Radical-scavenging Activity of Estrogen and Estrogen-like Compounds Using the Induction Period Method

The radical-scavenging activity of estrogens (estrone, 2-hydroxyestradiol), estrogen-like compounds (diethylstilbestrol, DES; bisphenol A, BPA) and the monophenolic compound 2,6-di-t-butyl-4-methoxyphenol (BMP) was investigated using the method of measuring the induction period for polymerization of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2′-azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO) at 70°C using differential scanning calorimetry (DSC). The stoichiometric factor (n, number of free radicals trapped by one mole of antioxidant moiety) for the AIBN system declined in the order BMP (2.0), 2-hydroxyestradiol (2.0)> DES (1.3) > BPA (1.2) > estrone (0.9), whereas that for the BPO system declined in the order BMP (2.0) >DES (1.9), BPA (1.9) > estrone (1.3) > 2-hydroxyestradiol (0.7). The inhibition rate constant (kinh × 10−3 M−1s−1) for the AIBN system declined in the order estrone (2.2) > BPA (2.0) > DES (1.9) > 2-hydroxyestradiol (1.2) > BMP (1.1), whereas that for the BPO system declined in the order 2-hydroxyestradiol (3.2) > estrone (1.4) > DES (1.2) > BPA (1.0) > BMP (0.9). The radical-scavenging activity for bioactive compounds such as estrogens should be evaluated using these two methods (the n and kinh) to elucidate the mechanism of a particular reaction. The great difference of the n and kinh for estrogens between the AIBN and BPO system suggested that their oxidation process is complex.


Introduction
Estrogens such as estrones and 2-hydroxyestradiols possess a phenolic hydroxy group and have a variety of beneficial effects in vivo, including protection against osteoporosis, coronary heart disease, Alzheimer's disease and stroke [1], and hormone replacement therapy appears to have many unexpected beneficial effects in the treatment of these diseases [2]. In contrast quinoids, quinoid radicals and phenoxy radicals formed from estrogens may be responsible for adverse effects such as carcinogenesis [3]. The biological effects of estrogens involve their dual role as prooxidants and antioxidants. Studies using models for active oxygen radicals (ROO . , RO . and HO . ) in biological systems have reported that estrogens are effective antioxidants [4,5]. Previously quantitative in vitro studies of the radical-scavenging activity of phenolic compounds [6,7] were performed by the induction period method under aerobic conditions. The oxygen tension under a 15 torr oxygen atomosphere is similar to that in many tissues [8,9], suggesting that oxygen is scarce in living cells and that the radical-scavenging activity of estrogens in vivo may differ considerably from that observed under aerobic conditions. We have previously reported the use of differential scanning calorimetry (DSC) to evaluate the radical-scavenging activity of estrones and antiestrogenes (tamoxifen, toremifene) [10] by the induction period method [10]. In the present study, we used this previously reported method induction with DSC to investigate the radical scavenging activity of estrogens (estrone, 2-hydroxyestradiol) and estrogen-like compounds (diethylstilbestrol, DES; bisphenol A, BPA) by determining the corresponding stoichiometric factors (n) and the inhibition rate constants of polymerization (k inh ).

Results and Discussion
The chemical structures of estrone [3-hydroxyesta-1,3,5 (10)-tren-17-one] and 2-hydroxyestradiol [estra-1,3,5 (10)-triene-2,3,17β-triol], respectively, are shown in Figure 1.   Curves were calculated from the findings shown in Figure 2. Abbreviations used are shown in Figure 2. Polymerization of the control was slightly inhibited, even though the reaction was carried out in a sealed DSC pan, because the pan contained a small amount of oxygen as it had been sealed in air.
Tangents were drawn to polymerization curves at an early stage in the run. The induction period (IP) of the test compounds was determined from the length of time between the zero point on the abscissa and the point of intersection of the tangents drawn to the curves at the early stage of polymerization. The IP was calculated from the difference between the induction period of specimens and that of controls. The n values can be calculated from Equation 1. We also examined the relationships between induction period [IP] and [IH]/R i for estrogens ( Figure 4). The IP was linearly related to the [IH]/R i . The n value was determined from each slope.  Induction period, sec (x10 -3

