Repair synthesis and sedimentation analysis of DNA of human cells exposed to dimethylnitrosamine and activated dimethylnitrosamine

Vol. 52, No. 3, 1973

REPAIR

BIOCHEMICAL

SYNTHESIS

EXPOSED

AND

AND BIOPHYSICAL

SEDIMENTATION

TO DIMETHYLNITROSAMINE

Brian Cancer

AND

A.

Research

ANALYSIS

Centre,

and

Received

April

HUMAN

CELLS

Stich of

8,

DNA OF

DIMETHYLNITROSAMINE

H.F.

University

Vancouver

OF

ACTIVATED

Laishes”

RESEARCH COMMUNICATIONS

British

Columbia

Canada

6,1973 SUMMARY

The capacity of dimethylnitrosamine(DMN), and of DMN activated by a NADPHfortified mouse liver microsomal preparation, ‘to elicit DNA alterations in cultured human iibroblasts was examined. A maximum induction of DNA repair synthesis, estimated by unscheduled incorporation of tritiated thymidine, occurred following 60-minute incubation of the human cells with DMN activated by a NADPHfortified mouse liver microsomal preparation. A low level of DNA repair activity followed exposure to DMN alone, or to DMN mixed with the microsomal preparation without NADPIH or without 02. The extent of DNA damage, estimated by velocity sedimentation of DNA through alkaline sucrose gradients, was maximum fol lowing treatment with DMN mixed with the NADPH-fortified microsomal preparation. The combined application of --in vitro activation systems and estimation of DNA repair synthesis in cultured cells may be exploited in the detection of precarcinogens.

INTRODUCTION DNA induced

repair

by

employed

to

isomers

of

synthesis

physical

(4).

and

chemical

between

between

l-hus,

in

cultured

the

identification

(2-5) high,

With

it

has

of human -the

Fellow

been

agents

weak,

and

potential

utilization

and

its

could

to

indicate

mammalian

active

DNA

ccl

non-oncogenic

4-nitropyridine-l-oxide

chemical

Is.

It

and between

N-hydroxy

and

K-region

epoxide

or

DNA

repair

its

that

the

measurement

be

developed

as

carcinogens

of

N-acetoxy

a screening with

was

derivatives (21,

and

changes

the

National

human

lymphocytes

Cancer

Institute

827

(5)

or

of

fibroblasts

Canada

the meta-

dihydrosynthe-

procedure distinct

for advantage

(6). of

the

in

and

proposed

cells

cells

of

applied

benzcajanthracene

mammalian

employing

Research

successfully

4-nitroquinoline-l-oxide

(3),

sis

*

or

been

Z-acetylaminofluorene

bolites

of

(1)

distinguish

precarcinogen

diol

has

(2-4)

as

BIOCHEMICAL

Vol. 52, No. 3, 1973

“receptor” stricted

in

not --in

that

require

--in

they

of metabolites

repair

synthesis In

in

in

can

we

following

vated

by

receptor

measured

as

sedimentation

the to

taken in

skin

from this

maintained fetal The

were in

calf

serum

cells

were

grown

the

activation

DNA

of the

deto

to

trigger

DNA

repair

in

cultured

(DMN)

human

and

(7,8). thymidine

to

DMN

DNA

repair

and

as

actiis

shifts

in

which

was

METHODS

cultured

from

4 or

5.

medium

skin-punch

200

mm glass

biopsy

woman.

Cel

Monolayers

of

f ibroblasts

supplemented

with

(MEM),

(penicillin

10x35

a

Caucasian

essential

antibiotics on

AND

passages

minimal

and

sufficient

tritiated

a 26-year-old

transfer

Eagle’s

utilization

precarcinogens

preparation

of

were of

of

do

(9-11).

fore-arm

study

that

therefore,

converting

of

microsome

incorporation

fibroblasts

the

the is,

alterations

level

MATER I ALS Human

for

dimethylnitrosamine

liver

profiles

re-

carcinogens

programs of

severely

cells.

compare

unscheduled

are

those

requisite

prescreening

DNA

RESEARCH COMMUNICATIONS

repair

only

capable

exposure

DNA

detecting

induce

a NADPH-fortified

for

A prime or

human

study,

fibroblasts

in

procedures which

this

systems

useful

screening

activation

active

test

activation.

systems

velopment

vitro

are

metabolic

vitro

DNA

the

ce I I s,

AND BIOPHYSICAL

U/ml,

coverslips

and

kept

I cultures

used were 15%

streptomycin

in

40

stoppered

pg/ml)

Leighton

tubes. For male

Swiss

buffered (0.25

mice

were

saline M).

