In Vitro Metabolic Enzyme Activity Characterization in
Cryopreserved, Freshly Isolated Hepatocytes and
Liver S9 Fraction from Rainbow Trout
Karla Johanning, Lisa Dungan, Rachel Whisnant, Kristen Lohnes, Cornelia Smith, Kirsten
Amaral, Rebecca Gauvin, Matthew Palmer, Kevin Lehnert and Jeanette Hill
Cell Systems Division • Life Technologies • 1624 Headway Circle • Austin, Texas 78754 • USA
A
•
Metabolic enzyme activities
(Phase I and II): results are
comparable in both fresh and
cryopreserved hepatocytes.
•
Likewise, trout liver S9 showed
similar results in metabolic
enzyme activities.
•
In vitro metabolism utilizing
trout liver S9 fraction and
hepatocytes can be used to
determine
the
BCF
and
therefore
bioaccumulation
potential of chemicals.
•
The rainbow trout liver S9
fraction metabolic assay for the
BCF determination is currently
under further optimization.
•
Fish (e.g. trout and carp)
hepatocytes may represent an
additional tool to determine
bioaccumulation as the human
hepatocytes are considered the
gold standard when assessing
drug metabolism.
Formation of Estradiol-3-Glucuronide (250 µM
Estradiol) in Cryopreserved Trout Hepatocytes
(Tr110)
0.35
Estradiol-3-Glucuronide
(µg/mL)
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0.3
0.25
0.2
0.15
0.1
0.05
T
te
H
ub
str
a
o
S
24
0
24
0
24
0
N
24
0
C
ell
s
o
N
N
o
24
0
H
24
0
60
0
T
Su
bs
tra
te
ell
s
24
0
C
24
0
N
o
60
12
0
0
30
12
0
0
0
30
Estradiol-3-Glucuronide
(µg/mL)
Conclusions
B
Formation of Estradiol-3-Glucuronide (250 µM Estradiol)
in Fresh Trout Hepatocytes (Tr110)
Time (minutes) or Control Treatment
Time (minutes) or Control Treatment
Incubation Conditions: 250 µM Estradiol,
0.5 x 106 cells/mL, pH 7.8, 12°C.
C
E 3 -G lu c u ro n id e
(µg /m L )
Formation of Estradiol-3-Glucuronide in S9 Trout
Liver Fractions (Tr007)
0
12
90
60
30
0
0.6
0.5
0.4
0.3
0.2
0.1
0
Time (minutes)
Figure 1 – Testosterone hydroxylation
results are comparable in both trout fresh
(A) and cryopreserved hepatocytes (B)
Formation of 6ß-Hydroxytestosterone (500 µM Testosterone) in Fresh Male Trout
Hepatocytes (Tr110)
600
500
400
300
Disappearance of Test Chemical A (0.5 µM) in
Trout Liver S9 Fraction
Use
d to
120
100
80
dete
rmin
Active S9
Heat Treated
ek
ME
T
60
No S9
40
20
12
0
0
90
Formation of 6ß-Hydroxytestosterone (500 µM Testosterone) in Cryopreserved Male
Trout Hepatocytes (Tr110)
800
140
0
tra
Su
bs
No
0
24
Time (minutes) or Control Treatment
% Remaining Parent
te
T
ls
H
0
Ce
l
24
0
N
o
24
0
0
24
60
12
0
30
0
60
200
100
B
Estradiol,
Figure 4 – Model for whole body
transformation rate and BCF determination
from in vitro metabolism data.
(Cowan-Ellsbery et al, 2008)
40
6ß-Hydroxytestosterone
(ng/mL)
700
Incubation Conditions: 250 µM
1mg/mL, pH 7.8, 12°C.
20
A
800
Time (minutes)
700
600
Incubation Conditions:
S9 protein: 2 mg/mL
T = 12 oC
500
400
300
200
S9 Hepatic Intrinsic Clearance Rate
of Parent Chemical CLm
100
tra
te
T
H
ls
Su
bs
24
0
N
o
24
0
N
o
24
0
Ce
l
24
0
60
12
0
0
30
0
Hepatic Clearance
CLH
Time (minutes) or Control Treatment
Incubation Conditions: 500 µM Testosterone,
Metabolic Transformation rate
kMET
0.5 x 106 cells/mL, pH 7.8, 12°C.
