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2-Hydroxylation of estradiol by rat kidney and lung
2-HYDROXYLATION RAT KIDNEY
OF ESTRADIOL AND LUNG”
MITSLJTERU NUMAZAWA, YOKO KIYONO
and
BY
TOSHIONAMBARA
Pharmaceutical Institute, Tohoku University, Sendai, Japan (Received 4 December 1979) SUMMARY The existence of estradiol2-hydroxylase
in rat kidney and lung has been demonstrated by a radiometric method using [2-3H]-estradiol as a substrate. The enzyme activity was principally localized in the microsomal fraction of the kidney and in the mitochondrial fraction of the lung. The specific activities of these subcellular fractions were found to be 194 and 97 pmol/mg protein/min, respectively. On the other hand no significant enzyme activity was observed in rat heart even when the radiometric assay method with a detection limit of 29 pmol per assay was employed. Typical saturation curves were obtained by varying the amount of the substrate with the enzyme preparations of the kidney and lung. The kinetic constants observed were as follows: the kidney mitochondria and microsomes, K, = 39.5 and 31.0 PM, Vmar= 26.7 and 63.3 pmol/mg protein/min; the lung mitochondria and microsomes, K, = 86.6 and 106PM, V,,, = 41.7 and 26.3 pmol/mg protein/min. 2-Hydroxyestradiol formed enzymatically from [6,7-3H]-estradiol was identified as the triacetate by means of the reverse dilution method. The physiological significance of estradiol 2-hydroxylase in the kidney and lung is briefly discussed.
MATERIALS AND METHODS Catechol metabolites
estrogens
have
of estradiol*
been
recognized
Animals
as major
since the first report
on the
occurrence of 2-methoxyestrone in human pregnancy urine [I+]. Extensive evidence has accumulated in recent years to reveal that catechol estrogens are active me~~lites, exhibiting their own biological and endocrine activities [7-123. Although 2-hydroxylation is a principal in vitro bioconversion of estrogens in liver [13, 141, placenta [is] and brain [16,17], little is known about this transformation by other tissues. Recently, the enzyme system involved in the formation of catechol in rat liver [18] and brain [17] has been shown to be cytochrome P450-dependent oxidase. Accordingly, it appears to be of particular interest to clarify the physiological role of other tissues having adrenoceptor in the biosynthesis of catechol estrogens. The present paper describes the formation of 2-hydroxyestra~ol from estradiol by rat kidney and lung and the subcellular distribution of the 2-hydroxylase activity in these tissues.
* Part CLVII of Studieson Steroids by T. Nambara; Part CLVI: K. Shimada and T. Nambara, Chem. Phurm. Butt. (Tokyo), 28 (1980) 1559-l 562. *The following trivial names are used in this paper: estrone = 3-hydroxy-1,3,5(10)-estratrien-17-one; estradiol = 1,3,5(10)-estratriene_3,17B_diol; 2-hydroxyestrone = 2,3-dihydroxy-1,3,5(10)-estratrien-17one; 2-hydroxyestradiol = 1,3,5(10)-estratriene_2,3,17ptriol.
Male Wistar rats (8-13-weeks old) weighing 200-300 g were used. The animals were fasted for the last 24 h prior to sacrifice.
NADPH was purchased f;om Sigma Chemical Co. (St. Louis, MO), silica gel HF from E. Merck AG (Darmstadt, West Germany) and [6,7-‘HI-estradiol (48 Ci/mmol) from the Radiochemical Centre (Amersham, England), respectively. [2-3HJ-Estradiol was chemi~lly synthesized from 2-iodoestradiol by methods developed in these laboratories 1191. The radiochemical purity of these 3H-labeled steroids was checked by thin-layer chromatography (t.1.c.) prior to use. 2-Hydroxyestradiol was prepared in the manner described by Gelbke et aI.[20].
