Vol.
95, No.
July
16. 1980
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
ELEVATION
OF HEPATIC EPOXIDE HYDRATASE ACTIVITY BY ETHOXYQUIN IS DUE TO INCREASED SYNTHESIS OF THE ENZYME Regine
Kahl
Department of Pharmacology, Obere Zahlbacher Strasse 67, Received
163-169
May
University of Mainz D-6500 Mainz, Germany
19,198O
SUMMARY. Feeding of the antioxidant ethoxyquin to rats leads to an increase The apparent half life of of epoxide hydratase activity in liver microsomes. the increase is 3-4 days. Elevation of epoxide hydratase activity is also obtained by intraperitoneal treatment of mice with ethoxy-quin. This elevation is prevented by concomitant treatment with cycloheximide. When radiolabelled leutine is incorporated intn microsomal protein by liver cell fractions from either gel electrophoresis reveals that ethoxyquin ethoxyquin-fed or untreated rats feeding increases incorporation into epoxide hydratase. These results suggest that the elevation of epoxide hydratase activity by ethoxyquin is due to increased biosynthesis of the enzyme, i.e. enzyme induction. Evaluation oxidants
of microsomal
ethoxyquin,
butylated
has been demonstrated other
Moreover, pounds
shown
phenobarbital-inducible ronosyl
cal
if
The mechanism
tase
in rat
of a polypeptide grates
with
elevation not merely
a partially
(7,8,1,4),
anti-
tissues
(5,6).
of foreign
com-
antioxidants: b5 (8),
(IO).
antioxidants
It
is
glucu-
has been
against
(11-14)
some chemi-
related
to such
elevate
We have demonstrated
that
liver
feeding
by antioxidant
purified
the
hydroxyanisole
cytochrome
hydrocarbons
dodecyl
these
S-transferase
the antioxidants
is due to increased to activation
and butylated
by feeding
enzymes.
in sodium
by feeding
in the metabolism
of these
polycyclic
by which
elucidated.
activity
to increase
effect
metabolizing
activity
and extrahepatic
involved
and glutathione
including
on drug
yet been
(9)
the protective
carcinogens
effects
(l-4)
monooxygenases
transferases
discussed
liver
activities
been
hydratase
hydroxytoluene
in rodent
enzyme
have also
epoxide
sulfate epoxide
concentration
of preexisting
enzyme activities
the
increase is
in epoxide
related
polyacrylamide hydratase.
This
However,
to the gels
comithat
in the it
hydraincrease
which
indicates
of the enzyme enzyme.
has not
is not
liver
the and
clear
if
0006-291X/80/130163-07$01.00/0 163
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Copyright 0 1980 rights of reproduction
by Academic Press, Inc. in any form reserved.
Vol.
95, No.
the
1, 1980
increase
AND
in enzyme concentration
antioxidants synthesis
BIOCHEMICAL
against
nication
to provide
in rat
and mouse liver
(i.e.
evidence after
(i.e.
induction). that
RESEARCH
is due to a protective
enzyme degradation
of the enzyme
BIOPHYSICAL
It
stabilization) is
the elevation
feeding
COMMUNICATIONS
effect
of the
or to increased
the aim of the present of epoxide
of ethoxyquin
is
hydratase
commuactivity
due to enzyme induction.
