Inhibition of Rat Ovarian 3Β-Hydroxysteroid Dehydrogenase
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Endocrinol. Japon. 1989, 36 (3), 387-394 Inhibition of Rat Ovarian 3ƒÀ-Hydroxysteroid Dehydrogenase (3ƒÀ-HSD), 17ƒ¿-Hydroxylase and 17,20 Lyase by Progestins and Danazol SATOKO ARAKAWA, MIZUE MITSUMA, MASATO IYO, RYOICHI OHKAWA, AKIRA KAMBEGAWA, SHoicm OKINAGA AND KIYOSHI ARAI Department of Obstetrics and Gynecology, Teikyo University School of Medicine 2-11-1 Kaga Itabashi-ku, Tokyo, Japan Abstract The site of action of synthetic progestins or danazol in the treatment of endometriosis is considered to be mainly the hypothalamo-pituitary level, but the direct action to the uterine endometrium and the ovary is also suggested. We investigated the effect of these synthetic steroids to rat ovarian steroido- genic enzymes. The effect of norethisterone, levonorgestrel, danazol, gestri- none, desogestrel and 3-keto-desogestrel was studied in vitro. The sources of the enzymes were prepared from ovaries of immature rats treated either with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) for 3ƒÀ-hydroxy steroid dehydrogenase (3ƒÀ-HSD), or with PMS for 17ƒ¿- hydroxylase and 17,20 lyase. The substrates used were pregnenolone (P5) for 3ƒÀ-HSD, progesterone (P4) for 17ƒ¿-hydroxylase, and 17ƒ¿-hydroxy-progesterone (17ƒ¿-OH-P4) for 17,20 lyase. The substrates were incubated with the enzyme sources and coenzymes, and the products formed were measured. All the steroids inhibited 3ƒÀ-HSD, and the inhibition by gestrinone (Ki= 3.0ƒÊM) and 3-keto-desogestrel (17.5ƒÊM) was particularly marked. Only desogestrel (Ki= 30.3ƒÊM) and danazol (168ƒÊM) inhibited 17ƒ¿-hydroxylase. All the steroids inhibited 17,20 lyase, and the inhibition by desogestrel (Ki= 0.70ƒÊM), danazol (0.80ƒÊM), and gestrinone (30ƒÊM) was particularly marked. The site of action of synthetic progestins and danazol (Barbieri et al.; 1977, Barbieri and danazol in the treatment of endo- et al.; 1981) inhibit ovarian enzymes di- metriosis is considered to be mainly the rectly. In the following study, we investi- hypothalamo-pituitary level (Franchimont gated the effect of synthetic steroids on rat and Cramilion, 1977; Franchimont et al., ovarian 3ƒÀ-hydroxy steroid dehydrogenase 1970; McDonald and Gilmore, 1971; (3ƒÀ-HSD), 17ƒ¿-hydroxylase, and 17,20 Cullberg et al., 1982; Garmendia et al., lyase. 1976). But the direct effect of synthetic steroids on ovarian enzymes was also re- ported. Norethisterone (Shinada et al., Materials and Methods 1978;Johansson, 1971), levonorgestrel (Mukherjee et al.; 1972, Johansson; 1971) Chemical Received January 20, 1989 [4-14C]-pregnenolone (P5) (57.2 mCi/mmol), Endocrinol. Japon. 388 ARAKAWA et al. June 1989 [4-14C]-progesterone (P4) (57.2 mCi/mmol),[4-14C]- hibitor. For assays of 17ƒ¿-hydroxylase and 17ƒ¿-hydroxy progesterone (17ƒ¿-OH-P4) (50 mCi/ 17,20 lyase, each incubation mixture (1ml) con- mmol), [1, 2, 6, 7-3H(N)]-P4 (106.8 Ci/mmol) and tained a preparation of tissue from PMS treated 17ƒ¿-[1, 2-3H(N)] hydroxy P4 (57.4 Ci/mmol) were rats (12-15ƒÊg protein), 0.5ƒÊmol of NADPH, 14C- or 3H-steroid (about 0 purchased from New England Nuclear Corp. .03ƒÊCi, 0.1-4 mol) (Boston, MA, USA). NAD and NADPH were and an inhibitor. Labelled P4 was employed the products of Boehringer (Mannheim, Ger- for a 17ƒ¿-hydroxylase assay, and labelled 17ƒ¿- many). Norethisterone, levonorgestrel, pregnant OH-P4 for a 17, 20 lyase assay. The mixture mare serum gonadotropin (PMS) and human was incubated in a Dubnoff-type incubator at chorionic gonadotropin (hCG) were provided 37•Ž for various time intervals. The incubation by Teikoku Hormone Co.