Inhibition of catechol estrogen formation in rat liver microsomes by hormonal steroids and related compounds

J. steroid Biochem. Vol. 31, No. 4A, pp. 421-426, 1988

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INHIBITION OF CATECHOL ESTROGEN FORMATION IN RAT LIVER MICROSOMES BY HORMONAL STEROIDS AND RELATED COMPOUNDS JENNIFERA. QUAIL, ANNE-MARIENEWCOMBEand PETERH. JELLINCK* Department

of Biochemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 (Received

13 November 1987)

Summary-The inhibitory action of a number of different hormonal steroids and related compounds on the 2-hydroxylation of estradiol by male rat liver microsomes was examined by a radiometric assay. Progesterone, Diethylstilbestrol, testosterone and 4-androstenedione were found to be the most potent of the compounds tested but inhibition was also observed with other steroids and a group of androgen analogs which are aromatization inhibitors. The kinetic constant K, for those steroids which gave linear double reciprocal plots when added to [2-3Hlestradiol was determined and the products from [r4C]estradiol in the presence of the inhibitors were examined by TLC and autoradiography. The addition of steroids with a 17-hydroxyl group such as testosterone or dihydroequilin resulted in the formation of mainly 2-hydroxyestradiol with smaller amounts of other metabolites while those with a reducible ketonic group such as progesterone, Candrostenedione, equilin or equilenin gave rise to considerable amounts of estrone in addition to the catechol estrogens. Further purification of the liver microsomes did not alter this effect. The possible role of progesterone and the catechol estrogens in the control of estrogen hydroxylation in liver as well as other aspects of steroid interaction are discussed.

INTRODUCTION The numerous reports on the biological activities of the catechol estrogens has stimulated much interest, particularly on their role in the central nervous system [l-3]. Both 2- and 4-hydroxyestradiol (2OHEr ,4-OHE,) have been isolated in approximately equal amounts from brain tissue but the catechol estrogens are formed mainly in the liver where 2-hydroxyestradiol is the major metabolite of Er [4]. This hepatic hydroxylation reaction is brought about by estradiol 2-hydroxylase associated with the microsoma1 fraction of the cell and the enzyme, like other mixed-function oxidases, is a cytochrome P-450 and shows NADPH specificity [5, 61. Higher levels of estrogen 2-hydroxyiase activity have been observed [7,8] in male rat liver compared to the female and sex-differences in the kinetics of the hydroxylation of estradiol has been reported [9]. Steroid inhibitors of estradiol 2-hydroxylase can provide information about the importance of the catechol estrogens in various physiological and biochemical processes and have been used successfully to probe the structural requirement of the active site of the enzyme [IO-141. In this paper, we have extended these studies by comparing the effect of a number of different types of steroids, as well as diethylstilbestrol (DES), on the

*To whom correspondence

should be addressed.

Michaelis-Menten kinetics of catechol estrogen formation by male rat liver microsomes as determined by a radiometric assay [15]. We have also examined the pattern of metabolites formed in the presence of these compounds and shown inhibition of the hydroxylase with a concurrent stimulation of the oxidation of E, to E, by ketosteroids such as 4-androstenedione, progesterone, equilin and equilenin. EXPERIMENTAL Materials

[2-3H]E,, kindly provided by Dr J. Fishman, Rockefeller University, NY, was prepared and handled as described previously [17]. [4-r4C]Ez (57 mCi/mmol; New England Nuclear Corp., Boston, MA) was shown by chromatography and autoradiography to be free of radioactive impurities. The steroids, including androsta-1,4,6-triene-3,17-dione (ATD), used as inhibitors, were purchased from Steraloids, Wilton, NH. 3-Methylene-androst-4-ene17-one (A-M), 17/?-hydroxy-3-methyleneandrost-4ene (T-M) [ 161, 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and lOt?-(2-propynyl)-estr-4-ene-3,17-dione (IO/I-PED) were generously provided by Drs S. Miyairi and J. Fishman (Rockefeller University, NY). Activated charcoal (Norit A) was supplied by Fisher Scientific (Waltham, MA). All chemicals were the purest available commercially. 421

422

JENNIFERA.

