BIOCHEMICAL

Vol. 63, No. 2, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

THE INHIBITION OF 16a-HYDROXYTESTOSTERONE AROMATIZATION Jacob Department

A. Canick

of Obstetrics

Received

January

and Kenneth

J. Ryan

and Gynecology,

Human Reproduction Harvard

BY CARBON MONOXIDE

Medical

and Laboratory

and Reproductive School,

Boston,

of

Biology, Mass.

02115

23,1975

SUMMARY 1'he aromatization of 16a-hydroxytestosteronel by human placental microsomal preparations is inhibited by carbon monoxide. At a CO:02 ratio of 19:1, the extent of inhibition was 45-60%. In the same experiments, CO inhibition of androstenedione aromatization was negligible (O-7.8%). In contrast to 19-nortestosterone, a poor substrate whose aromatization is inhibited by CO, the rates of aromatization of 16a-hydroxytestosterone and androstenedione are similar. It is possible that different cytochrome P-450 species or binding sites are involved in the aromatization of different substrates. INTRODUCTION Estriol, placenta

the major

through

the production than

the

system

of estriol

synthesis

placenta

estrogens,

three

As was first steroid

aromatization

It

more detail.

system.

synthesized

of 16a-hydroxylated

in the

of other

is

is little

androgens

quantitatively is

in the (l),

more

known about

human

the

Though

important enzyme

involved.

utilizing

all

of pregnancy,

the aromatization

The microsomal far

estrogen

involves

moles

a complex

mixed

of NADPH and O2 for

demonstrated

for

mixed-function The most

of androstenedione

widely

the

oxidases used basis

496

of estrone (4),

the NADPH-cytochrome

cytochrome

1 Trivial names: 16a-hydroxytestosterone 3-one; androstenedione = androst-4-en-3,17-dione; hydroxyestr-4-en-3-one.

Copyright o 19 75 by Academic Press, Inc. AN rights of reproduction in any form reserved.

oxidation

21-hydroxylase

contain for

function

each mole

adrenal

has been presented

P-450

in

(2) produced most

if

(3). not

P-450

involvement

= 16a-hydroxyandrost-4-en-176-Ol19-nortestosterone

has

= 178-

BIOCHEMICAL

Vol. 63, No. 2, 1975

been

the demonstration

However,

while

placental

of carbon

cytochrome

microsomes

comparison, not

placenta,

(7)

the aromatization

appear

In the

to be present

aromatization

by carbon

inhibited light

of the carbon

published

and since

placenta,

it

estriol

hydroxytestosterone

a steroid

In

which

does

of aromatization

studies

inhibition

predominant

to determine

is sensitive

monoxide.

in the

(6).

monoxide

is the

is of interest

to carbon

or intermediate

monoxide

19-

has been shown by Meigs

19-nortestosterone,

substrate

in human

of androstenedione,

to be insensitive of

to be a natural is

of enzyme activity.

and 19-oxoandrostenedione

6) and others

RESEARCH COMMUNICATIONS

inhibition

has been shown

microsomal

hydroxyandrostenedione, and Ryan (5,

monoxide

P-450

(5),

AND BIOPHYSICAL

estrogen

whether

to carbon

its

which

have

by the

synthesized synthesis

been

from

16~

monoxide.

METHODS Androstenedione-4-l4

C (New England

terone-4-14C

(Amersham-Searle,

matography.

Non-radioactive

Preparation delivered

containing

was centrifuged Aromatase and 7O$l

steroid

NADP+, 2 I.U. buffer, for

somal

protein,

Gassing. using bation

contained

substrate,

ran

for

to a final Mixtures

a gas proportioner flasks

were

were

from

at 25'.

from

The 15,000g

and washed

once with

freshly

prepared

microsomes

volume

dehydrogenase

of 5 ml.

The incubation,

up to 30 min at 37' concentration

equilibrated

Gas Co., with

(7-10

mg protein)

6-phosphate,

flasks

with

the

were

addition

and was terminated

2 nM

preincuof micro-

by the addition

of 70%.

E. Rutherford,

the gas phase

497

M

M KCl.

of 95% Np - 5% 02 and 95% CO - 5% 02 were (Matheson

0.25

and 50 n@l phosphate

Incubation begun

Inc.

supernatant

0.15

12 r&l glucose

chro-

freshly

in 3 parts

1 hr,

ethanol,

by paper

Steraloids,

prepared

was homogenized

6-phosphate

methanol

and 19-nortestos-

on receipt

purchased

pH 7.2

1% (v/v)

in a total

10 min at 37O.

of methanol

for

per ml glucose

pH 7.0,

bated

M Tris-HCl,

at 100,OOOg

incubations

were

Tissue

Corp.,)

purified

Microsomes

placentas.

0.01

were

steroids

and incubation.

human term

sucrose

Inc.)

Nuclear

for

N.J.). 15 min prior

obtained All to

incu-

BIOCHEMICAL

Vol. 63, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Gassing was continuous throughout the pre-

addition of microsomal protein. incubation and incubation. 14C estrogen assay. acetate, --et al.

The 70%methanol extract

was extracted

with ethyl

washed and subjected to phenolic separation as described by Nimrod The phenolic separation was repeated and the radioactivity

(8).

covered regarded as the total

estrogens.

paper chromatographic analysis re-

vealed that more than 90% of the radioactivity estradiol

re-

resided in the estrone and

zones.

Unlabeled estrogen assay.

The washed ethyl

as above, was subjected to radioimnunoassay. hydroxytestosterone, an estriol-specific

in which estriol antibody,

kindly

obtained

Incubations containing

was synthesized,

16a-

were assayed using

provided by Dr. David Watson of the

Worcester Foundation for Experimental Biology. hydroxytestosterone

acetate extract,

Cross-reactivity

with 16~

and estrone was ~0.1% and with estradiol-176,

0.22%.

Androstenedione incubations were assayed using a non-specific

estrogen anti-

body provided by Dr. Dan Tulchinsky,

Estrone and

estradiol-17B cross-reactivity

gave nearly identical

Harvard Medical School.

standard curves, with androstenedione