Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

Vol.

96, No.

September

2, 1980 30,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 946-952

1980

INDUCTION OF CHOLINE KINASE BY POLYCYCLIC AROMATIC HYDROCARBON CARCINOGENS IN RAT LIVER Kozo Medical

Ishidate,

Research

Michiko

Tsuruoka

Institute,

Tokyo Medical Tokyo

Received

August

and Yasuo Nakazawa

101,

and Dental

University,

Japan

15,198O

SUMMARY The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a)pyrene and B-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers(phenobarbita1, l,l,l-trichloro 2,2-bis(p-chlorophenyl) ethane(DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons. INTRODUCTION

It has been known for quite carcinogens, and/or

other

that

of other

of microsomal proposed chrome More

P-450 species

laboratories terization mechanism

environmental compounds

mixed-function

to represent

some time chemicals

via

oxygenase

two prototype

species

and the latter,

of this

cytochrome

of induction

forms process

a wide

and steroids

system(l,2).

inducers, so-called

progress

of cytochrome by individual

variety induce

of cytochrome

of drugs, their

P-450,

metabolism

a key enzyme

PB and 3-MC have been

the

former

cytochrome

have been reported(5-11).

have made considerable of multiple

induction

that

toward P-450(12-17), inducer

inducing P-448

the cytospecies(3,4).

Although

several

the purification the difference has not been fully

and characin the under-

stood. Abbreviations: naphthoflavone; DDT, l,l,l-trichloro

PB, phenobarbital; 3-MC, 3-methylcholanthrene; 8-NF, B3,4-BP, 3,4-benzo(a)pyrene; PCBs, polychlorinated biphenyls; 2,2-bis(p-chlorophenyl)ethane; HCB, hexachlorobenzene.

0006-291X/80/180946-07$01.00/0 Copyrighi All rights

0 I980 by Academic Press, of reproduction in any form

Inc. reserved.

946

Vol.

96, No.

2, 1980

BIOCHEMICAL

On the other philic

and expected

us to study lipid

the

the effect

between

with

of microsomal

kinase[EC

thesis;

3-MC caused

inducers, between

a first

a significant

typical

there

exists

has led

relation-

and modulation biosynthesis

in toward

in choline

kinase

in

the effect

of PB

the activity

of

key enzyme in phosphatidylcholine increase

lipo-

on membrane

a possible system

differences

highly

components,

of phosphatidylcholine

was seen

are

inducers

drug-metabolizing

metabolism

2.7.1.321,

biosyn-

activity

while

PB

the activity(20).

In the present stimulation

that

COMMUNICATIONS

inducers membrane

of the

One of the most striking

choline

RESEARCH

of these

intracellular

especially

and 3-MC on phosphatidylcholine

decreased

BIOPHYSICAL

most

We have found

metabolism,

liver(19,20).

that

of administration

induction

of phospholipid

fact

to interact

metabolism(l8,19).

ship

rat

hand,

AND

study,

of choline together cytochrome

with P-448

we examined

kinase the

activity time

what after

course

and choline

a mechanism

the administration

comparison

kinase

may be involved

activity

of 3-MC-type

of the induction in rat

in the

process

liver.

METHODS Inducer Treatment of Animals: Young Wister male rats(lOO-120 g) were used for al-experiments. 2 ztmhoused in one cage each, fed standard labora3-MC, B-NF, 3,4-BP, PCBs(Polychlotory chow and allowed free access to water. ro Biphenyl, tetra, from Wako Pure Chemical Industries Co. Ltd., Japan) and DDT were dissolved in hot corn oil while HCB was dispersed in corn oil under brief sonication. PB(Na salt) was dissolved in saline. All chemicals were injected i.p. to the experimental group at a dosage of 50 mg(3-MC, 3,4-BP), 40 mg(B-NF), 30 mg(DDT), 80 mg(PB) and 100 mg(PCBs, HCB) per kg, respectively. Control animals were given equivalent volumes(0.25 ml per 100 g body wt.) of vehicle. Injection was performed at 18:00 otherwise indicated and animals were killed 16 hr after the injection, i.e. at 10:00 in the next morning. Food was removed from the cage throughout experimental period. Cycloheximide was administered at a single i.p. dose of 2.5 mg per kg in saline at the time of 3-MC injection, while actinomycin D(1 mg per kg in saline, i.p.) was administered twice 1 hr before and 6 hr after the 3-MC injection. In the time course study, the experiment was started at 10:00 by a single i.p. dose of 3-MC injection. Food was removed for the first 24 hr and every night-time thereafter(22:OO through 10:00 in the next morning). Assay for Choline Kinase liver was prGrm.15%(W/V)Cl a Teflon pestle homogenizer. g for 15 min and the supernatant resulting supernatant was used was rehomogenized in a proper