Preparation of liver microsomes

Mature male (300-400 g) and female (180-220 g) Sprague-Dawley rats, (Charles River, St Constant, Quebec, Canada) were maintained under standard conditions of light (0600-2000 h) and temperature (21-22°C) on a diet of Purina laboratory chow (Ralston-Purina, St Louis, MO) and water ad fibiturn. The animals were sacrificed by cervical dislocation after CO, anesthesia and a 10% (w/v) homogenate of the liver was prepared in 0.25 M sucrose using a Potter-Elvejhem homogenizer with a Teflon pestle. The homogenate was centrifuged at 8000 g for 15 min, and a microsomal fraction was obtained from the supernatant by centrifuging at 105,OOOg for 1 h, washing with 0.5 ml of 0.25 M sucrose and resuspending the pellet in sucrose. The microsomes, derived from 200mg of original tissue/ ml, could be stored at -70°C without loss of hydroxylating activity for several months. Protein was determined by the method of Lowry et al. [18] using bovine serum albumin as standard. Conditions of incubation

[2-3H]E, (OS-10 PM; 2.9-6.0 x 10’ dpm) was incubated for various time periods with constant shaking at 37°C with resuspended microsomes (105,OOOg pellet) from 20 mg liver ( N 0.3 mg protein) and NADPH (0.28 mM) in 0.1 M Tris-HCl buffer, pH 7.4; total volume: 4 ml. The inhibitors were added at a concentration range of S-50 PM and the total volume of ethanol in the mixture was kept below 1%. At the appropriate time after incubation, a portion (1 ml) of the solution was mixed with a suspension of 1% (w/v) charcoal in buffer and kept on ice for 10 min before pelleting the charcoal by centrifugation at 1OOOgfor 10 min at 4°C. For the experiments with [4-14C]E2, the incubations were carried out in the presence of 2 mM ascorbic acid to prevent the further oxidation of labile products, and the solution extracted three times with equal volumes of diethyl ether. The extract, dried over anhydrous Na,SO,, was evaporated to dryness under N, at 40°C the residue was dissolved in ethanol and, after the addition of 10 pg 2-hydroxyestradiol (2-0HE2) as carrier, separated by TLC on silica gel using cyclohexaneethyl acetate-ethanol (10: 9: 1, by vol). After autoradiography, the areas containing E,, 2-0HE2 or other metabolites were scraped directly into counting vials for the determination of “C-radioactivity. Recovery of catechol estrogen and E, by this procedure was checked by adding different quantities of [4-14C]2OHE, and [4-14C]E2to the buffer and isolating them as described above. Recoveries varied between 4560% and 65-70%, respectively, depending on the amount of steroid used. Determination of 2-hydroxylase activity

This was determined by the radiometric assay of Fishman et al. [15]. Aliquots (0.5 ml) from the

QUAILet al. charcoal-treated incubation mixture were allowed to evaporate to dryness in a fume hood at room temperature and ‘H,O formation was determined from the difference between the 3H-radioactivity in the original sample and that in the dry residue redissolved in 0.5 ml of H20. This method gave results identical to those obtained by lyophilization [17]. Kinetic constants (K,,,, Ki) for the various tissue preparations were calculated from the values obtained by using the least square regression method [19] to determine the slope of the lines. RESULTS

