Only one type of benzo(a)pyrene-DNA adduct is detected in transformable mouse cells

Vol. 99, No. 3,198l April

BIOCHEMICAL

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BIOPHYSICAL

RESEARCH

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15, 1981

Pages 820-829

ONLY ONE TYPE OF BENZO(A)PYRENE-DNA ADDUCT IS DETECTED IN TRANSFORMABLE MOUSE CELLS Ko-yu Cell

Received

Lo and Takeo

Kakunaga

Genetics Section, Laboratory of Molecular National Cancer Institute, National Institute Bethesda, Maryland 20205

February

Carcinogenesis of Health

24,1981

Summary When highly transformable BALB/3T3-A31 clone 1-13 cells are exposed to benzo(a)pyrene for various lengths of time, only one type of carcinogen-DNA adduct is detected. No minor adducts were found as reported in other cell systems. High pressure liquid chromatography identified this persistent adduct as lo-trans-7R-benzo(a)pyrene diolepoxide Ideoxyguanosine. Formation of benzo(a)pyrene-deoxyguanosine adduct, therefore, appears sufficient to initiate the transformation process induced by benzo(a)pyrene in 1-13 cells. Introduction The prevailing that

covalent

initiation only

adducts

covalent

could

of 06-alkyl

formed to

be the

both

arylamidated

in

liver

DNA;

persistent

deoxyguanosine

susceptibility

of these

in

lesion certain

tissues

bound

process.

amide, rat

a crucial

of reports

remain

such

carcinogenesis

DNA is

number

which for

chemical to

A growing

be essential

were

in

carcinogens

adducts

aromatic

been found

theory

of

of neoplasia.

carcinogen,

the

binding

those

periods

genetic

tissues

DNA for

N2-and only

vivo

to carcinogenesis

the that

extended with

liver

C8-deoxyguanosine the

N2-adduct

(l-4). has

in

indicated

For examples,

however, in

step

have to

argues

has

The persistence

been

correlated

with

(5-8).

Abbreviations used: BP, benzo(a)pyrcne; BPCE 1,(&l-78, 8a-dihydroxy%,lOa-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, diol epoxide I, r-7,t-8dihydroxy-t-9,10-oxy-7,8,9,lO-tctrahydrobenzo(a)pyrene; BPDE II, (t)-7B,8adihydroxy-98,106-epoxy-7,8,9,lO-tetrahydrobenzo(a)pyrene, diol epoxide II, r-7,t-8-dihydroxy-c-?,lO-oxy-7,8,9,lO-tetrahydroxybenzo(a)pyrene, IO-trans7R-BPDE I-d&a, N2-(10S-[7R,8S,9R-trihydroxy-7,8,9,lO-tetrahydrobenzo(a)pyrene]yl-deoxyguanosine; dGua, deoxyguanosine; HPLC, high-pressure liquid chromatography; PBS, phosphate buffered saline; HEC, hamster embryo cells; MEF, secondary mouse embryo, cell; BHK, baby hamster kidney cells. 0006-291X/81/070820-10$01.00/0 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

820

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Vol.59,No.3,1981

Benzo(a)pyrene strong

(BP)

carcinogen. in mammalian

Although

BP-deoxyguanosine

examined

to the

human

than

deoxyguanosine

it

not

known

strongly We chose

BALB/3T3-A31 gives

high

susceptible provides

in the

investigate

clone

1-13

for

transformation system

produced

here

(i.e.

BPDE I-dGua)

line,

suggesting

that is

that

adducts

(13,18).

been

animals

(19).

formed

in all

rodent in

cells

nucleosites

At the

DNA modifications

BP-DNA

following (2)

present

are

induced

this

(1)

time

correlated

cell

by carcinogens

which

may play

produced This

the carcinogen-DNA

amount,

of cell

adducts

reasons:

Since

to detect

cell

line

is

(20),

it

adducts,

in line

highly

especially

an important

role

transformation.

only formed

BP

covalent

in minute

process

products

has

a

process.

the

to the

intact

transformable

minor

these

the

efficiency;

We report

Materials

of

the

produced

carcinogenic

to

initiation

transformation

types

plating

adducts

some

major

and

adducts

and in

the

from

were also

the

a sensitive

those

ranged

cells,

which

with

were

COMMUNICATIONS

contaminant

nucleoside

(9-18)

adducts

RESEARCH

environmental

BP-DNA

cultures

which

other

most

common of

tissue

systems,

resistant

is

a

Formation

detected

the

is

AND BIOPHYSICAL

one in

BPDE I-dGua

type

this itself

of highly is

persistent

BP-DNA

transformable sufficient

mouse to

initiate

adduct cell the

process.

and Methods.

