Vol. 91, No. 2, 1979 November

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

28, 1979

Pages 658-664

THE INHIBITION OF INDUCTION OF MICROSOMAL MONOOXYGENASE ACTIVITY BY 1,3-DIAMINO-Z-PROPANOL, AN INHIBITOR OF ORNITHINE DECARBOXYLASE Hannu Department Finland

Raunio

of Pharmacology,

and Olavi

Pelkonen

University

of Oulu,

SF-90220

Oulu

22

September 21,1979

Received

SUMMARY: The p.o. administration of 1,3-diamino-2-propanol, an indirect inhibitor of ornithine decarboxylase, to rats and mice inhibited in a dose-dependent manner the-induction of cytochrome P-450, benzo(a)pyrene hydroxylase, and 7-ethoxycoumarin O-deethylase by phenobarbital, B-naphtoflavone or Clophen C, a mixture of polychlorinated biphenyls. The results are consistent with the hypothesis that the induction of ornithine decarboxylase is a necessary step in the induction of microsomal monooxygenases. The induction

of

carboxy-lyase, of

cyclic

EC 4.1.1.17,

in

barbital,

1254

following

concentration

to the

and/or in

lizing

(1,2).

enzymes

Repeated inhibitor putrescine of

occurring --et

al.

and in

(1).

A single

@ I979

injection

1) increased

and 3) induction

of ODC prevented

the

and spermidine

(3,4)

the

stimulation

after

the

partial

showed that

a close administered

by Academic Press, Inc. in any form reserved.

658

kinase,

drug-metabo-

accumulation a profound

DNA synthesis

analog

CAMP

an indirect

and produced

resection

shown to

protein of

prereplicative

of liver

induction of pheno-

of CAMP-dependent

0006-291X/79/220658-07$01.00/0 of reproduction

subsequent

of 1,3-diaminopropane,

1,3-diamino-2-propanol,

Copyright All rights

the

suggested

has been

of events:

ODC activity

activation

has been

or 3-methylcholantrene

activation

injections

kinase

growth

sequence

2) an increase

Piik

ODC) in response

enzymes

Aroclor the

bition

(L-ornithine

protein

liver

drug-metabolizing

elicit

decarboxylase

3' ,5'-AMP-dependent

to be a key event of

ornithine

of

the

liver

of inhi-

normally (4,s).

of 1,3-diaminopropane, orally

inhibits

completely

BIOCHEMICAL

Vol. 91, No. 2, 1979

ODC activity If

in

ODC induction

livers is

drug-metabolizing

of partially indeed

enzymes,

by 1,3-diamino-2-propanol drug-metabolizing

AND BIOPHYSICAL

hepatectomized

a prerequisite then should

RESEARCH COMMUNICATIONS

the

for

the

inhibition

also

rats

(6).

induction

of

of ODC induction

inhibit

the

induction

of

enzymes.

MATERIALS AND METHODS: 1,3-diamino-2-propanol was purchased from Fluka AG Chemicals: (Buchs, Switzerland). t3-Naphtoflavone was obtained from Pfalz & C from Bayer (LeverBauer Inc. (Flushing, NY, USA), Clophen kusen, West Germany) and phenobarbital from E. Merck (Darmstadt, The lingonberry juice was purchased from a local West Germany). grocery. All other chemicals were of the highest purity commercially available. Animals and treatments: Male Wistar rats (120-160 g) and male and female mice of both the aryl hydrocarbon responsive (C57B1/6) and aryl hydrocarbon non-responsive (DBA/2) strains (20-30 g) were used in this study. The animals were fed standard rodent chow. Drinking water was replaced during the actual experiments by lingonberry juice to mask the taste of diaminopropanol. Various doses of diaminopropanol were given orally to the animals, the controls receiving the vehicle alone. 24 hours after the beginning of diaminopropanol administration the animals were injected with either 8-naphtoflavone (100 mg/kg i.p. in corn oil) or Clophen C (200 mg/kg i.p. in corn oil). The aryl hydrocarbon non-responsive mice received phenobarbital (250 mg/litre p.o.) for 7 days. Diaminopropanol was continued until the killing of animals. The mice were killed by cervical dislocation and the rats by decapitation, the livers of the animals were removed, chilled immediately on ice, weighed, and homogenized in 4 vol. of 0.1 M sodium potassium phosphate buffer, pH 7.4. The homogenate was used for enzymatic assays. Determination of monooxygenase activities were all performed on samples from individual livers. The livers from each group of 6 mice were pooled for measuring the cytochrome P-450 content in microsomes. Analytical methods: 7-Ethoxycoumarin 0-deethylase activity was measured as described by Aitio (7). Benzo(a)pyrene hydroxylase activity was determined by the method of Nebert and Gelboin (8). Cytochrome P-450 content was measured according to Omura and Sato (9). Microsomal protein concentrations were determined according to Lowry --et al. (lo), using bovine serum albumin as a standard. RESULTS: effect

In

the

first

of diaminopropanol

monooxygenase

activities

propane

caused

alone

experiment

with

C57B1/6

administration

on the

by B-naphtoflavone a significant

mice

decrease

(table

induction

was studied. in

1) the of Diamino-

benzo(a)pyrene

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1: Effect of 1,3-diamino-Z-propanol (DAP) on mouse hepatic 7-ethoxycoumarin 0-deethylase and benzo(a)pyrene hydroxylase activities and cytochrome P-450 content after the administration of B-naphtoflavone (BNF). 7-ethoxycoumarin 0-deethylase (nmol/min per g of liver)

Treatment

Control DAP 50 mM BNF DAP 10 mM+BNF DAP 50 mM+BNF

17.90 17.29 30.08 32.08 23.20

-I k f + +

Benzo(a)pyrene hydroxylase (nmol/min per g of liver)