Initially, the percentage decrease in the 2-hydroxylation of E, by male rat liver microsomes was determined at various concentrations of a number of different inhibitors (Table 1). The estradiol concentration chosen for the assay was 9.2pM which is approx 5 times the apparent K, of estradiol 2-hydroxylase for its substrate [9,23]. Progesterone, DES and the androgens, testosterone and Candrostenedione, were found to be the most potent but all the other compounds tested showed considerable estrogen hydroxylase inhibitory activity including a group of androgen analogs [16] which are aromatization inhibitors (Table 2). Similar inhibitory activities were observed with the first group of compounds when they were added to female rat liver microsomes (values not shown). The kinetics of inhibition of the 2-hydroxylation of E, by male rat liver microsomes was examined for a number of these compounds using the ‘H-release assay and a double reciprocal plot as illustrated for DES, progesterone and testosterone (Fig. 1). For the kinetic studies, incubations with [2-)H]E, were carried out for 10 min with low concentrations (0.07-0.12 mg/ml) of microsomal protein to obtain linearity of 3H20 formation and to maintain the Table 1. Relative potency of inhibitors of rat liver microsomal &radio1 2-hydroxylase % Inhibition of ‘H,O formation Concentration

of inhibitor (pM)

Inhibitors

5

10

25

50

Progesterone

54

74

90

97

DES

53

13

87

93

Testosterone 4-Androstenedione 2-Hydroxyestradiol CHydroxyestradiol 2-Methoxyestradiol Equilin Dihydroequilin Equilenin 2-Bromoestradiol 4-Bromoestradiol 2,4-Dibromoestradiol

51 46 -

51 63 24 21 33 30 28 15 41 31 52

66 70 51 52 46 57 59 64 68 53 63

75 68 72 14 59 68 72 69 71 68 14

-

[2-‘H]E, (6 x 105dpm. 9.2pM) was incubated for 10min at 37°C with male liver microsomes ( _ 0.3 mg protein) and NADPH (0.28 mM) in the presence or absence of inhibitor. The formation of ‘H,O was measured as described in Experimental. Values are averages of 2 or 3 experiments with a range of <6% from the mean.

Inhibition of catechol estrogen formation Table 2. Effect of aromatization inhibitors on the 2-hydroxylation estradiol by rat liver microsomes Inhibitors*

of

% Inhibition of ‘H20 formation

A-M T-M

68 57

4-OHA IOF-PED ATD

59 54 39

The incubations (conditions as in Table 1) were carried out with 9.2 p M [2-3H]E, and an equimolar concentration of inhibitor. *See Experimental for abbreviations.

amount of available substrate relatively constant. A concentration of inhibitor which produced approx 50% inhibition was chosen and, in many cases, this form of analysis was possible because the reaction followed classical Michaelis-Menten kinetics producing a linear Lineweaver-Burk plot. With the exception of DES, the compounds tested (Fig. 1 and Table 3) behaved as competitive inhibitors but 4bromoestradiol, 2,4-dibromoestradiol [ 1l] and equilenin gave non-linear double reciprocal plots and therefore an apparent Ki could not be assigned to them. 4-Bromo- and 2,4-dibromoestradiol showed upwardly-curving and equilenin a sigmoidal Lineweaver-Burk plot (not shown). Reliable kinetic studies with the catechol estrogens as inhibitors would be difficult to perform due to their chemical instability. r

y

[sl

Fig. 1. Lineweaver-Burk plots for the inhibition of estradiol 2-hydroxylase by progesterone, testosterone and diethylstilbestrol. Various concentrations of [2-‘H]E, (0.5 to 10 FM; [email protected] x lo5 dpm/tube) were incubated with NADPH (0.28 mM) and rat liver microsomes (-0.30 mg) for 10min at 38°C and )H,O formation measured as described in the text. The inhibitors were added at a concentration which produced approx 50% inhibition under standard incubation conditions (Experimental). No inhibitor (0); 5pM progesterone (0); 5 PM DES (A); 10pM testosterone (A). The velocity of the enzymatic reaction is expressed as nmol of product/mg of microsomal protein/min of incubation time. Values are the means of 3 experiments (SD < 5%). S.B. 31,4A--E

423

Table 3. Apparent Kis of DES and various steroids for the inhibition of estradiol 2-hydroxylase in male rat liver microsomes Inhibitor

Apparent K, @hi)

Progesterone DES Dihydroequilin Testosterone 2-Bromoestradiol 2-Methoxyestradiol Equilin Apparent K, for estradiol

0.47 0.56 2.00 2.65 5.33 13.01 13.26 1.69

Various concentrations of [2-‘I-IjE, were incubated with rat liver microsomes, NADPH and the inhibitors under the conditions used in Fig. 1and ‘Hz0 formation measured as described in the text. The apparent X;s were calculated from the slope of the line and the y intercept [I 81.

