Theophylline suppression of Δ-aminolevulinic acid synthetase induction in chick embryo and rat livers

BIOCHEMICAL

Vol. 53, No. 4,1973

THEOPHYLLINE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SUPPRESSION OF A-AMINOLEVULINIC

ACID SYNTHETASE INDUCTION

IN CHICK EMBRYO AND RAT LIVERS Josef Department

Korinek

of Pathology,

and Harold

School

of Medicine,

Nashville, Received

July

L. Moses

Tenn.

Vanderbilt

University

37232

5,1973

SUMMARY: Theophylline was found to be a potent inhibitor of induction of hepatic 6-aminolevulinic acid synthetase (ALAS). The inhibitory effect of theophylline was observed in chick embryo and rat livers with allylisopropylacetamide (AIA), a strong inducer of ALAS. The effect of other inducers of ALAS in chick embryo (phenobarbital and steroids) was also suppressed by theophylline, which exerted its effect when administered either simultaneously with the inducer or 3 to 4 hours following the induction. Theophylline had no direct inhibitory effect on ALAS activity in vitro. The effect. of xanthine derivatives other than theophylline on ALAS induction was also tested: however, theophylline and aminophylline were the only significantly potent suppressors of ALAS in both chick embryo and rat livers.

A-aminolevulinic enzyme

shown

porphyrias In the

latter,

and in

preexisting

to exhibit

carbohydrate

will

the course

during

induced

in the

of enzyme

The induction in

that

to have

an unexpected

of this

derivatives

report

Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

hepatic

chick

porphyria

(3).

to be due to of activation animals

administration

of has been

or a high

of adenosine-3',5'-cyclic glucose

effect

effect

the effect

1246

of human hepatic

experimental

suppressive

in

enzyme in liver

of ALAS (5).

effect

to describe

on ALAS induction

attacks

glucose

and the

marked

of this

instead

induction

on the

and rate-limiting

of ALAS appears

(induction)

block

of studies

first

acute

of ALAS in

effect"

partially

the

experimental

activity

(CAMP) on ALAS induction

the purpose

xanthine

(4).

a "glucose

diet

monophosphate was found

synthesis

enzyme

During

chemically

is

The activity

elevated

the increase

--de novo

(ALAS)

pathway.

to be markedly

(1,2)

increased

is

synthetase

in the heme biosynthetic

has been

shown

acid

(6),

theophylline

on ALAS induction.

of theophylline

embryo

and rat

livers.

and other

It

Vol. 53, No. 4, 1973

BIOCHEMICAL

MATERIALS

Chick

AND METHODS:

extraembryonic

part

were

then

were

pooled

10.0

mM Tris-HCl

assayed

sealed

through

immediately.

injected

from

5 animals

3 mg per

embryo;

11,20-dione

(P), AIA

derivatives

chick rats.

embryo,

The last

rats

embryo;

was

40-60

Aliquots

of livers

studies

compound

in

to rats

gms

(AIA),

(E), rats.

were

was administered

EDTA,

weighing

5 mg per

The xanthine

aminophylline, These

ti

5B-pregnan-3a-17a-diol

in

theophylline,

to be lethal

0.5

allylisopropylacetamide

and 180 mg/kg or in later

but was found

5 to 8 embryos

above.

enzyme induction

were

the

The eggs

ALAS activity

24 hours.

5 mg per

for

into

cavity.

and etiocholan-3a-ol-17-one

was used

embryo

air

containing

as described

with

injected

from

(v/v).

for

and 1-methyl-3-isobutylxanthine.

in

chick

5 mg per embryo;

the study

Livers

(CD strain)

fasting

(PB),

the

of a solution

River

embryos

phenobarbital

in

hours.

and homogenized

in chick

were

over

and 30% glycerol

after

RESEARCH COMMUNICATIONS

days old,

drilled

several

Charles

pooled

tested

of 9 mg per weight

pH 7.4,

(300 mg/kg)

theobromine,

hole

for

subcutaneously were

17-18

in 3 volumes

Male

ALAS was induced

embryo.

a small

and homogenized

were

embryos,

and incubated

buffer,

AND BIOPHYSICAL

administered

in doses

100 mg/kg

body

of 1.18

mg per

doses

even

caffeine,

in greatly

reduced

dosages. The steroids compounds

were

were dissolved

mine was injected

partially

in the

.J

purified

a 30 minute

ALA produced RESULTS :

