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Effects of flavonoids on aromatase activity, an in vitro study
J. SteroidBiochern.Molec.Biol.Vol.57, No. 3/4, pp. 215-223,1996
Pergamon 0960-0760(95)00261-8
Copyright© 1996ElsevierScienceLtd. All rights reserved Printed in GreatBritain 0960-0760/96$15.00+ 0.00
Effects of flavonoids on aromatase a n in v i t r o s t u d y
activity,
C. P e l i s s e r o , 1,z* M . J. P . L e n c z o w s k i , 3 D . C h i n z i , 1 B . D a v a i l - C u i s s e t , z J. P. S u m p t e r 4 a n d A. F o s t i e r 5 1ENITA Bordeaux, Ave GEnEral de Gaulle, 33175 Gradignan Cedex, France, 2Unit~ I N R A de Biologie de la Reproduction des Poisso~s, Universit~ Bordeaux I, Avenue des Facult~s, 33405 Talence Cedex, France, 3Department of Experimental Zoology, Research Group Comparative Endocrinology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands, 4Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, U.K. and 5Laboratoire I N R A de Physiologie des Poissons, UniversitE de Rennes I, 35042 Rennes, France In the study, the inh!ibitory effect of flavonoids, including isoflavonic p h y t o e s t r o g e n s , on the o v a r i a n a r o m a t a s e e n z y m e c o m p l e x f r o m t h e r a i n b o w t r o u t , O n c o r h y n c h u s m y k i s s , was a s s e s s e d in vitro. S o m e o f t h e c o m p o u n d s t e s t e d o n fish w e r e also t e s t e d o n h u m a n p l a c e n t a l a r o m a t a s e a c t i v i t y as a c o m p a r i s o n b e t w e e n t h e t w o s o u r c e s o f e n z y m e . It was f o u n d t h a t f l a v o n e , d / - a m i n o g l u t e t h i m i d e , apigenin, quercetin, 7,4'-dihydroxyflavone, at-naphthoflavone and equol were potent inhibitors of the o v a r i a n a r o m a t a s e a c t i v i t y in r a i n b o w t r o u t . R e l a t i v e p o t e n c i e s ( R P ) o f t h e s e c o m p o u n d s c o m p a r e d to f l a v o n e (assigned[ a n effect o f 1) w e r e , r e s p e c t i v e l y , 19.0, 8.7, 5.3, 3.7, 3.2 a n d 0.9. T w o o t h e r phytoestrogens, namely biochanin A and genistein, slightly inhibited aromatase activity. Finally, 7-hydroxyflavone, formononetin, daidzein, coumestrol, chrysin, flavanone and estradiol-17fl did not i n h i b i t o v a r i a n a r o m a t a s e a c t i v i t y a t d o s e s u p to 1 0 0 0 p M . E x p e r i m e n t s o n h u m a n p l a c e n t a l a r o m a t a s e s h o w e d i n h i b i t o r y effects o f d l - a m i n o g l u t e t h i m i d e , f l a v o n e , f l a v a n o n e a n d e q u o l w i t h R P v a l u e s o f 2.8, 1, 1.5 a n d 0.4, r e s p e c t i v e l y . T h e s e r e s u l t s a r e in a c c o r d a n c e w i t h p r e v i o u s s t u d i e s . T h e i n f l u e n c e o f t h e e x p e r i m e n t a l p r o c e d u r e o n ICs0 v a l u e s a n d R P is d i s c u s s e d . C o p y r i g h t © 1996 Elsevier Science Ltd
J. Steroid Biochem. Molec. Biol., Vol. 57, No. 3/4, pp. 215-223, 1996
of phytoestrogens, and has been used because these fish are easy to obtain and rear, and much information is Various in vitro studJLes, performed on microsomal already available on their biology. T h e possible effect preparations from human placental tissue, suggested an of flavonoids on fish aromatase was questioned when, inhibitory effect of various flavonoids on placental during studies on the pattern of sex steroid levels in tissue aromatase activity [1-4]. These flavones are maturing female sturgeon, it was surprisingly found sometimes found in large amounts in fruits and that a change from a diet based on animal proteins to vegetables [5]. In addition, some isoflavonoids possess- a diet based on vegetable proteins, provided by soya ing estrogenic potencies, the so called phytoestrogens, beans, led to a pronounced decrease in plasma estrogen are also found in large amounts in various Graminae levels, down to close to the detection limit (average frequently used in human and animal diets (e.g. soya) 1 ng/ml). Furthermore, an increase in plasma androgen [6]. However, up until now, only one paper [4] has levels (up to 120 ng/ml) was observed [10]. T h e great reported a clear inhibitory effect of a single phyto- change in the androgen:estrogen ratio caused by the estrogen, namely equc,1, on human placental aromatase diet change was unexpected, since androgens and estroactivity. gens are metabolically linked. Furthermore, estrogen On fish, the effects of phytoestrogens have only levels were no longer correlated with oocyte developrecently been documented [7-9]. In general, the trout ment. In addition, equol was detected by gas-chromamodel appears to be appropriate for studying the effects tography-mass spectrometry analysis in the plasma of sturgeon [11]. These observations, together with the data obtained by other authors on mammals, involving the correlation of a vegetarian diet with increased *Correspondence to C. Pelissero. androgen levels and decreased estrogen levels [12, 13], Received 9 Mar. 1995; accepted 11 Oct. 1995. 215 INTRODUCTION
C. Pelissero et al.
216
led to several hypotheses. T h e most likely of these hypotheses postulates that phytoestrogens, present in the vegetarian diet, might have had an inhibitory effect on the ovarian aromatase activity, and thereby reduce estrogen synthesis. T h e present study was set up, therefore, to determine whether flavonoids, including some isoflavonic phytoestrogens known to be present in soya, could inhibit ovarian aromatase activity in fish. T h e rainbow trout, Oneorhynehus mykiss, was chosen as a model for this study because aromatase activity had previously been investigated in vitro in this species [14, 15]. In trout, it has been demonstrated that estradiol-17fl is synthesized in granulosa cells by aromatization of androgens from the special thecal cells [16]. Other sites of aromatization in the brain have been shown by several authors [17, 18]. These sites seem to play a role in the transformation of androgens into estrogens which may be involved in feedback control of gonadotrophin synthesis. In the course of the present study, a few compounds were also tested on human placental aromatase, under the same incubation conditions as were used for the experiments conducted on rainbow trout ovarian aromatase. This was done to compare results obtained from experiments on rainbow trout ovarian aromatase with those previously obtained by other authors on human ovarian aromatase [1, 3, 4]. This study is the first to test the effects of flavonoids on the aromatase activity in fish. MATERIALS AND M E T H O D S
Materials T h e different flavones, as well as dl-aminoglutethimide, were purchased from Sigma Chemical Co. (Dorset, U.K.) T h e isoflavone biochanin A and coumestrol were obtained from, respectively, Aldrich Chemical Co. (Dorset, U.K.) and Apin Chemicals (Oxon, U.K.). Other isoflavones, including equol, were synthesized at the laboratory of Organic and Organometallic Chemistry in Bordeaux (France) [8]. [ lfl,2fl-3 H]Androstenedione (sp. act.: 50.5 Bq/mmol) was obtained from D u Pont (Hertfordshire, U.K.). T h e other chemicals were purchased from Sigma Chemical Co., except for Picofluor 40 ®, the scintillation fluid, which was obtained from Camberra Packard (Berkshire, U.K.). For the experiments, water was redistilled prior to use.
Buffers Standard phosphate buffer: 13 vol. of 200 m M NaH2PO4 were mixed with 87vol. of 2 0 0 r a M Na2HPO4. This mixture was diluted 10 times in water to obtain a 20 m M standard phosphate buffer. H o m ogenized buffer: standard 2 0 m M phosphate buffer containing sucrose (0.25M) and NaC1 (0.21M) (pH 7.55). Incubation buffer: standard 20 m M phos-
phate buffer containing sucrose (0.25M), (0.21 M) and KC1 (0.15 M) (pH 7.55).
NaC1
Animal tissue Ovaries from the rainbow trout, O. mykiss, were collected at a fish-farm in Chesham (England) in late October. Immediately after decapitation of the fish the ovaries were removed and put on ice, after which they were transported to the laboratory in Uxbridge. Here, they were kept at a temperature of - 7 0 ° C , until they were processed further. A human placenta was obtained from Hillingdon Hospital, Middlesex, U.K. After a normal full-term delivery, the placenta was kept on ice for 2 h before processing.
