Monitoring of endocrine disruption in different milieu matrices

1
(No Transcript)
2
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

3
Introduction
4
A few facts

• Man made chemicals are found everywhere on the
  planet
• Many of these xeno-biotics may interfere with the
  endocrine system
• Mainly with (anti-)estrogenic action
• These include PCBs, pesticides, plastics, heavy
  metals

5
Possible effects

• Cancers
• Obesity, diabetes
• Genital tract anomalies
• Pubertal disturbances
• Infertility

6
The spectrum of Testicular dysgenesis syndrome
Skakkebaek et al (2001), Hum Reprod 16 972978
7
Problems of analytical testing

• Number of chemicals is growing
• It is costly difficult to test each separately
• Frequently, no standard method is available
• Analytical tests do not detect mixture effects

8
Biological tests

• Receptor-based assays
• Sensitive (signal amplification), detect mixture
  effects
• Receptor activation ? Signal (color change etc)
• Cells expressing receptor (yeast, liver, ..)

9
Biological tests

• Cell-based assays ? possible false negative
  results (cell toxicity) in heavily polluted
  environmental samples
• Receptor test without a cell !!!

10
Objectives

• Develop screening tools
• affordable,
• sensitive, rapid
• biologically relevant
• Allow screening
• environmental samples
• Humans exogenous, endogenous substances
• Low doses of highly active substances (natural
  estrogens)

11
The Yeast assay (YES)
12
Estrogen inducible expression system in yeast
13
Yeast assay as developed by Routledge and Sumpter
(1996)
14
Validation of the Yeast assay
15
Detection limit ethanol / DMSO
Ethanol
Ethanol
Dimethylsulfoxide
Dimethyl sulfoxide


17b-estradiol (ng/l)










Days of incubation
Significantly different from previous
measurement, plt0.005 Significantly different
from day 2 measurement, plt0.01
16
Ringtest

• 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. 17ß-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)
17
Receptor test rationale

• 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

18
The Estrogen Receptor Based Assay (ERBA)
19
Principle of the ERBA
competitive binding test
Anti-GST
GST-ER
20
Competition of (xeno-) estrogens with
17b-Estradiol in ERBA
21
17b-Estradiol curve for ERBA
22
Relative induction efficiencies (RIE) of tested
compounds in the ERBA and YES (ngt3 independent
experiments)
EC50 (GM) YES IC50 (GM) ERBA RIE (AM) YES RIE (AM) ERBA
17b-estradiol 2.30E-10 6.96E-10 100 100
Bisphenol-A 2.83E-06 2.88E-05 112.6 93.9
4,4-Biphenol 9.87E-06 8.88E-05 106.0 87.5
4-n-Octylphenol 2.46E-06 3.32E-05 75.0 89.7
p-Nonylphenol 2.21E-06 2.79E-05 102.7 101.5
Lindane 1.10E-04 5.26E-05 92.2 32.0
ICI 182.780 5.98E-06 1.86E-08 98.3 101.1
Methoxy chlore 1.80E-05 NA 107.9
EC50 50 effect concentration RIE relative
induction efficiency GM Geometric mean, AM.
Arethmatic mean
23
Receptor Test vs YES

• 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 systems
• Substances with low binding affinity in the YES
  ERBA yield similar results

24
Environmental Samples in Different Test Systems
25
Competition of environmental sample with
17b-Estradiol for TER-GST
180.00
ERBA
160.00
YES
140.00
120.00
MVLN
100.00
80.00
60.00
40.00
20.00
0.00
01J 143
01J 142
01J 140
01J 141
01J 145
01J 144
02C045
02C169
02C172
02C046
02C171
02C044
02B015-2
02B011-2
02B011-3
02B015-3
26
Environmental samples in the ERBA test
conclusions

