Synthesis and accumulation of polyamines in rat liver regenerating after treatment with carbon tetrachloride

BIOCHEMICAL

Vol. 54, No. 1, 1973

SYNTHESIS

AND

LIVER

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACCUMULATION

REGENERATING

OF

AFTER

POLYAMINES

IN

TREATMENT

WITH

RAT

CARBON

TETRACHLORIDE

E.

Holtta,

Department

Received

Riitta

of Medical SF-00170

July

Sinervirta Chemistry, Helsinki

and

J.

Janne

University Finland

17,

of

Helsinki,

17,1973

A single intraperitoneal injection of carbon tetrachloride SUMMARY: into rats resulted within 12 hours in a marked accumulation of putrestine in liver with a concomitant decrease in the concentration of spermidine. The accumulation of putrescine apparently was partly due to an immense stimulation of ornithine decarboxylase activity occurring at the same time. However, in addition it was found that during the there was a marked incorporation maximal accumulation of putrescine of radioactivity from labelled spermidine to liver putrescine in vivo. The conversion of spermidine to liver putrescine was hardly detectable the treatment with carbon tetrachloride, inin control animals. Besides creased conversion of radioactive spermidine to liver putrescine in vivo also occurred after treatment with growth hormone, after partial hepatectomy and after treatment with thioacetamide, i. e. under cirof ornithine decarboxylase cumstances characterized by a stimulation activity and an increased accumulation of putrescine. The

formation

of

L-ornithine

decarboxylase

exclusively

located

straight as

putrescine

to

mammalian

(L-ornithine in

the

decarboxylation

yet

in

cytosol

carboxy-lyase, fraction

putrescine

in

no cell

cell

other

free

is EC

of various

of L-ornithine

synthesize

tissues

Besides

has

from

by

4.1.1.17),almost

types.

reaction

extracts

catalyzed

been

various

this

demonstrated mammalian

tissues. The tissues

is

synthesis greatly

known

examples

various

target

after rats

are

regenerating following

carboxylase

also

application rat

liver

after

(4),

the

in

cell

hepatectomy

activity to

of of

purified

Well (l-3),

such

prostate

The due

stimuli.

treatment

ventral

(5). cultures

mammalian

growth

partial

hormone

testosterone

enhanced

in various

of certain

appropriate hormone

with

putrescine

as

rat

castrated

ornithine

de-

epidermal

growth

(6). Usually

growth

growth

treatment is

of

after

organs with

following

carboxylase

accumulation

increased

treatment

factor

and

the

stimulus

first is

with

an

change immense

a concomitant

Cop.vri~hl @ 19 73 by Academic Press. Inc. AN rights of’ reproduction in any form reserved.

in

polyamine

increase

metabolism in

accumulation

350

the

activity of

putrescine

after

appropriate

of ornithine (7).

deThe

accumul-

liver

BIOCHEMICAL

Vol. 54, No. 1, 1973

ation

of putrescine of

tion

the

is

spermidine

from

spermidine

greatly

increased the

relatively putrescine

a common in

in

spermine partial

mone

in

(8).

from

The

conversion

normal

However,

liver

even to

conversion

to be

radioactivity

in

spermidine

of this

addition can

putrescine.

negligible

from

in

concentra-

but

during

liver

putrescine

to the

was

accumulation

of

we

found

agent,

that

into

rats

and or

hepatectomy

offers

during

synthesis

similar

injection

of carbon an

comparable

for

those

found

following

the

studying

the

In principal,

of putrescine,

to or

model

regeneration.

accumulation

(7,9,10)

useful

tissue

tetrachloride,

spermidine

in

rat

liver

treatment

and

regenerating

with

growth

hor-

(4,ll).

the

er

that,

of

liver

the

putrescine

incorporation

was

contribution

metabolism

the

were

reported

to

label

in

spermine,

regeneration of

study

polyamine

changes

the

increase

open.

present hepatotoxic

changes

liver

the

remained In the

from

early

and

(8) and

putrescine

incorporation

feable

Siimes

judged by 14 C-spermidine

to

at

regeneration

the

as

spermidine

slow

spermidine

injected

exogenous

a relatively

1967

In

between

intraperitoneally of

by

(1,4,7).