The pattern of [14C]estradiol metabolites formed by the liver microsomes after 30 min of incubation in the presence of inhibitors, and ascorbic acid was examined by TLC followed by visualization by autoradiography (Fig. 2). The addition of those with a 17-hydroxyl group such as testosterone or dihydroequilin gave rise to mainly 2-0HE2 with smaller amounts of “estriol” and unchanged substrate while those with a reducible ketonic group such as 4-androstenedione, or equilin resulted in the formation of considerable amounts of estrone and some of its 2-hydroxylated derivative. DES, estradiol-17a or 2bromoestradiol behaved like the first and progesterone or equilenin like the second group of inhibitors in this system. The addition of non-radioactive E, also increased the conversion of [14C]E, to [14C]E, while excess unlabeled E,, as expected, competed with [‘4C]E2 for the 2-hydroxylase and decreased the yield of [4-‘4C]2-OHE,. The ascorbic acid (2 mM) that was added to prevent further oxidation of 2-OHE, did not interfere with the formation of “C-metabolites of estradiol and had very little effect (-=z10%) on ‘H,O release. Microsomes, further washed by suspending the pellet in 0.05 M Tris-HCl buffer (pH 7.4) containing 0.5 mM ascorbic acid and then re-isolated by centrifugation at 105,OOOgfor 1 h, gave results that were identical with those observed with the original 0.25 M sucrose preparations. This indicates that, in addition to estradiol2-hydroxylase, other steroid metabolizing enzymes are firmly associated with the microsomal complex. DISCUSSION

The 2-hydroxylation of estradiol, one of the main reactions in the hepatic metabolism of estrogen [l] can be inhibited by a number of steroid hormones and has been investigated extensively [l&14] since the original kinetic studies with the rat microsomal enzyme by Brueggemeier [9]. In these experiments, either the radiometric [15] or a product isolation method was used and gave identical results with the haloestrogens as inhibitors [ll]. Although the radiometric assay also measures ‘HZ0 released by non-cytochrome P-450-catalyzed reactions which can

424

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A. QUAIL et al.

E2 2-01 ‘El

‘E2

8

E2

EI

T

Std

AD

Eq

DHEq

Fig. 2. Autoradiogram of products formed from [‘4C]estradiol-17/3 by male rat liver microsomes in the presence of various inhibitors. The inhibitors (25 PM) were added to [4-“‘C]E2 (ca 50,000 dpm, 10 PM) and incubated for 30 min as described in the text. E,: estrone, E,: estradiol, E,: estriol, 2-OHE,: 2_hydroxyestrone, 2-OHE,: 2-hydroxyestradiol, T: testosterone, AD: 4-androstenedione, Eq: equilin, DHEq: dihydroequilin.

introduce inaccuracies in assessing estrogen hydroxylation in tissues of low enzymatic activity such as the brain [20], it is still the method of choice for male rat liver, rich in estrogen 2-hydroxylase. The radioenzymatic assay [6] is more sensitive but presents several inherent problems with blanks, particularly because of possible contamination of the catechol-0 -methyl transferase preparations by estrogen 2/4-hydroxylase

PI.

