Vol. 91, No. 2, 1979 November

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

28, 1979

THE

Pages 490-497

INVOLVEMENT

OF

OF A NON-‘BAY-REGION’

BENZ(a)ANTHRACENE-DNA

A.D.

Chester

Received

ADDUCTS

MacNicoll, A.

Beatty Cancer

October

DIOL-EPOXIDE

C.S.

Hewer,

C.

Research Hospital,

IN A RAT-LIVER

Cooper,

0.

Walsh,

P.L.

Institute, Fulham

Road,

Ribeiro,

IN THE

FORMATION

MICROSOMAL

SYSTEM

P.G.

Grover

and

Institute London

Gervasi”,

P.

Sims

of Cancer SW3 6JB,

Research:Royal England

8, 1979

SUMMARY: The metabolic activation of benz(a)anthracene was investigated incubating [3H]-b enz(a)anthracene with DNA, a NADPH-generating system rat-liver microsomes. When hydrolysates of the DNA were chromatographed Sephadex LH20 columns, three hydrocarbon-nucleoside adduct peaks were resolved and these were further examined using HPLC. One adduct probably results from the reaction of the non-bay-region diol-epoxide r-8,t-9dihydroxy-t-l0,11-oxy-8,9,lO,ll-tetrahydrobenz(a)anthracene (%ti=BA-8,9diol lO,ll=oxide) with DNA. The other two adducts did not co-chromatograph with adducts formed from any of the four possible isomeric diolepoxides that can be formed in the 8,9,10,11-ring of benz(a)anthracene.

INTRODUCTION: biological may

and

systems

be

ation

further of

ic to

aromatic

metabolized

that to

hydrocarbon-nucleic

through with

the

nucleic

hydrocarbon

with

preparations adducts >: Present

(4,s) formed

address:

are

hydrocarbons

products

of

have

vicinal studies

nucleic

acids

in

shown

that

CNR,

to

Pisa,

those

shown

Studies

vivo

from

that

these

diol-epoxides

Other

similar

--in

that

the some

of

that

are

of the

in

several that

on

the

adducts the the

(1) probably

bay-region incubation

rat-liver

--in

type of

a

microsomal

hydrocarbon-nucleic formed

form-

benzo(a)pyrene

of

involved

presence

ized dihydrodiols

diol-epoxides.

acids.

have

metabol

non-K-region

adducts

(2) reaction

are

include

vicinal acid

7-methylbenz(a)anthracene

arise (3)

Polycycl

by and on

vivo

acid and

that

are

Italy

Abbreviations: Anti-BA-8,9-diol lO,ll-oxide, r-8,t-9-dihydroxy-t-lO,ll-oxy8,9,10,11-tetrahydrobenz(a)anthracene; z-BA=8,9=diol lO,ll-oxTde, r-8,t-9dihydroxy-~-10,11-oxy-8,9,10,11-tetrahydrobenz(a)anthracene; anti-BA~lO,ll-dial 8,9-oxide, t-10, I-ll-dihydroxy-t-8,9-oxy-8,9,1O,ll-tetrahydrobenz(a)anthracene; z-BA-lO,ll-diol 8,9-oxide, ~-l0,r-ll-dihydroxy-c-8,9-oxy-8,9,lO,ll-tetrahydrobenz(a)anthracene .

0006-291X/79/220490-08$01.00/0 Copyright All rights

@ I979 by Academic Press, Inc. of reproduction in anyform reserved.

490

Vol. 91, No. 2, 1979

BIOCHEMICAL

thought

to

be

involved

effects

of

the

parent

bay-region been

theory

obtained of

the

hydrocarbons.

shown

the

parent

found

with

that

the

that

this

ing

in

the

8,8,TO,ll-ring

not

of

the

bay-region

or

rats of

In

type. that

and

shown)

and

related

active

in

both

It

has

incubated

of

agreement

with

with

pretreated

hydrocarbon-DNA DNA

with

anti-BA-8,9-diol

in

the

mutagenic dihydrodiols

recently

been are

culture

the

formed

were

treated

spectrum

results adduct of

3-methylcholanthrene with

although

occurr-

diol-epoxide

presence

lO,ll-oxide

the

benz(a)anthracene

a vicinal this

systems from

fluorescence of

formation

vicinal

that

in

these

systems,

adducts

cells

activation

a major

these

(10)

embryo

of

derived

a phenanthrene-like

the

results

test

acid

hamster

(8,9)

in

carcinogenic.

