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Catechol estrogen adducts
J. steroid Eiochem. Vol. 31, No. 1, pp. 107-110, 1988 Printed in Great Britain. All rights reserved
CATECHOL
Copyright 0
ESTROGEN
0022-4731/88 S3.00 + 0.00 1988 Pergamon Press pk
ADDUCTS
YUSUF J. ABUL-HAJJ and PAUL L. CISEK Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, U.S.A. (Received25 Augu.rt 1987) SummPry-Reaction of estrone-3,4-o-quinone with ethanethiol and glutathione leads to the formation of 4-hydroxyestrone-2-thioethers. Incubations of [ l-‘Hlhydroxyestrone with rat liver microsomes and NADPH in the presence of glutathione results in the formation of Chydroxyestrone-S-glutathione with no release of tritium in the water indicating GSH addition to C-2 of Chydroxyestrone.
INTRODUCI’ION Estrone (E,) and estradiol (E2) are converted in the presence of glutathione (GSH) by rat liver preparations to the l- and 4-S-glutathione conjugates of the corresponding 2,3_catechol estrogens [ 1,2]. The conjugation is interpreted as the sequential reaction involving a hydroxylation at the 2-position, further oxidation to a semiquinone radical as a hypothetic intermediate, and non-enzymatic coupling with the sulthydryl group. The identity of the GSH adducts was established by comparing these metabolites with the chemical synthesis involving a 1,6-addition of GSH to the 2,3estrogen-o-quinones [3,4]. Although many studies have shown the formation of l-hydroxy estrogens by several tissues [5-81, the’ isolation and identification of Chydroxy estrogen glutathione adducts has not yet been observed. This may be due to the significantly lower amounts of Chydroxylations versus 2-hydroxylations in most mammalian tissues. Recent finding by Kalyanaraman et af.[9] showed indirect evidence for selective removal of 3,4-o-quinone through reaction with amino acids and proteins that led these workers to propose a 1,6addition of nucleophiles to 3,4-estrogen-o quinones leading to the formation of l-substituted 3,4-catechol estrogens. In view of the fact that 40H-E, is a more potent estrogen than 2-0HE2 [lo, 1l] and since 4-OHE, was shown to be carcinogenic in Syrian hamsters [12], we carried out this investigation to determine the adducts obtained from reaction of 4-OHE, using liver preparations and to establish the regioselectivity of the 3,4-catechol estrogen adducts. EXPERIMENTAL All chemicals were of analytical grade and were obtained from Sigma, St Louis, Missouri or from Aldrich, Milwaukee, WI. [18,2/I-‘HlCandrostenedione was purchased from New England Nuclear, Cambridge, MA and was used to prepare [1/?-3H]4-
androstenedione by alkaline equilibration. Male SpragueDawley rats (250-300 g) were used. Washed livers were homogenized and centrifuged sequentially to obtain microsomal fraction.
The preparation of [I-‘H’&OHE, was carried out using [l/3-3 H]4-hydroxyandrostenedione (4OH-A) as described previously [ 131. [ lfl -3H]COH-A (2.4 mCi, sp. act. 4.1 Ci/mmol) was dehydrogenated using 2,3dichloro-$6~dicyano-l&benzoquinone (DDQ) in dioxane and HCl. After refluxing for 18 h, the product was purified on silica1 gel followed by crystallization from methanol as described by Marsh et ul.[ 141. Reductive aromatization of [ 1-3H&OHandrostadienedione 17-ketal [ 151 followed by acid hydrolysis gave 4-OHE, in 26% yield. Several crystallizations from acetone-hexane gave pure [ 1-3H]4OHE, m.p. 265-266°C. Reaction of ethanethiol with estrone-3,4_oquinone
The method described previously in our laboratory [16] was used for synthesis of o-quinones from catechol estrogens. Manganese dioxide (100 mg) was added to a magnetically stirred solution of 4-OHE, (50 mg) in chloroform (10 ml) and the mixture stirred for 1 h at room temperature. Following centrifugation at l,OOOg for 10 min, ethanethiol (0.2 mM) was added to the red chloroform solution which resulted in instantaneous decoloration. Evaporation of the chloroform left a solid that showed the presence of at least two spots on TLC (benzene-ethyl acetate, 3 : 1, v/v) which were separated using preparative TLC. The spot having the lower Rf value was identified as 4-OHE,. The ethanethiol adduct was crystallized from acetone-hexane to give pure product: m.p. 195-196’C; ‘H-NMR (CDCI,): 6 0.92 (3H, s, 18CH,), 1.24 (3H, t, SCH,CH,), 2.76 (2H, q, SCH,CH,), 7.0 (lH, s, C-1H): m/e (%R.A.) 346 (lOO%), 284 (2.9), 261 (4.0), 222 (8.8) 183 (12.2), 97 (21.6), 41 (21.9).
