Hepatic microsomal mixed function oxygenase: Enzyme multiplicity for the metabolism of carcinogens to DNA-binding metabolites

Vol. 61, No. 2, 1974

BIOCHEMICAL

HEPATIC

MICROSOMAL

ENZYME

MULTIPLICITY

CARCINOGENS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MlXED

FUNCTION

OXYGENASE:

FOR THE METABOLISM

TO DNA-BINDING

OF

METABOLITES

H. L. Gurtoo and Natalie Bejba Department of Experimental Therapeutics and Grace Cancer Drug Center Roswell Park Memorial Institute New York State Department of Health Buffalo, New York 14203 Received

August

12,1974

SUMMARY: Microsome-mediated metabolic activation of aflatoxin Bl (AFB1) and benzo[a]pyrene (BP), as determined by the -m in vitro formation of DNA binding metabolites, was studied, using hepatic microsomes from untreated, phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated male rats. Contrasting results were obtained for the two substrates: in the case of AFB microsomes from PB-treated rats were twice as active as microsomes from un I’ reated and MC-treated rats, whereas, in the case of BP, microsomes from MC-treated rats were several fold more active than microsomes from untreated and PB-treated rats. These data strongly suggests enzyme multiplicity of microsomal mixed function oxygenase for the activation of carcinogens, especially AFBl and BP whose reactive metabolites are believed to be epoxides. Hepatic

endoplasmic

to as microsomal in the oxidation insecticides

of various

hydroxylase

widely

a group of enzymes

(MMFO)

and other differing

into two general

complex

categories:

of only a specific

group

system

phenobarbital

type,

and the activity

compounds

have

causing

an increase

in the

of several

3-methylcholanthrene cytochrome,Pq48,

one of which

is involved

foreign

in the activities

of a different

referred

than 200 drugs,

and in biological

In this regard,

(1).

of enzymes,

enzyme

both --in vivo and --in vitro

type (e . g.,

the content

commonly

(1) 0 More

in structure

PqFjo arad an enhancement hydrocarbon

. This

chemicals

are lmown to modify

(BP) ), increasing

activities

oxygenase

in the MMFO

aromatic

contains

carcinogens

activities,

of cytochrome

and polycyclic

Supported Research

of drugs,

enzymes

been divided

pyrene

function

and carcinogens,

pharmacological

content

mixed

reticulum

enzymes,

and benzo and also the

is arylhydrocarbon

(AHH) . by USPHS Core Pro&ram Grant CA-13038 and an allocation from General supported Grant RR-05648-08 and American Cancer Society IN-54-M5.

[a]

Vol. 61, No. 2, 1974

BIOCHEMICAL

AHH catalyzes into various lyrene

the conversion

metabolites

being

one them,

of the following

enzymatically

the initiation

Some polycyclic

hydrase

produced

potent hepatotoxic

of MM IQ in the metabolism

all aflatoxins

(ll),

DNA,

RNA and proteins

Examinations

furan

(13,14),

metabolites

interest

to see whether

involved

in the formation In this paper

AND METHODS:

Chemicals:

Chromatographically

was verified

dissolved

evidence

species

(10) D

which

aflatoxins

alkylate

(14). Since

bond in the

MMFO

compounds

or different

enzyme

from

and BP.

AFBl

to show the enzyme

(16,17).

have suggested

of C2-C3 double

bonds in various

metabolites

are

(15) and are mutagenic

compounds

system

flavus,

that some aflatoxins

metabolites

amon g various

pure A FB1 was obtained

by TLC and spectroscopically

in purifed

in

enzymes

(5), it was of

systems

multiplicity

are

of MMFO

in

of A FB1 and BP.

MATERIALS

purity

DNA,

the most potent of

it is only recently

by the oxidation

of double

of animal

Bl (A FBl),

in culture

and related

of reactive

with

may be critical

Aspergillus

into reactive

bacteria

the same enzyme

we present

to the activation

However,

are produced

the oxidation

(b) are acted

macromolecules,

which

in a number

relationships

ring of some aflatoxins

are known to catalyze

relation

kill

one

(c) are conjugated

reaction

of aflatoxin

by MMFO

of structure-activity

that the reactive first

(12).

to be converted

to p&MIS,

of the mold,

agents

Participation

have been reported

benzo[a]-

then undergo

and (d) alkylate

by some strains

lmown

hydrocarbons

(9).

and hepatocarcinogenic

is well

which

non-enzymatically

It is the latter

process

aromatic

hydrocarbons,

to form dihydrodiols,

(7,8).

of the neoplastic

Aflatoxins,

aromatic

or non-enzymatically,

in the cell

of polycyclic

by AHH into epoxides

(a) rearrange

epoxide

RNA and proteins,

of a number

are converted

fates (4-6):

upon by microsomal glutathione

(2,3).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dimethyl

sulfoxide

(DMSO)

from

as described and stored

Calbiochem previously

at -20” under

o Its (12).

