Vol.68,No.3,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RNA SYNTHESIS AND RNA POLYMERASE ACTIVITY IN HEPATICNUCLEI ISOLATED

FROM RATS FED THE CARCINOGEN 2-ACETYLAMINOFLUORENE Michael P. Adams and Jay I. Goodman Department of Pharmacology Michigan State University East Lansing, Michigan 48824

Received

December 6,1975 SUMMARY

An assessment was made of the activity of RNA polymerase I and the capacity for RNA synthesis, under conditions optimized for RNA polymerase I activity, in hepatic nuclei isolated from rats fed a diet containing the hepatic carcinogen AAF. Animals were maintained on the carcinogenic diet for either 4, 7 or 14 days. RNA polymerase activity progressively increased with time on the carcinogenic diet. However, the capacity for RNA synthesis remained quite constant. These results are suggestive of a progressive inhibition of DNA template activity during the early stages of AAF-induced hepatocarcinogenesis. The "permanance" of the increase in polymerase activity was examined by switching carcinogen fed animals to a control diet for either 2 or 5 days prior to making an assessment of the above parameters.

It is well nogens

established

that

become covalently

tissues

(1,2,3),

and it

of such binding time,

of the

(2).

investigators

macromolecular

carcinogen

er carcinogen

studies

their

or an inhibition

of the

capacity

Recently,

et al.

(4)

ase II activities,

in liver

nuclei,

not

as a result

at the

mechanism(s) N-OH-AAF,

aspects

of hepatic

directed

toward

the question

that

both inhibited

of RNA polymerase as a template

RNA polymerase 2 hours

present

been unequivocally

derivative,

in an inhibition

are

cancer

However,

carci-

in target

on various

of DNA to serve

reported

induce

the potential

N-hydroxy

effect

results

they

has (have)

have been primarily

administration

Herzog

target(s)

and its

that

of chemical

of the cells

macromolecules.

to elucidate

AAFl

have examined These

thesis.

has become axiomatic

In an effort

hepatic

of many classes

to DNA, RNA and proteins

to one or more of these

the critical

identified

bound

the members

after

for

of action several RNA synof whethactivity

RNA synthesis.

I and RNA polymera single

injec-

1Abbreviations: AAF, 2-acetylaminofluorene; N-OH-AAF, N-hydroxy-2-acetylaminofluorene; A-AAF, N-acetoxy-P-acetylaminofluorene; 3H-ATP, [2,8-3H] adenosine 5'-triphosphate; TCA, trichloracetic acid; Poly (dA-dT), a high molecular weight, double stranded, copolymer composed of alternating dA and dT units.

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

850

Vol. 68, No. 3,1976

tion

of N-OH-AAF.

from

the livers

results

BIOCHEMICAL

No inhibition of the

essentially

hibition

than

that the

that

plate

following

a single

Inhibition weeks

et al.

reports

injection

for

pacity

report

for

describes

studies

RNA synthesis

from

rats

view

of the

which

had been fed a diet

and by Zieve to inet al.

nucleolar

an affect

DNA isolated

from

DNA temon RNA poly-

rats

fed AAF for

has been reported

apparently

conflicting

reports

or the

activity

of nucleolar

template

optimized

for

polymerase,

an assessment RNA polymerase

through

feeding

tential

for

involved

as opposed

assessing

in the

by Troll

of the

Studies

biochemical

from

studies.

A control

U. S. Pharmacopiea p-aminobenzoic

(w/w)

Mills,

0.11

0.24

diet: Research basal

XIV salt

acid,

E acetate Teklad

Spartan

AAF, purchased

AAF.