metabolic by

is from

reaction

vivo

with

showing

benz(a)anthracene

the

weakly

the has

to

initiating

highly

of

tumourigenic

dihydrodiols

tumour

is

support

metabolized active

hydrocarbon-nucleic

--in

consistent

more

carcinogenic

hydrocarbons

and

further

other

the

The

(formula

possessed

evidence

are or

the

only

major

be

and

polycyclic (6,7)

may

type

on

is

skin

and

by

that

RESEARCH COMMUNICATIONS

in

hydrocarbons.

3,4-dihydrodiol

the

mouse

preparations

several

benz(a)anthracene

benz(a)anthracene

provide

of

of

mutagenic

hydrocarbons,

hydrocarbon

is

the

studies

of

either

on

of

evidence

activation

bay-region

parent

(11)

(12)

when

the

Thus,

activities

activation Further

dihydrodiols

of

either

metabolic

metabolic studies

that

diol-epoxides

have

of

dihydrodiols

show

than

the

hydrocarbon.

from

activities studies

in

AND BIOPHYSICAL

that presented

formed rat-liver probably

is here

when microsomal arises

DNA.

2

a

7

6

I MATERIALS AND METHODS: C3H]-Benz(a)anthracene unlabelled hydrocarbon by catalytic exchange Bucks, U.K.). The anti-BA-8,9-diol lO,ll-oxide

491

(1.5 Ci/mmol) was prepared from (Radiochemical Centre, Amersham, and the --BA-8,9-diol

Vol.

91,

lO,Tl-oxide 8,9-oxide Keith of Repository that was St. Louis,

No.

2, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

were synthesized as described (13,14) and the anti-BA-lO,ll-diol and the syn-BA-lO,ll-diol 8,9-oxide were kindly supplied by Dr.J.N. the I.I.T. Research Institute, Chicago, Ill., USA through the Chemical of the National Cancer Institute. DNA (salmon testes, type I II), deproteinized before use, was obtained from Sigma Chemical Co., MO., USA.

Chemical and enzymic reactions of benz(a)anthracene with DNA. Each of the four diol-epoxides of benz(a)anthracene named above were incubated with DNA and the DNA’ isolated and hydrolysed to nucleosides exactly as described previously (2). [3H]-B enz ( a ) ant h racene (2 pmol) was incubated at 37OC with DNA (125 mg), NADP, MgCl 2, glucose 6-phosphate and glucose 6-phosphate dehydrogenase in the presence of a microsomal fraction (2) prepared from 1 iver (209) from rats that had been pretreated with 3-methylcholanthrene. After 30 min, the incubation mixture was cooled to 4OC, the microsomes separated by centrifugation (1 h, 100,000 x g, 4’C) and DNA precipitated from the supernatant with ethanol (2 x 1 vol). The DNA was washed with ethanol, deproteinized and hydrolysed to nucleosides as described (2). Sephadex