Microsome preparation All of the following procedures were carried out on ice. After the ovaries had been defrosted in 1.0% NaC1 solution in water, they were cut into pieces and rinsed twice with homogenizing buffer. Afterwards, the ovaries were put into the fresh homogenizing buffer (100 g tissue/200 ml buffer) and roughly crushed in the presence of phenylmethyl sulfonylfluoride (PMSF: 1 mM). T h e tissue was again homogenized, this time in a motor driven g l a s s - T e f o n homogenizer. T h e final homogenate was then centrifuged three times at a temperature of 4°C. T h e first centrifugation was at 8 0 0 g in a M S E 18 centrifuge for 20min. After this, most of the yolk floating on the surface was removed, and the supernatant was separated from the pellet by filtering through aseptic gauze. This filtrate was then centrifuged at 10,000 g for 20 min. T h e remaining yolk was removed and the supernatant was separated from the pellet as already described. T h e filtrate was then centrifuged at 195,000 g in a M S E prepspin 65 centrifuge for 2 h. After this ultracentrifugation, the supernatant was carefully removed and the pellets were suspended in the incubation buffer (0.4 ml incubation buffer/1 g original tissue) in the presence of ~-monothioglycerol (11.6 mM) or d/-dithiothreitol (5/~M) depending on the batch. Finally, the pellets were stored at - 7 0 ° C (batch 1). A second batch ofmicrosomes was prepared from the same collection of ovaries. This time, instead of ~-monothioglycerol, dl-dithiothreitol (5 mM) was used. Batch 2 was also stored at - 7 0 ° C . Finally, a third batch of microsomes was prepared from a human placenta in the presence of dl-dithiothreitol (5 ~M).
Aromatase activity T h e technique used to measure the inhibition of the aromatase activity in trout was first described by De Mones [14] and Monod et al. [15], and was used with no major differences from those described in mammals [19, 20]. Incubations with [3H]androstenedione was carried out for 3 0 m i n at a temperature of 12°C in normal air. Before this, however, the microsome fraction was pre-incubated for 1 h at 12°C in the presence
I n h i b i t i o n of F i s h O v a r i a n A r o m a t a s e b y F l a v o n o i d s
217
Table 1. List of the aromatase inhibitors used in this study No. 1 .* 2.* 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Chemical name
Biological name
d/-Aminoglutethimide 1,4,9(11 )-Androstatriene-3,17-dione 1,3,5(10)-Estratrien-3,17fl-diol Flavone 7-Hydroxyflavone 7,4'-Dihydroxyflavone 5,7-Dihydroxyflavone 4',5,7-Trihydroxyflavone 3,3',4',5,7-pentahydroxyflavone 7,8-Benzoflavone Flavanone 7-Hydroxy-4'-methoxyisoflavone 4',7-Dihydroxyisoflavone 5,7-Dihydroxy-4'-methoxyisoflavone 4',5,7-Trihydroxyisofiavone Coumestrol 7-Hydroxy-3-(4'-hydroxyphenyl)-chroman
Androstatrienedione Estradiol- 17B Flavone
Chrysin Apigenin Quercetin -Naphthoflavone Flavanone Formononetin Daidzein Biochanin A Genistein Coumestrol Equol
*Used as a control.
of the flavonoids which were to be tested, and half of the final amount of N A D P H . Pre-incubations and incubations involving human placental microsomes were carried out at 37°C. T h e total volume of the final incubation mixture was 250#1 and contained the following: 220#1 of ~Ehe microsome solution [final concentration in incubation mixture: l l . 3 m g protein/ml (batch 1), or 9.4 mg protein/ml (batch 2) and 1.8 mg/ml (human batch)]; N A D P H dissolved in 20 #1 incubation buffer (final concentration: 0.57raM); [1/3,2//-3 H]androstenedione dissolved in 5 #1 of ethanol (final concentration: 13.1 nM). All the compounds tested on fish were tested on both batches of microsomes. N o significant differences were found between results obtained with the two batches. Compounds listed in Table 1 were dissolved in 5/~1 of dimethyl sulfoxide ( D M S O ) , and tested individually by adding increasing concentrations from 0.5 and
Flavone
120100- ..... ~ A
500 # M to the pre-incubation mixture, unless specified in the text. Control activities were obtained from tubes also preincubated for 1 h with 5 #1 of D M S O and half the final quantity of N A D P H . Both blank samples containing boiled microsomes and control samples with no inhibitor present were run at the same time. Moreover, the samples which did not receive any inhibitor did receive 5/~1 of D M S O . Incubations were ended by addition of trichloroacetic acid (50 #1; 33% solution in water) and 4 5 0 p l of water. After the samples had been vortexed, they were centrifuged at 3 0 0 0 g for 15 min at 4°C. T h e n , 500#1 of the aqueous supernatant was removed and treated with active charcoal (250/d; 50mg/250#1 water). After a 1-h incubation at room temperature, the samples were centrifuged again at 3000 g for 15 min at 4°C. Aliquots of 500 #1 were then added to 4.5 ml of Picofluor 40 ® to
120
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20
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100
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Estradiol concentration (I~M)
Fig. 1. Influence of t h e level of n a t i v e a c t i v i t y on t h e i n h i b i t i o n p a t t e r n w i t h flavone or estradiol.
. . . . . . . .
i
10000
Flavone DL-Aminoglutethimide Estradiol 7-Hydroxyflavone 7,4'-Dihydroxyflavone F or m o non eti n Daidzein Biochanin A Genistein E qu ol Coumestrol Qercetin Chrysin Flavanone A pig en in ~t-Naphthoflavone
8.0 -. --. . . . . . 12.0 0.5 8.0 1.2 0.07 .
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0.7 16.0 1.0 6.7 114.0
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20.0 5.0
1.0 4.5
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I b r a h i m and Abul-Hajj [3]'1Relative IC5o aromatase (#M) inhibition --. . . . > 1000 . . 150.0 . . . -. . . .
.
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.
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--
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Adlercreutz et al. [4]t Relative ICso aromatase (#M) inhibition
.
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.
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--
250.0
850.0
. .
375.0 130.0
1.5
0.4
--
1.0 2.8
Present study Relative ICso aromatase (#M) inhibition
731.0 39.0 > 1000 > 1000 200.0 ~> 1000 >>1000 (2200)~: (3500)~: 793.0 >>1000 139.0 >>1000 > 1000 84.0 227.0
1.0 19.0 <0.7 < 0.7 3.7 ~ 0.7 360.7 (0.3) (0.2) 0.9 ,~0.7 5.3 ,~0.7 <0.7 8.7 3.2
P r esen t study* Relative ICso aromatase (/~M) inhibition
R a i n b o w tr o u t ovarian aromatase
*Each incubation m i x t u r e wit h total volume of 250 ktl contained 0.57 m M N A D P H , 13.1 n M [lfl,2fl-3H]androstenedione and 11.3 mg p r o t e i n / m l incubation mixture. In cu bat io ns were carried out at 12°C for 30 min. t I n c u b a t i o n conditions: Kellis and Vickery, 4 0 n M androstenedione, 0.1 m g prot e i n/ ml incubation mixture, 2 . 5 r a m glucose-phosphate, 0.25 u n it of glucose-6-phosphate deshydrogenase and 0.1 m M N A D P H , incubations were carried out for 6 m i n at 37°C; I b r a h i m & Abul-Hajj, 40 n M androstenedione, 0.05 m g p r o t e i n / m l incubation m ix tur e, 2.5 m M glucose-6-phosphate, 1 unit of glucose-6-phosphate deshydrogenase, 10 m M E D T A and 0.5 m M N A D P H , incubation was carried out for 10 m i n at 37°C. ~Estimated figures obtained by extrapolation of the i n h i b i t i o n curve (cf. Fig. 2).
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Kellis and Vicker [1]l" Relative ICs0 aromatase (#M) inhibition
H u m a n placental aromatase
Table 2. C5o values obtained from studies on either human placental aromatase ( R e f s [ 1 ], [3], [4] and present study) or rainbow trout ovarian aromatase (present study)
O0
t,9
Inhibition of Fish Ovarian Aromatase by Flavonoids
219
determine the radioactivity due to the presence of [3H]H20. F o r this purpose a T r i - C a r b ® liquid scintillation counter was used (model 2000 CA, Packard). RESULTS A progressive decrease in the a m o u n t of aromatase activity was observed during the storage of microsomes at - 7 0 ° C . Tests were p e r f o r m e d on high and low activity batches o f m i c r o s o m e s in order to check for any potential influence of specific activity on the inhibitory effect, but no clear differences appeared between the two batches of microsomes. We thus assumed that this slow decrease of activity did not affect our results. As an illustration, Fig. 1 shows the effect of decreasing native activity on the inhibitory effect of estradiol and flavanone. I n T a b l e 2, ICs0 values (i.e. concentration of inhibitor needed for a 50% inhibition of the aromatase activity), obtained f r o m the present experiments on ovarian aromatase f r o m rainbow trout are presented, together with those obtained from studies on h u m a n placental aromatase [1L,3,4]. Also, results obtained f r o m our own experiments on h u m a n placental aromatase are summarized in T a b l e 2 for comparison.
