Danazol inhibits human adrenal 21-and 11β-hydroxylation in vitro

251

2531 DANAZOL INHIBITS HUMAN 118-HYDROXYLATION Robert

ADRENAL Pl-AND IN VITRO --

L. Barbieri, Rapin Osathanondh, Jacob A. Canick, Robert J. Stillman+, and Kenneth J. Ryan

Laboratory of Human Reproduction and Reproductive Biology and Department of Obstetrics and Gynecology Boston Hospital for Women Harvard Medical School 02115 Boston, Massachusetts and +Division of Reproductive Endocrinology Department of Obstetrics and Gynecology The George Washington University School of Medicine Washington, DC 20037 Received 7-30-79 ABSTRACT The effects of danazol on steroidogenesis in vitro in the 16-20 week old human fetal adrenal were examinedy studying: danazol binding to adrenal microsomal and mitochondrial1) cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the The addition of danazol to mitochondrial 118-hydroxylase. preparations of adrenal microsomes or mitochondria elicited Danazol bound a type I cytochrome P-450 binding spectrum. to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (KS) of 1 nM and with a Danazol bound to mitochondrial lower affinity K'S of 10 PM. cytochrome P-450 with a KS of 5 uM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant KI = 0.8 ~.IM)and the These findings mitochondrial 118-hydroxylase (KI = 3 PM). demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal -~ in vitro. INTRODUCTION Danazol

is being

used

endometriosis

(l-3),

chronic

cious

(6,7) and congenital

puberty

Although

the mechanisms

peutic

effects

stated

that danazol

minimal

androgenic

studies

in men

clinically cystic

by which

are poorly

danazol

S

TDROIDII

exerts

edema

antigonadotropin

women

However,

(8).

its thera-

it has been

(.l-5,7,8).

of

(3-5), preco-

angioneurotic

is a selective

(9), premenopausal

VoZwne 35, Number 3

mastitis

understood,

effects

in the management

repeatedly with

clinical

(10) and children

(6)

March, 1980

252

have

shown

gonadal

that danazol

steroids

LH or FSH.

without

These

can significantly significantly

observations

suggest

one of its primary

pharmacologic

inhibiting

steroidogenesis.

ported

gonadal

that danazol

of rat adrenal observations on human

competitively

and testicular

prompted

adrenal

suppress

serum

suppressing

serum

that danazol

effects

by directly

We have inhibits

recently

multiple

steroidogenesis

us to examine

exerts

enzymes

(11,lZ).

the effects

re-

These

of danazol

steroidogenesis. MATERIALS

AND METHODS

Adrenals were obtained from 16-20 week old human fetuses legally aborted with prostaglandin F2u. Unlabelled 17-hydroxyprogesterone, deoxycorticosterone and corticosterone urchased from Steraloids and recrystallized were before use. [1,2- t; HI-17-hydroxyprogesterone (SA = 40.4 Ci/mmol) and [1,2-3H]-deoxycorticosterone (SA = 46.8 Ci/mmol) were purchased from New England Nuclear and purified by thin layer chromatography using a solvent system of ehloroform:methanol Danazol was supplied by the Sterling-Winthrop c97:3 v/v). Research Institute. Preparation of mitochondrial an.d microsomal suspensions: Adrenals were homogenized in three parts 0.25 M sucrose The 750g supercontaining 0.01 M Tris-HGL, pH 7.2 at 25O. This pellet natant was centrifuged at 12,000g for 10 min. was resuspended in 0.15 M KCL containing 0.01 M 'Iris, and centrifuged at 12,OOOg for 10 min. The final pellet was homogenized in a small volume of KCL-Tris, sonicated and used The 12,000g supernatant as the mitochondrial suspension. This pellet was was centrifuged at 100,OOOg for 60 min. resuspended in 0.15 M KCL containing 0.01 M Tris and recentrifuged at 100,OOOg for 60 min. The final pellet was Protein concenhomogenized in a small volume of KCL-Tris. tration was determined by the method of Lowry -et al. (13) with bovine serum albumin as the standard, All spectral determinations Spectral determinations: were made usins a DW-2 UV-visible spectronhometer (American Instrument Co.; Silver Spring, MD).- Cytochrome P-450 concentrations were measured by the method of Estabrook et al. (14). To determine the spectral changes caused by the addition of steroids to microsomal or mitochondrial preparation, 6 ml of a 1 mg protein/ml microsomal or mitothondrial suspension was divided between sample and reference cuvettes, and a base-

