Pre­
Validation
of
the
Aromatase
Assay
using
Human
and
Bovine
Placental,
and
Human
Recombinant
Microsomes
Endocrine
Disruptor
Methods
Validation
Subcommittee
(
EDMVS)

Plenary
Session
June
5,
2003
Research
Triangle
Park,
North
Carolina
°
RTI
International
is
a
tradename
of
Research
Triangle
Institute.
Overview

Background:
Aromatase
(
CYP19)


Study
Goals

Substrate
characterization

Placenta
tissues
 
human,
bovine,
porcine

Methods

Results
Protein
yield
P450
Spectra
Aromatase
activities

Conclusions

Future
studies
Aromatase

Cytochrome
P450
enzyme
 
CYP19

Present
in
the
gonads
and
placenta

Responsible
for
the
biosynthesis
of
estrogen
steroid
hormones

Can
be
inhibited
at
the
level
of
gene
expression
(
e.
g.,
ethylhexylphthalate),
or
directly
at
enzyme
(
e.
g.
azoles)
Steroid
Hormone
Biosynthesis
by
Aromatase
O
O
H
O2,
NADPH
CYP19
O
O
HO
HO
O2,
NADPH
CYP19
androstenedione
19­
hydroxyandrostenedione
O
O
HO
19,19­
dihydroxyandrostenedione
O
HO
estrone
­
HCOOH
H2O
O
O
O
HO
O
O
O
O
Fe
3
+
CYP19
O2,
NADPH
peroxy
enzyme
intermediate
19­
oxoandrostenedione
­
H2O
Steroid
Hormone
Biosynthesis
by
Aromatase
(
cont'd)

O
H
O
O
OH
androstenedione
 
17
­
HSD
testosterone
aromatase
HO
OH
estradiol
+
H2O
+
HCOOH
Study
Goals
The
goal
of
this
work
is
to
identify
the
optimal
factors
and
conditions
for
the
assay
of
aromatase,
including:


Characterization
of
radiolabeled
substrate
(
androstenedione)


Selection
of
mammalian
placenta
allowing
sufficient
yield
of
catalytically­
active
microsomal
protein,
and
assessment
of
human
recombinant
CYP19

Optimization
of
assay
with
respect
to
concentration
of
protein,
cofactors,

substrate,
and
incubation
time
using
a
factorial
design

Using
this
optimized
assay,
determine
the
effect
of
selected
substances
on
aromatase
activity
Substrate
Characterization
Sources

Nonradiolabeled
4­
androstene­
3,17­
dione
(
99%):
Sigma
Chemical
Co.


Radiolabeled
[
1 ­
3H(
N)]
androst­
4­
ene­
3,17­
dione:
Perkin­
Elmer
Life
Science

Specific
activity:
25.3
Ci/
mmol

Radiochemical
purity:
98%
HPLC
Radiochromatogram
for
[
3H]
Androstenedione
Samp
leName
1
:
1
1
069
1­
43A:
4
1C
;
Vial
5;
In
jectio
n
1;
Cha
nn
el
2487C
ha
nne
l
1
;
D
ate
Acq
uire
d
2/
19
/
0
3
4:
36:
08
PM
SampleName
1
:
1
1
0691­
43A:
4
1C
;
Vial
5;
Injection
1;
C
hannel
SATIN
;
D
ate
Acquired
2
/
19/
03
4
:
36:
08
PM
­
0
.02
0.00
0.02
0.04
0.06
­
2
.00
0.00
2.00
4.00
6.00
8.00
10
.00
M
inu
tes
2.
00
4.
00
6.
00
8.
00
10
.00
12
.00
14
.00
16
.00
18
.00
20
.00
Column
:
Zorbax
SB­
C1
8,
U
SCL0
119
03
,
25
0
x
4.6
mm
Mob
ile
Phase
:
55:
15
:
30
ddH2O:
THF:
MeOH
10691
­
95A
Flow
R
ate:
1
mL/
m
in
Detecto
rs:
Waters
2487
at
240
nm
B­
RAM
with
250
u
l
LiGL
solid
cell,
#
115
90
1
2
N
am
e
RT
Area
H
eight
AS
DN
[
3H
]
A
SDN
14
.50
6
14
.62
5
29
3714
33
7219
11
441
11
235
Pea
k
Re
sults
Placental
Tissues
http://
www.
vivo.
colostate.
edu/
hbooks/
pathphys/
reprod/
placenta/
structure.
html
Placental
Tissues
(
con't)