Inhibition of the ovarian aromatase activity of rainbow trout Looking at the results obtained f r o m all our experiments (see T a b l e 2), it is clear that flavone, dl-aminoglutethimide, 7,4"-dihydroxyflavone, equol, quercetin, apigenin and ~-naphthoflavone, are all relatively strong inhibitors of aromatase activity when c o m p a r e d to the less potent inhibitors biochanin A and genistein, two phytoestrogens known to be present in soya. Estradiol-17fl, 7--hydroxyflavone, formononetin, daidzein, coumestrol, chrysin and flavanone seem to have hardly any or ilo inhibitory effect. 1,4,9(11)Androstatriene-3,17-dione totally blocked the aroma-
120]=_ 100
o¢o ~ 80" ¢= 60'
40'
o '<~
[] 7 minutes
20'
0
1
I
10
100
Concentration of Inhibitor
1000
10000
(pM)
Fig. 3. I n v i t r o i n h i b i t i o n o f the h u m a n p l a c e n t a l a r o m a t a s e activity by d l - a m i n o g l u t e t h i m i d e a f t e r two different i n c u b a t i o n t i m e s (7 rain a n d 30 rain).
tase activity at a concentration of 0.5/aM. I n addition, comparing our results to those obtained with h u m a n aromatase by other authors, we found that our ICs0 values for trout aromatase were about 100 times higher than those reported previously for m a m m a l i a n aromatase. T h i s could mean that fish are less sensitive to these compounds than m a m m a l s , or that our technique was less sensitive than that used by other authors. T o investigate these possibilities, we studied the influence of various parameters of the basic t e c h ~ q u e used in this work, compared to those previously described [14,15]. T o determine whether an extension of the preincubation time would lead to a stronger aromatase inhibition, i.e. better sensitivity, ovarian microsomes from rainbow trout were pre-incubated for 1, 2 or 3 h with either flavanone or coumestrol at four different concentrations (50/aM, 100/aM, 500/aM, 1000/aM). After this, normal inhibition studies were carried out. T h e results suggest that the degree of aromatase inhibition is not affected by a longer pre-incubation time (Results not shown). In vitro inhibition of human placental aromatase activity
200
Flavone Flawmone • Equol . . . . . . .
,Ot
~
,1
. . . . . . . .
\
=
1
. . . . . . . .
=
. . . . . . . .
10
Concentration of Inhibitor
i
100 (p.M)
. . . . . . . .
=
1000
Fig. 2. I n v i t r o i n h i b i t i o n o f t h e h u m a n p l a c e n t a l a r o m a t a s e a c t i v i t y . E a c h i n c u b a t i o n m i x t u r e with a total v o l u m e o f 250 pl contained 0.57 m M NADPH, 13.1 n M [1,,,2,,3 H ] a n d r o s t e n e d i o n e a n d 1.8 m g p r o t e i n / m l i n c u b a t i o n m i x t u r e . I n c u b a t i o n s w e r e c a r r i e d o u t at 37°C for 30 rain.
Results obtained from experiments on h u m a n placental aromatase are shown in T a b l e 2 and Fig. 2. Using the same incubation conditions as described for the experiments on rainbow trout aromatase, ICs0 values of > 5 0 0 # M , 3 7 5 p M , 1 3 0 # M and l l 0 # M were found for equol, flavone, flavanone and dlaminoglutethimide, respectively. Relative aromatase inhibition values for these compounds were, respectively, 0.4, 1.0, 1.5 and 2.8. T o determine the effect of a shorter incubation period on aromatase inhibition, h u m a n placental microsomes were incubated for either 30 min or 7 min according to the same incubation parameters as m e n tioned in Fig. 2. Results shown in Fig. 3 indicate that
C. Pelissero et al.
220
the shorter the incubation time, the lower the ICs0 values. After an incubation period of 7 rain, the ICs0 value for dl-aminoglutethimide was 33 p M , whereas after an incubation period of 30 rain the ICs0 value was 210pM.
DISCUSSION
Inhibition of trout aromatase I n the present study, several flavonoids, including some isoflavonic phytoestrogens, were tested for their ability to inhibit the ovarian aromatase activity of rainbow trout. It was found (Table 2) that flavone (RP = 1.0), dl-aminoglutethimide (RP = 19.0), 7,4'dihydroxyflavone (RP = 3.7), equol (RP = 0.9), quercetin (RP = 5.3), apigenin (RP = 8.7) and ~ - n a p h thoflavone (RP = 3.2) inhibited the aromatase activity. Biochanin A and genistein (estimated RP respectively 0.3 and 0.2) were less potent but still active, whereas estradiol-17/~, 7-hydroxyflavone, formononetin, diadzein, coumestrol, chrysin and flavanone had no effect at doses up to 1000/~M. 1,4,9(ll)-Androstatriene3,17-dione, a steroidal inhibitor used as a control, totally blocked the aromatase activity (ICs0 < 0.5 p M ; RP = 3600). It is interesting to note that this compound was more potent at inhibiting aromatase than d/-aminoglutethimide (ICs0 39 # M ; RP = 19), another c o m p o u n d often used as a potent inhibitor. RPs, as well as ICs0 values, for biochanin A and genistein were obtained by extrapolation of their inhibition curves. Although this m a y result in a reliable approximation, it is realized that this m a y lead to an overestimation of their effect on the inhibition of aromatase activity, because at higher concentrations these compounds m a y no longer be soluble. Furthermore, these relatively high concentrations may not be of physiological importance. oL-Aminoglutethimide is an inhibitor of aromatase activity in h u m a n full-term placental microsomes [21]. This c o m p o u n d is often used as a reference in aromatase inhibition studies in mammals. By testing this compound, it was thus possible to compare the results obtained from both fish and m a m m a l i a n aromatase inhibition studies. F u r t h e r m o r e , 1,4,9(ll)-androstatriene-3,17-dione is known to totally inhibit aromatase activity. Finally, estradiol-17/~ was tested to determine whether this estrogen could exert a negative-feedback effect on the aromatase activity. As shown in T a b l e 2, estradiol-17/~ did not inhibit the aromatase activity, suggesting that aromatase activity is inhibited by "estrogen-like" compounds not because of their structural similarity with estradiol.
Comparison of the inhibition offish and human aromatase T h e results obtained from our experiments on h u m a n placental aromatase, using the same incubation conditions as described for the experiments on rainbow
trout ovarian aromatase, showed that flavone ( R P = 0 . 1 ) , flavanone ( R P = I . 5 ) and dl-aminoglutethimide (RP = 2.8) were relatively potent inhibitors of h u m a n placental aromatase activity. Equol (RP = 0.4) was a less potent inhibitor (Table 2 and Fig. 2). T h e studies on h u m a n placental aromatase were undertaken to assess whether there was any difference in the sensitivity of trout aromatase to inhibition compared to h u m a n aromatase. Although only four chemicals were tested on both h u m a n and trout aromatase, it appears that the two aromatases are approximately equally sensitive.
Comparison of the ICso values obtained here and by other authors ICs0 values obtained from the present experiments on h u m a n placental aromatase were higher than those observed previously [1, 3, 4] (Table 2), indicating that the conditions used for the incubations may have a pronounced effect on the degree of inhibition, and hence on the ICs0 values. One of the conditions in the present experiments, which was different from those in previous studies, was the amount of proteins incubated. In the present study, the amount of microsomal protein incubated was up to 100 times greater than in experiments on h u m a n placental aromatase reported by others [1, 3,4]. By increasing the amount of microsomes, which leads to a higher amount of m e m b r a n e - b o u n d proteins (e.g. the aromatase enzyme complex), the lipid content (lipid bilayer of the m e m b r a n e s ) in the incubation mixture also increases. As a result, flavonoids may accumulate more extensively in the lipid part of the incubation mixture because of their non-polarity. As a consequence, a smaller amount of flavonoids will be present in the aqueous fraction. T h i s may affect ICs0 values for the compounds which were tested. In this context, it is relevant to mention that a large proportion of a drug called pleuromutilin was previously demonstrated to be confined to the m e m b r a n e part in rat liver microsomes, and that its presence there was a consequence of the lipid structure of the m e m b r a n e [22]. Another parameter which is different from those of some of the previous studies is the fact that we performed preincubations with the potential inhibitors. It was decided to do these preincubations in order to show all types of inhibitions, i.e. competitive or noncompetitive. Our reasoning was that in the in vivo situation, chemicals can be in contact with the enzyme and exert various types of actions, all of which end by inhibiting the enzyme. M a n y different chemical interactions m a y occur during this preincubation. A m o n g these we can list the strictly non-competitive interactions, the irreversible inhibitions, or even the metabolism of test compounds into another molecule which would be a more or less potent inhibitor than the parent compounds. With our technique, we cannot determine what mechanisms occur, but we can say that
Inhibition of Fish Ovarian Aromatase by Flavonoids an inhibition occurs. However, a point has to be raised relative to the length e f the preincubation. T h i s 1 h duration is quite long and it is possible that oxygen, which is necessary fi3r the reaction, becomes a limiting factor in our case. I f so, the inhibition could exhaust itself and consequently could appear less efficient. One of the ways to check this point would be to reduce the preincubafion time instead of prolonging it, as we did. Another parameter to examine is the length of the incubation time during which the enzyme can interact with the substrate. We used a 3 0 m i n incubation period, which implies that measurements were taken at a steady state level, whereas in previously published experiments [1-4], microsomes were incubated for, respectively, 6 and 10min, and measurements were p r e s u m a b l y taken before equilibrium was reached. In our conditions, the ava:ilability of neither androstenedione nor xenobiotics were limiting factors, as was verified in unpublished experiments. O u r approach can be justified in vivo since, in the microenvironment of the enzyme, within the cell, one can consider that the aromatase is in contact with the xenobiotics for some considerable time, especially if these compounds are present in large concent~ations. In addition, we decided to p e r f o r m our m e a s u r e m e n t s at the steady state level to achieve better reproducibility. A change in incubation time from 30 min to 7 min resulted in a m u c h lower ICs0 value for d/-.aminoglutethimide in the present study (Fig. 3). T h i s shows that by changing just one of the various parameters, the ICs0 value can alter markedly. Therefore, the discrepancy between our ICs0 values and those of other authors may be due to only the varying procedures used in the different studies. It is to be noted that, although the absolute ICs0 values for the compounds tested on h u m a n placental aromatase activity in our study were different from those previously obtained [1, 3, 4] (Table 2), relative potencies were very similar. T h i s tendency has also been found by other authors and can be observed comparing ICs0 values and RPs obtained by two authors on two modelis with two slightly different procedures. T h i s is the case when comparing results obtained on hamster ovarian tissue [23] and on h u m a n placental aromatase [24] with the same inhibitors. Absolute in vitro potencies obtained when the compounds were tested on h u m a n aromatase were ten times lower than the potencies obtained by other authors for the inhibition of h u m a n placental aromatase; RPs, however, were of the same order. Therefore, RPs rather than ICs0 values seem to be more reliable when comparing results obtaiLned from different studies.