line drawn. The steroid sample, dissolved in propylene glyThe spectrum co1 alone was added to the reference cuvette. The propylene glycol concenfrom 370 to 500 urn was scanned. Hanes tration in the cuvettes never exceeded 1.0% (v/v). plots were prepared as previously described (15). The assay Inhibition of enzymes of steroidogenesis: for the 21-hydroxylase and the llg-hydroxylase utilized a reaction mixture containing 0.15 M KCL, 50 mM Tris, pH 7.4 at 37O, 500 F\M NADPH, 50 mM glucose-6-phosphate, 5 units of glucose-6-phosphate dehydrogenase, 5 mM MgC12, 0.5 PCi of radiolabelled substrate and appropriate amounts of unlabelled substrate and danazol in a final volume of 0.3 ml. The reaction was started by the addition of 0.1 ml of microsomal or mitochondrial protein. Lineweaver-Burk plots were prepared as previously discribed (16). For the 21-hydroxylase assay the substrate was 17hydroxyprogesterone and the product was ll-deoxycortisol. Progesterone was not chosen as the substrate for the 21hydroxylase assay because mid-trimester human fetal adrenal microsomes can efficiently convert progesterone to deoxycorticosterone, 17-hydroxyprogesterone and 16cchydroxyprogesterone (17,18). In contrast, mid-trimester human fetal adrenal microsomes can only efficiently convert 17-hydroxyprogesterone to 11-deoxycortisol (19). For the 118-hydroxylase assay the substrate was deoxycorticosterone and the product was corticosterone. Samples in the il-hydroxylase assay were incubated for 3 and 6 minutes. For the 118-hydroxylase assay, samples were incubated for 6 and 12 minutes. After incubation, the reaction was stopped by the addition of 0.4 ml methanol. An aliquot of the reaction mixture was then applied to silica gel thin layer plates and the product and the substrate were separated using a solvent system of chloroform:methanol 97:3 v/v (17hydroxyprogesterone rf = 0.66; and corticosterone rf = 0.25). The product was scraped from the plate, eluted from the gel and the radioactivity measured by liquid scintillation spectrometry. The purity of the products of the two reactions was established by recrystallization of representative samples to constant specific radioactivity. In no case was the purity of the product isolated by initial chromatopraphy less than 90%. RESULTS Danazol P-450

interaction

concentrations

chondria

prepared

adrenals

ranged

with

obtained

cytochrome

from microsomes

from four pairs

from

0.50

P-450:

of pooled

to 0.60 nmoles/mg

Cytochrome

and mitohuman

fetal

protein

for the

microsomes

and 0.45

to 0.50 nmoles/mg

protein

for the mito-

chondria. Addition in a type

of danazol

I difference

to microsomal

spectrum

with

380 and 390 nm and a Soret minimum the titration

of a microsomal

demonstrated

a high

affinity

constant

(K'S) of 10 PM

difference minimum

spectrum

at 420 nm.

suspension

with

To a microsomal in the sample achieve This

(Pig. 1).

to microsomal with

suspension cuvette,

of 17-hydrox-

elicited

already

a type

I

at 388 and Soret

of the titration

J.7-hydroxyprogesterone

dissociation

dissociation

Addition

a Soret maximum

of

danazol

spectral

affinity

between

Analysis

with

preparations

Analysis

of a microsomal

yielded

containing

17-hydroxyprogesterone

a concentration

resulted

at 418 nm.

apparent

(KS) of 1 uM and a lower

resulted

a Soret maximum

suspension

Constant

yprogesterone

preparations

a KS of 0.8 PM. 100 pH danazol was

added

to

of 35 PM 17-hydroxyprogesterone,

in a 50% increase

in the type

I difference

spectrum. Addition gave

a type

between

I difference

to mitochondrial spectrum

with

suspensions

a Soret maximum

380 and 390 nm and a Soret minimum

Analysis with

of danazol

of the titration

danazol

corticosterone producing

demonstrated bound

of a mitochondrial

suspension

a KS of 5 I.IM(Pig. 21.

to mitocbondrial

a type, I difference

respectively.

at 419 nm.

spectrum

cytochrome

DeoxyP-450

at 390 nm and 420 nm

The KS of deoxycorticosterone

binding

to

mitochondrial drial

suspension

sample added

cytochrome

cuvette, to aehleve

already

I difference

was

containing

corticosterone

This

0.75 p&f.

To a mitochon-

3.5 pM danazol

in the

or deoxycorticosterone

a concentration

or deoxycorticosterone. type

P-450

were

of 30 ~119cortieosrerone

resulted

in no change

In the

spectrum.