LH20 column chromatography and high pressure liquid chromatoSamples of enzymic hydrolysates of DNA that had been incubated $$73 H ]- benz(a)anthracene in the presence of a rat-liver microsomal fraction were mixed with UV-absorbing amounts of nucleoside adducts obtained from the chemical reaction of one of four diol-epoxides of benz(a)anthracene with DNA and the mixture chrcmatographed on columns (70 x 1.5 cm) of Sephadex LH20 that were eluted with either water-methanol (15) or with sodium borate (O.O5M,pH 8.7)-methanol (16) gradients as described. Eluant fractions (5 ml) were collected and the absorbance (260 nm) and fluorescence (excitation 300 nm, emission 356 nm) determined for each fraction. 3H-Labelled products present in each fraction were determined by liquid scintillation counting of a sample (1 ml) of each fraction in PTX scintillant (10 ml) (Packard Instrument Co., Downers USA) using a Packard Tricarb 3320 scintillation counter. Grove, Illinois, MO., Fractions that contained hydrocarbon-nucleoside adducts that were to be further investigated were pooled and evaporated to dryness and the residue dissolved in methanol (0.5 ml). Samples (0.1 - 0.25 ml) of these solutions were examined by HPLC at 40°C on columns of Spherisorb 5 ODS (4 x 250 mm; HPLC Technology Ltd., Wilmslow, Cheshire, U.K.) that were eluted at a flow rate of 2 ml/min with a convex gradient, over 45 min, from O-35% methanol in water and then with a concave gradient, over 45 min, from 35-65% methanol in water using a Waters 660 Solvent Programmer (Waters Associates Ltd., Hartford, Cheshire, U.K.). Fractions (2 ml) of eluate were collected and hydrocarbon-nucleoside adducts detected by fluorescence as described above. Radiolabelled products in samples (1 ml) of each fraction were determined by liquid scintillation counting. In some cases, fractions from Sephadex LH20 evaporated to dryness and the residues incubated with co1 umns were pooled, acetic anhydride-pyridine (l:g, v/v; 0.5 ml, 40h at 4’C). The mixtures were evaporated to 100 ~1 volume with a stream of nitrogen and the residues examined by HPLC at 40°C on a column of Spherisorb 5 ODS that was eluted with a convex gradient, over 45 min, of O-60% methanol in water followed by a concave gradient over 45 min, of 60-80% methanol in water, at a flow rate of Fractions (2 ml) of eluant were collected and fluorescence and 2 ml/min. radioactivity determined as described above.

RESULTS

AND

DISCUSSION:

II 1

3 H -benz(a)anthracene

Sephadex

LH20

columns,

When

hydrolysates

and

a metabolizing

the

elution

of

DNA

system

profile

contained

492

that were

had

been

chromatographed three

peaks

incubated

with on

(products

BIOCHEMICAL

Vol. 91, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

25

0

1

I 25

2.0

1 50

Fraction

1 75

I 100

number

I 125

I 150

2.0

1.5

1.5 3 zei 2 5

& g

1.0

1.0

0.5

0.5

4 !s “0 s 2s “2

w”

0 25

50

75 Fraction

Fig.

111,

Sephadex A hydrolysate fraction a hydrolysate lO,ll-oxide. gradient fig. 16)

IV

and

V

fig.

LH20

with

in

and

and and

1)

that

Sephadex

in and

The

be

formed

which

wi th

columns

or in

the

region

150

of of

hydrolysates

occupied

these DNA

benz(a)anthracene of

DNA

that

that

had

493

been

benz(a)anthracene.

invest-

incubated

had

been

on

incubated

8,g-oxide,

8,+oxide, of

was

co-chromatographed

anti-BA-lO,ll-diol

8,9.10,11-ring

by

products

were

-syn-BA-lO,ll-diol the

normally

identity

hydrolysates

lO,ll-oxide,

lO,ll-oxide can

in

abelled

3H-1

anti-BA-8,Pdiol

8,9-diol that

experiments

LHZO

either

eluted

adducts.

microsomes

125

column chromatography of hydrocarbon-nucleoside adducts. of DNA that had been incubated with a rat-liver microsomal 3H-labelled benz(a)anthracene was co-chronatographed with of DNA that had been incubated with anti-BA-8,9-diol The Sephadex LH20 column was eluted with a water-methanol radioactivity (D---O, fig. lA), fluorescence (A+, absorbance (m, fig. 1B) were determined.

hydrocarbon-nucleoside igated

100 number

the

four

with ~JJ-BA-

diol-epoxides When

the

Vol. 91, No. 2, 1979

Sephadex

LH20

labelled

product

nucleoside

columns

were did

V

adducts

labelled

products

8,9-diol

lO,ll-oxide

diol-epoxides identity

in

which

not

formed III

and

products

3H-labelled

water-methanol

with these

did 1)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

co-elute

IV

not

of

eluted

from

(fig. (results

The ments

BIOCHEMICAL

and

any

four

the

with

hydrocarbon-

However,

adducts

adducts

jH-

non-labelled

diol-epoxides.

co-elute with

of

gradients,

formed

formed from

3H-

from the

anti-BA-

other

three

shown). III

and

products

was

IV

and

further

investigated

non-labelled

adducts

in that

experi-

co-eluted

0

10

Fig.