Comparison between trout and human aromatase F r o m a comparison of our results obtained using fish and h u m a n aromatase, it can be seen that for most compounds, RPs obtained f r o m experiments on
221
rainbow trout ovarian aromatase are different f r o m those obtained previously on h u m a n placental aromatase [1, 3, 4]. T h e s e differences may be due to structural differences between the rainbow trout and the h u m a n cytochrome P450 aromatase complex. T h e structure of the cytochrome P450 aromatase complex in man has been described by m a n y authors [25]. A recent study by T a n a k a and collaborators [26] has reported the sequence of the c D N A encoding the ovarian P450 aromatase from trout. T h i s work reports a 52% homology of the deduced polypeptide sequence of trout aromatase with that of human, rat and mouse, and a 53~o homology with that of chicken. T h i s means that the tertiary structure of the P450 aromatase complex may be somewhat different in trout and humans and therefore that the mechanism of inhibition may also be different.
Effects of phytoestrogens in vivo Finally, the great differences in ICs0 values obtained by changing only one parameter of the enzyme assay suggest that it could be very unwise to extrapolate the results obtained in vitro to the in vivo situation. T h i s means that additional work has to be done in vivo, injecting the potentially active compounds into live fish and following the production of estrogens from androgens by assay of plasma steroids, for instance. U n d e r the present in vitro incubation conditions, equol has an effect comparable to that of the other compounds tested; still, one must be aware of the relatively high consumption rate by vegetarians of precursors of this c o m p o u n d [6, 27]. Equol can be produced in the gastrointestinal tract of m a m m a l s by bacterial modifications of isoflavonic phytoestrogens [5, 27-29] and could thus be responsible for physiological effects in mammals. Several authors hypothesized that this c o m p o u n d may act as an anti-estrogenic c o m p o u n d by inhibiting the aromatase activity, and thus reduce the concentration of estrogens in the body fluids [12, 13, 30]. However, this has to be considered with caution since an in vitro effect cannot be extrapolated directly to the in vivo situation. In particular, very little is known as yet on the plasma levels of isoflavone. I f very high concentrations are encountered in urine, it is probably because a large part of the ingested p h y toestrogens are eliminated from the body by this means. T o draw a conclusion concerning the effect of these compounds in vivo we need to understand what happens to them after ingestion. Beside, phytoestrogens have been demonstrated to have an estrogenic effect in fish; this has been shown by their ability to induce vitellogenin ( V T G ) production by fish hepatocytes both in vivo and in vitro [31, 32]. Indeed, V T G synthesis is known to be under estrogen control in fish, amphibian and reptiles [33]. Until now, only equol has been identified in fish plasma [11]. In conclusion, the present results on ovarian aromatase from rainbow trout show that certain flavonoids,
C. Pelissero et al.
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including some phytoestrogens, are potential aromatase inhibitors, at least in vitro. At present, however, of the compounds tested, only equol has been detected in the blood plasma of a lower vertebrate, namely the Siberian sturgeon [11]. Even though no actual concentration of equol in plasma was given in that study [11], the results obtained in this study on equol support the hypothesis of inhibition ofestradiol synthesis by compounds in the diet. As already stated, one must bear in mind the fact that the present data represent in vitro values obtained using a particular incubation method. However, results obtained from the present experiments do suggest that, in fish, some of the compounds tested, when ingested via the diet in sufficient amounts, could have an inhibitory effect on aromatase activity. In this way, these compounds may affect plasma levels of both estrogens and androgens, and as a result they may have an effect on the reproductive cycle of the fish. Acknowledgements--This work was funded by two 1-year research grants (contract Nos 3250 and 5024) which were awarded to Drs Pelissero and Sumpter by the European Union.
12.
13.
14.
15.
16.
17.
18.
REFERENCES 1. Kellis J. T., Jr. and Vickery L. E.: Inhibition of human estrogen synthetase (aromatase) by flavones. Science 225 (1984) 1032-1034. 2. Moochhala S. M., Loke K. H. and Das N. P.: Spectral pertubation of human microsomal cytochrome P-450 by flavonoid binding. Biochem. Int. 17 (1988) 755-762. 3. Ibrahim A. R. and Abul-Hajj Y. J.: Aromatase inhibition by flavonoids. J. Steroid Biochem. Molec. Biol. 37 (1990) 257-260. 4. Adlercreutz H., Bannwart G., W~ih/il/i K., M~el/i T., Brunow G., Hase T., Arosemena P. J. and Kellis J. T., Jr.: Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens..7. Steroid Biochem. Molec. Biol. 44 (1993) 147-153. 5. Kaldras R. S. and Hughes C. L., Jr.: Reproductive and general metabolic effects of phytoestrogens in mammals. Reprod. Toxic. 3 (1989) 81-89. 6. Setchell K. D. R.: Naturally occurring non-steroidal estrogens of dietary origin. In Estrogens in the Environment H. Influences on Development (Edited by John A. McLachlan). Elsevier Science, New York (1985) pp. 69-85. 7. Pelissero C., Le Menn F. and Kaushick S.: Estrogenic effect of dietary soya bean meal on vitellogenesis in cultured Siberian sturgeon Acipenser baeri. Gen. Comp. Endocr. 83 (1991) 447--457. 8. Pelissero C., Bennetau B., Babin P., Le Menn F. and Dunogu6s J.: The estrogenic activity of certain phytoestrogens in the Siberian sturgeon Acipenser baeri. .7. Steroid Biochem. Molec. Biol. 3 (1991) 293-299. 9. Pelissero C., Foucher J. L., Bennetau B., Dunogu6s J., Flouriot G. and Sumpter J. P.: In vitro estrogenic activity of phytoestrogens on liver vitellogenin synthesis in the rainbow trout (Oncorhynchus mykiss). In Reproductive Physiology of Fish (Edited by A. P. Scott, J. P. Sumpter, D. E. Kime and M. S. Rolfe) FishSymp 91, Norwich (1991) pp. 247-249. 10. Pelissero C. and Le Menn F.: Evolution of sex steroid levels in males and first time maturing females of the Siberian sturgeon (Acipenser baeri) reared in a French fish farm. In Proceedings First Int. Syrup. Acipenser, Bordeaux 1989 (Edited by Williot P.) Cemagref, Paris (1991) pp. 87-97. 11. Pelissero C., Le Menn F. and Garrigues P.: Detection of equol in the steroid fraction from sturgeon (Acipenser baeri) plasma
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using capillary gas chromatography-mass spectrometry. Analyst 114 (1989) 1703-1705. Goldin B. R., Adlercreutz H., Gorbach S. L., Woods M. N., Dwyer J. T., Conlon T., Bohn E. and Gershoff S. N.: The relationship between estrogen levels and diet of caucasian American and Oriental immigrant women. Am..7. Clin. Nutr. 44, (1986) 945-953. Adlercreutz H., Hockerstedt K., Bannwart C., Bloigu S., H/im/ilainen E., Fotsis T. and Ollus A.: Effect of dietary components including lignans and phytoestrogens on entherohepatic circulation and liver metabolism of estrogen and on sex hormone binding globulin (SHBG). J. Steroid Biochem. 27 (1987) 1137-1144. De Mones A.: Contribution /l la connaissance du contr61e endocrinien de la ponte chez la tuite arc-en-ciel, Salmo gairdneri, par l'6tude des r6gulations au niveau ovarien, de la chute pr6ovulatoire des concentrations circulantes d'oestrog~nes: analyse in vitro des productions ovariennes d'oestradiol et de l'activit6 microsomale d'aromatisation des androg6nes. Th6se de Docteur Ing6nieur, ENSA de Rennes no. 8715,H,9 78p. Monod G., De Mones A. and Fostier A.: Inhibition of ovarian microsomal aromatase and follicular 0estradiol secretion by imidazole fungicides in rainbow trout. Mar. Environ. Res. 34 (1992) 153-157. Nagahama Y.: The functional morphology of teleost gonads. In Fish Physiology (Edited by S. Hoar, D. J. Randall and E. M. Donaldson). Academic Press, New York, Vol. IXA (1983) pp. 223-275. Callard G. V., Petro Z. and Ryan K. J.: Biochemical evidence for aromatization of androgen to estrogen in the pituitary. Gen. Comp. Endocr. 47 (1981) 359-364. Borg B., Timmers R. J. M. and Lambert J. G. D.: Aromatase activity in the brain of the three spined stickleback Gasterosteus aculeatus. II The effect of castration in winter. Exp. Biol. 47 (1987) 69-71. Rabe T., Rabe D. and Runnebaum B.: New aromatase assay and its application for inhibitory studies of aminogluthetimide on microsomes of human term placenta. J. Steroid Biochem. 17 (1982) 305-309. Pasanen M.: Human placental aromatase activity: use of a C TM reversed phase cartridge for separation of tritiated water or steroid metabolites in placentas from both smoking and non smoking mother, in vitro. Biol. Res. Pregn. 6 (1985) 94-99. Chakraborty J., Hopkins R. and Parke D. V.: Inhibition studies on the aromatization of androst-4-ene-3,17-dione by human placental microsomal preparations. Biochem. `7. 130 (1972) 19p-20p. Parry G., Palmer D. N. and Williams D. J.: Ligand partitioning into membranes: its significance in determining K mand K~ values for cytochrome P-450 and other membrane bound receptors and enzymes. F E B S Lett. 67 (1976) 123-129. H/iusler A., Schenkel L., Kr~ihenbfihl C., Monnet G. and Bhatnagar A. S.: An in vitro method to determine the selective inhibition of estrogen biosynthesis by aromatase inhibitors. `7. Steroid Biochem. 33 (1989) 125-131. Bhamagar A. S. and H/iusler A.: Fortschritte in der Entwicklung neuer wirksamer und selektiver Aromatasehemmer. In Aktuelle Onkologie (Edited by K. Possinger and W. R. Miller). Zuckschwerdt, Munich (1987) pp. 23-28. Thompson E. A., Jr. and Siiteri P. K.: Utilisation of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatisation of androstenedione. `7. Biol. Chem. 249 (1974) 5364-5372. Tanaka M., Telecky T. M., Kukada S., Adachi S., Chen S. and Nagahama Y.: Cloning and sequence analysis of the cDNA encoding P-450 aromatase (P-450arom) from a rainbow trout (Onchorhynchus mykiss) ovary; relationship between the amount of P450arom mRNA and the production of oestradiol- 17fl in the ovary. `7. Molec. Endocr. 8 (1992) 53--61. Setchell K. D. R. and Adlercreutz H.: Mammalian lignans and phyto-estrogens. Recent studies on their formation, metabolism and biological role in health and disease. In Role of the Gut Flora in Toxicity and Cancer (Edited by I. R. Rowhand). Academic Press, London (1988) pp. 315-345. Davies H. L. and Hill J. L.: The effect of diet on the metabolism in sheep of the tritiated isoflavones formononetin and biochanin A. Austr. .7. Agric. Res. 40 (1989) 157-163.
