Peripheral aromatization: Studies on controlling factors

ABSTRACT Using constant infusions of 3H-labeled androgens and I4C-labeled estrogens with measurements of radiolabeled estrogens in blood and/or urine we have carried out studies on the peripheral aromatization of androgens in humans, nonhuman primates, sheep, and rabbits. In the human, aromatization is increased in women as they become postmenopausal, although the mechanism remains uncertain. In humans and cynomolgus monkeys the administration of ACTH and/or glucocorticoids does not increase peripheral aromatization, but results in a slight decrease in the aromatization of androstenedione. The administration of &thyroxine to cynomolgus monkeys increases peripheral aromatization of androstenedione from basal, 1.16 t 0.153, to 1.71 t 0.14% probably due to increased tissue blood flow. The aromatization-of testosterone is not affected, probably due to an increase in sex hormone-binding globulin. Peripheral aromatization occurs to a similar degree in humans, rhesus and cynomolgus monkeys, and baboons, but is much lower in sheep and rabbits. The compound IO-(2-propynyl)-estr-4-ene-3,17-dione is an effective inhibitor of the peripheral aromatization of both androstenedione and testosterone.

INTRODUCTION 4lthounh West et.

(1) and Leach et

(2) suggested that andro-

gens could be aromatized to estrogens in peripheral, i.e.,

extra-

gonadal, tissues, it was not until the studies of MacDonald and coworkers (3,4) and Longcope and co-workers (5), using radiolabeled techniques, that the importance of this source of estrogens was fully realized. Since then it has been established that the peripheral aromatization of androgens is a major source of circulating estrogens in men (4,5) and post-menopausal women compared with younger women with menstrual cycles (4,6). Work by James -et al (7), Baker -et al (8), and Southren et al (9) have confirmed and extended these original reports

STEROIDS

50 / l-3

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254

Longcope

to show that peripheral aromatization is increased in cirrhosis and perhaps other diseases. Aromatization can be stimulated by follicle stimulating hormone (FSH) in ovarian granulosa cells (10) and by luteinizing hormone (LH) in testicular Leydig cells (ll), but the gonadotropins do not affect peripheral aromatization and the control of peripheral aromatization remains uncertain. We wish to report studies on periphera? aromatization, its stimulation, species differences, and its inhibition. MATERIALS AND METHOBS [7-3H]testosterone (30 Ci/mnol), [4-I4CJestradiol (54 mCi/mnol), and [4-I4C]estrone (54 mCi/mnol) were obtained from New England Nuclear Corp. (Boston, MA). [7-3Hlandrostenedione (5 mCi/~ol) was obtained from ~ersham/Searle Corp. (Arlington Heights, IL). All steroids were purified before use (5). All studies were done using constant infusions of 3H-labeled androgen (60 uCi) and I4C-labeled estrogen (3 uCi) in 15 mL 8% ethanolic saline after a priming dose of 3H-labeled androgen (40 $i) and I4Clabeled estrogen (2 uCi) in 10 mL 8% ethanolic saline (12,13). Blood samples were obtained at 2.5, 3, and 3.5 h of infusion, and the androgens and estrogens extracted and purified by multiple chromatographic and derivatization steps (12,13). The radioactivity in the purified steroids was measured and these data were used to calculate the extent of peripheral aromatization ([plpBd,Est = percent of androgen infused that is aromatized and measured in the blood as estrogen) (14). In our later studies we drew and analyzed blood as described, but in addition we collected all urine for 96 h and used the estrogen lucuronides f3,14) to calculate the extent of peripheral aromatization = percent of androgen infused that is aromatized to estrogen 9 tpl$d,Est in the body) (4,14,15). Studies in humans were done between 0800 and 1300 h with the subjects supine, and all protocols had been approved by the appropriate review boards of the institution and all subjects had given their informed consent. The studies in sheep were done with the subjects upright and awake; in rabbits and primates with the subjects under light ketamine anesthesia except for the rhesus monkeys, which were studied under nembutal anesthesia. Statistical analysis was done using Student's t test, paired t test, and Student-Ne~an-Keuls' test where appropriate. Non~ighted,

