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Effects of clomiphene on adrenal cortical steroidogenesis in dogs
Effects of clomiphene on adrenal cortical steroidogenesis PAUL
CUSHMAN,
JAMES
G.
HILTON,
in dogs JR.,
M.D.* M.D.**
New York, New York
The mechanism of action of clomiphene, the ovulation inducing agent, remains uncertain. Most of the euidence has been interpreted to reflect a weak estrogen-like action and an estrogen inhibitory influence at higher concentrations. Direct adrenal cortical effects have also been postulated. The present study was undertaken to test the latter hypothesis by perfusing the isolated adrenal of the hypophysectomized dog with clomiphene. No alteration in either the resting 17-OH corticosteroid or 17ketosteroid secretion rates was obserued after 3 doses of clomiphene. It appears unlikely that clomiphene acts directly on the adrenal cortex.
collected in 5 minute periods. After four consecutive initial collection periods to establish base-line secretion, clomiphene was administered for 15 minutes into the arterial circuit to produce 1 x 10V6 M concentration. Ten minutes after completion of the low dose administration, 1 x 1O-5 M clomiphene was injected for 15 minutes. ‘l’en minutes later 1 x 10m4 M clomiphene was similarly perfused for 15 minutes. The responses to each dose employed were calculated from the average of the steroid secretion rates in 4 consecutive collection periods. At the end of each experiment, 25 units of bovine ACTH was injected to insure the functional viability of each preparation, which was reflected in the mean of the steroid secretion rates in the next two collection periods. In control studies, similar isolated adrenal pouch preparations were used. Into the arterial circuit of the perfusion system was injected identical volumes of isotonic NaCl. The responses to saline administration were calculated from the mean of the steroid secretion rates measured in four consecutive collection periods 15 to 60 minutes after completion of the base-line collection periods. At the end of each control experiment, 25 units of bovine ACTH was similarly injected and its effect on steroid secretion similarly
THE ME C H A N I S M Of action Of ClOmiphene, the ovulation-inducing agent, remains uncertain despite considerable recent investihas been presented for: gation. l-7 Evidence (a) a weak estrogen-like actionl, ‘; (b) an estrogen inhibitory action at higher concenand (c) a direct ovarian intration?; fluence.“, 5 A direct hypothalamic hypophyseal action has been described33 o and adrenal cortical49 8-1o effects have been postulated also. The present study was undertaken to test the latter hypothesis by perfusing the isolated dog adrenal with clomiphene. Materials
and
methods
In acutely hypophysectomized dogs, the isolated in situ adrenal pouch was prepared by the method of Hilton and associatesl and perfused at constant rates with arterial blood obtained from other hypophysectomized dogs. The venous effluent was From the Department of Medicine, St. Luke’s Hospital Center. Supported in pal-t by grants from the National Institutes of Health and the American Heart Association. *Senior Investigator, New York Heart Association. **Career scientist of the Public Health Research Council of the City of New York (I-130) 958
Effects of clomiphene
0 BASELINE
25
I
50
on adrenal
cortical
steroidogencslr
Y5F
I
Fig. 1. Comparison of the effect of clomiphene and saline alone on the 17-OH c:orticosteroiG secretion rates in the dog adrenal before and after .I\CTH. The stippled bars represent the mean + SEM secretion rates before (base-line value; and after 3 doses of clomiphene and after ACTH. The open bars represent thP controls, thr baac-lirw p~~rinrl initiallv. thrt! aftt.r salin?: and after GCTH.