It was

conditions

n

detailed TLC,

elsewhere dissolved

Benzo[a]-pyrene Milwaukee,

(12).

‘H-AFBI

was obtained

in DMSO and used within (3-4-benzopyrene)

Wisconsin,

purified

a week.

was obtained by recrystallization

736

from

New England

Various

details

from Aldrich from

Nuclear,

have been described

Chemical

benzene

purified

Co “,

or ethanol

and

by (12).

Vol. 61, No. 2, 1974

then by TLC Germany) 3

gel plates

above,

and diluted

Ci/pmole).

Preparation

of other

phenobarbital-and

strain.

cholanthrene

of either

isolation

over a period

of microsomes

by the method

according

and the inductive the microsomes

to Lowry,

aminopyrine

(21) and cytochrome for microsomes

reductase

Binding

activities

and cytochrome

incubation

mixture

untreated,

phenobarbital-or DNA),was

(NADP,

Aldrich

Chemical

from

were

grams)

of the i. p. ) and 3-methy

given a total days, 4,

of seven

giving

Rats were

a single killed

(12).

Protein

in the microsomes

integrity

of microsomes

experiments

by assaying

(19), AHH (20), zoxazolamine

and the contents

of P450,

by

in each pool) and used for the

in preliminary

hydroxylase

of cytochromes

ratsj

P450

and b5(22).

demethylase

P450 content

Company,

untreated,

In general,

and cytochrome

by about two-fold.

C

The effect of

enzymes

was evidenced

by the

by the increase

in the activity

of AHH

and by a lack of an effect on the activities

of aminopyrine

C reductase.

Metabolites

(4.5 ml),

activity

isolated

The functional

of microsomal

Pqs8 instead

hydroxylase

of the Reactive

calf thymus

earlier

aminopyrine

on the activities

of cytochrome

demethylase

reported

and the cytochrome

3-methylcholanthrene

and zoxazolamine

(2 livers

from methylcholanthrene-treated

activities

presence

pooled

as

(12).

on days 2 through

was verified

was found to increase

by TLC,

specific

of four consecutive

demethylase

C reductase

Generally

(40 mg/kg/injection,

animals

--et al . (18).

effect of chemicals for

phenobarbital

daily

removed,

system,

from

were

sodium

i.pO) treated

compound

Darmsmdt,

male rats (190-260

Roth phenobarbital

on day 5, livers

was assayed

system

microsomes

on day 1 and two injections

decapitation

(~4~~

(98%) was obtained

: Hepatic

E. Merck,

to a required

have been described

(15 mg/kg/injection

injections injection

oxide

RESEARCH COMMUNICATIONS

it was purified

3-methylcholanthrene-treated

Sprague-Dawley

fran

(1:15 v/v) solvent

with unlabeled-BP

chemicals

of Microsomes

obtained

from Amersham/Searle;

Cyclohexene

and the sources

AND BIOPHYSICAL

(0.25 mm,

with benzene:hexane

BP was obtained

described (6.2~

on silica

developed

H-labeled

BIOCHEMICAL

of AFB,

containing

and BP to DNA:

3H-labeled

A FB1or

3- methylcholanthrene-treated incubated

in the presence

0.81 mM; DL-isocitrate,

17 mM;

737

In a standard 3 H-BP, rats,

and the absence isocitrate

assay,

miCrOSOmes

from

and DNA (native of an NADPH-generating

dehydrogenase,

150 pg

Vol. 61, No. 2, 1974

protein),

BIOCHEMICAL

in potassium

phosphate

and ethylenediamine mixture

tetraacetic

devoid of DNA,

3’7” to insure

buffer

the

thermoequilibration;

(0.07 M, pH 7.4) containing

acid disodium

microsomes

presence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(EDTA)

and AFBl

of an adquate

this was followed

amount

of NADPH

by the addition in that order.

additional

sulfate

of DNA,

essentially

The reisolated

according

Counting

extinction

coefficient

reisolated

DNA (23).

of DNA,

and used for counting

and AFB1

and Heidelberger

(8).

the incorporated

radio-

(X3).

