EFFECTS OF CYPROTERONE AND CYPROTERONE ACETATE ON THE ADRENAL GLAND IN THE RAT: STUDIES IN VIVO AND IN VITRO N. S. PANESAR, *
Division
D. G. HERRIES
AND
S. R. STITCH
of Steroid Endocrinology, Department of Chemical Pathology and f Department of Biochemistry, University of Leeds, Leeds, LS2 9LN
(Received 26 May 1978) SUMMARY
Male Wistar rats were treated for three weeks with cyproterone (1\m=.\7 or 5\m=.\1 mg/day) or cyproterone acetate (2 or 6 mg/day). The adrenal weights of animals treated with either dose of cyproterone acetate were significantly less (P < 0\m=.\001) than those of untreated animals. In contrast, the adrenal weights of animals treated with cyproterone did not differ from those
of the controls. The concentrations of corticosterone in the plasma were significantly less (P < 0\m=.\001) in both groups treated with cyproterone acetate compared with those of the controls; only the higher dose of cyproterone reduced the plasma concentration of corticosterone (P < 0\m=.\001). Cyproterone acetate inhibited the rat adrenal 3\g=b\-hydroxysteroiddehydrogenase\p=n-\5-ene,4\x=req-\ ene-isomerase complex in vitro, with both pregnenolone and dehydroepiandrosterone as substrates. Analysis of the reaction rates suggested an uncompetitive mode of inhibition. These results suggest that in rats the antiandrogens cyproterone and cyproterone acetate may provoke adrenal insufficiency by inhibition of steroid biosynthesis. Furthermore, indirect evidence from the mass and morphology of the adrenal suggests that cyproterone acetate may also suppress production or secretion of ACTH by the pituitary gland. INTRODUCTION
Both cyproterone (l,2a-methylene-6-chIoro-17a-hydroxy-4,6-pregnadiene-3,20-dione) and cyproterone acetate (l,2a-methylene-6-chloro-17a-acetoxy-4,6-pregnadiene-3,20-dione) are
inhibitors of androgen action (Neumann, von Berswordt-Wallrabe, Elger, Steinbeck, Hahn «fe Kramer, 1970). The latter steroid (Androcur, Schering A.G., Berlin) is used clinically for treatment of various androgen-dependent disorders such as hypersexuality, precocious puberty and hirsutism. The atrophie effects of cyproterone acetate on the adrenal glands of animals have been reported by several authors (Neri, Monahan, Meyer, Afonso «fe Tabachnik, 1967; Denef, Vendeputte «fe de Moor, 1968; Starka, Motlik «fe Schreiber, 1972). Zieger, Lux «fe Kubatch (1976) reported extensive cytological atrophy of the zona fasciculata and zona reticularis after administration of cyproterone acetate to hamsters. The reports concerning the effects of cyproterone acetate on the human pituitary-adrenal system are conflicting. For example, Girard & Baumann (1976) reported adrenal insufficiency after administration of cyproterone acetate to young children. In contrast Smals, Kloppenborg, Goverde & Benraad (1978) reported that cyproterone acetate had no effect on the pituitary-adrenal system of hirsute women.
In the present
study
the direct inhibition of the
3ß-hydroxysteroid dehydrogenase-5-
% Present address : Department of Pathology, University of Leeds, Leeds, LS2 9JT.