2

20

30

40 Fraction

50 number

Fraction

number

60

70

80

High pressure 1 iquid chromatography of deoxyribonucleoside-hydrocarbon adducts. 3H-Labelled products present in the hydrolysis of DNA that had been incubated with[sH)benz(a)anthracene in the presence of a rat-liver microsomal fraction and fluorescent amounts of non-labelled adducts formed from DNA that had been incubated with anti-BA-8,9diol lO,ll-oxide in vitro were co-chromatographed on Sephadex LH20 Eluted fractions Gnmng 9H-labelled product III and non-labelled product VI(fractions 74-90, fig. 1A.B) were pooled and subjected to HPLC on Spherisorb 9 ODS either before (fig. 2A) or after (fig. 28) acetylation. Eluted fractions were examined for the presence of fluorescent and radioactive material.

494

Vol. 91, No. 2, 1979

from

Sephadex

BIOCHEMICAL

LH20

were

3 H-labelled

system,

In

3 H-label

w-BA-lO,ll-diol (fig.

co-eluted Spherisorb

with

from

adducts

oxide

and

from

Under

formed

several

fractions

used

separated

these in

HPLC

III

were

on

was

separated

syn-BA-8,9-diol

of

with

a similar

position

to

the

position

that

of

the

is

not

lO,ll-

formed

non-labelled

from

8,9-oxide

was

nucleoside-hydrocarbon

adducts

However,

III

syn-BA-lO,ll-diol

the

in

this

study

the

been

suggest

or

conditions,

V are

has

ites

product

product

borate

and

Sephadex

with

to

that

lO,ll-oxide

8,9-oxide,

sodium

IV

by

and

adducts

prior

28);

lO,ll-

adducts

(0.05

M,

formed that of

pH 8.7)-methanol

from

in

which

elution

products

on

the

two

they of

and,in

syn-

eluted

in

product

III

borate,eluted

earlier.

from

non-polar

(fig.

from

z-BA-8,9-diol

anti-BA-8,9-diol

3H-1abe11ed

eluted

to

Products

III

formed anti-BA-8,9-diol

non-labelled

acetylated

from

diol-epoxides.

from

and

this

separated

adducts

formed

and

from

behaviour

gradients. relative

four

from

adducts

anti-BA-lO,ll-diol

the

eluted

changed

the

of

separated

were

lO,ll-oxide

water-methanol

was

8,9-oxide

columns

that

columns

diol-epoxides

and

Using

8,9-oxide.

examining

gradients.

5 ODS.

than

products

RESEARCH COMMUNICATIONS

Spherisorb

each

from

product

x-BA-8,9-dial

LH20

on

was

not

adducts

evidence

Sephadex

study

LH20

z-BA-lO,ll-diol

by

III

3 H-labelled

only

formed

obtained

metabol

from

3H-labelled

Additional

epoxide

formed

but

Sephadex

5 ODS,

co-eluted

attempt

earlier

product

HPLC

anti-BA-lO,ll-diol

when

from

or

eluted

led

2A),

However,

oxide.

HPLC

IV

8,g-oxide

lO,ll-oxide

epoxides

by

adducts

addition,

either

examined

product

nucleoside-hydrocarbon

AND BIOPHYSICAL

may DNA,

with that

probably because

non-labelled LH20 made

the

3 H-labelled

adducts

identify

arisen has

been

DNA

in

a similar III

(data

the

IV

reaction

observed

is

from

the

system

(4).

formed

from

495

from

of

these

not

shown).

and

V,

of

for

any

products

formed

products by

as

product

formed

chromatography

to

have

not

results

anti-BA-8,g-diol

IV

diol-

and

V were by

Although

a K-region

The

four

diol-epoxides

product

reaction

the

V is or

of

no

further

relatively

other

simple

benzo(a)pyrene presented lO,ll-oxide

in

this

Vol. 91, No. 2, 1979

because

BIOCHEMICAL

a hydrocarbon-nucleoside

indistinguishable after

from

acetylation,

consistent formed

in

graphic

the

finding

mouse

skin

and

fluorescence

region the

spectra

activity

that in

of

of

bay-region

dihydrodiols

initiation

studies

LH20

column

major embryo

those

of

and

diol-epoxides

by

HPLC,

which

III

possess

indicate

might and

diol-epoxide both

was

before

chromatography.