(Japan, Tokyo). was terminated by chilling in an ice bath. Desogestrel and 3-keto-desogestrel were a gift Steroids were extracted with ether (5ml). A from Organon Co.(Holland). Danazol was mixture of nonradioactive carrier steroids was provided by Tokyo Tanabe (Japan), and gestri- added to each extract, which was then applied none by Roussel Uclaf (Paris, France). All to a silica gel thin layer plate and developed chemicals and reagents used were of analytical in a solvent system of benzene-acetone (4:1, grade. v/v). Steroids which were not separable in this system were sub jected to further thin layer Preparation of enzymes chromatography in the following solvent system: Immature female rats of the Wistar-Imamichi cyclohexane-ethyl acetate (1:1, v/v) was employ- strain were used. Ten IU of PMS was injected ed for the separations of androstenedione from subcutaneously (s. c.) at 9:00 h, on the 23rd day 3ƒ¿-hydroxy-5ƒ¿-pregnan-20-one and of 17ƒ¿-OH- of life. PMS treated rats were decapitated at P4 from 3ƒÀ-hydroxy-5ƒ¿-pregnan-20-one. Andro- 17:00 h two days later. Another group of rats stenedione and 17ƒ¿-hydroxy-5ƒ¿-pregnane-3,20- were given an injection of 30 IU of hCG at dione were completely separated by developing 14:00 h two days after PMS injection, and killed the plate three times in ethyl acetate-n-heptane three hours after the last treatment. Ovaries (2:5, v/v). Spots of the carrier on thin layer were homogenized in a teflon glass homogenizer chromatograms were located under an UV light with 0.25 M sucrose/0.05 M Tris HC1 buffer and (254 nm) or by being exposed to iodine vapor. then centrifuged at •~ 800 g for 20 min. The The 14C-spots were detected by autoradiography. supernatant was used as enzyme sources. Pro- Each spot was scraped off the plate, added with tein in the supernatant was determined by the 0.1ml methanol and 3ml toluene scintillator, Bradford's method (Bradford, 1976). and the radio-activity was measured in a liquid scintillation spectrometer (Aloka LSC 703). The Enzyme assay recovery of steroids was 55-70%. For an assay of 3ƒÀ-HSD, each incubation The enzyme activity was expressed as the mixture (1ml) contained a preparation of tissue sum of metabolites formed from radioactive from PMS-hCG treated rats (2.5-100ƒÊl of the substrates. Some of the results were analyzed enzyme; 1.5-61.1ƒÊg protein), 0.5ƒÊmol of NAD, by the method of Lineweaver-Burk (Lineweaver 14C-P5 (about 0 .03ƒÊCi, 1-100 nmol), and an in- and Burk, 1934). Table 1. Title;Identification of radioactive metabolites formed by repeated recrystallizations to constant specific radioactivities. Regend; Methanol-chloroform or methanol-water were used as the recrystallization solvent. Vol. 36, No. 3 INHIBITION OF OVARIAN ENZYME 389 Fig. 1a Statistical