and Cytochrome P-450: A 2O%(W/V) homogenate of containing 10 mM Tris-HCl, pH 7.4, using The homogenate was centrifuged twice at 15,000 x was centrifuged at 105,000 x g for 60 min. The for choline kinase assay. The microsomal pellet amount of isotonic KC1 and recentrifuged at

947

Vol.

96, No.

2, 1980

8lOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

The washed microsomes were used for cytochrome P-450 105,000 x g for 30 min. determination according to the method of Omura and Sato(21). Choline kinase activity was assayed by a slight modification of the method of Weinhold and Rethy(22) as described elsewhere(20). RESULTS AND DISCUSSION Choline reaction

kinase

rate

preparation

is

was constant within

compared

in Fig.

ed on the basis The activity

animals

assayed

in the morning.

relatively fasting

resulted

Groener

et a1.(23)

also

activity

in rat

increase

in choline

nificant

as early

(100% increase)

became significant then

The overall

gradually

patterns

choline

kinase

were

similar

to each other

ing

that the increase

nism of induction

12 hr after

of induction

activity

in choline

when reffered kinase

by 3-MC of hepatic

activity

microsomal

948

to the maximum level representation

induction

as was clearly

of cytochrome reached

shown

by 3-MC administration

and microsomal

cytochrome

may be involved

to

in Fig. of both

P-448

to the function

mixed-function

kinase

by 3-MC was sig-

the 3-MC administration,

decreased,

Recently,

in choline

either

the

were

in a considerable

reached

hand,

was

that

kinase.

effect

at 96 hr with

in

group

indicating

a decrease

stimulatory

On the other

decrease

in the control control,

control

the activity

of choline

caused

respectively.

This

where

activities of O-time

represent-

at 12 hr in both

periods

the administration,

1A and B).

of enzyme

are

and D),

of 3-MC resulted

by 48 hr and was sustained

The

of 3-MC are

content

rhythm.

activity

This

cell.

and microsomal

administration

decrease

fasting

activity.

cytosolic very

that

as 6 hr after

amount

liver(B

later

later

to that

reported

to the

activity

diurnal

in a diminished

kinase

the maximum at 72 hr, 1C and 5.

the

The administration

liver.

of the activities(Fig. P-45O(P-448)

a marked

at the

low when compared

overnight

a single

due to its

However,

liver

and the cytochrome

showed

reversed

of the

incubation.

kinase

and C) and whole

probably

was partly

per

choline

the activity

kinase

groups

control

kept

of both

of mg protein(A

of choline

portion

30 min and proportional

up to 96 hr after

1, where

and experimental

soluble

of 0.6 mg protein

patterns

P-45O(P-448)

in the

for

the range

The induction cytochrome

located

in rat

of time,

liver

suggest-

in the mechaoxygenase

system.

Vol.

96,

No.

2, 1980

BIOCHEMICAL

61224

AND

48 72 96 Time after a single dose

BIOPHYSICAL

RESEARCH

61224 48 of 3-MC administration

COMMUNICATIONS

72

96

I 72

98

2000 tl 2 $1500 I t .f 1000 1 E 500

,’ 6 I2

1 24

48

72

Time after

Fig.

