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Effects of ethinylestradiol and norethisterone on liver microsomal metabolism of steroids in male and female rats
278
Biochimica @ Elsevier
et Biophysics Acta, Scientific Publishing
369 (1974) 278-293 Company, Amsterdam
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EFFECTS OF ETHINYLESTRADIOL AND NORETHISTERONE ON LIVER MICROSOMAL METABOLISM OF STEROIDS IN MALE AND FEMALE RATS
KURT EINARSSON, JAN L.E. ERICSSON, and EBERHARD ZIETZ* Department of Medicine, Sjukhus and Department
JAN-.&KE
GUSTAFSSON,
JAN SJijVALL
Serafimerlasarettet, Department of Pathology, Sabbatsbergs of Chemistry, Karolinska Instituted, S-l 04 01 Stockholm (Sweden)
(Received April 24th, 1974) (Revised manuscript received
August
9th, 1974)
Summary The effects of administration of ethinylestradiol (17e-ethinyl-1,3,5(10)estratriene-3,17/3-diol), 5 or 500 mg - kg-’ per day, and norethisterone (17P-hydroxy-l7a-ethinyl-4-estren-3-one), 0.5 or 50 mg - kg-’ per day, for two weeks on the hepatic microsomal metabolism of 5a-[4-l 4 C] androstane-3a,l7P-diol, 4-[4-l 4 C] androstene-3,17-dione, 4-[ 4-l 4 C] pregnene-3,20-dione and 7cY-hydroxy-4-[6/3- 3H] cholesten-3-one and on the fine structure of parenchymal cells were studied in male and female rats. The low dose of ethinylestradiol suppressed most hydroxylase levels and increased 5a-reductase levels in male rats in a manner consistent with a feminizing effect of the drug. Female rats reacted much less to the low dose of ethinylestradiol although 5a-reductase levels were slightly but significantly reduced. The high dose of ethinylestradiol suppressed most enzyme activities both in male and female rats. The low dose of norethisterone stimulated some hydroxylase and oxidoreductase activities in the metabolism of 4-androstene-3,17-dione, 4-pregnene3,20-dione and 7a-hydroxy-4-cholesten-3-one. In contrast, the high dose reduced these activities in both male and female rats. No alterations in the fine structure of hepatic parenchymal cells were observed following the administration of ethinylestradiol or norethisterone. It is concluded that ethinylestradiol affects hepatic steroid hydroxylation and reduction mainly in male rats and by virtue of its estrogenic, demasculinizing effect, and that it causes a general suppression of the steroid metabolizing enzymes in rats of both sexes when given in the large doses, 500 mg * kg-‘, commonly used in previous studies. Norethisterone, like progesterone, increases the activities of a few hydroxylases but generally suppresses * Permanent
address:
Organisch-Chemisches
Institut
der Universitit.
34 GSttingen,
G.F.R.
279
enzyme activities when given in larger correlated to changes in ultrastructure.
amounts.
None
of the effects
were
Introduction The extensive use of synthetic female sex hormones has stimulated many investigators to study the biochemical effects of these drugs on organs other than those related to reproductive functions. Interest has focussed on effects of estrogens and gestagens on the liver since use of these hormones may give intrahepatic cholestasis or other signs of disturbed liver function [l--3]. Effects on the activity of the hepatic microsomal drug-metabolizing hydroxylase system have been reported [4-lo], but the results are sometimes confusing and seemingly contradictory since both stimulating and suppressive effects have been observed [ 51. In order to characterize the effects in more detail it seemed essential to study the dose and sex dependence and to use a well-defined test system for microsomal hydroxylation. The present paper describes the influence of different doses of ethinylestradiol and norethisterone on liver microsomal metabolism of steroids in male and female rats. Materials and Methods Chemicals The sources of reference steroids have been given in previous publications [ 11,121. 4-[4-’ “C] Androstene-3,17-dione (spec. act., 1.0 pCi/mg) and 4-[4-l 4C] pregnene-3,20-dione (spec. act, 1.6 pCi/mg) were obtained from the Radiochemical Centre, Amersham, England. 5a(-14-l 4 C] Androstane-&,17&diol was prepared by incubation of 4-[ 4-l 4 C] androstene-3,17-dione (spec. act., 210 &i/mg) with minced testicular tissue from 4-week-old Sprague-Dawley rats [12]. Prior to incubation 5~[4- 1 “C] androstane-3a ,1’7@diol was diluted with unlabelled 5ol-androstane-3a,l,7&diol (the purity of which was assayed by gasliquid chromatography) to give a specific activity of 3.0 &i/mg. 7a-Hydroxy4-[6/3-3 H] cholesten-3-one (spec. act., 6.7 pCi/mg) was prepared as described by Bjdrkhem [ 131. NADP, NADPH, DL-isocitric acid (trisodium salt) and isocitric dehydrogenase (Type IV: 36 units/ml) were obtained from Sigma Chemical Co., St. Louis, MO. Conditions of animal experiments Five groups of five 200 g male and five 200 g female rats, age 56 days, of the Sprague-Dawley strain were treated for 14 days with intramuscular injections of ethinylestradiol (17a-ethinyl-1,3,5( lO)-estratriene-3,17&diol), (17fl-hydroxy-17a-ethinyl-4-estren-3-one) or vehicle only norethisterone (propylene glycol). Ethinylestradiol was given in daily doses of 1 pg (“low in daily doses of dose”, EL) or 100 c(g (“high dose”, EH) and norethisterone 100 c(g (“low dose”, NL) or 10 mg (“high dose”, NH): both drugs were administered in 0.5 ml of propylene glycol. The rats were fed a pellet diet ad
280
libitum tion.
