In
Vitro
Aromatase
Assay:

Prevalidation
Studies
Susan
Laws,
Ph.
D.

Endocrinology
Branch
Reproductive
Toxicology
Division
NHEERL
Office
of
Research
and
Development
U.
S.
EPA
In
Vitro
Aromatase
Assay:


A
cytochrome
P450
enzyme
complex
bound
in
endoplasmic
reticulum

Catalyzes
the
conversion
of
androgens
to
estrogens

Androstendione
Estrone

Testosterone
Estradiol

Present
in
ovary,
placenta,
testis,
brain,
bone,

vasculature
and
adipose
tissue

Present
in
all
vertebrates

Known
to
be
inhibited
by
EDCs
In
Vitro
Aromatase
Assay:

Prevalidation
Studies

Historical
Perspective

EDSTAC
recommended
as
alternative
assay

EDSP
Detailed
Review
Paper

Radiometric
method

Human
placental
microsomes

Initial
prevalidation
studies

DRP
protocol

Compared
tissue
sources
for
enzyme
Prevalidation
Studies:
Goals

Optimize
protocols

Enzyme,
substrate
and
cofactor
concentrations

Linear
time
course
response

Positive
control

Performance
Criteria

Intra­
and
inter­
assay
variation

Technician
variation

Compare
placental
and
recombinant
microsomes
(
11
test
chemicals)


Protocol
for
multi­
laboratory
studies
HO
O
O
O
androstenedione
O
O
estrone
HO
O2
NADPH
O
O
HO
O2
NADPH
HO
­
H2O
O
O
O
19­
hydroxyandrostenedione
19,19­
dihydroxyandrostenedione
19­
oxoandrostenedione
O2
NADPH
O
O
HO
O
O
+
3Fe
­
H2O
­
HCOOH
peroxy
enzyme
intermediate
Reaction
Mechanism:

Androstenedione
to
Estrone
­
Cytochrome
P450arom
and
NADPH­
cytochrome
P450
reductase
In
Vitro
Aromatase
Assay:

Radiometric
(
3H20)
Method
Microsomal
(
Arom(
CYP19))

Complex
3H­
Androstenedione
and
NADPH
3H2O
Estrone
+

Postitive
Control:
4­
OH­
androstenedione
(
100
nM)
Test
Chemical
(
0.1
nM
­
1.0
mM)

+
Indicators
of
Optimized
Protocol

Small
fraction
(
10­
15%)
substrate
converted
to
estrone

Estrone
production
linear
with
time
and
enzyme
concentration

Estrone
production
dependent
upon
presence
of
enzyme
and
NADPH

Estrone
formation
can
be
inhibited
Placental
Microsomes:
Product
Formation
versus
Protein
Figure
1:
Product
formation
vs.
[
protein]

0.0000
0.0010
0.0020
0.0030
0.0040
0.0050
0.0060
0
0.01
0.02
0.03
0.04
0.05
0.06
[
protein]
mg/
mL
nmol
estrone
formed/

min
Complete
Inhibited
(
9%)
(
18%)
(
38%)

(%)
conversion
substrate
to
product
Inhibited:
4OH­
androstenedione
(
100nM)

Intra­
assay
(
triplicates)
CV=
3%;
Inter­
assay
(
3
exp.)
CV=
7.4%

Aromatase
Optimization
Supplementary
Studies
(
pages
11,28,29)
Placental
Microsomes:
Product
Formation
versus
Time
Figure
1:
Activity
vs.
Time
R
2
=
0.96
R
2
=
0.78
0.0000
0.5000
1.0000
1.5000
2.0000
0
10
20
30
40
Time
(
min)

Product
formation
rate
(

nmol/

mg)
Complete
Inhibited
2.7,
5.1,
10,
14,
17.5%
Substrate
conversion
over
time
Inhibited=
4OH­
androstenedione
(
100
nM)

Estrogen
Production:
Human
Placental
Assay
Results
Quick
Response
Task
2
(
Table
1,
pages
2­
3)
Placental
Microsomes:
Inhibition
of
Aromatase
Activity
Estrogen
Production:
Human
Placental
Assay
Results
Quick
Response
Task
3
(
Figure
3)

Placental
Microsomes:
Inhibition
of
Aromatase
Activity
with
4­
OH­
Androstenedione
­
10
­
9
­
8
­
7
­
6
­
5
0
25
50
75
100
IC50=
42
nM
log[
4­
OH­
ASDN]

Percent
of
control
activity
Placental
Microsomes:

Intra­
and
Inter­
Assay
Variance

Intra­
assay
(
triplicates)

CV
1.4­
7.5%


Inter­
assay
(
3
days)

CV
1.7
 
11.5%

Comparison
of
QR1,
QR2
and
QR3
Aromatase
Activities
(
0.0125
mg/
mL
protein
and
15
min)

0.0000
0.0100
0.0200
0.0300
0.0400
0.0500
0.0600
Total
activity
Inhibited
QR1
QR2
QR3
Estrogen
Production:
Human
Placental
Assay
Results
Quick
Response
Task
3
(
Table
3,
Figure
4)
Human
Recombinant:
Protocol
Optimization
Experiments
Activity
vs.
Time
R2
=
0.96
R2
=
0.84
0.0000
2.0000
4.0000
6.0000
8.0000
10.0000
0
10
20
30
40
Time
(
min)

Product
formation
rate
(

nmol/

mg)
Complete
Inhibited
Recombinant:
Inhibition
of
Aromatase
Activity
w
ith
4­
OH­
Androstenedione
­
10
­
9
­
8
­
7
­
6
­
5
0
25
50
75
100
IC50=
25
nM
log[
4­
OH­
ASDN]

Percent
of
Control
Activity
Estrogen
Production:
Human
Placental
Assay
Results
Quick
Response
Task
4
and
5
(
Tables
4,
5,
Figures
5­
7)

7,
12,
25,
33,
42%
Substrate
conversion
over
time
Inhibited=
4OH­
androstenedione
(
100
nM)

Intra­
assay
(
triplicates):
CV
=
1­
3%

Inter­
assay
(
2
days):
CV
=
5
 
20%
Comparison
of
QR4
and
QR5
Aromatase
Activities
0.0000
0.0500
0.1000
0.1500
0.2000
0.2500
0.3000
0.3500
0.4000
0.4500
0.5000
Total
activity
Inhibited
QR4
QR5
In
Vitro
Aromatase
Assay:

Optimized
Assay
Conditions
0.283
+/­
0.0005
(
2)

0.053
+/­
0.001
(
3)

Activity
(
nmol/
mg/
min)
15
15
Incubation
time
(
min)
100
100
[
3H]
ASDN
(
nM)
0.3
0.3
NADPH
(
mM)
0.004
0.0125
Protein
(
mg/
mL)
Human
Recombinant
Human
Placenta
Assay
Type
Assay
Factor
Estrogen
Production:
Human
Placental
Assay
Results
Quick
Response
Task
4
Optimized
Protocols:
Variability
Between
Assay
Day
and
Technicians

Experiment
design

Three
technicians
conducted
each
assay
independently
over
3
days

Triplicate
assay
tubes

Maximum
aromatase
activity
determined

Comparison
of
coefficient
of
variations
Estrogen
Production:
Human
Placental
Assay
Results
Quick
Response
Task
4
Tables
7
and
8
Coefficient
of
Variation:
Intra­
assay,

Assay
Day,
and
Technician
Variability
53%
(
15%)*

47%
(
10%)*

Day
5
30%

29%

Day
4
17%

36%

Day
3
19%

12%

Tech
3
50%
(
11%)*

49%
(
12%)*

Tech
2
17%

22%

Tech
1
3.7%

4%

Triplicates
Recombinant
Placenta
Parameter
(%)*
CV
after
Tech
2,
Day
5
data
deleted
Estrogen
Production:
Human
Placental
Assay
Results
Quick
Response
Task
4,
Tables
7
and
8
In
Vitro
Aromatase
Assay:

Comparison
of
Test
Chemicals

Experiment
Design

Optimized
protocols
using
placental
and
recombinant
microsomes

Test
chemicals
(
11,
positives
and
negatives)


Complete
concentration
curve
for
each
chemical
ran
on
4
separate
days

Two
technicians
(
one
ran
placental,
the
other
recombinant)


Single
set
of
test
chemical
concentrations
shared
by
2
tech.
each
day
Test
Chemicals:


Inhibitors

4­
OH­
androstenedione

Chrysin

Ketoconazole

Aminoglutethimide

Econazole

Genistein
(?)

