348
Biochimica et Biophysica Acta, 1035(1990) 348-352
Elsevier BBAGEN 23381
Stopped-flow investigation of antioxidant activity of estrogens in solution Kazuo Mukai 1, Koji Daifuku 1, Satoshi Yokoyama 1 and Minoru Nakano 2 I Department of Chemistry, Faculty of Science, Ehime University, Matsuyama and 2 College of Medical Care and Technology, Gunma University, Gunma (Japan)
(Received 15 February 1990)
Key words: Estrogen;Antioxidant activity; Vitamin E radical; Lipid peroxidation A kinetic study of the reaction between estrogens (female hormone) and substituted phenoxyl radical has been performed, as a model for the reactions of estrogens with lipid peroxyl radical in biological systems. The rates of reaction of estrogens (estrone 1, estradiol 2, 2-methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5) with substituted phenoxyl radical in benzene have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants, k2, obtained are 84 M - t . s -1 for 1, 138 M - t . s - t for 2, 520 M - t . s -1 for 3, < 1 0 - 4 M - 1 . s - 1 for 4, and 2.6. l0 s M - l . s -1 for 5 at 25.0°C. 2-Hydroxyestrone 5 was found to be 2.9-times more active than a-tocopherol, which has the highest antioxidant activity among natural tocopherois. The order of magnitude of k 2 value (1 < 2 < 3 < a-Toc < 5) is in agreement with that of in vitro tests of their antioxidant activities, as measured by the inhibition of lipid peroxidation. Further, similar measurements have been performed for the reaction between the above estrogens 1 - 5 and tocopheroxyl 6 in benzene solution. It was found that the estrogens having an O H group at the aromatic ring have an ability to regenerate the tocopheroxyl 6 to tocopherol. Especially, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than ascorbic acid.
Introduction It is well recognized that tocopherols (vitamin E) are localized in cellular membranes and have functions as an antioxidant by protecting unsaturated lipids from peroxidation. The antioxidant actions of tocopherols have been ascribed to the oxidation reaction of phenolic hydroxyl group, producing corresponding tocopheroxyl radicals (reaction 1) [1,2]. kl
LOO'+ TocH ~ LOOH+Toc"
(1)
It has been found recently that estrogens (female hormones) which possess a phenolic hydroxyl group have an effective antioxidant action [3-6]. Yagi et al. [3] found that estrogens, such as estrone, estradiol and estriol, inhibit peroxidation of methyl linoleate caused by UV-irradiation and that of rat liver microsomal lipids by the Fea+-ADP system. Nakano (one of the present authors) et al. [4] reported that estrogens function as efficient inhibitors of microsomal phospholipid
Correspondence: K. Mukai, Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790, Japan.
peroxidation induced by Fe3+-ADP-adriamycin or Fe3+-ADP-ascorbate. The efficiencies in the microsomal system were in the order of estradiol > estriol > estrone [3,4]. Furthermore, catechol estrogen (2-hydroxyestrone and 2-hydroxyestradiol), a metabolite of estrogen, was found to have strong activities in inhibiting lipid peroxidation in in vivo and in vitro tests [5,6]. However, it is still obscure how these estrogens contribute to the inhibition of lipid peroxidation in biological systems. In the present work, in order to clear the antioxidant activity of these estrogens, we have first measured the reaction rates, k2, of five kinds of estrogens 1-5 (see Fig. 1 and Table I) with 2,6-di-tert-butyl-4-(4-methoxyphenyl)phenoxyl (PhO" (abbreviated to 'substituted phenoxyl' hereafter)) in benzene solution, as a model of active oxygen radical (LOO, LO, and HO') in biological systems, using stopped-flow spectrophotometer. From the results, the structure-activity relationship in the above radical-scavenging reaction has been discussed. The observed rates, k 2, were compared to those of the a-tocopherol, vitamin C (ascorbic acid), and 3 , 5 - d i - t e r t - b u t y l - 4 - h y d r o x y t o l u e n e (BHT), which are well known as popular antioxidants [2,7-10]. Secondly, we have measured the reaction rates, k 3, of estrogens 1 - 5
0304-4165/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
349 o
H
O
OH
Recently, several investigators have measured the second-order rate constants, k,, for H atom abstraction by active free radicals (poly(peroxystyryl)peroxyl, galvinoxyl and substituted phenoxyl) from a-, fl-, 3'and 6-tocopherols in homogeneous solution, by using different experimental methods such as 0 2 consumption [21, ESR [15] and stopped-flow spectrophotometry [7,8]. It was observed that the second-order rate constants, k s values, of tocopherols decrease in the order of et >/3 = 3' > 6-tocopherol; further, the relative magnitudes of the k s values, that is, the relative antioxidant activities of a-, fl-, 3'- and 6-tocopherols, obtained for three different oxyradicals agree well with each other [8]. Therefore, the relative k 2 values obtained in the present work will have relevance to the relative values of reaction rate between estrogens and peroxyl radical in biological systems.
