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The present work reports on the effect of oxidizing (OH, O2•−) and reducing free radicals (e−aq, H) on 17β-estradiol (17βE2) in respect to breast cancer initiation. The objectives of the study were based on the following premise: the ability of 17βE2 to emit electrons (e−aq) as well as to transfer them to other biological systems. Thereby, the resulting transient hormone products are leading to the formation of metabolites, some of which may initiate the neoplastic process. The present work considers the effect of the simultaneously generated oxidizing and reducing free radicals on the carcinogenic properties of the 17βE2 metabolites.
Water-soluble 17βE2 with incorporated 2-hydroxypropyl-β-cyclodextrin (HBC) in various aqueous media (pH ~7.4), saturated with air, N2O or argon, as well as HBC alone were exposed to the action of free radicals produced by γ-ray. Escherichia coli bacteria (AB 1157) were used as a model for living systems.
From the survival curves, obtained under different conditions, the derived ΔD37 values (representing the radiation dose at which N/N0 = 0.37; N/N0 ratio: N0 = starting number of colonies, N = number after irradiation treatment) illustrate that 17βE2 as well as HBC act as very powerful scavengers of OH and O2•− radicals. On the other hand, 17βE2 and HBC intermediates resulting from attack of the reducing species (e−aq, H) have strong anticancer properties.
It is stated that ΔD37 values strongly depend on the reactivity of the individual free radicals. Oxidizing free radicals lead to positive ΔD37 values, illustrating the strongly pronounced radiation protecting ability of the systems. On the contrary, the primary reducing free radicals result in negative ΔD37 values, indicating anticancer effect.
It is well recognized that the human body is permanently generating and consuming oxidizing (OH, O2•−, etc.) as well as reducing free radicals (e−aq, H, R•, etc.), which are involved in various biological processes (1). Free radicals are also involved in the aging process and may be responsible for the initiation of cancer (2). In this respect it was very recently observed for the first time that sexual hormones such as 17β-estradiol (17βE2), progesterone (3) and testosterone (4) can emit electrons in polar media when excited in the singlet state. The same property was also established for the phytohormone genistein (5) and for adrenaline (6). The electron yield was found to be dependant on the water content in the solvent mixtures, substrate concentration and temperature. Thereby, a proportion of the e−aq is scavenged by the hormones themselves because their reactivity with e−aq is very high (k=109-1010 Lmol−1s−1) (7). Hence, the bifunctional property of the hormones to emit as well as to react with electrons classifies them as electron mediators and enables them to communicate with other biological systems in the organism. As generally accepted, the specific intracellular receptors are involved as main transducers in this process. The metabolites formed from the transients resulting from this electron emission can have diverse biological behaviours. Some of them, e.g. from 17βE2, can initiate cancer (8-11). It should be mentioned that the hormone metabolites, similar to the initial hormones, can also eject as well as consume electrons. This effect was very recently investigated in the case of 4-hydroxyestrone (4-OHE1), a typical carcinogenic metabolite originating from 17βE2 (12).
It is noteworthy that very few experimental data are available concerning the effect of oxidizing and reducing free radicals on hormones. Of particular interest is the question of whether the hormone degradation products as well as metabolites resulting from the attack of free radicals generated in the organism can initiate cancer and/or proliferation of cancer cells. For this purpose, specifically designed experiments in vitro using bacteria as a model were performed. In the present study gamma rays were implemented as the energy input for free radical production. By irradiation of appropriate aqueous media and under suitable experimental conditions, it is possible to study the proposed phenomenon for hormones and metabolites. Since 17βE2 is not soluble in water, it was used as complex with HBC for two reasons: (i) The system 17βE2/HBC is water soluble, which simplifies the reaction mechanisms. (ii) HBC (a polysaccharide) is used as representative for everyday food, therefore it was of interest to learn its biological effect in respect to the subject matter.
Table 1 summarizes the generation of oxidizing and reducing free radicals by radiolysis of water.
All chemicals of the highest purity available (<99%; Sigma-Aldrich, Vienna, Austria) were used. Water soluble 17βE2, including 2-hydroxypropyl-β-cyclodextrin (HBC), with a content of 4.7% 17βE2 and 95.3% HBC was used in all experiments. Hence, the aqueous solutions contained 1×10−4 mol/l 17βE2 and 3.98×10−4 mol/l HBC. All solutions were prepared using triple distilled water. As a model for living systems, the well known Escherichia coli bacteria (AB1157; DSMZ. Braunschweig, Germany) were used. In order to study the specific action of the oxidizing and reducing free radicals, the aqueous media were accordingly saturated with air, N2O or argon (cf. Table I).
As irradiation source for the generation of free radicals a 60Co-gamma-irradiator (Nordion Inc., Canada) providing a dose-rate of 30.5 Gy/min was used. By means of a modified Fricke-Dosimeter the dose-rate of the γ-source was determined and permanently controlled (13).
The handling of the E. coli bacteria was performed by a standard method previously described (14, 15). All glassware etc. was sterilized before use (6 h at 250°C or by γ-ray at 30 kGy). All solutions were heated for 20 min at 120°C in a steam-pressure autoclave.