Optical

density

Molar

the concentration

of the DNA was also estimated

97% pure.

for an

by the extraction

previously

of 6600 at 260 nm was used for estimating

and it was found to be over

and to achieve

was continued

by Kuroki

was done as described

The concentration

needed)

microsomes

Incubation

described

in buffer

of the radioactivity

(where

for 10 min at

(0.020/a) was added followed

to the method

DNA was dissolved

activity.

(24,25)

lauryl

(3.3 mM)

salt (0.2 mM) . Incubation

or BP was preincubated

(in 20 1.11DMSO) or BP (50 ~1 acetone) 1 hr and then sodium

MgCl2

260/280

of

calorimetrically ratio

for the DNA

was about 1.9. RESULTS

AND DISCUSSION:

As shown in Table

I, it was found that MMFO-mediated

and 3H-BP

bind to DNA.

In the absence

observed.

These

are in agreement

and other

laboratories

103-fold

more

binding

results

AFB1

of the AFB1

complexes solvent

were

extraction

metabolite

more

active

some from --in vitro

microsomes

than microsomes

previously

reported

to separation

also differed,

to be covalent

on repeated

rats.

(G-25)

AFBl

rats.

The effects of various

metabolite(s)

738

from

gel filtration. microsomes

rats were more

their

from was used about g-fold

active

pretreatments

BP parallels

The

repeated

when 3 H-BP

rats and about 5-fold

BP.

as the resulting

as microsomes

By contrast,

this

from

and DNA,

as effective

was

at least

precipitation,

3-methylcholanthrene-treated

from untreated

of the reactive

between

at least twice

-treated from

from

bound to DNA than the metabolite(s) to DNA appears

rats were

phenobarbital-treated

formation

no such binding

of the binding

of the complex

and 3-methycholanthrene

as the substrate,

results

of ‘H-AFBl

or NADPH

ether and CHC13 and on Sephadex

the formation

phenobarbital-treated

untreated

became

and BP metabolites

with diethyl

with

The magnitude

found to be resistant

In catalyzing from

(7,X3,15).

of microsomes

metabolites

than microof rats on the

reported

effects on

to DNA of the reactive different sources.

metabolites

from

aflatoxin

I

Bl and benzo[a]pyrene

in the presence

of

1.418

‘19.44

(3)b,f

* 2.16 (3)b

(2)b

96. llh

MC, mcs used

8.10 * 1.84 (3)b

43.26

4.79

0.00

1:120,000

1:20,000

1:210,000

-v--m

-----

Benz0 [ alpyrene metabolite Stochiometry of binding DNA-P (Metabolite:DNA-P)

0.00 (2)b

p-mole

p-moles

a) hepatic microsomes; b) number of experiments given in the parentheses; (c,d, and e) hepatic microsomes from untreated, 3-methylcholanthrene-treated and phenobarbital-treated rats; f) significantly different from the corresponding control microsomes at p < 0.05; g) similar results were obtained with microsomes from phenobarbital-treated rats; h) results are representative of several experiments in which different concentrations of microsomal protein and BP were used.

+ Cyclohex.

mcs used

1:140

7.13 * 2.2 (4)bSf

PB, MCSe

Control

1:558

1:460

1.79 * 0.38 (6)b

(4)b

---em

-----

k tochiometry of binding (Metabolite:DNA-P)

B

MC, MCSd

oxide (0.66 mM)

2.17 * 0.14

MCSC

Control,

(2)b

0.17

- NADPH

DNA-P

Aflatoxin metabolite

0.04 (2)b

p-mole

n-moles

-MCSa

Conditions of Incubation Prior to Reisolation of DNA

Native calf thymus DNA (4 mg/incubation flask) was incubated with hepatic microsomes (2.5 mg protein) from different sources and 3H-aflatoxin BI (0.25 mM; specific activity, 0.41 pCi/pmole) or 3H-benzo[a]pyrene (0.12 mM; specific activity, 6. I8 pCi/pmole) in the presence of an NADPH-generating system as described in the text. An aliquot of reisolated DNA (> O,D. of 5 at 260 nm) was counted as described in the text.