and received
their last treatment
in the morning
the day before incuba-
Preparation of homogenates Animals were killed by cervical dislocation. The liver was excised immediately and chilled on ice. Liver homogenates, 20% (w/v), were prepared in a modified Bucher medium [ 141, pH 7.4, with a Potter--Elvehjem homogenizer equipped with a loosely fitting Teflon pestle. The 105 000 X g microsomal and supernatant fractions were prepared as described previously [ 111. The protein concentration of the microsomal fraction was determined according to Lowry et al. [15]. Incubation conditions 4-[ 4-l 4 C] Androstene-3,17-dione, 300 pg, in 50 ~1 of acetone, was added to a mixture of 0.30 (male rats) or 0.15 ml (female rats) of microsomal fraction and 2.5 ml of Bucher medium fortified with 3 pmoles of NADPH. 4-[4-l 4 ClPregnene-3,20-dione, 250 pg in 50 ~1 of acetone, was added to a mixture of 0.50 (male rats) or 0.25 ml (female rat) of microsomal fraction and 2.5 ml of Bucher medium fortified with 3 pmoles of NADPH. 5a-[4-l 4 C] Androstane3a,17P-diol, 200 pug, in 50 ~1 of acetone, was added to a mixture of 2.5 ml (both sexes) of microsomal fraction, 1.5 ml of Bucher medium, 0.03 pmoles of MnClz , 3 pmoles of NADP, 12.5 pmoles of isocitrate, and 10 1_t1isocitrate dehydrogenase solution. Incubations were carried out for 10 min at 37°C and were terminated as described before [ 11,121. 7a-Hydroxy-4-[6@-3 H] cholesten-3-one, 50 pg, in 50 ~1 of acetone, was added to a mixture of 2 ml (both sexes) of microsomal fraction and 1 ml of Bucher medium fortified with 3 pmoles of NADPH. Incubations were carried out for 12 min at 37°C. Analysis of incubation mixtures The incubation mixtures were extracted and analyzed by thin-layer chromatography [ 11,121. The radioactive zones were located by radioautography and were scraped off and eluted with methanol. Radioactivity was determined in a Packard liquid scintillation spectrometer, Model 3003, using Instagel@ as scintillation liquid. Part of the extracts were (trimethyl)silylated and analyzed by gas-liquid chromatography [11,12]. When a thin-layer zone contained more than one metabolite, the relative amounts of the metabolites were determined by gas chromatography. For identification purposes some of the silylated samples were analysed by gas chromatography-mass spectrometry [ 161. A compound was considered to be identified if it had the same mass spectrum and gas-liquid chromatographic behaviour as the reference compound. Preparation of tissue for electron microscopy Immediately after the animals had been killed, small cubes of liver tissue with a side of approximately 1 mm were excised with the aid of a razor blade and were rapidly immersed in 2 per cent s-collidine buffered 0s04 (pH 7.4) at +4”C. After fixation in the 0s04 solution for 2 hours the tissues were dehy-
281
drated at 4°C in ethanol solutions of increasing concentrations (starting with 30 per cent) and propylene oxide, and were subsequently embedded in Epon 812 epoxy resin. Sections cut at 0.5 p were stained with alkaline toluidine blue for light microscopy and were utilized for orientation in the blocks. The latter were then trimmed in such a way that the appearance of both centrolobular and periportal cells could be studied in the thin sections in the electron microscope (a Siemens Elmiskop I). Prior to investigation in the electron microscope the thin sections were stained with lead citrate in order to enhance the contrast in the tissue and enable visualization of particulate glycogen. Statistical analysis Student’s t-test was used and the significance
level was set at 0.05.
Results Incubations of 5a-[4-l 4 C!]androstane-3a,l7&diol The metabolites formed after incubations of 5a-[ 4-l 4 C] androstane3a,17fl-diol with the microsomal fraction of liver from male and female rats have been described in earlier studies [11,12]. Confirming the results of these studies the following compounds were identified: 5a-androstane-2&,3a,l7& triol, 5a-androstane-2/3,3t,l’7/3-triol, 5~-androstane-3ol,7a,17P-triol, 3a,7a-dihydroxy-%-androstan-17-one, 7a,17P-dihydroxy-5a-androstan-3-one, 5cY-androstane-3e,7/3,17&triol and 5a-androstane-3a(and 3/3),17/3,18-triol. The rates of formation of these compounds in microsomal preparations from the different groups of animals are shown in Fig. 1 A-E. The 2A-hydroxylase system (Fig. 1A) was much more active in male than in female control rats but male rats were completely “feminized” with respect to this enzyme activity already with the low dose of ethinylestradiol; no further reduction was obtained with the high dose. When male rats were treated 5a-Androstant
-2a,3a,l70- trio1
nmoks mg prot. x 10 min
C NHNLEHEL CT Fig. 1. (A), see P. 283 for legend.
C NHNLEHEL 0
282
C NHNLEHEL d
C NHNLEHEL 9
5a-Androstane-3u,l7t3,16Sa-Androstanc-3R
nmoks mg prot.xtOdn
t riol +
,170,18-trivt
A l(C)
10.
8.
6.
4. xx
-J-_
2.
_I==
x
XXJIUU
C NHNLEH u
EL
C NHNLEHEL 9
283
Ba-An&Mane
nmoles mg prot. x 10 min
-3a,7O,l70- trio1
l(D)
4
C NHNLEHEL
C NHNLEHEL
0”
9
3a,7a-Dihydroxy-5a-androstw-17-one
+
7a.l7O-Dihydroxy-5a-androstan-J-one
+
5a-Androstane-3&7a.-trtol
l-
l(E)
5
Fig. 1. Activities of liver microsomal active on 5or-androstane-3a,l’Ip_diol
2~ (A), 2fl- (B), 1% (C), 7p- (D). and 7~ (E) hydroxylase systems after 14 days of daily administration of 100 pg (NL) or 10 mg (NH)
of norethisterone and of 1 @g (EL) or 100 /.& (EH) of ethinylestradiol to male and female rats. The standard deviation is indicated by a vertical line at the top of the bars. Significant differences from control values (C) are indicated in the bars by X (P < U.05). XX (P < 0.01) or XXX (P < 0.001).
with the low dose of norethisterone the a-hydroxylase activity was not affected, whereas the high dose of norethisterone led to a marked reduction in enzyme activity. Neither ethinylestradiol nor norethisterone treatment significantly influenced the 2+hydroxylase activity in female rats. The 2/3-hydroxylase activity (Fig. 1B) in male rats was affected by ethinylestradiol and norethisterone in a similar way to the %-hydroxylase system.
284
However, female rats responded somewhat differently showing significant elevations in 2@-hydroxylase activity following treatment with the high dose of ethinylestradiol and with both doses of norethisterone. The activities of the 1%hydroxylase system (Fig. 1C) also decreased in male rats after low and high doses of ethinylestradiol and high dose of norethisterone whereas the levels in female rats were relatively constant in all experimental groups except in the group given the high dose of ethinylestradiol where a slight but significant reduction in activity was observed. A significant reduction in enzyme activity following administration of the high dose of ethinylestradiol to female rats was also seen in the case of the 7&hydroxylase system (Fig. 1D); the activity of this enzyme in male rats conformed to the same type of response as those of the 2a-, 2p- and l%hydroxylase activities. In contrast, the 7a-hydroxylase activity (Fig. 1E) remained essentially unchanged during steroid treatment (both in male and female rats) and the only effect seen was a slight reduction in activity following administration of the high dose of ethinylestradiol to male rats. Incubations
of 4-[4-l 4 C] androstene-3,17-dione
Confirming the result of a previous study [ll] the following metabolites were identified: 5a-androstane-3,17-dione, 3a(and 3@)-hydroxy-5a-androstan17-one, 17/3-hydroxy-4-androsten-3-one and 6/3-, SLY-and 16a!-hydroxy-4-androstene-3,17-dione. Fig. 2 A-D shows the effects of ethinylestradiol and norethisterone on the activities of the reducing enzymes. The 5a-reductase activity (Fig. 2A) Total
5a-reduced
mtabditrr
ot
4- androstmc-3.17-dlon,
2(A)
T
350.
300.
250.
Fig. 2. (A). see p. 286
for legend.
