Dimethylstilbestrol and 16-oxo-estradiol: Anti-estrogens or estrogens?

DIMETHYLSTILBESTROL AND 16-OXO-ESTRADIOL: ANTI-ESTROGENS OR ESTROGENS? by L. M a r t i n Division of Physiology, Imperial Cancer Research Fund, Lincoln's Inn Fields, London W.C.2. Received October 12, 1968 ABSTRACT

A dose of dimethylstilbestrol, which is antiestrogenic when g i v e n as a s i n g l e intravaginal injection, produces all the vaginal changes characteristic of estrogen a c t i o n when g i v e n as 1 or 2 series o f 10 1 - h o u r l y injections. Similar results were obtained with 16-oxo-estradiol given in 1 or 2 series of 5 2-hourly injections. It is suggested that competitive antagonists of estrogens themselves act as estrogens if they occupy receptor sites for a sufficient length of time. INTRODUCTION Dimethylstilbestrol response the to

to

binding

of

estrogens

estrogen

is are

estrogenic of

vaginal

estrogen the

(11,6,9). that

from receptors (4,8)

and

potency

receptor

at

DHS a p p e a r s

so p r e v e n t i n g

some e v i d e n c e lost

vaginal

intravaginally

(2).

occupying

briefly,

estrogen

estrogens

duration

the

albeit

There

potent

as

when g i v e n

competitively,

receptors,

that

estrogens

same t i m e act

(DMS) i n h i b i t s

it

might

has

less

potent

more r a p i d l y been

suggested

be d e t e r m i n e d

occupation.

If

this

than (8)

by the were

so,

2

S T E R O I D S

DMS c o u l d if

its

be

expected

level

at

the

a sufficient

length

experiments

explore

was is

included an

for

to

exhibit

receptors of this

The

since

anti-estrogen

in

activity

maintained

for

following

possibility.

comparison

effective

estrogenic

were

time.

13:1

16-Oxo-estradiol intravaginally

much t h e

it

same way as

DMS ( S ) . A N I t ~ L S AND METHODS Randomly-bred female albino QS m i c e w e r e u s e d two weeks after ovariectomy without estrogen priming. In each experiment mice within a 2g b o d y w e i g h t range were allotted to treatment groups at random. Intravagina] injections w e r e made w i t h an A g l a micrometer syringe i n 2 " 5 ~1 o f O ' 9 ~ s a l i n e containing 3~ v / v o f e t h a n o l . Tetrazolium reduction epithelial thickness and the number of mitoses in the vagina were estimated as described previously (5). In the vaginal cornification experiment the vaginae were fixed in Bouin's fluid; 5~ s e c t i o n s stained with Weigert's iron haematoxylin and eosin were examined under the microscope and scored as follows: O

:

no growth

or

stratification;

1

:

some g r o w t h

and

2

:

considerable thin layers

growth and of keratin;

3

:

extensive

stratification

but

stratification

no

keratin; with

keratinisation.

T h u s t h e maximum s c o r e be 18. The amount of significant differences

for a group of six mice ]eueoeytosis was noted, were observed.

would but no

Solutions were prepared immediately before each experiment from eth~nolic stock solutions stored in a refrigerator a t 5 C. Aliquots o~ t h e s t o c k solutions were dried u n d e r N 2 a t 70UC, t a k e n u p J n redistilled ethanol and made up t o t h e r e q u i r e d concentrations with 0"9~El in water. When t w o series of injections w e r e g i v e n 74 h r a p a r t , the

Jan. 1969

s T E R O I D S

solutions were The DMS ( B a t c h Professor C.W.

3

stored overnight i v a deep freeze. ~ P - S F m . p . 1 9 9 - 2 0 0 C) w a s a g i f t from Emmens.

The d o s e o f 50 n g DMS u s e d i n t h e s e e x p e r i m e n t s , given in a single intravaginal injection is not overtly estrogenic and completely inhibits 24 h r r e s p o n s e s to estrogen (5). 16-Oxo-estradiol, a more potent antagonist t h a n DMS d o e s s h o w s o m e e s t r o g e n i c activity at this dose level 17,8). The s c h e d u l e s were based on the estimated vaginal half lives of estradiol-17~ (~ h r ) , 16-oxo-estradiol ( ~ - 1 h r ) a n d D~:S (~ h r ) ( 8 ) . It was ttlought that 10 i n j e c t i o n s o f DMS o r 5 i n j e c t i o n s of 16-oxo-estradiol would maintain each compound at levels comparable to those obtained with a single intravaginal injection of estradiol-17~. In the mitosis, epithelial thickness and tetrazolium experiments the total dose o f e a c h c o m p o u n d ( 5 0 rig) w a s g i v e n i n o n e i n t r a vaginal injection, in 5 intravaginal injections at 2 hr intervals o r i n 10 i n j e c t i o n s at 1 hr intervals. The m i c e w e r e k i l l e d 2~ h r a f t e r the first or only injection. In the vaginal cornification experiment the same schedules of injections were given twice, 2~ h r a p a r t , so that each animal received a total dose of 100 ng. The m i c e w e r e k i l l e d ~8 h r a f t e r the first injection. In each experiment, additional groups o f mice were killed after similar injections or series of injections of vehicle alone. RESULTS Table i shows the effects of single and multiple injections o f

DMS a n d

vehicle,

16-ox___oo-estradiol

vaginal tetrazolium reduction. of

saline

almost

doubled

those

produced

~iultiple injections

tetrazolium

resulting optical densities w e r e b y DhS a n d

on

reduction

but

low compared with

16-oxo-estradiol

(see

below).

