Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b5 and epoxide hydrase in reconstituted phospholipid vesicles

vol. 95, July

No.

1. 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

16, 1980

BENZO(/,)PYREiIE

METABOLISM

MICROSOMAL HYDRASE

IN

Annika

P-450,

Brunstrom

Box

60

of

Chemistry, S-104

OF RABBIT b5 AND

LIVER

EPOXIDE

PHOSPHOLIPID VESICLES

and

400,

FORMS

CYTOCHROME

RECONSTITUTED

Department

Received

BY PURIFIED

CYTOCHROME

431-439

Magnus

Ingelman-Sundberg

Karolinska

Institutet,

Stockholm,

Sweden

01

May 19,198O

SUMMARY The roles of rabbit liver cytochrome b5, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450~~2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450 M4 primarily participated in the formation of 9,10-dihydroxy-9,l b -dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450~~3. Cytochrome b5 enhanced cytochrome P-450LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b5 was present in the membrane.

INTRODUCTION Polycyclic after

aromatic

metabolic

cellular

activation

constituents

ment,

and

its

concerning its

potent

the

of

genic

to

is

reactive

be

the fatal

metabolites

that benzo(a)pyrene

activity

have

prompted

polycylic

effects

bind

of

this

primarily

to

our

numerous and

only

critical in

hydrocarbon

metabolized

membrane-bound

effects.

undergo,

carcinogenic

occurrence

carcinogenic of

their

environ-

studies

identification

of

metabolites.

the

a result

exert

reactive The

metabolism

carcinogenic

action

to (l-3).

Benzo(a)pyrene

As

hydrocarbons

intermediates

Among one with

enzymes

leading regard

different to to

cytochrome are

often

metabolic

the

cell

P-450 formed

pathways

benzo(a)pyrene cell

in

by and

thay that

the

combined

epoxide may

exert

benzo(a)pyrene

7,8-dihydrodiol-9,10-oxide

transformation

431

(4-6),

whereas

hydrase. carcinomay seems

other

benzo(a)-

0006-291X/80/130431-09$01.00/0 Copyright 0 1980 by Academic Press. Inc. All righfs 01 reproduction in nn.v form reserved.

Vol.

95, No.

1, 1980

BIOCHEMICAL

pyrene

metabolites

system

contains

ties in

for

are multiple

a given

a cell

may

harmful

forms

substrate thus

Therefore,

it

is

fied

of

P-450

forms

more

interest

to

the

BIOPHYSICAL

the

cell

cytochrome

(8-10) the

in

to

of

determine of

AND

the

(7). P-450

presence

biological

Since with

of

the of

COMMUNICATIONS

the

monooxygenase

different

one

activity

examine

conversion

RESEARCH

specifici-

specific of

form

e.g.

specificity

benzo(a)pyrene

of

P-450

benzo(a)pyrene.

of

different

to

reactive

puriinter-

mediates. In and

previous

the

lipid

papers

rabbit

liver

vesicles

in

reconstituted

was P-450,

the

(ll?,

to

the

of

importance

of the

to b5

using

and this

AND

to

the

the

kinetics

these of

system. evaluate epoxide native

In

the

upon

type

of

view

of

of

of in

these

various the

reconstituted

metabolic

system

We obtained into

various

the

altered

vesicles

types

of

interactions the

findings

with

it of

was

cytochrome compared considered

cytochrome

transformation

as

cytochrome

membrane-bound

types

hydrase phospho-

a non-membranous

their in

epoxide

reconstituted

incorporation

reactions

roles

hydrase

of

(10-13).

proteins, the

in

in

specificities

of

properties

system

properties

enzymes

substrate

non-membranous

cytochrome

MATERIALS

the

the

dilauroylphosphatidylcholine

reducibility and

b5

pyrene

from

characterized hydroxylase

comparison

properties

evident

have

microsomal

with

catalytic

we

P-450, of

benzo(a)-

system.

