc.) (g/L)-13-13-12-12-11-11-10-10 -9-9 -8-8 -7-7 -6-6 -5-5 -4-4 -3-3 -2-2 -1-1 00
Validation of the Yeast assayValidation of the Yeast assay
Detection limit ethanol / DMSO
*Significantly different from previous measurement, p<0.005 ** Significantly different from day 2 measurement, p<0.01
Days of incubation
Dimethylsulfoxide
Ethanol
***
***
*
** **
**
**
1000
100
10
1
0.1
17-
estr
adio
l (ng
/l)
2 5 7 9 15
Dimethyl sulfoxide
Ethanol
****
*****
***
***
RingtestRingtest
• The YES was performed according to Routledge and Sumpter (1996). Test plates were incubated for 10 days and absorbances (540 / 620 nm) were measured at regular intervals. 17β-estradiol (E2) was used as a positive control.
• Relative Potency (RP) = EC50 (E2) / EC50 (test compound).
• Relative Induction Efficiency (RIE) = Amax (test compound) / Amax (E2), with Amax = maximal absorbance.
Variability (expressed as coefficient of variation)Intra-lab: 0.52 % - 8.2 % Intra-lab: 1.0 % - 7.3 %Inter-lab: 0.84 % - 7.9 % Inter-lab: 0.6 % - 17 %
(except for DDE & lindane) (except for endosulfan)
*not tested in lab 1 *not detected in lab 3
Solvent: ethanol
RP
Lab 1
Lab 2
Lab 3
* * *
Solvent: DMSO
RP
Lab 1
Lab 2
Lab 3
**
Relative potencyRelative potency
Problems with the Yeast assay • Toxicity (also with other tests using living organisms)• Cell wall permeability• Time consuming
Development of a receptor-based test system
Based on competitive binding of compounds to the ER alpha Receptor production: truncated human estrogen receptor coupled
to glutathione sulphotransferase (GST) for purification Large scale production of the protein
Receptor test: rationaleReceptor test: rationale
The Estrogen Receptor Based The Estrogen Receptor Based Assay (ERBA)Assay (ERBA)
Principle of the ERBAPrinciple of the ERBAcompetitive binding test competitive binding test
EE2
ERER ER
E2
Anti-GST GST-ER
Competition of Competition of (xeno-) estrogens with (xeno-) estrogens with 1717-Estradiol -Estradiol in ERBAin ERBA
%%
-12-12 -10-10 -8-8 -6-6 -4-4 -2-2 00
% b
ind
ing
% b
ind
ing
-20-20
00
2020
4040
6060
8080
100100
120120
140140
Estradiol
Coumestrol
Genisteine
Bisfenol-A
Bifenyl
Cortisol
log conc (E2 equiv M)log conc (E2 equiv M)
1717-Estradiol curve -Estradiol curve forfor ERBA ERBA
Estradiol curveEstradiol curve
log conc (M)log conc (M)
-13-13 -12-12 -11-11 -10-10 -9-9 -8-8 -7-7
cpm
cpm
00
2020
4040
6060
8080
100100
120120
140140
IC50
Relative induction efficiencies (RIE) of tested Relative induction efficiencies (RIE) of tested compounds in the ERBA and YES (n>3 compounds in the ERBA and YES (n>3
independent experiments)independent experiments)
EC50 (GM)EC50 (GM)
YESYESIC50 (GM)IC50 (GM)
ERBAERBARIE (AM)RIE (AM)
YESYESRIE (AM)RIE (AM)
ERBAERBA1717-estradiol-estradiol 2.30E-102.30E-10 6.96E-106.96E-10 100100 100100
Bisphenol-ABisphenol-A 2.83E-062.83E-06 2.88E-052.88E-05 112.6112.6 93.993.9
4,4’-Biphenol4,4’-Biphenol 9.87E-069.87E-06 8.88E-058.88E-05 106.0106.0 87.587.5
4-n-Octylphenol4-n-Octylphenol 2.46E-062.46E-06 3.32E-053.32E-05 75.075.0 89.789.7
p-Nonylphenolp-Nonylphenol 2.21E-062.21E-06 2.79E-052.79E-05 102.7102.7 101.5101.5
LindaneLindane 1.10E-041.10E-04 5.26E-055.26E-05 92.292.2 32.032.0
ICI 182.780ICI 182.780 5.98E-065.98E-06 1.86E-081.86E-08 98.398.3 101.1101.1
Methoxy chloreMethoxy chlore 1.80E-051.80E-05 NANA 107.9107.9