)
The initial rates (%/sec) of polymerization in the absence (Rp con ) and presence (Rp inh ) of estrogens were calculated from the slope of the plots of the first linear line of the conversion rate of MMA polymerization (tangent drawn at the early polymerization stage, Figure 3). The relationships between Rp inh /Rp con and concentration for estrogens are shown in Figure 5. As concentrations increased, Rp inh /Rp con values linearly decreased. For the AIBN system, but not for the BPO one, 2-hydroxyestradiol showed the greatest suppression of MMA radical growth, whereas estrone showed the greater suppression for the BPO system.  The measurement for antioxidant activities is described in the text. Values are the mean of three independent experiments. Standard error <10%. The determination of n and k inh is described in the text. The k t is approximately 3.7 x 10 7 M -1 s -1 [18].
The results of n and k inh values for the indicated compounds are summarized in Table 1 The n value for estrone for both AIBN and BPO was about 1. This value was similar to that mentioned in a previous report [10]. The estrone quinol 10β-hydroxyesta-1,4-diene-3,7-dione has been reported to be a product of the oxidation of estrone with 3-chloroperbenzoic acid in the presence of BPO as a radical initiator and under light irradiation in refluxing dry dichloromethane [11]. Oxidation of estrone produces steroidal quinols and syn-epoxyquinols [12]. Therefore, the fully oxidized n for estrone should be 2, due to the formation of estrone quinol, but estrone gave approximately n=1 in the present study. The n value found for estrone in the present study suggests that oxidation of estrogen is complex, and that quinoids, quinoid radicals and phenoxyl radicals may be formed from oxidation of estrones and may be responsible for carcinogenecity and/or inflammatory effects in biological systems [3]. The free radical-scavenging activity of estrones is a result of their A-ring phenolic hydroxy group, but the oxidation of estrones by hydrogen peroxide has shown to occur at the carbonyl group, with a cleavage of D-ring and production of a hydroxy acid; this reaction is essentially the oxidation of a cyclic ketone to a lactone [13]. The mechanism of the radical reaction is complicated and the chemical nature of the products derived from the radical-scavenging reaction remains unknown.
For AIBN, the n value for 2-hydroxyestradiol, a catechol estrogen, was approximately 2, whereas that for BPO was approximately 1. Cytochrome P450 oxidizes 2-hydroxyestradiol to 2,3-estradiol quinone [14]. This suggested that oxidation of 2-hydroxyestradiol may produce catechol quinones. Oxidation of catechols to semiquinones and quinones is a mechanism of tumor initiation, not only for endogenous estrogens, but also for synthetic estrogens such as DES and BPA, a human carcinogen, because quinone reacts with DNA [15]. The n values for DES and BPA for the AIBN and BPO system were about 1 and 2, respectively. These compounds preferentially scavenged PhCOO . radicals derived from BPO, which may result in the formation of quinone.
Rp inh /Rp con and k inh . Figure 5 shows the relation between Rp inh /Rp con and concentration of estrone and 2-hydroxyestradiol for both the AIBN and BPO system. In the both systems, Rp inh /Rp con for each estrogen decreased linearly with increasing concentration. 2-Hydroxyestradiol showed much stronger enhancement of the rate in the AIBN system. In contrast, estrone showed much more enhancement in the BPO system. The reduction of Rp inh /Rp con implies the retardation of polymerization. The oxidized products of estrogens produced by initiator radicals during the induction period could interact strongly with growing MMA radicals. The k inh values for estrogens, estrogen-like compounds and monophenol, BMP calculated from Eq. (5) are shown also in Table 1. The inhibition rate constant (k inh x 10 -3 M -1 s -1 ) for the AIBN system declined in the order estrone (2. Several studies are available in which each of these two methods (the n and k inh ) was applied to elucidate the mechanism of a particular reaction, but there is no comparative study with respect to the scope and limitation of these methods [16]. In the present study, we examined the radical-scavenging activity using both methods under nearly anaerobic conditions. The compounds having both large n and k inh values are good radical-scavengers.
Under aerobic conditions at 25 °C, k inh values for estrone and estradiol against 2,6-di-t-butyl-4-(4methoxyphenyl)phenoxyl radical (PhO . ) measured by the stopped flow technique have previously been reported by to be 84 M -1 s -1 and 138 M -1 s -1 , respectively [4]. In the present study, the k inh value for estrone was approximately one order of magnitude greater. The ratio k inh /k p for 2-hydroxyestradiol has previously been determined using the induction period method to be 2.4 x 10 3 for oxidation of methyl linoleate micells under aerobic conditions [5], from which a k inh value of approximately 2.4 x 10 5 M -1 s -1 can be calculated by estimating a k p value of about 100 M -1 s -1 at 30°C [7]. This value was approximately two-fold order of greater than that obtained in the present study. This considerable difference in the absolute values of k inh is probably caused by difference in methodology between the studies (PhO . radical, linoleate radical LOO . or growing MMA radical; stopped flow technique or induction period method, respectively). In vivo experiments are too complex to amenable to simple interpretation and, hence, we employed physical-chemical studies using the induction period method in the radical polymerization of MMA under nearly anaerobic conditions. We expect that the n and k inh values for estrogens and estrogen-like compounds determined in the present study will be relevant for the development of compounds that mimic their biological activity.

