Document ID: EPA-HQ-OPPT-2003-0016-0018
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2003-06-16T04:00Z

Optimization
of
the
Sliced
Testis
Steroidogenesis
Assay
Carol
S.
Sloan,
Amanda
B.
Goodman,

Carol
S.
Sloan,
Amanda
B.
Goodman,

Rochelle
Tyl
Rochelle
Tyl
RTI
International
RTI
International
June
2003
June
2003
Background

The
DRP
named
several
possible
procedures
to
The
DRP
named
several
possible
procedures
to
study
steroidogenesis
and
its'
ability
to
study
steroidogenesis
and
its'
ability
to
characterize
the
endocrine
effects
of
various
characterize
the
endocrine
effects
of
various
environmental
contaminants,
industrial
substances
environmental
contaminants,
industrial
substances
and
pesticides
and
pesticides

Tissue
culture
Tissue
culture

Purified
cell
preparations
Purified
cell
preparations

Cell
lines
Cell
lines

Ovarian
Ovarian
in
vitro
in
vitro
steroidogenesis
assay
steroidogenesis
assay

Testicular
Testicular
in
vitro
in
vitro
steroidogenesis
assay
steroidogenesis
assay
Why
Sliced
Testis
Assay?

Minimal
Cost
Minimal
Cost

Quick
Quick

Uses
Standard
Laboratory
Equipment
Uses
Standard
Laboratory
Equipment

Basic
Laboratory
Training
Basic
Laboratory
Training

Stable
Preparation
Stable
Preparation

Uses
Reduced
Number
of
Animals
Uses
Reduced
Number
of
Animals

Can
be
Standardized
Can
be
Standardized

Well
Well­
defined
endpoint
in
testosterone
defined
endpoint
in
testosterone
concentration
concentration

Can
be
modified
to
use
intermediate
hormonal
Can
be
modified
to
use
intermediate
hormonal
endpoints
endpoints
Optimization
of
the
Sliced
Testis
Assay
This
was
planned
to
be
Implemented
This
was
planned
to
be
Implemented
in
Two
Phases
in
Two
Phases
Prototypical
Sliced
Testis
Assay

Testes
are
weighed
and
placed
in
DPBS
Testes
are
weighed
and
placed
in
DPBS
buffer
buffer

Each
testis
is
sliced
along
the
longitudinal
Each
testis
is
sliced
along
the
longitudinal
axis
into
slices
of
proper
weight
axis
into
slices
of
proper
weight

Slices
are
placed
with
5mL
of
media
Slices
are
placed
with
5mL
of
media

Incubated
at
34
Incubated
at
34oC
in
5%
CO
C
in
5%
CO
2
and
95%
air
and
95%
air
on
a
shaker
on
a
shaker
Sliced
Testis
Assay

At
first
time
At
first
time­
point
point
 
baseline
baseline
 
media
is
removed
media
is
removed
and
discarded
and
discarded

Fresh
media
is
added
and
an
aliquot
is
collected
Fresh
media
is
added
and
an
aliquot
is
collected

Half
of
the
samples
are
challenged
with
a
Half
of
the
samples
are
challenged
with
a
stimulant,
such
as
hCG
stimulant,
such
as
hCG

Aliquots
are
collected
at
1,
2,
3
and
4
hours
post
Aliquots
are
collected
at
1,
2,
3
and
4
hours
post­

challenge
challenge

Samples
are
analyzed
for
testosterone
Samples
are
analyzed
for
testosterone
concentration
in
a
RIA
assay
concentration
in
a
RIA
assay
Technical
Flow
Illustration
of
Sliced
Testis
Assay
Phase
1:
Preliminary
Experimental
Phase

Establish
whether
a
given
level
of
each
Establish
whether
a
given
level
of
each
factor
affects
assay
performance
factor
affects
assay
performance

The
factors
are
unlikely
to
have
an
The
factors
are
unlikely
to
have
an
interaction
or
at
best
a
minimal
interaction
interaction
or
at
best
a
minimal
interaction
with
another
experimental
factor
with
another
experimental
factor

