Inactivation of hepatic cytochrome P-450 by allenic substrates

Vol. May

94, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 443-449

30, 1980

INACTIVATION

OF HEPATIC CYTOCHROME P-450

Paul '

R. Ortiz

March

SUBSTRATES

and Kent L. Kunze

Department

of Pharmaceutical School of Pharmacy and Liver Center, of California, San Francisco,

University Received

de Montellano

BY ALLENIC

Chemistry California

94143.

21,19%0

SUMMARY Hepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats is destroyed by 17-u-propadienyl-19-nortestosterone, l-propadienylcycloSubstantial activity is also exhibited by hexanol, and l,l-dimethylallene. 1-propadienylcyclohexanol against the enzymes in microsomes from 3-methylcholanthrene pretreated rats. The destructive process requires NADPH, is not inhibited by glutathione, and is paralleled by equimolar loss of hepatic heme but not by --in vivo accumulation of identifiable "green" pigments. Attempted metabolism of allenes can thus result in destruction of cytochrome P-450. Cytochrome bital,

are

certain

P-450

destroyed

enzymes, during

substrates.

This

propyl-4-pentenamide unsaturated fluroxene

(8),

synergists

the potential

of unsaturated heteroatomic

bonds

a property

reported

vinyl

with with

chloride

and acetylene,

conveyed

Z-isoother

insecticide

of gaseous

P-450

in

(5-7),

of propargylic

activity

cytochrome

bonds

shown to occur

(3,4),

olefin

by phenobar-

ethylene clearly

(13) demon-

is an intrinsic

property

by molecular

placement

and acetylenes

requires

or

substitution.

and molecular

12-16).

to destroy than

first

been

destructive

induced carbon-carbon

and a number

quintessential

rather

The destruction

and is

the

of unsaturated

secobarbital

the

forms

interaction,

(9,10),

In fact,

(12,14),

those

has subsequently

including

(11,lZ).

that

destructive

norethisterone

and acetylene strates

metabolism

(1,2),

structures

particularly

of cytochrome

oxygen,

accompanied

P.R. Ortiz A.Y.H. Lu,

inhibited

decrease

enzyme after

agents

de Montellano, in preparation.

is B.A.

by olefins

by carbon

by a parallel

Loss of the

of the destructive

is

P-450

in microsomal

--in vivo

associated Mica,

monoxide

administration with

K.L.

accumulation

Kunze,

J.M.

but

not

by glutathione,

heme content of some, but of abnormal Matthews,

NADPH

(2,3,7,9, not

all,

hepatic

G. Miwa,

and

0006-291X/80/100443-07$01.00/0 443

Copyrigllr E:, 1980 by Academic Press, Inc. AN rights of reproducfion in any form reserved.

Vol.

94, No.

("green")

2. 1980

BIOCHEMICAL

pigments.

destruction

nistic

(12).

of the cytochrome

gical

exploitation is

oxygenases

and acetylenic P-450

however,

three groups, enzymes

for

the

attempting

the pigments

are

covalent

which

functions

references of allenes

functional reveals

can bring

to metabolize

the porphyrin

about

framework

func-

pharmacolo-

19-22).

Essentially

with

groups. that,

1:l

carbon

have made their

example

of these

MATERIALS

mecha-

of unsaturated

interaction

substrates

allenic

important

(10,12,17,18).

(see

the metabolism

provides

heme group

a class

after

not by disubstituted,

IX,

and acetylenes,

about

allenic

but

that

COMMUNICATIONS

example,

protoporphyrin

of allenes,

from olefins

or about

using

with

of interest

known,

for

when observed,

prosthetic

properties

distinct

nothing

a study

agents

RESEARCH

by monosubstituted,

we have established

P-450

The chemical

BIOPHYSICAL

such pigments,

formation,

since

of destructive

tionalities

P-450

Pigment

information

adducts

chrome

We have isolated

of cytochrome

acetylenes

AND

hepatic

mono-

We now report

as with destruction

olefinic of cyto-

them.

