Rusyn2_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Procedures
Data
Definitions
References
Workflow
and
sampling
Workflow
|
Procedure
|
|
Equipment |
Data
collected |
|
Male
mice
(unfasted)
of
all
cohorts
are
weighed
|
pre-treatment
|
|
body
weight |
|
Control
and
treated
cohorts
are
given
(via
gavage)
vehicle
(control)
or
TCE |
0 |
|
-
|
|
Naive,
control,
and
TCE
treated
mice
are
weighed
and
blood
sample
collected;
mice
are
euthanized
for
liver
and
kidney
histology |
2,
8,
24
hrs
|
|
body
and
liver
weight
|
|
Frozen
serum
are
thawed
and
analyzed
for
biological
markers
of
tissue
injury |
- |
|
serum
ALT,
AST,
BUN |
|
Frozen
serum
are
thawed
and
analyzed
for
TCE
metabolites
|
- |
|
serum
DCA,
TCA,
DCVC,
and
DCV
|
*High-performance
liquid
chromatography/electrospray
ionization
tandem
mass
spectrometry
Equipment
- Small
rodent
dissection
pack
(scalpel,
forceps,
scissors,
etc.)
- Centrifuge,
tabletop
- Freezer:
-80°C
•
Blood
chemistry
analyzer:
Automatic
Chemical
Analyzer,
Johnson
&
Johnson's
VT250
•
High-performance
liquid
chromatography/electrospray
ionization
tandem
mass
spectrometry
(HPLC-ESI-MS/MS)
with
a
Finnigan
Surveyor
autosampler
and
pump
coupled
to
a
Finnigan
TSQ
Quantum
triple-quadruple
mass
spectrometer
(Thermo
Finnigan,
San
Jose,
CA)
•
High-performance
liquid
chromatography/electrospray
ionization
tandem
mass
spectrometry
(HPLC-ESIMS/MS)
with
an
Acquity
UPLC®
(Waters,
Milford,
MA)
system
coupled
to
a
TSQ
Quantum
Ultra
triple
quadruple
mass
analyzer
(Thermo
Finnigan,
San
Jose,
CA)
Reagents,
supplies,
solutions
- Gavage
system
for
mice
- Pressurized
CO2
gas
for
euthanasia
- Trichloroethylene
(TCE)
(2100
mg/kg)
in
corn
oil
(vehicle,
10
mL/kg)
- Corn
oil
(vehicle)
- Needles
and
syringes
- Z-gel
tubes
(Sarstedt,
Germany)
Acclimation
to
test
conditions
Mice
are
acclimated
to
the
new
facility
for
at
least
2
wks.
Procedures
I.
Treatment,
euthanasia,
blood
collection,
and
kidney
and
liver
dissection
a.
Unfasted
male
mice
are
treated
(at
9
am)
orally
by
gavage
with
TCE
(2100
mg/kg)
in
corn
oil
(vehicle,
10
mL/kg).
b.
Final
body
weight
is
obtained
before
mice
are
euthanized.
c.
Mice
are
sacrificed
at
2,
8
and
24
hrs
after
treatment.
The
experimental
design
is
selected
based
on
the
previous
pharmacokinetic
analysis
of
TCE
metabolism
in
male
B6C3F1
mice
(Kim
et
al.,
2009).
d.
The
mice
are
euthanized
by
fully
trained
personnel
using
CO2
gas
asphyxiation.
e.
Blood
samples
are
collected
via
the
vena
cava
for
determination
of
tissue
damage
using
serum
biomarkers
and
TCE
metabolites.
f.
Blood
samples
collected
without
anti-coagulant
are
allowed
to
clot
for
~30
min
before
they
are
centrifuged.
g.
Serum
is
prepared
by
centrifugation
using
Z-gel
tubes
according
to
the
manufacturer
instructions,
separated,
stored
frozen
at
-80°C
until
ready
to
be
analyzed.
h.
The
frozen
serum
is
completely
thawed
before
testing
for
serum
alanine
transaminase
(ALT),
aspartate
aminotransferase
(AST),
and
blood
urea
nitrogen
(BUN)
levels
using
standard
blood
chemistry
analyzer
according
to
manufacturer's
instruction.
II.
Determination
of
TCE
metabolites
in
the
serum
(see
Kim
et
al.,
2009
for
greater
details)
a.
Concentrations
of
DCA
and
TCA
are
measured
using
HPLC-ESI-MS/MS
with
autosampler
and
pump
coupled
to
triple-quadruple
mass
spectrometer.
b.
Determination
of
DCVC
and
DCVG
is
also
conducted
using
HPLC-ESI-MS/MS
with
an
Aquity
UPLC®
system
coupled
to
a
TSQ
Quantum
Ultra
triple
quadruple
mass
analyzer
using
a
heat-assisted
electrospray
ionization
source
in
positive
ion
mode.
c.
Quantification
is
based
on
peak
areas
relative
to
the
stable
isotope-labeled
internal
standards
and
the
calibration
curve
is
prepared
in
each
batch.
d.
Limits
of
detection
are
determined
by
a
signal-to-noise
ratio
of
3:1,
such
that
TCA
-
0.4
nmol/mL;
DCA
-
0.01
nmol/mL;
DCVG
and
DCVC
-
0.001
nmol/mL.
Data
collected
by
investigator
•
Serum
alanine
aminotransferase
(ALT),
aspartate
aminotransferase
(AST),
and
blood
urea
nitrogen
(BUN)
in
naive,
control,
and
TCE-treated
groups
at
2
h,
8
h,
and
24
h
post-treatment.
•
TCE
metabolites
in
the
serum:
trichloroacetic
acid
(TCA),
dichloroacetic
acid
(DCA),
S-(1,2-dichlorovinyl)-L-cysteine
(DCVC),
and
S-(1,2-dichlorovinyl)-L-glutathione
(DCVG)
in
naive,
control,
and
TCE-treated
groups
at
2
h,
8
h,
and
24
h
post-treatment.
•
Body
and
liver
weights
in
naive,
control,
and
TCE-
treated
groups
at
2
h,
8
h,
and
24
h
post-treatment.
Definitions
&
formulas
dichloroacetic
acid
(DCA):
a
major
metabolite
of
TCE.
S-(1,2-dichlorovinyl)-L-cysteine
(DCVC):
oxidative
metabolite
of
TCE.
S-(1,2-dichlorovinyl)
glutathione
(DCVG):
glutathione-conjugated
metabolite
of
TCE.
trichloroacetic
acid
(TCA):
a
major
metabolite
of
TCE.
trichloroethylene
(TCE):
is
widely
used
as
an
industrial
chlorinated
organic
solvent.
It
is
found
in
a
variety
of
sources
such
as
indoor
and
ambient
air,
soil
and
groundwater.
Human
exposure
can
lead
to
hepatotoxicity,
neurotoxicity,
nephrotoxicity,
and
auto-immune
disorders.
|
|
only
Test age:
7wks