EC50: 50% effect concentration; RIE: relative induction efficiency; GM: Geometric mean, AM. Arethmatic meanEC50: 50% effect concentration; RIE: relative induction efficiency; GM: Geometric mean, AM. Arethmatic mean
Similar results
Negative tests are negative in all systems
Positives are positive including:
Anti-estrogens
Methoxychlor and permethrin (not shown)
Absolute sensitivity (EC50 values) are 3-10x lower than YES
Possible toxic effects in cell systemsPossible toxic effects in cell systems
Substances with low binding affinity in the YES & ERBA yield similar results
Receptor Test vs YESReceptor Test vs YES
Environmental Samples in Different Test SystemsEnvironmental Samples in Different Test Systems
CodeCode SampleSample ERBAERBA YESYES MVLNMVLN01J 143 S water 9.32 3.47 3.48
01J 142 S water 0.97 0.46 0.67
01J 140 S water 1.51 13.72 14.99
01J 141 S water 1.57 2.42 1.9
01J 145 S water 6.46 6.21 6.72
01J 144 S water out of range 40.61 34.7
02B015-2 Industry 93.64 0.33 Toxic
02C045 S water 0.93 1.46 1.42
02C169 S water 2.14 0.63 0.19
02C172 S water 10.78 16.21 5.28
02B011-2 Industry 165.20 3.38 Toxic
02C046 S water 1.45 1.76 0.82
02C171 S water 107.70 69.73 28.51
02B011-3 Industry 0.50 <LOD <LOD
02C044 S water 11.56 2.12 3.18
02B015-3 Industry 0.83 <LOD <LOD
Competition of environmental Competition of environmental sample with 1sample with 177--Estradiol Estradiol forfor TER-GST TER-GST
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
01J 1
43
01J 1
42
01J 1
40
01J 1
41
01J 1
45
01J 1
44
02B01
5-2
02C04
5
02C16
9
02C17
2
02B01
1-2
02C04
6
02C17
1
02B01
1-3
02C04
4
02B01
5-3
ERBA
YES
MVLN
Environmental samples in the ERBA testEnvironmental samples in the ERBA test: : conclusionsconclusions
ERBA-test can be used for pure substances AND environmental samples
Test results are mostly in the same order of magnitude as the YES and MVLN
For some samples discripancies may be due to: Cell toxicity Mixture of estrogens & anti-estrogens
Non-specific binding in ERBA: less likely in view of shape of binding curves
Toxicity-guided fractionationsToxicity-guided fractionations
Environmental sampleEnvironmental sample
filtration
dissolved phase
Identification of estrogensIdentification of estrogensin active fractions viain active fractions via
LC-MS/MSLC-MS/MS
Particulate material
YES
YES
Fractionation procedure protocolFractionation procedure protocol
Investigate relationship between concentration of compounds and
estrogen activity in different fractions
SPE Extract: 250 µl
Fractionation of environmental Fractionation of environmental samplessamples
4 56
7
8
910
11
1213
14
15
16
1718
2627
0
20
40
60
80
100
120
45
6
7
8
910
11
12
13
14
15
16
1718
19
2021
22
23
24
252627
2829
30
Fraction number
6
1 5 10 15 20 25 30
Fraction number
A B
% e
str
og
en
ac
tiv
ity
re
lati
ve
to
ma
x E
2 s
tan
da
rd c
urv
e
1 5 10 15 20 25 300
20
40
60
80
100
120
1
2 3
4
5
6
7
8
910
2627282930
0
20
40
60
80
100
1 5 10 15 20 25 30
A
% e
stro
gen
acti
vity
rel
ativ
e to
max
E2
stan
dard
cur
ve
910
2627282930
5
Fraction number
LC-MS/MS LC-MS/MS
fr4-6: Polar fraction?fr4-6: Polar fraction?
fr 7&8: Methyl, ethyl & propylparabenfr 7&8: Methyl, ethyl & propylparaben
fr9&10: Estron, E2, EE2, Propylparabenfr9&10: Estron, E2, EE2, Propylparaben
fr16-19: 4-n-octylphenol, 4-n-fr16-19: 4-n-octylphenol, 4-n-nonylphenol, 4-tertiair octylphenolnonylphenol, 4-tertiair octylphenol
fr 22-29: apolar substances ?fr 22-29: apolar substances ?
