LETTER
REPORT
ON
PHASE
I
PRE­
OPTIMIZATION
EXPERIMENTS
FOR
SUBSTRATE
CHARACTERIZATION
FOR
BOVINE
MICROSOMES
EPA
Contract
Number
68­
W­
01­
023
WA
2­
24
May
8,
2003
PREPARED
FOR
GARY
E.
TIMM
WORK
ASSIGNMENT
MANAGER
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
ENDOCRINE
DISRUPTOR
SCREENING
PROGRAM
WASHINGTON,
D.
C.

BATTELLE
505
KING
AVENUE
COLUMBUS,
OH
43201
Letter
Report
Work
Assignment
2­
24
Pre­
Validation
of
the
Aromatase
Assay
using
Human,
Bovine,
and
Porcine
Placental
Microsomes,
and
Human
Recombinant
Microsomes
Phase
I
Pre­
Optimization
Experiments
for
Substrate
Characterization
for
Bovine
Microsomes
1
Introduction
The
pre­
optimization
experiments
were
designed
to
assess
the
chemical
and
biological
properties
of
the
critical
components
that
are
used
in
the
aromatase
assay.
These
experiments
included
characterizing
the
radiolabeled
substrate
and
preparation
of
placental
microsomes.
In
addition,
each
of
the
four
microsomal
preparations
(
human,
bovine,
and
porcine
placental
microsomes
and
the
human
recombinant
microsomes)
was
analyzed
for
protein
concentration,
cytochrome
P450
(
P450)
content,
and
aromatase
activity.
The
P450
content
measurement
confirms
that
the
enzyme
is
present
(
and
in
what
concentration/
preparation
type)
prior
to
beginning
the
more
elaborate
aromatase
activity
assay.
Finally,
a
single
aromatase
activity
assay
determination
using
each
type
of
microsomal
preparation
was
included
as
a
pre­
optimization
experiment
in
order
to
determine
whether
the
preparations
are
of
sufficient
activity
to
conduct
the
definitive
optimization
experiments.

This
report
includes
the
results
of
the
pre­
optimization
experiments
related
to
bovine
placental
microsomes.

2
Materials
and
Methods
2.1
Chemicals
Non­
radiolabeled
4­
androstene­
3,17­
dione
(
ASDN)
was
received
through
Battelle
from
Sigma
(
St.
Louis,
MO).
[
1$­
3H(
N)]
Androst­
4­
ene­
3,17­
dione
([
3H]
ASDN)
was
obtained
from
Perkin
Elmer
Life
Science,
Boston,
MA.
NADP,
glucose
6­
phosphate,
glucose
6­
phosphate
dehydrogenase,
glycerol,
niacinamide,
dithiothreitol
and
bovine
serum
albumin
(
BSA)
were
purchased
from
Sigma.
Sodium
phosphate
monobasic,
sodium
phosphate
dibasic,
sucrose
and
propylene
glycol
were
from
JT
Baker
(
Phillipsburg,
NC).
Ultima
Gold
scintillation
cocktail
was
purchased
from
Packard
Instruments
(
Meriden,
CT).
DC
Protein
assay
kit
was
purchased
from
Biorad
(
Hercules,
CA)
.
Bovine
Phase
1
Letter
Report,
Page
2
2.2
Placental
Microsome
Preparation
A
bovine
placenta
was
received
from
NCSU's
Reedy
Creek
Field
Laboratories
on
February
27,
2003.
Upon
delivery,
the
placenta
was
placed
in
a
plastic
bag
and
left
at
ca.
0
NC.
The
placenta
was
transported
on
ice
to
RTI
about
1.5
h
after
delivery.
The
tissue
was
placed
in
a
tub
that
was
nestled
in
a
tub
of
ice
and
the
cotyledons
were
dissected
away
from
the
membrane.
Cotyledons
were
rinsed
of
soil
with
chilled
isotonic
saline
prior
to
their
transfer
into
beakers
containing
ice­
cold
buffer
(
2:
1
tissue
weight:
buffer;
0.25
M
sucrose,
0.05
M
sodium
phosphate
(
pH
7.0),
0.04
M
niacinamide).
The
cotyledons
were
minced
with
scissors
and
then
were
homogenized
in
portions
using
a
Polytron
homogenizer.
The
homogenate
was
transferred
to
centrifuge
tubes
and
centrifuged
at
a
setting
of
10,000g
for
30
min
at
4
NC
in
an
IEC
B­
22M
centrifuge.
The
supernatant
was
transferred
to
ultracentrifuge
tubes
and
was
centrifuged
at
a
setting
of
35,000
rpm
(
which
is
equivalent
to
approximately
100,000g)
in
a
refrigerated
Beckman
L5­
50B
Ultracentrifuge
for
1
h
to
obtain
the
crude
microsomal
pellet.
The
supernatant
was
decanted
and
discarded
and
the
microsomal
pellet
was
resuspended
in
a
chilled
buffer
containing
0.1
M
sodium
phosphate
buffer,
pH
7.4.
The
sample
was
centrifuged
again
at
a
setting
of
35,000
rpm
in
the
Beckman
L5­
50B
for
1
h
to
wash
the
microsomes.
This
washing
procedure
was
repeated
one
additional
time.
The
twice­
washed
microsomal
pellet
was
resuspended
in
chilled
0.1
M
sodium
phosphate
buffer
(
pH
7.4)
containing
0.25
M
sucrose,
20%
glycerol
and
0.05
M
dithiothreitol.
The
microsomal
suspension
(
total
volume
ca.
15
mL)
was
divided
among
20
vials,
flash
frozen
in
liquid
nitrogen,
and
stored
at
ca.
­
70
NC.

