Differential Effects of Environmental Chemicals and Selected Pharmaceuticals on
Aromatase Activity and Hormone Concentrations
Eric Higley1, John Newsted2, John Giesy1,3 , and Markus Hecker1,2
1 Department
of Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada; 2Entrix, Inc., Okemos, MI; 3City University of Hong Kong, Hong Kong, China.
Introduction
Objectives
Studies have suggested links between the exposure to natural and man-made
substances in the environment and alterations in endocrine and reproductive systems
in wildlife and humans. One process that is currently discussed as an important and
relevant target for endocrine disrupting chemicals is the synthesis of sex steroids,
specifically the production of 17β-estradiol, by the enzyme aromatase. As a
consequence, a number of assays have been developed to test for potential effects of
chemicals on aromatase as a predictor for subsequent alterations of hormone
homeostasis. Most of these assays, however, only measure a specific endpoint such
as aromatase gene expression or enzyme activity, and it is unclear whether the
observed changes are truly predictive of effects at the hormone level. Therefore, with
this study we wished to investigate in parallel the effects of a number of known
inducers and inhibitors of steroidogenic pathways on both aromatase enzyme activity
(direct and indirect changes) and sex steroid concentrations. This was done using an
in vitro screening assay developed to measure alterations in hormone production, the
H295R cell steroidogenesis assay (1). Previous studies have indicated that this cell
line was useful in screening for effects on gene expression of steroidogenic enzymes,
aromatase enzyme activity and steroid hormones (1, 2, 3).
1. Identify effects of nine known or suspected endocrine disrupting chemicals on sex
steroid production and aromatase enzyme activity in H295R cells.
CYP11B1
Cortisol
17β-HSD
CYP19
17β-estradiol
fold-change
fold-change
+
+
10
100
4. Remove medium from cells and
extract with ether
5. Conduct cell viability assay
immediately after removal of medium
(MTT (6) and live/dead assays)
6. Measure hormone concentrations in
medium extract
Acknowledgments:
3
6
4
@
2
@
0.1
*
*
*
+
1
10
Aromatase activity after 48 hrs with chemical
.
Aromatase activity -- direct effects
2.5
100
2
1.5
1
0.5
0
0.001
*
*
*
0.01
0.1
1
10
Aromatase Activity
Estradiol
Testosterone
Fadrozole
3
2.5
2
*
*
1.5
1
0.5
0
0.01
+
@
@
0.1
@
*+
@
1
@
@
+
+
10
3
2.5
2
*
*
100
*
1
+
+
0.1
0.01
*
+
*
*
1.5
1
0.5
0
100
0.001
Fadrozole (uM)
*
+
*
0.01
0.1
1
*
@
*
100
Aromatase activity after 48 hrs with chemical
Aromatase activity -- direct effects
*
10
*
1
@
+
*
*
+
0.1
1
*
*
*
* *
10 * 100
Aromatase activity after 48hrs
Estradiol
Testosterone
10
Aromatase activity after 48 hrs with chemical
Aromatase activity -- direct effects
@
Aromatase Activity
No Change
Trenbolone
↑↑↑
↑↑↑
Trilostane
↑↑↑
↑↑↑
↓↓
↓↓
↓↓
↓↓
↑↑↑
↑↑↑
↑↑↑
Direct
Aromatase
Activity
Direct
Aromatase
Activity
(Chemical
added only to
the assay)
(Pre-exposed +
chemical added
to the assay)
↑↑
↑↑↑
No change
↑ then ↓↓↓
↑↑↑
↑↑↑
↑↑
↓↓↓
↓↓↓
↓↓↓
↓
↓
No change
No change
NA
NA
↓↓
NA
NA
↑↑ then ↓
No change
↑
↑↑↑
↓↓↓
↓↓↓
↓↓
↑↑
Figure 3. Changes in the production of estradiol and testosterone, and activity of
aromatase in H295R cells after exposure to all 9 chemicals studied. Magnitude of relative
changes compared to solvent controls is represented by increasing number of arrows:
One arrow: 1- to 2-fold change; Two arrows; 2- to 5-fold change; Three arrows: >5-fold
change. Downward arrows indicate suppression; Upward arrows indicate increase. Only
significant changes are listed (p<0.05).
