Preferential binding of 2-acetylaminofluorene to rat liver rRNA during early stages of hepatocarcinogenesis

BIOCHEMICAL

Vol. 47, No. 5, 1972

PREFERENTIAL

BINDING DURING

OF 2-ACETYLAMINOFLUORENE

EARLY

Charles Cancer Research of Biochemistry, Received

May 1, 19’72

Summary

:

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

STAGES

rRNA

OF HEPATOCARCINOGENESIS

C. Irving

Laboratory, University

TO RAT LIVER

and Richard

A. Veazey

Veterans Administration of Tennessee, Memphis,

Hospital, and Department Tennessee 38104

During continuous feeding of 2-acetylaminofluorene-9J4C (AAF-9-14C) under conditions known to induce a high incidence of liver tumors ln male rats, there is preferential binding of the carcinogen to liver rRNA. Furthermore, simultaneous administration of 3-methylcholanthrene, which inhibits liver tumor induction by AAF, resulted in a 35-40s decrease in binding to liver rRNA as opposed to a 50% increase in the binding of AAF-9J’C to liver tRNA. These data support the concept that binding of AAF to liver rRNA might be involved in hepatocarcinogenesis. Administration metabolite

of the carcinogen

to rats leads to base substitution

ment of 2-acetylaminofluorenyl position

of guanine

greater

than that of rRNA

speculation

injection

(4,s).

of AAF

the continuous

additional

experiments

or N-hydroxy-AAF. feeding

rRNA during

@ 1972, by Academic

Press, Inc.

is several-fold

of AAF-9-I’C

liver

or N-

tRNA has led to of action

of

in the interpretation tumors

of hepatocellular

for 8-12 weeks.

following

a

carcinoma As a result

of binding

was preferential

stages of hepatocarcinogenesis.

attach-

at the C-8

to tRNA in the mechanism

but that there

1159 Copyright

tRNA

(6), we found that there was no correlation

initial

by covalent

to rat liver

Induction

of these compounds

or its N-hydroxy

primarily

injection

binding

of binding

acids

One fact that must be considered

tRNA and hepatocarcinogenicity

to rat liver

of liver

a single

The preferential

(AAF)

residues,

of these data is that rats do not develop

requires

to liver

nucleic

radioactivity

or DNA following

the involvement

these hepatocarcinogens of the significance

The specific

(1,2,4).

regarding

in liver

and 2-aminofluorenyl

(l-3).

hydroxy-AAF-9J4C

single

2-acetylaminofluorene

binding

of

of AAF of AAF

Vol. 47, No. 5, 1972

Methods

BIOCHEMICAL

: J4C was fed to Holtzman

2-Acetylammofluorene-9 solution

in dimethylsulfoxide/corn

isolated

as follows.

a .pellet

3 hours Ribosomal

rpm

potassium

converted

determined

to equivalent

by liquid

picomoles

scintillation

were

yielding

(7).

S1 was centrifuged

pfor

(P2) and supernatant

was isolated

from S2, then puri-

salt and re-conversion (dpm carbon-14 residues

(S2).

to the

bound per mg nucleic bound per mg)

were

(2).

and Discussion:

DNA from

male

acids

with 25 ml of cold 6% sodium

of fluorenyl

counting

as a

SS-34 rotor),

to give a pellet

radioactivities

administration

DNA of male rats increased

AAF,

P1 was stirred

from P2 (7) and tRNA

The specific

Upon continuous liver

rpm (Sorvall

to the cetyltrimethylammonium

salt (8).

Nucleic

i.p.

in cold 0.9% NaCl and the homo-

at 10,000

No. 40 rotor)

rats or injected

vol/vol).

and DNA was isolated

(Spinco

RNA was obtained

fied by conversion

Results

(Sl).

for 45 minutes

at 38,000

(l/6,

was homogenized

for 30 minutes

(Pi) and supernatant

aminosalicylate

oil solution

The liver

genate was centrifuged

acid,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2-8 weeks.

binding

to liver

rats (300-400

both sexes. at l-3

to tRNA

of female

8 weeks.

DNA increased

bound to liver

rRNA.