I n h i b i t i o n of F i s h O v a r i a n A r o m a t a s e b y F l a v o n o i d s 29. Axelson M., Kirk D. N., Farrant R. D., Cooley G., Lawson A. M. and Setchell K. D. R.: The identification of the weak oestrogen equol [7-hdyroxy-3-(4'-hydroxyphenyl)-chroman] in human urine. Biochem. ft. 201 (1982) 353-357. 30. Vitt6 C.: Influence des phytoestrog6nes sur l'expression biologique des oestrog6nes. In DEA: Biologie de la Cellule Normale et Pathologique. Universit6 Paris V (1991) 28 pp. 31. Pelissero C. and Sumpter J. P.: Steroids and 'steroid-like' substances in fish diets. Aquaculture 107 (1992) 283-301.
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32. Pelissero C., Flouriot G., Foucher J. L., Bennetau B., Dunogu6s J., Le Gac F. and Sumpter J.: Vitellogenin synthesis in cultured hepatocytes an in vitro test for the estrogenic potency of chemicals, ft. Steroid Biochem. Molec. Biol. 44 (1993) 263-272. 33. Wallace R. A.: Vitellogenesis and oocyte growth in nonmammalian vertebrates. In Developmental Biology (Edited by L. Browder). Pergamon Press, New York, Vol. 1, pp. 127-177.
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Effects of flavonoids on aromatase a n in v i t r o s t u d y
activity,
C. P e l i s s e r o , 1,z* M . J. P . L e n c z o w s k i , 3 D . C h i n z i , 1 B . D a v a i l - C u i s s e t , z J. P. S u m p t e r 4 a n d A. F o s t i e r 5 1ENITA Bordeaux, Ave GEnEral de Gaulle, 33175 Gradignan Cedex, France, 2Unit~ I N R A de Biologie de la Reproduction des Poisso~s, Universit~ Bordeaux I, Avenue des Facult~s, 33405 Talence Cedex, France, 3Department of Experimental Zoology, Research Group Comparative Endocrinology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands, 4Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex UB8 3PH, U.K. and 5Laboratoire I N R A de Physiologie des Poissons, UniversitE de Rennes I, 35042 Rennes, France In the study, the inh!ibitory effect of flavonoids, including isoflavonic p h y t o e s t r o g e n s , on the o v a r i a n a r o m a t a s e e n z y m e c o m p l e x f r o m t h e r a i n b o w t r o u t , O n c o r h y n c h u s m y k i s s , was a s s e s s e d in vitro. S o m e o f t h e c o m p o u n d s t e s t e d o n fish w e r e also t e s t e d o n h u m a n p l a c e n t a l a r o m a t a s e a c t i v i t y as a c o m p a r i s o n b e t w e e n t h e t w o s o u r c e s o f e n z y m e . It was f o u n d t h a t f l a v o n e , d / - a m i n o g l u t e t h i m i d e , apigenin, quercetin, 7,4'-dihydroxyflavone, at-naphthoflavone and equol were potent inhibitors of the o v a r i a n a r o m a t a s e a c t i v i t y in r a i n b o w t r o u t . R e l a t i v e p o t e n c i e s ( R P ) o f t h e s e c o m p o u n d s c o m p a r e d to f l a v o n e (assigned[ a n effect o f 1) w e r e , r e s p e c t i v e l y , 19.0, 8.7, 5.3, 3.7, 3.2 a n d 0.9. T w o o t h e r phytoestrogens, namely biochanin A and genistein, slightly inhibited aromatase activity. Finally, 7-hydroxyflavone, formononetin, daidzein, coumestrol, chrysin, flavanone and estradiol-17fl did not i n h i b i t o v a r i a n a r o m a t a s e a c t i v i t y a t d o s e s u p to 1 0 0 0 p M . E x p e r i m e n t s o n h u m a n p l a c e n t a l a r o m a t a s e s h o w e d i n h i b i t o r y effects o f d l - a m i n o g l u t e t h i m i d e , f l a v o n e , f l a v a n o n e a n d e q u o l w i t h R P v a l u e s o f 2.8, 1, 1.5 a n d 0.4, r e s p e c t i v e l y . T h e s e r e s u l t s a r e in a c c o r d a n c e w i t h p r e v i o u s s t u d i e s . T h e i n f l u e n c e o f t h e e x p e r i m e n t a l p r o c e d u r e o n ICs0 v a l u e s a n d R P is d i s c u s s e d . C o p y r i g h t © 1996 Elsevier Science Ltd
J. Steroid Biochem. Molec. Biol., Vol. 57, No. 3/4, pp. 215-223, 1996
of phytoestrogens, and has been used because these fish are easy to obtain and rear, and much information is Various in vitro studJLes, performed on microsomal already available on their biology. T h e possible effect preparations from human placental tissue, suggested an of flavonoids on fish aromatase was questioned when, inhibitory effect of various flavonoids on placental during studies on the pattern of sex steroid levels in tissue aromatase activity [1-4]. These flavones are maturing female sturgeon, it was surprisingly found sometimes found in large amounts in fruits and that a change from a diet based on animal proteins to vegetables [5]. In addition, some isoflavonoids possess- a diet based on vegetable proteins, provided by soya ing estrogenic potencies, the so called phytoestrogens, beans, led to a pronounced decrease in plasma estrogen are also found in large amounts in various Graminae levels, down to close to the detection limit (average frequently used in human and animal diets (e.g. soya) 1 ng/ml). Furthermore, an increase in plasma androgen [6]. However, up until now, only one paper [4] has levels (up to 120 ng/ml) was observed [10]. T h e great reported a clear inhibitory effect of a single phyto- change in the androgen:estrogen ratio caused by the estrogen, namely equc,1, on human placental aromatase diet change was unexpected, since androgens and estroactivity. gens are metabolically linked. Furthermore, estrogen On fish, the effects of phytoestrogens have only levels were no longer correlated with oocyte developrecently been documented [7-9]. In general, the trout ment. In addition, equol was detected by gas-chromamodel appears to be appropriate for studying the effects tography-mass spectrometry analysis in the plasma of sturgeon [11]. These observations, together with the data obtained by other authors on mammals, involving the correlation of a vegetarian diet with increased *Correspondence to C. Pelissero. androgen levels and decreased estrogen levels [12, 13], Received 9 Mar. 1995; accepted 11 Oct. 1995. 215 INTRODUCTION
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led to several hypotheses. T h e most likely of these hypotheses postulates that phytoestrogens, present in the vegetarian diet, might have had an inhibitory effect on the ovarian aromatase activity, and thereby reduce estrogen synthesis. T h e present study was set up, therefore, to determine whether flavonoids, including some isoflavonic phytoestrogens known to be present in soya, could inhibit ovarian aromatase activity in fish. T h e rainbow trout, Oneorhynehus mykiss, was chosen as a model for this study because aromatase activity had previously been investigated in vitro in this species [14, 15]. In trout, it has been demonstrated that estradiol-17fl is synthesized in granulosa cells by aromatization of androgens from the special thecal cells [16]. Other sites of aromatization in the brain have been shown by several authors [17, 18]. These sites seem to play a role in the transformation of androgens into estrogens which may be involved in feedback control of gonadotrophin synthesis. In the course of the present study, a few compounds were also tested on human placental aromatase, under the same incubation conditions as were used for the experiments conducted on rainbow trout ovarian aromatase. This was done to compare results obtained from experiments on rainbow trout ovarian aromatase with those previously obtained by other authors on human ovarian aromatase [1, 3, 4]. This study is the first to test the effects of flavonoids on the aromatase activity in fish. MATERIALS AND M E T H O D S
Materials T h e different flavones, as well as dl-aminoglutethimide, were purchased from Sigma Chemical Co. (Dorset, U.K.) T h e isoflavone biochanin A and coumestrol were obtained from, respectively, Aldrich Chemical Co. (Dorset, U.K.) and Apin Chemicals (Oxon, U.K.). Other isoflavones, including equol, were synthesized at the laboratory of Organic and Organometallic Chemistry in Bordeaux (France) [8]. [ lfl,2fl-3 H]Androstenedione (sp. act.: 50.5 Bq/mmol) was obtained from D u Pont (Hertfordshire, U.K.). T h e other chemicals were purchased from Sigma Chemical Co., except for Picofluor 40 ®, the scintillation fluid, which was obtained from Camberra Packard (Berkshire, U.K.). For the experiments, water was redistilled prior to use.
