Inhibition of DNA repair by cocarcinogens

vol. 48,

NO.

BIOCHEMICAL

4, 1972

INHIBITION

OF DNA REPAIR BY COCARCINOGENS

David

Laboratory

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Gaudin, Robert S. Gregg and K. Lemone Yielding

of Molecular Biology, University of Medicine in Birmingham,

July

of Alabama Alabama

School

7,1972

Summary: DNA repair replication in normal human lymphocytes has been inhibited by every cocarcinogen tested to date, including anthralin, 12-O-tetradecanolyphorbol-H-acetate, and the neutral fraction from cigarette smoke. Such inhibition of DNA repair is proposed as the general mechanism of action of cocarcinogens. Introduction Cocarcinogens various

have the

carcinogens

stage

process

tumorigenic response. ability

(1).

the proximate

response

and the

is

already

to interact of DNA is

the repair

process

of the

cocarcinogens

may act

hypothesis, were

tested

in normal

our

earlier

experiments

80,

Span 80, Arlacel

Copyright AN rights

are

carcinogen

most,

with

DNA (2)

to protect

thought

acting

that

for

the tumorigenic to act

as a promoter

if

all,

and also

of the of that

carcinogens that

have

the

such chemical

the excision

repair

the

DNA genome against

cellular

of

in a two

as the initiator

acting not

action

enzymes

(3,4).

Thus, potentially

damage.

facts

cited

by inhibiting

a number

replication

steroids,

substances

a substrate

or carcinogenic

In light

structures

known

serves

to enhance

cocarcinogen

covalently

alteration

this

These

with

It

mutagenic

ability

above,

it

the DNA repair

of cocarcinogens

by us and found

A, vitamin

and acetylaminofluorene

0 1972 by Academic Press, Inc. of reproduction in any form reserved.

a wide

to be potent (5).

a methanol A alcohol, derivatives. 945

process.

having

human lymphocytes included

seemed reasonable

to suggest In order

diversity

inhibitors

of croton

diethylstilbestrol, Our earlier

to test

of chemical of DNA repair

The cocarcinogens extract

that

tested oil,

in

Tween

various work

was per-

Vol. 48, No. 4, 1972

formed

with

in this

crude

mixture

extended oil

BIOCHEMICAL

extracts were

of croton

responsible

our earlier

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

oil

for

observations

and did

second

only

to include

cigarette

has been reported

to the phorbol

esters

smoke has been

carcinogenic to test

activity

(7).

the consistancy

cocarcinogenic cigarette

shown

DNA repair

to have

ingredient

to be a very

strong

cocarcinogen

the neutral

below

hypothesis,

of anthralin

fraction

carcinogenic

reported

as that

of croton anthralin

only

in croton

We have

substance,

not

present

compounds

Another

Also,

of our original

as well

smoke,

(6).

which

inhibition.

an active

The experiments

activities

indicate

the DNA repair

- 12-O-tetradecanoylphorbol-13-acetate.

(l,&dihydroxyanthrone)

not

of

but

also

were

co-

carried

and to show that

oil,

the neutral are

all

out the

fraction

of

due to inhibition

of

replication. Methods

The assay and Norman normal

procedures

(8)

are based

to demonstrate

uptake

These

of tritiated

thymidlne

Addition

of hydroxyurea

measurement

of tritiated

thymidine

the higher

vative

synthesis

was incubated essential were

of DNA. for

medium

then washed

The uptake amount

into

of uptake

containing

background

two hours containing

DNA repair

in the W irradiated

lymphocytes

experimental

have

in detail

methods

already

otherwise

W irradiation,

in

controls.

The activities

added

been published

here.

946

preparation minimal

as a percentage inhibitors. (5)

The cells

by scintillation

was corrected

without

replication

thymidine.

determined

expressed

permits

Eagle's

and tritiated

of

from semiconser-

lymphocytes

are

the presence

lymphocyte

modified

in

of the W

mixture

result

the

Spinner

uptake

unirradiated inhibitors

measurement

due to DNA repair

would

hydroxyurea

the UV irradiated into

uptake

out by Evans

capability

by lymphocytes

at 37'C in

and the thymidine

involve

to the incubation

which

After

carried

of a DNA repair

procedures

hydroxyurea.

without

experiments

the presence

human lymphocytes.

stimulated

on the

and will

for

counting. a small

in samples of the activity Details

of the

not be repeated

BIOCHEMICAL

Vol. 48, No. 4, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.5 concentration Figure

1.

The effect

13-acetate

(curve

of anthralin

B) on DNA repair

The activity

in the UV-irradiated

is

as a percentage

expressed

the drugs.

A small

subtracted

(curve

from

amount

all

5 x lCf5 M

A) and of 12-O-tetradecanoylphorbol-

replication samples

in normal containing

of the activity of incorporation

repair

inhibiting

in UV-irradiated into

of the UV-irradiated

human lymphocytes.

unirradiated

samples

before

drugs

samples

without

controls

was

calculating

the

percentages.

Since

the materials

in water,

stock

(DMSO) just

solutions

before

any detectable Cigarette The neutral

showed

this

120 cigarettes

from

with

assay mixture 1, it

this

whole

mixture

soluble

in dimethylsulfoxide

the assay mixture. was 1%.