Figure 1. Danazol binding to human adrenal microsomal. cytochrome P-450. Concentrations of danazol: 11 0.33 pi%, 2) 1.00 pM, 4) 3.00 pM, 5) 4.33 PM, 6) 7.00 PM, 7) 20.33 p%, 8) 17.0 j.tM,9) 23.67 YEI, 10) 37 yM, and 11) 63.67 PM. Inset: Hanes plot of danazol bjlnding to micxosomal cytochxome P-45#. % = 3. y&2, K'S fz 10 pM.

S

256

I ,

TDEIBOXDB

6 I

380

I

400

420

I

440

Wavelength

1

I

460

480

500

(nm)

Figure 2. Danazol binding to human adrenal mitochsndrial Concentrations of danazol: cytochrome P-450. 1) 1.6 NJ, 2) 4.27 I.IM,3) 10.93 PM, 4) 17.6 pM, 5) 3Q.93 JAM. Inset: Hanes plot of danazol binding to mitochondrial cytochrome P-450 Kg = 5 I.IM. Enzyme

the 21-hydroxylase The apparent danazol

!JM.

was

minute

constant

(Fig. 3,4). (KT) for

was 0.8 PM. (KM) of 17-

in the Zl-hydroxylase inhibition

inhihited

assay was

assay was

incubations

2.8 PM.

4

of the 118-hydrox-

The KM for deoxycorticosterone

3 PM.

ll$-hydroxylase

constant

of the 21-hydroxylase

The KT for danazol

ylase

inhibition

Michaelis-Menton

hydroxyprogesterone

competitively

and the llfi-hydroxylase

enzymatic

inhibition

The apparent

Danazol

inhibition:

The three

for the 21-hydroxylase

in the and six

assay

gave

S similar

reaction

incubations reaction product

257

The six and twelve

velocities.

for the lib-hydroxylase

velocities. formation

The values concentration,

These

over

assay

results

similar

to values fetal

P-450

which

reported

obtained

have been Cytochrome

(20).

binding

in these reported P-450

C-23). When

added

enzymatic

inhibition

enzymatic Addition suspension

suggests

of deoxycorticosterone already

saturated

spectrum.

competitively

llg-hydroxylase

with

(KT = 3 pM)

dissociation

adrenal

(21).

of adrenal

1 difference

that danazol. binds

site of the human

adrenal

(22) and the lib-

constant

spectral

to values

as the terminal

that danazol

mitochondrial

s.lmilar to the apparent (4s = 5 pM)

a type

P-450

to cortico-

to a preparation

and the observation

apparent

elicits

adult

implicated

This

the adrenal

mitochondrial.

for the human

for

the Kg for

are similar

21-hydroxylase

are

reported

conversion

experiments

danazof

inhibits

been

to adrenal

mitochondria finding

P-450

in this paper

In addition,

has been

of the adrenal

hydroxylaae

cytochrome

previously

and the KM for deoxycorticosterone

oxidase

of

studied.

microsomal

have

adrenal

deoxycorticosterone

which

linearity

and the KS for 17-hydroxyprogesterone

to microsomal

sterone

minute

had similar

indicate

the time period

for adrenal

binding

the human

TEIROXDS

constant

to the active

lie-hydroxylase.

to a mitochondrial with

danazol

did not

change

an

the size

of the type I difference

it can be inferred chondrial

spectrum.

that danazol

cytochrome

P-450

can bind

sites

Therefore,

to all the mito-

available

to deoxycor-

ticosterone. Human eytochrome

fetal

adrenal

P-450

involved

roid hydroxylations: ylase,

micrasomes

in at least

adrenal

microsomes

difference

complicates

spectra

elicited

microsomaf F3

P-450

= 1 PM, RrS = 10 PM)

the active

The results

@omen been

reported

never

to have Adrenal

(24).

the adrenal

than the serum suggest daily,

that

adrenal

here

adrenal

serum

concentration

in women

the adrenal

to the enzymatic

orally

However,

taking

the

to adrenal

I spectrum bfnds

that

to

danazol

is

in vitro. --

per day have

concentrations

as high

of danasol. have

However,

in humans.

of

21-hydroxylase.

steroidogenesfs

concentrations

concentration

a type

indicate

danazol

fetal

inhibits

that danazol

400 mg of danazof

been measured

animals

eliciting

suggests

presented

of human

receiving

as 2 PM

the interpretation

competitively

site of the human

sn inh%bitor

in human

fK, = 0.8 $IM) and that it binds

cytochrome

(17,X8).

in these microsomes,

the ob.servation that danazol 2X-hydroxylase

P-450

ste-

the Pl-hydrox-

and the 17,20-lyase

of the cytochrome

of

four different

the Z7a-hydroxylase,

the Iba-hydroxylase,

The heterogeneity

species

contain

in laboratory

of danasol. is higher

(25).