0
(measured as the total amount of %-reduced metabolites formed, i.e. &Iandrostane-3,17-dione and 301- and 3fl-hydroxy-5a-androstan-17-one) was markedly affected in both male and female rats. The normal sexual difference in 5a-reductase activity was partly eliminated following treatment with the low dose of ethinylestradiol: the enzyme activity was stimulated in male rats and suppressed in female rats. The most pronounced reduction in 5a-reductase activity was observed in female rats treated with high doses of ethinylestradiol and norethisterone: the high dose of norethisterone also reduced the 5cy-reductase activity significantly in male rats. The formation of 3a-hydroxy-5a-androstan-17-one (Fig. 2B) and 3/3-hydroxy-5a-androstan-17-one (Fig. 2C) reflects 3a-Hydroxy-5a-ondrostan-17-one
MnOlcS
mg prot x 10min
2(B)
t
1
C NHNLEHEL
C NH NL EH EL
CF
nmoles mg prot x 10 min
0
30-Hydroxy-Sa-androstan-J-one
C NHNLEHEL 0’
C NH NL EH EL 0
286 nmoles mg prot. x 10 min
170-Hydroxy-C-androstenJ-one
2
2(D)
C NH NL Eli EL ol
C NH NL EH EL 0
Fig. 2. Formation of 5a-reduced metabolites (A), 3a-hvdroxy-5a-androstan-17-one (B). 3p-hydroxy-5aandrostan-17-one (C) and 17P-hydroxy-4-androsten-3-one (D) after incubation of 4-androstene-3,17-dione with liver microsomal preparations from male and female rats treated with norethisterone and ethinylestradiol. For further explanations. see legend to Fig. 1.
activities of 3~- and 3P-hydroxysteroid oxidoreductases, although their formation does not give an accurate estimate of these enzyme activities since the incubated 4-androstene-3,17-dione is not the immediate precursor. Male control rats formed significantly more 3/3-hydroxy&-androstan-l7-one and significantly less 3a-hydroxy-5a-androstan-17-one than female control rats. A feminization of the male liver with respect to these enzyme activities was observed following treatment with the low (and high) dose of ethinylestradiol. Treatment of male rats with the high dose of norethisterone led to a significant decrease in the formation of 3/3-hydroxy-5a-androstan-17-one. In this respect female rats were much less influenced by steroid treatment but significant reductions in the production of 3a- and 3/3-hydroxy-5a-androstan-17-one were seen following administration of high doses of ethinylestradiol and norethisterone. The effects of steroids on the formation of 17&hydroxy-4-androsten-3one (Fig. 2D) were of another type: in this case norethisterone treatment seemed to have the most marked effects: the low dose of norethisterone significantly stimulated the formation of 17P-hydroxy-4-androsten-3-one in both male and female rats. The effects of ethinylestradiol and norethisterone on the activities of the 16a-, 6p- and 7a-hydroxylase systems active on 4-androstene-3,17-dione are shown in Fig. 3 A-C. The 16a-hydroxylase activity (Fig. 3A) was only measurable in male rats: a significant suppression i.e. feminization, of this enzyme activity was observed following treatment with ethinylestradiol. The low norethisterone dose caused a three-fold stimulation of the 16a-hydroxylase activity. The activity of the G/3-hydroxylase system (Fig. 3B) seemed to be regulated in a similar way since suppression of enzyme activity was noted following low and high doses of ethinylestradiol whereas the low dose of norethisterone tended to stimulate the enzyme activity. Female rats were affected relatively little by
287
steroid treatment with respect to the activity of the Go-hydroxylase system. The 7P-hydroxylase system (Fig. 3C) was more active in female than in male rats. Administration of ethinylestradiol tended to feminize, i.e. stimulate, this enzyme activity in male animals, but the changes were not statistically signifinmalr mg prot a l0 min
16a-fi@mxy-&-a-drostcnc-3,17-dione 3(A)
t 60_
50_
I
L
40_
a
30_ Q: No l6a-hydrcuy-kmdrostene3,17- dionc found in any
20_
lo_
of
the
female
animals
I
C NiNLEHEL d
6R- Hydrory-C-ondrortcOc-3.17-dion*
nrnola
mg
prot.xtomh t 3(B) 70..
60.
50.
LO.
3a
20.
10.
C NH NLEHEL cf
C tWt&EHEL 0
-
288
Fig. 3. Activities of liver microsomal 16~ 4-androstene-3,17-dione after administration tions, see legend to Fig. 1.
(A). 6p- (B), and 7Or- (C) hydroxylase systems active on of norethisterone and ethinylestradiol. For further explana-
cant. Norethisterone treatment had no effects in male rats. The 7a-hydroxylase activity was suppressed in female rats following low and high doses of ethinylestradiol and the high dose of norethisterone. Incubations
of 4/4-l
4 Clpregnene-3,20-dione
Confirming results of the previous study [ 111 the following metabolites were identified: 5a-pregnane-3,20-dione, 3a- and 3&hydroxy-5a-pregnan20-one, 2@,3a-dihydroxy-5a-pregnan-20-one and 6@-hydroxy-4-pregnene-3,20dione. Steroid treatment did not change the production rate of 2&3a-dihydroxy-5a-pregnan-20-one. The effects on the formation of ELI- and 3/3-hydroxy501-pregnan-20.one were almost identical to the corresponding effects on the formation of 3/3- and 3a-hydroxy-5a-androstan-17-one from 4-androstene-3,17dione. Fig. 4A shows the influence of ethinylestradiol and norethisterone on the 5a-reductase activity. The normal sexual difference of this activity disappeared following treatment with the low dose of ethinylestradiol. The high dose of ethinylestradiol reduced the 5oreductase activity in both male and female rats when compared to animals given the low dose of ethinylestradiol. Likewise, the high dose of norethisterone suppressed the 5a-reductase activity in both male and female rats. The activity of the Go-hydroxylase system active on 4-pregnene-3,20-dione (Fig. 4B) was stimulated by the low dose of norethisterone in male rats and by the high doses of ethinylestradiol and norethisterone in female rats. Incubation
of 7a-hydroxy-4-[6fl-3
H] cholesten-3-one
The main metabolites formed from 7a-hydroxy-4-[6fl-3 H] cholesten-3-one were 7a,lti-dihydroxy-4-cholesten-3-one, 7a-hydroxy-5a-cholestan-3-one and 5a-cholestane-3P,7a-diol [ 17,181. The 5a-reductase active on 7a-hydroxy-4cholesten-3-one (Fig. 5A) (estimated from the total formation of 7a-hydroxy&-cholestan-3-one and 5a-cholestane-3P,7a-diol) was more active in female than in male control rats but this sexual difference decreased following treatment with the low dose of ethinylestradiol. High doses of ethinylestradiol and norethisterone suppressed the 5a-reductase activity in both male and female rats when compared to animals treated with low doses of ethinylestradiol and norethisterone, respectively. The activity of the la-hydroxylase system (Fig.
289
Total
5a-reduced
nwtobdiies ol
nmoles
mg prot.x 10 mill 300
1 250.
200..