ene,4-ene-isomerase complex (3ß-HSD-5-ene,4-ene-isomerase) in the
rat adrenal
gland by
cyproterone acetate in vitro, the effects of cyproterone and cyproterone acetate on the adrenal
glands of rats treated in vivo and the plasma concentrations of corticosterone in rats treated with cyproterone and cyproterone acetate have been investigated. The study was restricted to male rats to avoid the complication of interpretation of results which might have arisen from using female rats. MATERIALS
Reagents
(NAD) was obtained from Sigma (London) Chemical Company Ltd, Kingston-upon-Thames, Surrey. [la-2a(n)-3H]Corticosterone (sp. act. 45 Ci/mmol), [7(n)-3H]pregnenolone (sp. act. 18-6 Ci/mmol), [7(n)-3H]dehydroepiandrosterone (DHA, sp. act. 500 Ci/mol), [4-14C]progesterone (sp. act. 61 Ci/mol) and [4-14C]androstenedione (sp. act. 60 Ci/mol) were all obtained from The Radiochemical Centre, Amersham, Bucks. Labelled steroids were purified by paper chromatography with petroleum ether (b.p. 60-80 °C) : methanol : water (10 : 8 : 2, by vol., Bush A system). Non-radioactive androstenedione, corticosterone, pregnenolone and progesterone were obtained from Steraloids, Croydon, London, dehydroepiandrosterone was purchased from Nicotinamide adenine dinucleotide
Organon Laboratories Ltd, Morden, Surrey and cyproterone and cyproterone acetate were gifts from Schering A.G., Berlin, Germany. Organic solvents of analytical grade were used as supplied. Scintillators a consisted of 0-4% solution of 2-(4-/-butylphenyl)-5(4-biphenyl) 1,3,4Butyl PBD-scintillator oxidoazole in toluene. Butyl PBD was purchased from Ciba Ltd, Duxford, Cambridge. Triton X-100 scintillator cocktail contained 1 g p-bis [2-(5-phenyloxazole)]-benzene (POPOP), 15 g 2,5-diphenyloxazole (PPO), 1250 ml Triton X-100 and 3750 ml xylene. This scintillator was used for counting aqueous samples. Both fluors were purchased from Packard Instruments Ltd, Reading. Materials for preparation of adrenal cytology The fixing solution was 10% formalin in saline. Paraffin wax was used for sections were stained with haematoxylin and eosin.
embedding and
Buffer Phosphosaline buffer (pH 7-4) contained equal parts of 0-1 mol phosphate buffer and 0-154 mol NaCl/1. Animals Animal Male Wistar rats bred locally at the House, Medical School, Leeds were used in these studies. The animals were maintained on a lighting schedule of 12 h light : 12 h darkness and supplied with food and water ad libitum. METHODS
Effects of cyproterone acetate on the 3$-hydroxysteroid dehydrogenase-5-ene,4-ene-isomerase complex in vitro The effects of cyproterone acetate on the 3ß-HSD-5-ene,4-ene-isomerase complex were determined by the method of Goldman (1967) for the spectrophotometric assay (study i). This involved conversion of a 5-ene-3ß-steroid by the 3ß-HSD-5-ene,4-ene-isomerase complex to a u.v.-absorbing (244 nm) 4-ene-3-oxosteroid. To increase the accuracy of the assay further studies (see below) were carried out with radioactively labelled substrates (studies ii and iii).
Preparation of the tissue homogenate The adrenals from three Wistar rats were excised, weighed and homogenized in a PotterElvehjem glass homogenizer for 5 min at 0-4 °C. The concentration of tissue in the homo¬ genate was adjusted to 5 mg/ml phosphosaline buffer before centrifugation at 2500 g for 10 min at 0 °C. The supernatant fraction was decanted and used for the reaction. The assay The assay system (2-0 ml) comprised 0-25 ml adrenal homogenate, 8-0 µ NAD and various quantities of either pregnenolone or DHA (7-9, 15-8, 23-7, 31-6, 47-4 or 63-1 nmol) dissolved in 25 µ ethanol. To improve the accuracy of the assay for studies (ii) and (iii), radioactive steroids were added to obtain final specific activities of 3-22 Ci/mol for pregneno¬ lone and 3-34 Ci/mol for DHA. The quantities of cyproterone acetate (solubilized in 25 µ ethanol) employed were: study i, 0, 8 nmol (4 µ / ) and 16 nmol (8 µ / ); study ii, 0, 3 nmol (1·5µ >1/1), 6 nmol (3 µ / ) and 12 nmol (6 µ / ); study iii, 0, 1-2 nmol (0-6 µ / ) and 2-4 nmol (1·2µ 1/1). Control assays were run without cyproterone acetate. (In setting up the assay using Goldman's (1967) method, it was found necessary to employ supraphysiological quantities of substrates to detect the products with a
spectrophotometer.)