Cell Cultures: Clone 1-13 isolated from mass culture of BALB/3T3-A31 was used (20). Cells were plated at 1~10~ cells per lo-cm tissue culture dish and were grown in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum. Binding of [6-3H]BP to DNA: [6-3~1 BP (Amersham-Searle) was diluted with cold BP (Chemical Repository, NCI) to specific activity at 6-20 ci/mmoleie;;; iyrified by a gravity flow silica gel column (21) before use. 70% confluency were treated with [6-3~1 BP for the lengths of time specified. At the end of incubation, cells were washed once with ice-cold phosphate buffered saline (PBS) and collected by scraping. After harvesting, cells were again washed with PBS, suspended in 1OmM Tris-HCl (pH7.5), 1OmM EDTA, 1OmM NaCl, lysed by 0.5% sodium dodecyl sulfate (SDS) and treated with proteinase K at

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Vol. 99, No. 3,198l

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50ug/ml overnight. DNA was extracted twice with buffered saturated phenol containing 0.1% 8-hydroxyquinoline, and was then precipitated with two volumes of cold 95% ethanol. The precipitated DNA was further purified by digestion with RNase, phenol extraction and ethanol precipitation. DNA solutions were then heated to 100°C for 15 min to remove the possible intercalated hydrocarbon, passed through a short Sephadex LH20 (pharmacia) column (lxl5cm) and lyophilyzed to dryness. The amount of BP bound to DNA was determined by counting of samples of treated DNA in a Beckman LS8100 scintillation counter. Concentration of samples was determined by Burton reaction (22). Intracellular BP Pools: BP treated cells from two lo-cm culture dishes were rinsed twice with MEM, harvested with trypsin and resuspended in PBS at 2x106 cells/ml. 2 ml of cell suspension were lysed with 2ml of acetone and then extracted with 4 ml of ethyl acetate. Following removal of the organic phase, the aqueous phase was extracted with equal volume of acetone/ethyl acetate (1:2 v/v). The organic phases were combined, dehydrated, dried, and finally dissolved in 0.5 ml of benzene. BP was separated from its metabolites on a silica gel column as described by Okano et al (23). Analysis of [6-3H]BP-DNA Adducts: BP modified DNA samples were enzymatlcally hydrolyzed and the nucleoside mixture was chromatographed on Sephadex LH20 (24). The isolated nucleoside adducts were analyzed by high pressure liquid chromatography (HPLC, Waters Associated) fitted with a Zorbax ODS column (6.2 x 250mm, DuPont). Samples were eluted with a linear gradient from 40-88% Methanol-water over a 50-min period. Preparation-of BP-7,8-dihydrodiol 9,10-oxide (BPDE)-polynucleotide products: [JH]BPDE I (Chemical Repository, NCI) was reacted with poly d(G) and poly d(A) (P-L Biochemicals) for 18 hrs at 37°C in a tetrahydrofuranwater mixture (1:5, v/v, PH 7.0). After extraction with ethyl acetate, the polynucleotides were precipitated twice with ethanol. The BPDE Ipolynucleotide products were enzymatically hydrolyzed, separated on Sephadex LH20 column, and analyzed by HPLC as authentic markers.

Results

and Discussion

Previously,

we reported

BP produced

25 transformed

examined,

at

to DNA and of BP to

this

1-13

This for level

could

foci

of

cellular

DNA is

(or

even

suggest

DNA binding

that at

of DNA binding

104

of

carcinogen,

formed. shown

(0.5uM),

before)

in the

18 hr

of

no remaining

later seen

time in

l-13

surviving

The Fig.1. amount

cells

with

cells the

amount

time

course

with

Alternatively,

could

822

be due to equal

BP bound

of

binding

were

of BP bound

of

We then

of

When cells

incubation

0.5uM

(25).

exposed

to DNA reached the

BP or BP metabolites

points. Fig.1

of

per

BP adducts

of carcinogen at

treatment

concentration

types

to low level a plateau

that

carcinogen.