Conclusions
The n and k inh for estrone, 2-hydroxyestradiol, DES, BPA and BMP were determined using the induction period method under nearly anaerobic conditions at 70°C. The n values for these compounds were 1-2 and their k inh were 0.9 x 10 3 -3.2 x 10 3 M -1 s -1 . 2-Hydroxyestradiol scavenged R . radicals preferentially, whereas DES and BPA preferentially scavenged PhCOO . radicals. The k inh of 2-hydroxyestradiol for the BPO system was the greatest, whereas that of estrone for the AIBN system was the greatest among the indicated compounds.

Experimental
General DSC (differential scanning calorimeter) measurements were similar to those in our previous publication [17]. In brief, the experimental resin consisted of MMA and AIBN (or BPO) with or without additives. AIBN (or BPO) were added at 1.0 mol%, and the additives were used at 0, 0.02, 0.05 and 0.1 mol%. Approximately 10 µL of the experimental resin (MMA: 9.12-9.96 mg) was loaded into an aluminum sample container and sealed by applying pressure. The container was placed in a differential scanning calorimeter (model DSC 3100; MAC Science Co., Tokyo, Japan) kept at 70°C, and the thermal changes induced by polymerization were recorded for the appropriate periods. The heat due to polymerization of MMA was 13.0 kcal/mole in this experiment. The conversion of all samples, as calculated from DSC thermograms, was 91-96%.

Measurement of stoichiometric factor (n)
The relative n value in Eq. (1) can be calculated from the induction period in the presence of inhibitors: where [IP] is the induction period in the presence of an inhibitor. The number of moles of peroxy radicals trapped by the antioxidant is calculated with respect to 1 mole of inhibitor moiety unit. The R i values for AIBN and BPO were 5.66 x 10 -6 Ms -1 and 2.28 x 10 -6 Ms -1 , respectively [17].

Measurement of the inhibition rate constant (k inh )
When R i is constant, i.e. when new chains are started at a constant rate, a steady-state treatment can be applied and the initial rate of polymerization of MMA is given by Eq. (2) where MMA represents methyl methacrylate and k p and k t are the rate constants for chain propagation and termination, respectively. The k p /(2k t ) 1/2 rate of polymerization of MMA (9.4 M) by AIBN (1 mol%) and BPO (1 mol%) at 70°C was a constant value, 9.86 x 10 -2 M -1/2 s -1/2 [18]. The Rp inh rates are determined by Eq. (3): in which Rp inh is the initial rate of inhibited polymerization, [MMA], n, [IH] and k p are defined above, and k inh is the rate constant for scavenging (inhibiting) of MMA radicals by an antioxidant. From Eq.