The reaction was started by the addition of 0-25 ml of the homogenate. The incubations carried out for 20 min at 37 °C, with constant shaking. The reaction was stopped by placing the incubating vessel in ice and adding glacial acetic acid (0-2 ml). Tracer amounts (approximately 18 000 disintegrations/min) of [14C]progesterone and [14C]androstenedione were added (studies ii and iii) to appropriate incubations, together with excess unlabelled steroids which acted as carriers. The steroids were extracted with 10 ml ethyl acetate and the extract was dried overnight over anhydrous sodium sulphate. The solvent was decanted and evaporated under N2. The residue was chromatographed with the Bush A system. In studies ii and iii the products were further chromatographed in a Bush A system, and then in a t.l.c. system (benzene : ethanol, 98 : 2, v/v) on the impregnated t.l.c. plates (Gelman Hawkesley). The products were located with u.v. light and with a radiochromatogram strip scanner (Nuclear Chicago Actigraph III) or a spark-chamber (Birchover Instruments Ltd). The steroids were eluted from the chromatograms and 3H : 14C ratios determined (3H : 14C ratios remained constant within + 2% throughout Chromatographie purifications), and the amount of product formed was calculated. In separate studies it was found that in the presence of NAD and NADPH, DHA was mainly metabolized to androst-4-ene-3,17-dione (35-37%). Very little (< 1%) testosterone was produced. With pregnenolone as substrate and in the presence of NAD and NADPH, progesterone (35%) and deoxycorticosterone (25%) were the major metabolites. However, in the absence of NADPH (the present studies) progesterone was the only product detected. were
Protein determination Protein estimated by determining the amount of nitrogen liberated from the micro-Dumas method (Gustin, 1960). a using was
sample,
Analysis of kinetic data examined in the form of double-reciprocal (LineweaverInitial velocity data were first rate law (Cleland, 1963a, b, c). The data were then determine the to Burk) plots appropriate fitted to the corresponding rate equations using a computer program written by one of us for use in the ICL 1906A computer installation at Leeds University. These programs performed calculations based on the methods of Cleland (1967). The initial velocity data for various concentrations of an inhibitor and a single substrate conforming to linear uncompetitive and
linear
non-competitive inhibition were fitted to equations (1) and (2) respectively: VA VA V=
(1+ / + (1+
«
°°™°mP^>
where A and /are the concentrations of substrate and inhibitor respectively, Kis the maximum velocity, K& is the Michaelis constant for A, and Kit Ka are inhibitor constants for a particu¬ lar inhibitor, respectively affecting the slope and intercept of double reciprocal plots. The rate equation giving the best fit to the experimental data was chosen as that with least variance, calculated as the average square of the difference between an experimental velocity and the corresponding value derived from rate equations incorporating the values of the kinetic constants. In studies involving labelled substrates, it was found that data fitted to the non-competitive inhibition equation (2) yielded K¡ values less than 0 which are theoretically impossible. However, when fitted to the uncompetitive inhibition equation (1) all values were greater than 0, thus favouring uncompetitive inhibition.
Studies in vivo Fifty Wistar rats (180-220 g) were randomly divided into five groups often animals (housed in pairs) and designated and treated as follows : group A, control rats injected with 0-2 ml vehicle daily (vehicle =4:1 (v/v) castor oil : benzyl benzoate); group B, 1-7 mg (4-8 µ ) cyproterone/day; group C, 5-1 mg (14-4 µ ) cyproterone/day ; group D, 2 mg (4-8 µ )
cyproterone acetate/day; group E, 6 mg (14·4 µ
) cyproterone acetate/day.