were the

available

steady

rates

state

of forma-

BIOCHEMICAL

Vol.99,No.3,1981

0

AND BIOPHYSICAL

10

20

30

RESEARCH

40

TIME AFTER ADDITION

50

COMMUNICATIONS

60

70

OF [sH]BPlhrsl

Time course of binding of [6-3H]BP to DNA in BALB/3T3-A31 Figure 1. cells were exposed to 0.5uM of [6-3H]BP. clone 1-13 cultures. -o-e-, After 18 hr incubation with the carcinogen, hydroxyurea was added to medium (final concentration, ImM) to inhibit semiconservative DNA -o-o-, "posttreatment incubation". Cells were exposed to synthesis. 0.5uM of [6-3~16~ for 18 hr. after which cells were rinsed with warm medium and then incubated in fresh BP free medium containing 1mM of -&-A, cells were exposed to Z.OuM of [6-3H]BP. DNA was hydroxyurea. isolated and the amount of BP bound to DNA was determined as described in Materials and Methods.

tion

and removal

of

been

reported

many mammalian

in

between

these

and in

intracellular

treatment in Table only the

BP-DNA adducts

possibilities,

1,

1% of

sequestering

1-13

cells

free

medium

were for

we studied

of

after BP

systems

BP pools.

incubation

of first various

the

incubation

in the

in

the

medium.

exposed lengths

to

fate

833

of

cells

18hr it

BP both

in

DNA repair carcinogen. 0.5uM

studies

of also

Furthermore,

and then

incubated the

medium

by postAs shown

cells.

followed

DNA has

To distinguish

with

pool

inside

of times,

BP damaged

of

monitored

BP

BP for

of

(11,13,18,26).

absence of

carcinogen

excision

the

We also

cells

18 hrs

remained

since

[6-3H]BP, suggested when with

same binding

BP

BIOCHEMICAL

Vol. 99, No. 3,198l

Table

I

Duration treatment (hr)

of

Intracellular

AND

Intracellular pools b plel 10 cell)

2/4

0.5 /Al

2 /AM

100.0 37.0 24.4 13.4

96.5 ND ND 1.0

;D” ND

::; 0.3

12 :: 40 65

lY7

a values obtained from two determinations b mean of 5 independent experiments c values in parentheses represent BP bound with BP free medium

as that

findings

support

of BP or its decrease

in

specific

activity

excised

cell

whether

the

susceptibility

when cells

were

of BP to

cellular

that

repair

in

medium

capacity

to

DNA increased or

order

in

to

the obtain

cells

interactions

by this

it

carcinogen,

gen in both

types

has to

used

be

is

better

of experiments. to

detect

with

the

and that

cells

of

1,

correlation and

the

cells

to

These

between

transformation

a higher

biochemical

824

in its

other

hand,

products,

the

binding available

data the

suggest study

process

concentration

and

role

as BP was

1).

two

determine

(2.OuM),

to use the same concentration If

over

On the

carcinogen

Table

indicated

a crucial

as long

is

low efficiency.

required

play

lack

small

incubation

extremely in the

These

the

Our results

are

time

(Fig.

relevant

chemical-biological

with

1).

is due to the

posttreatment

intact

level

(Fig.

reactions,

transformation.

high

of culture

of DNA binding

from DNA.

of 1-13

t.! 2.9 (3.1 F 3.0 (2.8) 2.9 (2.6)

incubation

was used

studies

BP-induced

exposed

ND

posttreatment

adducts

Further

to

6.7 5.0 6.6b ND 14.1” ND

after

of BP adducts

Extent of bindir$ (A mole BP/mole of nucleotides)

ND ON:4 0:14 0.15 0.12

further

BALB/3T3-

0.5 AM

6.5 ND ND ND

plateau

remained

BP

to

2rM

of DNA during

lesions

BP binding

COMMUNICATIONS

0.5 FM

medium

for

generations. low

the

carcinogen

80% of the

a half

in the

that

metabolites

cells

More than

high

idea

to the removal

1-13

to DNA

BP containing

active

attributable that

when the

RESEARCH

BP pools and extent of A31 clone 1-13 DNA.

% of BP remained in medium

ii

kinetics

BIOPHYSICAL

of

induced

of carcinoof carcinogen importance

of

BIOCHEMICAL

Vol.99,No.3,1981

these

products

in

relation

AND

BIOPHYSICAL

the

transformation

to

RESEARCH

COMMUNICATIONS

process

should

be

established.

DNA isolated was subjected

from to

chromatographed by high

relativey

large

enzymatic

total

cell

cells

in total with

and of

authentic

revealed

only

with

second

one

first

peak

accord skin in the

found

l-3%

of

in

relative in

in

abundance

of

7s enantiomer.

In the

--in

types

adduct

formed

of

BP-DNA

7R-BPDE I-dGua explants

(14).