150.
x)0_
C NHNLEliEL
C WNLEllEL
Cf
nmoles mg
0
6R-Hydroxy-C-pregnene-3,20-dione
prot. x 10 min 4
I;
C NHNLEHEL
I:
C NH NL Eli EL
o*
9
Fig. 4. Activities of liver microsomal 5mreductase (A) and Gphydroxylase (ES) active on 4-pregnene-3.20dione after administration of norethisterone and ethinylestradiol. For further explanations, see legend to Fig. 1.
5B) was relatively little influenced by steroid cantly stimulated in male rats after treatment terone and the high dose of ethinylestradiol.
administration but was signifiwith the low dose of norethis-
Electron microscopy No alterations in fine structure of the liver cells were observed in any of the experimental groups. The appearance of portions of hepatic parenchymal cells from a control rat and a rat which had received the high dose of ethinylestradiol is illustrated in Fig. 6.
290
5a-Cholcstanc-30,7a-diol nll-lOlt35 mg
+
la-Hydroxy-5a-cholestan-3-one
prd. x IOmin
+
C NH NL EHEL
C NH NL EH EL
0”
0
rimless mg
prot
x 10mm
61
C NH NL EH EL a’
Fig. 5. Activities of liver microsomal cholesten-S-one after administration legend to Fig. 1.
C NH NL EH EL 0
5cweductase (A) and 12c+hydroxylase of norethisterone and ethinylestradiol.
(B) active on ?‘a-hydroxy-4For further explanations, see
Discussion
The present investigation has demonstrated highly significant effects of ethinylestradiol and norethisterone- administration on the liver microsomal metabolism of steroids in rats. Since intact animals were used, it is possible that some of the effects noted are indirect, e.g. mediated via effects on gonads or pituitary-hypothalamus. Administration of daily doses of 1 pg of ethinylestradiol to male rats for 14 days resulted in an almost total feminization of the microsomal metabolism of Ekx-androstane-3c,l7@-diol, 4-androstene-3,17-dione, 4-pregnene-3,20-dione
Fig. 6. Picture illustrating the fine structural appearance of portions of hepatic parenchwnal Control X 12 500. B. High dose of ethinylestradiol (100 pg ner day for 2 weeks) X 15 000.
cells. A.
292
and 7a-hydroxy-4-cholesten-3-one. In general, this meant a reduction of hydroxylase activities and a stimulation of 5a-reductase activity. Similar treatment of female rats (which generally had lower hydroxylase activities than male rats) had a limited effect. However, in contrast to the result with male rats the 5a-reductase activity in female rats was significantly reduced. This effect was even more pronounced after daily administration of 100 pg of ethinylestradiol and with this dose the enzyme activity was also depressed in male rats below control values. This means that a low dose of ethinylestradiol stimulated (hormonal effect) and a high dose suppressed (“pharmacological” effect) the 5a-reductase activity in male rats whereas both low and high doses of ethinylestradiol suppressed (“pharmacoiogical” effect) the enzyme activity in female rats. This illustrates the necessity of using both male and female rats and both low and high doses for evaluation of drug effects on hepatic microsomal enzyme systems. The effects of norethisterone administration upon steroid metabolism in rat liver microsomal preparations are more complex. When animals were treated with daily doses of 100 pg of norethisterone for 14 days the effects were usually limited. However, the 16a-hydroxylase active on 4-androstene-3,17dione was stimulated threefold (I’ < 0.05) and also the G/3-hydroxylase system active on the same substrate tended to increase in activity in male rats. Furthermore, the formation of 3a-hydroxy-5a-androstan-17-one in male and of 3fl-hydroxy-5a-androstan-17-one in male and female rats was significantly stimulated following norethisterone administration. Finally, the 6/?-hydroxylase active on 4-pregnene-3,20-dione and the 12a-hydroxylase active on 7a-hydroxy-4-cholesten-3-one were significantly stimulated after norethisterone treatment. When animals were treated with 10 mg of norethisterone per day for 14 days the effects observed were usually similar to those observed with a daily dose of 100 E_cg of ethinylestradiol: the enzyme activities were generally suppressed (“pharmacological” effect). In conclusion, therefore, the results indicate that ethinylestradiol influences liver microsomal hydroxylase activities in male rats mainly through its estrogenic, i.e. feminizing effect, but that such a small daily dose as 1 pug of ethinylestradiol administered for a 14-day period suppresses at least the 5cu-reductase activity in female rats. The higher dose of 100 pg of ethinylestradiol reduced almost all steroid metabolizing enzyme activities in the microsomal preparations, from both male and female rats. Norethisterone administered in a daily dose of 100 (ug, exerts a stimulatory influence upon certain hydroxylase and oxidoreductase activities in male rats, especially those active on 4-androstene-3,17-dione. The higher norethisterone dose of 10 mg suppresses almost all liver microsomal steroid metabolism in both male and female rats. These results permit a reevaluation of results obtained by other authors in studies of the effects of contraceptive steroids on the liver. Thus, Juchau and Fouts reported that treatment of male rats with norethynodrel for three weeks stimulated microsomal hexobarbital metabolism but that combined treatment of male rats with norethynodrel and mestranol suppressed this metabolism [ 51. Our results indicate that the suppression caused by the combined treatment was due to the “pharmacological” effect of a large dose of mestranol. The stimulating effect of norethynodrel may be due to induction of hepatic cyto-
293
chrome P-450 as observed after administration of large amounts of progesterone to rats (for ref., see 3). Watanabe investigated the effect of ethinylestradiol on the biliary metabolites of 17/3-[6,7-3 Hz ] estradiol in bile from female rats and observed a significant decrease in the normally major metabolites 2-methoxyestrone and 2-hydroxyestrone [ 191. This could be analogous to our finding of decreased activities of a number of hydroxylase systems caused by a large dose of ethinylestradiol. No morphologic alterations were observed in the parenchymal cells of the livers despite the fact that the treatment induced marked changes in enzyme activities. In contrast to this finding, it appears that large doses of progesterone of the will produce alterations in fine structure, i.e. adaptive proliferation smooth endoplasmic reticulum in liver cells of hamsters [20]. Our findings indicate that increased as well as decreased levels of hydroxylases (under the present experimental conditions) may occur unrelated to the fine structural frame-work within which they exert their functions. The extent to which contraceptive drugs affect the hepatic microsomal hydroxylase system and thus the metabolism of steroid hormones in women is not known. Crawford and Rudofsky have demonstrated that women metabolize pethidine and promazine more slowly after taking oral contraceptives [4] and O’Malley et al. have recently reported an increased plasma half-life time of antipyrine and phenylbutazone in women taking contraceptive steroids [lo] . Thus, evidence is accumulating that the drug-metabolizing capacity of the human liver is affected by contraceptive steroids even if this is not usually revealed by conventional clinical liver function tests [ 211. Acknowledgments This investigation was supported by grants from the Swedish Medical Research Council (13X-219) and from W.H.O. One of us (E.Z.) thanks the Deutsche Forschungsgemeinschaft for financial support. References 1
Song,
2
Roman.
C.S.,
Rifkind,
W.
and
3
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4
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M.R.
and
6
Tephly,
T.R.
and
7
Blackham,
8
Riimke.
C.L.
9
Jori,
Bianchetti,
J.S.
O’Malley.