Counting was carried out on a Packard Tri-Carb Model 3380 liquid scintillation spectrometer employing the Bray’s scintillator [21]. Correction for quenching was made by the automatic external standard method. Preparation of sffbcellu~~rfractions The rats were sacrificed by stunning and decapitation, and the liver, lung, heart and kidney were immediately removed and chilled on ice. All the subsequent procedures were carried out at 04°C. The
1101
1102
MITSUTERUNUMAZAWA,YOKOKIYONO and
tissue was weighed, finely minced with scissors, and homogenized with a 4-fold volume of an ice-cold 0.25 M sucrose by a Potter-Elvejhem homogenizer with a Teflon pestle. The homogenate was centrifuged at 6009 for 10min and the supernatant was in turn centrifuged at 8000g for 10 min. The sediment was washed twice with 0.25 M sucrose and used as a mitochondrial fraction. The 8CQOg supematant was then centrifuged at 20,OOOg for 10min. The lysosomal sediment thus obtained was discarded and the supernatant was centrifuged at 105,OOOgfor 60 min by a Beckman Model L5-65 ultracentrifuge. The sediment was washed with 0.25 M sucrose and used as a microsomal fraction. The supernatant was again centrifuged at 105,OOOg for 90min. The mitochondrial and microsomal fractions obtained were gently resuspended in 0.25 M sucrose. The amount of protein was determined by the method of Lowry et aI.[22] using bovine serum albumin as a reference. Electron microscopy
Microsomal and mitochondrial pellets were fixed with osmium tetraoxide, dehydrated, and embedded in the epoxy resin. These sections were stained with uranium acetate and lead, and photographed at random with a Hitachi Model H 500 electron microscope. Enzyme assay
The incubation studies were carried out with two standard systems: (A) an enzyme preparation (0.5 ml), NADPH (3.3 wol), [6,7-3H]-estradiol (7.4 nmol, 4.5 @i) dissolved in 50% (v/v) aq. methanol (0.1 ml), and sufficient 0.05 M Tris-HCl buffer (pH 7.4) to make the final volume 1.1 ml. The amounts of protein in subcellular fractions used were as follows: kidney microsomes and mitochondria 2 mg, lung microsomes 0.5 mg, lung mitochondria 0.25 mg. Incubations were carried out for 60 min with an exception of the experiment for kidney microsomes (15 min) at 37°C under aerobic conditions. After a definite period ascorbic acid (2 mg) was added to terminate the reaction, and the incubation mixture was cooled immediately in an ice-bath. 2-Hydroxyestradiol (1 mg) and estradiol (1 mg) dissolved in methanol (0.2 ml) were added to the mixture, and the resulting solution was centrifuged at 3000 g for 20 mm to separate denatured protein. The sediment was washed successively, with 1 N HCl (1 ml), methanol (0.5 ml), and ethyl acetate (2 ml). The supernatant was extracted with ethyl acetate (4 ml x 2), and 2-hydroxyestradiol (30 mg) was then added to the extract. The organic layer was washed with water, dried over anhydrous sodium sulfate and evaporated to give a crude steroid fraction; (B) an enzyme preparation (0.5 ml), NADPH (3.3 mol), [2-3H]-estradiol (35 nmol, 1 PCi) dissolved in 50% (v/v) aq. methanol (0.1 ml) and sufficient 0.05 M Tris-HCl buffer (pH 7.4) to make the final volume 1.1 ml. Incubation was carried out at 37°C under aerobic conditions. After addition of 1 N HCl (2 ml)
TOSHIONAMBARA
to terminate the reaction, the incubation mixture was allowed to stand at 4°C overnight and centrifuged at 30009 for 20min to remove denatured protein. The sediment was washed with water (1.5 ml x 3) and washings were combined with the supernatant. The radioactivity of tritiated water released into the aqueous phase was measured to determine the activity of estradiol 2-hydroxylase by the method developed in these laboratories [19]. Isolation of 2-hydroxyestradiol triacetate
The crude steroid fraction obtained by incubation using system A was dissolved in pyridine (1.5 ml)acetic anhydride (0.8 ml) and allowed to stand at room temperature overnight. After evaporation of the solvent under reduced pressure, the crude product was purified by preparative t.1.c. using n-hexane-ethyl acetate (5: 1, v/v) as a developing solvent. The zone corresponding to 2-hydroxyestradiol triacetate (RF0.35) was scrapped off and eluted with ethyl acetate. The organic layer was evaporated under reduced pressure to provide radioactive 2-hydroxyestradiol triacetate (28-34 mg). The constant specific activity of the triacetate was attained after four crystallizations (three times from methanol followed by one from aq. acetone or two times from methanol followed by two from aq. acetone) for each case. RESULTS
When [2-3H]-estradiol was incubated respectively, with rat liver, lung, kidney and heart homogenates in the presence of NADPH, 2-hydroxyestradiol was produced by the former three tissues but not by the heart homogenate to any detectable extent. The enzyme activities of kidney and lung homogenates were approx. 2.4% and 1.0% of that of the liver homogenate, respectively. Effects of incubation time and protein concentration on the formation of 2-hydroxyestradiol by the subcellular fractions of lung and kidney were examined. The reaction rate was linearly raised with an increasing amount of the microsomal or mitochondrial preparation of both tissues (kidney: up to 2 mg of protein, lung: up to 0.5 mg of protein) and with the incubation time (kidney: up to 20min, lung: up to 30 min). Based upon these data the best conditions for the assays were chosen as follows: for kidney 2 mg of protein and 15 min of incubation time; for lung 0.5 mg (microsomes) or 0.25 mg (mitochondria) of protein and 20 min of incubation time. In the case of liver the previously established conditions [IS] were used. The subcellular distribution of estradiol 2-hydroxylase in the kidney and lung was determined by the standard assay procedure (Table 1). Enzymatic activity of kidney was localized in the microsomal fraction (105,OOOgpellet) as previously observed in liver. In sharp contrast the activity of the mitochondrial fraction (SOOOgpellet) was approx. 250% of that of the microsomal fraction in the lung. No detectable ac-
2-Hydroxylation of estradiol Table 1. Subcellular distribution of estradiol 2-hydroxylase in rat kidney, lung and liver*
1103
Table 3. Reverse isotope dilution analysis of 2-hydroxyestradiol formed from [6,7-‘HI-estradiol by incubation with subcellular fractions of rat kidney and lung under the condition of system A in the text*
3650, 3160 3130 1150
5390 4800 4620
4
180 600 650 630
4
1100 900
[2-3H]Estradiol was incubated with various subcellular fractions under the condition of system B in the text. ** Mean k SD. (n = 6). *** N.D. = not detected.