The binding

slowly

pmoles/mg

(loo-150

In male rats,

by 2 weeks,

of AAF-92%

to liver

rRNA

1160

tumor

induction

the value

obtained

by in

tRNA was much less in

tRNA of male rats occurred

2-8 weeks. pmoles/mg)

was observed binding

to

pmoles/mg

of bound carcinogen

to liver

then fell gradually to liver

off at 300-400

to liver

to liver

from

sex difference

of the carcinogen

but reached

Binding

of AAF-9J4C

was constant

significant

rRNA

more

are resistant

tRNA) but the amount

at 100-150

rat liver

which

binding

then leveled

at 4-8 weeks.

binding

The most

to 350 pmoles/mg

rats,

pmoles/mg

and remained

cinogen

In female

The maximum

rapidly,

8 weeks.

for 2 weeks,

pmoles/mg)

days (200-250

of AAF-9J4C,

Binding

of AAF-9%

between

1 day and

in the amount rRNA

to 100-150

of female

declined

of the car-

increased pmoles/mg

rats increased

rapidly by very

BIOCHEMICAL

Vol. 47, No. 5, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

.. rRNA:

DNA

tRNA:rRNA

2

0 Weeks

12345676 Weeks

Figure

Fed 0.04%

and reached

data presented

in Fig. binding

The specific

radioactivity

1 day of feeding ratios

were

the apparent

The specific

value of 100-150

1A illustrate

of AAF-914C

change from

an initial

tRNA to preferential

In male rats,

at 1 day the tRNA/DNA

respectively.

binding

However,

after

of the carcinogen

of tRNA relative to rRNA

For female

rats,

(1 day)

1161

0 from

to rRNA.

or DNA after and tRNA/

these ratios

were

one week of feeding

AAF-

to liver

tRNA disappeared.

to that of DNA was about 0.4-O.

was 0.4-l.

The

binding

than that of rRNA

and 3.0 (tRNA/rRNA).

that relative

at 4-8 weeks.

tRNA was greater

to rats.

preferential

pmoles/mg

the dramatic to rat liver

of liver

3.6 and 3.1,

radioactivity

2-8 weeks while

and female (------) rats were fed a grain diet containing 4 animals were sacrificed and hepatic At weekly intervals, sulfotransferase was determined (9).

AAF-9-‘4C

1.6 (tRNA/DNA) 9”C,

6

AAF

AAF-6-14C

a maximum

preferential

rRNA

0.04%

and female (-------) ra t s were fed a grain diet (14) con1. (A) Male (-) taining 0.04% AAF-9-14C (1.91 dpm/pmole). At 1 and 3 days, then at weekly intervals for 8 weeks, 2 animals were sacrificed and tRNA, rRNA The specific radioactivity (dpm/mg) and DNA were isolated from the liver. of each nucleic acid was determined and the data expressed as the ratio of specific radioactivities indicated. (B) Male (-) 0.04% AAF. N-hydroxy-AAF

slowly

6

4 Fed

2-8 weeks

(Fig.

5 from 1A).

Vol. 47, No. 5,1972

BIOCHEMICAL

AAF-N-sulfate,

an extremely

hydroxy-AAF

by the action

homogenates

(9).

early

of AAF-9%

binding

activity in hepatic

(Fig.

1B).

1) suggest

gens to liver

in the soluble

fraction

N-

of rat liver

drop in the specific

during

parallels

radio-

the decrease

continuous

to a significant

feeding

of AAF

extent in the binding

DNA (10) and the results

may not be involved

from

for most of the

precisely

observed

is not involved

that AAF-N-sulfate

is formed

is responsible

to DNA and rRNA

to rat liver

of AAF,

The dramatic

sulfotransferase

AAF-N-sulfate

presented

in binding

here

of these carcino-

rRNA.

Approximately

10% of the labeled

DNA after administration pletely

tRNA.

IA) relative

of AAF or N-hydroxy-AAF (Fig.

metabolite

that AAF-N-sulfate

to liver

N-hydroxy-AAF

to rats (Fig.

reactive

of a sulfotransferase

We believe

of tRNA

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

disappears

of a single

from

liver

rRNA

carcinogen

remained

dose of AAF-9-%, by 4 weeks

(2).

firmly

whereas

bound to liver the label

Bound carcinogen

com-

(single

dose)

100

P p 75 ii E .-iix $ 50 2 2 s ,j

25

Weeks

Figure

after

Injection

of

AAF-Q-14C

2. Twelve male rats (180-200 g) were each given a single i.p. injection of 2-acetylaminofluorene -9 -14C (dose, 30 mg/kg; specific radioactivity, 8.90 dpm/pmole). At 1, 3, 7, 14, 28 and 56 days, 2 animals were sacrificed and liver tRNA, rRNA and DNA were isolated and the specific radioactivities of the nucleic acids were determined. The ordinate is expressed as percent of the maximum binding (attained at day 1) for each nucleic acid. Values for the amount of carcmogen bound at day 1 were: tRNA, 116 pmoles/mg; rRNA, 49; and DNA, 26.

1162

Vol. 47, No. 5,1972

disappears

from

BIOCHEMICAL

liver

tRNA at a much faster

in Fig.