STEROIDS 50 I l-3 1987

PERIPHERAL

ARO~TI~TION

least squares linear regression was used to calculate correlation coefficJents and partial correlations, using BMDP programs (BMDP, UCLA, Los Angeles, CA). RESULTS In post-menopausal women the mean 2 SE

tplft El, 2.33 + 0.16%, is BB

greater (pt0.05) than in pre-~nopausal women, 1.48: 0.08X;,and men, I.612 0.16% (Table 1). EplT,E2 is greater fpc0.05) in post-menopausal women, 0.47 + 0.06X, than in pre-menopausal women, 0.28 f 0.02%, but not greater than in men, 0.34 + 0.13%. Because the presence of obesity could have influenced the results, this analysis included only subjects less than 125% of ideal body weight. Table 1 Ar~atization ([lintel

and CP]&$?Z) in Men and Pre-Menopausal

qp

C~l&h

%

% Pre-menopausal women Post-menopausal women Men

-aAll subjects (125% of ideal body weight. bMean + SE. c#umbeF in parentheses = number of subjects. dSignif~cantly greater than other groups (p
Cpl,TbQand CplA El, we analyzed the data for peri-

BB and post-menopausal women between 42 and 57 years old.

The women were

placed into one of four groups according to their menstrual status and FSH levels, and the results are shown in Table 2. nor

Cpl/jhE~ was

STEROIDS

For neither Cpl&E2

there a significant difference across the groups.

50 I 1-3 1987

255

256

Longcope

Table 2 Aromatization ([PI&El and [p]ihEZ) in Peri-Menopausal and ---___-_~-.

Menstrual Status

Post-Menopausal .Womena . . --...-. .~~..~ - .- .“. FSH mIU/mL

~~._

[&&El &

CPIT¶Q BM %

%

12 2.04 + 0.16b Regular (40 13 2.12 T 0.27 Irregular >40 >40 2.13 + 0.09 Amenorrheic trlyr 2: 2.06 z 0.07 ~enorrheic >l yr >40 __....".__ .. aAll subjects were ~125% of ideal body weight. bMean + SE.

0.40 0.40 0.34 0.42 ____~

+ y T E

0.03 0.04 0.05 0.02

In order to focus more closely on the events surrounding the menopause, we compared the aromatization in 14 of these women who were having cycles at the time of the first study and who were amenorrheic for more than 1 year

at the time of the second study. As shown in Table

3, there was a significant increase fpt0.01) in tion.

There was also an increase in

Cpl&El in the transi-

Cpl&Q, but this was not

significant. The,Jncreases were not altered when the results were corrected for body weight. Table 3 Peripheral Aromatization ([pItiE and [pj&E2) in 14 Womena _._____-_--_-

t PI&El

qp

%

%

1.84 + 0.09b Pre-menopausal 0.39 + 0.03 2.34 2 0.14c Post-menopausal 0.46 5 0.06d _. _.._...__ ._._. -.._-,__--.--.------.__._____~._..__ ._~.----aInitial measurements made when subjects were pre-menopausal and .repeat studies done two years later when they were post-menopausal. bMean + SE. cSigniTicantly different from pre-menopausal, peO.01. dNot different from pre-menopausal, paO.1.

STEROIDS

50 f l-3 1987

To see whether the aromatization was correlated with a number of varjables which included weight, percent free steroid, sex hormonebinding levels, or metabolic clearance rates of the androgens or estrogens, the appropriate values were analyzed by skpfe The results are shown in Table 4.

linear regression.