800
*
tivity was observed even in the subcellular fractions of the heart. The subcellular fractions were clearly characterized by means of electron microscopy. The saturation curves were obtained by varying the
amount of substrate for each enzyme preparation. A Lineweaver-Burk plot of the reaction velocity against the concentration of estradiol gave the apparent K, and I$,,, values (Table 2). In order to identify 2-hydroxyestradiol formed from estradiol the 3H-labeled substrate was incubated with the subcellular fractions at 37°C under the conditions using system A. The steroid fraction obtained from the incubation mixutre was acetylated in the usual manner and the resulting 2-hydroxyestradiol triacetate was isolated by preparative t.1.c. The radio-
830
4
*The steroid fraction was mixed with nonradioactive 2-hydroxyestradiol (30 mg) and then acetylated with acetic anhydride and pyridine. The resulting 2-hydroxyestradiol triacetate was separated by t.1.c. and was crystallized repeatedly up to constant specific activity.
chemical purity of the isolated triacetate is given in Table 3. The conversion rates of [6,7-3H]-estradiol to radioactive 2-hydroxyestradiol were comparable to those obtained by the experiments using [2-3H]estradiol. A portion of the acetylated steroid fractions obtained in each experiment was applied to t.1.c. where only two radioactive peaks corresponding to estradiol diacetate and 2-hydroxyestradiol triacetate were detected on the chromatogram. DISCUSSION
Table 2. Apparent KM and V,,, values of estradiol 2-hydroxylase in subcellular fractions of rat kidney and lung* -_ Subcellular fraction Km “max (pmollmg protein/mIn) (WI Kidney Mitochondria
39.5
26.7
Micrasomes
31.0
63.3
86.6
41.7
Lung Mitochondria Microsomes
106
26.3
* The estradiol 2-hydroxylase activity was assayed with various concentrations of estradiol (2.5-100 pM) under the standard condition (system B, see text).
The radiometric method which involves the determination of tritiated water enzymatically formed from [2-3H]-estradiol, has been used for measurement of estradiol 2-hydroxylase activity in rat kidney, lung and heart. It has been demonstrated that both kidney and lung exhibited 2-hydroxylase activity while the heart showed no detectable activity when the assay method with a detection limit of 29 pmol was employed. The formation of 2-hydroxyestradiol by the kidney and lung was confirmed by the experiments using [6,7-3H]-estradiol as a substrate. In the previous papers it has been reported that the 2-hydroxylase activity is localized mainly in the microsomal fractions of rat brain [17] and human placenta [15] and that the specific activity of the brain
1104
MITSUTERU NUMAZAWA, YOKOKIYONO and
microsomes is comparable to that of the liver microsomes. The subcellular distribution of the enzyme activity in rat kidney appears to be similar to that of the tissues described above. In sharp contrast to these findings the 2-hydroxylase activity of the lung is distributed mainly in the mitochondrial fraction. The subcellular fractions used in the present study were unambiguously identified by means of electron microscopy. The specific activities of the kidney microsomes and lung mitochondria were found to be only 11% and 5% of that of the liver microsomes, respectively, but somewhat higher than that of the rat brain microsomes reported by Paul et aL[l7]. The lung with a large blood supply [23] and capillary surface area [24] may have a potential function to form a large amount of catechol estrogens in the pulmonary tissue in spite of the relatively low enzyme activity. Estradiol 2-hydroxylase in rat lung is peculiar and interesting in that the enzyme activity is localized in the mitochondrial fraction, although oxygenase which participates in the biosynthesis of steroid hormones in the adrenal cortex is also distributed in the mitochondrial fraction [25,26]. Determination of the kinetic constant with a heterogeneous system does not permit the precise analysis of the enzymatic reaction. The data obtained in this study appear to follow the Michaelis-Menten kinetics and hence comparison of the kinetic constant between the subcellular fractions in the lung and kidney is profitable. The Knr values were nearly identical between the microsomal and mitochondrial fractions in each tissue. The V,,, values of the kidney mitochondria and lung microsomes were approximately half of that of the kidney microsomes and two-third of that of the lung mitochondria. These results imply that the relatively