2, and is completely

tration

of AAF-9%.

found here (Fig.

Normal

to note the greatly liver

rRNA

that disappearance

half-life

from

(2).

and tRNA

each have half-lives

of bound carcinogen

from

are unknown,

of approximately

was

to 3 days for rRNA

involved

(or turnover

is not due simply

after adminis-

radioactivity

in contrast

Mechanisms

removal

or DNA as seen

by 2 weeks

tRNA and rRNA

accelerated

rRNA

of the tRNA-bound

3 and 14 days),

reported

bound derivatives

rate than from

( < 1% of maximum)

from data between

2) and previously

of the carcinogen interesting

removed

The biological

1.5 days (calculated

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in the removal but it is of) the tRNA.

5 days (1 l-13)

to normal

turnover

of the

rRNA and tRNA. 3-Methylcholanthrene (see ref.

(MC) inhibits

14) and also inhibits

liver

the binding

TO RAT LIVER

pmoles Nucleic

in male rats by AAF

to rat liver

rRNA and DNA

I

OF 3-METHYLCHOLANTHRENE OF AAF-92%

induction

of AAF-9-14C

TABLE

INFLUENCE

tumor

bound/mg

(MC) ON THE BINDING NUCLEIC

nucleic

ACIDS?

acid

acid AAF-9-I% diet

AA-99”G4C + MC diet

Influence of MC (%)

DNA

358 I!I 30

232 f 29

- 36

rRNA

328 + 24

200 f

6

- 39

tRNA

148 f 10

224 f

9

+158

a_Male rats were fed a grain diet containing 0.04% AAF-914C (1.91 dpm/pmole) without or with MC (O.OOSp7,) for 2 weeks before sacrifice. Mean + S.E. (3 rats). The average amounts of AAF-9% ingested during the 2-week period were: AAF-91% diet, 0.32 mmoles; and AAF-9% + MC diet, 0.40 mmoles.

1163

so

BIOCHEMICAL

Vol. 47, No. 5, 1972

following

a single

injection

(0.005%)

was fed simultaneously

to liver

rRNA

to liver

tRNA was increased

rat. initial

of the carcinogen

several

lines

to liver

2-5 weeks

almost

50% under of evidence

of AAF-9

of carcinogen

feeding,

phase of hepatocarcinogenesis

by AAF

for 2 weeks,

40% (Table

suggest

binding

When 3-methylcholanthrene

1).

these conditions

tRNA is not correlated

The preferential

(14).

with AAF-9-Z4C

and DNA was inhibited

Thus, AAF-914C

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

binding

of AAF-9-1”C

However,

the binding

(Table

1).

that the extent of covalent

with hepatocarcinogenicity J’C to liver

rRNA

the time at which begins

(15,16),

binding

of AAF

in the male the early

of

in the

rat during

the

proliferative

may be of significance.

Acknowledgment: These from

the National

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

studies

were Cancer

supported

in part by USPHS Research

Grant

CA -05490

Institute.

:

Irving, C. C. , Veazey, R. A. and Williard, R. F. Cancer Res. 27: 173 (1967). Irving, C. C. and Veazey, R. A. Cancer Res. 29: 1799 (1969). Kriek, E. , Miller, J. A., Juhl, U. and Miller, E. C. Biochemistry 6: 177 (1967). Agarwal, M. K. and Weinstein, I. B. Biochemistry 9: 503 (1970). Fink, L. M., Nishimura, S. and Weinstein, I. B. Biochemistry 9: 946 (1970). Irving, C. C. and Veazey, R. A. Proc. Am. Assoc. Cancer Res. 12: 54 (1971). Irving, C. C. and Veazey, R. A. Biochim. Biophys. Acta 166: 246 (1968). Irving, C. C., Janss, D. H. and Russell, L. T. Cancer Res. 31: 387 (1971). DeBaun, J. R., Miller, E. C. and Miller, J. A. Cancer Res. 30: 577 (1970). L. T. Chem. -Biol. Interactions 1: Irving, c. c., Veazey, R. A. and Russell, 19 (1969/70). Hanoune, J. and Agarwal, M. K. FEBS Letters 11: 78 (1970). Hirsch, C. A. and Hiatt, H. H. J. Biol. Chem. 241: 5936 (1966). Loeb, J. N., Howell, R. R., and Tompkins, G. M. Science 149: 1093 (1965). Irving, C. C., Peeler, T. C., Veazey, R. A. and Wiseman, R., Jr. Cancer Res. 31: 1468 (1971). Farber, E. Cancer Res. 16: 142 (1956). Jackson, C. D. and Irving, C. C. Cancer Res. 32: in press.

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