107
108
YUSIJFJ. ABUL-HAJIand PAULL.
CISEK
NADPH t RSH Scheme Reaction of glutathione with e&one-3,4_oquinone Glutathione (100 mg) dissolved in a mixture of water (1.5 ml) and acetic acid (1.5 ml) was added to a stirred solution of the chloroform solution of the estrogen 3,4-o-quinone (50 mg, see above). Decoloration took place within 20 s and stirring continued for an additional 30 s. The aqueous phase was diluted with water and extracted 3 times with chloroform. The water extract was lyophilized and crystallized from water to yield an amorphous compound m.p. 209-212°C (decomp.); ‘H-NMR (DMSO): 6 0.83 (3H, S, 18CH3), 1.9 (2H, m, CHICH2CH), 2.3 (2H, m, CH,CH,CH), 3.0 (2H, S, SCH,CH), 3.4 (lH, m, HOOCCHNH,), 3.7 (2H, S, NHCH,COOH), 4.29 (2H, m, SCH,CH), 6.77 (lH, S, C-1H); FAB mass spectrum (% R.A.) 592 (42.3), 308 (49.9) 279 (23.5) 217 (62.4), 199 (23.5), 181 (76.1) 149 (39.8) 126 (lOO%), 109 (85.5). Incubation of [I-‘H]4-OHE,
with rat liver microsomes
The procedure of Numazawa et a1.[17] for incubations with microsomes in the presence of NADPH
1.
was used. Incubations were carried out in the presence of glutathione and the proteins precipitated by addition of 1 ml of 2N HCl and left overnight at 0°C. The protein precipitate was centrifuged and the supernatant solution was applied to a column of Amberlite XAD-2 resin as described by Bradlow[l8]. The column was eluted with H,O (60-80ml) and then methanol (2&30 ml). Radioactivity was determined in all fractions. Methanol fractions containing radioactive tubes were pooled, evaporated, and the residue dissolved in a small volume of methanol and chromatographed over TLC in N-butanol-acetic acid-water (4:1:1, by vol.). The radioactive areas corresponding to standard thioether conjugates of 4-OHE, were scraped, diluted with unlabeled 4-OHE, glutathione conjugate, and recrystallized from water to constant specific activity. RESULTSAND
DISCUSSION
Previous studies on thiol additions to 2,3-estrogeno-quinones [ 191 gave a mixture of C-l and C-4 thio-
Catechol estrogen adducts Table 1. Reaction of [l-3Hlestrone-3,4-oquinone
substrate [L3Hj4-OHE, [l-‘H]3,4_EQ [I-‘H]3,4-EQ [I-%J4-OHE, [I-sH]Q-OHE, [13H]4-OH&
(3,4-EQ) with thiols and [I-‘H&OHE, somes*
Reaction conditions
% Radioactivity released as ‘H,O
-
-
MnOz EtSH GSH Microsomes Microsomes + GSH
2.8 3.2 3.0 11.6 12.3
[I-‘I-I&OHE,
109
% Radioactivity in product 95.5 94.1 94.9 72.9
with mictospecitic activity (wm/mmoW
1.34 7.06 (7.11) 5.75 (6.96) 3.37 (6.99) 7.69 6.84 2.72 (5.65)
*Chemical reactions were carried out using 2l~~rn/~ of [I-‘H&OHE,. Microsomal i~u~tio~ were carried out using 1.1 x IO”cpm of [I-‘HP-OHE, followed by dilution with 50 mg of unlabeled 60HE, or 4-OHE,-2-SG. +Values in brackets were normalized to reflect increased molecular weight of catochol estrogen adducts.