BPDE II-dGua

was the In

vivo

only

adduct

addition

to

was formed

in

the

removed

from

explants

825

1-13

samples coincided

corresponded

cells

(HEC,13).

to This

radioactivity

rewith

the

of BPDE I-dGua,l3). by

1-13 cells

not as

have

system

produced this

DNA digest

all

to

cells

is

(18),

Mouse

observed that

at a faster

which

of were

which

lOT1/2

isomers

systems vary

purine

total

by

fraction

position

We did

two

is

either

DNA digests

of

isomer

(27).

I-dGuo

1-13

enantiomer

embryo

these

prevent

was associated

BPDE I

mouse

microsomes

7R-BPDE

the

were

may be produced

elution

radioactivity

liver

which

of

(7s

to

[14C]

embryo

was

We have employed

which

adducts,

adducts

finding

adducts

digests

hamster

of

nucleoside

of

Its

97-99%

synthesis

and

HEC (13),

in

mixture

by a negligible

The

deoxyguanosine

remaining

the

(19),

2). peak.

of deoxyguanosine

with

peaks

BPOE I modified

contributed

The stereoselective

minor

[6-3H]BP

nucleoside

HPLC studies

HPLC profiles

(Fig.

and ~.OJJM of

(HPLC).

be produced

markers

of

isolated

for

the

of

I-dGua

The

samples

profiles

peak

the

chromatography

radioactive

isomer

covered.

of

to 0.5&M and

The

population.

the

lo-trans-7R-BPDE predominant

liquid

or might

cell

compared

LH20.

overestimating

population

exposed

hydrolysis

pressure amount

or

the

cells

on Sephadex

analyzed

overlooking

1-13

changes

reported rate

been

in human and bovine

major

adduct,

small

of

human colon

(15),

in

than

its

studied,

system.

in

the

lo-transbronchial amount lung

of (16),

Vol. 99, No. 3,198l

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RESEARCH

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HPLC profiles of-DNA adducts formed in BALB/3T3-A31 clone l-13 %%%%incubated-with [6-JH]BP. (a) vitro marker from digest of --in BPDE I modifi ed ["C] purine DNA. (b) in vitro marker formed by reaction of C3H] BPDE I with polv d(G) and uolv7dx fispectively. (c)-(e) DNA adducts isolated from'l-i3 ckl'ls after i2 hr (c), 18 hr (d), and 40 hr (e) exposure to Z.OuM of [6-3H] BP. DNA isolated frown cells exposed to D.!%M of [6-3H]BP exhibits an identical elution pattern.

826

BIOCHEMICAL

Vol. 99, No. 3.1981

and in

secondary

mouse embryo

was also

detected

dGua at

comparative

(BHK,II)

and

of BPDE

I-dGua

both

in

and

are

readily

of these

cells

(18).

tumor

cells

transformed

baby

hamster

kidney

(17).

mouse

adducts,

small

are

also

carcinogen.

cells

skin

(19).

amounts

to

transforcells

(32)

It is not known which

types

most strongly

lOT1/2

of

Cells

found.

resistant and

BPDE II-

7s enantiomers

in

HEC (28-31)

are correlated

The

were

human cells,

by this

BP-DNA adducts

and

adducts

However,

BP.

adduct

BPDE I-

and BPDE II-dGua

like

COMMUNICATIONS

Both

produced

deoxycytidine

systems, by

in

were

RESEARCH

The deoxycytidine

found

II-dGua

BPDE Iand

induced

(MEF,ll).

were

BPDE

besides

of some of these

cells

human alveolar

deoxyadenosine

mation

lOT1/2 level

A549

In HEC (13),

AND BIOPHYSICAL

with

the carcinogenic

process.

Minor

adducts

have been systems

(11,13,17). the

Our assays

are

is

extented

provided step

in

l-13

that

periods. for

that

in chemical

the the

in all

they

probably

represent

minor

transformation

them to be the

of binding

is

than major

transformation

carcinogenesis.

827

carcinogen

in

some

not

the of

examined.

as little Should less

as minor

than

are

0.1%

a thousand

deoxyguanosine

cause of transformation.

persistent

formation

of

adducts

adducts

one type

at quantities

these

is

the DNA samples

cells

Unless

this only

produced.

Therefore,

covalent

since

adducts

BPDE I-dGua

induction

that

cells,

adducts

processes

total

formed.

for

of multiple

the

in inducing

show

indicate

1-13

minor

of excision

transformation

was observed

of

impossible

also

and clearly

detect

that

adducts

responsible

results

to

more efficient

Our data over

of

total

it

Our

rate

transformable

able

be formed

adduct,

mutagenic

(BPDE I-dGua)

one thousandth

fold

in

highly

BP-DNA adduct

adducts

and differential

implicated

case with

of the

(33)

of by

in

BP-treated

this

adduct

BP in

to DNA is

1-13

cells appears cells,

a necessary

Vol.99,No.3,1981

BIOCHEMICAL

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ACKNOWLEDGEMENTS The authors wish to thank Drs. M. Osborne and P. Brookes for providing [14C] purine DNA, and Dr. R.S. Day III for critically reviewing this manuscript.

