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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 Presentation

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Page 1: Monitoring of endocrine disruption in different milieu matrices
Page 2: Monitoring of endocrine disruption in different milieu matrices

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

Page 3: Monitoring of endocrine disruption in different milieu matrices

IntroductionIntroduction

Page 4: Monitoring of endocrine disruption in different milieu matrices

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

Page 5: Monitoring of endocrine disruption in different milieu matrices

““Possible” effectsPossible” effects

CancersCancers

Obesity, diabetes Obesity, diabetes

Genital tract anomaliesGenital tract anomalies

Pubertal disturbancesPubertal disturbances

InfertilityInfertility

Page 6: Monitoring of endocrine disruption in different milieu matrices

Skakkebaek et al (2001), Hum Reprod 16: 972–978

The spectrum of Testicular dysgenesis syndromeThe spectrum of Testicular dysgenesis syndrome

Page 7: Monitoring of endocrine disruption in different milieu matrices

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

Page 8: Monitoring of endocrine disruption in different milieu matrices

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, ..)

Page 9: Monitoring of endocrine disruption in different milieu matrices

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 !!!

Page 10: Monitoring of endocrine disruption in different milieu matrices

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)

Page 11: Monitoring of endocrine disruption in different milieu matrices

The Yeast assay (YES)The Yeast assay (YES)

Page 12: Monitoring of endocrine disruption in different milieu matrices

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

Page 13: Monitoring of endocrine disruption in different milieu matrices

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

Page 14: Monitoring of endocrine disruption in different milieu matrices

Validation of the Yeast assayValidation of the Yeast assay

Page 15: Monitoring of endocrine disruption in different milieu matrices

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

****

*****

***

***

Page 16: Monitoring of endocrine disruption in different milieu matrices

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

Page 17: Monitoring of endocrine disruption in different milieu matrices

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

Page 18: Monitoring of endocrine disruption in different milieu matrices

The Estrogen Receptor Based The Estrogen Receptor Based Assay (ERBA)Assay (ERBA)

Page 19: Monitoring of endocrine disruption in different milieu matrices

Principle of the ERBAPrinciple of the ERBAcompetitive binding test competitive binding test

EE2

ERER ER

E2

Anti-GST GST-ER

Page 20: Monitoring of endocrine disruption in different milieu matrices

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)

Page 21: Monitoring of endocrine disruption in different milieu matrices

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

Page 22: Monitoring of endocrine disruption in different milieu matrices

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

Page 23: Monitoring of endocrine disruption in different milieu matrices

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

Page 24: Monitoring of endocrine disruption in different milieu matrices

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

Page 25: Monitoring of endocrine disruption in different milieu matrices

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

Page 26: Monitoring of endocrine disruption in different milieu matrices

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

Page 27: Monitoring of endocrine disruption in different milieu matrices

Toxicity-guided fractionationsToxicity-guided fractionations

Page 28: Monitoring of endocrine disruption in different milieu matrices

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

Page 29: Monitoring of endocrine disruption in different milieu matrices

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

Page 30: Monitoring of endocrine disruption in different milieu matrices

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

Page 31: Monitoring of endocrine disruption in different milieu matrices

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

Page 32: Monitoring of endocrine disruption in different milieu matrices

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

Page 33: Monitoring of endocrine disruption in different milieu matrices

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)

Page 34: Monitoring of endocrine disruption in different milieu matrices

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?

Page 35: Monitoring of endocrine disruption in different milieu matrices

Studies on Human serumStudies on Human serum

Page 36: Monitoring of endocrine disruption in different milieu matrices

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)

Page 37: Monitoring of endocrine disruption in different milieu matrices

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

Page 38: Monitoring of endocrine disruption in different milieu matrices

The adolescents’ studyThe adolescents’ study

550 adolescent males550 adolescent males

Hormones (classical methods)Hormones (classical methods)

Total estrogen load (YES)Total estrogen load (YES)

Page 39: Monitoring of endocrine disruption in different milieu matrices

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

Page 40: Monitoring of endocrine disruption in different milieu matrices

Prediction of mixture effectsPrediction of mixture effects

Page 41: Monitoring of endocrine disruption in different milieu matrices

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)

Page 42: Monitoring of endocrine disruption in different milieu matrices

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

Page 43: Monitoring of endocrine disruption in different milieu matrices

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

Page 44: Monitoring of endocrine disruption in different milieu matrices

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

Page 45: Monitoring of endocrine disruption in different milieu matrices

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