11
Einarsson.
and
K..
12
Berg.
13
Bjsrkhem,
A. and
14
Bergstriim,
15
Lowry,
16
Reimendal.
Noordhoek.
Gustafsson. Gloor,
BjGrkhem, Danielsson.
19
Watanabe.
20
Emans,
J.B.
21
Carter.
A.C.,
U.
I. and
SjGvall,
H. (1971)
J. Biol.
A.L.
B. and
Acta Anal.
Eur.
(1966)
Biophys. (1968)
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BBA 56512
EFFECTS OF ETHINYLESTRADIOL AND NORETHISTERONE ON LIVER MICROSOMAL METABOLISM OF STEROIDS IN MALE AND FEMALE RATS
KURT EINARSSON, JAN L.E. ERICSSON, and EBERHARD ZIETZ* Department of Medicine, Sjukhus and Department
JAN-.&KE
GUSTAFSSON,
JAN SJijVALL
Serafimerlasarettet, Department of Pathology, Sabbatsbergs of Chemistry, Karolinska Instituted, S-l 04 01 Stockholm (Sweden)
(Received April 24th, 1974) (Revised manuscript received
August
9th, 1974)
Summary The effects of administration of ethinylestradiol (17e-ethinyl-1,3,5(10)estratriene-3,17/3-diol), 5 or 500 mg - kg-’ per day, and norethisterone (17P-hydroxy-l7a-ethinyl-4-estren-3-one), 0.5 or 50 mg - kg-’ per day, for two weeks on the hepatic microsomal metabolism of 5a-[4-l 4 C] androstane-3a,l7P-diol, 4-[4-l 4 C] androstene-3,17-dione, 4-[ 4-l 4 C] pregnene-3,20-dione and 7cY-hydroxy-4-[6/3- 3H] cholesten-3-one and on the fine structure of parenchymal cells were studied in male and female rats. The low dose of ethinylestradiol suppressed most hydroxylase levels and increased 5a-reductase levels in male rats in a manner consistent with a feminizing effect of the drug. Female rats reacted much less to the low dose of ethinylestradiol although 5a-reductase levels were slightly but significantly reduced. The high dose of ethinylestradiol suppressed most enzyme activities both in male and female rats. The low dose of norethisterone stimulated some hydroxylase and oxidoreductase activities in the metabolism of 4-androstene-3,17-dione, 4-pregnene3,20-dione and 7a-hydroxy-4-cholesten-3-one. In contrast, the high dose reduced these activities in both male and female rats. No alterations in the fine structure of hepatic parenchymal cells were observed following the administration of ethinylestradiol or norethisterone. It is concluded that ethinylestradiol affects hepatic steroid hydroxylation and reduction mainly in male rats and by virtue of its estrogenic, demasculinizing effect, and that it causes a general suppression of the steroid metabolizing enzymes in rats of both sexes when given in the large doses, 500 mg * kg-‘, commonly used in previous studies. Norethisterone, like progesterone, increases the activities of a few hydroxylases but generally suppresses * Permanent
address:
Organisch-Chemisches
Institut
der Universitit.
34 GSttingen,
G.F.R.
279
enzyme activities when given in larger correlated to changes in ultrastructure.
amounts.
None
of the effects
were
Introduction The extensive use of synthetic female sex hormones has stimulated many investigators to study the biochemical effects of these drugs on organs other than those related to reproductive functions. Interest has focussed on effects of estrogens and gestagens on the liver since use of these hormones may give intrahepatic cholestasis or other signs of disturbed liver function [l--3]. Effects on the activity of the hepatic microsomal drug-metabolizing hydroxylase system have been reported [4-lo], but the results are sometimes confusing and seemingly contradictory since both stimulating and suppressive effects have been observed [ 51. In order to characterize the effects in more detail it seemed essential to study the dose and sex dependence and to use a well-defined test system for microsomal hydroxylation. The present paper describes the influence of different doses of ethinylestradiol and norethisterone on liver microsomal metabolism of steroids in male and female rats. Materials and Methods Chemicals The sources of reference steroids have been given in previous publications [ 11,121. 4-[4-’ “C] Androstene-3,17-dione (spec. act., 1.0 pCi/mg) and 4-[4-l 4C] pregnene-3,20-dione (spec. act, 1.6 pCi/mg) were obtained from the Radiochemical Centre, Amersham, England. 5a(-14-l 4 C] Androstane-&,17&diol was prepared by incubation of 4-[ 4-l 4 C] androstene-3,17-dione (spec. act., 210 &i/mg) with minced testicular tissue from 4-week-old Sprague-Dawley rats [12]. Prior to incubation 5~[4- 1 “C] androstane-3a ,1’7@diol was diluted with unlabelled 5ol-androstane-3a,l,7&diol (the purity of which was assayed by gasliquid chromatography) to give a specific activity of 3.0 &i/mg. 7a-Hydroxy4-[6/3-3 H] cholesten-3-one (spec. act., 6.7 pCi/mg) was prepared as described by Bjdrkhem [ 131. NADP, NADPH, DL-isocitric acid (trisodium salt) and isocitric dehydrogenase (Type IV: 36 units/ml) were obtained from Sigma Chemical Co., St. Louis, MO. Conditions of animal experiments Five groups of five 200 g male and five 200 g female rats, age 56 days, of the Sprague-Dawley strain were treated for 14 days with intramuscular injections of ethinylestradiol (17a-ethinyl-1,3,5( lO)-estratriene-3,17&diol), (17fl-hydroxy-17a-ethinyl-4-estren-3-one) or vehicle only norethisterone (propylene glycol). Ethinylestradiol was given in daily doses of 1 pg (“low in daily doses of dose”, EL) or 100 c(g (“high dose”, EH) and norethisterone 100 c(g (“low dose”, NL) or 10 mg (“high dose”, NH): both drugs were administered in 0.5 ml of propylene glycol. The rats were fed a pellet diet ad
280
libitum tion.