All drugs were administered subcutaneously in 0-2 ml vehicle for 3 weeks. The animals were allowed free access to water and food. All animals were weighed at the start and the end of the treatment period. Twenty-four hours after the last injection the animals were decapi¬ tated within 5 s of stunning. Blood was collected into cold (0 °C) heparinized tubes main¬ tained in an ice bath. The blood was centrifuged (800 g) at 4 °C, and the plasma stored as small samples at —20 °C until assayed for corticosterone. Adrenal glands were dissected and weighed. They were then fixed in 10% formalin, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Determination
of corticosterone in plasma Plasma corticosterone was determined by radioimmunoassay according to the method of Underwood «fe Williams (1972) using antiserum raised against corticosterone-21-hemisuccinate. The intra-assay coefficient of variation was < 4% and the interassay coefficient of variation was < 8%. Data
were
significant.
analysed statistically using
Statistics Student's r-test.
values of
1/1; A, 6-0 µ / ) of cyproterone acetate. The points on the graph are actual values obtained in the reactions, is the initial velocity; A is the substrate concentration. For DHA, the broken line is perhaps a truer representation of the reaction, because the inhibitor concentration may be higher than the value used by the computer, i.e. 3 µ -cyproterone acetate.
J-
'
_L 1/µ A
1/µ 1
Text-fig. 3. Computer-derived Lineweaver-Burk plots for the reactions pregnenolone to progesterone (a) and dehydroepiandrosterone (DHA) to androst-4-ene-3,17-dione (b) using labelled substrates (study iii) in the absence [ (a), + (b)] or presence [ (a), A (b), 0-6 µ / ; A (a) · (b), 1 -2 µ / ] of cyproterone acetate. The points on the graphs are actual values obtained in the reactions, is the initial velocity; A is the substrate concentration. See Table 1 for Ku values.
Table 1. Values ofKa and Ku
(mean ± S.D.) as calculated by the computer for the 3$-HSD-5-
ene,4-ene-isomerase complex of the rat adrenal gland
Study no.*
Assay Spectro-
pregnenolone
Inhibitor cyproterone
Radiolabel
pregnenolone
cyproterone
photometric
Substrate
9-1+2-98
105 Ku (mol/1) 86 ±0-023
6-45 ±2-01
0-045 ±0-011
1·04±0·22
0·18±0 24
25-5 ±12-5
0-031 ±0-015
105
Ä^mol/l)
acetate acetate
Radiolabel Radiolabel
cyproterone dehydroepiandrosterone acetate cyproterone pregnenolone acetate
Radiolabel
l-36±0-32 dehydrocyproterone epiandrosterone acetate K& is the Michaelis constant for the substrate. is the inhibitor constant. A^ii * See text for details of studies.
0-33±0-14
yielded a series of parallel lines, which are characteristic of the uncompetitive type of inhibition. The Kn values for each of the assays are given in Table 1. acetate
Effects of cyproterone and cyproterone acetate on the adrenal glands Aa shows the effects of cyproterone and cyproterone acetate on the weight of glands. Both doses of cyproterone acetate caused a marked reduction in adrenal weight (P< 0-001) compared with that of the controls. However, cyproterone had very little effect on adrenal weight (Text-fig. Aa). The Plate shows cross-sections through the adrenal cortices of rats with or without cyproterone or cyproterone acetate treatment. Table 2 gives relative widths of cortex and zona glomerulosa, and the number of nuclei per unit area in the inner two zones of a typical section from each of the groups. Cyproterone acetate but not cyproterone caused atrophy of the zona fasciculata and zona reticularis (zones normally under adrenocorticotrophic hormone (ACTH) control) whereas the zona glomeru¬ losa (normally independent of ACTH control Bergner «fe Deane, 1948) appeared to be
Text-figure
the adrenal
unaffected.
