Suppression of hepatic ribonuclease during phenobarbital stimulation of drug metabolism

Vol. 38, No. 4, 1970

BIOCHEMICAL

SUPPRESSION PHENOBARBITAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF HEPATIC STIMULATION

RIBONUCLEASE DURING OF DRUG METABOLISM’:

R. T. Louis-Ferdinand and G. C. Fuller Department of Pharmacology and Toxicology University of Rhode Island, Kingston, Rhode Island

Received

January

02881

6, 1970

Summary Hepatic microsomal oxidative demethylase activity of phenobarbitaltreated animals 1 or 3 days following 6 daily administrations of phenobarbital (100 mg/kg) was significantly (p 4 0.05) greater than that of controls. Conversely hepatic microsomal ribonuclease activity of phenobarbital-treated rats was significantly (p 4 0. 05) reduced in rats killed on days 7 and 9 by 99% and 94% respectively. Administration of phenobarbital (50 mg/kg) for 5 days resulted in significant (p < 0.05) inhibition (45%) of ribonuclease and increased microsomal oxidative demethylase activity. Phenolpthalein B-glucuronidase activity of hepatic microsomal fractions obtained from phenobarbital-treated animals was not significantly (p 7 0.05) different from controls. Phenobarbital (2.3 mg/ml) did not inhibit ribonuclease activity following in to the assay system. These data suggest the -- vitro additions possibility that induction of drug -metabolizing enzymes by phenobarbital may be mediated in part through suppression of enzymes capable of degrading RNA. Introduction Increased of inducing increased

drug

agents RNA

microsomal

1965)

drug

*This Grant-In-Aid

incorporation

(Kato

by Pb is prevented

enzymes

e al., &al.,

1967)

through

Induction

of drug

D (Orrenius

et&.

DNA-dependent indicate

with

with

and enhanced

1965).

investigation

by Pb is associated

administration

associated

by actinomycin

is mediated of this

following

(Pb) has been (Gelboin

Results

(RNase)

activity

activity

Pb induction

activity.

metabolizing

ribonuclease

acid

that

enzyme

as phenobarbital

polymerase

enzymes

indicating

polymerase

such

amino

metabolizing

metabolizing

that

reduction

,

RNA

induction

of

of hepatic

activity.

work was supported from the University

by an NDEA pre-doctoral of Rhode Island Research 811

fellowship and a Committee.

Vol. 38, No. 4,197O

Materials

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

Methods

Chemicals

used

polymerized

yeast

California

or Adult

fast.

RNA

from

were

5 volumes

of ice-cold

2 hrs

and

was

The

O°C.

assayed

for

Protein

content

method

of Lowry

10 J.&I,

natant

(8. 8 mg

MgC12

phosphate

RNase

activity

described

by

containing

as

aminopyrine

of Ap

previously

I.E.C.

was

centrifuged

was

for

Ap

were

minute

with

centrifuged

at

preparative

with

at

105,000

0.25M

x g

sucrose

demethylase

activity.

estimated

according

to the

yeast

protein/ml.

50 uM,

by

The

RNA

and

for

Easton,

contained:

-6 -phosphate 0.8

ml

suspension

1962).

15 PM,

105,000

x g super-

(6 mg

protein),

and

7.4.

estimated

0.2

assaying

determinations

glucose

microsomal

pH

by

(McMahon

45 JIM,

ml

(1969).

estimated

demethylase

nicotinamide

0.2

was

was

described

semicarbazide

buffer

mg

B-60

and

one

were

(Ap)

suspensions

contained:

18-39

for

rinsed

overnight

sucrose

RPM

Angeles,

City.

following

Homogenates

was

Los

York

0.25M

1000

supernate

and

50 JIM,

polymerized

ice-cold

x g pellet

(5 ml)

Tsukada

determinations

Calbiochem,

guillotined

a Model

Highly

(1951).

protein),

0. 1M

highly

al.

medium

2 uM,

1 ml

resulting

equivalent.

New

at

in

demethylation

incubation

NADP

O°C

105,000

formed

with

sucrose.