of 2-OWE,. These studies showed that thiol addition was primarily dependent on the nature of the thiol compounds and less on steric hindrance- Results obtained from this report show that thiol additions to 3,4-estrogen-o-quinone lead to the formation of only one thiol adduct which has been characterized using *H-NMR data as the C-2 thioether conjugate of 4-OHE, indicating a 1,6addition of the thiol group to the 3,4-o-quinone (Scheme 1). Further support for the identification of the 3,4catechol estrogen adduct was obtained by reaction of the thiols with ~~0~~~~1~ labeled 4-OHE,. [I -3H]40HEi (Scheme 2) was prepared by reaction of (l/I-‘H]COH-A with DDQ in dioxane leading to the formation of [ 1fl-3 H]4-OH- 1,4*androstadienedione which was reductively aromatized resulting in the formation of [1-3H]4-OHE,. In all these chemical reactions the specific activity of the isolated products remained the same indicating no loss of radioactivity. Table 1 shows that reaction of ethanet~ol and glutathione with [l-3~strone-3,4-oq~none results in essentially no release of radioactivity in water indicating that thiol addition to 3,4-o-quinone did not occur by a 1,4-addition at the C-l position. Furthermore, Table 1 shows that incubations of [1-3H]4-OHE1 with GSH in the presence of microsomes results in the formation of 4-OHE,-2&glutathione (4-OHEi-2-SG) with most of the radioactivity retained in the conjugate indicating glutathione addition at C-2. The identity of the glutathione adduct formed during microsomal incubations in the presence of GSH was established by dilution of labeled compound with authentic standard compounds followed by recrystallization to constant specific activity
ether conjugates
Table 2. R~~i~~tion of [‘HJ4-OHE,-SG obtained from microsomal incubations to constant specific activity Specific activity
After adding carrier 1st Recrystalliition 2nd Recrystalliition 3rd Recrystallization 4th Recrystalllltion
cpm/mg
cpmtmmol
1731 1632 1603 1612 1610
2.92 2.76 2.71 2.72 2.72
(Table 2). About 1l-12% of tritium was released as 3H,0. This release is most likely not due to release from C-2 but rather from C-l as shown recently by Jellinck et aL[20]. About 15% of radioactivity was found to bind to ~~roso~l proteins. Attempts at obtaining 4-OHE,-2-SG from incubations of estrone with rat liver microsomes in the presence of glutathione have not yet been successful mainly because of very low levels of 4-hydroxylations as compared to 2-hydroxylations of estrogens. However, the fact that in Y&-Omicrosomal incubations of 4-OHE1 with glutathione show the formation of 4-OHE,-2-SG suggest that these estrogen adducts are formed but have not yet been detected. In view of the fact that the estrogen 3,4+quinones are selectively removed by reaction with amino acids and proteins as compared to estrogen 2,3-o-quinones [9] and since 4-OH estrogens were found to be carcinogenic in Syrian hamsters while 2-OH estrogens are not [12] suggests that the 4-OH catechol estrogen may play a more significant role in its binding to cellular macromolecules and therefore its potentially higher genotoxic effects. Acknowledgement-This work was supported CA 41269.
by
NIH grant
RRFERRNCES 1. Jellinck P. H., Lowis J. and Boston F.: Further evidence of au estrogeu-peptide conjugate by rat liver in t&o.
Steroids 10 (1967) 329346. 2. Kuss E.: Wasserlosliche Metabolites des 17fl-Oestradiol. Hoppe-Seyler’s 2. physiol. Chem. 348 (1967) 1707-1708. 3. Jellinck P. H. and Eke J. S.: Synthesis of estrogen glutathione and cysteine derivatives. Steroids 13 (1969) 711-718. 4. Kuss E.: Wa~rlosliche Metabolite de Oestradiol 178. III. Trennung und identi~e~g der I- and 4-glutathionthioethcr von 2,3~ihydroxy~~at~~en. Hoppe seyter’s Z. physiol, Gem. 350 (1969) 95-97. 5. Ball P. and Knuppen R.: Formation of 2- and 4-hydroxyestrogens by brain, pituitary, and liver of the human fetus. J. clin. Endoer. Met& 47 (1978) 732-737. 6. Nelson S. D., Mitchell J. R., Dybing E. and Sasame H. E.: Q&chrome P-450 mediated oxidation of 2-hydroxyestrogens to reactive intermediates. Biochem. biophys. Res. Commun. 70 (1976) 1157-l 165.