These

observations

400 mg or more

concentration

of the drug

KT 's for danazol

inhibition

of danasol is similar of the adre-

nal 21-hydroxylase data

suggest

that

and Ll@-hydroxylase. danazol

sis few investigators steroidogenesis et al.

increase

metyrapone synthetic

have

that

in women

in urinary

stimulation

1

adrenal

receiving

examined

adrenal

danazol.

receiving

response

was within

these

steroidogene-

Went2

danazol

17-hydroxycorticoids

and the incremental ACTH

alter

carefully

in patients

(26) reported

percent

may

Although

after

of cortisol

normal

the

to

limits.

2bHYDROXYLASE

i I/(i7+iydroxyprogesteronef fpM_‘)

plots of danazol inhibition of Figure 3. Lineweaver-Burk Zl-hydroxylase substrate: 17-hydroxyprogesterone, product: ll-deoxycortisol) protein concentration 12.5 ug/ml. Danazol concentrations: O-O,+1 pM,A-2 nM,A-5 JIM. Enzymatic inhibition constant = 0.8 nM. These

studies

present

suggest

in women

that no g1ucocoricoS.d

receiving

danazol.

deficiency

However,

is

s.tudies b*y

Stillman itative et

al.

et

(27)

of

after

the

studied once

on as

Danazol

ficant

change

sulted

in

effect

the

of

danazol

before Five

6th

of

day

and

and

of

danazol

increase

control

orally

menagain per

in no

progesterone,

androstenedione

con-

cosyntro-

and

concentration, in

serum

consecutive

mg

qual-

menstruating

resulted

cortisol

the

after

two

600

the

Stillman

on

normally

administration

a significant

alters

synthesis.

a pre-treatment

in baseline

droepiandrosterone

danazol

steroid

steroids

administration

6 days.

that

stimulation.

cycles; the

adrenal

of various

were

strual

suggest

measured

(AcTH~_~~~)

women

for

(27)

pattern

centration pin

al.

day

signibut

re-

dehy-

concentrations.

li/3-HYDROXYLASE

I

2

I/(Desoxycorticosterone)

(PM)-’

Figure 4. Lineweaver-Burk plot of danazol inhibition of llf3-hydroxylase (substrate: deoxycorticosterone, product: corticosterone) protein concentration 625 pg/ml. Danazol concentrations: @-0,&-l uM,m-5 uM. Enzymatic inhibition constant = 3 @I. The

administration

of

cosyntropin

during

the

danazol

treat-

ment

period

resulted

in greater

and deoxycortisol

and smaller

and cortisol

that seen

findings

than

suggest

ylase

sufficient

to assess

hydroxylase.

centration,

the effects danazol

does not

but may

adrenal

steroids.

danazol

on human

further

study.

produces

obtained

In summary

800 mg per day)

in progesterone

alter

alter

adrenal

These

inhibition

and the 118-hydrox-

in the study were of danazol

baseline

serum

(600cortisol

concentration

elucidation

steroidogenesis

not

on the 21-

administration

the serum

Complete

period.

a partial

oxidoreductase

The data

in vivo. --

increments

in pregnenolone

in the control

that danazol

of the 3S-hydroxysteroid

increments

of other

of the effects in vivo --

con-

of

awaits

ACKNOWLEDGEMENTS Supported by United States HD07923-06 and Rockefeller SYSTEMIC

EQUIVALENTS

androstenedione corticosterone cortisol danazol dehydroepiandrosterone deoxycorticosterone 11-deoxycortisol 16a-hydroxyprogesterone 17-hydroxyprogesterone pregnenolone progesterone

Public Health Service Grant Foundation Grant RF77045.

OF TRIVIAL

NAMES

AND ABBREVIATIONS

4-androstene-X,17-dione llS,Zl-dihydroxypregn-4ene-3,20-dione llfi,l7,21-trihydroxypregn4-ene-3,20-dione 17B-pregn-4-en-20-yno(2.3-d)isoxazol-17-01 ?B-hvdroxv-5-androsten-1'78-one Zl-hydroxypregn-4-ene-3, 20-dione 17,21-dihydroxypregn-4-ene3,20-dione 16a-hydroxypregn-4-ene-3, 20-dione 17-hydroxypregn-4-ene-3, 20-dione 3$-h@ro~-+preffnen-2O-one pregn-4-ene-3,20-dione

262

KS KI

S Apparent Apparent

T13EOIJ3_

spectral dissociation constant enzymatic inhibition constant

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