high activities of the kidney microsomes and lung mitochondria as compared with other subcellular fractions in tissues may be in part attributable to the V,,, value. The kidney plays an important role in the biotransformation of estrogens, in particular glucoronidation [27,28] and oxidoreduction of the functional group at C-17 [28]. On the other hand, the activities of dehydroepiandrosterone 7cc-hydroxylase, 17fi-hydroxysteroid oxidoreductase, 3jShydroxysteroid oxidoreductase and 1l/I-hydroxysteroid oxidoreductase have been observed in human lung [29-311. TO the best of our knowledge this is the first recorded existence of estradiol 2-hydroxylase in the lung and kidney. Ichikawa et al.[32] reported the presence of cytochrome P450 in the rat kidney microsomes and also possibly in the lung [33,34]. It is substantiated that 2-hydroxylation of estradiol by the rat liver [18] and brain [17] microsomes is catalyzed by a cytochrome P450-dependent oxygenase but 2-hydroxylase in the liver microsomes is somewhat different from that in the brain microsomes and other steroid hydroxylases with respect to the sensitivity to carbon monoxide [18]. These results suggest that 2-hydroxylase in the kidney and lung may also be a cytochrome P4SO-dependent oxygenase. Recently, it has been
TOSHIONAMBARA
demonstrated that the lung tissue has /3-adrenoceptor [35] and the nerve tissue of the histochemically adrenargic characteristics is abundant in rat kidney [36]. Considering the biological potency of catechol estrogens, these findings strongly imply that the enzyme system involved in the formation of catechol estrogens in tissues may play an important role in the neuroendocrine regulation. Acknow[edgemenIs-This work was supported in part by a Grant-in-Aid for the Scientific Research from the Ministry of Education, Science and Culture of Japan. We thank Dr H. Watanabe, Tohoku University, School of Medicine, for his help in the electron microscopic study. REFERENCES
1. Kraychy S. and Gallagher T. F.: 2-Methoxyestrone, a new metabolite of estradiol-17b in man. J. hiol. Chem. 229 (1957) 519-526. 2. Fishman J.: Role of 2-hydroxyestrone in estrogen metabolism. J. c/in. Endocr. Metab. 23 (1963) 207-210. 3. Gelbke H. P., Bottger M. and Knuppen R.: Excretion of 2-hydroxyesterone in urine throughout human pregnancies. J. clin. Endocr. Metab. 41 (1975) 744-750. 4. Ball P., Gelbke H. P. and Knuppen R.: The excretion of 2-hydroxyesterone during the menstrual cycle. J. clin. Endocr. Metab. 40 (1975) 406-408. 5. Yoshizawa I. and Fishman J.: Radioimmunoassay of 2-hydroxyestrone in human plasma. J. clin. Endocr. Metah. 32 (1971) 34. 6. Ball P., Emons G., Haupt O., Hoppen H.-O. and Knuppen R.: Radioimmunoassay of 2-hydroxyestrone. Steroids 31 (1978) 249-258. 7. Ball P., Knuppen R., Haupt M. and Breuer H.: Interactions between estrogens and catechol amines III. Studies on the methylation of catechol estrogens, catecho1 amines and other catechols by the catechol-Omethyltransferase of human liver. J. clin. Endocr. Metab. 34 (1972) 736-746. 8. Knuunen R.. Wennrich W.. Ball P. and Breuer H.: Wechselwirkingen zwishen bstrogenen and Catecholaminen beider Ratte. Hoppe-Seyler’s Z. physiol. Chem. 353 (1972) 1209-1216. 9. Parvizi N. and Ellendorff F. : 2-Hydroxyestradiol- 17/I as a possible link in steroid brain interaction. Narure 256 (1975) 59-60. 10. Naftolin F., Morishita H., Davies I. J., Todd R., Ryan K. J. and Fishman J.: 2-Hydroxyesterone induced rise in serum luteinizing hormone in the immature male rat. Biochem. biophys. Res. Commun. 64 (1975) 905-910. 11. Gethmann U. and Knuppen R.: Effect of 2-hydroxyesterone on lutropin (LH) and follitropin (FSH) secretion in the ovariectomized primed rat. HoppeSeyler’s Z. physiol. Chem. 357 (1976) 101l-1013. 12. Luttge W. G. and Jasper T. W.: Studies on the possible role of 2-OH-estradiol in the control of sexual behavior in female rats. Lifr Sci. M (1977) 419325. 13. Kuss E.: Mikrosomale Oxidation des Ostradiols-17P. 2-Hydroxylierung und 1-bzw. 4-Thioltherbildung mit und ohne 17b-Hydroxy-Dehydrogenierung. HoppeSeyler’s Z. physiol. Chem. 352 (1971) 817-836. 14 Hoppen H.-O., Siekmann L. and Breuer H.: Stoffwechse1 von &tron in der Mikrosomenfraktion der Rattenleber. Hoppe-Seyler’s Z. physiol. Chem. 355 (1974) 1305~1315. 15 Fishman J. and Dixon D.: 2-Hydroxylation of estradiol by human placental microsomes. Biochemistry 6 (1967) 1683-1687.
16 Fishman J. and Norton B.: Catechol estrogen formation in the central nervous system of the rat. Endocrinology % (1975) 10541059.