http://
www.
vivo.
colostate.
edu/
hbooks/
pathphys/
reprod/
placenta/
ruminants.
html
Placenta
Tissues
(
con't)
Methods
Preparation
of
microsomes
from
tissues

Iced­
down
(
not
frozen!)
within
10
min
of
delivery

Soft
tissue
harvested,
homogenized
in
cold
buffer

Homogenate
centrifuged
@
10,000g
for
30
min,
4
°
C

Supernatant
centrifuged
@
100,000g
for
60
min,
4
°
C

Pellet
resuspended
in
buffer,
centrifuged
@
100,000g
for
60
min,
4
°
C

Pellet
"
washed"
by
repeating
above

Resuspended
in
buffer,
protein
concentration
determined
Spectral
P450
content
determination:
difference
spectrum,
400
 
500
nm,
of
CO
vs.

CO/
dithionite
reduced
microsomes,
quantitation
using
extinction
coefficient
for
the
450
nm
absorbance
of
100
mM­
1
cm­
1
Addition
of
the
following
into
16
x
100
mm
test
tubes:

0.1
mL
propylene
glycol
50.0
nM
3H­
androstenedione
(
0.1
µ
Ci)

1.7
mM
NADP+

2.8
mM
glucose­
6­
phosphate
1.0
u
glucose­
6­
phosphate
dehydrogenase
Dilute
to
1.0
mL
volume
with
phosphate
buffer,
pH
7.4,

and
warm
to
37
°
C
Preparation
of
placental
or
recombinant
microsomal
pellet
Suspension
of
placental
or
recombinant
microsomes
and
dilution
to
~
0.1
mg/
mL
in
phosphate
buffer
Warm
microsomes
to
37
°
C
Add
1.0
mL
placental
microsomal
suspension
to
each
test
tube
Incubate
at
37
°
C
for
30
minutes
in
shaking
water
bath
Methods
(
cont'd)
Add
2.0
mL
of
CH2Cl2
to
quench
enzyme
reaction;
vortex
for
30
seconds;
centrifuge
for
10
minutes
at
500
rpm
Transfer
organic
layer
to
a
capped
vial.

Add
2.0
mL
of
CH2Cl2
to
test
tube
containing
aqueous
layer;
vortex
for
30
seconds;
centrifuge
for
10
minutes
at
500
rpm
Transfer
organic
layer
to
a
capped
vial.

Add
2.0
mL
of
CH2Cl2
to
test
tube
containing
aqueous
layer;
vortex
for
30
seconds;
centrifuge
for
10
minutes
at
500
rpm
Transfer
organic
layer
to
a
capped
vial.
Prepare
aliquots
of
each
organic
extract
for
analysis
by
LSC.

Transfer
aqueous
layer
to
a
capped
vial;
transfer
0.5
mL
to
a
LSC
vial;
add
10.0
mL
scintillation
cocktail;
count
in
LSC.