The

tissue

fraction

immediately

used

8.5

adjustment each umoles

was

Homogenizer

supernatant

system,

pooled

containing

Potter-Elvejhem

and

procedure,

of

the

Leighton NADPH,

the pH

homogenized

calcium

and

10

operating

at

1000

tube 25

umoles

7.0

with

contained magnesium

a

decapitated

ice-cold

magnesium by

from

of in

homogenized

activation to

livers

and

resulting for

the

12

(PBS;

rev./min.

experiments

following

from

chloride,

and

7.2)

the

sucrose of

min,

4O)

of

complete

activation

ymoles

a

post-mitochondrial

addition

ZOO-300 20

and

strokes

(10

For

microsomes

828

pH

The

centrifugation

M NaOH.

phosphate-

up-and-down

9,000xg

0.1

4 month-old

Dulbecco’s

ions

to

3 to

mg

I iver

glucose-Gphos-

was

co-factors

plus

BIOCHEMICAL

Vol. 52, No. 3, 1973

phate

(7,8).

tion

A solution

with

1.0

the

fortified

The

ml.

tightly

of

DMN

MEM was

activation

contents

were

stoppered

in

added

system

mixed

without

AND BIOPHYSICAL

to

give

thoroughly,

further

to

RESEARCH COMMUNICATIONS

the total

flushed

agitation

at

cultures

in

reaction

volumes

with

37’

for

0, the

gas,

conjuncof

and

required

left incubation

period. For

analysis

of

tritiated

thymidine,

deficient

medium

order

to

with

20

Ci/mmol;

ADM

to

the

Fol

lowing

of

regular

M EDTA/0.075

buffer,

vortexed in ~1. (0.3

cell

the

pH 0.9

SW-40

10)

M NaCl An

cells at

0.03

and room

rotor

and

0.1

of

linear

top 0.3

temperature model

tube

was

a

L2-658

at

activity for

autora-

to

an

alkaline

aliquot

of

filled

with

ultracentrifuge

ml

mineral

gradients

3H-TdR

were

rinsed

4

0.5

ml (2

~1) overlay

to

were

hours. with

lOOOxg), 5-10~10~

ml)

gradient

oil.

20-24

min,

sucrose

lysing

added

EDTA/NaCI

and

a one

was

times

give (0.3

Cox,

of

buffer,

over

R.

Institute,

for

into

centri-

by

Research

uCi/ml

(Beckman

829

Farber

l-2

prepared 0.1

gradient

E.

pelletted

M Tris-HCI

and

the

specific

carcinogen,

(100-300

M NaOH,

(25O),

Dr.

scraped

added

M EDTA,

incubated

prepared

Fels

the

cultures

buffer was

additional

the

on

on

the

aggregates,

EDTA/NaCI

then

sucrose

of

7.51,

disperse

suspension

M NaCl,

M sucrose.

period an

sufficient

of to

cell

(pH

in

carcinogen

and

then

of

a concentration

buffer

carcinogen,

c3H-TdR,

alkaline

laboratory Sarma

the

to

by

exposure

at

treatment,

The

containing

of

medium

lightly

the

to

arginine-

(12).

repair

D.S.R.

Prior

M NaCl

sulfate,

2.3

used.

and

ADM

were

described

in

with

cultures

the

into

Following

thymidine

The

and

to

(1’2).

3 times

of

transferred

exposure

replication

tritiated

damage

incorporation

were

to

rinsed

previously

Abanobi,

carcinogen

solution

were

unscheduled

cultures prior

DNA

DNA

growth

0.024

100

days,

developed

was

resuspended

2-3

Nuclear).

as

S.E.

Philadelphia,

decyl

England

by

cell

10 $i/ml

el technique

Damjanov,

per

for

containing

analysis

fugation,

synthesis

coverslip

cultures

analysis For

I.

cell

New

diographic

repair

semiconservative

the

37O

the (ADM)

block

treatment,

DNA

and cells

of

0.5%

cold

a

lysing

sodium

do-

(5-20$, ml

solution After centrifuged Instruments

cushion was

a

added

lo-minute in

buckets Inc.,

5 ml) of on

top

lysis of Palo

Vol. 52, No. 3, 1973

Table

BIOCHEMICAL

AND BIOPHYSICAL

1.