2.3
Protein
determination
The
protein
concentration
of
the
bovine
placental
microsome
preparation
was
determined.
A
6­
point
standard
curve
was
prepared
using
BSA,
ranging
from
0.13
to
1.5
mg
protein/
mL.
Protein
concentration
was
determined
by
using
a
DC
Protein
Assay
kit.
To
a
25
:
L
aliquot
of
unknown
or
standard,
125
:
L
of
BioRad
DC
Protein
Kit
Reagent
A
was
added
and
mixed.
Next,
1
mL
of
BioRad
DC
Protein
Kit
Reagent
B
was
added
to
each
standard
or
unknown
and
the
samples
were
vortex
mixed.
The
samples
were
allowed
to
sit
at
room
temperature
for
at
least
15
min
to
allow
for
color
development.
The
absorbances
are
stable
for
about
1
h.
Each
sample
(
unknowns
and
standards)
was
transferred
to
disposable
polystyrene
cuvettes
and
the
visible
absorbance
(@
750
nm)
was
measured
using
a
spectrophotometer.
The
protein
concentration
of
the
microsomal
sample
was
determined
by
extrapolation
of
the
absorbance
value
using
the
curve
developed
from
the
absorbance
of
the
protein
standards.

2.4
P450
Content
P450
content
was
determined
for
the
bovine
placental
microsome
preparation.
Using
the
carbon
monoxide
(
CO)
spectral
assay
of
Omura
and
Sato
(
1964),
a
single
experiment
was
conducted
as
described
below.
Bovine
Phase
1
Letter
Report,
Page
3
A
sample
of
the
microsomal
preparation
was
diluted
1:
20
in
0.1
M
phosphate
buffer
(
pH
7.4).
The
diluted
sample
was
gently
bubbled
with
carbon
monoxide
for
approximately
10
s
and
then
was
divided
between
a
pair
of
matched
cuvettes
(
1
mL/
cuvette).
Next,
a
few
grains
of
solid
sodium
dithionite
was
added
to
the
sample
cuvette
with
gentle
mixing.
The
visible
spectrum
was
then
recorded
from
400
to
500
nm
using
an
Aminco
split­
beam
spectrophotometer.

The
concentration
(
nmol/
mL)
of
P450
was
calculated
according
to
Beer's
Law
using
an
extinction
coefficient
value
for
P450
of
100
mM­
1
cm­
1.
The
specific
content
(
nmol/
mg
protein)
was
calculated
by
multiplying
the
P450
concentration
(
nmol/
mL)
times
the
dilution
factor
and
dividing
this
product
by
the
protein
content
(
mg/
mL)
of
the
original
sample.

2.5
Aromatase
Activity
Aromatase
activity
was
determined
for
the
bovine
placental
microsome
preparation.
A
single
experiment
was
conducted
using
only
the
substrate
([
3H]
ASDN/
ASDN).
The
assay
was
conducted
as
described
in
the
following
paragraph.

The
[
3H]
ASDN/
ASDN
substrate
solution
was
prepared
by
combining
solutions
of
[
3H]
ASDN
and
ASDN.
A
1
mg/
mL
solution
of
ASDN
was
prepared
in
ethanol.
Serial
dilutions
of
this
solution
were
prepared
in
assay
buffer
to
yield
a
solution
containing
ca.
1
:
g
ASDN/
mL.
The
[
3H]
ASDN
stock
was
diluted
1:
100
in
assay
buffer
to
yield
a
solution
containing
ca.
10
:
Ci/
mL.
The
substrate
solution
was
prepared
by
combining
275
:
L
of
the
1
:
g
ASDN/
mL
solution,
100
:
L
of
the
10
:
Ci
[
3H]
ASDN/
mL
solution
and
625
:
L
buffer.