H O ~ Estrodlol (2)
~
Estrone (I) o
o
HO 2-Nethoxyestrone(5)
H3CO 3-Methoxyestrone (Ii)
:oO 2-Hydroxyestrone (5)
HO
a-TocoPherol
OH
~.~-c.~)~o OH OH Ascorblc ACIO
BHT
Experimental procedures
Second-order rate constants (k 2 and k3) for the reaction of estrogens 1-5 with substituted phenoxyl (PhO') and 5,7-diisopropyltocopheroxyl 6 radicals, respectively, in benzene at 25.0oC.
Commercial estrogens (estrone 1, estradiol 2, 2methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5), L-ascorbic acid and 3,5-di-tert-butyl-4-hydroxy-toluene (BHT) were used as received. D-aTocopherol was kindly supplied from Eisai. The 5,7-diisopropyltocopheroxyl radical 6 was prepared by the PbO 2 oxidation of corresponding 5,7-diisopropyltocopherol in benzene solution under nitrogen atmosphere [8,16]. 2,6-di-tert-butyl-4-(4-methoxyphenyl)phenoxyl (PhO') was prepared according to the method of Miiller et al. [17]. The stopped-flow data were obtained on a Unisoku stopped-flow spectrophotometer Model RS-450 by mixing equal volumes of solutions of estrogen and substituted phenoxyl or tocopheroxyl radicals [7]. The oxidation reactions were studied under pseudo-firstorder conditions, and the observed rate constants, kob,d, were calculated in the usual way using a standard least-squares analysis. All measurements were performed at 25.0 + 0.5 o C.
Estrogen
k2 a (M-I.s -1) (PhO')
k3 a (M-Ls -1) (Toc" 6)
Results and Discussion
Estrone 1 Estradiol 2 2-Methoxyestrone3 3-Methoxyestrone4 2-Hydroxyestrone5 a-Tocopherol Ascorbic acid BHT
84 138 520 < 10- 4 2.6.105 9.0.104 61 b 84
49 53 29 < 10- 4 6.0.105 550 b 940
Tocopheroxyl(6)
PhO.
Fig. 1. Molecular structures of estrogens (female hormones) 1-5, a-tocopherol, ascorbic acid, BHT, substituted phenoxyl (PhO') and tocopheroxyl6.
with 5,7-diisopropyltocopheroxyl 6 in the same solvent, in order to clear whether the estrogens can regenerate the tocopheroxyl to tocopherol [10-14]. k2
PhO'+ estrogen~ PhOH+ estrogen"
(2)
k3
Toc"+ estrogen--*TocH+ estrogen"
(3)
TABLE I
Rate constants
a Data are means of four or five experiments. For each estrogen, experimental error in k 2 and k 3 values was less than approx. 10%. For a plot of kobsa vs. [estrogen], the correlation coefficient was more than + 0.99. b Measured in benzene/ethanol (2 : 1, v/v) solution [10].
Antioxidant activity of estrogens 1-5 in benzene solution The oxidation rates of estrogens 1 - 5 by substituted phenoxyl (PhO') were studied spectrophotometrically by the stopped-flow technique in the presence of excess estrogen in benzene solution (reaction 2). The details of these experiments are reported in a previous paper [7]. The rate was measured by following the decrease in absorbance at 377 and 572 nm of the substituted phenoxyl radical. The values of the second-order rate constants, k 2, at 25.0 ° C are listed in Table I, together with those of a-tocopherol, ascorbic acid and BHT, which are well known as popular antioxidants.
350 The results listed in Table I demonstrate that estradiol 2 is 1.6-times as reactive as estrone 1. The 2methoxyestrone 3 having a methoxy substituent at the ortho position of the OH group was also found to be 6.2-times more active than the estrone 1. Further, the k 2 value of 2-hydroxyestrone 5 (k 2 = 2.6.105 M -1. s -1) having two hydroxy substituents at the aromatic ring is approx, three orders of magnitude larger than that of estrone 1 ( k 2 = 84 M -1. s-l). On the other hand, 3methoxyestrone 4 without an OH group at the aromatic ring did not react with the PhO" radical. As reported in previous papers, absolute reactivities (k 2 and kx) of tocopherols to PhO" and L O O , respectively, increase as the total electron-donating capacity of the alkyl substituents at the aromatic ring increases [2,8,18]. For the tocopherol derivatives the log of the second-order rate constants (k 2 and kl) was found to roughly correlate with the sum of the Hammett's o constants (Eo) or the Brown's o + constants (Eo+), although the two cases could not be distinguished. As described above, the 2-methoxyestrone 3 having a methoxy substituent at the ortho position of the 3-OH group is 6.2-times as reactive as estrone 1. The methoxy group can be a powerful electron-donating group (o = -0.27), if it is ortho or para to the hydroxy group and has the correct orientation for conjugative interaction of its oxygen lone pair with the aromatic rr-orbital. Otherwise, it is electron-withdrawing (o = +0.12). The high reactivity observed for 3 suggests that the methoxy group acts as an electron-donating group in this compound. Further, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than the 2-methoxyestrone 3. The 2-OH group (o = - 0 . 3 7 ) has higher electron-donating property than C H 3 0 group (o = - 0 . 2 7 ) and thus we can expect that 5 shows higher antioxidant activity than 3. In addition, the 2-hydroxyestrone 5 has two hydroxy substituents at the aromatic ring. Consequently, these two OH groups will contribute to the high reactivity of 2-hydroxyestrone 5. How0.50
o
0,25
0,00
i
i
i
10
20
30
t40
Time ( sec ) Fig. 2. The decay of tocopheroxyl 6 for the reaction of 6 with estradiol 2 in benzene solution at 25.0°C. [Toc'],= 0 approx. 0.10 m M and [estradiol],-0 0.70 mM. At 425 nm.