RNase

--et

formaldehyde

aminopyrine

situ

of microsomal

Microsomal

The

the

from

were

homogenizer

at

or

Laboratories,

in --

15 minutes

grade

obtained

rats

0.25M

and at

as

Research

a coaxial

ultracentrifuge, for

used

perfused

with

x g for

was

reagent

Sprague-Dawley

homogenized

14,000

analytical

Mann

male

Livers

were

a modification

incubation

ml

0.2M

and

lo-25 RNA

medium

~1

buffer

of microsomal added

a procedure

(1 ml)

Tris-HCl

was

812

of

last

for

pH

RNase

7.6,

1.0

mg

suspension and

the

incubation

was

Vol. 38, No. 4, 1970

carried

out in air

the incubation the mixture

was

for

shaken

mn vs.

Readings

after

distilled

RPM

obtained

were

The

was

soluble

15 minutes.

dilution

with

using

2-5

for

added

rotor

The

Following

as a precipitant was

separated

absorbance

by

of 1 ml of the

water

double-beam

absorbances

and

of an I. E. C.

ml of distilled

a Beckman

corrected

incubator.

fraction

at OoC in the #874 for

water

in a Dubnoff

in 76% ethanol

thoroughly.

ultracentrifuge

supernatant

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20 minutes

1M HCl

at 10,000

preparative clear

at 37’C

1.0 ml

centrifugation

260

BIOCHEMICAL

was

read

at

spectrophotometer.

of suitable

tissue

and

RNA blanks. Phenolpthalein-B by Talal.ay

-glucuronidase

activity

was

estimated

as described

--et ~$1. (1946).

TABLE

I

INHIBITION OF HEPATIC FUBONUCLEASE DURING PHENOBARBITALa (100 mg /kg) INDUCTION OF MICROSOMAL AMINOPYRINE DEMETHYLASE

RNaseb Group

(OD 260 /mg protein) t S.E.

N

Control

Aminopyrine demethylase urn formaldehyde formed/mg proteinihr

0.313

$ 0.056

17.7

+-

1.7

Killed

on day 7

3

0.004

: o.oo2c

93.4

‘r 10. 9c

Killed

on day 9

3

0.018

$ 0. O1!jc

79.2

+

aTwo bl.

groups controls

of rats 400-550 gm treated received distilled water.

0 ml of incubation medium vs. distilled water.

‘Significantly from

different each other.

from

was controls

with

diluted (p

813

100 mg/kg

with ( 0.05)

5 ml HOH

Pb for

7.6c

6 days

and read

but not statistically

at 260 IIU~ different

Vol. 38, No. 4, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Results Table days

on

Ap

fractions Ap -fold

99%

to

sacrificed activity

was

RNase

activities

demethylase

killed

day

Hepatic

(p

day

above

Ap

demethylase

to

by

increase

toward

94%

while

control

repeated

HEPATIC

relative

reduced

0. 05)

with

(OD 260/mg ? S.E.

0.357

t 0.039

Phenobarbital Treated

4

0.197

? 0. oo7c

bl.

0 ml of incubation read at 260 mu

%ignificantly

different

gms was received

medium was vs. distilled from

controls

approximately of <

microsomal

0.05)

reduced of

by

rats

to

controls

while

of

control

activity.

hepatic

protein)

5

375-410 Controls 6.

micro-

RNase

Ap

microsomal

fractions

II

Control

group of rats for 5 days. killed on day

Hepatic

values.

DURING OF

Aminopyrine mnm formedlmg

N

aOne

6

microsomal

corresponding

RIBONUCLEASE mg/kg) INDUCTION DEMETHYLASE

(50 AMINOPYRINE

Pb.

activity

microsomal -fold

liver

increased

RNase

TABLE

RNaseb

7 was

(p

was

Group

of

significantly

decrease

INHIBITION OF PHENOBARBITALa MICROSOMAL

day

i. p. ) for

with

was

9 appear

experiment

mg/kg

administration

four

<

(100

associated

on

increased

significantly

on

last

animals

9 was

treatment

Conversely

controls.

activities

The

rats

Pb activity

of

values. on

RNase the

Pb-treated

control

of

following

demethylase

from of

influence and

3 days

relative

fractions

the

demethylase 1 or

somal five

I shows

demethylase formaldehyde proteinlhr

8.7 78.6

treated distilled

with 50 mg/kg water. All

diluted water. (p

814

with

(

0.05).

2.0

ml

t t

3.2 10.

6c

phenobarbital animals were

distilled

water

and

Vol. 38, No. 4,197O

of rats with

BIOCHEMICAL

sacrificed

the dose

RNase

one day following reduced

demethylase

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

administration

to 50 mg/kg

i.p.