Negative
for
Inhibition

Nonylphenol

Atrazine

Bis­(
2­
ethylhexyl)
phthlate

Lindane

Dibenz(
a,
h)
anthracene
In
Vitro
Aromatase
Activity:

Comparison
of
Inhibition
Recombinant
Microsomes:

4­
OH­
androstenedione
­
10
­
9
­
8
­
7
­
6
­
5
­
4
­
3
­
2
0
25
50
75
100
Day
1
Day
2
Day
3
Day
4
Log
(
4­
OH­
androstenedione)

Percent
of
Control
Activity
Human
Placenta:

4­
OH­
Androstenedione
­
10
­
9
­
8
­
7
­
6
­
5
­
4
­
3
­
2
0
25
50
75
100
Day
1
Day
2
Day
3
Day
4
Log
(
4­
OH­
androstenedione)

Percent
of
Control
Activity
CV
(
20%)
CV
(
54%)

Figure
10­
Placenta
aromatase
response
curves
Figure
11­
Recombinant
aromatase
response
curves
In
Vitro
Aromatase
Activity:

Comparison
of
Inhibition
Recombinant
Microsomes:

Aminoglutethimide
­
10
­
9
­
8
­
7
­
6
­
5
­
4
­
3
­
2
0
25
50
75
100
Day
1
Day
2
Day
3
Day
4
Log
(
Aminoglutethimide)

Percent
of
Control
Activity
Human
Placenta:
Aminoglutethimide
­
10
­
9
­
8
­
7
­
6
­
5
­
4
­
3
­
2
0
25
50
75
100
Day
1
Day
2
Day
3
Day4
log
(
Aminoglutethimide)

Percent
of
Control
Activity
CV
(
47%)
CV
(
14%)

Figure
10­
Placenta
aromatase
response
curves
Figure
11­
Recombinant
aromatase
response
curves
In
Vitro
Aromatase
Activity:

Examples
of
Data
Human
Placenta
Genistein
­
10
­
9
­
8
­
7
­
6
­
5
­
4
­
3
­
2
0
25
50
75
100
4­
OH­
Androstenedione
Log
(
genistein)

Percent
of
Control
Activity
Placental
Microsomes
Econazole
and
Atrazine
­
10
­
9
­
8
­
7
­
6
­
5
­
4
­
3
­
2
0
25
50
75
100
4­
OH­
androstenedione
Atrazine
Econazole
Log
(
Test
Chemicals)

Percent
of
Control
Activity
Figure
10­
Placenta
aromatase
response
curves
Figure
11­
Recombinant
aromatase
response
curves
Conclusions:
Test
Chemical
Experiment

Variability
between
reps.
is
greater
than
expected
for
both
assays

IC50s
for
inhibitors
(
CVs
ranged
7
 
49%)


Technician
error
rather
than
inadequate
protocol
method
is
likely
cause
of
variability

Despite
variability,
both
protocols
correctly
identified
inhibitors
In
Vitro
Aromatase
Assay:

Next
Steps

Identify
source
of
variability

Substrate
concentration

Technician
training

Conduct
additional
experiment
to
evaluate
day­
to­
day
and
technician
variability
(
e.
g,

better
estimate
of
performance
criteria)


2
Tech.,
3
test
chemicals
(
8­
9
concentrations
in
triplicates),
4
days,
both
protocols
In
Vitro
Aromatase
Assay:

Next
Steps

Rerun
assays
for
test
chemicals
with
incomplete
curves

econazole,
ketoconazole

Evaluate
the
usefulness
of
estrone
measurement
rather
than
3H
2
0
for
recombinant
protocol

Prepare
updated
protocols
for
validation

Broader
concentration
range
for
test
chemicals

Guidelines
for
data
analysis
and
interpretation
In
Vitro
Aromatase
Assay:

Summary

Protocols
optimized
for
placenta
and
recombinant
assays

Assays
produce
similar
data

Assays
differ
in
advantages/
disadvantages

High
throughput
assays

KGN
cell
line

CYP19/
Fluorescent
substrate
(
HTP)
kit
available
Acknowledgements:

Battelle
Memorial
Institute
Columbus,
OH
°
David
Houchens
°
Paul
Feder
°
Terri
Pollock
Chemical
and
Life
Sciences
Research
Triangle
Institute
RTP,
NC
°
Sherry
Black
°
RTI
Technical
Staff
°
James
Mathews
°
Marcia
Phillips
°
Rochelle
Tyl
Endocrinology
Branch
RTD,
NHEERL,
ORD
U.
S.
EPA
RTP,
NC
°
Ralph
Cooper
°
Earl
Gray
°
Tammy
Stoker
°
Vickie
Wilson
°
Jerome
Goldman
OSCP,
U.
S.
EPA
Washington,
DC
°
Gary
Timm
°
Jim
Kariya
°
Jane
Scott­
Smith