110
YUSUFJ. ABUL-HAJJand PAUL L. CISEK
7. Numazawa M., Soeda N., Kiyono Y. and Nambara T.: Properties of estradioi 2-hydroxylase and 2-hydroxy3-deoxy estradiol 3-hydroxylase in rat liver. 1. steroid Biochem. 10 (1979) 227-233. 8. Williams J. G., Longcope C. and Williams K. I. H.:
4-Hydroxyestrone: a new metabolite of estradiol-17/I from humans. Steroids 24 (1974) 687-701. 9. Kalyanaraman B., Hintz P. and Sealy R. C.: An electron spin resonance study of free radicals from catechol estrogens. Fe&. Proc. 45 (1986) 2477-2484. 10. Martucci C. P. and Fishman J.: Impact of ~ntinuously administered catechol estrogens on uterine growth and luteinizing hormone secretion. Endocrinology 105 (1979) 1288-1292.
11. Ball P. and Knuppen R.: Catecholestrogens: Chemistry, biogenesis, metabolism, occurrence and physiological significance. Acfa endocr., Copenh. 232 (1986) l-127. 12. Liehr J. G., Fana W. F., Sirbasku D. A. and Ariulubelen A.: Carc&ogenicity of catechol estrogens in Syrian hamsters. J. steroid Biochem. 24 (1986) 353-356. 13. Abul-Haij Y. J.: Studies on the mechanism of action of the aromatase inhibitor, 4-hydroxy-androstenedione. Steroids 41 (1983) 783-790.
14. Marsh D. A., Brodie H. J., Garrett W., Tsai-Morris C. and Brodie A. M. H.: Arotnatase inhibitors. Synthesis and biological activity of androstenedione derivatives. J. med. Chem. 28 (1985) 788-795. 15. Dryden H. L., Webster G. W. and Wieczorek J. J.: The reductive aromatization of steroidal dienones. A new method for the preparation of estrone. J. Am. them. Sot. 86 (1964) 742-743.
16. Abul-Hajj Y. J.: Synthesis of 3,4-estrogen-a-quinone. J. steroid 3iochem. 21 (1984) 621-622. 17. Numazawa M., Shirao R., Soeda N. and Nambara T.: Properties of enzyme systems involved in the formation of catechol estrogen glutathione conjugates in rat liver microsomes. Biochem. Pharmac. 27 (1978) 1833-1838. 18. Bradlow H. L.: Extraction of steroid conjugates with a neutral resin. Steroids 11 (1968) 265285. 19. Abul-Hajj Y. J. and Cisek P. L.: Regioselective reaction of thiols with catechol estrogens and estrogen-oquinones. J. sieroid Biochem. 25 (1986) 245-247. 20. Jellinck P. H., Hahn E. F. and Fishman J.: Absence of reactive intermediates in the formation of catechol estrogens by rat liver microsomes. J. biol. Chem. 261 (1986) 7729-7732.
CATECHOL
Copyright 0
ESTROGEN
0022-4731/88 S3.00 + 0.00 1988 Pergamon Press pk
ADDUCTS
YUSUF J. ABUL-HAJJ and PAUL L. CISEK Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, U.S.A. (Received25 Augu.rt 1987) SummPry-Reaction of estrone-3,4-o-quinone with ethanethiol and glutathione leads to the formation of 4-hydroxyestrone-2-thioethers. Incubations of [ l-‘Hlhydroxyestrone with rat liver microsomes and NADPH in the presence of glutathione results in the formation of Chydroxyestrone-S-glutathione with no release of tritium in the water indicating GSH addition to C-2 of Chydroxyestrone.