ever, the reason is not clear, at present, why 2-hydroxyestrone 5 shows three orders of magnitude higher reactivity than the 2-methoxyestrone 3. By comparing the second-order rate constant (k2 = 84 M -1. s -1) observed for estrone 1 with that (k2 = 61 M-~ • s -~) for ascorbic acid, the former is 1.4-times as reactive as the latter. The rate constant of estrone 1 is very similar to that of BHT. However, estrone ! (k2 = 84 M - 1 . s - l ) showed three orders of magnitude less reactivity than a-tocopherol (k 2 = 9.0-10 4 M - 1 - s - ] ) . On the other hand, 2-hydroxyestrone 5 was found to be 2.9-times more active than a-tocopherol, which has the highest antioxidant activity among natural tocopherols [2,7]. Thus, this compound has the highest antioxidant activity among natural lipid-soluble, chain-breaking antioxidants in solution [8,13,14]. As described in a previous section, Nakano et al. [4,5] found that estrogens (estrone 1, estradiol 2, and 2-hydroxyestrone 5) inhibit peroxidation of phospholipid induced by Fe3+-ADP-adriamycin. The antioxidant activity measured by the induction period of oxygen absorption decreased in the order of 2-hydroxyestrone 5 > a-tocopherol > estradiol 2 > estrone 1. Yagi and Komura [3] reported that estrogens (estradiol, estrone and estriol) inhibit peroxidation of methyl linoleate caused by UV irradiation and that of rat liver microsomal fipids by the Fe3+-ADP system. The efficiencies in the microsomal system were in the order of estradiol 2 > estriol > estrone 1. In the present work, we have measured the rate constants k 2 for the reactions of estrogens 1-5 with substituted phenoxyl radical, and found that the reactivities of estrogens decrease in the order of 5 > a-Toc > 3 > 2 > 1 > 4. These results show that the relative reactivities of estrogens are in agreement with in vitro tests of their relative antioxidant activities, as measured by the inhibition of lipid peroxidation [3-5]. The reaction between estrogens 1-5 and tocopheroxyl 6 in solution The 5,7-diisopropyltocopheroxyl 6 is fairly stable and shows absorption peaks at ~'max= 425 and 405 nm in benzene solution [10,14,16]. By adding benzene solution of excess estrogen to benzene solution of tocopheroxyl (1 : 1, v/v), the absorption spectrum of the tocopheroxyl immediately disappeared. The rate was measured by following the decrease in absorbance at 425 nm of tocopheroxyl. The time dependence of the decrease in absorbance at 425 nm observed when approx. 0.20 mM benzene solution of tocopheroxyl is mixed with 1.40 mM benzene solution of estradiol 2 (1:1, v / v , final concentration of 2 is 0.70 mM) is shown in Fig. 2. The pseudo-first-order rate constants, kobsd values, were obtained by varying the concentration of estrogen. Tocopheroxyl shows a very slow natural decay in a similar solution. Therefore, the pseudo-first-order rate
351 x10-3 1,5
I
I
1,0
0,5
0.0
I
1 [ Estradl01 ]
I
2 xlO-3M
Fig. 3. The dependence of the pseudo-first-order rate constant, kobsd, on the concentration of estradiol 2 in benzene solution.
constant, kobsd , for tocopheroxyl bleaching is given by Eqn. 4: kobsd = k 0 + k3[estrogen ]
(4)
where k 0 is the rate constant for the natural decay of tocopheroxyl in the medium, and k 3 is the second-order rate constant for the reaction of tocopheroxyl with added estrogen. These rate parameters are obtained by plotting kobsd against [estrogen], as shown in Fig. 3. The second-order rate constants, k3, obtained for estrogens 1-5 at 25.0°C are summarized in Table I. Data are means of four or five experiments. The experimental errors in the k 3 values were less than approx. 10% in every case. As listed in Table I, the estrone 1 and estradiol 2 reacted with the tocopheroxyl radical at a similar rate. The reaction rate of 2-methoxyestrone 3 is approx. 40% lower than that of estrone 1. On the other hand, 3methoxyestrone 4 having no hydroxy group at aromatic ring could not reduce the tocopheroxyl to tocopherol as expected. The result indicates that estogen regenerates tocopherol by donating a hydrogen atom of the O H group at aromatic ring to tocopheroxyl radical. Among these estrogens, 2-hydroxyestrone 5 was found to have the highest activity in regenerating the tocopheroxyl to tocopherol. The k 3 value obtained for 2-hydroxyestrone 5 is 6.0.105 M - t • s -1 in benzene solution at 25.0°C. This is approx, four orders of magnitude higher than that observed for estrone 1. The reaction between atocopheroxyl and ascorbic acid is well known as usual regeneration reaction of tocopheroxyl in biomembrane systems [11,12]. Therefore, the above reaction rate, k3, obtained for 5 was compared with that ( k 3 = 550 M -a • s -a) reported for the reaction between ascorbic acid and 5,7-diisopropyltocopheroxyl 6 in benzene-ethanol (2 : 1, v / v ) [10,19]. The former is about three orders of magnitude higher than the latter.