2-Hydroxylation of estradiol 17. Paul S. M., Axelrod J. and Diliberto E. J. Jr.: Catechol estrogen forming enzyme of brain: Demonstration of a cytochrom P450 monooxygenase. Endocrinology 101 (1977) 1604-1610. 18. Numazawa M., Soeda N., Kiyono Y. and Nambara T.: Properties of estradiol 2-hydroxylase and 2-hydroxy-3deoxyestradiol 3-hydroxylase in rat liver. J. steroid Biochem. 10 (1979) 227-233. 19. Numazawa M., Kiyono Y. and Nambara T.: A simple radiometric assay for estradiol 2-hydroxylase activity. Analyt. Eiochem. accepted for publication. 20. Gelbke H. P., Haupt 0. and Knuppen R.: A simple method for the synthesis of catechol extrogens. Steroids 21 (1973) 205-218. 21. Bray G. A.: A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Eiochem. 1 (1960) 279-285. 22. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193 (1951) 265-275. 23. Bakhle Y. S. and Vane J. R.: Pharmacokinetic function of the pulmonary circulation. Physiol. Reo. 54 (1974) 1007-1045. 24. Fishman A. P. and Pietra G. G. : Handling of bioactive materials by the lung. New Engl. J. Med. 291 (1974) 884890. 25. Hayano M., Dorfman R. I. and Yamada E. Y.: The conversion of desoxycorticosterone to glycogenic material by adrenal homogenates. J. biol. Chem. 193 (1951) 1755181. 26. Raman P. B., Sharma D. C. and Dorfman R. I. : Studies on aldosterone biosynthesis in vitro. Biochemistry 5 (1966) 1795-1804. 27. Kirdani R. Y., Sampson D., Murphy G. P. and Sandberg A. A.: Studies on phenolic steroids in human sub-
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jects. XVI. Role of the kidney in the disposition of estriol. J. c/in. Endocr. Metab. 34 (1972) 54&557. 28. Honjo H., Ishihara M., Osawa Y., Kirdani R. Y. and Sandberg A. A.: In oivo and in vitro conjugation and metabolism of estrogens by the baboon kidney. Endocrinology 99 (1976) 1054-1062. 29. Milewich L., Winters A. J., Stephens P. and MacDonald P. C.: Metabolism of dehydroisoandrosterone and androstenedione by the human lung in vitro. .I. steroid Biochem. 8 (1977) 277-284. 30. Milewich L., Stephens P. and MacDonald P. C.: Testosterone metabolism by human lung tissue. J. steroid Biochem. 9 (1978) 29-32. 3 I. Murphy B. E. P.: Cortisol production and inactivation
by the human lung during gestation and infancy. J. clin. Endocr. Metab. 47 (1978) 243-248. 32. Ichikawa Y. and Yamano T.: Electron spin resonance of microsomes, correlation of the amount of CObinding species with so-called microsomal Fex in microsomes of normal tissues and liver microsomes of Sudan III-treated animals. Arch. biochem. Biophys. 121 (1967) 742-749. 33. Hook G. E. R., Bend J. R., Hoe1 D., Fouts J. R. and
Gram T. E.: Preparation of lung microsomes and a comparison of the distribution of enzymes between subcellular fractions of rabbit lung and liver. J. pharmacol. Exp. Ther. 182 (1972) 474490. 34. Smith B. R., Maguire J. H., Ball L. M. and Bend J. R.: Pulmonary metabolism of epoxides. Fed. Proc. 37
(1978) 248t&2484. 35. Barnett D. B., Rugg E. L. and Nahorski S. R.: Direct evidence of two types of 1 adrenoceptor binding site in lung tissue. Nature 273 (1978) 166168. 36. Barajas L.: Innervation of the renal cortex. Fed. Proc. 37 (1978) 1192-1201.
OF ESTRADIOL AND LUNG”
MITSLJTERU NUMAZAWA, YOKO KIYONO
and
BY
TOSHIONAMBARA
Pharmaceutical Institute, Tohoku University, Sendai, Japan (Received 4 December 1979) SUMMARY The existence of estradiol2-hydroxylase
in rat kidney and lung has been demonstrated by a radiometric method using [2-3H]-estradiol as a substrate. The enzyme activity was principally localized in the microsomal fraction of the kidney and in the mitochondrial fraction of the lung. The specific activities of these subcellular fractions were found to be 194 and 97 pmol/mg protein/min, respectively. On the other hand no significant enzyme activity was observed in rat heart even when the radiometric assay method with a detection limit of 29 pmol per assay was employed. Typical saturation curves were obtained by varying the amount of the substrate with the enzyme preparations of the kidney and lung. The kinetic constants observed were as follows: the kidney mitochondria and microsomes, K, = 39.5 and 31.0 PM, Vmar= 26.7 and 63.3 pmol/mg protein/min; the lung mitochondria and microsomes, K, = 86.6 and 106PM, V,,, = 41.7 and 26.3 pmol/mg protein/min. 2-Hydroxyestradiol formed enzymatically from [6,7-3H]-estradiol was identified as the triacetate by means of the reverse dilution method. The physiological significance of estradiol 2-hydroxylase in the kidney and lung is briefly discussed.