-
(b) 150
(a)
30
100 oo
20
«
50 •o
10-
Division
D. G. HERRIES
AND
S. R. STITCH
of Steroid Endocrinology, Department of Chemical Pathology and f Department of Biochemistry, University of Leeds, Leeds, LS2 9LN
(Received 26 May 1978) SUMMARY
Male Wistar rats were treated for three weeks with cyproterone (1\m=.\7 or 5\m=.\1 mg/day) or cyproterone acetate (2 or 6 mg/day). The adrenal weights of animals treated with either dose of cyproterone acetate were significantly less (P < 0\m=.\001) than those of untreated animals. In contrast, the adrenal weights of animals treated with cyproterone did not differ from those
of the controls. The concentrations of corticosterone in the plasma were significantly less (P < 0\m=.\001) in both groups treated with cyproterone acetate compared with those of the controls; only the higher dose of cyproterone reduced the plasma concentration of corticosterone (P < 0\m=.\001). Cyproterone acetate inhibited the rat adrenal 3\g=b\-hydroxysteroiddehydrogenase\p=n-\5-ene,4\x=req-\ ene-isomerase complex in vitro, with both pregnenolone and dehydroepiandrosterone as substrates. Analysis of the reaction rates suggested an uncompetitive mode of inhibition. These results suggest that in rats the antiandrogens cyproterone and cyproterone acetate may provoke adrenal insufficiency by inhibition of steroid biosynthesis. Furthermore, indirect evidence from the mass and morphology of the adrenal suggests that cyproterone acetate may also suppress production or secretion of ACTH by the pituitary gland. INTRODUCTION
Both cyproterone (l,2a-methylene-6-chIoro-17a-hydroxy-4,6-pregnadiene-3,20-dione) and cyproterone acetate (l,2a-methylene-6-chloro-17a-acetoxy-4,6-pregnadiene-3,20-dione) are
inhibitors of androgen action (Neumann, von Berswordt-Wallrabe, Elger, Steinbeck, Hahn «fe Kramer, 1970). The latter steroid (Androcur, Schering A.G., Berlin) is used clinically for treatment of various androgen-dependent disorders such as hypersexuality, precocious puberty and hirsutism. The atrophie effects of cyproterone acetate on the adrenal glands of animals have been reported by several authors (Neri, Monahan, Meyer, Afonso «fe Tabachnik, 1967; Denef, Vendeputte «fe de Moor, 1968; Starka, Motlik «fe Schreiber, 1972). Zieger, Lux «fe Kubatch (1976) reported extensive cytological atrophy of the zona fasciculata and zona reticularis after administration of cyproterone acetate to hamsters. The reports concerning the effects of cyproterone acetate on the human pituitary-adrenal system are conflicting. For example, Girard & Baumann (1976) reported adrenal insufficiency after administration of cyproterone acetate to young children. In contrast Smals, Kloppenborg, Goverde & Benraad (1978) reported that cyproterone acetate had no effect on the pituitary-adrenal system of hirsute women.
In the present
study
the direct inhibition of the
3ß-hydroxysteroid dehydrogenase-5-
% Present address : Department of Pathology, University of Leeds, Leeds, LS2 9JT.