AND METHODS

Samples of 17-a-propadienyl-19-nortestosterone (A) were provided by Ca.) and by Sandoz (Hanover, N.J.); l-propadienylSyntex Research (Palo Alto, cyclohexanol (2J was obtained from Dr. Alf Claesson (Upsalla, Sweden) (21), and l,l-dimethylallene (2 was purchased from Pfaltz and Bauer Co. These allenes were shown to be at least 99% pure by thin layer and gas chromatographic analyses. Unstarved Sprague-Dawley male rats weighing 200-250 g were Incubations. injected intraperitoneally once a day for four days with either sodium phenobarbital (80 mgfkg, aqueous solution) or 3-methylcholanthrene (20 mg/kg as a 5 mglml solution in corn oil). The rats were sacrificed 24 hr after the last dose of inducing agent and hepatic microsomal preparations were obtained as Incubations contained the following in addition previously reported (3,18). to allenic substrates (1 mM): microsomal protein (1 mg/ml), NADPH (1 mM), KC1 (150 mM), and EDTA (1.5 mM), all in 0.1 M Na/K phosphate buffer (pH 7.4). The incubation procedure and the assay for cytochrome P-450 content have been reported (12,lS). Control incubations in the absence of NADPH, to measure NADPHindependent cytochrome P-450 loss, and in the absence of substrate, to measure substrate-independent (lipid-peroxidative) loss, were performed with each set of experiments. As long as unstarved rats were used, peroxidative and NADPHindependent enzyme losses after '&P min-1 of incubation did not exceed 2%. An extinction coefficient of 100 cm mM was used to calculate molar cytochrome Except where indicated otherwise, the values in the tables P-450 losses (27). Standard deviaare the average of at least three independent determinations. tions are given where appropriate. Difference Binding Spectra. Binding of allenes to oxidized cytochrome P-450 from phenobarbital-pretreated rats was examined spectroscopy (13,23) using an Aminco-Chance DW-2 instrument.

444

microsomal by difference

Vol.

94, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Groups of three phenobarbital-induced rats were inPigment Isolation. jected intraperitoneally (ZOO mg/kg) with either steroidal allene & (dose dissolved in 1 ml propylene glycol), 1-propadienylcyclohexanol (in 0.1 ml ethanol), The rats were sacrificed or l,l-dimethylallene (in 0.1 ml ethanol). four hours later and their livers were extracted as previously described (10, 12,13). The characteristic green (red-fluorescing) bands we have observed with other agents (10,12,13) were not detectable on thin layer chromatographic analysis of the extracts except for a faint band in the experiment with l,ldimethylallene. Microsomal Heme Determinations. Small (1 ml) aliquots drawn from incubation mixtures with and without NADPH were diluted with 2 ml of a 20% pyridine in 0.1 N NaOH solution. Heme content in each aliquot was calculated from the 557-600 nm absorbance different -f bef,yeen dithionite reduced and unreduced samr&l for the extinction coefficient (24,27). ples, using a value of 34.4 cm Values given are the average of three determinations. RESULTS AND DISCUSSION Incubation barbital

pretreated

cytochrome sence

P-450

substrates

rats

resulted

(Table

1).

of NADPH, suggesting

quired

for

its

confirmed P-450? than

of allenic

that

so long

as microsomes

as shown

in Table

1, addition

vention

the

analogous

steroid,

be demonstrated

Type I binding

did which

metabolite

was not

argues

CH=C=C~

Ho

spectroscopy with

Fig.

CH=C=CH,

\/

C

ki,

1.

2

Structure

of allene

445

no more

incubation

rats.

Furthermore,

against

the

3

substrates.

(23),

destruc-

the interprocess. (Fig.

the allenic

i

b ’ i

that

to the

not moderate

OH

0&

fact

cytochrome

in the destructive

obtained

pre-

enzyme has been

and l,l-dimethylallene

by difference

spectrum

added

from unstarved

a result

electrophilic

by the

was not

obtained

on the

reconstituted

was excluded

of 1-propadienylcyclohexanol

enzyme could

of the

of 5 mM glutathione

of the allenic

of a diffusible, Binding

were

pheno-

of the enzyme was re-

purified,

a substrate

from

dependent

participation

enzyme loss

mixture,

activity

was completely

using

if

microsomes

of spectroscopically-measured

participation

to be published

2% of the enzyme was lost

tive

loss

catalytic

Catalytic

Lipid-peroxidative

1) with

in rapid

Enzyme loss

destruction.

in studies

k&(Fig.

2) to

although steroid.

an

Vol.

94, No.

TABLE

2, 1980

1.

BIOCHEMICAL

Destruction phenobarbital

by allenes (Pb)

AND

BIOPHYSICAL

of cytochrome P-450 and 3-methylcholanthrene

RESEARCH

enzymes (MC) Percent at

Substrate (1 mw

COMMUNICATIONS

in microsomes induced rats. P-450 given

destruction time: 20 min

Inducer

NADPH (1 UN

Pb

Yes Yesa NO

43+5 -0

57+6 55+4 0-

Yes No

29+3 0-

35+3 0-

Pb

Yes NO

28+4 0-

52+2 0-

lb

MC

Yes NO

8 0

8 0

2

MC

Yes NO

25+3 0-

36+2 0-

3b

MC

Yes NO

1 0

3 0

1

aGlutathione (5 UN) was also b These experiments were only less than 1% in each instance.

10 min

added done

to in

this

from

incubation.

duplicate.

The

two

values

differed

by

350IO Wavelength Fig.

2.

(nm)

Difference spectrum caused by addition of2 phenobarbital-pretreated rats. The lower 2 and the upper curve with 3 (the baseline The two curves can be clearly distinguished).