Recently, Yoshino et al. [6] reported that when estradiol 2 was administered intraperitoneally to female mice, serum and liver lipid peroxide levels were significantly decreased, while in the case of male mice they only tended to decrease. On the other hand, the intraperitoneal administration of 2-hydroxyestradiol (catechol estrogen), a major metabolite of estradiol 2 in the liver, brought about a decrease in liver lipid peroxide levels in both male and female mice. Accordingly, they supposed that the remarkable effect of estradiol 2 found in female mice is due to the 2-hydroxyestradiol formed in them. In the present paper, we measured the reaction rates of estrogens 1-5 with substituted phenoxyl radical and 5,7-diisopropyltocopheroxyl radical 6 in benzene, and found that the reaction rates observed for 2-hydroxyestrone 5 (catechol estrogen) were approx, three or four orders of magnitude higher than those of estrone (or estradiol). Further, 2-hydroxyestrone 5 was found to be 2.9-times more active than a-tocopherol, which has the highest antioxidant activity among natural tocopherols. Such a high reactivity of 2-hydroxyestrone 5 may explain the fact that only the intraperitoneal administration of 2-hydroxyestradiol brought about a decrease in liver lipid peroxide levels in both male and female mice.
Acknowledgments We are very grateful to Professor Yoichi Kitamura for use of stopped-flow spectrophotometer. We also wish to thank Miss Yuko Fujii for her kind help in measuring the rate constants.
References 1 Mukai, K., Tsuzuki, N., Ouchi, S. and Fukuzawa, K. (1982) Chem. Phys. Lipids 30, 337-345. 2 Burton, G.W., Doba, T., Gabe, E.J., Hughes, L., Lee, F.L., Prasad, L. and Ingold, K.U. (1985) J. Am. Chem. Soc. 107, 7053-7065. 3 Yagi, K. and Komura, S. (1986) Biochem. Int. 13, 1051-1055. 4 Sugioka, K., Shimosegawa, Y. and Nakano, M. (1987) FEBS Lett. 210, 37-39. 5 Nakano, M., Sugioka, K., Naito, I., Takekoshi, S. and Niki, E. (1987) Biochem. Biophys. Res. Commun. 142, 919-924. 6 Yoshino, K., Komura, S., Watanabe, I., Nakagawa, Y. and Yagi, K. (1987) J. Clin. Biochem. Nutr. 3, 233-240. 7 Mukai, K., Watanabe, Y., Uemoto, U. and Ishizu, K. (1986) Bull. Chem. Soc. Jpn. 59, 3113-3116. 8 Mukai, K., Kageyama, Y., Ishida, T. and Fukuda, K. (1989) J. Org. Chem. 54, 552-556. 9 Mukai, K., Okabe, K. and Hosose, H. (1989) J. Org. Chem. 54, 557-560. 10 Mukai, K., Nishimura, M., Nagano, A., Tanaka, K. and Niki, E. (1989) Biochim. Biophys. Acta 993, 168-173. 11 Packer, J.E., Slater, T.F. and Willson, R.L (1979) Nature 278, 737-738. 12 Scarpa, M., Rigo, A., Maiorino, M., Ursini, F. and Gregolin, C. (1984) Biochim. Biophys. Acta 801, 215-219. 13 Burton, G.W. and Ingold, K.U. (1986) Acc. Chem. Res. 19, 194-201.
352 14 Niki, E. (1987) Chem. Phys. Lipids 44, 227-253. 15 Niki, E., Tsuchiya, J., Yoshikawa, Y., Yamamoto, Y. and Kamiya, Y. (1986) Bull. Chem. Soc. Jpn. 59, 497-501. 16 Mukai, K., Takamatsu, K. and Ishizu, K. (1984) Bull. Chem. Soc. Jpn. 57, 3507-3510. 17 MiJller, E., Schick, A. and Scheffler, K. (1959) Chem. Ber. 92, 474-482.
18 Mukai, K., Fukuda, K., Tajima, K. and Ishizu, K. (1988) J. Org. Chem. 53, 430-432. 19 Mukai, K., Fukuda, K., Ishizu, K. and Kitamura, Y. (1987) Biochem. Biophys. Res. Commun. 146, 134-139.