The
assay
was
performed
in
duplicate
in
13x100
mm
test
tubes
maintained
at
37
±
1
°
C
in
a
shaking
water
bath.
An
aliquot
(
100
:
L)
of
propylene
glycol
was
added
to
the
tubes
to
serve
as
a
co­
solvent.
The
substrate,
[
1$­
3H]­
androstenedione
(
0.1
:
Ci,
50
nM),
was
added
to
the
tubes.
An
NADPH­
generating
system
comprised
of
NADP
(
1.7
mM),
glucose­
6­
phosphate
(
2.8
mM)
and
glucose­
6­
phosphate
dehydrogenase
(
1.0
units)
was
added
to
each
tube.
The
tubes
were
placed
at
37
±
1
°
C
in
the
water
bath
for
5
min
prior
to
initiation
of
the
assay
by
the
addition
of
the
diluted
microsomal
suspension
(~
0.1
mg
microsomal
protein/
mL).
The
total
volume
was
2.0
mL,
and
the
tubes
were
incubated
for
30
min.
The
incubations
were
stopped
by
the
addition
of
methylene
chloride
(
2.0
mL);
the
tubes
were
vortex­
mixed
for
about
30
s.
The
tubes
were
then
centrifuged
using
a
Beckman
GS­
6R
centrifuge
with
a
GH­
3.8
rotor
for
10
min
at
a
setting
of
1000
rpm
(
which
is
approximately
equivalent
to
230g).
The
methylene
chloride
layer
was
removed
to
a
vial
and
weighed;
the
aqueous
layers
were
extracted
again
with
methylene
chloride
(
2.0
mL).
This
extraction
procedure
was
performed
one
additional
time,
each
time
reserving
and
weighing
the
methylene
chloride
layer
in
a
separate
vial.
The
aqueous
layers
were
transferred
to
vials,
weighed,
and
duplicate
aliquots
(
0.5
mL)
were
weighed
into
20­
mL
liquid
scintillation
counting
vials.
Duplicate
aliquots
of
each
methylene
chloride
fraction
were
weighed
into
scintillation
vials.
Liquid
scintillation
cocktail
(
Ultima
Gold,
Packard,
10
mL)
was
added
to
each
counting
vial
and
shaken
to
mix
the
solution.
Bovine
Phase
1
Letter
Report,
Page
4
The
radiochemical
content
of
the
substrate
solution
was
determined
by
analyzing
5
weighed
aliquots
by
LSS.
The
substrate
solution
specific
activity
was
determined
by
dividing
the
radiochemical
content
of
the
substrate
solution
(
dpm/
g)
by
the
total
concentration
of
ASDN
in
the
solution
(
ASDN
+
[
3H]
ASDN;
nmol/
g
solution).

Analysis
of
the
samples
was
performed
using
LSS
as
described
in
SOP
METAB­
610.
Radiolabel
found
in
the
aqueous
fractions
represents
3H2O
formed,
and
that
in
the
methylene
chloride
fractions
represents
unreacted
substrate.

The
amount
of
estrogen
product
formed
was
determined
by
dividing
the
total
amount
of
3H2O
formed
by
the
specific
activity
of
the
[
3H]
ASDN
substrate
solution
(
expressed
in
dpm/
nmol).
The
activity
of
the
enzyme
reaction
was
expressed
in
nmol
(
mg
protein)­
1
min­
1
and
was
calculated
by
dividing
the
amount
of
estrogen
formed
by
the
product
of
mg
microsomal
protein
used
times
the
incubation
time,
e.
g.
30
min.

3
Results
and
Discussion
A
bovine
placenta
was
obtained
from
a
local
research
farm
and
microsomes
were
prepared.
A
sample
of
the
microsomes
was
thawed
rapidly
in
a
37
NC
water
bath
and
rehomogenized
prior
to
assay
for
protein
and
P450
content
and
aromatase
activity.
The
protein
content
of
the
bovine
placental
microsomes
was
determined
to
be
ca.
45
mg/
mL.
The
total
protein
yield
for
the
preparation
was
calculated
to
be
ca.
675
mg.
This
exceeds
the
250
mg
of
protein
criteria
set
in
the
protocol.
P450
content
of
the
bovine
placental
microsomes
was
determined
to
be
ca.
0.031
nmol/
mg
protein.
The
aromatase
activity
of
the
bovine
placental
microsomes
was
ca.
3
pmol
estrogen
formed/
mg
protein/
min;
this
is
in
good
agreement
with
the
3.62
±
0.78
pmol
estrogen
formed/
mg
protein/
min
reported
by
Tsumagari
et
al.
(
1993).

4
Conclusion
The
bovine
placental
microsomes
have
sufficient
protein
and
P450
content
for
the
conduct
of
these
studies.
The
aromatase
activity
for
the
preparation
was
similar
to
that
reported
in
the
literature
and
is
sufficient
to
proceed
with
the
optimization
phase
of
the
study.

5
References
Omura,
T.;
Sato,
R.
The
carbon
monoxide
binding
pigment
of
liver
microsomes.
I.
Evidence
for
its
hemoprotein
nature.
J.
Biol.
Chem.
1964,
239,
2370­
2378.

Tsumagari,
S.,
Kamata,
J.,
Takagi,
K.,
Tanemura,
K.,
Yosai,
A.,
Takeishi,
M.
Aromatase
activity
and
oestrogen
concentrations
in
bovine
cotyledons
and
caruncles
during
gestation
and
parturition.
J.
Reprod.
Fert.
1993,
98,
631­
36.