References 1. ;: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Epstein, S.M., McNary, J., Bartus, B. and Farber, E. Science, 162, 907-908 (1968). 29 1799-1804 (1969). Irving, C;C., -and Veazey, R.A. Cancer Res., Kriek, E. Cancer Res.. 32 2042-2m 19f2): Kriek, E. and Hengev e1d.G.M. Chem-Bioi. Interact., 21, 179-201 (1978). Goth, R. and Rajewsky, M.F. oroc. Natl. Acad. Sci. Un, 71, 639-643 (1974). Biochem,J., 148, 521-525 (1974). Margison, G.P. and Kleihues, P. Cox, R. and Irving, C.C. Cancer Lett., 3, E5-m(1977). Cox, R. and Irving, C.C. Cancer Lett., 5, 273-278 (1979). Grover, P.L., Hewer, A., Pal, K. and Sims, P. Int. J. Cancer, 3, l-6 (1976). Jeffrey, A.M., Jennette, K.W., Blobstein, S.H., Weinstein, I.B., Beland, F.A., Harvey, R.G., Kasai, H., Miura, I. and Nakanishi, K. J. Am. Chem. Sot., 98, 5714-5715 (1976). Shinohara, K. and Terutti, P.A. Proc. Natl. Acad. Sci. USA, -74 979-983 (1977). Baird, W.M. and Diamond, L. Biochem. Biophys. Res. Commun., 11, 162-167 (1977). Ivanovic, V., Geacintov, N.E., Yamasaki, H. and Weinstein, I.B. &gq# '-'w;i+"t;;1,60: g978ynette K w Grzeskowiak, K., Nakani;hi, c', Harvey, R:G., Asrup, H. aid H&-;s, C.C. Nature, 269, 348-350 (1977). C.C., Trump, B.F. and Jeffrey, A.M. Cancer Res., Autrup, H., Harris, 38. 3689-3696 119781. Feldman, G., Remsen,J., Shinohara, K. and Cerutti, P.A. Nature,=, 796-798 (1978). Cerutti, P.A., Sessions, F., Hariharam, P.V. and Lusky, A. Cancer m., 38, 2118-2124 (1978). Brown, 7i;S., Jeffrey, A.M. and Weinstein I.B. Cancer Res., 2, 1673-1677 (1979). Koreeda, M.; Moore, P.D., Wislocki, P.G., Levin, W., Conney I, A.H., Yagi, H. and Jerina, D.M. Science, 199, 778-781 (1978). Kakunage, T. and Crow, J.D.,Sciencexx, 505-507 (1980). Selkirk, J.K., Plotkin,E.V. and Gelboin, H.V. Cancer Yang, S.K., (1975). &. , 35, 3642-3650 Burton, K. Biochem. J., 62, 315-323 (1956). Cancer Okano, P., Miller, H.N., Robinson, R.C. and Gelboin, H.V. &., 2, 3184-3193 (1979). Lo, K. and Kakunaga, T. in preparation. Kakunaga, T., Lo, K., Crow, -J.D., and Augl, C. proc. Am. Assoc. Cancer Res., 2J, 350 (1980). Mutation Res., 50, Day, R.S., Scudiero, D. and Dimattina, M., 384-394 (1978). P.P. and Gelboin, H.V. Proc. Yang, S.K., McCourt, D.W., Roller, Natl. Acad. Sci., USA, 3, 2594-2598 (1976).

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28. 29. 30. 31. 32. 33.

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RESEARCH

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Berwald, Y. and Sachs, L. J. Natl. Cancer Inst.,%, 641-661 (1965). Dipaolo, J.A., Donovan, P. and NmR.1. Cancer Inst., 2, 867-874 (1969). Heidelberger, C. Chemical Carcinogenesis. Ann. Rev. Biochem., 44, 79-121 (1975). Mager, R., Huberman, E., Yang, S.K., Gelboin, H.V. and Sachs, L. Int. J. Cancer, 19, 814-817 (1977). Mondal, S., Bra&w, D.W. and Heidelberger, C. Cancer Res., 36, 2254-2260 (1976). Kakefuda, T. And Yamamoto, H. Proc. Natl. Acad. Sci. USA, 3, 415-419 (1978).

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