Significant correlations were noted

between aromatizatation and several variables, including weight, IUICR62, and sex hormone-binding globulin. However, when the effect of weight was removed, the partial correlations were not significant except between SHBG and ~pl~~E1. There was no correlation between FSH or LH levels and the extent of peripherul aromatizat5on. Table 4 Simple and Partial Correlation Coefficients as Determined by Unear Regression for fpIik62 and fplA# ------.~---.-~."-_t

Independent Variable

Partial Correlation Coefficients Correlation Coefficients _LPJl t2 Bi

Wejght ;$y 62

0.389a,b 0.184 0.331c

8.321c 0.120 0.022

-0.156 0.125

ET"

0.317c 0.307c

0.198 0.301c

0.238 0.188

*

Sex hormonebinding globulin FSH LH ._--

-0.391b -8,GJO 8.027

-E$b 0:011

-G,Ofl -0.821 0.046

c :y;;d 0.205 0:lss

-%: Lo39

acorrelatfon coefficient. bpp>0.001. ~O,O6>p>0.01. Studies were also carried out with ACTH and cortisol, and, as shown in Table 5, neither the acute administratfon of ACTS nor of cortisol had an effect on ar~tizat~on. signfficant.

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50 I f-3 1987

The decrease of fp3A El after ACTH was not ss

258

Longcope

Table Effects

of ACTH and Cortisol

Control

5

on Peripheral

After

Control

After Cortisolc

0.23 + 0.04

0.26 + 0.06

ACTHb

CPI&EZ,

%

0.32 _+ 0.06d

0.32 + 0.04

CPl;~El,

%

2.55 _+ 0.35

1.75 + 0.15

Aromatizationa

ND

ND

z Data from Pratt and Longcope (16). ACTH gel 60 units every 12 h for 4 doses. c Cortisol 50 mg every 12 h for 4 doses. d Mean + SE. When the ---_ Macaca

fascicularis

150 ug a day for 8 weeks, 0.19%),

CPI~~E~ did

but there was a slight

1.16% to 1.71%

monkeys

were treated

not change

but significant

with

t-thyroxine

appreciably

(0.23% to

increase

in [pIliE

from

(Table 6). Table

6

Effects of r-Thyroxine, and Acute and Chronic Administration on Peripheral Aromatization Cynomolgus Monkeys

CPlT9E2 _I_Treatment,

Control -%

a-Thyroxine 150 pg 0.23fP.03a per day x 8 weeks Dexamethasone 8 mg 0.29+0.05 oer 8 h x 3 doses 0.3820.05 Dexamethasone 1 mg per day x 8 days __-._-----..-*

Dexamethasone in Male

CPly

BM

AfterTreatment -%

---Control

After Treatment %

%

0.19~0.04

1.1620.15

1.7120.14

0.27+0.06

1.2820.22

0.8320.16

0.4520.04

1.4120.25

0.68+0.03

_ ..__.--_-----

a Mean -+ SE. Dexamethasone or chronically

Cpl&C!

administered

acutely

(8 mg every

(1 mg per day for 8 days)

and a slight,

but not sjgnificant,

resulted

8 h or 3 doses)

in no change

decrease

in

in CpYkEl.

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50 / 1-3 1987

PERIPHERAL ARO~TI~TIO~

259

In order to determine the extent of aromatization in other species, we measured aromatization in sheep, rabbits, two species of macaques, and in baboons. The results are shown in Table 7.

In the human and

non-human primates there is a remarkable similarity for [pIikE2 and C~l~~51, with ranges of 0.18-0.34% and 1.01-1.472, respectively. In the sheep both

[pIA El and [pJT 52 were very low, 0.13% and 0.01X, BB

B8

respectively. However, in rabbits, although the mean value for Ep3&22 was close to the lower primate range, [pl;~El was barely detectable, 0.05%. Table 7 Peripheral Aromatization in Humans, Sheep, Rabbits, Macaca mulatta and -M. --_ fascicularis Monkeys and in .papi anubisBabo&s ---__--ll_-___-_-_

Human, male female

0.34 T+ 0.13a 0.28 0.02

M. -_.--mulatta, male female M. male

0.28 + 0.03

:$j; .

anubis, female fasciculari?, S;leep,female Rabbits, male

0.18 z 0.02 0.01 0.12 + 0.06

1.23 + 0:13 0.13 7: 0.03 0.05 z 0.01

1147 z 0:14

?