and received
their last treatment
in the morning
the day before incuba-
Preparation of homogenates Animals were killed by cervical dislocation. The liver was excised immediately and chilled on ice. Liver homogenates, 20% (w/v), were prepared in a modified Bucher medium [ 141, pH 7.4, with a Potter--Elvehjem homogenizer equipped with a loosely fitting Teflon pestle. The 105 000 X g microsomal and supernatant fractions were prepared as described previously [ 111. The protein concentration of the microsomal fraction was determined according to Lowry et al. [15]. Incubation conditions 4-[ 4-l 4 C] Androstene-3,17-dione, 300 pg, in 50 ~1 of acetone, was added to a mixture of 0.30 (male rats) or 0.15 ml (female rats) of microsomal fraction and 2.5 ml of Bucher medium fortified with 3 pmoles of NADPH. 4-[4-l 4 ClPregnene-3,20-dione, 250 pg in 50 ~1 of acetone, was added to a mixture of 0.50 (male rats) or 0.25 ml (female rat) of microsomal fraction and 2.5 ml of Bucher medium fortified with 3 pmoles of NADPH. 5a-[4-l 4 C] Androstane3a,17P-diol, 200 pug, in 50 ~1 of acetone, was added to a mixture of 2.5 ml (both sexes) of microsomal fraction, 1.5 ml of Bucher medium, 0.03 pmoles of MnClz , 3 pmoles of NADP, 12.5 pmoles of isocitrate, and 10 1_t1isocitrate dehydrogenase solution. Incubations were carried out for 10 min at 37°C and were terminated as described before [ 11,121. 7a-Hydroxy-4-[6@-3 H] cholesten-3-one, 50 pg, in 50 ~1 of acetone, was added to a mixture of 2 ml (both sexes) of microsomal fraction and 1 ml of Bucher medium fortified with 3 pmoles of NADPH. Incubations were carried out for 12 min at 37°C. Analysis of incubation mixtures The incubation mixtures were extracted and analyzed by thin-layer chromatography [ 11,121. The radioactive zones were located by radioautography and were scraped off and eluted with methanol. Radioactivity was determined in a Packard liquid scintillation spectrometer, Model 3003, using Instagel@ as scintillation liquid. Part of the extracts were (trimethyl)silylated and analyzed by gas-liquid chromatography [11,12]. When a thin-layer zone contained more than one metabolite, the relative amounts of the metabolites were determined by gas chromatography. For identification purposes some of the silylated samples were analysed by gas chromatography-mass spectrometry [ 161. A compound was considered to be identified if it had the same mass spectrum and gas-liquid chromatographic behaviour as the reference compound. Preparation of tissue for electron microscopy Immediately after the animals had been killed, small cubes of liver tissue with a side of approximately 1 mm were excised with the aid of a razor blade and were rapidly immersed in 2 per cent s-collidine buffered 0s04 (pH 7.4) at +4”C. After fixation in the 0s04 solution for 2 hours the tissues were dehy-
281
drated at 4°C in ethanol solutions of increasing concentrations (starting with 30 per cent) and propylene oxide, and were subsequently embedded in Epon 812 epoxy resin. Sections cut at 0.5 p were stained with alkaline toluidine blue for light microscopy and were utilized for orientation in the blocks. The latter were then trimmed in such a way that the appearance of both centrolobular and periportal cells could be studied in the thin sections in the electron microscope (a Siemens Elmiskop I). Prior to investigation in the electron microscope the thin sections were stained with lead citrate in order to enhance the contrast in the tissue and enable visualization of particulate glycogen. Statistical analysis Student’s t-test was used and the significance
level was set at 0.05.
Results Incubations of 5a-[4-l 4 C!]androstane-3a,l7&diol The metabolites formed after incubations of 5a-[ 4-l 4 C] androstane3a,17fl-diol with the microsomal fraction of liver from male and female rats have been described in earlier studies [11,12]. Confirming the results of these studies the following compounds were identified: 5a-androstane-2&,3a,l7& triol, 5a-androstane-2/3,3t,l’7/3-triol, 5~-androstane-3ol,7a,17P-triol, 3a,7a-dihydroxy-%-androstan-17-one, 7a,17P-dihydroxy-5a-androstan-3-one, 5cY-androstane-3e,7/3,17&triol and 5a-androstane-3a(and 3/3),17/3,18-triol. The rates of formation of these compounds in microsomal preparations from the different groups of animals are shown in Fig. 1 A-E. The 2A-hydroxylase system (Fig. 1A) was much more active in male than in female control rats but male rats were completely “feminized” with respect to this enzyme activity already with the low dose of ethinylestradiol; no further reduction was obtained with the high dose. When male rats were treated 5a-Androstant
-2a,3a,l70- trio1
nmoks mg prot. x 10 min
C NHNLEHEL CT Fig. 1. (A), see P. 283 for legend.
C NHNLEHEL 0
282
C NHNLEHEL d
C NHNLEHEL 9
5a-Androstane-3u,l7t3,16Sa-Androstanc-3R
nmoks mg prot.xtOdn
t riol +
,170,18-trivt
A l(C)
10.
8.
6.
4. xx
-J-_
2.
_I==
x
XXJIUU
C NHNLEH u
EL
C NHNLEHEL 9
283
Ba-An&Mane
nmoles mg prot. x 10 min
-3a,7O,l70- trio1
l(D)
4
C NHNLEHEL
C NHNLEHEL
0”
9
3a,7a-Dihydroxy-5a-androstw-17-one
+
7a.l7O-Dihydroxy-5a-androstan-J-one
+
5a-Androstane-3&7a.-trtol
l-
l(E)
5
Fig. 1. Activities of liver microsomal active on 5or-androstane-3a,l’Ip_diol
2~ (A), 2fl- (B), 1% (C), 7p- (D). and 7~ (E) hydroxylase systems after 14 days of daily administration of 100 pg (NL) or 10 mg (NH)
of norethisterone and of 1 @g (EL) or 100 /.& (EH) of ethinylestradiol to male and female rats. The standard deviation is indicated by a vertical line at the top of the bars. Significant differences from control values (C) are indicated in the bars by X (P < U.05). XX (P < 0.01) or XXX (P < 0.001).
with the low dose of norethisterone the a-hydroxylase activity was not affected, whereas the high dose of norethisterone led to a marked reduction in enzyme activity. Neither ethinylestradiol nor norethisterone treatment significantly influenced the 2+hydroxylase activity in female rats. The 2/3-hydroxylase activity (Fig. 1B) in male rats was affected by ethinylestradiol and norethisterone in a similar way to the %-hydroxylase system.
284
However, female rats responded somewhat differently showing significant elevations in 2@-hydroxylase activity following treatment with the high dose of ethinylestradiol and with both doses of norethisterone. The activities of the 1%hydroxylase system (Fig. 1C) also decreased in male rats after low and high doses of ethinylestradiol and high dose of norethisterone whereas the levels in female rats were relatively constant in all experimental groups except in the group given the high dose of ethinylestradiol where a slight but significant reduction in activity was observed. A significant reduction in enzyme activity following administration of the high dose of ethinylestradiol to female rats was also seen in the case of the 7&hydroxylase system (Fig. 1D); the activity of this enzyme in male rats conformed to the same type of response as those of the 2a-, 2p- and l%hydroxylase activities. In contrast, the 7a-hydroxylase activity (Fig. 1E) remained essentially unchanged during steroid treatment (both in male and female rats) and the only effect seen was a slight reduction in activity following administration of the high dose of ethinylestradiol to male rats. Incubations
of 4-[4-l 4 C] androstene-3,17-dione
Confirming the result of a previous study [ll] the following metabolites were identified: 5a-androstane-3,17-dione, 3a(and 3@)-hydroxy-5a-androstan17-one, 17/3-hydroxy-4-androsten-3-one and 6/3-, SLY-and 16a!-hydroxy-4-androstene-3,17-dione. Fig. 2 A-D shows the effects of ethinylestradiol and norethisterone on the activities of the reducing enzymes. The 5a-reductase activity (Fig. 2A) Total
5a-reduced
mtabditrr
ot
4- androstmc-3.17-dlon,
2(A)
T
350.
300.
250.
Fig. 2. (A). see p. 286
for legend.