Biochimica et Biophysica Acta, 1035(1990) 348-352
Elsevier BBAGEN 23381
Stopped-flow investigation of antioxidant activity of estrogens in solution Kazuo Mukai 1, Koji Daifuku 1, Satoshi Yokoyama 1 and Minoru Nakano 2 I Department of Chemistry, Faculty of Science, Ehime University, Matsuyama and 2 College of Medical Care and Technology, Gunma University, Gunma (Japan)
(Received 15 February 1990)
Key words: Estrogen;Antioxidant activity; Vitamin E radical; Lipid peroxidation A kinetic study of the reaction between estrogens (female hormone) and substituted phenoxyl radical has been performed, as a model for the reactions of estrogens with lipid peroxyl radical in biological systems. The rates of reaction of estrogens (estrone 1, estradiol 2, 2-methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5) with substituted phenoxyl radical in benzene have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants, k2, obtained are 84 M - t . s -1 for 1, 138 M - t . s - t for 2, 520 M - t . s -1 for 3, < 1 0 - 4 M - 1 . s - 1 for 4, and 2.6. l0 s M - l . s -1 for 5 at 25.0°C. 2-Hydroxyestrone 5 was found to be 2.9-times more active than a-tocopherol, which has the highest antioxidant activity among natural tocopherois. The order of magnitude of k 2 value (1 < 2 < 3 < a-Toc < 5) is in agreement with that of in vitro tests of their antioxidant activities, as measured by the inhibition of lipid peroxidation. Further, similar measurements have been performed for the reaction between the above estrogens 1 - 5 and tocopheroxyl 6 in benzene solution. It was found that the estrogens having an O H group at the aromatic ring have an ability to regenerate the tocopheroxyl 6 to tocopherol. Especially, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than ascorbic acid.
Introduction It is well recognized that tocopherols (vitamin E) are localized in cellular membranes and have functions as an antioxidant by protecting unsaturated lipids from peroxidation. The antioxidant actions of tocopherols have been ascribed to the oxidation reaction of phenolic hydroxyl group, producing corresponding tocopheroxyl radicals (reaction 1) [1,2]. kl
LOO'+ TocH ~ LOOH+Toc"
(1)
It has been found recently that estrogens (female hormones) which possess a phenolic hydroxyl group have an effective antioxidant action [3-6]. Yagi et al. [3] found that estrogens, such as estrone, estradiol and estriol, inhibit peroxidation of methyl linoleate caused by UV-irradiation and that of rat liver microsomal lipids by the Fea+-ADP system. Nakano (one of the present authors) et al. [4] reported that estrogens function as efficient inhibitors of microsomal phospholipid
Correspondence: K. Mukai, Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790, Japan.
peroxidation induced by Fe3+-ADP-adriamycin or Fe3+-ADP-ascorbate. The efficiencies in the microsomal system were in the order of estradiol > estriol > estrone [3,4]. Furthermore, catechol estrogen (2-hydroxyestrone and 2-hydroxyestradiol), a metabolite of estrogen, was found to have strong activities in inhibiting lipid peroxidation in in vivo and in vitro tests [5,6]. However, it is still obscure how these estrogens contribute to the inhibition of lipid peroxidation in biological systems. In the present work, in order to clear the antioxidant activity of these estrogens, we have first measured the reaction rates, k2, of five kinds of estrogens 1-5 (see Fig. 1 and Table I) with 2,6-di-tert-butyl-4-(4-methoxyphenyl)phenoxyl (PhO" (abbreviated to 'substituted phenoxyl' hereafter)) in benzene solution, as a model of active oxygen radical (LOO, LO, and HO') in biological systems, using stopped-flow spectrophotometer. From the results, the structure-activity relationship in the above radical-scavenging reaction has been discussed. The observed rates, k 2, were compared to those of the a-tocopherol, vitamin C (ascorbic acid), and 3 , 5 - d i - t e r t - b u t y l - 4 - h y d r o x y t o l u e n e (BHT), which are well known as popular antioxidants [2,7-10]. Secondly, we have measured the reaction rates, k 3, of estrogens 1 - 5
0304-4165/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
349 o
H
O
OH
Recently, several investigators have measured the second-order rate constants, k,, for H atom abstraction by active free radicals (poly(peroxystyryl)peroxyl, galvinoxyl and substituted phenoxyl) from a-, fl-, 3'and 6-tocopherols in homogeneous solution, by using different experimental methods such as 0 2 consumption [21, ESR [15] and stopped-flow spectrophotometry [7,8]. It was observed that the second-order rate constants, k s values, of tocopherols decrease in the order of et >/3 = 3' > 6-tocopherol; further, the relative magnitudes of the k s values, that is, the relative antioxidant activities of a-, fl-, 3'- and 6-tocopherols, obtained for three different oxyradicals agree well with each other [8]. Therefore, the relative k 2 values obtained in the present work will have relevance to the relative values of reaction rate between estrogens and peroxyl radical in biological systems.