-_.__-__.____.

i-f:,; ;*A;

..- .^.__.__” _-.-.-_

.__.-._-.

8.:;

-...--

a Mean

* SE. b Not d;ne. The administration of the propargylic steroid [(lo-2-propynyl)-estr4-ene-3,17-dione (MOL-18,962)l results in significant inhibition of aromatization in female baboons (Table 8).

The aromatization of andro-

stenedione was inhibited at doses of MOL-18,962 from 0.01 to 4 mg/kg IV. Although tested at only 4 mg/kg IV, the aromatization of testosterone was also markedly inhibited as shown.

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Longcope

Table 8 Effect of Intravenous MDL-18,962 on Aromatization of Androgens in Female -P. anubis ~_...__.__..._....

Cd&E2 _.--_

-_--n

CPIA$

Control

4

0.182

HDL-18,962 4 rag/kgIV MDL-18,962 0.4 mulka IV

4

0.04 2 0.02 (8lX)b

MDL-18,962" 0.1 mg/kg IV

__.-^_“___-

---

n---

%

0.03a

4

1.36 + 0.05

4

0.112

NOC

2

0.16 + 0.03 (88%)

ND

2

0.18 + 0.06 (87%)

0.01 (92%)

___. _.__._ ..___..----_ . .. __.___ ._.... _..._.. _.._“.__I._____

a Mean + SE. b Figure in parentheses = % inhibition compared with control. c Not done. MDL-18,962 was also effective in inhibiting the aromatization of androstenedione when given orally (Table 9), but this was more marked after 4 rag/kghad been given daily for 5 days po (84% inhibition) compared with a single dose of 4 mg/kg po (67% inhibition). Table 9 Effect of Orally-Administered MDL-18,962 on Aromatization of Androstenedione in Female -II P. anubis

~___.__

_ __.._ . -_.

.-__~.-....l_l_

.

I_

Treatment

n

CplA,El BM %

Control MDL-18,962 4 mg~kg po MDL-18,962 4 mg/kg po per day for 5 days

3 3

1.35 + 0.06a 0.43 z 0.12 (67%)b

3

0.21 + 0.01

(84%)

a Mean + SE. b NumbeFs in parentheses = % inhibition compared with control. DISCUSSION As noted,

in our early studies we used radioactivity data from

blood samples and calculated [p]&d,Est, but in our later studies we

STEROIDS

50 f 1-3 1987

PERIPNERAL AROMATIZATION

used radioactivity data from urine and calculated

261

C~l~~d,Est, Edman and

RaeDonald (15) and we (14) have discussed the differences in the two measurements. Using infusions lasting 3-4 h the two methods appear to yield similar data in subjects of normal weight; in overweight subjects the [P&B

will

underestimate the extent of perjphera~ ar~atization

unless the infusion is carried on for a longer (i.e., 24-36 h) time interval (15). Therefore, in certain of our analyses, as noted, only data from subjects ~125% of ideal body weight were used. The increase in peripheral ar~atjzation seen in post-~nopausal women compared with younger pre-menopausal women was reported by Hemsell et al (14) and ourselves (6). The initial studies primarily fnvolved measurements of

LplA,El,

but it is apparent that [pJT,Q also increases.

However, because the production rate of testosterone is small in postmenopausal women, the actual amount of estradiol formed by the peripheral aromatization of testosterone will not be large.

In order to determine when this increase in peripheral aromatization might occur we studied a group of peri- and post-menopausal women. When the analysis was done stratifying by cycle regularity and FSH levels, we could detect no difference in the percent of peripheral ar~atjzat~on.