ene,4-ene-isomerase complex (3ß-HSD-5-ene,4-ene-isomerase) in the
rat adrenal
gland by
cyproterone acetate in vitro, the effects of cyproterone and cyproterone acetate on the adrenal
glands of rats treated in vivo and the plasma concentrations of corticosterone in rats treated with cyproterone and cyproterone acetate have been investigated. The study was restricted to male rats to avoid the complication of interpretation of results which might have arisen from using female rats. MATERIALS
Reagents
(NAD) was obtained from Sigma (London) Chemical Company Ltd, Kingston-upon-Thames, Surrey. [la-2a(n)-3H]Corticosterone (sp. act. 45 Ci/mmol), [7(n)-3H]pregnenolone (sp. act. 18-6 Ci/mmol), [7(n)-3H]dehydroepiandrosterone (DHA, sp. act. 500 Ci/mol), [4-14C]progesterone (sp. act. 61 Ci/mol) and [4-14C]androstenedione (sp. act. 60 Ci/mol) were all obtained from The Radiochemical Centre, Amersham, Bucks. Labelled steroids were purified by paper chromatography with petroleum ether (b.p. 60-80 °C) : methanol : water (10 : 8 : 2, by vol., Bush A system). Non-radioactive androstenedione, corticosterone, pregnenolone and progesterone were obtained from Steraloids, Croydon, London, dehydroepiandrosterone was purchased from Nicotinamide adenine dinucleotide
Organon Laboratories Ltd, Morden, Surrey and cyproterone and cyproterone acetate were gifts from Schering A.G., Berlin, Germany. Organic solvents of analytical grade were used as supplied. Scintillators a consisted of 0-4% solution of 2-(4-/-butylphenyl)-5(4-biphenyl) 1,3,4Butyl PBD-scintillator oxidoazole in toluene. Butyl PBD was purchased from Ciba Ltd, Duxford, Cambridge. Triton X-100 scintillator cocktail contained 1 g p-bis [2-(5-phenyloxazole)]-benzene (POPOP), 15 g 2,5-diphenyloxazole (PPO), 1250 ml Triton X-100 and 3750 ml xylene. This scintillator was used for counting aqueous samples. Both fluors were purchased from Packard Instruments Ltd, Reading. Materials for preparation of adrenal cytology The fixing solution was 10% formalin in saline. Paraffin wax was used for sections were stained with haematoxylin and eosin.
embedding and
Buffer Phosphosaline buffer (pH 7-4) contained equal parts of 0-1 mol phosphate buffer and 0-154 mol NaCl/1. Animals Animal Male Wistar rats bred locally at the House, Medical School, Leeds were used in these studies. The animals were maintained on a lighting schedule of 12 h light : 12 h darkness and supplied with food and water ad libitum. METHODS
Effects of cyproterone acetate on the 3$-hydroxysteroid dehydrogenase-5-ene,4-ene-isomerase complex in vitro The effects of cyproterone acetate on the 3ß-HSD-5-ene,4-ene-isomerase complex were determined by the method of Goldman (1967) for the spectrophotometric assay (study i). This involved conversion of a 5-ene-3ß-steroid by the 3ß-HSD-5-ene,4-ene-isomerase complex to a u.v.-absorbing (244 nm) 4-ene-3-oxosteroid. To increase the accuracy of the assay further studies (see below) were carried out with radioactively labelled substrates (studies ii and iii).
Preparation of the tissue homogenate The adrenals from three Wistar rats were excised, weighed and homogenized in a PotterElvehjem glass homogenizer for 5 min at 0-4 °C. The concentration of tissue in the homo¬ genate was adjusted to 5 mg/ml phosphosaline buffer before centrifugation at 2500 g for 10 min at 0 °C. The supernatant fraction was decanted and used for the reaction. The assay The assay system (2-0 ml) comprised 0-25 ml adrenal homogenate, 8-0 µ NAD and various quantities of either pregnenolone or DHA (7-9, 15-8, 23-7, 31-6, 47-4 or 63-1 nmol) dissolved in 25 µ ethanol. To improve the accuracy of the assay for studies (ii) and (iii), radioactive steroids were added to obtain final specific activities of 3-22 Ci/mol for pregneno¬ lone and 3-34 Ci/mol for DHA. The quantities of cyproterone acetate (solubilized in 25 µ ethanol) employed were: study i, 0, 8 nmol (4 µ / ) and 16 nmol (8 µ / ); study ii, 0, 3 nmol (1·5µ >1/1), 6 nmol (3 µ / ) and 12 nmol (6 µ / ); study iii, 0, 1-2 nmol (0-6 µ / ) and 2-4 nmol (1·2µ 1/1). Control assays were run without cyproterone acetate. (In setting up the assay using Goldman's (1967) method, it was found necessary to employ supraphysiological quantities of substrates to detect the products with a
spectrophotometer.)