INTRODUCI’ION Estrone (E,) and estradiol (E2) are converted in the presence of glutathione (GSH) by rat liver preparations to the l- and 4-S-glutathione conjugates of the corresponding 2,3_catechol estrogens [ 1,2]. The conjugation is interpreted as the sequential reaction involving a hydroxylation at the 2-position, further oxidation to a semiquinone radical as a hypothetic intermediate, and non-enzymatic coupling with the sulthydryl group. The identity of the GSH adducts was established by comparing these metabolites with the chemical synthesis involving a 1,6-addition of GSH to the 2,3estrogen-o-quinones [3,4]. Although many studies have shown the formation of l-hydroxy estrogens by several tissues [5-81, the’ isolation and identification of Chydroxy estrogen glutathione adducts has not yet been observed. This may be due to the significantly lower amounts of Chydroxylations versus 2-hydroxylations in most mammalian tissues. Recent finding by Kalyanaraman et af.[9] showed indirect evidence for selective removal of 3,4-o-quinone through reaction with amino acids and proteins that led these workers to propose a 1,6addition of nucleophiles to 3,4-estrogen-o quinones leading to the formation of l-substituted 3,4-catechol estrogens. In view of the fact that 40H-E, is a more potent estrogen than 2-0HE2 [lo, 1l] and since 4-OHE, was shown to be carcinogenic in Syrian hamsters [12], we carried out this investigation to determine the adducts obtained from reaction of 4-OHE, using liver preparations and to establish the regioselectivity of the 3,4-catechol estrogen adducts. EXPERIMENTAL All chemicals were of analytical grade and were obtained from Sigma, St Louis, Missouri or from Aldrich, Milwaukee, WI. [18,2/I-‘HlCandrostenedione was purchased from New England Nuclear, Cambridge, MA and was used to prepare [1/?-3H]4-
androstenedione by alkaline equilibration. Male SpragueDawley rats (250-300 g) were used. Washed livers were homogenized and centrifuged sequentially to obtain microsomal fraction.
The preparation of [I-‘H’&OHE, was carried out using [l/3-3 H]4-hydroxyandrostenedione (4OH-A) as described previously [ 131. [ lfl -3H]COH-A (2.4 mCi, sp. act. 4.1 Ci/mmol) was dehydrogenated using 2,3dichloro-$6~dicyano-l&benzoquinone (DDQ) in dioxane and HCl. After refluxing for 18 h, the product was purified on silica1 gel followed by crystallization from methanol as described by Marsh et ul.[ 141. Reductive aromatization of [ 1-3H&OHandrostadienedione 17-ketal [ 151 followed by acid hydrolysis gave 4-OHE, in 26% yield. Several crystallizations from acetone-hexane gave pure [ 1-3H]4OHE, m.p. 265-266°C. Reaction of ethanethiol with estrone-3,4_oquinone
The method described previously in our laboratory [16] was used for synthesis of o-quinones from catechol estrogens. Manganese dioxide (100 mg) was added to a magnetically stirred solution of 4-OHE, (50 mg) in chloroform (10 ml) and the mixture stirred for 1 h at room temperature. Following centrifugation at l,OOOg for 10 min, ethanethiol (0.2 mM) was added to the red chloroform solution which resulted in instantaneous decoloration. Evaporation of the chloroform left a solid that showed the presence of at least two spots on TLC (benzene-ethyl acetate, 3 : 1, v/v) which were separated using preparative TLC. The spot having the lower Rf value was identified as 4-OHE,. The ethanethiol adduct was crystallized from acetone-hexane to give pure product: m.p. 195-196’C; ‘H-NMR (CDCI,): 6 0.92 (3H, s, 18CH,), 1.24 (3H, t, SCH,CH,), 2.76 (2H, q, SCH,CH,), 7.0 (lH, s, C-1H): m/e (%R.A.) 346 (lOO%), 284 (2.9), 261 (4.0), 222 (8.8) 183 (12.2), 97 (21.6), 41 (21.9).
107
108
YUSIJFJ. ABUL-HAJIand PAULL.
CISEK
NADPH t RSH Scheme Reaction of glutathione with e&one-3,4_oquinone Glutathione (100 mg) dissolved in a mixture of water (1.5 ml) and acetic acid (1.5 ml) was added to a stirred solution of the chloroform solution of the estrogen 3,4-o-quinone (50 mg, see above). Decoloration took place within 20 s and stirring continued for an additional 30 s. The aqueous phase was diluted with water and extracted 3 times with chloroform. The water extract was lyophilized and crystallized from water to yield an amorphous compound m.p. 209-212°C (decomp.); ‘H-NMR (DMSO): 6 0.83 (3H, S, 18CH3), 1.9 (2H, m, CHICH2CH), 2.3 (2H, m, CH,CH,CH), 3.0 (2H, S, SCH,CH), 3.4 (lH, m, HOOCCHNH,), 3.7 (2H, S, NHCH,COOH), 4.29 (2H, m, SCH,CH), 6.77 (lH, S, C-1H); FAB mass spectrum (% R.A.) 592 (42.3), 308 (49.9) 279 (23.5) 217 (62.4), 199 (23.5), 181 (76.1) 149 (39.8) 126 (lOO%), 109 (85.5). Incubation of [I-‘H]4-OHE,
with rat liver microsomes
The procedure of Numazawa et a1.[17] for incubations with microsomes in the presence of NADPH
1.