However, when a sub-group of women was studied when they

were having cycles and again 3 years later when they had been amenorrheic for more than one year, a different picture emerges. With the cessation of cycles and entry into the menopausal state, an increase in ar~atization was noted, although the increase was significant only for

Clrl#~El. These data thus indicate that menopause is associated with a relatively abrupt alteration in peripheral aromatization. This has been suggested before, using indirect data (6); but repeat studies on a group of women have not been reported heretofore.

STEROIDS

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Longcope

262

The reason been

suggested

factor

released

aromatization the

In analyzing

aromatization

would

Weight

levels

(19,20),

we examined

zation

removing

the effect

[pIikE

was not significant,

with

[pl&El

remained.

of this correlation and CplA,El,

is not likely ciated

and co-workers

women

when measured

jects

could

several

with peripheral

correlation

correla-

weight

and SHBG

of SHBG to aromati-

The partial

correlation correlation

of SHRG of SHBG

A nor EI is bound to SHBG the explained.

is an important

However,

for

factor

but

controlling

role in the increased

several

have still

of ACTH and/or

(21) noted that days after major

been under

aromatization

cortisol,

we examined

on aromatization

and found

decrease

in Cpl&EI

ACTH

in [o]iBB1

after after

(16).

Cp]&El

was

surgery.

some stress

cortisol

change

of our subjects,

asso-

with the menopause.

Rizkallah

levels

be lost

between

is not readily

weight

to play a major

would

but the partial

Since neither

peripheral

the strongest

correlation

of weight.

If this

all follicles,

factor

correlated showed

the partial

with

[PI&Q

(18).

increase.

and SHBG levels is a strong

of an

from ovarian

inhibit

by this

the data from a large number

but since there

both

locally

it might

aromatization

aromatization

tions,

persistence

a factor

it has

at that time,

of the removal

Then with the loss of essentially

were found to be significantly

aromatization.

and while

is stimulated

reported

into the circulation

of peripheral

and the peripheral

variables

inhibits

as well.

inhibition

uncertain,

is the result

et al have recently --

fluid which

were

remains

aromatization

that the increase

Campeau

follicular

increase

that peripheral

it is possible inhibitor.

for this

no change

Since these

with higher

the effects

increased

in sub-

than normal

of ACTH and

or, if anything,

a

Reed --et al (22) also found

no

ACTH and cortisol.

STEROIDS

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PERIPHERAL

AROMATIZATION

263

Similarly, in male cynomolgus monkeys treated with dexamethasone either acutely or chronically, we could detect no significant effect of glucocorticoids on peripheral aromatization. Mendelsohn et al (23), using an adipose stromal cell culture system, and Folkerd and James (24), using adipose tissue slices, showed a marked effect of glucocorticoids on aromatization. However, in both humans and the cynomolgus model, CplA,El was, coids.

if anything, decreased after ACTH or glucocorti-

Therefore, there seems to be a disparity between the effects of

glucocorticoids measured using an -in vivo or an -in vitro system.

It is

possible that endogenous inhibitors of aromatization block the effects of glucocorticoids-in vivo. We had previously reported (25) that hyperthyroidism had little effect on peripheral aromatization. These findings were in contradistinction to the report of Southren et al (26) who reported a marked increase in both [p]T 62 and [p]A 61. BB BS

Our present studies carried out

in cynomolgus monkeys indicate the induction of hyperthyroidism results in no change in

CpI&k?, as we also noted in humans (25). However, we

did find a slight but significant increase in Cpl~~El after

the monkeys

became hyperthyroid. It should be noted that in our previous study both hyperthyroid men, but only one of seven women, had values for [p]A El ,A above, the normal range. In our monkeys, as has been reported for humans (20), we found a marked increase in sex hormone-binding globulin levels. The slight increase in

CplA,El in hyperthyroidism probably is

the result of the increased peripheral blood flow, but the increase in SHBG counteracts the increase in blood flow and prevents more testosterone from reaching the aromatization sites and hence Cp]T,E2 remains the same.