assessed iri the two followimg collection periods. The 17-OH corticostcroids content of the adrenal venous effluent was determined by the method of Peterson, Karrer, and Guerra.12 After subtraction of the steroid present irl the donor arterial blood, the secretion rates were then calculated. The plasma 17ketosteroids were measured according to the modified Drckter procedure,l” and the secretion rates similarly derived. Results
The wsults of 6 control experiments, with use of perfusion of saline alone, are presented in Fig. 1. It is evident that the mean cortirostcroid secretion rates fell gradually during the course of the experiments reachlng 74.1 per cent of the base-line level (p <0.05) and 65.9 per cent of the base-line at 30 to .45 minutes after level (p
base-line secretion rates were c~omparatlle to those of the control experiments. After each succrssivcx clorniphenr dose, small but conslstcnt clecrraws in 17-OH cortic-aster-aid secretion rvere obserl-(%d. These decreases r~rxre of similar magnitude to those lneasurcd in the c.ontrol rqwrimrnts. The incwases in 17OH corticosteroid secretion alter. AC’IH irl the clomiphene treated animals also WC’W not significantly different from the controls. ‘l’he responses of tfle 17-ketosteroid SWJTtion rates in thf* same plasmas, in whirlI the 17-OH corticostcroid secrrtion rates ww(’ measured. before and after clorniphenc~ anti .4GTH are giver1 ill Table I. No rha:~qv iI1 the mean 17-kctosteroid secretion raw afrct clonliphrnc or A(:‘rH \vas cvidcnt in ~‘oJI~paris \Gth the nlc’an baw-lincx src r.~~tioll rate. Comment
There arc some lines of cvidcncr \;hic,h sqgest that clomiphene may cxer‘t ;~n aiticm directly on ttlc adrrnal cortex. ‘The prilli:ipal argunlents for such an action might reslrlt fron I the folloMinq data :
5’60
Cushman
Table I. Effect
and
Apil 1, 1967 Am. J. Obst. & Gynec.
Hilton
of clomiphene
and ACTH
on corticosteroid
and ketosteroid
secretion
in dogs No. of dogs I. 17-OH corticosteroid Control studies Clomiphene II.
17-Ketosteroid Clomiphene
No. of observations
Base line
secretion rates (pg/min.) 6 4
24 16
‘p Cc 0.01 compared tp cc 0.05. :Not significant.
3
13
1 x IO-5 M Saline
1 x IO-4 M
1.80 + 0.30* 2.16+0.20*
2.00k0.28’
8.24 +- 1.15” 7.532 l.lO*
1.88?0.41#
1.83+0.53+
1.95+0.10?
ACTH
(mean It S.E.M.)
2.73 2 0.45 2.981T0.31
secretion rates (pg/min.)
I x IO-e M
2.40~0.19*
(mean ? S.E.M.) 2.16kO.40
2.28C0.51+
to base line.
ject may excrete more 17-ketosteroid and more 17-ketogenic steroids in the 24 hour urine after clomiphene therapy.g 2. The high incidence of inducing ovulation in patients with anovulation as a part of the Stein-Leventhal syndrome.6 Since, in the latter disorder, at least in some instances, abnormalities in adrenal cortical as well as ovarian function can be demonstrated, alteration of ovarian function might be a secondary result of an adrenal cortical effect. 3. The recent observation of Hagerman, Smith, and DayI that in placental tissue in vitro, enhancement of the availability of reduced triphosphopyridine nucleotide (TPNH) may occur with clomiphene. Since TPNH has long been recognized as an important and rate-limiting co-factor in steroid secretion,15 this observation might support a direct adrenal cortical action on steroid secretion if it were applicable to the adrenal cortex as well as to the placenta. Because of the difficulty of the intact subject in separating those actions which result from direct adrenal cortical influences from all others, isolated adrenal pouches of hypo-
physectomized dogs were employed. In this way the arteriovenous differences in steroid concentrations across the adrenal should reflect any acute adrenal cortical action of clomiphene. The mean 17-OH corticosteroid rates fell the control experiments progressively in probably as a consequence of reduction of some residual ACTH activity related to the surgery. No change was observed in this reduction in the mean 17-OH corticosteroid rates in the clomiphene experiments. Therefore, these studies indicate a lack of effect of clomiphene on this parameter of adrenal cortical function in the resting gland. No similar modifying effect of clomiphene was observed on the response to maximal ACTH stimulation. The lack of significant change in 17-ketosteroid secretion following clomiphene or ACTH is also noteworthy, since in the 17ketosteroid determination both the free and conjugated forms were measured. It appears unlikely, therefore, that clomiphene acts directly on the adrenal cortex.