DNA Repair Exposure Incomplete

Synthesis in Cultured DMN (~xIO-~M) and Activation Systems

to

Human Fibroblasts DMN (5x10-*M)

to

Table

GRA I NS/NUCLEUS

Contro I (No Treatment) DMN alone Activation System alone DMN plus Activation System DMN plus Activation System with N2 flush DMN plus Activation System without NADPH

0.0 0.5 0.3 23.2

UV UV

85.5

of

DMN Concentration Human Fibroblasts

(a) (b)

if.)

at

approximately

on the (60-minute

OF

DMN plus DMN alone

25,000

acid

acid

(TCA),

pierced insoluble collected

tubes,

and

assayed

radioactivity on

membrane

of DNA exposure)

(a)

(b)

3.6 8.4 14.2 16.8 17.6 21.3 19.3 17.2

0.1 0.1 0.2 0.2 0.3 0.5 0.6 0.7

activation

fractions and

Level

Repair

Synthesis

in

GRA INS/NUCLEUS

complete

rev./min

15 sequential

(11):

1.0

85.7

5.0x10-4 5.0x10-3 7.5x10-3 1 .ox1o-2 2.5x10-* 5.0x10-2 7.5x10-2 1.0x10-1

of

1.2

(900 ergs/mm*) (900 ergs/mm2) plus Activation System

CONCENTRATION DMN (mol/l)

bottom

60-Minute and

2.

Effect Cultured

Cal

Following Complete

plus

TREATMENT

Alto,

RESEARCH COMMUNICATIONS

20’

for

30

of

16

drops

for was

system

min.

Following

each

radioactivity precipitated

filters,

washed

830

were as

with with

centrifugation, collected

from

previously 8-10s

TCA,

described trichloroacetic

dried,

and

counted.

the

Vol. 52, No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

30

. .... .... . :’ .. ‘..... ‘L..._ ..’ . ... / .‘...., ,’ ...i .-...... ..:..... ‘.......

25

/Y

/ ,:;c ,...g .:’ ;. .... :

?.I;:: ...Ai.: Z

+=B=fj

0

20

A

60

40 EXPOSURE

SO

TIME

RESULTS

ating

human

fied

mouse

ment

with

duced

in

toxic

exposed plete

ccl

plus

level

to

900

microsomal

DNA

of

DNA

repa

the obtain

system.

with

non-prolifera NADPH-forti-

treatment

NADPH

alone

or

This

without

against

further

information

on and

cells

the the

120

time min

the

question

DNA

repair

problem,

subsequently exhibited

831

the

for

raised

mixture

(12)

with

Exposure

1).

The

DMN

increased

activation

preparation.

in

incubated

without

i r synthesis

W-light

alone system in culthe

or

treat-

oxygen

in-

synthesis.

(Figure

ergs/mm2

occurred

DMN 1).

mixture

activation

activity

to

(Table

repair

to 5x10-‘M DMN complete activation DNA synthesis detection of

synthesis

exposure

activation

120

DISCUSSION

repair

fraction

DNA the

in of

To

the

of

a drop

Is.

of

following

level

plus

effects

level

microsomal

low

DMN

high

broblasts

liver

a very

5x10m2M

the

f

DMN

The

sult

vely

AND

100

I mlnuter1

Figure 1. Effect of duration of exposure and to 5x10e2M DMN plus (01O,A-A), (0 ..-. , A --- A) , on the level of unscheduled tured human fibroblasts. (Autoradiographic unscheduled incorporation of 3H-TdR)

A relati

.‘...

cell incubated

no

detectable

of

exposure

appeared of

to

to

re-

possible

mechanism

of

cultures with inhibition

were the

comof

Vol.

52,

No.