A single injection of DMS had no effect.

Five

injections

slightly

but

not

vehicle,

increased significantly

tetrazolium more

than

the

reduction did

5 injections

of

llowever i0 injections produced a large and

q.

ST ER O I D S

TABLE

Vaginal

tetrazolium

injections

13:1

I

reduction

after

single

o f DMS, 1 6 - o x o - e s t r a d i o l

and multiple

and saline

The r e s u l t s are expressed as mean optical densities w i t h 12 a n i m a l s ~ g r o u p . The transformation IOO l o g t n (1OOO x O . D . ) was used for the analysis 5T v s r i a n c e .

Fiean O p t i c a l

of

NO.

injections

Saline

16-oxo

DFIS

1

0.080

0.221

0-084

5 10

0.128

0.411

O-164

o.15o

0.346

o.351

Anal~sis

Source No.

of V a r i a t i o n

of injections

Replicates IXS

1

led/

v.16-oxo

(R) a v. d a v. e by. d b v. e

Within

Interactions groups

(error)

*O.Ol
Square

F

(I)

Substances (S) DMS v . s a l , DMS, sal.

o f Variance D_ff M e a n

5

I,I0 v.

Residual

Density

313

I'0

1

~,608

1

30,435

1

293

1 I 1 1

4,351 2,033 176 1,901J

8

378

1.2

90

326

-

**O-O01
14-I*** 93-14 ***

0.9 13"3"** 6-2" 0-5 5"8"

***P<0.O01

Jan. 1969

s T ER O I D S

significant with

Similar

16-oxo-estradiol.

produced larger was

increase.

response

given

was

response,

produced

in 5 injections.

I0 i n j e c t i o n s p r o d u c e d

results

Although

a significant

5

a single

obtained

injection

a much

when

the same

Splitting

no further

were

dose

the dose

increase

into

in

response. Epithelial of e s t r o g e n epithelial that

the

action

given

with

greater

sensitivity

and

and

of 1 6 - o x o - e s t r a d i o l (Table

2).

Again, when

also

consistent

of the mitosis

with

response;

no effect w h e r e a s

5 injections

produced

maximal

pattern.

In this

16-oxo-estradiol single

case,

injections Multiple epithelial

but of

(Table however,

produced

injections.

ineffective,

the

the

produced

data

Five

injections thickness.

of saline

both

IO

The

2) show a s i m i l a r neither

injections

16-oxo-estradiol

a

effects.

an increase

10 i n j e c t i o n s

however,

The

injection

thickness

and

so it was not u n e x p e c t e d

in 5 or I0 injections.

DMS were

epithelial

measure

reduction

increased s i g n i f i c a n t l y

results

single

sensitive

tetrazolium

response

was

dose was

is a more

injection

a large

the r e s p o n s e same

than

thickness

single

produced

mitosis

DMS n o r

when g i v e n

in

o f DMS w e r e a l s o

o f DMS and 5 and lO produced increase

As i n t h e

maximal effects. mitosis

tetrazolium

and test,

6

ST E R O I D S

TABLE

Vaginal

epithelial

multiple

injections

mitosis

13:1

2

and thickness

after

o f DMS, 1 6 - o x o - e s t r a d i o l

single

and

and

saline.

The r e s u l t s a r e e x p r e s s e d a s mean n u m b e r s o f m i t o s e s and a s mean e p i t h e l i a l thickness (arbitrary units) with six animals/group. The d a t a w e r e n o t t r a n s f o r m e d for analysis. Mean No. Mitoses

No. Of injections

Saline

Mean

Epith.

Thickness

16-oxo

DMS

Saline

16-oxo

DMS

1

3"7

13"9

2.6

17"2

17"9

16.2

5

3.8

19.4

19.5

18-4

33"4

17.1

10

7"5

22.8

19.8

19.1

34.1

33"8

Analysis Source

of Variation

No.

injections 1 v. I0

of

1 , 1 0 v. Substances

of Variance Df

Mean Mitosis

Squares Ep. Thick

(I)

5

(a)

i

(b)

1

893***

75

5

i

1021"**

784***

1 1 1 I

269*** 5 204*** 13

307*** 103"* 65* 269***

1178"**

(S)

DMS, sal.

v. 16-oxo

IXS a a h b Within

v. d v. e v. d v. e

group *O.OI
43 **O-O01
19

15 ***P
Jan. 1969

s T E R O I D S

however, with

the

those

increases

produced

Extensive

all

10 i n j e c t i o n s estradiol. little

growth

with

of

the

of

DES o r

injections

growth,

stratification

epithelium. only

found

in

cornification

response

that

to

found

twice of

in

but treated

single

produced

extensive

to

the

mice

nor

any

layers

of

the

of

keratin

treated

with

Although

DES w a s this

than

with

16-oxo-

organs

16-oxo-estradiol.

rather

in

keratinisation

estradiol-17~,

a quantitative

found

Neither

some o f

of

and

stratification

vehicle

and

10 i n j e c t i o n s

with

and

or

Thick

were treated

DES. of

compared

16-oxo-estradiol.