METHODS

Materials. G-3H Benzo(a)pyrene (specific activity 26 Ci per mmol) was from the Radiochemical Centre, Amersham, England. Reference benzo(a)metabolites were supplied by the ITT Research Institute, Chicago, USA. Assay and purification methods. Microsomal phospholipids were prepared according to Bligh and Dyer (15). Rabbitliverepoxide hydrase was prepared as described previously (12). The preparation had a specific activity of 434 nmol of styrene oxide hydrolyzed per mg of the protein and min and was homogeneous according to SDS-polyacrylamide gel electrophoresis. Electrophoretically homogeneous preparations of liver microsomal VADPH-cytochrome P-450 reductase, cytochrome P-450~~2, P-450~~3 and P-45OLM4 were prepared from phenobarbitaltreated rabbits as previously described (13,14). The procedures are based on

obtained pyrene Illinois,

1

Abbreviations used: BP, benzo(a)pyrene; BP-9,10-diol, 9,10-dihydroxy-9,10dihydrobenzo(a)pyrene; BP-4,5-diol, r,5-dihydroxy-4,5-dihydrobenzo(a)pyrene; BP-7,8-diol, 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene; P-450~~1, liver microsomal cytochrome P-450; P-45DLM2, P-45OLM3 and P-450~~4, forms of P-450~ designated according to their electrophoretic properties; SDS, sodium do !I4ecyl sulphate;

432

Vol.

95, No.

BIOCHEMICAL

1, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

those presented by Yasukochi and Masters (16) and Haugen and Coon (17), respectively. The specific contents of the proteins were: P-450~~2, 12.5-14.2 10.5 nmol/mg; NADPH-cytochrome P-450 nmol/mg; P-45OLM3, 9.5 nmol/mg; P-450~~4, reductase 12.5 nmol of flavin/mg when flavin was detected by the absorption at 456 nm of the protein, using the absorption coefficient 10.7 mM-lcm-1 (18). Cytochrome b5 was purified as described elsewhere2 to a specific content of 28.2-34.5 nmol/mg and the preparations were electrophoretically homogeneous. Cytochrome P-450 was measured according to Omura and Sato (19) using 91 mM-1 cm-l as absorption coefficient. Cytochrome b5 was detected using the absorptiot coefficient 100 mM-lcm-1 for the absorbance difference between the reduced minus the oxidized form of the protein at 424 nm (20). Protein was determined according to Lowry (21). Preparation of vesicles. Unilamellar phospholipid vesicles containing the various proteins were prepared from microsomal phospholipids using the cholate gel filtration technique previously described (12,14,22). The vesicles were usually prepared devoid of cytochrome b5. The subsequent addition of cytochrome b5 to the vesicles at 370C resulted in a rapid and complete incorporation of the protein into the vesicles as judged from results obtained by Sepharose 48 chromatography of the preparations. Analysis of benzo(a)pyrene (BP) metabolism. Incubations with benzo(a)pyrene were performed using vesicles corresponding to 0.2 LIM of P-450 having a molar ratio of cytochrome P-450:NADPH-cytochrome P-450 reductase:epoxide hydrase:phospholipid of 3:1:3:100 in 1 ml 50 mM potassium phosphate buffer, pH 7.4. Benzo(a)pyrene (25 nmol) with 500 000 dpm 3H-BP in 40 ~11 of methanol was added and after 3 min preincubation at 37oC, the reactions were started by the addition of 0.5 mg NADPH in 100 ~1 of water. Control incubations were performed in the absence of NADPH. The incubations were terminated after 10, 20 or 30 min by the addition of 1 ml of acetone. The incubation mixtures were extracted with 2 x 2 ml of ethyl acetate, containing 0.08% (w/v) of butylated hydroxytoluene. The combined organic layers were dried with 0.5 g of anhydrous sodium sulphate and the ethyl acetate was removed under a stream of nitrogen. The incubations were stored in the dark under nitrogen at -200C until HPLC analysis. Separation of the different benzo(a)pyrene metabolites was performed on an LDC High Pressure Liquid Chromatograph equipped with two Constametric pumps and a Bondapack Cl8 column (1.52 x 25 cm). The samples were dissolved in 50 ~1 of acetone and 20 1~1 aliquots were injected onto the column. The metabolites were eluted with a linear 50 min gradient of elutent (2.2 ml/min) from 60'2 methanol in water to 100% methanol. Fractions were collected each 40 set directly into counting vials for an Intertechnique SL-30 liquid scintillation spectrometer. Counting was performed using 3 ml of LumagelR as scintillator liquid. RESULTS Benzo(a)pyrene

Metabolism

Two

major

containing tic pyrene diol, bolites 2

9-

the and

phenolic

the

Reconstituted

fractions

reconstituted

were vesicles

3-hydroxybenzo(a)pyrene

quinones BP-4,5-diol by

in

the

Ingelman-Sundberg,

and and

different

BP-7,8-diol,

reconstituted M.,

isolated and

from

three

and

Johansson,

from

were the

I.,

433

incubation together

HPLC-column.

detected. was

the

eluting

benzo(a)pyrene were

vesicles

Vesicles

linear submitted

In

mixtures with

addition

dihydrodiols, The for

for

benzo(a)i.e.

formation 20-30

authen-

of min

publication.