RNA polymerase

were

nucleolar

RNA

to carcinogens

compound,

alterations

In

(4,7)

conditions

exposure

of the

which

offer are

the

likely

poto be

diet

AND METHODS

Male

Sprague-Dawley

Animals, (11)

Inc.,

g/kg

of diet;

from

Eastman

This

control

Kodak,

851

MI,

basal long

0.11

units)

weighing

Haslett,

for

inositol,

international

Wisconsin.

rats

(carcinogenic

mix and supplemented,

g (121

Madison

isolated

administration

and the

involving

ca-

process.

and carcinogenic

20 g, obtained

acute

our experimental

injection

MATERIALS Animals

nuclei

carcinogen

nucleolar

RNA synthesis

to a single

progressive

in hepatic

the hepatic

DNA (8)

I (10).

was made of the

as to whether

of nucleolar

carcinogenic

activity

containing

in an inhibition

0.05%

than

a determination

and RNA polymerase

results

from

These

Grunberger

of the

RNA synthesis

in which

of a carcinogen

vitamin

(4).

(5,6),

However,

impairment

of hepatic

isolated

was more susceptable

of N-OH-AAF rather

as a template

be detected et al.

of the enzyme.

of the capacity

to serve

by Glazer

is preferential

of chromatin

could

RNA polymerase

form

there

activity

(9). This

these

template animals

nucleolar

nucleoplasmic

have reported

several

earlier

the

(8)

merase.

of the

carcinogen-treated

confirm

(7) who indicated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/kg diet

Rochester,

g/kg

were

diet)

term

used for

containing

feeding,

of diet;

of diet,

175 f

with

and dry

was purchased

was supplemented New York.

with

Food inges-

Vol.68,No.3,1976

tion

BIOCHEMICAL

was regulated

lights

by maintaining

are on from

Animals

were

7 p.m.

maintained

AND BIOPHYSICAL

the

until

animals

7 a.m.

on the

diet

on the AAF-containing

diet.

The animals

diet

8 a.m.

and all

at approximately Isolation

sucrose,

of hepatic

as described

retention

of both

these

I.

were

dithiothreitol,

mM, 20 pCi/umole;

0.2

ml.

were

The reaction

to being

measured (isolab

with Inc.,

tions

were

portional

run

for

This

Ohio) 3 min.

amount

validity assayed

the

basic

and the

Nuclear),

(14).

scintillation

the

rate

of at

5% TCA. percipitate

(4:l

spectrometer, fluor.

was linear

twice

v/v)

Radioactivity

as the scintillation time

volume

was washed

ethanol-ether

acid

6.0 mM;

and incubated

The percipitate

was multisol

All

reac-

and directly

pro-

added. of RNA polymerase template,

poly

DNA was blocked

by the

addition

reaction

described

(New England

was added

in

RNA poly-

100 mM; MgC12,

an exogenous

been verified

employed

of 2.0 ml of ice-cold

The activity

has been successfully

(13).

resistant,

of nuclei

5 min.

liquid

which

of nuclei

employing

has recently using

addition

addition

was employed during

of the endongenous procedure

3380

in the

(70 * 20 pg DNA) in a final

ml of 88% formic

I activity:

was measured

activity

model

results

of that

3H-ATP

5% TCA and once with

in 0.5

+ 1 hour.

conditions

HCl (pH 8.2),

placed

in hypertonic

procedure

a-amanitin

mM each;

at 1000 x g for

a Packard

to the

by the

isolated,

The assay

Tris

to being

on the carcinogenic

of RNA polymerase

0.5 ml of a 0.5% solution,

dissolved

Akron,

This

the

of the day.

at 9 a.m.

was a modification

by the

of ice-cold

RNA polymerase nuclei

initiated

by centrifuging

2.0 ml portions

prior

and contained

started

were

nucleolar,

employed

was stopped

serum albumin,

collected with

(8)

the

and 50~1 of nuclei

Reactions

Bovine

nuclei

forms

12 hours

killed

(12).