H O ~ Estrodlol (2)
~
Estrone (I) o
o
HO 2-Nethoxyestrone(5)
H3CO 3-Methoxyestrone (Ii)
:oO 2-Hydroxyestrone (5)
HO
a-TocoPherol
OH
~.~-c.~)~o OH OH Ascorblc ACIO
BHT
Experimental procedures
Second-order rate constants (k 2 and k3) for the reaction of estrogens 1-5 with substituted phenoxyl (PhO') and 5,7-diisopropyltocopheroxyl 6 radicals, respectively, in benzene at 25.0oC.
Commercial estrogens (estrone 1, estradiol 2, 2methoxyestrone 3, 3-methoxyestrone 4, and 2-hydroxyestrone 5), L-ascorbic acid and 3,5-di-tert-butyl-4-hydroxy-toluene (BHT) were used as received. D-aTocopherol was kindly supplied from Eisai. The 5,7-diisopropyltocopheroxyl radical 6 was prepared by the PbO 2 oxidation of corresponding 5,7-diisopropyltocopherol in benzene solution under nitrogen atmosphere [8,16]. 2,6-di-tert-butyl-4-(4-methoxyphenyl)phenoxyl (PhO') was prepared according to the method of Miiller et al. [17]. The stopped-flow data were obtained on a Unisoku stopped-flow spectrophotometer Model RS-450 by mixing equal volumes of solutions of estrogen and substituted phenoxyl or tocopheroxyl radicals [7]. The oxidation reactions were studied under pseudo-firstorder conditions, and the observed rate constants, kob,d, were calculated in the usual way using a standard least-squares analysis. All measurements were performed at 25.0 + 0.5 o C.
Estrogen
k2 a (M-I.s -1) (PhO')
k3 a (M-Ls -1) (Toc" 6)
Results and Discussion
Estrone 1 Estradiol 2 2-Methoxyestrone3 3-Methoxyestrone4 2-Hydroxyestrone5 a-Tocopherol Ascorbic acid BHT
84 138 520 < 10- 4 2.6.105 9.0.104 61 b 84
49 53 29 < 10- 4 6.0.105 550 b 940
Tocopheroxyl(6)
PhO.
Fig. 1. Molecular structures of estrogens (female hormones) 1-5, a-tocopherol, ascorbic acid, BHT, substituted phenoxyl (PhO') and tocopheroxyl6.
with 5,7-diisopropyltocopheroxyl 6 in the same solvent, in order to clear whether the estrogens can regenerate the tocopheroxyl to tocopherol [10-14]. k2
PhO'+ estrogen~ PhOH+ estrogen"
(2)
k3
Toc"+ estrogen--*TocH+ estrogen"
(3)
TABLE I
Rate constants
a Data are means of four or five experiments. For each estrogen, experimental error in k 2 and k 3 values was less than approx. 10%. For a plot of kobsa vs. [estrogen], the correlation coefficient was more than + 0.99. b Measured in benzene/ethanol (2 : 1, v/v) solution [10].
Antioxidant activity of estrogens 1-5 in benzene solution The oxidation rates of estrogens 1 - 5 by substituted phenoxyl (PhO') were studied spectrophotometrically by the stopped-flow technique in the presence of excess estrogen in benzene solution (reaction 2). The details of these experiments are reported in a previous paper [7]. The rate was measured by following the decrease in absorbance at 377 and 572 nm of the substituted phenoxyl radical. The values of the second-order rate constants, k 2, at 25.0 ° C are listed in Table I, together with those of a-tocopherol, ascorbic acid and BHT, which are well known as popular antioxidants.
350 The results listed in Table I demonstrate that estradiol 2 is 1.6-times as reactive as estrone 1. The 2methoxyestrone 3 having a methoxy substituent at the ortho position of the OH group was also found to be 6.2-times more active than the estrone 1. Further, the k 2 value of 2-hydroxyestrone 5 (k 2 = 2.6.105 M -1. s -1) having two hydroxy substituents at the aromatic ring is approx, three orders of magnitude larger than that of estrone 1 ( k 2 = 84 M -1. s-l). On the other hand, 3methoxyestrone 4 without an OH group at the aromatic ring did not react with the PhO" radical. As reported in previous papers, absolute reactivities (k 2 and kx) of tocopherols to PhO" and L O O , respectively, increase as the total electron-donating capacity of the alkyl substituents at the aromatic ring increases [2,8,18]. For the tocopherol derivatives the log of the second-order rate constants (k 2 and kl) was found to roughly correlate with the sum of the Hammett's o constants (Eo) or the Brown's o + constants (Eo+), although the two cases could not be distinguished. As described above, the 2-methoxyestrone 3 having a methoxy substituent at the ortho position of the 3-OH group is 6.2-times as reactive as estrone 1. The methoxy group can be a powerful electron-donating group (o = -0.27), if it is ortho or para to the hydroxy group and has the correct orientation for conjugative interaction of its oxygen lone pair with the aromatic rr-orbital. Otherwise, it is electron-withdrawing (o = +0.12). The high reactivity observed for 3 suggests that the methoxy group acts as an electron-donating group in this compound. Further, the 2-hydroxyestrone 5 showed about three orders of magnitude higher reactivity than the 2-methoxyestrone 3. The 2-OH group (o = - 0 . 3 7 ) has higher electron-donating property than C H 3 0 group (o = - 0 . 2 7 ) and thus we can expect that 5 shows higher antioxidant activity than 3. In addition, the 2-hydroxyestrone 5 has two hydroxy substituents at the aromatic ring. Consequently, these two OH groups will contribute to the high reactivity of 2-hydroxyestrone 5. How0.50
o
0,25
0,00
i
i
i
10
20
30
t40
Time ( sec ) Fig. 2. The decay of tocopheroxyl 6 for the reaction of 6 with estradiol 2 in benzene solution at 25.0°C. [Toc'],= 0 approx. 0.10 m M and [estradiol],-0 0.70 mM. At 425 nm.