REFERENCES
1. Roy, 2.
S., Mahesh,
B.: Acta endocrinol. Roy, S., Greenblatt, B.: Acta endocrinol.
V. B., and Greenblatt, 47: 645,
R. B., and Mahesh, 47: 657,
R.
5.
V.
6.
1964. 1964.
3. Roy, S., Mahesh, V. B., and Greenblatt, R. B.: Acta endocrinol. 47: 669, 1964. 4. Smith, 0. W., Smith, G. V., and Kistner, R. W.: J. A. M. A. 184: 878, 1963.
SMITH; 0. W.: AM. J. ORST. & GYNEC. 94: 440, 1966. Shearman, R. P., and Cox, R. I.: Obst. & Gynec. SUIT. 21: 1, 1966.
7. Cushman, P., Alter, 8, and Hilton, J. G.: Acta endocrinol. 50: 329, 1965. 8. Loraine, J. A.: Proc. Roy. Sot. Med. 59: 28,
1966.
9. 10.
1 I.
Effects
of
clomiphene
Pildrs. R. K.: AM. 1. ORST. & GYNEC. 166, 1965. Harkness, R. A., Bell, E. T., Loraine, J, and Morse, W. I.: J. Endocrinol. 31: 1964. Hilton, J. G., Weaver, D. C., Muelheims, Glaviano, V. V., and Wegria, R.: Am. I’hvsiol. 192: 525, 1958.
91:
12.
A., 53,
13. 14.
G., J.
15.
on
adrenal
cortical
steroidooetww
?tc;
Peterson. K. E., Karmr, A.. and (;tt~.rrn. 5. I,.: i\r~al. Chrm. 29: 144, 1957. Gohel, 0. J., Sobel, C., and tImw: K. .J: J. Clin. Endocrinol. 18: 522, 1958. Hagerman. D. P., Smith. 0. \V.. an i I):t) C!. F.: .4cta endocrinol. 51: 591, 19hti Hilf, R.: Ntw England J. Mrd. 27’i: -‘18. 196.5
CUSHMAN,
JAMES
G.
HILTON,
in dogs JR.,
M.D.* M.D.**
New York, New York
The mechanism of action of clomiphene, the ovulation inducing agent, remains uncertain. Most of the euidence has been interpreted to reflect a weak estrogen-like action and an estrogen inhibitory influence at higher concentrations. Direct adrenal cortical effects have also been postulated. The present study was undertaken to test the latter hypothesis by perfusing the isolated adrenal of the hypophysectomized dog with clomiphene. No alteration in either the resting 17-OH corticosteroid or 17ketosteroid secretion rates was obserued after 3 doses of clomiphene. It appears unlikely that clomiphene acts directly on the adrenal cortex.
collected in 5 minute periods. After four consecutive initial collection periods to establish base-line secretion, clomiphene was administered for 15 minutes into the arterial circuit to produce 1 x 10V6 M concentration. Ten minutes after completion of the low dose administration, 1 x 1O-5 M clomiphene was injected for 15 minutes. ‘l’en minutes later 1 x 10m4 M clomiphene was similarly perfused for 15 minutes. The responses to each dose employed were calculated from the average of the steroid secretion rates in 4 consecutive collection periods. At the end of each experiment, 25 units of bovine ACTH was injected to insure the functional viability of each preparation, which was reflected in the mean of the steroid secretion rates in the next two collection periods. In control studies, similar isolated adrenal pouch preparations were used. Into the arterial circuit of the perfusion system was injected identical volumes of isotonic NaCl. The responses to saline administration were calculated from the mean of the steroid secretion rates measured in four consecutive collection periods 15 to 60 minutes after completion of the base-line collection periods. At the end of each control experiment, 25 units of bovine ACTH was similarly injected and its effect on steroid secretion similarly
THE ME C H A N I S M Of action Of ClOmiphene, the ovulation-inducing agent, remains uncertain despite considerable recent investihas been presented for: gation. l-7 Evidence (a) a weak estrogen-like actionl, ‘; (b) an estrogen inhibitory action at higher concenand (c) a direct ovarian intration?; fluence.“, 5 A direct hypothalamic hypophyseal action has been described33 o and adrenal cortical49 8-1o effects have been postulated also. The present study was undertaken to test the latter hypothesis by perfusing the isolated dog adrenal with clomiphene. Materials