Control

incubation

was quantitated

The administration

(8).

of its

were

period

all

received

and resulted

(ImM)

of succinyl

provided in

calorimetrically

(9).

of 3.0 mg ALA to

chick

1247

(v/v)

from Rhodopseudomonas

modifications

generally

embryos

alone.

of Marver

phosphate

1 umole

theobro-

vehicle

of the method

of pyridoxal

other

of solubility,

the use of 3Q% glycerol

to generate These

whereas lack

animals

CoA synthetase

(sufficient

medium

glycol,

by a modification

the presence

succinyl

mutant

incubation

Because

- employed

solution

ALA-requiring

saline.

was assayed

The modificationc

homogenizing

over

in

in propylene

as a suspension.

ALAS activity (7).

dissolved

in

et al. the

and

spheroides CoA in

a linear higher

caused

30 min.)

reaction activities.

a marked

BIOCHEMICAL

Vol. 53, No. 4, 1973

AND BIOPHYSICAL

+-

T

500

AIA

RESEARCH COMMUNICATIONS

3mgfembryo.

control

-~~~~~~~~ AIA 3mg/embryp

+ saline

d---d

t Theophyllins

AIA

Imp/embryo

.E 400 E

Smg/embryo

SALINE or THEOPHYLLINE

8 \ -0 300 2 4’ 200 9 E =

0

;I, EE

100

01

AI,

4IA ‘hto

PB PB Thea

Fia. embryos with 3a-17a-dial mean &SD of

P

P Thea

Theophylline (Theo) suppression of hepatic ALAS induced in chick allylisopropylacetami.de @IA), phenobarbital (PB), 5&pregnan11, 20-dione (P) and etiocholan-3a-o1-17-one (E). Each bar is a three to five groups of pooled livers. after 8 hours of induction.

Fig. 2. Theophylline in chick embryos caused injected and undisturbed of pooled livers.

increase

ThW

administration 3 hours after ALAS induction by AIA a significant drop in enzyme activity over saline Each point is a mean + SD of three groups groups.

in ALAS activity

reaching

a peak at about

enzyme activity

was 10 to 20 times

The concomitant

administration

prominent

inhibition

obtained

being

was produced including

ALAS,

of ALAS induction

20 to 30% of that

phenobarbital

No inhibition

with

was placed

of ALAS activity

results.

The inhibitory

and theophylline shows stered hours

that three after

were

injection

of the effect

effectively after

in

the

(Fig. with

metabolites

time

embryos

given

embryo) levels

1).

the saline.

produced

a

of activity

A similar

other

inducers

(Fig.

1).

had a direct the incubation

at which

of ALAS

inhibitory mixture

phenomenon

effect

during

on

ALAS assay.

was observed.

administered

theophylline hours

AIA alone

.f theophylline

The mode of administration the

(9 mg per

when administered

to determine

in control

by AL4 with

and two steroid

1 mM theophylline

than

of theophylline

by theophylline

In order

greater

8 hours

induction

inducer

and theophylline

of theophylline

was present

in one injection suppressed

enzyme induction

of embryos

1248

chick

received

not

when the

or separately.

of ALAS by AIA in

of AIA one group

did

affect inducer

Figure

2

when admini-

embryo

livers.

Three

9 mg of theophylline

Vol. 53, No. 4,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1000

AIA 30~~/10Og

o.--c

.--AIA

900

+ THEOPHYLLINE

30mq/lOOg

I6mg/lOO9

+ SALINE I

Fip. 3. Theophylline administered 4 hours after AIA in rats inhibited the ALAS induction. Each point is a mean + SD of three groups of pooled

livers.

Fip. 4. Effect of theophylline, caffeine, theobromine and 1-methyl-3-isobutylxanthine on ALAS induction with AIA in chick embryos. Bars are means + SD of three or four groups of pooled livers after 8 hours of induction.

dissolved

in 1 ml of saline,

third

group

remained

group

exhibited

undisturbed

a diminished

phylline

injection

decrease

in enzyme activity

injected

embryos

to undisturbed it

could

in chick for

embryos.

12 hours.

caused

a 70 to 80% inhibition

2).

carried

a marked

control

after for

administration of ALAS induction.