The reaction was started by the addition of 0-25 ml of the homogenate. The incubations carried out for 20 min at 37 °C, with constant shaking. The reaction was stopped by placing the incubating vessel in ice and adding glacial acetic acid (0-2 ml). Tracer amounts (approximately 18 000 disintegrations/min) of [14C]progesterone and [14C]androstenedione were added (studies ii and iii) to appropriate incubations, together with excess unlabelled steroids which acted as carriers. The steroids were extracted with 10 ml ethyl acetate and the extract was dried overnight over anhydrous sodium sulphate. The solvent was decanted and evaporated under N2. The residue was chromatographed with the Bush A system. In studies ii and iii the products were further chromatographed in a Bush A system, and then in a t.l.c. system (benzene : ethanol, 98 : 2, v/v) on the impregnated t.l.c. plates (Gelman Hawkesley). The products were located with u.v. light and with a radiochromatogram strip scanner (Nuclear Chicago Actigraph III) or a spark-chamber (Birchover Instruments Ltd). The steroids were eluted from the chromatograms and 3H : 14C ratios determined (3H : 14C ratios remained constant within + 2% throughout Chromatographie purifications), and the amount of product formed was calculated. In separate studies it was found that in the presence of NAD and NADPH, DHA was mainly metabolized to androst-4-ene-3,17-dione (35-37%). Very little (< 1%) testosterone was produced. With pregnenolone as substrate and in the presence of NAD and NADPH, progesterone (35%) and deoxycorticosterone (25%) were the major metabolites. However, in the absence of NADPH (the present studies) progesterone was the only product detected. were
Protein determination Protein estimated by determining the amount of nitrogen liberated from the micro-Dumas method (Gustin, 1960). a using was
sample,
Analysis of kinetic data examined in the form of double-reciprocal (LineweaverInitial velocity data were first rate law (Cleland, 1963a, b, c). The data were then determine the to Burk) plots appropriate fitted to the corresponding rate equations using a computer program written by one of us for use in the ICL 1906A computer installation at Leeds University. These programs performed calculations based on the methods of Cleland (1967). The initial velocity data for various concentrations of an inhibitor and a single substrate conforming to linear uncompetitive and
linear
non-competitive inhibition were fitted to equations (1) and (2) respectively: VA VA V=
(1+ / + (1+
«
°°™°mP^>
where A and /are the concentrations of substrate and inhibitor respectively, Kis the maximum velocity, K& is the Michaelis constant for A, and Kit Ka are inhibitor constants for a particu¬ lar inhibitor, respectively affecting the slope and intercept of double reciprocal plots. The rate equation giving the best fit to the experimental data was chosen as that with least variance, calculated as the average square of the difference between an experimental velocity and the corresponding value derived from rate equations incorporating the values of the kinetic constants. In studies involving labelled substrates, it was found that data fitted to the non-competitive inhibition equation (2) yielded K¡ values less than 0 which are theoretically impossible. However, when fitted to the uncompetitive inhibition equation (1) all values were greater than 0, thus favouring uncompetitive inhibition.
Studies in vivo Fifty Wistar rats (180-220 g) were randomly divided into five groups often animals (housed in pairs) and designated and treated as follows : group A, control rats injected with 0-2 ml vehicle daily (vehicle =4:1 (v/v) castor oil : benzyl benzoate); group B, 1-7 mg (4-8 µ ) cyproterone/day; group C, 5-1 mg (14-4 µ ) cyproterone/day ; group D, 2 mg (4-8 µ )
cyproterone acetate/day; group E, 6 mg (14·4 µ
) cyproterone acetate/day.