interconversions

formed

followed

AND BIOPHYSLCAL RESEARCH COMMUNICATIONS

Shortly

after

activity

of

putrescine.

ornithine

At

concentration

of

creased

above

the

to

accumulation

dine

liver

Later

animals

the

to

hormone,

injection

partial

conversion

of

hardly

any time

intraperitoneally

in

spermidine was

in-

of the

maximal

appeared

putrescine that

In addition and

a marked

to

carbon

thioacetamide

injected

livthe

injected

incorporation

of it

of

decrease of

putrescine. hepatectomy

stimulation

accumulation transient

spermidine

at the

liver

immense

concentration

was

tetrachloride

an

a remarkable

radioactive

there

converted

was

a striking

however,

carbon was

there

was

When

putrescine,

enhanced

liver

Ccl4 and

there

values.

control

growth an

to

time

spermidine.

control

dose

tetrachloride,

of

decarboxylase

following of the

injection

same

to

radioactivity

caused

the liver

intraperitoneally

portion

a single

also

radioactive

spermi-

putrescine.

MATERIALS

AND Female

METHODS

rats

of the

Wistar

strain

(weighing

loo-130

g)

were

used

in

all

experiments. Unlabelled scribed

earlier

mmole)

and

purchased 50-H+ obtained

and

labelled

S-adenosylmethionine 14 Putrescine-1,4C (specific

(12). spermidine-l,4

from columns from

the

-f4C New

before E. Merck

England use AG

(7).

( s p ecific Nuclear Carbon

(Darmstadt,

activity Corporation

tetrachloride Germany).

351

were

prepared

radioactivity 10.22

as 17.5

mCi/mmole) and

and Carbon

purified thioacetamide tetrachloride

demCi/ were

on

Dowex were was

Vol.

BIOCHEMICAL

No. 1, 1973

54,

dissolved

in

olive

oil

intraperitoneally. Sweden)

The

dissolved

fractions

of The

liver,

as

the

after

butanol

with

-pyridine-water In each

spermine,

was

and

(16). really

cytosol

spermine

To

ensure

was

in

the

the

radio-

putrescine, paper

following

-glacial

n-propanol-concentrated

the

resi-

systems

:

chromato-

solvent

n -butanol

radioactive

were

that

to ascending in

(50:25:25),

only

de-

undialyzed

acetic

acid

-

HCl-water

fraction,

in

addition

to

spermidine

anaesthesia

by

putrescine.

Protein

was

Partial

hepatectomy

method

intraperitoneally.

using

spermidine Cohen

subjected

and the

Malmo,

(13-15).

acid-water

case

injected

Ferring,

injected

assayed

standard

(40:10:10:20)

(3O:lO:lO). and

were

putrescine

and

S-adenosylmethionine

were

earlier

extraction

acetic

and

decarboxylase,

of putrescine,

authentic

n-butanol-glacial

acid

of Raina and 14 1,4 C-spermidine

from

saline R ,

(Somacton

acetic

synthase

described

method

derived

M

ornithine

spermidine

by

graphy

of

and

physiological

hormone

0.04

concentrations

measured

in

growth in

activities

carboxylase

dues

thioacetamide

Porcine was

activity

and

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

measured

of Higgins

by

the

was

and

method

Anderson

Lowry

of

performed

under

--et al.

light

(17).

ether

the

(18).

RESULTS The

effect

(0.2

ml

/lOO

ities

of

ornithine

Fig.

1.

The

1.2 h

after

ornithine

liver.

injection

concentration it

drop reached

sharply

concentration

in

spermine

slowly

increased

towards

jection

of

tetrachloride

carbon

Its

concentration

the

putrescine

time

the

As activity

seen

content

in

the

of

S-adenosylmethionine

figure,

in

resulted

At

also

at

markedly until

control

pmoles

normal

rat

was

exceeding

liver. was

a transient decarboxylase

352

the

the

g wet

the

On only

the about

values.

in

after

ten

at

same

50%) the

the

the

about the

of the in injection

The

thereafter

12 h after

fourth

(about 4 h

is

hand,

one

where-

and

polyamine

other

of

a striking,

control

injection

which

decrease at

activity also

Spermidine

at

wt,

maximum

tetrachloride

the

main

in

its

was

Surprisingly, was

activ-

accumulation

spermidine.

h after

values.