(Fig.

The theophylline

this

after 2).

in livers

saline

the

theo-

A slight of saline

injection

saline

the

in

effect

is

comparison

obscure;

changes. gave results

of a single

increase

incubation.

observed

or volume

out in rats

whereas

at 1, 3 and 5 hours

3 hours

The reason

The administration

1 ml saline,

the 8 hour

was consistently

of stress

The concomitant

received

to the undisturbed

(Fig.

result

studies

24 hours

during

at 1 and sometimes group

group

ALAS activity

compared

be the

Similar

another

in hepatic

comparable

to those

dose of AIA to rats ALAS reaching

of theophylline

with

1249

starved

a peak

at about

AIA resulted

As can be seen in Figure

seen

3, theo-

in

Vol. 53, No. 4, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

200 .E E Fs \ m T 4 100 a % 0 E c m JNE

AIA THEOPHYCLINE

AIA AWINOPHYLLINE

AIA CAFFEINE

of theophylline, aminophylline, caffeine Fig. 5. Effect on ALAS induced with AIA in rat livers during 8 hours.Bars three groups of pooled livers.

phylline

also

markedly

suppressed

ALAS when

IA ROUINE

THE

administered

and theobromine are means + SD of

4 hours

after

injection

of AIA. To determine induction,

if

we compared-the

isobutylxanthine embryo

bromine

(Fig.

(Fig.

theobromine

derivatives

effects

of caffeine,

of theophylline

Theophylline

Caffeine

4).

DISCUSSION:

certain

having Theophylline

inhibitory that of induction

of this effect the

compound

effects

marked

in chick enzyme.

(increased

were

suppressive

embryos

and rats

which

cause

1250

of its

caffeine

and

determined

(Fig.

of ALAS with

significantly after

5).

theophylline

that

there

is

increase

the

increase

administration

shown

Since

--de novo synthesis

because

effects.

to reduce

was further

theo-

of ALAS

of aminophylline, livers

marked while

in rats

of inhibition

on ALAS in vitro.

causing

in no reduction

shown

It

by AIA in chick

suppression,

tested

of ALAS

and l-methyl-3-

compound

was not

some degree

equally

compounds

only

(ZO-30%)

by AIA in rat

has been

ALAS occuring inducers

to indicate

caused

inhibition

on ALAS induction

slight

the

cause

theobromine

resulted

however,

compounds

also

was the

caused

latter

on ALAS induction

and aminophylline

in hepatic

This

of toxicity;

of these

process

4).

xanthine

and 1-methyl-3-isobutylxanthine

degree

direct

that

of ALAS.

induction

All

with

livers

suppression

high

other

theophylline considerable

in hepatic of enzyme),

of has no evidence

ALAS do so by a it

seems

BIOCHEMICAL

Vol. 53, No. 4, 1973

reasonable

to conclude

by suppressing that

induction

theophylline

during

not

clear.

the

enzyme which

Exogenously

administered

implicated

in

the effect

of cyclic

diesterase

alone.

per

First,

does not

to enhance

strated

in this

potent

ALAS induction Recent to its

rat

not

diaphragms,

hormone.

These

of basal

rate

that

CAMP did

theophylline The doses high

in

chick

embryo

livers.

studies

1-methyl-3-isobutylxanthine,

suggested

other

of phosphodiesterase. the

whereas authors

not

uptake

on the

that

phosphodiesterase

Secondly,

theophylline

derivative they

to be 15 times (12),

were

as demon-

did

not

cell

of theophylline Assuming

of theophylline

that

more

suppress

division

delayed

cell

in

Mains

synchronized

dibutyryl hormone

effect

of

in growth

a 30% inhibition et al.