(Table

by Pb was

associated

with

a significant

relative

to controls.

activity

Phenolpthalein-B-glucuronidase obtained

from

Pb-treated

controls

thereby

not a factor assay

of 5 daily

system

activity animals

was

that

differential

indicating

in these

II).

studies.

The

did not inhibit

RNase

Stimulation

of

fractions

different

from

contamination

of Pb 2.3

activity

reduction

of microsomal

lysosomal

of Pb

of Ap

p < 0.05

not significantly

addition

doses

was

mg to the RNase

of the microsomal

fractions.

Discussion -These induction

data demonstrate

of microsomal

Pb appears ment

activity

reduction

daily

for

while

of hepatic

Enhanced

1967)

et&., (Kato

polymerase ectomized

and increased

Reduction

with for

Pb

of RNase

6 days

by

by Pb treat-

a 45% reduction resulte

(Barnabei

microsomal

A reciprocal

has been

shown

rats

from

of

in a 99%

amino

acid

has been

aminotransferase

acid

(Barnabei

1968;

Gelboin

activity nuclear

RNA

and Ottolenghi,

and alkaline

correlated activities

815

of DNA-

incorporating liver

agents

in cortisone-treated-adrenal-

workers

of hepatic

stimulation

between

hydrolysis

by other

of inducing

and Ottolenghi,

relationship

RNA

in triamcinolone-treated and alanine

administration

activity

reduction

in aspartate

following to result

Furthermore,

1968).

of Pb daily

is reported

and microsomal rats

during

of Ap demethylation

is associated

synthesis

polymerase

et &. , 1965).

5 days

RNase

RNase.

protein

RNA

activity.

Induction

100 mg/kg

such as Pb or cortisone dependent

of hepatic

Ap demethylase

to be dose-related.

(50 mg/kg)

RNase

suppression

with

RNase

simultaneous

(Sarkar,

1969).

activity increases Loss

of

BIOCHEMICAL

Vol. 38, No. 4, 1970

microsomal treated

amino rats

(Gelboin,

has been

1964).

production

incorporating

reported

lysosomal

these

with

suppression

1961)

cannot

activity

untreated

and the failure

suggest

microsomes disruption media

that

in these

studies

of lysosomes. did not inhibit

of RNase

activity

the inducing may

these

in RNase

in vitro --

addition

activity,

thereby

However,

x g liver

differences

activities

inhibitor

results.

with

attributable

of Pb (2.3 excluding

super-

in B-glucu-

associated

is not due to contamination

The RNase

RNase

in the 105,000 to detect

to altered

mg)

the to

to incubation

direct

inhibition

one of the resultant

effects

of

which

in

by Pb.

In conclusion

turn

the changes

RNA

by Pb is attributable

from

natant

to controls

RNase.

be ascertained activity

relative

stimulated

or increased

RNase

ronidase

that

turnover

of measurable rats

3 methylcholanthrenerate

of liver

the absence from

suggest

suppression

RNase

from

at a lower

data

of RNase

or microsomal (Shortman,

activity

to occur

Collectively

is associated

Whether

levels

acid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

agent be indirectly

these

data

Pb is its

suggest

that

suppression

responsible

of ribonuclease

for

enhanced

synthesis

activity

of enzyme

protein.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Barnabei, 0. and Ottolenghi, C., in Advances in Enzyme Regulation, Vol. VI, p. 189, Pergamon Press, New York(1968). Gelboin, H. V. , Wortham, J.S. and Wilson, R. G., Nature 2, 281 (1967). Gelboin, H. V., Biochim. Biophys. Acta 91, 130 (1964). Kato, R. , Loeb, L. and Gelboin, H. V., Nature 205, 668 (1965). N. J., Farr, Lowry, 0. H. , Rosebrough, A.L. and Randall, R. J., J. Biol. Chem. 193, 265 (1951). MC Mahon, R. E. and Easton, N. R., J. Pharmacol. 135, 128 (1962). Orrenius, S., Ericsson, J.L.E. and Ernster, L., J. Cell. Biol. 25, 627 (1965). Sarkar, N. K. , FEBS Letters 2, 37 (1969). Biophys. Acta 51, 37 (1961). Shortman, K., Biochim. Talalay, P. , Fishman, W. and Huggins, C., J. Biol. Chem. 3, 757 (1946). Tsukada, K., Biochim. Biophys. Acta 186, 21 (1969). 816