All drugs were administered subcutaneously in 0-2 ml vehicle for 3 weeks. The animals were allowed free access to water and food. All animals were weighed at the start and the end of the treatment period. Twenty-four hours after the last injection the animals were decapi¬ tated within 5 s of stunning. Blood was collected into cold (0 °C) heparinized tubes main¬ tained in an ice bath. The blood was centrifuged (800 g) at 4 °C, and the plasma stored as small samples at —20 °C until assayed for corticosterone. Adrenal glands were dissected and weighed. They were then fixed in 10% formalin, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin. Determination
of corticosterone in plasma Plasma corticosterone was determined by radioimmunoassay according to the method of Underwood «fe Williams (1972) using antiserum raised against corticosterone-21-hemisuccinate. The intra-assay coefficient of variation was < 4% and the interassay coefficient of variation was < 8%. Data
were
significant.
analysed statistically using
Statistics Student's r-test.
values of
1/1; A, 6-0 µ / ) of cyproterone acetate. The points on the graph are actual values obtained in the reactions, is the initial velocity; A is the substrate concentration. For DHA, the broken line is perhaps a truer representation of the reaction, because the inhibitor concentration may be higher than the value used by the computer, i.e. 3 µ -cyproterone acetate.
J-
'
_L 1/µ A
1/µ 1
Text-fig. 3. Computer-derived Lineweaver-Burk plots for the reactions pregnenolone to progesterone (a) and dehydroepiandrosterone (DHA) to androst-4-ene-3,17-dione (b) using labelled substrates (study iii) in the absence [ (a), + (b)] or presence [ (a), A (b), 0-6 µ / ; A (a) · (b), 1 -2 µ / ] of cyproterone acetate. The points on the graphs are actual values obtained in the reactions, is the initial velocity; A is the substrate concentration. See Table 1 for Ku values.
Table 1. Values ofKa and Ku
(mean ± S.D.) as calculated by the computer for the 3$-HSD-5-
ene,4-ene-isomerase complex of the rat adrenal gland
Study no.*
Assay Spectro-
pregnenolone
Inhibitor cyproterone
Radiolabel
pregnenolone
cyproterone
photometric
Substrate
9-1+2-98
105 Ku (mol/1) 86 ±0-023
6-45 ±2-01
0-045 ±0-011
1·04±0·22
0·18±0 24
25-5 ±12-5
0-031 ±0-015
105
Ä^mol/l)
acetate acetate
Radiolabel Radiolabel
cyproterone dehydroepiandrosterone acetate cyproterone pregnenolone acetate
Radiolabel
l-36±0-32 dehydrocyproterone epiandrosterone acetate K& is the Michaelis constant for the substrate. is the inhibitor constant. A^ii * See text for details of studies.
0-33±0-14
yielded a series of parallel lines, which are characteristic of the uncompetitive type of inhibition. The Kn values for each of the assays are given in Table 1. acetate
Effects of cyproterone and cyproterone acetate on the adrenal glands Aa shows the effects of cyproterone and cyproterone acetate on the weight of glands. Both doses of cyproterone acetate caused a marked reduction in adrenal weight (P< 0-001) compared with that of the controls. However, cyproterone had very little effect on adrenal weight (Text-fig. Aa). The Plate shows cross-sections through the adrenal cortices of rats with or without cyproterone or cyproterone acetate treatment. Table 2 gives relative widths of cortex and zona glomerulosa, and the number of nuclei per unit area in the inner two zones of a typical section from each of the groups. Cyproterone acetate but not cyproterone caused atrophy of the zona fasciculata and zona reticularis (zones normally under adrenocorticotrophic hormone (ACTH) control) whereas the zona glomeru¬ losa (normally independent of ACTH control Bergner «fe Deane, 1948) appeared to be
Text-figure
the adrenal
unaffected.
-
(b) 150
(a)
30
100 oo
20
«
50 •o
10-