(Fig.

BP-9,10the

meta1).

Vol.

95. No.

1. 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

3H0 ,*’ . I

. .’

/

/

I 0 /

I

I

Qui

45 9HO

/

I

I

I

I

,

I

I

’

I

/

/

//

/

P-

7.0 9,lO

0

10

20

30

TIME,min

Fig. 1. Time courses of cytochrome P-45DLM2-dependent oxygenations of benzo(a)pyrene in reconstituted phospholipid vesicles. The vesicles were prepared by the cholate gel filtration technique from microsomal phospholipids, cytochrome P-45DLM2, NADPH-cytochrome P-450 reductase, cytochrome bg and epoxide hydrase in a molar ratio of 1000:2:1:0.8:3. Incubations were performed as described under "Methods". Further explanations in the text.

The various

metabolism forms

of

Table

I.

Cytochrome

P-450

in

transforming

epoxide

hydrase

dials;

the

P-45OLM much

Only

very

by

the

was

4,5-diol,

active

into

more in

from

comprising

of

in

metabolizing

small

amounts

of

and

from

the

II HPLC

of

the

cytochromes

products.

formation

form

of

column)

together was

containing whereas

P-450, phospholipid

with

isolated

dihydro-

formed.

cytochrome the

of

various

metabolites, were

in

(eluting

of

the

in

Inclusion

mixtures

BP-7,Sdiol

benzo(a)pyrene

phenol

summarized

polar

15% of

a non-inducible

inactive

is

form

the

containing

P-450

incubation

about

BP-9,10-diol

P-45OLM3,

hydroxybenzo(a)pyrene

most

resulted isolated

vesicles

cytochrome

the

vesicles

metabolite

amounts

phospholipid

microsomal

benzo(a)pyrene

the

Cytochrome

liver

P-45OLM2

in

was

benzo(a)pyrene

rabbit

major

2 smaller

markedly

of

was vesicles.

authentic from

3-

P-450LM

-con3

taining

incubation The

different

mixtures.

regiospecificity compared

of to

cytochrome

cytochrome

P-450LM 4 . Although

P-450LM 2

434

against the

benzo(a)pyrene overall

activity

was was

= less

than

0.5

pmol

of

+ b5

LM4

13

1

5.5

+ EPH

product

was

formed

4.1

2.3

a

a

a

13.4

a

LM4

a

1.5 0.8

38

a

Catalytic

BP 4,5dihydrodiol

6.1

LM4

+ EPH

+ EPH + b5

l"3 LM3

EPH

f b5

LM2

t

+ EPH

LM2

1.1

a

a

+ b5

BP 9,10dihydrodiol

L"2

of

LM2

Composition vesicles

per

nmol

P-450,

3.7

1.1

a

a

a

14

2

a

a

BP 7,8dihydrodiol

activity,

pmol

min.

product/nmol

8.5

3.2

3.5

a

a

76

15.8

87

19

BP Quinones

of

P-450,

5.8

2.3

3.5

a a

45

8.2

56

20

BP phenols (9 HO)

of I

min.

35

8.5

14.4

2 20

255

48

196

90

BP phenols (3 HO)

II

Table I. Metabolism of benzo(a)pyrene in reconstituted phospholipid vesicles containing NADPH-cytochrome P-450 reductase, different forms of cytochrome P-45OLM and, if indicated, epoxide hydrase (EPH) and cytochrome b5. Incubations were performed as described under "Methods" using vesicles corresponding to 0.2 nmol P-450. The molar ratio of P-450:P-450-reductase:cytochrome b5:epoxide hydrase (EPH):phospholipid used was 3:1:2:3:1000. The values represent means from 2-6 experiments and were calculated from the time curves obtained from incubations performed for 10, 20 and 30 min.