Buffers Standard phosphate buffer: 13 vol. of 200 m M NaH2PO4 were mixed with 87vol. of 2 0 0 r a M Na2HPO4. This mixture was diluted 10 times in water to obtain a 20 m M standard phosphate buffer. H o m ogenized buffer: standard 2 0 m M phosphate buffer containing sucrose (0.25M) and NaC1 (0.21M) (pH 7.55). Incubation buffer: standard 20 m M phos-
phate buffer containing sucrose (0.25M), (0.21 M) and KC1 (0.15 M) (pH 7.55).
NaC1
Animal tissue Ovaries from the rainbow trout, O. mykiss, were collected at a fish-farm in Chesham (England) in late October. Immediately after decapitation of the fish the ovaries were removed and put on ice, after which they were transported to the laboratory in Uxbridge. Here, they were kept at a temperature of - 7 0 ° C , until they were processed further. A human placenta was obtained from Hillingdon Hospital, Middlesex, U.K. After a normal full-term delivery, the placenta was kept on ice for 2 h before processing.
Microsome preparation All of the following procedures were carried out on ice. After the ovaries had been defrosted in 1.0% NaC1 solution in water, they were cut into pieces and rinsed twice with homogenizing buffer. Afterwards, the ovaries were put into the fresh homogenizing buffer (100 g tissue/200 ml buffer) and roughly crushed in the presence of phenylmethyl sulfonylfluoride (PMSF: 1 mM). T h e tissue was again homogenized, this time in a motor driven g l a s s - T e f o n homogenizer. T h e final homogenate was then centrifuged three times at a temperature of 4°C. T h e first centrifugation was at 8 0 0 g in a M S E 18 centrifuge for 20min. After this, most of the yolk floating on the surface was removed, and the supernatant was separated from the pellet by filtering through aseptic gauze. This filtrate was then centrifuged at 10,000 g for 20 min. T h e remaining yolk was removed and the supernatant was separated from the pellet as already described. T h e filtrate was then centrifuged at 195,000 g in a M S E prepspin 65 centrifuge for 2 h. After this ultracentrifugation, the supernatant was carefully removed and the pellets were suspended in the incubation buffer (0.4 ml incubation buffer/1 g original tissue) in the presence of ~-monothioglycerol (11.6 mM) or d/-dithiothreitol (5/~M) depending on the batch. Finally, the pellets were stored at - 7 0 ° C (batch 1). A second batch ofmicrosomes was prepared from the same collection of ovaries. This time, instead of ~-monothioglycerol, dl-dithiothreitol (5 mM) was used. Batch 2 was also stored at - 7 0 ° C . Finally, a third batch of microsomes was prepared from a human placenta in the presence of dl-dithiothreitol (5 ~M).
Aromatase activity T h e technique used to measure the inhibition of the aromatase activity in trout was first described by De Mones [14] and Monod et al. [15], and was used with no major differences from those described in mammals [19, 20]. Incubations with [3H]androstenedione was carried out for 3 0 m i n at a temperature of 12°C in normal air. Before this, however, the microsome fraction was pre-incubated for 1 h at 12°C in the presence
I n h i b i t i o n of F i s h O v a r i a n A r o m a t a s e b y F l a v o n o i d s
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Table 1. List of the aromatase inhibitors used in this study No. 1 .* 2.* 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Chemical name
Biological name
d/-Aminoglutethimide 1,4,9(11 )-Androstatriene-3,17-dione 1,3,5(10)-Estratrien-3,17fl-diol Flavone 7-Hydroxyflavone 7,4'-Dihydroxyflavone 5,7-Dihydroxyflavone 4',5,7-Trihydroxyflavone 3,3',4',5,7-pentahydroxyflavone 7,8-Benzoflavone Flavanone 7-Hydroxy-4'-methoxyisoflavone 4',7-Dihydroxyisoflavone 5,7-Dihydroxy-4'-methoxyisoflavone 4',5,7-Trihydroxyisofiavone Coumestrol 7-Hydroxy-3-(4'-hydroxyphenyl)-chroman
Androstatrienedione Estradiol- 17B Flavone
Chrysin Apigenin Quercetin -Naphthoflavone Flavanone Formononetin Daidzein Biochanin A Genistein Coumestrol Equol
*Used as a control.
of the flavonoids which were to be tested, and half of the final amount of N A D P H . Pre-incubations and incubations involving human placental microsomes were carried out at 37°C. T h e total volume of the final incubation mixture was 250#1 and contained the following: 220#1 of ~Ehe microsome solution [final concentration in incubation mixture: l l . 3 m g protein/ml (batch 1), or 9.4 mg protein/ml (batch 2) and 1.8 mg/ml (human batch)]; N A D P H dissolved in 20 #1 incubation buffer (final concentration: 0.57raM); [1/3,2//-3 H]androstenedione dissolved in 5 #1 of ethanol (final concentration: 13.1 nM). All the compounds tested on fish were tested on both batches of microsomes. N o significant differences were found between results obtained with the two batches. Compounds listed in Table 1 were dissolved in 5/~1 of dimethyl sulfoxide ( D M S O ) , and tested individually by adding increasing concentrations from 0.5 and
Flavone
120100- ..... ~ A
500 # M to the pre-incubation mixture, unless specified in the text. Control activities were obtained from tubes also preincubated for 1 h with 5 #1 of D M S O and half the final quantity of N A D P H . Both blank samples containing boiled microsomes and control samples with no inhibitor present were run at the same time. Moreover, the samples which did not receive any inhibitor did receive 5/~1 of D M S O . Incubations were ended by addition of trichloroacetic acid (50 #1; 33% solution in water) and 4 5 0 p l of water. After the samples had been vortexed, they were centrifuged at 3 0 0 0 g for 15 min at 4°C. T h e n , 500#1 of the aqueous supernatant was removed and treated with active charcoal (250/d; 50mg/250#1 water). After a 1-h incubation at room temperature, the samples were centrifuged again at 3000 g for 15 min at 4°C. Aliquots of 500 #1 were then added to 4.5 ml of Picofluor 40 ® to
120
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Estradiol
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•
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~ o -~o 60 E"5 ~,~°o 40
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Lowcontr Io activity
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20
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. . . . . . . .
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1000
|
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. . . . . . . .
01
,
1
. . . . . . . .
,
10
. . . . . . .
|
. . . . . . . .
100
i
1000
Estradiol concentration (I~M)
Fig. 1. Influence of t h e level of n a t i v e a c t i v i t y on t h e i n h i b i t i o n p a t t e r n w i t h flavone or estradiol.
. . . . . . . .
i
10000
Flavone DL-Aminoglutethimide Estradiol 7-Hydroxyflavone 7,4'-Dihydroxyflavone F or m o non eti n Daidzein Biochanin A Genistein E qu ol Coumestrol Qercetin Chrysin Flavanone A pig en in ~t-Naphthoflavone
8.0 -. --. . . . . . 12.0 0.5 8.0 1.2 0.07 .
.
.
. .
.
.
.
.
0.7 16.0 1.0 6.7 114.0
---
1 --
.
. .
.
.
.
.
. . 8.0 . .