METHODS For feeding experiments, male Sprague-Dawley rats with an initial body weight of 120 g received a powdered Altromin @ diet with I % ethoxyquin (kindly donated by Lohmann Tierernahrung, Cuxhaven). The animals were maintained on this diet for 14 days and were sacrificed at various intervals after stop of ethoxyquin feeding. For experiments on inhibition of protein synthesis male NMRI mice (20 g) received 25 mg cycloheximide/kg (in 0.9 % NaCl) i.p. at 8 a.m., 2 p.m., 4 p.m. and IO p.m.; animals were sacrificed at 8 a.m. Preparation of microsomes (2) and determination of e oxide hydratase activity (15, 16) were performed as described previously. oxide and[3HFbenzo[a]r148 styrene pyrene 4,5-oxide were a generous gift from Professor F. Oesch, Mains. Cell free protein synthesis was measured by the incorporation of PHlleucine into hepatic microsomal protein from either ethoxyquin-fed or untreated rats. The reaction mixture contained in a total volume of 0.5 ml: potassium phosphate buffer (pH 7.4), 12 mM; freshly prepared microsomal protein, 2 mg; freshly prepared 100 000 x g supernatant from the respective livers, 50 ~1 (approx. ATP, 1.5 mM; GTP, 0.3 mM; phosphoenol pyruvate, 15 mM; 1 4; MgC12, 12 ti; pyruvate kinase, 50 ug; sucrose, 75 mM; 2-mercaptoethanol, 6 mM;[3H$leucine, 16 UM (0.2 mCi). This incubation was similar to that described by Weksler and Gelboin (17) who used the creatine phosphokinase system instead of the pyruvate kinase system. The incubation was carried out for 60 min at 37 OC and stopped by the addition of 1.0 ml IO % trichloro acetic acid. The protein precipitate was centrifuged down and washed with acetone/O.1 M acetic acid, ethanol and ethyl ether. After drying it was dissolved in 1.0 ml of gel electrophoresis sample buffer (consisting of 0.5 M Tris, pH 6.8, 10 % glycerol, 1 % sodium dodecyl sulfate and 1 % 2lnercaptoethanol); a few drops of 1 N NaOH were added to facilitate dissolving. The solution was heated to 100 'C for a few minutes. 0.05 % bromphenol blue were added before electrophoresis. Discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out according to Laemmli (18); a partially purified rat liver epoxide hydratase (kindly donated by Professor F. Oesch, Mainz) was included as a standard. For densitometricmeasurement of epoxide hydratase subunit concentration the gels were stained with Coomassie B$e and scanned in a Gilford 2400 spectrophotometer. For measurement of pa-1 eucine incorporation four walls each of a 10 wall slab gel contained the protein from the ethoxyquin experiment and four walls that of the control experiments while two walls contained the standard epoxide hydratase. After electrophoresis, the standard walls and one wall each from the ethoxyquin and control walls were stained for protein while the remaining three walls of each preparation were cut into 0.9 mm slices. The slices were resolved in 30 % H202 by incubation at 60 'C for 36 hr and radioactivity was quantitated by liquid scintillation counting. RESULTS The time the substrates
dependence styrene
of the elevation oxide
of epoxide
and benzo[a]pyrene
164
hydratase
4,5-oxide
activity and increase
towards of the
vol.
95, No.
1. 1980
BIOCHEMICAL
O
4-1’ -EO-
AND
i-I
3
1 71
5
I
COMMUNICATIONS
stop of EO
Decline of the elevation of epoxide hydratase activity in rat liver microsomes and of the epoxide hydratase-containing band in polyacrylamide gels of rat liver microsomes after stop of ethoxyquin feeding. Means of two experiments are plotted. Upper part: enzyme activity towards styrene oxide (SO). Middle part: Enzyme activity towards benzo[alpyrene 4,5-oxide (BPO). Lower part: Epoxide hydratase (EH)-containing band in sodium dodecyl sulfate polyacrylamide gels given in percentage of total microsomal protein found in the molecular weight region between 200 000 and 15 000. The molecular weight of the standard epoxide hydratase was 49 000.
epoxide
hydratase-containing
protein
band
ted
rat
in Fig.
1 The elevated
for
within life
RESEARCH
1L days after
1% ECI ?L days
Fig.
BIOPHYSICAL
liver
3-4
days after
of the enzyme.
values
are obtained
Two types activity
is
hydratase
stop
of ethoxyquin.
Increased
levels
are
after
stop
at 14 days
of experiments
were
due to enzyme induction,
due to the antioxidant:
of protein
synthesis,
liver
fractions
cell
microsomes
2) in vitro from
in polyacrylamide
This still
activity
may reflect
observed
after
to test increased
if
the
biosynthesis
incorporation
of radiolabelled
165
the half
7 days;
elevation
treatment
animals.
by 50 %
basic
feeding.
1) ethoxyquin
ethoxyquin-treated
is demonstra-
decreases
decrease
of ethoxyquin
performed i.e.
gels
during
of enzyme of epoxide inhibition leucine
by
Vol.
95, No.
1, 1980
Feeding
of ethoxyquin
of epoxide survive
BIOCHEMICAL
hydratase
leads
activity
within
However,
period
the mouse but was much less
shows
ethoxyquin
that
there
mouse liver
true
plus
enzyme This
this
induction
conclusion
either
microsomes
assay.
proteins teins.
shows
the molecular
identical
distance
ethoxyquin
band
has a molecular inducible
form
Fig.