0
(measured as the total amount of %-reduced metabolites formed, i.e. &Iandrostane-3,17-dione and 301- and 3fl-hydroxy-5a-androstan-17-one) was markedly affected in both male and female rats. The normal sexual difference in 5a-reductase activity was partly eliminated following treatment with the low dose of ethinylestradiol: the enzyme activity was stimulated in male rats and suppressed in female rats. The most pronounced reduction in 5a-reductase activity was observed in female rats treated with high doses of ethinylestradiol and norethisterone: the high dose of norethisterone also reduced the 5cy-reductase activity significantly in male rats. The formation of 3a-hydroxy-5a-androstan-17-one (Fig. 2B) and 3/3-hydroxy-5a-androstan-17-one (Fig. 2C) reflects 3a-Hydroxy-5a-ondrostan-17-one
MnOlcS
mg prot x 10min
2(B)
t
1
C NHNLEHEL
C NH NL EH EL
CF
nmoles mg prot x 10 min
0
30-Hydroxy-Sa-androstan-J-one
C NHNLEHEL 0’
C NH NL EH EL 0
286 nmoles mg prot. x 10 min
170-Hydroxy-C-androstenJ-one
2
2(D)
C NH NL Eli EL ol
C NH NL EH EL 0
Fig. 2. Formation of 5a-reduced metabolites (A), 3a-hvdroxy-5a-androstan-17-one (B). 3p-hydroxy-5aandrostan-17-one (C) and 17P-hydroxy-4-androsten-3-one (D) after incubation of 4-androstene-3,17-dione with liver microsomal preparations from male and female rats treated with norethisterone and ethinylestradiol. For further explanations. see legend to Fig. 1.
activities of 3~- and 3P-hydroxysteroid oxidoreductases, although their formation does not give an accurate estimate of these enzyme activities since the incubated 4-androstene-3,17-dione is not the immediate precursor. Male control rats formed significantly more 3/3-hydroxy&-androstan-l7-one and significantly less 3a-hydroxy-5a-androstan-17-one than female control rats. A feminization of the male liver with respect to these enzyme activities was observed following treatment with the low (and high) dose of ethinylestradiol. Treatment of male rats with the high dose of norethisterone led to a significant decrease in the formation of 3/3-hydroxy-5a-androstan-17-one. In this respect female rats were much less influenced by steroid treatment but significant reductions in the production of 3a- and 3/3-hydroxy-5a-androstan-17-one were seen following administration of high doses of ethinylestradiol and norethisterone. The effects of steroids on the formation of 17&hydroxy-4-androsten-3one (Fig. 2D) were of another type: in this case norethisterone treatment seemed to have the most marked effects: the low dose of norethisterone significantly stimulated the formation of 17P-hydroxy-4-androsten-3-one in both male and female rats. The effects of ethinylestradiol and norethisterone on the activities of the 16a-, 6p- and 7a-hydroxylase systems active on 4-androstene-3,17-dione are shown in Fig. 3 A-C. The 16a-hydroxylase activity (Fig. 3A) was only measurable in male rats: a significant suppression i.e. feminization, of this enzyme activity was observed following treatment with ethinylestradiol. The low norethisterone dose caused a three-fold stimulation of the 16a-hydroxylase activity. The activity of the G/3-hydroxylase system (Fig. 3B) seemed to be regulated in a similar way since suppression of enzyme activity was noted following low and high doses of ethinylestradiol whereas the low dose of norethisterone tended to stimulate the enzyme activity. Female rats were affected relatively little by
287
steroid treatment with respect to the activity of the Go-hydroxylase system. The 7P-hydroxylase system (Fig. 3C) was more active in female than in male rats. Administration of ethinylestradiol tended to feminize, i.e. stimulate, this enzyme activity in male animals, but the changes were not statistically signifinmalr mg prot a l0 min
16a-fi@mxy-&-a-drostcnc-3,17-dione 3(A)
t 60_
50_
I
L
40_
a
30_ Q: No l6a-hydrcuy-kmdrostene3,17- dionc found in any
20_
lo_
of
the
female
animals
I
C NiNLEHEL d
6R- Hydrory-C-ondrortcOc-3.17-dion*
nrnola
mg
prot.xtomh t 3(B) 70..
60.
50.
LO.
3a
20.
10.
C NH NLEHEL cf
C tWt&EHEL 0
-
288
Fig. 3. Activities of liver microsomal 16~ 4-androstene-3,17-dione after administration tions, see legend to Fig. 1.
(A). 6p- (B), and 7Or- (C) hydroxylase systems active on of norethisterone and ethinylestradiol. For further explana-
cant. Norethisterone treatment had no effects in male rats. The 7a-hydroxylase activity was suppressed in female rats following low and high doses of ethinylestradiol and the high dose of norethisterone. Incubations
of 4/4-l
4 Clpregnene-3,20-dione
Confirming results of the previous study [ 111 the following metabolites were identified: 5a-pregnane-3,20-dione, 3a- and 3&hydroxy-5a-pregnan20-one, 2@,3a-dihydroxy-5a-pregnan-20-one and 6@-hydroxy-4-pregnene-3,20dione. Steroid treatment did not change the production rate of 2&3a-dihydroxy-5a-pregnan-20-one. The effects on the formation of ELI- and 3/3-hydroxy501-pregnan-20.one were almost identical to the corresponding effects on the formation of 3/3- and 3a-hydroxy-5a-androstan-17-one from 4-androstene-3,17dione. Fig. 4A shows the influence of ethinylestradiol and norethisterone on the 5a-reductase activity. The normal sexual difference of this activity disappeared following treatment with the low dose of ethinylestradiol. The high dose of ethinylestradiol reduced the 5oreductase activity in both male and female rats when compared to animals given the low dose of ethinylestradiol. Likewise, the high dose of norethisterone suppressed the 5a-reductase activity in both male and female rats. The activity of the Go-hydroxylase system active on 4-pregnene-3,20-dione (Fig. 4B) was stimulated by the low dose of norethisterone in male rats and by the high doses of ethinylestradiol and norethisterone in female rats. Incubation
of 7a-hydroxy-4-[6fl-3
H] cholesten-3-one
The main metabolites formed from 7a-hydroxy-4-[6fl-3 H] cholesten-3-one were 7a,lti-dihydroxy-4-cholesten-3-one, 7a-hydroxy-5a-cholestan-3-one and 5a-cholestane-3P,7a-diol [ 17,181. The 5a-reductase active on 7a-hydroxy-4cholesten-3-one (Fig. 5A) (estimated from the total formation of 7a-hydroxy&-cholestan-3-one and 5a-cholestane-3P,7a-diol) was more active in female than in male control rats but this sexual difference decreased following treatment with the low dose of ethinylestradiol. High doses of ethinylestradiol and norethisterone suppressed the 5a-reductase activity in both male and female rats when compared to animals treated with low doses of ethinylestradiol and norethisterone, respectively. The activity of the la-hydroxylase system (Fig.
289
Total
5a-reduced
nwtobdiies ol
nmoles
mg prot.x 10 mill 300
1 250.