0.5 2.0
10.0 2.2
.
. .
.
.
. .
. . 1.3
20.0 5.0
1.0 4.5
.
. .
.
.
. .
. .
I b r a h i m and Abul-Hajj [3]'1Relative IC5o aromatase (#M) inhibition --. . . . > 1000 . . 150.0 . . . -. . . .
.
. .
.
.
. .
. . --
--
--
--. .
Adlercreutz et al. [4]t Relative ICso aromatase (#M) inhibition
.
. .
.
.
. .
. .
--
250.0
850.0
. .
375.0 130.0
1.5
0.4
--
1.0 2.8
Present study Relative ICso aromatase (#M) inhibition
731.0 39.0 > 1000 > 1000 200.0 ~> 1000 >>1000 (2200)~: (3500)~: 793.0 >>1000 139.0 >>1000 > 1000 84.0 227.0
1.0 19.0 <0.7 < 0.7 3.7 ~ 0.7 360.7 (0.3) (0.2) 0.9 ,~0.7 5.3 ,~0.7 <0.7 8.7 3.2
P r esen t study* Relative ICso aromatase (/~M) inhibition
R a i n b o w tr o u t ovarian aromatase
*Each incubation m i x t u r e wit h total volume of 250 ktl contained 0.57 m M N A D P H , 13.1 n M [lfl,2fl-3H]androstenedione and 11.3 mg p r o t e i n / m l incubation mixture. In cu bat io ns were carried out at 12°C for 30 min. t I n c u b a t i o n conditions: Kellis and Vickery, 4 0 n M androstenedione, 0.1 m g prot e i n/ ml incubation mixture, 2 . 5 r a m glucose-phosphate, 0.25 u n it of glucose-6-phosphate deshydrogenase and 0.1 m M N A D P H , incubations were carried out for 6 m i n at 37°C; I b r a h i m & Abul-Hajj, 40 n M androstenedione, 0.05 m g p r o t e i n / m l incubation m ix tur e, 2.5 m M glucose-6-phosphate, 1 unit of glucose-6-phosphate deshydrogenase, 10 m M E D T A and 0.5 m M N A D P H , incubation was carried out for 10 m i n at 37°C. ~Estimated figures obtained by extrapolation of the i n h i b i t i o n curve (cf. Fig. 2).
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Kellis and Vicker [1]l" Relative ICs0 aromatase (#M) inhibition
H u m a n placental aromatase
Table 2. C5o values obtained from studies on either human placental aromatase ( R e f s [ 1 ], [3], [4] and present study) or rainbow trout ovarian aromatase (present study)
O0
t,9
Inhibition of Fish Ovarian Aromatase by Flavonoids
219
determine the radioactivity due to the presence of [3H]H20. F o r this purpose a T r i - C a r b ® liquid scintillation counter was used (model 2000 CA, Packard). RESULTS A progressive decrease in the a m o u n t of aromatase activity was observed during the storage of microsomes at - 7 0 ° C . Tests were p e r f o r m e d on high and low activity batches o f m i c r o s o m e s in order to check for any potential influence of specific activity on the inhibitory effect, but no clear differences appeared between the two batches of microsomes. We thus assumed that this slow decrease of activity did not affect our results. As an illustration, Fig. 1 shows the effect of decreasing native activity on the inhibitory effect of estradiol and flavanone. I n T a b l e 2, ICs0 values (i.e. concentration of inhibitor needed for a 50% inhibition of the aromatase activity), obtained f r o m the present experiments on ovarian aromatase f r o m rainbow trout are presented, together with those obtained from studies on h u m a n placental aromatase [1L,3,4]. Also, results obtained f r o m our own experiments on h u m a n placental aromatase are summarized in T a b l e 2 for comparison.
Inhibition of the ovarian aromatase activity of rainbow trout Looking at the results obtained f r o m all our experiments (see T a b l e 2), it is clear that flavone, dl-aminoglutethimide, 7,4"-dihydroxyflavone, equol, quercetin, apigenin and ~-naphthoflavone, are all relatively strong inhibitors of aromatase activity when c o m p a r e d to the less potent inhibitors biochanin A and genistein, two phytoestrogens known to be present in soya. Estradiol-17fl, 7--hydroxyflavone, formononetin, daidzein, coumestrol, chrysin and flavanone seem to have hardly any or ilo inhibitory effect. 1,4,9(11)Androstatriene-3,17-dione totally blocked the aroma-
120]=_ 100
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40'
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[] 7 minutes
20'
0
1
I
10
100
Concentration of Inhibitor
1000
10000
(pM)
Fig. 3. I n v i t r o i n h i b i t i o n o f the h u m a n p l a c e n t a l a r o m a t a s e activity by d l - a m i n o g l u t e t h i m i d e a f t e r two different i n c u b a t i o n t i m e s (7 rain a n d 30 rain).
tase activity at a concentration of 0.5/aM. I n addition, comparing our results to those obtained with h u m a n aromatase by other authors, we found that our ICs0 values for trout aromatase were about 100 times higher than those reported previously for m a m m a l i a n aromatase. T h i s could mean that fish are less sensitive to these compounds than m a m m a l s , or that our technique was less sensitive than that used by other authors. T o investigate these possibilities, we studied the influence of various parameters of the basic t e c h ~ q u e used in this work, compared to those previously described [14,15]. T o determine whether an extension of the preincubation time would lead to a stronger aromatase inhibition, i.e. better sensitivity, ovarian microsomes from rainbow trout were pre-incubated for 1, 2 or 3 h with either flavanone or coumestrol at four different concentrations (50/aM, 100/aM, 500/aM, 1000/aM). After this, normal inhibition studies were carried out. T h e results suggest that the degree of aromatase inhibition is not affected by a longer pre-incubation time (Results not shown). In vitro inhibition of human placental aromatase activity
200
Flavone Flawmone • Equol . . . . . . .
,Ot
~
,1
. . . . . . . .
\
=
1
. . . . . . . .
=
. . . . . . . .
10
Concentration of Inhibitor
i
100 (p.M)
. . . . . . . .
=
1000
Fig. 2. I n v i t r o i n h i b i t i o n o f t h e h u m a n p l a c e n t a l a r o m a t a s e a c t i v i t y . E a c h i n c u b a t i o n m i x t u r e with a total v o l u m e o f 250 pl contained 0.57 m M NADPH, 13.1 n M [1,,,2,,3 H ] a n d r o s t e n e d i o n e a n d 1.8 m g p r o t e i n / m l i n c u b a t i o n m i x t u r e . I n c u b a t i o n s w e r e c a r r i e d o u t at 37°C for 30 rain.
Results obtained from experiments on h u m a n placental aromatase are shown in T a b l e 2 and Fig. 2. Using the same incubation conditions as described for the experiments on rainbow trout aromatase, ICs0 values of > 5 0 0 # M , 3 7 5 p M , 1 3 0 # M and l l 0 # M were found for equol, flavone, flavanone and dlaminoglutethimide, respectively. Relative aromatase inhibition values for these compounds were, respectively, 0.4, 1.0, 1.5 and 2.8. T o determine the effect of a shorter incubation period on aromatase inhibition, h u m a n placental microsomes were incubated for either 30 min or 7 min according to the same incubation parameters as m e n tioned in Fig. 2. Results shown in Fig. 3 indicate that
C. Pelissero et al.
220
the shorter the incubation time, the lower the ICs0 values. After an incubation period of 7 rain, the ICs0 value for dl-aminoglutethimide was 33 p M , whereas after an incubation period of 30 rain the ICs0 value was 210pM.
DISCUSSION
Inhibition of trout aromatase I n the present study, several flavonoids, including some isoflavonic phytoestrogens, were tested for their ability to inhibit the ovarian aromatase activity of rainbow trout. It was found (Table 2) that flavone (RP = 1.0), dl-aminoglutethimide (RP = 19.0), 7,4'dihydroxyflavone (RP = 3.7), equol (RP = 0.9), quercetin (RP = 5.3), apigenin (RP = 8.7) and ~ - n a p h thoflavone (RP = 3.2) inhibited the aromatase activity. Biochanin A and genistein (estimated RP respectively 0.3 and 0.2) were less potent but still active, whereas estradiol-17/~, 7-hydroxyflavone, formononetin, diadzein, coumestrol, chrysin and flavanone had no effect at doses up to 1000/~M. 1,4,9(ll)-Androstatriene3,17-dione, a steroidal inhibitor used as a control, totally blocked the aromatase activity (ICs0 < 0.5 p M ; RP = 3600). It is interesting to note that this compound was more potent at inhibiting aromatase than d/-aminoglutethimide (ICs0 39 # M ; RP = 19), another c o m p o u n d often used as a potent inhibitor. RPs, as well as ICs0 values, for biochanin A and genistein were obtained by extrapolation of their inhibition curves. Although this m a y result in a reliable approximation, it is realized that this m a y lead to an overestimation of their effect on the inhibition of aromatase activity, because at higher concentrations these compounds m a y no longer be soluble. Furthermore, these relatively high concentrations may not be of physiological importance. oL-Aminoglutethimide is an inhibitor of aromatase activity in h u m a n full-term placental microsomes [21]. This c o m p o u n d is often used as a reference in aromatase inhibition studies in mammals. By testing this compound, it was thus possible to compare the results obtained from both fish and m a m m a l i a n aromatase inhibition studies. F u r t h e r m o r e , 1,4,9(ll)-androstatriene-3,17-dione is known to totally inhibit aromatase activity. Finally, estradiol-17/~ was tested to determine whether this estrogen could exert a negative-feedback effect on the aromatase activity. As shown in T a b l e 2, estradiol-17/~ did not inhibit the aromatase activity, suggesting that aromatase activity is inhibited by "estrogen-like" compounds not because of their structural similarity with estradiol.