was used. Incubations were carried out in the presence of glutathione and the proteins precipitated by addition of 1 ml of 2N HCl and left overnight at 0°C. The protein precipitate was centrifuged and the supernatant solution was applied to a column of Amberlite XAD-2 resin as described by Bradlow[l8]. The column was eluted with H,O (60-80ml) and then methanol (2&30 ml). Radioactivity was determined in all fractions. Methanol fractions containing radioactive tubes were pooled, evaporated, and the residue dissolved in a small volume of methanol and chromatographed over TLC in N-butanol-acetic acid-water (4:1:1, by vol.). The radioactive areas corresponding to standard thioether conjugates of 4-OHE, were scraped, diluted with unlabeled 4-OHE, glutathione conjugate, and recrystallized from water to constant specific activity. RESULTSAND
DISCUSSION
Previous studies on thiol additions to 2,3-estrogeno-quinones [ 191 gave a mixture of C-l and C-4 thio-
Catechol estrogen adducts Table 1. Reaction of [l-3Hlestrone-3,4-oquinone
substrate [L3Hj4-OHE, [l-‘H]3,4_EQ [I-‘H]3,4-EQ [I-%J4-OHE, [I-sH]Q-OHE, [13H]4-OH&
(3,4-EQ) with thiols and [I-‘H&OHE, somes*
Reaction conditions
% Radioactivity released as ‘H,O
-
-
MnOz EtSH GSH Microsomes Microsomes + GSH
2.8 3.2 3.0 11.6 12.3
[I-‘I-I&OHE,
109
% Radioactivity in product 95.5 94.1 94.9 72.9
with mictospecitic activity (wm/mmoW
1.34 7.06 (7.11) 5.75 (6.96) 3.37 (6.99) 7.69 6.84 2.72 (5.65)
*Chemical reactions were carried out using 2l~~rn/~ of [I-‘H&OHE,. Microsomal i~u~tio~ were carried out using 1.1 x IO”cpm of [I-‘HP-OHE, followed by dilution with 50 mg of unlabeled 60HE, or 4-OHE,-2-SG. +Values in brackets were normalized to reflect increased molecular weight of catochol estrogen adducts.
of 2-OWE,. These studies showed that thiol addition was primarily dependent on the nature of the thiol compounds and less on steric hindrance- Results obtained from this report show that thiol additions to 3,4-estrogen-o-quinone lead to the formation of only one thiol adduct which has been characterized using *H-NMR data as the C-2 thioether conjugate of 4-OHE, indicating a 1,6addition of the thiol group to the 3,4-o-quinone (Scheme 1). Further support for the identification of the 3,4catechol estrogen adduct was obtained by reaction of the thiols with ~~0~~~~1~ labeled 4-OHE,. [I -3H]40HEi (Scheme 2) was prepared by reaction of (l/I-‘H]COH-A with DDQ in dioxane leading to the formation of [ 1fl-3 H]4-OH- 1,4*androstadienedione which was reductively aromatized resulting in the formation of [1-3H]4-OHE,. In all these chemical reactions the specific activity of the isolated products remained the same indicating no loss of radioactivity. Table 1 shows that reaction of ethanet~ol and glutathione with [l-3~strone-3,4-oq~none results in essentially no release of radioactivity in water indicating that thiol addition to 3,4-o-quinone did not occur by a 1,4-addition at the C-l position. Furthermore, Table 1 shows that incubations of [1-3H]4-OHE1 with GSH in the presence of microsomes results in the formation of 4-OHE,-2&glutathione (4-OHEi-2-SG) with most of the radioactivity retained in the conjugate indicating glutathione addition at C-2. The identity of the glutathione adduct formed during microsomal incubations in the presence of GSH was established by dilution of labeled compound with authentic standard compounds followed by recrystallization to constant specific activity
ether conjugates
Table 2. R~~i~~tion of [‘HJ4-OHE,-SG obtained from microsomal incubations to constant specific activity Specific activity