446

and 2 to microsomes from curve was obtained with is displaced so that

Vol.

94, No.

2. 1980

BIOCHEMICAL

The absence

of a defined

the

of such a spectrum

absence

acetamide),

a compound

competition

experiments

trum

remains

three

activity treated

steroidal

allene

Table are

for

destruction only

allenes,

steroid

COMMUNICATIONS

is

reminiscent

this

of

(allylisopropylbe demonstrated

absence

by

of a binding

spec-

cytochrome

rats

(Table

may only

1 clearly

P-450

1).

particularly

enzymes

The marginal

reflect

show that

of the phenobarbital-inducible

1-propadienylcyclohexanol

destruction

3-methylcholanthrene

induced

decrease of minor

microsomes.

to destruction

in both

which

as fluroxene,

forms

also

types

3-methylcho-

about

by the

of cytochrome

In any case,

by allenes.

unambi-

from

brought

forms

of 1-propadienylcyclohexanol

the same class

exhibited

in microsomes

phenobarbital-inducible

susceptible

the activity in

the

can nevertheless

The reason

(25).

against

lanthrene

in

with

RESEARCH

2-isopropyl-4-pentenamide

binding

to the marked

enzyme by all

present

spectrum with

whose

BIOPHYSICAL

to be established.

In contrast

guous

binding

AND

P-450

the data

in

of cytochrome

P-450

On the other

hand,

of microsomes

is characterized

by this

places

it

ambidextrous

activity. A close vitro .-in ___ (Table of

quantitative

loss 2).

ever,

The 1:l

studies, despite

TABLE

Substrate

molar

protocol strongly

using

of these avoids

independently P-450

two processes,

that

they

are

currently

pigments

between lass of loss of microsomal

were

particularly

Heme loss (nmoles)

employed

linked. in

this

detected

cytochrome

Loss ratio P-450/heme --__

1

683+91 -

650+43 -

1.0/0.95

4a7+41 693+50 -

473+45 606+130 -

l.O/l.O

447

1.0/0.9

How-

laboratory

in the livers

2 z3

in view

mechanistically

not

heme

of analogous

phenobarbital-inducible hem.

P-450 loss (nmoles)

measured

and microsomal

some of the ambiguities

methodology

heme-derived

Relationship P-450 and

between

cytochrome

suggests

abnormal

2.

ratio which

a search

(10,12,13,18),

exists

of phenobarbital-inducible

the -___ in vitro

in vivo -__

relationship

Vol.

94, No.

of rats

2, 1980

BIOCHEMICAL

treated

--in vivo

of such a pigment little

of it

can not

was present

therefore

but

obtained most not

cause

in

formation

participation

distinction

between

process

tionality that

is

a trace too

artifactual

formation

nevertheless

and from the P-450

set

clear,

from

features

other

inactivity is

similar

during

catalytic

are consistent

studies

with

product

obtained

related

do

of such pigments

by our

the

thus

also

oxidative

the lack than

to those

during

species

reactive

heme,

process which

the data

as reaction

of closely

destroyed

pigments

unstable

possible

the heme and apoprotein

of microsonml

In the absence

or a transient

as the

loss

acetylenes,

formed

catalytic

not

The destructive

the highly (26),

requires

enzyme forms,is

although

The precedent

sequence,

allenes

hepatic

(12).

species

epoxide,

on structural

cytochrome

Its

three

by internal

be defined,

of an allene

epoxidation

It

Although

l,l-dimethylallene,

by an equimolar

pigments

functionality.

enzymatic

specific

of abnormal

of a reactive

oxidation

for

mediated

can not

chemical

by the

and acetylenes.

of hepatic

an exo-methylene

tive

formation

that

suggests

be made.

with

P-450

selective

olefins

event

of the allene

treatment

is matched

resembles

the destructive with

is

terminal

closely

COMMUNICATIONS

substrates.

and characterize.

of cytochrome

result

with

RESEARCH

be excluded.

by glutathione,

does not

after

to isolate

of the enzyme,

attenuated

BIOPHYSICAL

any of the allenic

was observed

The destruction turnover

with

AND

metabolism acetylenes

formed

on

during

the

agent,

although

a

targets

can not

now

of the

destruc-

of dependence presence saturated

of an allene systems

interaction

with

func(9,13),

allenic

functionalities. We thank Dave McLendon for experimental assistance, and Acknowledgements gratefully acknowledge the generous gift of substrates by Dr. Alf Claesson, Syntex Research, and Sandoz Corporation, P.R.O.M. is an Alfred P. Sloan Research Fellow. This research was supported by N.I.H. grants GM-25515 and p-50 m-18520. REFERENCES 1.

Wada, O., k7,

2. 3.

Yano,

Y.,

Urata,

G.,

and Nakao,

K. (1968)

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Pharmacol.

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94, No.

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BIOCHEMICAL

AND

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