In a previous experiment Brueggemeier et al. [12] determined the amount of irreversible binding of radiolabeled estradiol to microsomal protein and also examined by reverse-phase HPLC the nature of the products formed from estradiol by hepatocytes in the presence of inhibitors. In our current paper we used TLC to separate the ether-soluble metabolites of [4-‘4C]Ez formed by liver microsomes in the presence and absence of inhibitors and visualized them by autoradiography. It is apparent, that even after washing the microsomes with sucrose and Tris-HCl/ascorbit acid, considerable conversion of estradiol to products other than the catechol estrogens occurs and that the formation of some of these metabolites is affected differently by different types of inhibitors. Most inhibitors tested decreased primarily the formation of 2-hydroxyestradiol but others like progesterone, 4-androstenedione and equine estrogens possessing a keto group, gave rise to substantial amounts of estrone which was not hydroxylated rapidly to 2-OHE, under the incubation conditions because of its accumulation in the system. It is probable that, in the presence of excess NADPH, a dehydrogenase associated with the microsomes,

catalyzes the exchange of hydrogen between the keto group of the inhibitor and the 17/_?-hydroxyl group of the substrate resulting in the formation of E,. This would account for the absence of detectable E, when either testosterone or dihydroequilin replaces 4-androstenedione or equilin respectively. The inhibitors would presumably also decrease the 2-hydroxylation of E, resulting in its accumulation. However, even though 2-OHE, would be produced in smaller amounts, it would be protected from further metabolism by the ascorbic acid added to the incubation medium. It is also possible that the 2-OHE, is formed independently by oxidation of 2-OHE, at C-17 in the same way that Ez is converted to E, . In the absence of inhibitor, we observed a rate of E, metabolism by rat liver microsomes very similar to that of E, with virtually no accumulation of E, (unpublished data). Brueggemeier [lo] however, reported that steroids with a 17p-hydroxyl group (estradiol derivatives) had a higher affinity for the active site of estrogen 2-hydroxylase than those with a 17-ketone (estrone derivatives). He also found very little inhibition with 2-OHE, but, like us, found that 4-OHE, and 2-methoxyestradiol were relatively strong inhibitors of estradiol 2-hydroxylase. It is of interest that Theron et al. [22] found an NADPHdependent component of estrogen-2/4-hydroxylase in rat brain that was highly specific for E, with the relative activity with E, being 7% of that observed with E,. It should also be borne in mind that, overall, the mammalian metabolism of the female sex hormone is oxidative in nature with the rapid conversion of the 17/?-hydroxy function to the 17-ketone [2].

Inhibition of catechol estrogen formation The present studies point to a potential control of estrogen 2-hydroxylation by progesterone which was shown by us and also Brueggemeier [lo] to be the most potent naturally-occurring inhibitor of all the steroids tested. Thus, the in uiuo levels of progesterone in the female, or administered progestins, may act to regulate the rate of estradiol oxidation to catechol estrogens in the liver if sufficient amounts were concentrated in this tissue. .Progesterone might also influence gonadotropin and prolactin release if it inhibited the formation of catechol estrogens in the hypothalamus or pituitary in the same way as in the liver. The secretion of both these pituitary hormones has been shown to be affected by the catechol estrogens [24-261. On the other hand, 2-methoxyestradiol which is a major metabolite of the catechol estrogens [I] and is a relatively strong inhibitor of estradiol 2-hydroxylase [IO], was found to have no effect on LH release [27]. However, both 2-methoxyestradiol and the catechol derivatives of E, could have a regulatory role on estrogen metabolism by feedback inhibition. These results demonstrate the complex interaction between different types of steroids and other hormones and emphasize the need to consider the metabolic changes brought about by the liver microsomal system to not only the radioactive steroid substrate being studied but also the inhibitor. They also point to the desirability of using a more highly purified enzyme preparation to carry out kinetic studies on steroid hydroxylation although this can introduce other problems with a membrane-bound multienzyme complex and is also more remote from the in oiuo situation. Other aspects of steroid interaction to be considered are the feedback control of the 2-hydroxylation of E, by the catechol estrogens and their methoxy derivatives, the specific physiological changes that occur as a result of inhibiting catechol estrogen synthesis and the consequence of reducing this pivotal reaction in estradiol metabolism by pharmacological doses of DES or equine estrogens. Acknowledgement-This

work was supported by the

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