ever, the reason is not clear, at present, why 2-hydroxyestrone 5 shows three orders of magnitude higher reactivity than the 2-methoxyestrone 3. By comparing the second-order rate constant (k2 = 84 M -1. s -1) observed for estrone 1 with that (k2 = 61 M-~ • s -~) for ascorbic acid, the former is 1.4-times as reactive as the latter. The rate constant of estrone 1 is very similar to that of BHT. However, estrone ! (k2 = 84 M - 1 . s - l ) showed three orders of magnitude less reactivity than a-tocopherol (k 2 = 9.0-10 4 M - 1 - s - ] ) . On the other hand, 2-hydroxyestrone 5 was found to be 2.9-times more active than a-tocopherol, which has the highest antioxidant activity among natural tocopherols [2,7]. Thus, this compound has the highest antioxidant activity among natural lipid-soluble, chain-breaking antioxidants in solution [8,13,14]. As described in a previous section, Nakano et al. [4,5] found that estrogens (estrone 1, estradiol 2, and 2-hydroxyestrone 5) inhibit peroxidation of phospholipid induced by Fe3+-ADP-adriamycin. The antioxidant activity measured by the induction period of oxygen absorption decreased in the order of 2-hydroxyestrone 5 > a-tocopherol > estradiol 2 > estrone 1. Yagi and Komura [3] reported that estrogens (estradiol, estrone and estriol) inhibit peroxidation of methyl linoleate caused by UV irradiation and that of rat liver microsomal fipids by the Fe3+-ADP system. The efficiencies in the microsomal system were in the order of estradiol 2 > estriol > estrone 1. In the present work, we have measured the rate constants k 2 for the reactions of estrogens 1-5 with substituted phenoxyl radical, and found that the reactivities of estrogens decrease in the order of 5 > a-Toc > 3 > 2 > 1 > 4. These results show that the relative reactivities of estrogens are in agreement with in vitro tests of their relative antioxidant activities, as measured by the inhibition of lipid peroxidation [3-5]. The reaction between estrogens 1-5 and tocopheroxyl 6 in solution The 5,7-diisopropyltocopheroxyl 6 is fairly stable and shows absorption peaks at ~'max= 425 and 405 nm in benzene solution [10,14,16]. By adding benzene solution of excess estrogen to benzene solution of tocopheroxyl (1 : 1, v/v), the absorption spectrum of the tocopheroxyl immediately disappeared. The rate was measured by following the decrease in absorbance at 425 nm of tocopheroxyl. The time dependence of the decrease in absorbance at 425 nm observed when approx. 0.20 mM benzene solution of tocopheroxyl is mixed with 1.40 mM benzene solution of estradiol 2 (1:1, v / v , final concentration of 2 is 0.70 mM) is shown in Fig. 2. The pseudo-first-order rate constants, kobsd values, were obtained by varying the concentration of estrogen. Tocopheroxyl shows a very slow natural decay in a similar solution. Therefore, the pseudo-first-order rate
351 x10-3 1,5
I
I
1,0
0,5
0.0
I
1 [ Estradl01 ]
I
2 xlO-3M
Fig. 3. The dependence of the pseudo-first-order rate constant, kobsd, on the concentration of estradiol 2 in benzene solution.
constant, kobsd , for tocopheroxyl bleaching is given by Eqn. 4: kobsd = k 0 + k3[estrogen ]
(4)
where k 0 is the rate constant for the natural decay of tocopheroxyl in the medium, and k 3 is the second-order rate constant for the reaction of tocopheroxyl with added estrogen. These rate parameters are obtained by plotting kobsd against [estrogen], as shown in Fig. 3. The second-order rate constants, k3, obtained for estrogens 1-5 at 25.0°C are summarized in Table I. Data are means of four or five experiments. The experimental errors in the k 3 values were less than approx. 10% in every case. As listed in Table I, the estrone 1 and estradiol 2 reacted with the tocopheroxyl radical at a similar rate. The reaction rate of 2-methoxyestrone 3 is approx. 40% lower than that of estrone 1. On the other hand, 3methoxyestrone 4 having no hydroxy group at aromatic ring could not reduce the tocopheroxyl to tocopherol as expected. The result indicates that estogen regenerates tocopherol by donating a hydrogen atom of the O H group at aromatic ring to tocopheroxyl radical. Among these estrogens, 2-hydroxyestrone 5 was found to have the highest activity in regenerating the tocopheroxyl to tocopherol. The k 3 value obtained for 2-hydroxyestrone 5 is 6.0.105 M - t • s -1 in benzene solution at 25.0°C. This is approx, four orders of magnitude higher than that observed for estrone 1. The reaction between atocopheroxyl and ascorbic acid is well known as usual regeneration reaction of tocopheroxyl in biomembrane systems [11,12]. Therefore, the above reaction rate, k3, obtained for 5 was compared with that ( k 3 = 550 M -a • s -a) reported for the reaction between ascorbic acid and 5,7-diisopropyltocopheroxyl 6 in benzene-ethanol (2 : 1, v / v ) [10,19]. The former is about three orders of magnitude higher than the latter.