5.87 3.40 2.59 2.99 5.67 a

13.41 5.28 37.06 43.98 26.15

? f f + f

Cytochrome P-450 (nmol/mg of microsomal protein)

2.91 3.34 3.78 9.62 12.61

1.34 1.20 1.62 1.65 0.83

The mice received lingonberry juice alone (controls) or containing 10 mM or 50 mM1,3-diamino-2-propanol for 48 hours. After 24 hours some of the animals were injected with 8-naphtoflavone (100 mg/kg i.p. in corn oil). Liver samples were prepared and enzyme assays carried out as described in "Materials and methods". Each group consisted of 6 animals. The results are expressed as the mean + SEM. a Significantly

different

hydroxylase

activity,

pretreatment

with

oxygenase

drinking

from

had no effect

f3-naphtoflavone

on other

increased

up to 276 per cent

. The 10 mM concentration water

decreased

but

activities

hydroxylase)

compared to the induced value

had no effect

both

cytochrome

the induced

value)

activities.

different

(aryl

hydrocarbon

induction,

content

whereas

(50 per cent

and monooxygenase

The

mono-

of diaminopropanol

on the P-450

(~~0.05)

in 50 mM

decrease

activities

(about

26

(table

2) the effect

per cent). In the

second experiment

of diaminopropanol Concentrations toxic

propanol cent

of diaminopropanol

during inhibited

decrease

DBA/Z mice

on the phenobarbital

to mice and only

be studied

with

from

this

the

of

lowest

one-week

induction

50 and 100 mM were too

concentration experiment.

the

induction

of

the

induced

value),

660

was studied.

of

10 mM could

As seen,

cytochrome

P-450

benzo(a)pyrene

diamino(50 per hydroxy-

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2: Effect of 1,3-diamino-Z-propanol (DAP) on mouse hepatic 7-ethoxycoumarin 0-deethylase and benzo(a)pyrene hydroxylase activities and cytochrome P-450 content after the administration of phenobarbital (PhB).

Treatment

7-ethoxycoumarin 0-deethylase (nmol/min per g of liver)

Benzo (a)pyrene hydroxylase (nmol/min per g of liver)

Cytochrome P-450 (nmol/mg of microsomal protein)

18.04 f 3.85 36.58 + 4.48

19.83 t 4.37 25.96 f 3.84

0.99 1.88

22.31 i 7.49 a

16.02 ?r 4.70 b

0.95

Control (6) PhB (6) DAP 10 mM + PhB (4)

The mice received lingonberry juice alone (controls) or containing 250 mg/l of phenobarbital or phenobarbital and 10 mM 1,3-diamino-2propanol for 7 days. The number of animals in each group is given in parenthesis. The results are expressed as the mean f SEM. a Significantly b Significantly lase

different different

(38 per

cent).

cent)

Apparently

duction

of

Clophen pyrene

and 7-ethoxycoumarin diaminopropanol

(~~0.01) (~~0.02)

0-deethylase

prevented

(39 per

completely

the

in-

caused by phenobarbital.

In the third effect

compared to the induced value compared to the induced value

experiment

diaminopropanol

C was studied. hydroxylase

oxygenases.

with

male Wistar

on the

induction

Diaminopropanol

activity,

but

(table

alone

decreased

mono-

decreased

in drinking

water

by

benzo(a)-

on other

enzyme levels

of diaminopropanol

3) the

of monooxygenases

had no effect

The Clophen-inducible

the concentration

rats

when

was in-

creased. In all

experiments,

the extent

than what has been earlier “suppression” containing reminiscent

of vehicle of

inducibility

observed

lingonberry

effect”

tories.

661

was usually

in our laboratory.

may be related

(50 per cent

the”glucose

of induction

observed

to juice)

lower

This

the sugarand is

in many labora-

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 3: Effect of 1,3-diamino-Z-propanol (DAP) on rat hepatic 7-ethoxycoumarin 0-deethylase and benzo(a)pyrene hydroxylase activities and cytochrome P-450 content after the administration of a polychlorinated biphenyl (PCB). 7-ethoxycoumarin 0-deethylase (nmol/min per g of liver)

Treatment

Control DAP 50 mM

11.41 f 2.01 11.70 ? 1.32

PCB

28.10 f 2.07

Benzo(a)pyrene hydroxylase (nmol/min per g of liver) 18.67 12.25 49.67 46.37 37.76 18.84

DAP 10 mM+PCB 21.08 f 1.97 a DAP 50 mM+PCB 16.37 f 2.56 a DAP 100 mM+PCB 16.54 + 1.56 a

+ f f f + +

Cytochrome P-450 (nmol/mg of microsomal protein)

5.62 2.80 3.77 2.49 5.01 b 2.08 a

0.86 0.83 1.87 1.25 1.28 1.18

f 5 f f + 2

0.14 0.14 0.18 0.37 ' 0.18 b 0.52 '

The rats received lingonberry juice alone (controls) or 10 mM, 50 mMor 100 mM1,3-diamino-2-propanol for 48 hours. After 24 hours some of the animals were injected with a polychlorinated biphenyl, Clophen C (200 mg/kg i.p. in corn oil). The number of animals in each group is 6. The results are expressed as the mean f SEM. a Significantly b Significantly ' Significantly

DISCUSSION:

propanol crease

different different different

The present

to mice

and rats

inducers.

credence

to the

biochemical

hypothesis

sequence

experimental

system

strains

responsive

that

resulting

of

ODC is in

with

the

several

the Ah locus the

662

the

in-

administration

the induction

(1).

(C57B1/6),

diamino-

a key enzyme

In

of

in

studied

inducibility,

enzymes (12). induction

the

of microsomal

the best

of monooxygenase

(ll),

(p