and
methods
In acutely hypophysectomized dogs, the isolated in situ adrenal pouch was prepared by the method of Hilton and associatesl and perfused at constant rates with arterial blood obtained from other hypophysectomized dogs. The venous effluent was From the Department of Medicine, St. Luke’s Hospital Center. Supported in pal-t by grants from the National Institutes of Health and the American Heart Association. *Senior Investigator, New York Heart Association. **Career scientist of the Public Health Research Council of the City of New York (I-130) 958
Effects of clomiphene
0 BASELINE
25
I
50
on adrenal
cortical
steroidogencslr
Y5F
I
Fig. 1. Comparison of the effect of clomiphene and saline alone on the 17-OH c:orticosteroiG secretion rates in the dog adrenal before and after .I\CTH. The stippled bars represent the mean + SEM secretion rates before (base-line value; and after 3 doses of clomiphene and after ACTH. The open bars represent thP controls, thr baac-lirw p~~rinrl initiallv. thrt! aftt.r salin?: and after GCTH.
assessed iri the two followimg collection periods. The 17-OH corticostcroids content of the adrenal venous effluent was determined by the method of Peterson, Karrer, and Guerra.12 After subtraction of the steroid present irl the donor arterial blood, the secretion rates were then calculated. The plasma 17ketosteroids were measured according to the modified Drckter procedure,l” and the secretion rates similarly derived. Results
The wsults of 6 control experiments, with use of perfusion of saline alone, are presented in Fig. 1. It is evident that the mean cortirostcroid secretion rates fell gradually during the course of the experiments reachlng 74.1 per cent of the base-line level (p <0.05) and 65.9 per cent of the base-line at 30 to .45 minutes after level (p
base-line secretion rates were c~omparatlle to those of the control experiments. After each succrssivcx clorniphenr dose, small but conslstcnt clecrraws in 17-OH cortic-aster-aid secretion rvere obserl-(%d. These decreases r~rxre of similar magnitude to those lneasurcd in the c.ontrol rqwrimrnts. The incwases in 17OH corticosteroid secretion alter. AC’IH irl the clomiphene treated animals also WC’W not significantly different from the controls. ‘l’he responses of tfle 17-ketosteroid SWJTtion rates in thf* same plasmas, in whirlI the 17-OH corticostcroid secrrtion rates ww(’ measured. before and after clorniphenc~ anti .4GTH are giver1 ill Table I. No rha:~qv iI1 the mean 17-kctosteroid secretion raw afrct clonliphrnc or A(:‘rH \vas cvidcnt in ~‘oJI~paris \Gth the nlc’an baw-lincx src r.~~tioll rate. Comment
There arc some lines of cvidcncr \;hic,h sqgest that clomiphene may cxer‘t ;~n aiticm directly on ttlc adrrnal cortex. ‘The prilli:ipal argunlents for such an action might reslrlt fron I the folloMinq data :
5’60
Cushman
Table I. Effect
and
Apil 1, 1967 Am. J. Obst. & Gynec.
Hilton
of clomiphene
and ACTH
on corticosteroid
and ketosteroid
secretion
in dogs No. of dogs I. 17-OH corticosteroid Control studies Clomiphene II.
17-Ketosteroid Clomiphene
No. of observations
Base line
secretion rates (pg/min.) 6 4
24 16
‘p Cc 0.01 compared tp cc 0.05. :Not significant.
3
13
1 x IO-5 M Saline
1 x IO-4 M
1.80 + 0.30* 2.16+0.20*
2.00k0.28’
8.24 +- 1.15” 7.532 l.lO*
1.88?0.41#
1.83+0.53+
1.95+0.10?
ACTH
(mean It S.E.M.)
