Elevation of hepatic epoxide hydratase activity by ethoxyquin is due to increased synthesis of the enzyme

Elevation of hepatic epoxide hydratase activity by ethoxyquin is due to increased synthesis of the enzyme

Vol. 95, No. July 16. 1980 BIOCHEMICAL 1, 1980 AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ELEVATION OF HEPATIC EPOXIDE HYDRATASE ACTIVI...

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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.

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