• 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

27
Toxicity-guided fractionations
28
Fractionation procedure protocol
Environmental sample
filtration
Particulate material
dissolved phase
SPE Extract 250 µl
YES
Identification of estrogens in active fractions
via LC-MS/MS
YES
Investigate relationship between concentration of
compounds and estrogen activity in different
fractions
29
Fractionation of environmental samples
30
Fractionation of environmental samples
LC-MS/MS fr4-6 Polar fraction? fr 78 Methyl,
ethyl propylparaben fr910 Estron, E2, EE2,
Propylparaben fr16-19 4-n-octylphenol,
4-n-nonylphenol, 4-tertiair octylphenol fr 22-29
apolar substances ?
100
9
A
9
10
10
80
60
estrogen activity relative to max E2 standard
curve
40
7
8
4
20
5
1
26
26
6
27
29
27
29
2
28
30
28
30
3
0
1
5
10
15
20
25
30
5
Fraction number
31
Correlation between estrogen activity in
fractions chemical concentration
A
B
pg E2/L in fr 78
pg E2/L in fr 1718
conc octyl phenol (as pg E2/L)
conc methyl parabene (ng/L)
32
Fractionation of Environmental samples
Relation between estrogen activity in fraction
78 fr 1718 with substances present in these
fractions
A
9
10
16
estrogen activity relative to max E2 standard
curve
7
Results YES correlates with methyl parabene
octyl phenol
17
18
8
11
15
13
14
12
6
26
6
27
4
5
Fraction number
33
Toxicity-guided fractionations

• Industrial samples alkyl phenols up to 54 of
  the total estrogenic activity
• This is performed on 250µl of the extract
• Parabens alkyl phenols related to surface water
  estrogenic activity (has never been demonstrated
  before)

34
Summary fractionation

• The developed methods are sensitive, reproducible
  effectively detect the cause of estrogen
  activity (EA).
• The most active fractions fr910 natural
  synthetic estrogens. No quantitative relation
• Interesting Significant relation between
  estrogen activity in fr 78 methyl paraben
  fr 1718 octyl phenol
• The concentrations measured explain 50 of the
  EA maximum
• Further research other substances? Matrix
  effects?

35
Studies on Human serum
36
The aromatase study

• Placebo-controlled study
• Aromatase inhibitor (letrozole)
• Testosterone ? estradiol
• Hormones (classical methods)
• Total estrogen load (YES)

37
The aromatase study
Mean ng E2 equiv /LStdev Stdev
E2 load before 48.1 29.4
E2 load after 6.7 3.6
Difference 41.4 29.2
Percentage decline 83.0 10.3
Correlation with decline in E2 0.58 (p0.003)
Detection limit (E2 equivalent) 5
38
The adolescents study

• 550 adolescent males
• Hormones (classical methods)
• Total estrogen load (YES)

39
The adolescents study
Age Weight BMI E2 (pg/ml) frE2 (pg/ml)
logYES-corrected 0.21a 0.12 b 0.05 ns 0.40 a 0.39 a
Age 0.24 a 0.06 ns 0.42 a 0.41a
Weight 0.84 a 0.45 a 0.53 a
Height 0.21 a 0.48 a 0.51 a
BMI - 0.27 a 0.37a
a plt0.00001, b plt0.01
40
Prediction of mixture effects
41
Prediction of mixture effects

• Data from actual combination experiments were
  compared to theoretical curves assuming additive
  combination effects (112)
• Deviation from additivity suggests interaction
  between compounds
• (113, synergism)

42
Effect summation

• Only applicable with linear dose response
  relationships

Arbitrary units
Cell count
Observed effect 0.2 mM
Expected effect 0.2 mM
Effect 0.1 mM
0.01
0.1
1
10
Conc. (mM)
43
MCF7 (Br ca) cell growth with a mixture of low
level chemicals
44
Observed response after 3 days of incubation
compared to the predicted response
For p,p-DDE/E2 (41,7 pM) lindane/E2 mixtures
observed effect is higher than predicted But for
bisphenol A
45
Special thanks to

• The team of Milieu en Gezondheid (UGent, UIA,
  VUB, KUL, VITO, ....)
• A. Bossier, W. Verstraete, LabMeT
• S. Stuyvaert, Nick Hendryckx, labo Andrology UZ
  Gent
• Hormonology lab UZ Gent
• T. Benijts/ Prof. W. Lambert Labo Toxicologie
  FFW Ugent
• A. De Winter M. Van Oost VMM Gent