Recently, Yoshino et al. [6] reported that when estradiol 2 was administered intraperitoneally to female mice, serum and liver lipid peroxide levels were significantly decreased, while in the case of male mice they only tended to decrease. On the other hand, the intraperitoneal administration of 2-hydroxyestradiol (catechol estrogen), a major metabolite of estradiol 2 in the liver, brought about a decrease in liver lipid peroxide levels in both male and female mice. Accordingly, they supposed that the remarkable effect of estradiol 2 found in female mice is due to the 2-hydroxyestradiol formed in them. In the present paper, we measured the reaction rates of estrogens 1-5 with substituted phenoxyl radical and 5,7-diisopropyltocopheroxyl radical 6 in benzene, and found that the reaction rates observed for 2-hydroxyestrone 5 (catechol estrogen) were approx, three or four orders of magnitude higher than those of estrone (or estradiol). Further, 2-hydroxyestrone 5 was found to be 2.9-times more active than a-tocopherol, which has the highest antioxidant activity among natural tocopherols. Such a high reactivity of 2-hydroxyestrone 5 may explain the fact that only the intraperitoneal administration of 2-hydroxyestradiol brought about a decrease in liver lipid peroxide levels in both male and female mice.
Acknowledgments We are very grateful to Professor Yoichi Kitamura for use of stopped-flow spectrophotometer. We also wish to thank Miss Yuko Fujii for her kind help in measuring the rate constants.
References 1 Mukai, K., Tsuzuki, N., Ouchi, S. and Fukuzawa, K. (1982) Chem. Phys. Lipids 30, 337-345. 2 Burton, G.W., Doba, T., Gabe, E.J., Hughes, L., Lee, F.L., Prasad, L. and Ingold, K.U. (1985) J. Am. Chem. Soc. 107, 7053-7065. 3 Yagi, K. and Komura, S. (1986) Biochem. Int. 13, 1051-1055. 4 Sugioka, K., Shimosegawa, Y. and Nakano, M. (1987) FEBS Lett. 210, 37-39. 5 Nakano, M., Sugioka, K., Naito, I., Takekoshi, S. and Niki, E. (1987) Biochem. Biophys. Res. Commun. 142, 919-924. 6 Yoshino, K., Komura, S., Watanabe, I., Nakagawa, Y. and Yagi, K. (1987) J. Clin. Biochem. Nutr. 3, 233-240. 7 Mukai, K., Watanabe, Y., Uemoto, U. and Ishizu, K. (1986) Bull. Chem. Soc. Jpn. 59, 3113-3116. 8 Mukai, K., Kageyama, Y., Ishida, T. and Fukuda, K. (1989) J. Org. Chem. 54, 552-556. 9 Mukai, K., Okabe, K. and Hosose, H. (1989) J. Org. Chem. 54, 557-560. 10 Mukai, K., Nishimura, M., Nagano, A., Tanaka, K. and Niki, E. (1989) Biochim. Biophys. Acta 993, 168-173. 11 Packer, J.E., Slater, T.F. and Willson, R.L (1979) Nature 278, 737-738. 12 Scarpa, M., Rigo, A., Maiorino, M., Ursini, F. and Gregolin, C. (1984) Biochim. Biophys. Acta 801, 215-219. 13 Burton, G.W. and Ingold, K.U. (1986) Acc. Chem. Res. 19, 194-201.
352 14 Niki, E. (1987) Chem. Phys. Lipids 44, 227-253. 15 Niki, E., Tsuchiya, J., Yoshikawa, Y., Yamamoto, Y. and Kamiya, Y. (1986) Bull. Chem. Soc. Jpn. 59, 497-501. 16 Mukai, K., Takamatsu, K. and Ishizu, K. (1984) Bull. Chem. Soc. Jpn. 57, 3507-3510. 17 MiJller, E., Schick, A. and Scheffler, K. (1959) Chem. Ber. 92, 474-482.
18 Mukai, K., Fukuda, K., Tajima, K. and Ishizu, K. (1988) J. Org. Chem. 53, 430-432. 19 Mukai, K., Fukuda, K., Ishizu, K. and Kitamura, Y. (1987) Biochem. Biophys. Res. Commun. 146, 134-139.