Aromatase activity after 48hrs
Aminoglutethimide
100
Prometon
Aminoglutethimide
Aromatase activity after 48hrs
@
Ketoconazole
Prochloraz
Atrazine (uM)
fold-change
2. Replace medium and dose cells
Hormone and
Enzymatic Analyses
+
*
8
0
0.01
H295R Steroidogenesis Assay (5)
1.Seed at a density of 300,000 cells/mL
into 24 well plates, and incubate at
37°C and 5% CO2 for 24h
cells
*
Aromatase Activity
Estradiol
Testosterone
10
17β-HSD
Testosterone
3. Incubate at 37°C and 5% CO2 for 48h
medium
+
1
Atrazine
Estrone
1. Indirect measurement: Aromatase activity was measured after exposure to a
chemical for 48 hours. The chemical was not added to tritium release assay.
2. Direct measurement: Chemical was added directly to the tritium release
assay with no pre-exposure of cells.
3. Combined measurement: Aromatase activity was measured after chemical
exposure for 48 hours and chemical was added directly to the tritium release
assay.
Cell
viability
*
+
*
Fadrozole
Forskolin (uM)
• Aromatase activity was measured using a tritium release assay (4). Three
separate measurements for Aromatase activity was conducted involving seven
different chemicals.
Incubate at
37oC for 48 h
*+
0.1
*
3β-HSD
AndrosteneCYP19
dione
• Nine chemicals, including two selective aromatase inhibitors (fadrozole and
aminogutethimide), one fungicide (prochloraz), one herbicide (atrazine) and one
general inducer of steroidogenesis (forskolin) were tested in the H295R
steroidogenesis assay.
Dose cells after 24 h
at 37oC
@
DHEA
CYP17
Methods
Seed plate at
300,000 cells/mL
@
fold-change
17α-OHProgesterone
CYP21
11-Deoxycortisol
@
fold-change
17α-OHPregnenolone
3β-HSD
@
@
Forskolin
Aromatase activity after 48 hrs with chemical
Aromatase activity -- direct effects
18
16
* * *
14
*
12
* *
*
10
8
6
*
4
*
2
0
0.1
1
10
100
0.001 0.01
fold-change
CYP11B2
Aldosterone
CYP17
CYP17
45
40
35
30
25
20
15
10
5
0
0.01
↑
↑↑
↓↓↓
↓↓↓
↓↓↓
↓↓↓
Atrazine
Aromatase activity after 48hrs
CYP17
fold-change
3β-HSD
Progesterone
CYP21
11-Deoxycorticosterone
CYP11B2
Corticosterone
Figure 1. Steroidogenic
pathway.
Enzymes are
indicated
in
red,
hormone
products
in
black.
Purple arrows:
Direction of synthesis.
Aromatase Activity
Estradiol
Testosterone
Forskolin
fold-change
CYP11A
Cholesterol
Pregnenolone
Chemical
2. Distinguish between direct and indirect acting compounds by means of differential
aromatase activity analysis.
3. Evaluate the potential of using differential measurements of aromatase enzyme
activity to predict effects on the homeostasis of T and E2.
Aromatase
Testosterone Estradiol
Activity
0.1
10
100
0.01
0.1
Aminoglutethimide (uM)
1
*
*
*
*
10
*
*100
Figure 2.
Relative change of testosterone, estradiol and aromatase activity of H295R cells
exposed to Atrazine, Forskolin and Fadrozole. Symbols indicate significant differences
from controls ( * = Aromatase activity;+ = Testosterone; @ = Estradiol)
Conclusion
• Changes in aromatase activity in response to exposure with forskolin and fadrozole were
predictive of changes in estradiol production.
• All general inducers did not directly affect aromatase activity (i.e. Forskolin and Atrazine).
• Changes observed at the level of aromatase activity when either measured in the indirect
or direct aromatase activity experiments were not always predictive of effects on
estradiol and testosterone concentrations.
• However, integration of all endpoints measured in this study (testosterone, estradiol,
direct, indirect and combined aromatase activity) provided a better insight into the
potential mode of chemical interaction with the steroidogenic pathway investigated here..
• For compounds that do not directly compete with aromatase, the concentration of
estradiol is likely to also be affected by upstream events as well as by other feedback
mechanisms.
• Overall, measurement of aromatase activity and estradiol production can provide some
insight into the mechanism of action of a chemical on endocrine and reproductive
systems. However, further studies are needed to more specifically investigate whether a
chemical is active at the gene expression level, protein level or is part of a feedback loop.
References
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5. Hecker et al. 2007. Environ. Sci. Pol. Res. 14 (Special 1), 23-30.
6. Mosman et al. (1983). J. Immunol. Methods 100, 45-50.
Funding for this project was provided by USEPA, ORD Service Center/NHEERL. Specifically, we would like to thank Mr. Gary Timm, Dr. Ralph Cooper, Dr. Jerome Goldman, Dr Robert Kavlock for their support.