Comparison of the inhibition offish and human aromatase T h e results obtained from our experiments on h u m a n placental aromatase, using the same incubation conditions as described for the experiments on rainbow
trout ovarian aromatase, showed that flavone ( R P = 0 . 1 ) , flavanone ( R P = I . 5 ) and dl-aminoglutethimide (RP = 2.8) were relatively potent inhibitors of h u m a n placental aromatase activity. Equol (RP = 0.4) was a less potent inhibitor (Table 2 and Fig. 2). T h e studies on h u m a n placental aromatase were undertaken to assess whether there was any difference in the sensitivity of trout aromatase to inhibition compared to h u m a n aromatase. Although only four chemicals were tested on both h u m a n and trout aromatase, it appears that the two aromatases are approximately equally sensitive.
Comparison of the ICso values obtained here and by other authors ICs0 values obtained from the present experiments on h u m a n placental aromatase were higher than those observed previously [1, 3, 4] (Table 2), indicating that the conditions used for the incubations may have a pronounced effect on the degree of inhibition, and hence on the ICs0 values. One of the conditions in the present experiments, which was different from those in previous studies, was the amount of proteins incubated. In the present study, the amount of microsomal protein incubated was up to 100 times greater than in experiments on h u m a n placental aromatase reported by others [1, 3,4]. By increasing the amount of microsomes, which leads to a higher amount of m e m b r a n e - b o u n d proteins (e.g. the aromatase enzyme complex), the lipid content (lipid bilayer of the m e m b r a n e s ) in the incubation mixture also increases. As a result, flavonoids may accumulate more extensively in the lipid part of the incubation mixture because of their non-polarity. As a consequence, a smaller amount of flavonoids will be present in the aqueous fraction. T h i s may affect ICs0 values for the compounds which were tested. In this context, it is relevant to mention that a large proportion of a drug called pleuromutilin was previously demonstrated to be confined to the m e m b r a n e part in rat liver microsomes, and that its presence there was a consequence of the lipid structure of the m e m b r a n e [22]. Another parameter which is different from those of some of the previous studies is the fact that we performed preincubations with the potential inhibitors. It was decided to do these preincubations in order to show all types of inhibitions, i.e. competitive or noncompetitive. Our reasoning was that in the in vivo situation, chemicals can be in contact with the enzyme and exert various types of actions, all of which end by inhibiting the enzyme. M a n y different chemical interactions m a y occur during this preincubation. A m o n g these we can list the strictly non-competitive interactions, the irreversible inhibitions, or even the metabolism of test compounds into another molecule which would be a more or less potent inhibitor than the parent compounds. With our technique, we cannot determine what mechanisms occur, but we can say that
Inhibition of Fish Ovarian Aromatase by Flavonoids an inhibition occurs. However, a point has to be raised relative to the length e f the preincubation. T h i s 1 h duration is quite long and it is possible that oxygen, which is necessary fi3r the reaction, becomes a limiting factor in our case. I f so, the inhibition could exhaust itself and consequently could appear less efficient. One of the ways to check this point would be to reduce the preincubafion time instead of prolonging it, as we did. Another parameter to examine is the length of the incubation time during which the enzyme can interact with the substrate. We used a 3 0 m i n incubation period, which implies that measurements were taken at a steady state level, whereas in previously published experiments [1-4], microsomes were incubated for, respectively, 6 and 10min, and measurements were p r e s u m a b l y taken before equilibrium was reached. In our conditions, the ava:ilability of neither androstenedione nor xenobiotics were limiting factors, as was verified in unpublished experiments. O u r approach can be justified in vivo since, in the microenvironment of the enzyme, within the cell, one can consider that the aromatase is in contact with the xenobiotics for some considerable time, especially if these compounds are present in large concent~ations. In addition, we decided to p e r f o r m our m e a s u r e m e n t s at the steady state level to achieve better reproducibility. A change in incubation time from 30 min to 7 min resulted in a m u c h lower ICs0 value for d/-.aminoglutethimide in the present study (Fig. 3). T h i s shows that by changing just one of the various parameters, the ICs0 value can alter markedly. Therefore, the discrepancy between our ICs0 values and those of other authors may be due to only the varying procedures used in the different studies. It is to be noted that, although the absolute ICs0 values for the compounds tested on h u m a n placental aromatase activity in our study were different from those previously obtained [1, 3, 4] (Table 2), relative potencies were very similar. T h i s tendency has also been found by other authors and can be observed comparing ICs0 values and RPs obtained by two authors on two modelis with two slightly different procedures. T h i s is the case when comparing results obtained on hamster ovarian tissue [23] and on h u m a n placental aromatase [24] with the same inhibitors. Absolute in vitro potencies obtained when the compounds were tested on h u m a n aromatase were ten times lower than the potencies obtained by other authors for the inhibition of h u m a n placental aromatase; RPs, however, were of the same order. Therefore, RPs rather than ICs0 values seem to be more reliable when comparing results obtaiLned from different studies.
Comparison between trout and human aromatase F r o m a comparison of our results obtained using fish and h u m a n aromatase, it can be seen that for most compounds, RPs obtained f r o m experiments on
221
rainbow trout ovarian aromatase are different f r o m those obtained previously on h u m a n placental aromatase [1, 3, 4]. T h e s e differences may be due to structural differences between the rainbow trout and the h u m a n cytochrome P450 aromatase complex. T h e structure of the cytochrome P450 aromatase complex in man has been described by m a n y authors [25]. A recent study by T a n a k a and collaborators [26] has reported the sequence of the c D N A encoding the ovarian P450 aromatase from trout. T h i s work reports a 52% homology of the deduced polypeptide sequence of trout aromatase with that of human, rat and mouse, and a 53~o homology with that of chicken. T h i s means that the tertiary structure of the P450 aromatase complex may be somewhat different in trout and humans and therefore that the mechanism of inhibition may also be different.
Effects of phytoestrogens in vivo Finally, the great differences in ICs0 values obtained by changing only one parameter of the enzyme assay suggest that it could be very unwise to extrapolate the results obtained in vitro to the in vivo situation. T h i s means that additional work has to be done in vivo, injecting the potentially active compounds into live fish and following the production of estrogens from androgens by assay of plasma steroids, for instance. U n d e r the present in vitro incubation conditions, equol has an effect comparable to that of the other compounds tested; still, one must be aware of the relatively high consumption rate by vegetarians of precursors of this c o m p o u n d [6, 27]. Equol can be produced in the gastrointestinal tract of m a m m a l s by bacterial modifications of isoflavonic phytoestrogens [5, 27-29] and could thus be responsible for physiological effects in mammals. Several authors hypothesized that this c o m p o u n d may act as an anti-estrogenic c o m p o u n d by inhibiting the aromatase activity, and thus reduce the concentration of estrogens in the body fluids [12, 13, 30]. However, this has to be considered with caution since an in vitro effect cannot be extrapolated directly to the in vivo situation. In particular, very little is known as yet on the plasma levels of isoflavone. I f very high concentrations are encountered in urine, it is probably because a large part of the ingested p h y toestrogens are eliminated from the body by this means. T o draw a conclusion concerning the effect of these compounds in vivo we need to understand what happens to them after ingestion. Beside, phytoestrogens have been demonstrated to have an estrogenic effect in fish; this has been shown by their ability to induce vitellogenin ( V T G ) production by fish hepatocytes both in vivo and in vitro [31, 32]. Indeed, V T G synthesis is known to be under estrogen control in fish, amphibian and reptiles [33]. Until now, only equol has been identified in fish plasma [11]. In conclusion, the present results on ovarian aromatase from rainbow trout show that certain flavonoids,
C. Pelissero et al.
222
including some phytoestrogens, are potential aromatase inhibitors, at least in vitro. At present, however, of the compounds tested, only equol has been detected in the blood plasma of a lower vertebrate, namely the Siberian sturgeon [11]. Even though no actual concentration of equol in plasma was given in that study [11], the results obtained in this study on equol support the hypothesis of inhibition ofestradiol synthesis by compounds in the diet. As already stated, one must bear in mind the fact that the present data represent in vitro values obtained using a particular incubation method. However, results obtained from the present experiments do suggest that, in fish, some of the compounds tested, when ingested via the diet in sufficient amounts, could have an inhibitory effect on aromatase activity. In this way, these compounds may affect plasma levels of both estrogens and androgens, and as a result they may have an effect on the reproductive cycle of the fish. Acknowledgements--This work was funded by two 1-year research grants (contract Nos 3250 and 5024) which were awarded to Drs Pelissero and Sumpter by the European Union.
12.
13.
14.
15.
16.
17.
18.
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19.
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22.
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26.
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