per

also

carbon

the

illustrated

a rapid

there

12 h after

24

and

reached

time

of liver

decreased

0.5

there

same

concentration

spermidine

in

Furthermore,

putrescine

of

the

tetrachloride

is

of putrescine

to values

the

exceeded

concentration

drug.

a minimum

increased of

carbon

polyamines

decarboxylases

maximal. the

of of

concentration

of the was

transient,

injection

concentrations

tetrachloride

The

decarboxylase

although

after

of carbon

the

the

on the

S-adenosylmethionine

injection in

intraperitoneal

weight)

and

of putrescine at

of a single

g body

liver. times

normal. the of

in-

BIOCHEMICAL

Vol. 54, No. 1, 1973

Fig. 1 Effect of carbon tetrachloride and on the activities of ornithine The animals received in rat liver. 0.2 ml of CC14 per 100 g body The concentrations of polyamines as described in the text. Three

carbon

tetrachloride.

ase

swiftly

and

remained

ity

of

at this

spermidine The

12 h

after

been

an

This

was

rapid

enhanced

In fact,

least

partly idea

did in

has

to

not

the

about

whole

change

after

a rather

light

long

half-life

accumulation

greatly

increased

might

have

the

rat

of

liver

within

activ-

first

might

injection

have

of the

that,

at

e.

several

-- i. putrescine,

drug.

least

exodays

though

decarboqlase to

the

there

liver,

converted

values The

treatment.

that

observations

ornithine been

spermidine

in

control

tetrachloride

after

of the

the

observation.

suggested

polyamine

in the

of

carbon of

decarboxyl

3-fold

period

concentration

of this

S-adenosylmethionine

was

the

tetrachloride

concomitant

spermidine

of

activity for

the

plausible

the

activity

the

carbon

degradation

due that

level

of

especially spermidine

the

12 h,

injection

(8,

19).

At

decrease

the

the

higher

synthase

genous

on the concentrations of polyamines S-adenosylmethionine decarboxylases a single intraperitoneal injection of and were killed at time points indicated. wt. and the enzyme activities were measured animals in each group. and

Thereafter

increased.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at

activity,

putrescine

awaked

under

these

conditions.

As of the into

in

Table

the

liver

1,

injected

treatment

putrescine of

shown

intraperitoneally with

control fraction. spermidine

indeed

fact,

was the

at 4 h after

hardly

to

When

labelled

any

detectable

radioactivity the

a drastically

spermidine

tetrachloride. there

In

was

radioactive

carbon

animals

there

of

injection

353

of

putrescine radioactive

increased putrescine

conversion at

spermidine

was

radioactivity was

12 h after injected in

about

spermidine

the

half in

of that carbon

Vol. 54, No. 1, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect

of

carbon

radioactive

Treatment

tetrachloride to

Conversion 14 C-spermidine to putrescine

50t16

360+53

cpm/g

2 150+880

ccl4

on the

spermidine

Activity of ornithine deca rboxyla se pmole s/ mg protein per 30 min

Control

1

liver

of in

putrescine

spermidine nmole

2.90

of

s/g

540+220

mulation able

rats.

of putrescine, stimulation

might

also

spermidine

tine in

810+52

65Otll

3 70282

510+2

2,

increased

after

putrescine

ment,

evaluate

the

also the

other

of

far

been

there

was

of

spermidine

in

excretion

the

a marked

treated

during

the

accu-

and

remark-

animals.

It

of radioactivity

from

treatment

of carbon

radioactive

by

spermidine

tetra-

as

opposed

the

to

the

remained conversion from

carbon

case

an

carbon

As

putrescine

of

increase

in

ornithine

decarboxy treat-

unchanged.

spermidine

to

putrescine

tetrachloride-treated

rats

unsuccessful.

