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Estrogenic potential of estriol
Estrogenic potential of estriol A comparison solvents
with
ARTHUR J.
of estrone,
and estriol
in aqueous
and lipoid
bioassay
L.
RICHARD
Baltimore,
estradiol,
HASKINS, HEBEL,
M.D. PH.D.
Maryland
The comparative estrogenicity of estriol, estronc, and estradiol in a lipoid solvent wax determined using the mouse modification of the Astwood uterine imbibition test. Estriol, es&one, and estradiol from doses of 0.001 mcg. to 20 mcg. induced similar uterine weight increases, thereby indicating similar or identical estrogenic potentials among the hormones. Cornparson of estriol in oil with estriol in water indicated that at dosage levels from 0.001 mcg. to 0.1 mcg. yielded similar uterine responses. In doses above 0.1 mcg. the effectiveness of aqueous estriol is significantly less than estriol in peanut oil.
estriol when in lipid and aqueous vents,4, 5, B this aspect of the problem also investigated.
f N A RE c E N T dinid study,l the eStrOgenicity of estriol and the specific polarity of the estrogenic effect on the human reproductive tract2 was evaluated. Estrogenicity was determined by vaginal cytology, ferning of the cervical mucous, estrogen and progestin withdrawal bleeding, and endometrial stimulation. Estriol was shown to have a low order of estrogenicity which was comparable to 1/I0 the estrogen effect of stilbestrol and yIoo of ethinyl estradiol. A specific polar response in the human reproductive tract to the estrogen could not be demonstrated. As a part of this study to re-evaluate the estrogenicity of estriol, comparative bioassays of estriol, estrone, and estradiol were carried out in our laboratories. This study was motivated by conflicting reports concerning the comparative estrogenicity of estriol, estrone, and estradiol.3 Since there are also contradictory reports concerning the effect of
From the Department Gynecology, University School of Medicine
of Obstetrics of Maryland
sol-
was
Methods and materials A modification of the Astwood 6 hour uterine response test was used for estrogen bioassay.? This modification consisted of the substitution of 21-day-old albino Swiss mice for rats. Control and test animals received 0.1 ml. of solvent by a single subcutaneous injection. Six hours following the administration of the test hormone the animal was killed. The uterus, tubes, and ovaries were removed and weighed. Crystalline estrone, estradiol, and estriol were utilized in the study. Each was assayed for chemical purity by ultraviolet spectroscopy. The oil solutions were made by the introduction of the steroid into peanut oil followed by vigorous agitation. Aqueous solutions of e,strone, estradiol, and estriol were obtained by the introduction of the steroid crystals into distilled water. After standing for 10 days at room texnperature,
and
202
Volume Number
106 2
Estrogenicity
5
6
7
8
9
10
of estriol
203
11
Body Wt. - gm. Fig. 1. Variation mature mouse.
c
in mouse uterine weight
0.001 Dose
of
compared to variation
0.01 hormone
Fig. 2. Variation estriol.
0.1
in body weight of the im-
1.0
10
- mcg.
in estimated uterine weight compared
the undissolved crystalline material was removed by filtration. The solutions were analyzed by ultraviolet spectroscopy. Findings Two hundred and seven mice were used in the study. The animals weighed between 5.4 and 11.9 grams. The uterine weights of 73 control mice were found to vary directly
to the dose of estrone, estradiol, and
with the body weight. This relationship is shown in Fig. 1. The curve was fitted by least squares. It may be represented by the formula y = 1.90 + 1.67 x, where y is the uterine weight and milligrams and x is the body -weight in g-rams. One hundred and eighty-six mice were given varied amounts of estrone, estradiol, and estriol in oil by subcutaneous injection.
204
Haskins
and
Hebel
0.001 Doseof hormone-mcg.
Amel-.
-
Januar-y 15, 1970 .I, Ohsrtt. Gym.
~~~0.01
0.1
1.0
IO
Fig. 3. Comparison of estimated uterine weight response to varying doses of estriol in water and estriol in peanut oil. Cross marks indicate 95 per cent confidence limits.
The estimated uterine weight gain resulting from steroid administration was obtained by subtracting the predicted, untreated uterine weight from the observed uterine weight. Using this basis for comparison, a dose-responsecurve was determined for each hormone (Fig. 2). An analysis of variance indicated that the uterine responsedid not differ significantly among the three steroids tested at dosagelevels of 0.001 mcg. to 10 mcg. Forty-eight mice were injected with aqueous sohrtions of estriol. Their uterine
REFERENCES
1. Naskins, A. L., Moszkowski, E. F., and Whitelock, V. P.: AM. J. OBST. & GYNEC. In press. 2. Puck, A. : Munchen med, Wchnschr. 99: 1, 1957. 3.
Merrill,
responseis compared to the uterine response to estriol in oil in Fig. 3. There is no significant difference in responseuntil a dose of 0.1 mcg. Beyond this dose level, estriol in oil is more effective than estrio1in water. The aqueous solubility of estrio1 at room temperature was found to be 23 mcg. per mi’lliliter by ultraviolet spectroscopy based on the optical density at 280 mp. The solubility of estriol and estrone was less than 1 mcg. per milliliter.
4. Burn, J. II., and Elphick, G. K.: Quart. J. Pharmacol. 5: 192, 1932. 5. Szego, C. M.: Fed. Proc. 9: 124, 1950. 6. Zondek, B., and Sulman, F.: Endocrinology 49: 417,
R. Cl.: Physiol. Rev. 38: 463, 1958.
1951.