STEROIDS

50 / l-3 1987

264

Longcope

In many of our studies, we have used a nonhuman primate model to study

peripheral

peripheral

aromatization

fat content which

lack of adipose

generally

data)

it would

phenomenon

sites of aromatization

Although limited androgen

aromatization although

appear

birds

that

to increasing

and skin,

in vivo. ---

tissue

(C. Longcope,

5a-reductase

system.

aromatization

of the aromatase blood

is not a

to primates

there

peripheral

aromatization

inhibitor

in the baboon

are a number

of aromatase

When administered,

rapidly

to inactivate

that

may be

increasing of compounds

aromatization.

Testololac-

have all been shown

of androgens

indicate

system

flow and/or

lo-(2-propynyl)-estr-4-ene-3,17-dione

been shown to be a potent Our results

evidence

We were also unable

and 4-hydroxyandrostenedione

the peripheral

tissues

(28).

to make up for the

but may be restricted

tissue

to the tissue,

aminoglutethimide

enter

muscle

find little

tissue.

peripheral

for stimulation

tone,

inhihitor

15-20%

the fact that these

in rat adipose

significant

have been shown to inhibit

35).

(27),

(29).

availability

compound,

despite

there was an active

which

Another

we could

adipose

in all species

mechanisms

primarily

to decrease

man's

in monkeys

to the primate,

have considerable

that occurs

and, perhaps,

10% of his body weight

other than fat, probably

in sheep and rabbits,

to find significant

Thus

The body

tissue.

of aromatization

unpublished

to find that the

species.

less than that of the average

In contradistinction

animals

striking

among these

is only about

imply that tissues

be very active

It is perhaps

is so similar

of the monkey

is considerably

This would must

aromatization.

(30,31,32). (MDL-18,962)

in -in vitro systems

has (33-

it is also an effective

MDL-18,962

the peripheral

must circulate aromatase

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50 / l-3

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PERIPHERAL AROMATIZATION

265

since 90% inhibition can be achieved by a single 4 mgikg dose given as an IV pulse. Although all the tissues containing the aromatase system are not known, the tissues containing the major activity are probably adipose tissue, muscle, and skin. Despite the widespread nature of the aromatase, a dose as little as D.l ngikg given as an IV pulse results in *90% inhibition. Not only is it effective when given IV, but also when given in a single pulse orally. This latter finding is interesting since there appears to be considerable 'first pass" metabolism (tongcope, C. and Johnston, O., unpublished data).

Nevertheless, the pulse administration

of 4 mgikg was not so effective when given orally as when given IV.

How-

ever, there appears to be a cumulative effect, since repetitive dosing by the oral route results in near total inhibition of aromatization. In summary, the peripheral aromatization of androgens is present in humans as well as in some species of nonhuman primates.

Increases in

tissue blood flow can cause an increase in aromatization by providing more androgen to the aromatase system. For testosterone, at least, SHBG increases may counteract these effects of blood flow, but there are several compounds that are inhibitors of peripheral aromatization and MDL-18,962 appears to be among the most potent in this regard.

The author would like to thank C. Flood, C. Bourget and A. Femino for their excellent assistance in these studies, and Dr. W. Webster and the Department of Animal Medicine for their expert help. A portion of these studies were done at the New England Regional Primate Center, which is supported by hrant RR-00168-17 from the National Institutes of Health. I would like to acknowledge the assistance of Dr. C. Conrad Johnston, Jr., in performing studies on peri- and post-menopausal women. Many of these were done in the Clinical Research Center of the Indiana University Hospital, Indianapolis, Indiana, supported by grant RR-750 from the National Institutes of Health. I would also like to acknowledge the assistance and advice of Dr. O'Neal Johnston in the studies on MDL-18,962. This work was supported by grants HO-15443 and AG-02927 from the National Institutes of Health and a grant from the Merrell-Dow Research Institute, Cincinnati, Ohio.

STEROIDS 50 /’ l-3 1987

266

Longcope

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50 / 1-3 1987

PERIPHE~LARO~TI~TION

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50 / l-3 1987