Methods
(
cont'd)
Tissue
Procurement
Issues
Human
placenta

Caesarian
section
allows
for
1)
timed
delivery
and
optimal
collection
conditions,
and
2)

less
chance
of
disease
transmission
Bovine
and
porcine

Requires
farms
close
to
laboratories,
assistance
of
farm
staff

Deliveries
seasonal,
and
any
time
of
day
P450
Difference
Spectrum
Human
Recombinant
CYP19
Results
(
cont'd)

Preoptimization
Results
Enzyme
Source
Microsomal
protein
yield
(
mg/
g
wet
tissue
processed)
P450
Content
(
nmol/
mg
protein)
Aromatas
e
Activity
(
nmol/
mg
 
min)

Human
placenta
900/
511.79
(
1.76
mg/
g)
0.048
0.015a
Human
Recombinant
­­
0.38
0.022
Bovine
placenta
675/
748.99
(
0.9
mg/
g)
0.031
0.003b
Porcine
placenta*
126/
257.44
(
0.49
mg/
g)
0.053
0.003
*
Only
1
of
5
porcine
placentas
yielded
microsomes
with
acceptable
aromatase
activity
a
Acceptance
criteria:
0.005
nmol
product/
mg
protein/
min
b
Literature
value:
0.0036
±
0.00078
nmol
estrone
formed/
mg
protein/
min
(
Tsumagari
et
al.
(
1993).
J.
Reprod.
Fert.
1993,
98,
631­
36.)
Conclusions
Collection
conditions
used
for
placentas
are
crucial
to
activity
Human
Placentas

easiest
to
collect
under
optimal
conditions

well­
defined
morphology
and
good
yield
of
microsomal
protein

high
activity

Human
recombinant,
comparable
activity
with
best
placental
preparations
However,


SOPs
must
be
in
place
for
handing
potentially
infectious
materials

Although
Caesarian
vs.
birth
canal
delivery
minimizes
infection
of
the
placental
tissue,

information
regarding
screening
for
HIV,
hepatitis,
etc.
should
be
obtained
if
available.
Future
Studies
Optimization
of
conditions
using
a
factorial
design
Summary
of
Experimental
Factors
and
Levels
to
be
Optimized
Experimental
Factor
Levels
Experimental
Factors
Units
1
2
3
4
5
NADP+
mM
0.1
0.5
1
2
4
Glucose­
6­
Phosphate
mM
0.1
1
2
3
4
Glucose­
6­
Phosphate
Dehydrogenase
units
0.1
0.5
1
2
4
Androstenedione
(
substrate)
nM
10
25
50
100
500
Protein
mg/
mL
0.01
0.02
0.1
0.5
1
Incubation
Time
min
10
15
30
60
120
Future
Studies
Determination
of
variance
of
the
optimized
assay

Using
the
optimized
conditions
determined
for
each
preparation,

three
technicians
independently
conduct
the
assay
on
three
separate
days

The
results
are
assessed
for
technician­
to­
technician
and
day­

today
variance.
Future
Studies
Determination
of
IC
50
for:


aminoglutethimide
(
non­
steroidal
aromatase
Inhibitor)


4­
hydroxyandrostenedione
(
potent
steroidal
aromatase
inhibitor

chrysin
(
potent
flavonoid)


genistein
(
weak
isoflavonoid)


ketoconazole
(
weak
imidazole
anti­
fungal)


econazole
(
potent
imidazole
anti­
fungal)


atrazine
(
affects
aromatase
gene
expression;
no
aromatase
inhibition)


bis­(
2­
ethylhexyl)
phthlate
(
affects
aromatase
gene
expression;
no
aromatase
inhibition)


nonylphenol
(
affects
AR/
ER;
no
aromatase
inhibition)


lindane
(
affects
StAR
and
cholesterol
metabolism;
no
aromatase
inhibition)


dibenz(
a,
h)
anthracene
Acknowledgements
Study
Plans
Dr.
Robert
Brueggemeier
(
Ohio
State
University)

Dr.
Jerry
Johnson
(
Battelle)

Placental
Tissues
Dr.
Amy
Murtha
(
Duke
University)

Mr.
Rick
Kern
(
North
Carolina
State
University)

Mr.
Curtis
Powell
(
North
Carolina
State
University)

RTI
International
Ms.
Sherry
Black
Ms.
Purvi
Patel