MATERIALS AND METHODS Catechol metabolites
estrogens
have
of estradiol*
been
recognized
Animals
as major
since the first report
on the
occurrence of 2-methoxyestrone in human pregnancy urine [I+]. Extensive evidence has accumulated in recent years to reveal that catechol estrogens are active me~~lites, exhibiting their own biological and endocrine activities [7-123. Although 2-hydroxylation is a principal in vitro bioconversion of estrogens in liver [13, 141, placenta [is] and brain [16,17], little is known about this transformation by other tissues. Recently, the enzyme system involved in the formation of catechol in rat liver [18] and brain [17] has been shown to be cytochrome P450-dependent oxidase. Accordingly, it appears to be of particular interest to clarify the physiological role of other tissues having adrenoceptor in the biosynthesis of catechol estrogens. The present paper describes the formation of 2-hydroxyestra~ol from estradiol by rat kidney and lung and the subcellular distribution of the 2-hydroxylase activity in these tissues.
* Part CLVII of Studieson Steroids by T. Nambara; Part CLVI: K. Shimada and T. Nambara, Chem. Phurm. Butt. (Tokyo), 28 (1980) 1559-l 562. *The following trivial names are used in this paper: estrone = 3-hydroxy-1,3,5(10)-estratrien-17-one; estradiol = 1,3,5(10)-estratriene_3,17B_diol; 2-hydroxyestrone = 2,3-dihydroxy-1,3,5(10)-estratrien-17one; 2-hydroxyestradiol = 1,3,5(10)-estratriene_2,3,17ptriol.
Male Wistar rats (8-13-weeks old) weighing 200-300 g were used. The animals were fasted for the last 24 h prior to sacrifice.
NADPH was purchased f;om Sigma Chemical Co. (St. Louis, MO), silica gel HF from E. Merck AG (Darmstadt, West Germany) and [6,7-‘HI-estradiol (48 Ci/mmol) from the Radiochemical Centre (Amersham, England), respectively. [2-3HJ-Estradiol was chemi~lly synthesized from 2-iodoestradiol by methods developed in these laboratories 1191. The radiochemical purity of these 3H-labeled steroids was checked by thin-layer chromatography (t.1.c.) prior to use. 2-Hydroxyestradiol was prepared in the manner described by Gelbke et aI.[20].
Counting was carried out on a Packard Tri-Carb Model 3380 liquid scintillation spectrometer employing the Bray’s scintillator [21]. Correction for quenching was made by the automatic external standard method. Preparation of sffbcellu~~rfractions The rats were sacrificed by stunning and decapitation, and the liver, lung, heart and kidney were immediately removed and chilled on ice. All the subsequent procedures were carried out at 04°C. The
1101
1102
MITSUTERUNUMAZAWA,YOKOKIYONO and
tissue was weighed, finely minced with scissors, and homogenized with a 4-fold volume of an ice-cold 0.25 M sucrose by a Potter-Elvejhem homogenizer with a Teflon pestle. The homogenate was centrifuged at 6009 for 10min and the supernatant was in turn centrifuged at 8000g for 10 min. The sediment was washed twice with 0.25 M sucrose and used as a mitochondrial fraction. The 8CQOg supematant was then centrifuged at 20,OOOg for 10min. The lysosomal sediment thus obtained was discarded and the supernatant was centrifuged at 105,OOOgfor 60 min by a Beckman Model L5-65 ultracentrifuge. The sediment was washed with 0.25 M sucrose and used as a microsomal fraction. The supernatant was again centrifuged at 105,OOOg for 90min. The mitochondrial and microsomal fractions obtained were gently resuspended in 0.25 M sucrose. The amount of protein was determined by the method of Lowry et aI.[22] using bovine serum albumin as a reference. Electron microscopy
Microsomal and mitochondrial pellets were fixed with osmium tetraoxide, dehydrated, and embedded in the epoxy resin. These sections were stained with uranium acetate and lead, and photographed at random with a Hitachi Model H 500 electron microscope. Enzyme assay
The incubation studies were carried out with two standard systems: (A) an enzyme preparation (0.5 ml), NADPH (3.3 wol), [6,7-3H]-estradiol (7.4 nmol, 4.5 @i) dissolved in 50% (v/v) aq. methanol (0.1 ml), and sufficient 0.05 M Tris-HCl buffer (pH 7.4) to make the final volume 1.1 ml. The amounts of protein in subcellular fractions used were as follows: kidney microsomes and mitochondria 2 mg, lung microsomes 0.5 mg, lung mitochondria 0.25 mg. Incubations were carried out for 60 min with an exception of the experiment for kidney microsomes (15 min) at 37°C under aerobic conditions. After a definite period ascorbic acid (2 mg) was added to terminate the reaction, and the incubation mixture was cooled immediately in an ice-bath. 2-Hydroxyestradiol (1 mg) and estradiol (1 mg) dissolved in methanol (0.2 ml) were added to the mixture, and the resulting solution was centrifuged at 3000 g for 20 mm to separate denatured protein. The sediment was washed successively, with 1 N HCl (1 ml), methanol (0.5 ml), and ethyl acetate (2 ml). The supernatant was extracted with ethyl acetate (4 ml x 2), and 2-hydroxyestradiol (30 mg) was then added to the extract. The organic layer was washed with water, dried over anhydrous sodium sulfate and evaporated to give a crude steroid fraction; (B) an enzyme preparation (0.5 ml), NADPH (3.3 mol), [2-3H]-estradiol (35 nmol, 1 PCi) dissolved in 50% (v/v) aq. methanol (0.1 ml) and sufficient 0.05 M Tris-HCl buffer (pH 7.4) to make the final volume 1.1 ml. Incubation was carried out at 37°C under aerobic conditions. After addition of 1 N HCl (2 ml)