An
effect
of
one
of
these
factors
would
An
effect
of
one
of
these
factors
would
require
additional
verification
experiments
require
additional
verification
experiments
after
sensitivity
analysis
after
sensitivity
analysis
Phase
I
Design
Phase
II:
Primary
Experimental
Phase

Factors
that
may
affect
assay
performance
Factors
that
may
affect
assay
performance
were
tested
were
tested

These
factors
were
divided
into
four
These
factors
were
divided
into
four
sections
where
each
section
was
composed
sections
where
each
section
was
composed
of
factors
that
might
produce
interactions
of
factors
that
might
produce
interactions
Phase
II
Design
Testosterone
Radioimmunoassay

RIA
RIA

Used
to
measure
testosterone
concentration
Used
to
measure
testosterone
concentration
of
the
aliquots
taken
at
various
time
of
the
aliquots
taken
at
various
time­
points
points

Commercial
kit
Commercial
kit

Utilizes
125
I
Utilizes
125
I­
testosterone
and
a
testosterone
and
a
testosterone
testosterone­
specific
antibody
affixed
to
specific
antibody
affixed
to
tubes
tubes
Testosterone
RIA

Verification
of
the
assay
using
Media
199
Verification
of
the
assay
using
Media
199
without
phenol
red
without
phenol
red

Determination
of
assay
accuracy,

Determination
of
assay
accuracy,

sensitivity,
precision
and
parallelism
sensitivity,
precision
and
parallelism
Testosterone
RIA
Validation
with
M­
199
Lactate
Dehydrogenase
(
LDH)

Assay

Validation
of
the
assay
using
Media
Validation
of
the
assay
using
Media­
199
199
without
phenol
red
without
phenol
red

Measuring
the
assay
accuracy,
sensitivity,

Measuring
the
assay
accuracy,
sensitivity,

and
precision
and
precision

Performed
at
Laboratory
Corporation
of
Performed
at
Laboratory
Corporation
of
America
America
Phase
I

Tested:

Tested:


Media
Type
Media
Type
 
Eagles
MEM
Eagles
MEM
 
RPMI
RPMI­
1640
1640
 
Medium
Medium­
199
199

Gaseous
Atmosphere
Gaseous
Atmosphere
 
5%
CO
5%
CO
2
/
95%
air
/
95%
air
 
5%
CO
5%
CO
2
/
95%
O
/
95%
O
2
 
Air
Air

Rat
Age
Rat
Age
 
11
weeks
11
weeks
 
15
weeks
15
weeks
 
22
weeks
22
weeks
PHASE
I:
Experimental
Design
Phase
I
Optimizations

Medium
Medium­
199
without
phenol
red
199
without
phenol
red

Gaseous
atmosphere
of
5%
CO
Gaseous
atmosphere
of
5%
CO
2
/
95%
O
/
95%
O
2

Rats
that
were
11
weeks
of
age
showed
Rats
that
were
11
weeks
of
age
showed
results
similar
to
those
that
were
15
weeks
results
similar
to
those
that
were
15
weeks
of
age,
therefore
rats
11
of
age,
therefore
rats
11­
15
weeks
can
be
15
weeks
can
be
used
in
the
assay
used
in
the
assay
Phase
II

To
be
tested:

To
be
tested:


Incubation
Temperature
Incubation
Temperature

Incubation
Vessel
Type
Incubation
Vessel
Type

Incubation
Shaker
Speed
Incubation
Shaker
Speed

Incubation
Media
Volume
Incubation
Media
Volume

hCG
Concentrations
hCG
Concentrations

Testicular
Fragment
Size
Testicular
Fragment
Size

Time
Delay
before
starting
the
assay
Time
Delay
before
starting
the
assay
preparation
preparation

Organ
Preparation
Technique
Organ
Preparation
Technique

Sample
Aliquot
Volume
Sample
Aliquot
Volume
Technical
Flow
of
Sliced
Testis
Assay
Phase
II
Optimization
Currently
Being
Analyzed
Currently
Being
Analyzed