7. Astwood, E. B.: Endocrinology
23: 25, 1938.
with
ARTHUR J.
of estrone,
and estriol
in aqueous
and lipoid
bioassay
L.
RICHARD
Baltimore,
estradiol,
HASKINS, HEBEL,
M.D. PH.D.
Maryland
The comparative estrogenicity of estriol, estronc, and estradiol in a lipoid solvent wax determined using the mouse modification of the Astwood uterine imbibition test. Estriol, es&one, and estradiol from doses of 0.001 mcg. to 20 mcg. induced similar uterine weight increases, thereby indicating similar or identical estrogenic potentials among the hormones. Cornparson of estriol in oil with estriol in water indicated that at dosage levels from 0.001 mcg. to 0.1 mcg. yielded similar uterine responses. In doses above 0.1 mcg. the effectiveness of aqueous estriol is significantly less than estriol in peanut oil.
estriol when in lipid and aqueous vents,4, 5, B this aspect of the problem also investigated.
f N A RE c E N T dinid study,l the eStrOgenicity of estriol and the specific polarity of the estrogenic effect on the human reproductive tract2 was evaluated. Estrogenicity was determined by vaginal cytology, ferning of the cervical mucous, estrogen and progestin withdrawal bleeding, and endometrial stimulation. Estriol was shown to have a low order of estrogenicity which was comparable to 1/I0 the estrogen effect of stilbestrol and yIoo of ethinyl estradiol. A specific polar response in the human reproductive tract to the estrogen could not be demonstrated. As a part of this study to re-evaluate the estrogenicity of estriol, comparative bioassays of estriol, estrone, and estradiol were carried out in our laboratories. This study was motivated by conflicting reports concerning the comparative estrogenicity of estriol, estrone, and estradiol.3 Since there are also contradictory reports concerning the effect of
From the Department Gynecology, University School of Medicine
of Obstetrics of Maryland
sol-
was
Methods and materials A modification of the Astwood 6 hour uterine response test was used for estrogen bioassay.? This modification consisted of the substitution of 21-day-old albino Swiss mice for rats. Control and test animals received 0.1 ml. of solvent by a single subcutaneous injection. Six hours following the administration of the test hormone the animal was killed. The uterus, tubes, and ovaries were removed and weighed. Crystalline estrone, estradiol, and estriol were utilized in the study. Each was assayed for chemical purity by ultraviolet spectroscopy. The oil solutions were made by the introduction of the steroid into peanut oil followed by vigorous agitation. Aqueous solutions of e,strone, estradiol, and estriol were obtained by the introduction of the steroid crystals into distilled water. After standing for 10 days at room texnperature,
and
202
Volume Number
106 2
Estrogenicity
5
6
7
8
9
10
of estriol
203
11
Body Wt. - gm. Fig. 1. Variation mature mouse.
c
in mouse uterine weight
0.001 Dose
of
compared to variation
0.01 hormone
Fig. 2. Variation estriol.
0.1
in body weight of the im-
1.0
10
- mcg.
in estimated uterine weight compared
the undissolved crystalline material was removed by filtration. The solutions were analyzed by ultraviolet spectroscopy. Findings Two hundred and seven mice were used in the study. The animals weighed between 5.4 and 11.9 grams. The uterine weights of 73 control mice were found to vary directly
to the dose of estrone, estradiol, and
with the body weight. This relationship is shown in Fig. 1. The curve was fitted by least squares. It may be represented by the formula y = 1.90 + 1.67 x, where y is the uterine weight and milligrams and x is the body -weight in g-rams. One hundred and eighty-six mice were given varied amounts of estrone, estradiol, and estriol in oil by subcutaneous injection.
204
Haskins
and
Hebel
0.001 Doseof hormone-mcg.
Amel-.
-
Januar-y 15, 1970 .I, Ohsrtt. Gym.
~~~0.01
0.1
1.0
IO
Fig. 3. Comparison of estimated uterine weight response to varying doses of estriol in water and estriol in peanut oil. Cross marks indicate 95 per cent confidence limits.
The estimated uterine weight gain resulting from steroid administration was obtained by subtracting the predicted, untreated uterine weight from the observed uterine weight. Using this basis for comparison, a dose-responsecurve was determined for each hormone (Fig. 2). An analysis of variance indicated that the uterine responsedid not differ significantly among the three steroids tested at dosagelevels of 0.001 mcg. to 10 mcg. Forty-eight mice were injected with aqueous sohrtions of estriol. Their uterine
REFERENCES
1. Naskins, A. L., Moszkowski, E. F., and Whitelock, V. P.: AM. J. OBST. & GYNEC. In press. 2. Puck, A. : Munchen med, Wchnschr. 99: 1, 1957. 3.
Merrill,
responseis compared to the uterine response to estriol in oil in Fig. 3. There is no significant difference in responseuntil a dose of 0.1 mcg. Beyond this dose level, estriol in oil is more effective than estrio1in water. The aqueous solubility of estrio1 at room temperature was found to be 23 mcg. per mi’lliliter by ultraviolet spectroscopy based on the optical density at 280 mp. The solubility of estriol and estrone was less than 1 mcg. per milliliter.
4. Burn, J. II., and Elphick, G. K.: Quart. J. Pharmacol. 5: 192, 1932. 5. Szego, C. M.: Fed. Proc. 9: 124, 1950. 6. Zondek, B., and Sulman, F.: Endocrinology 49: 417,
R. Cl.: Physiol. Rev. 38: 463, 1958.
1951.
7. Astwood, E. B.: Endocrinology
23: 25, 1938.