TOSHIONAMBARA
to terminate the reaction, the incubation mixture was allowed to stand at 4°C overnight and centrifuged at 30009 for 20min to remove denatured protein. The sediment was washed with water (1.5 ml x 3) and washings were combined with the supernatant. The radioactivity of tritiated water released into the aqueous phase was measured to determine the activity of estradiol 2-hydroxylase by the method developed in these laboratories [19]. Isolation of 2-hydroxyestradiol triacetate
The crude steroid fraction obtained by incubation using system A was dissolved in pyridine (1.5 ml)acetic anhydride (0.8 ml) and allowed to stand at room temperature overnight. After evaporation of the solvent under reduced pressure, the crude product was purified by preparative t.1.c. using n-hexane-ethyl acetate (5: 1, v/v) as a developing solvent. The zone corresponding to 2-hydroxyestradiol triacetate (RF0.35) was scrapped off and eluted with ethyl acetate. The organic layer was evaporated under reduced pressure to provide radioactive 2-hydroxyestradiol triacetate (28-34 mg). The constant specific activity of the triacetate was attained after four crystallizations (three times from methanol followed by one from aq. acetone or two times from methanol followed by two from aq. acetone) for each case. RESULTS
When [2-3H]-estradiol was incubated respectively, with rat liver, lung, kidney and heart homogenates in the presence of NADPH, 2-hydroxyestradiol was produced by the former three tissues but not by the heart homogenate to any detectable extent. The enzyme activities of kidney and lung homogenates were approx. 2.4% and 1.0% of that of the liver homogenate, respectively. Effects of incubation time and protein concentration on the formation of 2-hydroxyestradiol by the subcellular fractions of lung and kidney were examined. The reaction rate was linearly raised with an increasing amount of the microsomal or mitochondrial preparation of both tissues (kidney: up to 2 mg of protein, lung: up to 0.5 mg of protein) and with the incubation time (kidney: up to 20min, lung: up to 30 min). Based upon these data the best conditions for the assays were chosen as follows: for kidney 2 mg of protein and 15 min of incubation time; for lung 0.5 mg (microsomes) or 0.25 mg (mitochondria) of protein and 20 min of incubation time. In the case of liver the previously established conditions [IS] were used. The subcellular distribution of estradiol 2-hydroxylase in the kidney and lung was determined by the standard assay procedure (Table 1). Enzymatic activity of kidney was localized in the microsomal fraction (105,OOOgpellet) as previously observed in liver. In sharp contrast the activity of the mitochondrial fraction (SOOOgpellet) was approx. 250% of that of the microsomal fraction in the lung. No detectable ac-
2-Hydroxylation of estradiol Table 1. Subcellular distribution of estradiol 2-hydroxylase in rat kidney, lung and liver*
1103
Table 3. Reverse isotope dilution analysis of 2-hydroxyestradiol formed from [6,7-‘HI-estradiol by incubation with subcellular fractions of rat kidney and lung under the condition of system A in the text*
3650, 3160 3130 1150
5390 4800 4620
4
180 600 650 630
4
1100 900
[2-3H]Estradiol was incubated with various subcellular fractions under the condition of system B in the text. ** Mean k SD. (n = 6). *** N.D. = not detected.
800
*
tivity was observed even in the subcellular fractions of the heart. The subcellular fractions were clearly characterized by means of electron microscopy. The saturation curves were obtained by varying the
amount of substrate for each enzyme preparation. A Lineweaver-Burk plot of the reaction velocity against the concentration of estradiol gave the apparent K, and I$,,, values (Table 2). In order to identify 2-hydroxyestradiol formed from estradiol the 3H-labeled substrate was incubated with the subcellular fractions at 37°C under the conditions using system A. The steroid fraction obtained from the incubation mixutre was acetylated in the usual manner and the resulting 2-hydroxyestradiol triacetate was isolated by preparative t.1.c. The radio-
830
4
*The steroid fraction was mixed with nonradioactive 2-hydroxyestradiol (30 mg) and then acetylated with acetic anhydride and pyridine. The resulting 2-hydroxyestradiol triacetate was separated by t.1.c. and was crystallized repeatedly up to constant specific activity.