Comparison of the binding hepatic microsomes from

TABLE

Vol. 61, No. 2, 1974

the activity

BIOCHEMICAL

of AHH (26), in which

3-hydroxy

BP [ZO] ),believed

This observation the formation

to be arising

to DNA,

inhibitor

that the reactive

BP metabolites

taken together

to DNA and RNA is derived

the hydrolysis of AFB~ with

of RNA-AFB1

is most probably

chromatographic

From the knowledge

A FBl

can be explained

the presence

metabolite 2,3-epoxide.

available

bonds in various

only in terms hydrase

the enzyme

of “BP epoxide” and Wright mediated

epoxide

to DNA,

is converted

mutagenesis

il failed

of the epoxide

of the reactive

AFBI

from

epoxide

hydrase

103-lo4

and (b) epoxide

corresponding

dihydrodiol

of microsomal

epoxide

a similar

inhibitor,

greater

binding

involved

may be ineffective is well

suggestion

metabolite

in the catalysis for “A FBl

740

(30).

derives

Finally,

by binding

the microsome then the the concentration in several support

to DNA than BP to the

‘I; the multiplicity a possibility

in

1). Garner

of A FBr

of “HP-epoxide(s)”

- epoxide,

oxide,

to enhance

to increase

of

RNA or DNA

and subsequent

intermediate

this

of AFBl

documented

cyclohexene

oxide failed

of

which

alkylates

assay could be explained

and unstable,

of A FBI.

of 2,3-dihydrodiol

or which

oxide,

of AFBf

oxidation

effect with A FBI (Table

cyclohexene

metabolite

intermediate,

accumulation

is the reactive

in the DNA-binding

hydrase

hydrases

increased

following

some metaholites

Although

that cyclohexene

If epoxide

may be very reactive

the data showing

metaholite;

reported

metabolite

caused

metabolite(s)

that the reactive

as the metabolic

the dihydrodiol.

studies

of C2-C3

of A FBI

formation

to 2, 3-dihydrodiol

to produce

of A FBI.

ineffectiveness

(a) AFBl

yields

oxidation

of MMFO-catalyzed

metabolic

of 2,3-epoxide

hydrase,

(17) have recently

(5,27),

Recent

of 2,3-dihydrodiol

the mechanisms

compounds

and the adduct on acid hydrolysis inhibiting

to the derivatives

suggests

that the reactive

2,3-dihydrodiol

(29) has reported

hydrase

(4-9)

epoxides.

MMFO-mediated

Garner

similar

reports

suggest

and have suggested

by increased

an epoxide

other

adduct

concerning

organic

of epoxide

from

--et al . (28) have isolated

properties

double

oxide,

several

aflatoxins

in

(5,27).

of BP, as measured

with

among various

Swenson,

bond (13,14).

be intermediates

which bind to DNA are most likely

of AFBI double

hydrocarbons

(predominantly

(5), are measured.

might

of cyclohexene

relationships

binds

epoxides

metabolite(s)

on structure-activity which

metabolites

that epoxides

in the presence

I); this observation

phenolic

intermediate

of aromatic

of the reactive

was increased

(Table

from

the contention

metabolites

The concentration binding

assay the terminal

tends to support of phenolic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

remains

ways:

-

Vol. 61, No. 2, 1974

that the reactive

metabolite

aromatic

hydrocarbons

oxidation

of C2-C3

support

~lOOWvfKAL

of AFBI,

(31),

double

AND BlOPHYSlCAL

like

reactive

metabolites

might be a free radical

bond in AFBi,

RESEARCH COMMUNICATIONS

although

formed

from

some polycyclic

by the microsome-mediated

at present

no data are available

to

this hypothesis. Whatever

the nature

of this reactive

the effect of MMFO-inducers systems

in MMFO

DNA-binding

(Table

complex

for a different

AFBI

enzyme

I) clearly

are involved

metabolites:

AFBI

metabolite,

suggest

that at least two different

in the conversion

serves

our data concerning

as a substrate

of polycyclic for one enzyme

enzyme

compounds system

to

and BP

system.

ACKNOWLEDGEMENTS

:

We wish to acknowledge

the skilled

also wish to thank MS, L. Caballes

technical

assistance

for her assistance

of Mr.

in radiochemical

L. Motycka

and

experiments.

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