2
activity
provides
in
with
intact
for
50 000, the
protein
syn-
evidence
for
assay.
weight
of about
of cytochrome
a cell
most
tissue
for
is incorporated
true
of the pro-
two bands from ethoxy-
bands
finding
can be identi-
indicates epoxide
enzyme
after
52 000 and may thus
protein
to the migration
to synthetize
evidence
free
into
fractions
one of these This
of
of the separated
By comparison
enzyme subunit.
the livers
hydratase
induction.
ethoxyquin
contain
that
The treatment
the phenobarbital-
P-450. DISCUSSION
Evidence liver
dant
is
that
derived
by an inhibitor
a
3. For
electrophoresis
when cell
the
more label
for
is observed
hydratase
which
gel
into
of Fig.
from
used
after
epoxide
further
experiment
and were
incorporation
has enabledliver
and provides into
in
In the rat,
treatment
that
prepared
incorporation
around
used
one containing
more rapidly other
region
vitro
sap were rats
leucine
authentic
feeding
not
of enzyme
protocol.
hydratase
and thus
profile
label
were
of the
as the
and cell
increased
liver
this
indicates
will
of the antioxidant
to a combined
effect
or untreated
weight
quin-treated
after
increase
animals
by feeding.
in epoxide
by the in
The radioactivity
However,
developing
Elevation
obtained
finding
elevation
COMMUNICATIONS
mechanism.
ethoxyquin-fed
synthesis
the
doses
subjected
This
was stressed
experiment,
fied
for
i.p.
that
an increase
cycloheximide.
is necessary
than
were
slowly
24 hr.
was observed
was no longer
when the animals
ethoxyquin thesis
effect
but
than
by three
marked
RESEARCH
cycloheximide-treated
much longer
24 hr was achieved
no significant
BIOPHYSICAL
to a very marked
activity.
an experimental
AND
ethoxyquin from
the
leads inhibition
of protein
to induction
of epoxide
of the elevating synthesis
166
on one hand
hydratase
effect (Fig.
in rodent
of the antioxi2) and from
in-
in
vol.
No.
95,
BIOCHEMICAL
1, 1980
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Front
CDrn at
f
-
Fig.
2
Prevention mouse liver by inhibition
of the elevation of epoxide hydratase activity in after intraperitoneal administration of ethoxyquin of protein synthesis. Values are means + S.E. * P-Z= 0.05. The substrate was benzo[a]pyrene 4,5-oxide. (n = 3), BP: benzo[a]pyrene, CO: control, EQ: ethoxyquin, CH: cycloheximide.
Fig.
3
SDS polyacrylamide gel electrophoresis of labelled proteins after incorporation of[3$leucine in vitro hepatic microsomal protein from ethoxyquin-fed and untreated rats. Gels were sliced and processed for measurement of radioactivity. Identification of epoxide hydratase (EH) was achieved by comparison with the migration of an authentic epoxide hydratase present in another wall of the same slab gel. CO: control incubation with microsomes and cytosol of the livers of untreated rats. EQ: incubation with microsomes and cytosol from the livers of ethoxyquin-fed rats.
creased
incorporation
hand
(Fig.
also
been
tase, by
P-450
leucine
(19),
into by
and
epoxide
inhibition
inducer
of
incorporation
employed
induction
(20) effects
mulation
of
periment
since 1).
Uitro
elevation another
oxide
method
ference
(Fig.
for
in
of
it
is
hydratase of
hepatic
likely
on
the
other
protein
synthesis
has
microsomal
epoxide
hydra
that
this
inducer
also
acts
induction.
measure
viuo
of
demonstrated
enzyme
leucine
Prevention
trans-stilbene
To in
3).
of
to on
newly
by
we
Dehlinger
avoid
Schmassmann
enzyme life
and
of
of Oesch
but
the
induced
(19)
have
167
in
Schimke
Stabilization
turnover.
half
and
an
reutilization
synthetized the
used
for
within
study
also
of
short
epoxide
found
than
to in
the
cytochrome and
lead
was the
rather
leucine
the
activity tested
the
labelled should
not
method
Vitro
interthe
accu-
vitro
ex-
to hydratase
be
3-4
days inducer
Vol.
95, No.
BIOCHEMICAL
1, 1980
trans-stilbene (12 days) effect
to that
of about
It
should
leucine gel
oxide
be an advantage
in
P-450 these
oxide
hydratase
been
synthetized
with
its
weight
from rat properties, molecular
a similar
half
life
and that
of
weight.
It
acid
methods
decided machinery identical
iz
of three
different These
weight,
if
and with with
that
cytochrome
microsomal
forms forms
its
to provide
the its
formed
study
membranes
of epoxide
differ
by immunological specificity
by differential
postulated
The able
technical
assistance
supported
by the Deutsche
of Ms. Ll. Folz
is
epoxide
but not
hydratase
acknowledged.