DISCUSSION A

single

injection

of carbon

tetrachloride

354

into

rat

is

known

seen greatly

tetrachloride

practically of

studied.

liver

activity

to

of putres-

were

each

of the

spermidine

accumulation

to

In

stimulation

spermidine

homogenates

an

of

activity,

thioacetamide. the

demonstrate

liver

conversion

characterized

However, of

of the

decarboxylase

with and

concentration to

the

universality

ornithine

conversion

observed.

using

activity

that

situations, of

treatment

Attempts --in vitro have so

1,

concentration

unchanged

the

concentration

was

the

Table

g body wt.) 12h and The concentrations were measured as deviations obtained

shown).

stimulation

Table

in

in

decarboxylase

remained

not

a few

and

ase

urine

further

putrescine

seen

of mentioning

(results To

ornithine

worth

into

chloride

also

decrease

of be

As

spermine

liver

The rats received carbon tetrachloride (0.2 ml per 100 14C-spermidine (1 PC, 0.05 pmoles) 4 h before sacrifice. of polyamines and the activity of ornithine decarboxylase described in the text. The values are means 2 standard from three rats in each group.

tetrachloride-treated

vivo

Concentration

of

liver

13 10045

conversion

putrescine

to

cause

BIOCHEMICAL

Vol. 54, No. 1,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect

of growth

the

hormone,

conversion

of

radioactive

Activity of ornithine decarboxylase pmoles/ mg protein per 30 min

Treatment

2

partial

hepatectomy

and

spermidine

to

on

putrescine

in

vivo

Concentration

Conversion of 14 C - spe rmidine to putrescine cpm/g

liver

thioacetamide

of

spermidine

puti-escine

n moles/g

liver

spermine

liver --

Control

37216

Growth hormone

230t

5126

650254

54Oi2

150+17

690+42

570214

76

1560+410

1

Partial hepa tectomy

680t

Thioacet amide

230

2110+280

2 91021

620

140+37

710+57

590233

4 88021

240

410281

640+29

510214

The rats received growth hormone (5 I.U. per 100 g body wt.) 4 h or thioacetamide (15 mg per 100 g body wt.) 24 h before killing, or were partially hepatectomized 12 h before sacrifice. l4C-spermidine (1 KC, 0.05 pmoles) was injected intraperitoneally 3 h before sacrifice. The concentration of polyamines and the activity of ornithine decarboxylase were measured as described in the text. The values are means 2 standard deviations obtained from four rats in each group.

an

acute

liver

changes

in

necrosis the

tetrachloride

were

partial

phases

after

the

or can

be

poison,

putrescine in

spermidine)

and

putrescine.

At

of

to rise

in

appears

also

time

to

involve

to The

and

in

second

phase,

355

rapid

ornithine

dif-

accumulation activity,

of

in

the

to

after

synthesis

spermidine

to

the

steadily

and of

several

(block

due starting

activity

after

metabolism

accumulation,

spermidine,

activity

that

conversion

minimum,possibly

the

seems

activity

its

decarboxylase

instance,

a dramatic

putrescine

of liver

for

decarboxylase

an increased

The

of carbon

of polyamine

ornithine

of maximal

concentration

of putrescine

It

changes

(20).

injection

found,

decarboxylase

putrescine. the

the

increased

decreased

S-adenosylmethionine centration

of

those

an

treatment.

in

phase

process

after

to

hormone

obviously the

spermidine

conversion rapid

first

a result

of

tion

comparable growth

regenerative

polyamines

adenosylmethionine

er

a rapid

distinguished

The as

a decrease

by

of liver

roughly

hepatectomy

ferent

of

followed

metabolism

the

increased

12 h,involved elevated

decrease decarboxylase.

liv-

concentra-

in

a levels

the

conThe

of

Vol. 54, No. 1, 1973

concentration

of

increase

creased

in

polyamine

there

appears

that

as

a precursor

is

known

for

as

yet

putrescine.

It is

--i. e. the methionine

the

transfer

and

cumstances.

This,

formation

are

aldehyde

hog

used

group

for

spermidine

in

the

blood kidney

spermidine

energy

contains is

be

also

of oxygen

In

to

form

an

and

putrescine

and

is reaction cirSecondly,

addition

to

growth.

Nothing to

spermidine,

S-adenosylto

yield

spermidine,

some

special

transfer

cirbecause

(decarboxylated in

mammalian type and

has

that

tissues

found

in

aminopropyl-

been

reported

amine

that

oxidase

from

aminopropylaldehyde

from

(22). The

evaluation

putrescine

as

currently

started

of the

well

as in

importance

attempts this

to

of the

characterize

conversion the

of

enzymic

spermidine

to

pathway(s)

are

laboratory.

ACKNOWLEDGEMENTS The

skilful

technical

assistance

of Mrs.