2 mM; GTP, CTP, UTP, 0.4

0.4

30".

for

mixture

et al.

always were

Hepatic

other

room in which

3 to 5 days prior

RNA synthesis:

optimum

The reaction

by Grunberger

for

for

animals

and engaged

capacity

experiments

merase

free

the

were

by Yu and Feigelson

the

Assay of the

nuclei:

in a windowless

and off

control

RESEARCH COMMUNICATIONS

employed

by Grunberger

by Yu (13). mixture

described

852

I in isolated

(dA-dT).

The template

of actinomycin-0. et al.

RNA polymerase above containing

(8)

and its

I activity

was

20 ~1 of

Vol.68,No.3,1976

nuclei

(28 + 6 ug DNA), 8 pg poly

and omitting time

BIOCHEMICAL

All

GTP and CTP.

the rate

was linear

AND BIOPHYSICAL

(dA-dT)

(Miles),

reactions

and directly

will

RESEARCH COMMUNICATIONS

4 ug actinomycin-D

run for

proportional

10 minutes

(Sigma)

during

to the amount

which

of nuclei

add-

ed. RESULTS AND DISCUSSION A single synthesis did tion

injection

in vivo

not affect of AAF did

of N-OH-AAF has been reported

(5).

this cause

Acute

process

administration (5).

a transient

of AAF (40 mg/kg)

However, inhibition

0

at very

OFF

high

of hepatic

2 DAYS

to inhibit

doses

hepatic

RNA

to normal (160 mg/kg)

RNA synthesis

rats injec-

in male

5 THE

AAF

DlET

Fig. 1.: Effect of AAF ingestion on RNA synthesis and RNA polymerase I ac.tivity in isolated hepatic nuclei. RNA synthesis (u ) and RNA polymerase activity (u) were determined, as described under Materials and Methods, in nuclei isolated from animals fed a diet containing 0.05% (w/w) AAF for 4 (A), 7 (B), or 14 (C) days. Groups of animals were killed at either the end of the period of carcinogen feeding (zero time on the chart) or at 2 and 5 days after being returned to the control diet. Each point represents the mean f S.E. of the data obtained from 3 animals. The control values (mean ? S.E. of the data obtained from 12 animals) were 2,2028 + 151 DPM/mg DNA/min and 488 + 67 DPM/mg DNA/min for RNA synthesis and RNA polymerase I activity, respectively.

853

Vol. 68, No. 3, 1976

mice

(15).

prepared

BIOCHEMICAL

The capacity from

rats (Fig.

prepared

animals

level

but

ed which

indicates

of RNA polymerase, can account

for

is easily tonic the

lost

buffer

were all

activity

(8)

activity

isolation

the

nuclear

nuclei,

isolation

procedure

favored

loss

the

1C).

acute

ef-

have con-

has been present populations population activity

and

the use of an iso-

studies

of free

(Fig.

nuclei

polymerase

involves

those

the

with

RNA polymerase

procedure

findings

diet

of two distinct

nuclear

(Fig.

to the control

evidence

The free

total

4 days

hepatic

Recent

(13,16).

of these

dealing

in nuclei

40%, in nuclei

carcinogenic

in isolated

in hepatic

either

and returned

of the studies

as much as 50% of the

In view

off

for

approximately

14 days,

taken

and engaged

when the

slightly, for

unchanged

diet

was inhibited.

presence,

free

(13).

nuclei

not

the

carcinogenic

the AAF diet

on RNA polymerase

polymerase

was essentially

was elevated

the animals

of N-OH-AAF that

It

fed

Some (4,5,6,7)

cluded

on the

1B).

5 days after

fect

RNA synthesis

maintained

1A) or 7 days from

for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(4,5,6,7)

in which

RNA polymerase

may have

of the

of RNA

to be reevaluated. The synthesis polymerase

activity

RNA synthesis. chromatin

of RNA in isolated and the The primary

relative

merase

binding

(17).