and

do

benz(a)anthracene

this

cells

findings

that

RESEARCH COMMUNICATIONS

the in

not

is

adduct have

chromato-

phenanthrene-like

that

major

routes

necessarily

explain

the

high

activity

mutagenesis

and

This

benz(a)anthracene-DNA

peak

benz(a)anthracene

a suggestion

from

examined

the

These

formed

when

hamster to

(12).

activation

III

Sephadex

similar

diol-epoxide, low

by

with

properties

metabolic

adduct product

and

AND BIOPHYSICAL

involve discrepancy of (11)

the and

of a baybetween related

tumour

(10).

ACKNOWLEDGEMENTS The authors thank Mr. C.J. Cadwallader for technical assistance. This research was supported in part by grants to the Chester Beatty Research Institute, Institute of Cancer Research:Royal Cancer Hospital, from the Medical Research Council and the Cancer Research Campaign, and in part by grant CA21959 from the National Cancer Institute, USPHS. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10

11.

Sims, P., Grover, P.L., Swaisland, A., Pal, K. and Hewer, A. (1974) Nature 252, 326-328 Tierney, B., Hewer, A., Walsh, C., Grover, P.L. and Sims, P. (1977) Chem.-Biol.lnteractions 9, 179-153 Jerina, D.M. and Daly, J.W. Oxidation at Carbon. In D.V. Parke and R.L. Smith (eds), Drug Metabolism, from Microbe to Man, Taylor E Francis Ltd., London (1977) pp 13-32 King, H.W.S., Thompson, M.H. and Brookes, P. (1975) Cancer Res. 35, 1263-1269 Thompson, M.H., King, H.W.S., Osborne, M.R. and Brookes, P. (1976) Int.J;Cancer ‘7, 270-274 Malaveille, C., Bartsch, H., Tierney, B., Grover, P.L. and Sims, P. (1978) Biochem.Biophys.Res.Commun. 83, 1468-1473 Wood, A.W., Levin, W., Ryan, D., Thomas, P.E., Yagi, H., Mah, H.D., Thakker, D.R., Jerina, D.M. and Conney, A.H. (1977) Biochem.Biophys.Res.Commun. 2, 847-854 Chouroul inkov, I., Gentil, A., Tierney, B., Grover, P.L. and Sims, P. (1977) Cancer Lett. 3, 247-253 Chouroulinkov, I., Gentil, A., Grover, P.L. and Sims, P. (1876) Br.J.Cancer 2, 523-532 Wood, A.W., Levin, W., Chang, R.L., Lehr, R.E., Schaefer-Ridder, M., Karle, J.M., Jerina, D.M. and Conney, A.H. (1977) Proc.Natl.Acad.Sci.USA 2, 3176-3179 Ertsch,H., Malaveil le, C., Tierney, B., Grover, P.L. and Sims, P. (1979) Chem.-Biol. interactions 26, 185-186

Vol. 91, No. 2, 1979

12.

13. 14.

16.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vigny, P., Kindts, M., Duquesne, M., Cooper, Sims, P. (1979) (submitted for publication) Lehr, R.E., Schaefer-Ridder, M. and Jerina, Tetrahedron Lett. pp 539-542 Lehr, R.E., Schaefer-Ridder, M. and Jerina,

42, 15.

BIOCHEMICAL

C.S.,

Grover,

D.M.

(1977)

D.M.

(1977)

P.L.

and

J.Org.Chem.

736-744

Gird, (1973) King,

(1976)

W.M., Dipple, A., Grover, P.L., Sims, P. and Brookes, Cancer Res. 22, 2386-2392 H.W.S., Osborne, M.R., Beland, F.A., Harvey, R.G. and Proc.Natl.Acad.Sci.USA 2, 2679-2681

497

P. Brookes,

P.