(14)

Tetrahymena,

found whereas

division.

we used in the equal

this

related

that

by growth

produced

protein.

not

found

abolished

theophylline into

(13) acid

completely

incorporation

inhibit

effects

of a-aminoisobutyric

found

significantly

(9 mg/embryo).

through

known

Payne et al.

theophylline also

of leucine

indicate

embryos.

have

affect

hepatic

contrary

ALAS induction

chick

in dose of 10 mg

dibutyryl

(6);

in

of phospho-

cAMP or its

embryos

than

and in some cases

mediated

chick

(CAMP).

shown to be

of evidence

in

inhibition

CAMP did

Two bits

shown that

of phosphodiesterase

were

increased

ALAS induction

inhibitor

monophosphate

theophylline

is not necessarily

study,

of induction

by an inhibitor

intraperitoneally in rats.

available.

phosphodiesterase

derivative

(-11) f ound that

10 fold

to inhibit

mimicked

structure

inhibition

enzymes $w,

was fully

we have previously

inhibit

found

hepatic

the possibility

currently

',5'-cyclic

dibutyryl

administered

of theophylline

inhibition.

adenosine-3

nucleotide Beck et al.

cause

is known

in ALAS activity

enzyme protein of data

could

Theophylline

of several

decarboxylase

the effect

theophylline

increase

However,

or alter

on the basis

CAMP or its

100 gm body weight

ornithine

degradation

catabolizes

induction

this

of the enzyme.

he excluded

by which

yet

inhibits

(synthesis)

cannot

The mechanism

(la),

theophylline

may increase

synthesis

of ALAS is

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

distribution,

1251

embryonated this

eggs were

dose would

give

relatively a

Vol. 53, No. 4,1973

concentration with

of approximately

this

concentration

occasional However,

for

the periods

of rats

on doses

in later

studies

we have

and this

enzyme suppression Our preliminary

in concentrations

the egg. of time

of 180 mgfkg used

ultrastructure

of ph.osphodiesterase

studies in chick 2

vitro,

The embryos

remained

We have

of body weight

the

inhibition

microscopic

RESEARCH COMMUNICATIONS

studied.

100 mgfkg

seem to decrease

(75 to 80 percent electron

to hepatic

an inhibitor

did not

AND BIOPHYSICAL

1.0 mM in

death

mortality,

damage

BIOCHEMICAL

embryos

shown

and rats.

theophylline

to eliminate

of theophylline

of ALAS induction have not

had an

of theophylline.

of body weight effect

viable

on

in rat

livers).

any evidence

of

When employed is

frequently

as

used

of 1Q mM.

ACKNOWLEDGEMENTS: J.K. is the recipient of a Special 54746) from Career Development Branch, NIH. H.L.M. is Research Career Development Award (1 K04 AM 46415-01) Supported by U.S.P.H.S. Grant No. AM 12977-05.

Fellowship (1 F03 GM the recipient of a from U.S;P.H.S.

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Tachudy, D.P., Perlroth, M.G., Marver, H.S., Collins, A., Hunter, G., and Rechcigl, M. (1965) Proc. Natl. Acad. Sci. U.S. 53, 841-847. Stanbury, J.B., Wyngaarden, J.B., and Fredrickson, D.S., eds. (1972) Metabolic Fiasis of Inherited Disease, pp. 1087-1140, McGraw Hill, New York. Tschudy, D.P., and Bonkowsky, H.L. (1972) Fed. Proc. 3l, 147-159. Marver, H.S., Collins, A., Tschudy, D.P., and Rechcigl, M. (1966) J. Biol. Chem. 241% 4323-4329. Rose, J.A., Hellman, E.S., and Tschudy, D.P. (1961) Metabolism lo, 514-521. Korinek, J., and Moses, H.L. (1973) Fed. Proc. 32, 865Abs. Marver, H-S., Tschudy, D.P., Perlroth, M.G., Collins, A. (1966) J. Biol. Chem. 241, 2803-2809. Lascelles, J., and Altshuler, T. (1969) J. Bacterial. 98, 721-727. Mauzerall, D., and Granick, S. (1956) J. Biol. Chem. 219, 435-446. Weiss, B., Davies, J.I., and Brodie, B.B. (1966) Biochem. Pharmacol. L-i, 1553-1561. Beck, W.T., Bellantone, R.A., and Canellakis, E.S. (1972) Biochem. Biophys. Res. Commun. 48, 1649-1655. Beavo, J.A., Rogers, N.L., Crofford, O.B., Hardman, J.G., Sutherland, E.W., and Newman, E.V. (1970) Molec. Pharmacol. 6, 597-603. Payne, S.G., and Kostyo, J.L. (1970) Endocrinology 87, 1186-1191. Mains, C.W., and Whitson, G.L. (1972) J. Cell. Biol. 55, 162a.

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