5 injections

were

of

multiple

5)

vaginae

no keratin

small

stratification,

(Table

5 injections

were

and

Some g r o w t h

or

very

b y DES a n d

some k e r a t i n i s a t i o n virtually

were

7

submaximal appears

to

a qualitative

the compared reflect

difference.

DISCUSSION Pollard

and Martin

intravaginal

dose

of

(12)

DMS w h i c h

in

2~ h r

mitosis

and

the

2 hr

vaginal

nucleolor

but

actually

These

and

competitive estradiol

present

a similar

antagonists are

anti-estrogenic,

like

themselves the

tests

response

results

final

that

an

was a n t i - e s t r o g e n i c

tetrazol±um

stimulated

the

found

to

did

inhibit

estradiol,

response

indicate

not

itself.

that

DMS a n d 1 6 - o x o -

estrogenic outcome

as well of

as

an experiment

8

ST ER O I D S

depending target the

on the

cells

are

response

dose

is

exposed

(1)

with

or

ratio

two doses

in

the

inactive

not

case

it

are

Thus

alter must

On t h e s e

the

criteria

explanation;

that

itself

estrogenic

within

the

Since

would

corresponding

metabolized the

route

effective

with is

which

about

1.

active

estrogens

administration

dose

since

exerting

in its

a pro-estrogen.

provide

either effect. However

an alternative

DMS g i v e n

subcutaneously

is

depot

body maintains

levels

for

sufficient

a measurable or

systemic

estrogenic

effect.

administration, have

an adequate

depot

in

to

the

be given body,

the

to S/L ratio

unity.

mode o f

action

correlation

would

between

anti-estrogenic

potencies

for

so far,

failure

the

themselves to

of

before

true

its

approach

the

into

pro-estrogens

would

remarkable

on

intravaginal

ratio)

material

This

and

because

by local

produce

compound,

simply

produce

sufficient

the

(S/L

DMS i s

results

to

the

estrogens

of

circulate

present

time

the

which

pro-estrogens

that

but

body.

does

the

and

suggested

the

for

effective

less

dose

are

to

the

subcutaneous

Emmens

time

classified

which

1/lOOth

of

of

measured.

Emmens estrogens

length

13:1

of to

account the

for

the

estrogenic

some anti-estrogens

find

a competitive

and (3),

Jan. 1969

s T ER O I D S

antagonist and f o r

totally

the

devoid

failure

which in all

inhibits

all

the

of estrogenic

to find

estrogen

effects

epithelial

mice treated

with

completely

3

and c o r n i f i c a t i o n

or multiple

16-oxo-estradiol

injections

in

o f DMS,

and s a l i n e

The r e s u l t s are expressed as total scores 6 mice. The s c o r i n g s y s t e m i s d e s c r i b e d maximum s c o r e p o s s i b l e i s 18.

Total

of injections

anti-

of estrogens.

stratification single

activity,

a competitive

circumstances,

TABLE

Vaginal

9

No.

Saline

for groups of in the text:

Score

16-oxo

DMS

1

0

~

O

5

0

12

6

IO

1

13

10

ACKNOWLEDGEMENT I w i s h to thank Mr. advice.

P.C.

Williams

for his help

and

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Emmens, C.W.

2.

Emmens, C.W.

5.

Emmens, C.W.,

J.

ENDOCR. ~ ,

and Cox, Cox,

R.I.

R.I.

4~4 J.

(1941)o ENDOCR. 17,

and M a r t i n ,

HOtel. RES. 1_~8, ~ i 5

(i962).

L.

265

(1958).

RECENT PkOG.

lO

S T E R O I D S

4.

Jensen,

E.V. Part

13:1

PROC. SECOND INTER. CONGR. ENDOCRINOL. 1,.p.~20. EXC. MED. INT. CONGR. SER.

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L.

J. ENDOCR.

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L.

J.

7.

Martin,

L. i n ESTROGEN ASSAYS IN CLINICAL MEDICINE, E d i t o r C.A. P a u l s e n , U n i v e r s i t y of Washington Press, Seattle, (1965), p.128.

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Martin,

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Martin,

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10.

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11.

Martin,

L.,

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Pollard,

D

50, 21 (196~a).

ENDOCR. 5 0 ,

Cox,

R.I.

B.

537 ( 1 9 6 ~ b ) .

J. P.J.

ENDOCR. 50, 41 ( 1 9 6 4 ) . J.

ENDOCR. 2 0 ,

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J.

173

ENDOCR. 22,

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L.

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