In

in rat which

1.

a single

dose

of 3-MC

I

1 48

administration

(hr)

Induction patterns of cytosolic choline kinase activity(A and B) and microsomal cytochrome P-45O(C and D) in rat liver following 3-MC administration. The experimental details are described in 'Methods' section. Open and closed circles represent the values from 3-MC-treated and control animals, respectively. Bars are means + S.E. of triplicate measurements from three animals. The number in parenthesis represents Z of control value(average).

order

liver

to

clarify

the

nature

of the

by 3-MC administration,

a well-known

inhibitor

sis(actinomycin shown

11 61224

96

in Table

I,

either

increase

in choline

indicating

that

several

of protein

D) was injected of the

increase

to both

experiments

were

kinase

activity

performed,

in

synthesis(cycloheximide)

or mRNA synthe

control

animals.

inhibitors

and experimental

completely

kinase

activity

in liver

3-MC could

elevate

a formation

949

in choline

105,000

blocked

the

x g supernatant

As

3-MC-mediated fraction,

of the new enzyme protein,

and

Vol.

96, No.

BIOCHEMICAL

2, 1980

AND

TABLE Effect

of

cycloheximide in

and

choline

BIOPHYSICAL

D on 3-MC-mediated

activity

in

Choline nmol/min mg protein Expt.

rat

liver

kinase

activity

per

nmol/min per whole liver

1. 2.36

+ 0.36

722

f

70

Cycloheximide

1.95

+ 0.36

600

it

28

3-MC

3.87

f 0.65*

1153

f

83*

2.27

i 0.32

707

f

50

2.24

+ 0.23

643

f

47

2.37

i 0.15

670

+

35

3.62

f 0.69"

1075

+ 205*

2.24

+ 0.36

646

f 102

Expt.

+ Cycloheximide 2.

Control Actinomycin

D

3-MC 3-MC

+ Actinomycin

D

Treatment of animals and other experimental details in 'Methods' section. Values are means + S.E. of ments from four animals. * Significantly (p< 0.01) different from untreated

the site

synthesis has also

increase

Control

3-MC

that

COMMUNICATIONS

I

actinomycin

kinase

RESEARCH

could

of action

of 3-MC leading

be beyond

been supported

to the

the transcriptional by the following

induction level.

criteria(data

0

are described triplicate measurecontrol

values.

of choline The former not

shown);

kinase possibility 1) the in

1

1

I

t

PB-Type

3-MC-Type

Fig.

2.

The effect of different types of inducers of microsomal mixedfunction oxygenase system on cytosolic choline kinase activity in rat liver. Treatment of animals and other experimental details are described in 'Methods' section. Bars represent means + S.E. of triplicate measurements from three or four rats.

950

Vol.

96, No.

vitro

2, 1980

BIOCHEMICAL

addition

kinase

at all.

min per mg protein) the

inducers,

oxygenase 2, it is

event

for

kinase

have been proposed

of choline

activity

so-called

induction

type

when compared

30-45%

kinase

is

amnonium

of microsomal

mixed-function

kinase

inducers

of inducer

for

cytochrome

As shown

of choline P-448

specific

i.e.

was investigated.

cytochrome

to be a mixed

activity(nmol/

hydrocarbons,

inducers

the

the specific

choline

chromatography).

aromatic

that

stimulate

preparation(through

induction

of well-known

for

in

not

COMMUNICATIONS

was much higher

column

polycyclic

demonstrated

corrmon event

increase

kinase

the

on choline

was clearly

which

whether

the effect

system

a very

choline DEAE-cellulose

to define

3-MC or a general

of

RESEARCH

did

3-MC administration

purified

precipitation, Next,

P-448

2) a degree

following

partially

sulphate

BIOPHYSICAL

of 3-MC to the enzyme preparation

activity

with

AND

of both

in Fig.

in rat

including

liver PCBs,

PB and 3-MC(24-

27). The overall are

involved

system ological carbon under

caused

results

strongly

in the mechanism by 3-MC-type

significance carcinogens investigation

of induction

but

of choline in

relation

in this

suggest

not

that

induction

of microsomal

by PB-type

kinase

the

induction

to hepatic

inducers

mixed-function in rat

by polycyclic

phosphatidylcholine

of choline

liver. aromatic metabolism

kinase oxygenase The physihydrois now

laboratory.