Sirkka

Kanerva

is

gratefully

acknowledged.

REFERENCES 1. 2. 3. 4. 5.

J&nne, J., Acta Physiol. Stand., Suppl. 300, 1 (1967). Stand 22 1349 (1968). Jbnne, J., and Raina, A., Chl em--Acta Lk’ -’ Natl. Acad. Sci., U.S., Russell, D., and Snyder, S.H., --Pr oc. 1420 (1968). JLnne’, J. ,* Raina, A., and Siimes, M., Biochim. Biophys. Acta, -419 (1968). and Williams-Ashman, H. G. , Pegg, A.-E., Lockwood, D.H., 117, 17 (1970). Biochem. J.,

356

serve

spermidine of

energy

of the

it

in-

under

of

putrescine fact,

an

a novel

in

under

that

activities

(21).

be

inducible

putrescine

with

possible

to

vivo,

synthesis

compound

spermidine

to

--in

tissue

reversible

aminopropylaldehyde,

capable

to

is

decarboxylated

coupled

fairly enzyme

converts

presence

be

sulphonium

oxidizing

which

to

activity.

the

to

should

out

conversion

from

might

least

is

during of the

pathway

a proton,

It is

starting converted

there

function,

spermidine,

propylamine

of a high

Pseudomonas

human

the

however,

S-adenosylmethionlne). there

specific

mechanism

that

that

at

decarboxylase

some

of

turn

enzyme(s)

(S-methyladenosylhomocysteamine)

thiomethyladenosine

of the

synthesis

of the

is

putrescine

might

ornithine

have

enzymic

possible

thereafter spermine

putrescine, it

of

might

of the

liver

by

a stimulation

putrescine

days

phase

of

Firstly,

catalyzed

is

two

this

accumulation to

respects.

metabolism

cumstances

the

spermidine

several

about

during

(8).

during

of

for

that

rate

that

conversion

interesting

possible

increased

finding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

decreased

It is

at The

it

spermine

slowly.

spermidine

in

BIOCHEMICAL

-’60 166,

Vol. 54, No. 1, 1973

6. 7. 8. 9.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Stastny, M., and Cohen, S., Biochim. Biophys. 204, --Acta, Raina, A., Janne, J., Hannonen, P., and Holtta, E., ---Ann. Sci ., 171, 697 (1970). Acta Physiol. Stand., Siimes, M., Suppl. 298, 1 (1967). Hannonen, P., Raina, A., and Janne, J., Biochim. Biophys.

578 N.Y.

(1970). Acad.

Acta,

273,

84 (1972). 10. 11.

12. 13.

Holtta, E., and Janne, J., FEBS Letters, 23, 117 (1972). Raina, A., and Holtta, E., in “Growth and?rowth Hormone”, A . , and Miiller, E.E. (eds.) Excerpta Medica International Series NO. 244. Amsterdam, p. 143 (1972). Pegg, A.E., and Williams-Ashman, H.G., Chem., ---J. Biol. Janne, J. , and Williams-Ashman, 11. G., 3. Biol. Chem ., --

(1971). 14.

JXnne,

42, 15. 16. 17.

18. 19.

Gina, Raina, Lowry, Chem Higgins, Russell,

6732 20. 21. 22.

J.,

and

Williams-Ashman,

I-I. G.,

Biochem.

Pecile, Congress - 244,

g4A,

Biophys.

--Res.

222 (1971). A., and Hannonen, A., and Cohen, O.H., Rosebrough,

.,

193, G.H., D.H.,

P., S.S.,

---FEBS Proc. NrFKAx

68.’

Letters 16, 1 (1971). Natl. Acad. Sci., U.S., andRandall,

Commun.,

55, 1587 R. J., J.

265 (1951). and Anderson, Medina, V. J.,

R.M., and

Arch. Snyder,SE.

Pathol.,

--

(19701.

Villefa, G:G., Biochem. Padmanabhan, R., and 19, 1 (1965). huash; G. ,’ and Taylor,

---Pharmacol., Kim, K., D.R.,

-’13 Biochem.

Clin. ----

Chim.

357

665 (1964). Biophys. Acta,

30,

12, B%l. -

Res. 17

186 (1931). Chem.,

Commun., (1970).

(i969).

1725

245,

(1966). Biol.