Alkylation

a result

of reacting

due to blocking Therefore,

while

the

inhibition

capacity

increase

manifestation

a reduction

chain those for

in

RNA polymerase

the

synthesis

elongation

A-AAF,

RNA synthesis

in which remains

as a template

in the

number

in the

ability

of the

RNA polymerase unchanged

its

appears with

polymer

of DNA, as

decreases

interfering

in whole

elongation

Arylamidation

markedly

for

of RNA poly-

of RNA chain

of RNA and this than

level

of gene repression

(18,19).

rather

situations

to serve

the rate

in a decrease

with

a function

of lo-fold

decrease

--in vitro

for

of template

Increasing

for it

as a template

21).

--in vitro

of DNA results

as a template

to act

of chromatin

and 3-fold

to serve

is

capacity

to DNA is sites

nuclei

capacity

to be primarily initiation

activity

is

may be indicative

(20, increased of an

activity.

the duration

in RNA polymerase

of carcinogen I activity

(Fig.

854

exposure 1, zero

resulted time

in a progressive

values).

At the end

Vol. 68, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of 4, 7 and 14 days of carcinogen

feeding,

to 145%,

values,

155% and 230% of control

synthesis, quite

under

constant

(Fig.

of the hepatic city

conditions

cess

amount

role

are

reports

were

to occur, tial

in rat

hepatectomy

was variable being

to the control

in those

14 days

(Fig.

1, A & C). which

The evoking

intact

chemical

However,

there

may not

for

would

capa-

was an ex-

the

enzyme on

not play

a limit-

have been the case. between

RNA synthesis.

cortisone

administration

There

increased This

levels

has been shown

(12)

(28,29).

However,

and after

there

(30,31,32). death,

activity

had been fed the

AAF diet

par-

These mutation

not readily

appears products corrected

This

855

to the

either

4 or

elevated (Fig.

process

and it

in 1B).

has been

has also

in target

organs,

repair

been in the

process

to be an error

free

formed

chemical

between

carcinogenesis.

damaging

(32),

in

process

by the DNA excision

to be potentially

and possibly

returned

is one of the early

of the DNA excision

and this

appear

(24)

DNA repair,

are some reaction are

(23).

days af-

7 days

repair)

in culture

can evoke

for

activity

Five

for

remained

diet

carcinogens

The fidelity

which

activity

(excision

cells

has been accessed

exposure.

RNA polymerase

synthesis

carcinogens

in polymerase

of carcinogen

the carcinogenic

in mammalian

and DNA bases

to cell

alteration

polymerase

to chemical

(25,26,27).

cells,

may lead

it

ingestion

RNA synthesis.

sites

correlation

diet,

had been fed

examined

process

this

that

of the

experiments

ribosomal

which

of DNA repair

mammalian

pair

animals

of exposure

animal

cinogens

initiation

upon the duration

value

shown that

all

of the carcinogen-induced

control

extensively

in our

RNA

remained

inhibition for

for

(22).

switched

consequences

indicate

as a template

of a direct

following

depending

animals

results

and consequently

I and increased liver,

I activity,

that

However,

literature

The stability

those

saturated)

in gene expression.

RNA polymerase these

I (i.e.,

was increased

The capacity

in a progressive

is

activity

respectively.

to serve

organ,

of RNA polymerase

of RNA polymerase

together,

explanation

in the

for

AAF results

target

chromatin

ing

Taken

carcinogen

alternative

nucleolar

ter

1).

of DNA, in the

A possible

optimized

RNA polymerase

carre-

i.e.

Vol.68,No.3,1976

There

BIOCHEMICAL

is

a need to determine

lesions,

both

excision

process.

indicate

that

meter

those

These

in those

of carcinogen-DNA

the

consequences

are and those experiments

an assessment

to examine

quences

which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

which

studies

are

represent

of DNA template aimed

of carcinogen-induced not readily

a step activity

repaired

in that

direction,

may be an important

at elucidating

the

functional

DNA by the and paraconse-

interactions.

ACKNOWLEDGEMENT This Institutes

investigation of Health,

was supported National Cancer

by a grant Institute.

(CA-13,344)

from

the

National

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