Figure 2 – Testosterone hydroxylation in
liver S9 fraction
Formation of 6ß-Hydroxytestosterone (500 µM
Testosterone) in S9 Trout Liver Fractions (Tr007)
Bioconcentration Factor
BCF
500
400
300
200
100
0
12
90
60
30
0
0
Methods
Rainbow trout were maintained
under
natural
photoperiod
(16L:8D) and temperature (about
17 oC) at the ABC Labs (Columbia,
MO) (for hepatocytes and liver S9
fraction). Fish were acclimated for
a week prior to sacrifice. Fish were
euthanized with MS-222, livers
flushed with HBSS (10 mM HEPES,
2.3 mM EDTA; pH 7.8), excised
and transported to one of our
facilities for further perfusion using
collagenase. In addition, another
set of trout were utilized for liver
S9 fraction preparation. Briefly,
whole livers were homogenized in
50 mM Tris-HCl pH 7.8, 150 mM
KCl, 2 mM EDTA, 1 mM DTT and
spun at 13,000 x g. Livers were
homogenized in buffer with or
without 250 mM sucrose and
stability overtime was determined
(presented elsewhere).
Figure 3 – Estradiol glucuronidation in trout
fresh (A) and cryopreserved hepatocytes (B)
as well as in liver S9 (C) is similar
Results
6ß-Hydroxytestosterone
(ng/mL)
Assessment of bioaccumulation for
all chemical substances with log
Kow ≥3 and produced in quantities
of >100 tons / year will be required
by regulatory agencies. We are in
the process of pre-validating an in
vitro assay utilizing rainbow trout
liver S9 fraction to determine the
metabolic
fate
for
different
categories of test chemicals. The
in vitro metabolism data generated
will be used in a bioconcentration
factor (BCF) assessment model.
Besides the trout liver S9 fraction,
hepatocytes may provide additional
information of the metabolic
turnover of the chemicals in
question. The objective of the
present study was to determine
and
compare
the
metabolic
enzymatic
activities
in
cryopreserved, freshly isolated
hepatocytes and liver S9 fraction
from rainbow trout. The results
may provide insight of any changes
in enzymatic activities as a function
of cryopreservation procedures.
Livers were flushed with HBSS,
excised and transported to another
facility for further perfusion and
processing. Batches of hepatocytes
were separated according to sex. A
portion of each lot was used to
assess enzyme activities in fresh
hepatocytes and the remainder was
subjected to cryopreservation. In
addition, livers were also collected
and S9 fraction obtained for further
analysis of enzyme activities.
Metabolic enzymes activities (Phase
I and II) were determined in fresh,
cryopreserved hepatocytes and
liver
S9
fractions.
Metabolic
stability of test chemicals was
determined in order to assess
metabolic rates. The
results
indicate that there are no
significant differences between
fresh
and
cryopreserved
hepatocytes
when
metabolic
enzyme activities are compared.
Liver S9 fraction enzyme activities
are being further optimized to
attain high enzyme activities.
Rainbow trout hepatocytes and
liver fractions may be considered
powerful tools to determine the
BCF
using
metabolic
rates
in
vitro
generated
from
metabolism assays.
A portion of the resulting supernatant (S9) was stored at
-80°C. Protein determination was performed using the
Micro Lowry. Different batches of liver S9 fractions were
prepared and separated according to sex. Metabolic
enzymes: Phase I: lauric acid (data not shown) and
testosterone hydroxylation; Phase II: 7OHCMN
glucuronidation and sulfation (data not shown) and
estradiol glucuronidation were determined in fresh and
cryopreserved (one month after cryopreservation)
hepatocytes as well as in trout liver S9 fractions.
Hepatocytes incubations: Trout hepatocytes were
diluted with DMEM (pH 7.8) containing 10% fetal bovine
serum (FBS), centrifuged at 700 rpm for five min at
approximately 4 oC and resuspended in culture media
without FBS. Cells were centrifuged and decanted again
and resuspended in fresh culture media without FBS.
Cells were diluted to a final density of 0.5 x 106 cell/mL.
Hepatocytes were incubated up to 240 min. Liver S9
incubations: All incubations were performed in 100 mM
phosphate buffer pH 7.8 up to 120 min according to the
specific assay. All incubations (hepatocytes and S9)
were performed at T= 12 oC mimicking the trout natural
environmental conditions.
6 ßH yd rox y te s to ste ro n e
(n g /m L )
Introduction
Time (minutes)
Incubation Conditions: 500 µM Testosterone,
1mg/mL, pH 7.8, 12°C.
Acknowledgments
We would like to thank the ABC Labs (Columbia, MO),
specifically Jon Rhodes, Christine Lehman, John
Aufderheide and Paul Cohle for their collaboration and
assistance in the sampling procedures and for the trout
maintenance and acclimation prior to the liver perfusions.
References
Cowan-Ellsberry, C., Dyer, S.D., Erhardt, S.,
Bernhard, M.J., Roe, A.L., Dowty, M.E., and
Weisbrod, A.V. 2008. Approach for
extrapolating in vitro metabolism data to refine
bioconcentration factor estimates.
Chemosphere 70:1804-1817.