chemical purity of the isolated triacetate is given in Table 3. The conversion rates of [6,7-3H]-estradiol to radioactive 2-hydroxyestradiol were comparable to those obtained by the experiments using [2-3H]estradiol. A portion of the acetylated steroid fractions obtained in each experiment was applied to t.1.c. where only two radioactive peaks corresponding to estradiol diacetate and 2-hydroxyestradiol triacetate were detected on the chromatogram. DISCUSSION
Table 2. Apparent KM and V,,, values of estradiol 2-hydroxylase in subcellular fractions of rat kidney and lung* -_ Subcellular fraction Km “max (pmollmg protein/mIn) (WI Kidney Mitochondria
39.5
26.7
Micrasomes
31.0
63.3
86.6
41.7
Lung Mitochondria Microsomes
106
26.3
* The estradiol 2-hydroxylase activity was assayed with various concentrations of estradiol (2.5-100 pM) under the standard condition (system B, see text).
The radiometric method which involves the determination of tritiated water enzymatically formed from [2-3H]-estradiol, has been used for measurement of estradiol 2-hydroxylase activity in rat kidney, lung and heart. It has been demonstrated that both kidney and lung exhibited 2-hydroxylase activity while the heart showed no detectable activity when the assay method with a detection limit of 29 pmol was employed. The formation of 2-hydroxyestradiol by the kidney and lung was confirmed by the experiments using [6,7-3H]-estradiol as a substrate. In the previous papers it has been reported that the 2-hydroxylase activity is localized mainly in the microsomal fractions of rat brain [17] and human placenta [15] and that the specific activity of the brain
1104
MITSUTERU NUMAZAWA, YOKOKIYONO and
microsomes is comparable to that of the liver microsomes. The subcellular distribution of the enzyme activity in rat kidney appears to be similar to that of the tissues described above. In sharp contrast to these findings the 2-hydroxylase activity of the lung is distributed mainly in the mitochondrial fraction. The subcellular fractions used in the present study were unambiguously identified by means of electron microscopy. The specific activities of the kidney microsomes and lung mitochondria were found to be only 11% and 5% of that of the liver microsomes, respectively, but somewhat higher than that of the rat brain microsomes reported by Paul et aL[l7]. The lung with a large blood supply [23] and capillary surface area [24] may have a potential function to form a large amount of catechol estrogens in the pulmonary tissue in spite of the relatively low enzyme activity. Estradiol 2-hydroxylase in rat lung is peculiar and interesting in that the enzyme activity is localized in the mitochondrial fraction, although oxygenase which participates in the biosynthesis of steroid hormones in the adrenal cortex is also distributed in the mitochondrial fraction [25,26]. Determination of the kinetic constant with a heterogeneous system does not permit the precise analysis of the enzymatic reaction. The data obtained in this study appear to follow the Michaelis-Menten kinetics and hence comparison of the kinetic constant between the subcellular fractions in the lung and kidney is profitable. The Knr values were nearly identical between the microsomal and mitochondrial fractions in each tissue. The V,,, values of the kidney mitochondria and lung microsomes were approximately half of that of the kidney microsomes and two-third of that of the lung mitochondria. These results imply that the relatively high activities of the kidney microsomes and lung mitochondria as compared with other subcellular fractions in tissues may be in part attributable to the V,,, value. The kidney plays an important role in the biotransformation of estrogens, in particular glucoronidation [27,28] and oxidoreduction of the functional group at C-17 [28]. On the other hand, the activities of dehydroepiandrosterone 7cc-hydroxylase, 17fi-hydroxysteroid oxidoreductase, 3jShydroxysteroid oxidoreductase and 1l/I-hydroxysteroid oxidoreductase have been observed in human lung [29-311. TO the best of our knowledge this is the first recorded existence of estradiol 2-hydroxylase in the lung and kidney. Ichikawa et al.[32] reported the presence of cytochrome P450 in the rat kidney microsomes and also possibly in the lung [33,34]. It is substantiated that 2-hydroxylation of estradiol by the rat liver [18] and brain [17] microsomes is catalyzed by a cytochrome P450-dependent oxygenase but 2-hydroxylase in the liver microsomes is somewhat different from that in the brain microsomes and other steroid hydroxylases with respect to the sensitivity to carbon monoxide [18]. These results suggest that 2-hydroxylase in the kidney and lung may also be a cytochrome P4SO-dependent oxygenase. Recently, it has been
TOSHIONAMBARA
demonstrated that the lung tissue has /3-adrenoceptor [35] and the nerve tissue of the histochemically adrenargic characteristics is abundant in rat kidney [36]. Considering the biological potency of catechol estrogens, these findings strongly imply that the enzyme system involved in the formation of catechol estrogens in tissues may play an important role in the neuroendocrine regulation. Acknow[edgemenIs-This work was supported in part by a Grant-in-Aid for the Scientific Research from the Ministry of Education, Science and Culture of Japan. We thank Dr H. Watanabe, Tohoku University, School of Medicine, for his help in the electron microscopic study. REFERENCES
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