The highest

Control

experiments

of DMSO does not

was collected smoke condensate A weighed

The DMSO extract

to test for its DNA repair Results and Discussion

can be seen that

readily

cause

replication.

of Day (9). DMSO.

are not

prepared

concentration

smoke from

was extracted

From figure

in the assay that

were

into

of DNA repair

to the procedure

lymphocyte

diluted

inhibition

fraction

according fraction

reached

DMSO alone

of repair

of the compounds

use and then

DMSO concentration run with

used as inhibitors

both

947

the phorbol

in a dry was then

amount

ice

trap.

prepared

of the neutral

was then

added

to the

inhibiting

capability.

ester

and the anthralin

Vol. 48, No. 4, 1972

BIOCHEMICAL

are potent inhibitors

of the W stimulated uptake of tritiated

normal humanlymphocytes.

fraction

to

mechanismagainst agents which can cause a

cellular

from cigarette

thymidine into

Thus, these cocarcinogens appear to act by over-

coming the normal protective chemical alteration

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DNA. Similarly,

smokecan also inhibit

DMSOextracts

of the neutral

DNArepair replication.

The DNArepair was reduced to 50% of the control value by dilution neutral

fraction

to less than 0.01%. Since the neutral

cigarette

smokeis a complex mixture,

structure

of the repair inhibitor

than one repair inhibiting identification

of the inhibitory

are not known. Also, there may be more Therefore,

compoundor compoundsand the concentrations

must remain the subject of future

is hoped that these results will

of the harmful effects

from

and chemical

compoundpresent in the mixture.

at which they are effective It

the concentration

fraction

of the

of cigarette

help to lead to a better

experiments. understanding

smoke.

Since the phorbol esters stimulate DNAsynthesis (10,11,12) while the anthralin

is cytostatic

important factor

(13), DNA repair inhibition

is probably a more

in the mechanismof cocarcinogenesis than whether or not

DNAsynthesis is stimulated in the affected

cells.

However, stimulation

of

DNAsynthesis by phorbol esters suggest that these compoundsplay a dual role in cocarcinogenesis,

first

by direct

inhibition

of the repair process and

secondly by decreasing the length of time available

to the cells

for carrying

out repair before they attempt to replicate

the DNAwhich had been chemically

altered.

is also demonstrated by the fact

The importance of the time factor

that cells which have been stimulated to undergo cell division of wound healing (12) show an increased susceptibility mycin D, which has the ability RNA and subsequently the protein the cocarcinogenic effects of irradiated time available

as a result

to carcinogens.

Actino-

to prevent this DNA synthesis by inhibiting

the

synthesis necessary for cell division,

decreases

of croton oil

(L&,15).

Similarly,

the survival

cells in the plateau phase of growth where there is a greater for repair

is increased over those cells in log phase growth (16).

948

Vol. 48, No. 4, 1972

Anthralin

BIOCHEMICAL

has been shown to improve the response of.psoriasis

treated with W light

(17).

the fact that anthralin (la),

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

it seemslikely

Although the picture

alone is beneficial

patients

is somewhat confused by

in the treatment of psoriasis

that its repair inhibitory

properties

also play an

important role in improving the W treatment. REFERENCES 1.

Clayson, D. B., Incomplete carcinogenesis and the two stage theory of carcinogenesis, in "Chemical Carcinogens", Little, Brown and Co., Boston, 1962, p. 290.

2.

Miller,

3.

Lieberman, H., Sell, 12, 5 (1971).

4.

Witschi,

5.

Gaudin, D., Gregg, R. S. and Yielding, Commun.,45, 630 (1971).

6.

Segal, A., Katz, C., and Van Duuren, B. L., J. Med. Chem., 14, 1152 (1971).

7.

Roe, F. J. C., Peto, R., Kearns, F., and Bishop, D., Brit. 24, 788 (1970).

8.

Evans, R. G., and Norman, A., Rad. Res., 36, 287 (1968).

9.

Day, T. D., Brit.

J. A., Cancer Res., 30, 559 (1970). S., and Farber, E., Proc. Am. Assoc. Can. Res.,

H., Epstein, S. M. and Farber, E., Cancer Res., 31, 270 (1971).

J. V.,

K. L., Biochem. Biophys. Res.

J. Cancer,

J. Cancer, 21, 56 (1967).

10.

Frei,

and Harsono, T., Cancer Res., 27, 1482 (1967).

11.

Hennings, H., Bowden, G. T., and Boutwell, (1969).

12.

Hennings, H., and Boutwell, R. K., Cancer Res., 30, 312 (1970).

i3.

Krebs, A., and Schaltegger, H., Experientia,

14.

Bates, R. R., Wortham, J. S., Counts, W. B., Dingman, C. W., and Gelboin, H. V., Cancer Res., 28, 27 (1968).

15.

Hennings, H., and Boutwell,

16.

Little,

17.

Bowers, R. E., Dalton, D., and Knowlden, J., Brit. 273 (1966).

J. Dermatol.,

18.

Farber, E. M., and Harris,

101, 381 (1970).

R.

K.,

R. K., Cancer Res., 29, 1773

Life Sci.,

21, 128 (1965).

6, 173 (1967).

J. B., Nature, 224, 804 (1969).

D. M., Arch. Dermatol.,

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78,