Fractionation of environmental Fractionation of environmental samplessamples
Correlation between estrogen activity in Correlation between estrogen activity in fractions & chemical concentrationfractions & chemical concentration
y=0.32+0.81x (r ²: 0.61) y= 0.06+1.61x (r ²:0.52)
A B
conc octyl phenol (as pg E2/L)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
pg
E2
/L in
fr
17
&1
8
conc methyl parabene (ng/L)
1 10 100
pg
E2
/L in
fr
7&
8
10-1
100
101
102
103
104
105
10-1
100
101
102
103
104
105
Fractionation of Environmental Fractionation of Environmental samplessamples
4 56
7
8
910
11
1213
14
15
16
1718
2627
0
20
40
60
80
100
120
6
1 5 10 15 20 25 30
Fraction number
A
% e
stro
gen
act
ivit
y re
lati
ve t
o m
ax E
2 st
and
ard
cu
rve Relation between Relation between estrogen activity in estrogen activity in fraction 7&8 & fr fraction 7&8 & fr 17&18 with 17&18 with substances present substances present in these fractionsin these fractions
Results YES Results YES correlates with correlates with methyl parabene & methyl parabene & octyl phenoloctyl phenol
Toxicity-guided fractionationsToxicity-guided fractionations
Industrial samples: alkyl phenols up to 54 Industrial samples: alkyl phenols up to 54 % of the total estrogenic activity% of the total estrogenic activity
This is performed on 250µl of the extractThis is performed on 250µl of the extract
Parabens & alkyl phenols related to surface Parabens & alkyl phenols related to surface water estrogenic activity (has never been water estrogenic activity (has never been demonstrated before)demonstrated before)
Summary fractionationSummary fractionation
The developed methods are sensitive, reproducible The developed methods are sensitive, reproducible & effectively detect the cause of estrogen activity & effectively detect the cause of estrogen activity (EA).(EA).
The most active fractions: fr9&10: natural & The most active fractions: fr9&10: natural & synthetic estrogens. No quantitative relationsynthetic estrogens. No quantitative relation
InterestingInteresting: Significant relation between estrogen : Significant relation between estrogen activity in fr 7&8 & methyl paraben; & fr 17&18 & activity in fr 7&8 & methyl paraben; & fr 17&18 & octyl phenoloctyl phenol
The concentrations measured explain 50% of the The concentrations measured explain 50% of the EA maximumEA maximum
Further research: other substances? Matrix effects?Further research: other substances? Matrix effects?