2.73 2 0.45 2.981T0.31
secretion rates (pg/min.)
I x IO-e M
2.40~0.19*
(mean ? S.E.M.) 2.16kO.40
2.28C0.51+
to base line.
ject may excrete more 17-ketosteroid and more 17-ketogenic steroids in the 24 hour urine after clomiphene therapy.g 2. The high incidence of inducing ovulation in patients with anovulation as a part of the Stein-Leventhal syndrome.6 Since, in the latter disorder, at least in some instances, abnormalities in adrenal cortical as well as ovarian function can be demonstrated, alteration of ovarian function might be a secondary result of an adrenal cortical effect. 3. The recent observation of Hagerman, Smith, and DayI that in placental tissue in vitro, enhancement of the availability of reduced triphosphopyridine nucleotide (TPNH) may occur with clomiphene. Since TPNH has long been recognized as an important and rate-limiting co-factor in steroid secretion,15 this observation might support a direct adrenal cortical action on steroid secretion if it were applicable to the adrenal cortex as well as to the placenta. Because of the difficulty of the intact subject in separating those actions which result from direct adrenal cortical influences from all others, isolated adrenal pouches of hypo-
physectomized dogs were employed. In this way the arteriovenous differences in steroid concentrations across the adrenal should reflect any acute adrenal cortical action of clomiphene. The mean 17-OH corticosteroid rates fell the control experiments progressively in probably as a consequence of reduction of some residual ACTH activity related to the surgery. No change was observed in this reduction in the mean 17-OH corticosteroid rates in the clomiphene experiments. Therefore, these studies indicate a lack of effect of clomiphene on this parameter of adrenal cortical function in the resting gland. No similar modifying effect of clomiphene was observed on the response to maximal ACTH stimulation. The lack of significant change in 17-ketosteroid secretion following clomiphene or ACTH is also noteworthy, since in the 17ketosteroid determination both the free and conjugated forms were measured. It appears unlikely, therefore, that clomiphene acts directly on the adrenal cortex.
REFERENCES
1. Roy, 2.
S., Mahesh,
B.: Acta endocrinol. Roy, S., Greenblatt, B.: Acta endocrinol.
V. B., and Greenblatt, 47: 645,
R. B., and Mahesh, 47: 657,
R.
5.
V.
6.
1964. 1964.
3. Roy, S., Mahesh, V. B., and Greenblatt, R. B.: Acta endocrinol. 47: 669, 1964. 4. Smith, 0. W., Smith, G. V., and Kistner, R. W.: J. A. M. A. 184: 878, 1963.
SMITH; 0. W.: AM. J. ORST. & GYNEC. 94: 440, 1966. Shearman, R. P., and Cox, R. I.: Obst. & Gynec. SUIT. 21: 1, 1966.
7. Cushman, P., Alter, 8, and Hilton, J. G.: Acta endocrinol. 50: 329, 1965. 8. Loraine, J. A.: Proc. Roy. Sot. Med. 59: 28,
1966.
9. 10.
1 I.
Effects
of
clomiphene
Pildrs. R. K.: AM. 1. ORST. & GYNEC. 166, 1965. Harkness, R. A., Bell, E. T., Loraine, J, and Morse, W. I.: J. Endocrinol. 31: 1964. Hilton, J. G., Weaver, D. C., Muelheims, Glaviano, V. V., and Wegria, R.: Am. I’hvsiol. 192: 525, 1958.
91:
12.
A., 53,
13. 14.
G., J.
15.
on
adrenal
cortical
steroidooetww
?tc;
Peterson. K. E., Karmr, A.. and (;tt~.rrn. 5. I,.: i\r~al. Chrm. 29: 144, 1957. Gohel, 0. J., Sobel, C., and tImw: K. .J: J. Clin. Endocrinol. 18: 522, 1958. Hagerman. D. P., Smith. 0. \V.. an i I):t) C!. F.: .4cta endocrinol. 51: 591, 19hti Hilf, R.: Ntw England J. Mrd. 27’i: -‘18. 196.5