After adding carrier 1st Recrystalliition 2nd Recrystalliition 3rd Recrystallization 4th Recrystalllltion
cpm/mg
cpmtmmol
1731 1632 1603 1612 1610
2.92 2.76 2.71 2.72 2.72
(Table 2). About 1l-12% of tritium was released as 3H,0. This release is most likely not due to release from C-2 but rather from C-l as shown recently by Jellinck et aL[20]. About 15% of radioactivity was found to bind to ~~roso~l proteins. Attempts at obtaining 4-OHE,-2-SG from incubations of estrone with rat liver microsomes in the presence of glutathione have not yet been successful mainly because of very low levels of 4-hydroxylations as compared to 2-hydroxylations of estrogens. However, the fact that in Y&-Omicrosomal incubations of 4-OHE1 with glutathione show the formation of 4-OHE,-2-SG suggest that these estrogen adducts are formed but have not yet been detected. In view of the fact that the estrogen 3,4+quinones are selectively removed by reaction with amino acids and proteins as compared to estrogen 2,3-o-quinones [9] and since 4-OH estrogens were found to be carcinogenic in Syrian hamsters while 2-OH estrogens are not [12] suggests that the 4-OH catechol estrogen may play a more significant role in its binding to cellular macromolecules and therefore its potentially higher genotoxic effects. Acknowledgement-This work was supported CA 41269.
by
NIH grant
RRFERRNCES 1. Jellinck P. H., Lowis J. and Boston F.: Further evidence of au estrogeu-peptide conjugate by rat liver in t&o.
Steroids 10 (1967) 329346. 2. Kuss E.: Wasserlosliche Metabolites des 17fl-Oestradiol. Hoppe-Seyler’s 2. physiol. Chem. 348 (1967) 1707-1708. 3. Jellinck P. H. and Eke J. S.: Synthesis of estrogen glutathione and cysteine derivatives. Steroids 13 (1969) 711-718. 4. Kuss E.: Wa~rlosliche Metabolite de Oestradiol 178. III. Trennung und identi~e~g der I- and 4-glutathionthioethcr von 2,3~ihydroxy~~at~~en. Hoppe seyter’s Z. physiol, Gem. 350 (1969) 95-97. 5. Ball P. and Knuppen R.: Formation of 2- and 4-hydroxyestrogens by brain, pituitary, and liver of the human fetus. J. clin. Endoer. Met& 47 (1978) 732-737. 6. Nelson S. D., Mitchell J. R., Dybing E. and Sasame H. E.: Q&chrome P-450 mediated oxidation of 2-hydroxyestrogens to reactive intermediates. Biochem. biophys. Res. Commun. 70 (1976) 1157-l 165.
110
YUSUFJ. ABUL-HAJJand PAUL L. CISEK
7. Numazawa M., Soeda N., Kiyono Y. and Nambara T.: Properties of estradioi 2-hydroxylase and 2-hydroxy3-deoxy estradiol 3-hydroxylase in rat liver. 1. steroid Biochem. 10 (1979) 227-233. 8. Williams J. G., Longcope C. and Williams K. I. H.:
4-Hydroxyestrone: a new metabolite of estradiol-17/I from humans. Steroids 24 (1974) 687-701. 9. Kalyanaraman B., Hintz P. and Sealy R. C.: An electron spin resonance study of free radicals from catechol estrogens. Fe&. Proc. 45 (1986) 2477-2484. 10. Martucci C. P. and Fishman J.: Impact of ~ntinuously administered catechol estrogens on uterine growth and luteinizing hormone secretion. Endocrinology 105 (1979) 1288-1292.
11. Ball P. and Knuppen R.: Catecholestrogens: Chemistry, biogenesis, metabolism, occurrence and physiological significance. Acfa endocr., Copenh. 232 (1986) l-127. 12. Liehr J. G., Fana W. F., Sirbasku D. A. and Ariulubelen A.: Carc&ogenicity of catechol estrogens in Syrian hamsters. J. steroid Biochem. 24 (1986) 353-356. 13. Abul-Haij Y. J.: Studies on the mechanism of action of the aromatase inhibitor, 4-hydroxy-androstenedione. Steroids 41 (1983) 783-790.
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