70.1

22.9

22.4

2 20

434.1

87.2

339

130.5

TOTAL

5 ii

P 3

I

4 n

I

I% % 9

5 I 2 i=l 9

6

6

F

?

5 E

Vol.

95, No.

1, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I-

E a

“oui -es>; ,

-o----0.5

--------o

- -

’

,

1.0

MOLAR

RATIO

0

2.0

CVT. b5/

CVT

E P

P-450LM 2

Fig. 2. Cytochrome bg-dependent stimulation of cytochrome P-450LM2-catalyzed oxygenations of benzo(a)pyrene in reconstituted phospholipid vesicles. The vesicles were prepared from microsomal phospholipids, cytochrome P-450~~2 and NADPH-cytochrome P-450 reductase in a molar ratio of 1000:4:1 and indicated amounts of cytochrome bg were subsequently added to the vesicles at 370C. After 3 min. incubations with benzo(a)pyrene were performed as described under “Methods”.

only

about

25,;

BP-9,10-diol

of

that

were

addition

lesser

epoxide

hydrase

exhibited

produced

by

amounts was

by

of

P-450LM

, six 2 vesicles

phospholipid

BP-4,5-diol

present.

In

and

contrast

times

higher

containing

BP-7,8-diol to

the

were

situation

amounts

of

P-450LM

. In 4 when

isolated when

using

P-450LM

, 2

as

much

as

40%

hydrodiols,

more

Effects ly

benzo(a)pyrene 60:

of

activities

and

epoxide

hydrase

stimulations

were

seen

in

all

I the the

case

of

comprised

introduction

cytochrome

cytochrome

-catalyzed 4 , NADPH-cytochrome

2 cytochrome

with P-450LM

di-

-dependent

b5

of

2

reactions P-450

(Fig.

activities

near-

P-450-dependent P-450LM

P-450LM P-450LM

of

BP-9,10-dial.

Table

enhanced

cytochrome

titrated in

from

b5

containing were

isolated to

seen

5-fold when

vesicles

As

cytochrome

benzo(a)pyrene

When

attributed

b5.

of

3 times

metabolites

being

cytochrome

amounts

metabolism

examined.

the than

of

equimolar

and

of

2)

-dependent were reductase

concomitant towards

benzo(a)-

vesicles.

These

2 pyrene

as

the

amount

of

cytochrome

b5

was

436

increased

in

the

vol.

95, No.

1, 1980

results,

in

BIOCHEMICAL

addition

to

stereospecificity in

the

vesicles.

molar at

in

ratio

of

results',

the

Maximal of

a ratio

b5

to

the

in reactions

stimulation

by

the in

of 2 formation

the

RESEARCH

Table

I,

b5 P-450

This

of

is

no

change

cytochrome

cytochrome

1:0.25.

COMMUNICATIONS

indicate

when

b5:NADPH-cytochrome

P-450LM

proteins

presented

BIOPHYSICAL

oxygenation

cytochrome

indicating

between

those

AND

was

in

in

b5

accordance

with

the

types

the

present at

vesicles,

active

is

obtained

the

a functionally

of

a 1:l i.e.

previous

ternary

complex

vesicles.

DISCUSSION The P-450

results

have

markedly

(a)pyrene.

The

primarily as

indicate

different

the

formation

in

inducible of

did

benzo(a)pyrene have

been

system,

thereby

not

making

bolites

possible.

metabolism the

it

for

the

action

catalyzed ciently

was

to

the of

than

by

absence of

for

and

any

in

donating

in

P-450

P-450,

may

the

liver

epoxide

microsomal

by

changes previously

meta-

cytochrome

in raised

NADPH-supported

cytochrome

the

second

to

thereby

the

benzo(a)pyrene

the

reductase,

in

benzo(a)pyrene

of

electron

in

, an un3 investiga-

hydratase

primary

significant

,

2 where-

P-45OtM

stimulation

the

P-450LM

Previous

rabbit

benzo-

, preferentially 4 thus participate

Cytochrome

of

of

BP-4,5-diol

reconstitution

Obviously, b5

BP.

a 3-5-fold

without

cytochrome

NADPH-cytochrome

purified

obtain

reactions.

oxygenations

by

detection

vesicles

in

substrate

cytochrome

metabolism

P-45OtM

benzo(a)pyrene.