REFERENCES

:: 3. 4. 5. 6. ;: 1;:

Conney, A.H. (1967) Pharmacol. Rev., 4, 317-366. Ullrich, V., Roots, I., Hildebrandt, A., Estabrook, R.W., and Conney, A.H. eds (1977) Microsomes and Drug Oxidations, Pergamon Press, Oxford. Sladek, N.E. and Mannering, G.J. (1966) Biochem. Biophys. Res. Commun.,

24, 668-674. C.J. and Shoeman, D.W. (1969) in Mannering, G.J., Sladek, N.E., Parli, Microsomes and Drug Oxidations, (Gillette, J.R., Conney, A.H., Cosmides, G.J. eds) pp. 303-330. G.J., Estabrook, R.W., Fouts, J.R. and Mannering, Chem., Haugen, D.A., van der Hoeven, T.A. and Coon, M.J. (1975) J. Biol.

250, 3567-3570.

Welton, A.F., O'Neal, F.O., Chaney, L.C. and Aust, S.D. (1975) J. Biol. Chem., 250, 5631-5639. Gibson, G.G. and Schenkman, J.B. (1978) J. Biol. Chem., 253, 5957-5963. Agosin, M., Morello, A., White, R., Repetto, Y. and Pedemonte, J. (1979) J. Biol. Chem., 254, 9915-9920. Negishi, M. and Nebert, W. (1979) J. Biol. Chem., 254, 11015-11023. Elshourbagy, N.A. and Guzelian, P.S. (1980) J. Biol. Chem., 250, 12791285.

951

Vol.

11. K: E: 16. 17. ii: 20. 21. f :: 2: 26. 27.

96, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Sugiyama, T., Miki, N. and Yamano, T. (1980) J. Biochem., 87, 1457-1467. Imai, Y. and Sato, R. (1974) Biochem. Biophys. Res. Cornnun., 60, B-14. Hashimoto, C. and Imai, Y. (1976) Biochem. Biophys. Res. Comnun., 68, 821827. Haugen, D.A. and Coon, M.J. (1976) J. Biol. Chem., 251, 7929-7939. Guengerich, F.P. (1978) J. Biol. Chem., 253, 7931-7939. Botelho, L.H., Ryan, D.E. and Levin, W. (1979) J. Biol. Chem., 254, 56355640. Masuda-Mikawa, R., Fujii-Kuriyama, Y., Negishi, M. and Tashiro, Y. (1979) J. Biochem., 86, 1383-1394. Ishidate, K. and Nakazawa, Y. (1976) Biochem. Pharmacol., 25, 1255-1260. Ishidate, K., Yoshida, M. and Nakazawa, Y. (1978) Biochem. Pharmacol., 3, 2595-2603. Ishidate, K., Tsuruoka, M. and Nakazawa, Y. (1980) Biochim. Biophys. Acta, in press (Lipids and Lipid Metabolism). Omura, T. and Sato, R. (1964) J. Biol. Chem., 239, 2370-2378. Weinhold, P.A. and Rethy, V.B. (1974) Biochemistry, 13, 5135-5141. Groener, J.E.M., Klein, W. and van Golde, L.M.G. (1979) Arch. Biochem. Biophys., 198, 287-295. Arvares, A.P. and Kappas, A. (1977) J. Biol. Chem., 252, 6373-6378. Yoshimura, H., Ozawa, N. and Saeki, S. (1978) Chem. Pharm. Bull., 26-, 1215-1221. Goldstein, J.A., Hickman, P., Bergman, H., McKinney, J.D. and Walker, M.P. (1977) Chem.-Biol. Interact., n, 69-87. Poland, A. and Glover, E. (1977) Mol. Pharmacol., 13, 924-938.

952