REFERENCES I. Kahl, R., and Netter, K.J. (1977) Toxicol. appl. Pharmacol. 40, 474-483. B., and Klaus, E. (1978) Biochem. biophys. 2. Kahl, R., Decker-s-Schmelzle, Res. Commun. 85, 938-945. 3. Cha, Y.-N., Martz, F., and Bueding, E. (1978) Cancer Res. 38, 4496-4498.
168
by
purification
Forschungsgemeinschaft.
gratefully
are
hydratase
by ethoxyquin.
was financially
is
a molecu-
ACKNOWLEDGEMENTS This
own
P-448
of the enzyme with
to test
of these
of ep-
of 59 000 in conven-
and by substrate
be interesting which
that
weight
vitro
synthetized
composition
should
We therefore
form
have been
Had the enzyme
molecular
processing
the
synthesis
the identification
differing
the active
free
systems
weight.
protein
into
enzyme before
on cell
assay
a molecular
(23).
antibody
labelled
have demonstrated
form with
only
the
molecular
of a final
has been reported
and by innaunological induced
to be similar
translation
with
protein
(21)
assay. the purification
by amino
on its
impossible.
synthesis
of 53 000 is
liver
and found
hydratase
in our
protein
own microsomal
germ system
added to the Recently,
COMMUNICATIONS
in experiments
However,
have been
the
epoxide to prepurify
dependent
in a precursor
wheat
RESEARCH
with
and heterologous
Kumar and Padmanaban
tional
is
(21,22),
as a precursor would
synthetized
lar
in order
experiments.
sap to ensure viuo.
effect
ethoxyquin,
has been done
was solely
identification
in
for
of its
to include
assay This
of cytochromes
messenger
here
BIOPHYSICAL
4 days.
incorporation
employed
the duration
reported
electrophoresis.
cell
for
AND
forms
Vol.
95, No.
1, 1980
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
4. Kahl, R., and Wulff, U. (1979) Toxicol. appl. Pharmacol. 47, 217-227. 5. Benson, A., Cha, Y.-N., Bueding, E., Heine, H.S., and Talalay, P. (1979) Cancer Res. 39, 2971-2977. 6. Kahl, R. (1980) Cancer lett. 8, 323-328. 7. Allen, J.R., and Engblom, J.F. (1972) Food Cosmet. Toxicol. 10, 769-779. 8. Parke, D.V., Rahim, A., and Walker, R. (1974) Biochem. Pharmacol. 23, 1871-1876. 9. Bock, K.W., Kahl, R., and Lilienblum, W. (1980) Naunyn-Schmiedeberg's Arch. Pharmacol. 310, 249-252. 10. Benson, A.M., Batzinger, R.P., Ou, S.-Y.L., Bueding, E., Cha, Y.-N., and Talalay, P. (1978) Cancer Res. 38, 4486-4495. 11. Wattenberg, L.W. (1972) J. Natl. Cancer Inst. 48, 1425-1430. 12. Slaga, F.J., and Bracken, W.M. (1977) Cancer Res. 37, 1631-1635. 13. Weisburger, E.K., Evarts, R.P., and Wenk, M.L. (1977) Food Cosmet. Toxicol. 15, 139-141. 14. Wattenberg, L.W., Jerina, D.M., Lam, L.K.T., and Yagi, H. (1979) J. Nat. Cancer Inst. 62, 1103-1106. 15. Oesch, F., Jerina, D.M., and Daly, J. (1971) Biochim. Biophys. Acta 228, 685-691. 16. Schmassmann, H.U., Glatt, H.R., and Oesch, F. (1976) Anal. Biochem. 74, 94-104. 17. Weksler, M.E., and Gelboin, H.V. (1967) Biochim. biophys. Acta 145, 184-187. 18. Laemmli, U.K. (1970) Nature (Land.) 227, 680-685. 19. Schmassmann, H.U., and Oesch, F. (1978) Mol. Pharmacol. 14, 834-847. 20. Dehlinger, P-J., and Schimke, R.T. (1972) J. biol. Chem. 247, 1257-1264. G. (1980) J. biol. Chem. 255, 522-525. 21. Kumar, A., and Padmanaban, 22. Colbert, R.A., Bresnick, E., Levin, W., Ryan, D.E., and Thomas, P.E. (1979) Biochem. biophys. Res. Commun. 91, 886-891. 23. Guengerich, F.P., Wang, P., Mason, P.S., and Mitchell, M.B. (1979) J. biol Chem. 254, 12255-12259.
169