the

reactions

the

the

non-toxic

metabolize

hampered

possible

the

of

phospholipid

oxygenation

specificities

of

of

cytochrome

carcinogen

metabolism

(23,24)

using

not

of

P-450-form,

of

P-450:s

By

formation

ultimate

P-450-form,

tions

in

the

inducible

the

in

form

9,10-epoxidation of

various

stereospecificities

3-methylcholantrene

catalyzes

that

phenobarbital-inducible

participates

the

the

presented

the

b5

regio-

explanation P-450-

P-450

more

enhancing

the

effioverall

n

hydroxylation pyrene

rated as

in

the

vesicles,

seems

substrate.

431

plausible

also

in

case

of

benzo(a)-

Vol.

95, No.

1. 1980

BIOCHEMICAL

In conclusion,

the

of benzo(a)pyrene cytochrome

humans

Metabolism

P-450

of secondary

present

importance

in aryl

here being

in the in this

hydrocarbon

in susceptibility

to variations

BIOPHYSICAL

presented

vesicles,

whereas

respect.

the

COMMUNICATIONS

regiospecificity

associated

with

cytochrome

b5 and epoxide

The interindividual

hydroxylase

activities

action

in the

RESEARCH

indicate

preferentially

to carcinogenic

be attributed in a given

results

AND

of e.g.

composition

(25,26) cigarette of the

the type

variations and,

are

in

furthermore,

smoke,

cytochrome

of

may well P-450

enzymes

tissue.

Acknowledgements We are

indebted

enzymes.

This

Swedish

Cancer

work

to MS Ann-Louise was supported

Edvardsson

by grants

from

for

purification

Svenska

Tobaks

of the AB and the

Society.

References 1.

14.

Miller, E.C., and Miller, J.A. (1974) in the Molecular Biology of Cancer Academic Press, New York. (Busch, H., ed) pp. 377-402, Jerina, D.M., and Grover, P.L. (1974) Science 185, 573-582. P.L. (1974) Adv. Cancer Res. 20, 165-274. Sims, P., and Grover, Malaveille, C., Bartsch, H., Grover, P.L., and Sims, P. (1975) Biochem. Biophys. Res. Commun. 66, 693-700. Huberman, E., Sachs, L., Yang, S.K.,and Gelboin, H.V. (1976) Proc. Natl. Acad. Sci. 70, 2281-2285. Wood, A.W., Goode, R.L., Chang, R.L., Levin, W., Conney, A.H., Yagi, H., D.M. (1975) Proc. Natl. Acad. Sci. 72, 3176Dansette, P.M., and Jerina, 3180. Levin, W., Wood, A.W., Wislocki, P.G., Chang, R.L., Kapitulnik, J., Mah, H.D., Yagi, H., Jerina, D.M., and Conney, A.H. (1978) in Polycyclic Hydrocarbons and Cancer Vol. 1 (Ts'o, P.O.P. and Gelboin, H.V. eds.) pp. 189-202, Academic Press, New York. Coon, M.J., Vermilion, J.L., Vatsis, K.P., French, J.S., Dean, W.L.,and Haugen, D.A. (1977) ACS Symp. Ser. 44, 46-71 Haugen, D.A., and Coon, M.J. (1976) J. Biol. Chem. 251, 7929-7939 Ingelman-Sundberg, M., and Johansson, I. (1980) Biochemistry, in press. I. (1980) in MicroIngelman-Sundberg, M., Glaumann, H., and Johansson, somes, Drug Oxidations and Chemical Carcinogenesis (Coon, M.J., Conney, A.H., Estabrook, R.W., Gelboin, H.V., Gillette, J.R. and O'Brien, P.J., eds.) Academic Press, New York, in press. M. (1979) J. Biol. Chem. Halpert, J., Glaumann, H., and Ingelman-Sundberg, 254, 7434-7441. Ingelman-Sundberg, M., Johansson, I., and Hansson, A. (1979) Acta Biol. Med. Germ. 38, 379-388. H. (1980) Biochem. Biophys. Acta, Ingelman-Sundberg, M., and Glaumann,

15.

i?i!$:S::G.,and

2. 3. 4. 5. 6. 7.

8. 9. 10. 11.

12. 13.

Dyer,

W.J.

(1959)

Can. J. Biochem.

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37, 911-917.

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BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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439