200..
150.
x)0_
C NHNLEliEL
C WNLEllEL
Cf
nmoles mg
0
6R-Hydroxy-C-pregnene-3,20-dione
prot. x 10 min 4
I;
C NHNLEHEL
I:
C NH NL Eli EL
o*
9
Fig. 4. Activities of liver microsomal 5mreductase (A) and Gphydroxylase (ES) active on 4-pregnene-3.20dione after administration of norethisterone and ethinylestradiol. For further explanations, see legend to Fig. 1.
5B) was relatively little influenced by steroid cantly stimulated in male rats after treatment terone and the high dose of ethinylestradiol.
administration but was signifiwith the low dose of norethis-
Electron microscopy No alterations in fine structure of the liver cells were observed in any of the experimental groups. The appearance of portions of hepatic parenchymal cells from a control rat and a rat which had received the high dose of ethinylestradiol is illustrated in Fig. 6.
290
5a-Cholcstanc-30,7a-diol nll-lOlt35 mg
+
la-Hydroxy-5a-cholestan-3-one
prd. x IOmin
+
C NH NL EHEL
C NH NL EH EL
0”
0
rimless mg
prot
x 10mm
61
C NH NL EH EL a’
Fig. 5. Activities of liver microsomal cholesten-S-one after administration legend to Fig. 1.
C NH NL EH EL 0
5cweductase (A) and 12c+hydroxylase of norethisterone and ethinylestradiol.
(B) active on ?‘a-hydroxy-4For further explanations, see
Discussion
The present investigation has demonstrated highly significant effects of ethinylestradiol and norethisterone- administration on the liver microsomal metabolism of steroids in rats. Since intact animals were used, it is possible that some of the effects noted are indirect, e.g. mediated via effects on gonads or pituitary-hypothalamus. Administration of daily doses of 1 pg of ethinylestradiol to male rats for 14 days resulted in an almost total feminization of the microsomal metabolism of Ekx-androstane-3c,l7@-diol, 4-androstene-3,17-dione, 4-pregnene-3,20-dione
Fig. 6. Picture illustrating the fine structural appearance of portions of hepatic parenchwnal Control X 12 500. B. High dose of ethinylestradiol (100 pg ner day for 2 weeks) X 15 000.
cells. A.
292
and 7a-hydroxy-4-cholesten-3-one. In general, this meant a reduction of hydroxylase activities and a stimulation of 5a-reductase activity. Similar treatment of female rats (which generally had lower hydroxylase activities than male rats) had a limited effect. However, in contrast to the result with male rats the 5a-reductase activity in female rats was significantly reduced. This effect was even more pronounced after daily administration of 100 pg of ethinylestradiol and with this dose the enzyme activity was also depressed in male rats below control values. This means that a low dose of ethinylestradiol stimulated (hormonal effect) and a high dose suppressed (“pharmacological” effect) the 5a-reductase activity in male rats whereas both low and high doses of ethinylestradiol suppressed (“pharmacoiogical” effect) the enzyme activity in female rats. This illustrates the necessity of using both male and female rats and both low and high doses for evaluation of drug effects on hepatic microsomal enzyme systems. The effects of norethisterone administration upon steroid metabolism in rat liver microsomal preparations are more complex. When animals were treated with daily doses of 100 pg of norethisterone for 14 days the effects were usually limited. However, the 16a-hydroxylase active on 4-androstene-3,17dione was stimulated threefold (I’ < 0.05) and also the G/3-hydroxylase system active on the same substrate tended to increase in activity in male rats. Furthermore, the formation of 3a-hydroxy-5a-androstan-17-one in male and of 3fl-hydroxy-5a-androstan-17-one in male and female rats was significantly stimulated following norethisterone administration. Finally, the 6/?-hydroxylase active on 4-pregnene-3,20-dione and the 12a-hydroxylase active on 7a-hydroxy-4-cholesten-3-one were significantly stimulated after norethisterone treatment. When animals were treated with 10 mg of norethisterone per day for 14 days the effects observed were usually similar to those observed with a daily dose of 100 E_cg of ethinylestradiol: the enzyme activities were generally suppressed (“pharmacological” effect). In conclusion, therefore, the results indicate that ethinylestradiol influences liver microsomal hydroxylase activities in male rats mainly through its estrogenic, i.e. feminizing effect, but that such a small daily dose as 1 pug of ethinylestradiol administered for a 14-day period suppresses at least the 5cu-reductase activity in female rats. The higher dose of 100 pg of ethinylestradiol reduced almost all steroid metabolizing enzyme activities in the microsomal preparations, from both male and female rats. Norethisterone administered in a daily dose of 100 (ug, exerts a stimulatory influence upon certain hydroxylase and oxidoreductase activities in male rats, especially those active on 4-androstene-3,17-dione. The higher norethisterone dose of 10 mg suppresses almost all liver microsomal steroid metabolism in both male and female rats. These results permit a reevaluation of results obtained by other authors in studies of the effects of contraceptive steroids on the liver. Thus, Juchau and Fouts reported that treatment of male rats with norethynodrel for three weeks stimulated microsomal hexobarbital metabolism but that combined treatment of male rats with norethynodrel and mestranol suppressed this metabolism [ 51. Our results indicate that the suppression caused by the combined treatment was due to the “pharmacological” effect of a large dose of mestranol. The stimulating effect of norethynodrel may be due to induction of hepatic cyto-
293
chrome P-450 as observed after administration of large amounts of progesterone to rats (for ref., see 3). Watanabe investigated the effect of ethinylestradiol on the biliary metabolites of 17/3-[6,7-3 Hz ] estradiol in bile from female rats and observed a significant decrease in the normally major metabolites 2-methoxyestrone and 2-hydroxyestrone [ 191. This could be analogous to our finding of decreased activities of a number of hydroxylase systems caused by a large dose of ethinylestradiol. No morphologic alterations were observed in the parenchymal cells of the livers despite the fact that the treatment induced marked changes in enzyme activities. In contrast to this finding, it appears that large doses of progesterone of the will produce alterations in fine structure, i.e. adaptive proliferation smooth endoplasmic reticulum in liver cells of hamsters [20]. Our findings indicate that increased as well as decreased levels of hydroxylases (under the present experimental conditions) may occur unrelated to the fine structural frame-work within which they exert their functions. The extent to which contraceptive drugs affect the hepatic microsomal hydroxylase system and thus the metabolism of steroid hormones in women is not known. Crawford and Rudofsky have demonstrated that women metabolize pethidine and promazine more slowly after taking oral contraceptives [4] and O’Malley et al. have recently reported an increased plasma half-life time of antipyrine and phenylbutazone in women taking contraceptive steroids [lo] . Thus, evidence is accumulating that the drug-metabolizing capacity of the human liver is affected by contraceptive steroids even if this is not usually revealed by conventional clinical liver function tests [ 211. Acknowledgments This investigation was supported by grants from the Swedish Medical Research Council (13X-219) and from W.H.O. One of us (E.Z.) thanks the Deutsche Forschungsgemeinschaft for financial support. References 1
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