Analysis Statistical analysis of the results was done by Student's t-test, and a P value of less than 0.05 was considered significant. Results Autoradiography 3ƒÀ-HSD: A metabolite formed from P5 was only P4 under the experimental condi- tions used and the 3ƒÀ-HSD activity was expressed by the amount of P4 17ƒ¿-hy- droxylase: Metabolites formed from P4 were Fig. 1b 17ƒ¿-OH-P4, androstenedione and a small amount of other steroids. The 17ƒ¿-hy- droxylase activity was expressed as the sum of 17ƒ¿-OH-P4 and androstenedione formed. 17, 20 lyase: Metabolites from 17ƒ¿-OH-P4 were androstenedione and a small amount of other steroids. The 17, 20 lyase activity was expressed by the amount of andros- tenedione. These metabolites were crystallized re- peatedly with the corresponding standard steroids. Constant specific radioactivity of the crystals is shown in Table 1. Effect of incubation time and volume of the tissue preparation Fig. 1c Fig. la shows the relation between the product (P4) formed and the amount of enzyme in the 3ƒÀ-HSD assay. The con- centration of the substrate was 10ƒÊM, and the incubation time was 20 min. Fig. 1b and c show the relation between the amount of products formed and the incubation time in 17ƒ¿-hydroxylase and 17, 20 lyase. The concentration of the substrate was 1ƒÊM and the amount of the enzyme was 25ƒÊl (16.2ƒÊg protein). A subsequent study was performed in which the enzymatic reaction was not saturated. For the experiment on 3ƒÀ-HSD, 25ƒÊl of the enzyme (15.3ƒÊg Fig. 1. Title; Effects of the amount of the tissue preparation used for a) 3ƒÀ-HSD protein) was incubated for 20 min. For assay and of incubation time for b) 17ƒ¿- 17ƒ¿-hydroxylase, 25ƒÊl of the enzyme was hydroxylase and c) 17, 20 lyase. incubated for 10 min. For 17, 20 lyase, the Endocrinol. Japon. 390 ARAKAWA et al. June 1989 *P<0 .05 Fig.2 c **P<0. 01 ***P<0 .001 *P<0 .05 Fig.2 b **P<0 .01 *P<0 .05 Fig .2 a Fig. 2. Title; Inhibition of a) 3ƒÀ-HSD, b) 17 ƒ¿-hydroxylase and c) 17,20 lyase by various steroids. Regend; Vertical bars indicate mean•}standard error. 391 Vol.36, No.3 INHIBITION OF OVARIAN ENZYME Fig. 3a Fig. 3c LEVONORGESTREL PROGESIERONE NORETHISTERONE NOFIETHISTERONE 100μM 20μM 20μM 100μM LEVONORGESTREL DESOGESTREL 3KETO-DESOGESTREL 20μM 20μM DESOGESTREL 100μM 2μM GESTRINONE 3KETO-DESOGESTREL 2μM 20μM DANAZOL GESTPINONE 2μM 100μM DANAZOL 20μM Fig. 3. Title; Lineweaver Burk's plot in the inhibition study of a) 3ƒÀ-HSD, b) 17ƒ¿-hydroxylase and c) 17,20 lyase by various steroids. 3b DESOGESTREL DANAZOL ficantly when the concentration was 20μM enzyme volume was 20 or 25ƒÊl and the incubation time was 16 or 20min. (Fig. 2b). Most of the steroids inhibited 17,20 lyase significantly (Fig. 2c). Inhibition of enzymes by steroids Fig. 3 shows Lineweaver Burk's plot Fig. 2a shows the effect of various indicating the inhibitory effects of steroids steroids on 3ƒÀ-HSD activity. All steroids on enzymes. Table 2 summarizes the inhibited 3ƒÀ-HSD significantly when 20ƒÊM results of the kinetic studies on steroida- was used. However, no steroid, except genic enzymes. The inhibition of 3ƒÀ-HSD desogestrel, inhibited 17ƒ¿-hydroxylase signi- by gestrinone (Ki = 3.0ƒÊM) and 3-keto- Endocrinol.