3, 1973

unscheduled for

BIOCHEMICAL

3H-TdR

up

to

120

The tivation A peak

incorporation

AND

by

BIOPHYSICAL

exposure

to

RESEARCH

the

complete

COMMUNICATIONS

activation

system

min.

effect

of

DMN

concentration,

system,

on

the

induction

activity

occurred

at

both of

5~10~~

DNA

with

and

repair

without

the

synthesis

M DMN mixed

with

was

the

complete

ac-

examined

complete

(Table

2).

activation

system. The shift

in

weight the

application the

following

3H-DNA

exposure

not

activation

tivation

the

and

to

of

DNA

DNA

show

damage

centrifugation

of

vation cultured detection

of

activated

DNA

DMN

cultured

mammalian chemical

be to

may

in

of exposure

also

to

that

the

no

induction

of

sucrose

incubations of

ac-

produced

alkaline

(Figure

converted

-~in that

cells.

the

gradients,

of lower

ZB),

in

the

fibro-

molecular

thus

is

it

an

utilized

832

DNA

weight

verifying

is

of

estimation a

changes

that

inducing

by

DNA

repair

economic

lead

to

after the

unscheduled

to The

a deto

profiles

(8).

and

NADPH-fortified

seem

interest

of rapid

the of

and

typhimurium

as

precarcinogens.

capable

gradients

connection, 2.

by

sedimentation

sucrose

in

vitro

These

shifts

with be

alone

shifts

a form

mutations

combination

cells,

can

by

this

with

occurring.

alkaline In

induced in

revealed

incubated

a heat-inactivated

show

weight

human

through

3H-TdR.

procedure,

fact

system

as

DMN

of

that

in

breaks

only

in

molecular

a one-hour

DMN

a

profiles

with

5x10m2M

produced

lower DMN

following

Post-treatment

molecular

activation

single-strand

incorporation

is

DMN.

revealed

sedimentation

sedimentation

medium

higher

damage

results

of

of

5x10-*M

observations

velocity

culture of

microsomal

tectable

by

to

DMN mixed to

These

activation

regular

a

liver

measured an

regions

28).

technique

a region

The

5x10A2M

Exposure

(Figure

to

The

position

only

profiles repair

28).

system

to

ceils

(Figure control

ZA).

gradient

3H-DNA

cultured

the

on

in

the

from

as

dependent

of

system

effect

blasts

of

(Figure

damage,

sucrose

change

system

discernible

is

alkaline profile

activation

did

a DNA

the

sedimentation

complete

the

of

note

--in

that

vitro

acti-

synthesis tool

in for

the

Vol. 52, No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Alkaline sucrose gradient sedimentation profiles of Figure 2. 3H-DNA released from cultured human fibroblasts exposed for 60 minutes to: A) No treatment (control) (0-O); activation system only or 5x10w2M DMN plus heat-inactivated activation system (superimposable profiles) (A**-=A); B) 5x10w2M DMN alone (0-O); 5x10m2M DMN plus complete activation system (A----A) ; 5x10m2M DMN plus complete activation system with a 4-hour post-treatment incubation in fresh medium co- -0). The horizontal bar designates the position of control DNA peaks.

Acknowledgements We are grateful to Drs. J.A. Miller and E.C. Miller, McArdle Laboratory for Cancer Research, University of Wisconsin, and to Drs. E. Farber and D.S.R. Sarma, Fels Research Institute, Temple University School of Medicine, for technical advice. This study was supported by the National Cancer Institute of Canada and the National Research Council of Canada. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Rasmussen, R.E., and Painter, R.B. J. Cell Biol. 2, 11 (1966). Stich, H.F., San, R.H.C., and Kawazoe, Y. Nature 229, 416 (1971). Stich, H-F., San, R.H.C., Miller, J.A., and Mi I ler, E.C. Nature New Biology 238, 9 (1972). Stich, H.F., and San, R.H.C. Proc. Sot. Exp. Biol. and Med. 142, 155 (1973). Lieberman, M.W., Baney, R.N., Lee, R.E., Sell, S., and Farber, E. Cancer Res. 31, 1297 (1971). Stich, H.F. In: Topics in Chemical Carcinogenesis, Takayama, S., Sugimura, T., and OdashimaTS. (Editors). University Park Press, Baltimore, pp 17-28 (1972) Garner, R.C., Miller, E.C., Miller, J.A., Garner, J.V., and Hanson, R.S. Biochem. Biophys. Res. Commun. 5, 774 (1971). Mal Iing, H.V. Mutation Res. 13, 425 (1971). McGrath, R.A., and Wi I I iams, R.W. Nature 212, 534 (1966). Lett, J.T., Caldwell, I., Dean, C.J., and Alexander, P. Nature 214, 790 (1967). Laishes, B.A., and Stich, H.F. Can. J. Biochem. (1973, in press). Stich, H.F., and San, R.H.C. Mutation Res. lo, 389 (1970).

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