Studies on Human serumStudies on Human serum
The aromatase studyThe aromatase study
Placebo-controlled studyPlacebo-controlled study Aromatase inhibitor (letrozole)Aromatase inhibitor (letrozole) Testosterone Testosterone estradiol estradiol Hormones (classical methods)Hormones (classical methods) Total estrogen load (YES)Total estrogen load (YES)
The aromatase studyThe aromatase study
Mean ng EMean ng E2 2
equiv equiv /LStdev/LStdevStdevStdev
E2 load beforeE2 load before 48.148.1 29.429.4
E2 load afterE2 load after 6.76.7 3.63.6
Difference %Difference % 41.441.4 29.229.2
Percentage Percentage declinedecline
83.0%83.0% 10.3%10.3%
Correlation with Correlation with decline in E2decline in E2
0.58 (p=0.003)0.58 (p=0.003)
Detection limit Detection limit (E2 equivalent) (E2 equivalent)
55
The adolescents’ studyThe adolescents’ study
550 adolescent males550 adolescent males
Hormones (classical methods)Hormones (classical methods)
Total estrogen load (YES)Total estrogen load (YES)
The adolescents’ studyThe adolescents’ study
AgeAge WeightWeight BMIBMI E2 E2 (pg/ml)(pg/ml)
frE2 frE2 (pg/ml)(pg/ml)
logYES-logYES-correctedcorrected
0.21a0.21a 0.12 b0.12 b 0.05 ns0.05 ns 0.40 a0.40 a 0.39 a0.39 a
AgeAge 0.24 a0.24 a 0.06 ns0.06 ns 0.42 a0.42 a 0.41a0.41a
WeightWeight 0.84 a0.84 a 0.45 a0.45 a 0.53 a0.53 a
HeightHeight 0.21 a0.21 a 0.48 a0.48 a 0.51 a0.51 a
BMIBMI -- 0.27 a0.27 a 0.37a0.37a
a: p<0.00001, b: p<0.01
Prediction of mixture effectsPrediction of mixture effects
Prediction of mixture effectsPrediction of mixture effects
Data from actual combination experiments Data from actual combination experiments were compared to theoretical curves were compared to theoretical curves assuming additive combination effects assuming additive combination effects (1+1=2)(1+1=2)
Deviation from additivity suggests Deviation from additivity suggests interaction between compounds interaction between compounds
(1+1=3, synergism)(1+1=3, synergism)
0
20
40
60
80
100
120
Estradiol
o,p-DDT
Summ
ation
Mixture
Arbitrary unitsArbitrary units
00
0.50.5
11
1.51.5
22
2.52.5
0.010.01 0.10.1 11 1010
Observed effect 0.2 mM
Expected effect0.2 mM
Effect 0.1 mM
Conc. (mM)
Effect summationEffect summation Only applicable with linear dose response Only applicable with linear dose response
relationshipsrelationshipsCell countCell count
00.10.20.30.40.50.60.70.80.9
1
E2 1 2 3 4 5 6 7 8 Mix
Background
19 nM 39 nM72 nM
32 nM16 nM
98 nM72 nM
52 nM
0.3 pM
After Kortenkamp et al., (1999)
MCF7 (Br ca) cell growth with a MCF7 (Br ca) cell growth with a mixture of low level chemicalsmixture of low level chemicals
For p,p’-DDE/E2 (41,7 pM) & lindane/E2 mixtures observed effect is higher than predicted
But for bisphenol A
Observed response after 3 days of incubation compared to the Observed response after 3 days of incubation compared to the predicted response predicted response
Y A
xis 2
0.0
0.4
0.5
0.6
0.7
0.8
0.9
Cor
rect
ed A
bsor
banc
e U
nits
bisphenol - A
p,p’ - DDElindane
no E2no E2no E2
+ E2
+ E2
+ E2RA
RA
RA
CA
Cor
rect
ed A
bsor
banc
e U
nits
Molar Concentration
10 -7 10 -6 10 -5 10 -4 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4
0.0
0.3
0.6
0.9
1.2
0.0
0.4
0.5
0.6
0.7
0.8
0.9
Cor
rect
ed A
bsor
banc
e U
nits
bisphenol - A
p,p’ - DDElindane
no E2no E2no E2
+ E2
+ E2
+ E2RA
RA
RA
CA
Cor
rect
ed A
bsor
banc
e U
nits
Special thanks to:Special thanks to:
The team of Milieu en Gezondheid (UGent, UIA, The team of Milieu en Gezondheid (UGent, UIA, VUB, KUL, VITO, ....)VUB, KUL, VITO, ....)
A. Bossier, W. Verstraete, LabMeTA. Bossier, W. Verstraete, LabMeT S. Stuyvaert, Nick Hendryckx, labo Andrology UZ S. Stuyvaert, Nick Hendryckx, labo Andrology UZ
GentGent Hormonology lab UZ GentHormonology lab UZ Gent T. Benijts/ Prof. W. Lambert: Labo Toxicologie FFW T. Benijts/ Prof. W. Lambert: Labo Toxicologie FFW
UgentUgent A. De Winter M. Van Oost VMM GentA. De Winter M. Van Oost VMM Gent