Five ml of a batch culture of bacteria (OD = 0.4 at λ = 580 nm) were harvested by centrifugation (25°C, 10 min, 4,000 rpm) and washed once with buffer (8 g/l NaCl, 0.2 g/l KCl, 0.25 g/l KH2PO4, 1.46 g/l Na2HPO4.2H2O). The obtained pellets were resuspended in 10 ml buffer, as well as in 10 ml buffer with added substrate (HBC or 17βE2+HBC). HBC was examined in the range of 1×10−7 to 3×10−4 mol/l HBC (Figure 1, curve A), whereas for the complex of 17βE2+HBC the concentration was varied from 1×10−7 up to 4×10−4 mol/l 17βE2, containing in each case a 4 times higher concentration of HBC. After 1 h of incubation at room-temperature the samples were diluted (3 μl sample in 250 ml buffer) and 100 μl of each sample were spread onto Petri dishes in triplicates. The surviving colonies formed after overnight incubation at 37°C were counted and the N/N0-ratio was calculated. Mean values of 4-5 individual measurements were obtained.
The survival curves represent the relative survival rate (N/N0 ratio: N0 = starting number of colonies, N = number after irradiation treatment) presented as function of the absorbed radiation dose. The experiments were performed by using a number of samples containing either buffer alone, 3.98×10−4 mol/l HBC or 1×10−4 mol/l 17βE2 containing 3.98×10−4 mol/l HBC. Before treatment with radiation the samples were saturated with air, N2O or argon, in order to study the action of the specific free radicals. The characteristic ΔD37 values represent the radiation dose at which N/N0 = 0.37, obtained by subtracting the D37 buffer value from each individual D37 value: D37(sample) – D37(buffer) = ΔD37(sample). Thereby, positive ΔD37 values indicate a radiation protective ability of the system, whereas negative ones demonstrate cytostatic property.
The toxicity curves of substrates HBC and 17βE2+HBC have an unusual course (Figure 1). They show a very strong dependence on the concentration, passing a maximum at ca. 2×10−5 mol/l for HBC and at 1×10−6 mol/l for 17βE2+HBC, respectively. In the investigated range up to 3×10−4 mol/l for HBC practically no toxic effect was observed, but above this concentration, the N/N0 of the curve of HBC (A) tends to <1, indicating the beginning of toxicity. Simultaneously, curve B (17βE2+HBC) however, increases again, N/N0 >1, tending to a second maximum above 5×10−4 mol/l 17βE2+HBC. This effect shows that 17βE2 enhances bacterial proliferation at higher concentrations. It shows once more the bifunctional property of this hormone in dependence of its concentration as mentioned above.
For better comparison of the effect initiated by oxidizing and/or reducing free radicals on 17βE2, the experiments in vitro were carried out in aqueous media (pH ~ 7.5) saturated with air, N2O or argon. Under these conditions one can generate different types and concentrations of desired free radicals, which can attack the 17βE2-molecules (cf. Table I). The resulting hormone transients are expected to interact with the bacteria and interfere with their proliferation.
Figure 2 shows the survival curves (N/N0 ratio) of bacteria cultured in buffer alone (A), HBC (B) and 17βE2+HBC (C) as a function of absorbed radiation dose (Gy), which is proportional to the free radicals produced. Hence, each desired radical concentration can be achieved by applying the appropriate radiation dose. In the present case, oxidizing radicals only (46% OH, 54% O2•−) were involved in the process. Based on the yield of each type of free radical it is possible to calculate the percentage of oxidizing and reducing radicals acting under given conditions (cf. Table I).
Curve B shows a maximum for N/N0 ratio at 50 Gy, indicating the very strong protective effect given by HBC from oxidizing radicals. This property is best expressed by the ΔD37 = +110 shown in the inset of Figure 2. The behaviour of 17βE2 against the action of the oxidizing species is illustrated by the corresponding ΔD37 = +40 (curve C). This value, which is lower compared to that for HBC, indicates that the hormone transients resulting from radical attack on 17βE2 appear to have a cytostatic property in the present media. This effect is, however, superimposed by the opposite action of HBC.
In medium saturated with N2O, all solvated electrons are converted into the strong oxidizing OH radicals (90%) and only 10% H reducing species are involved in the process. The reaction rate constant (k) of the OH radicals with respect to the molecular structure of 17βE2 is expected to be very high (k ~ 109-1010 lmol−1s−1). Hence, 17βE2 should act as a very efficient OH-scavenger. Under the same conditions the k (OH + HBC) = 7.6×108 lmol−1s−1 (7), which also illustrates this substrate's ability to act as an OH-scavenger.
The obtained survival curves are presented in Figure 3. The calculated characteristic ΔD37 values are positive (inset in Figure 3) as was the case in the presence of air. Whereas the ΔD37 value of HBC is slightly higher, that of 17βE2+HBC is more than 6 times higher than the corresponding value in aerated medium. This is attributed to the very high oxidizing ability of the OH radicals, which were in the present case predominant.
Based on available evidence, it can be stated that the OH as well as the O2•− radicals mainly attack the double bonds of the A-ring of 17βE2. The formed OH-adducts can split off water, resulting in a phenoxyl-type transient (R1), which has several mesomeric structures. Each of them can lead to the formation of a metabolite. A simplified presentation of the involved reactions is given in Table II. Each of the given intermediates in Table II can react with each other and with intermediates present in the medium, leading to formation of final products. About 30% of the OH-adducts of the hormone can undergo disproportion, regenerating the starting molecule and forming pyrocatechol and water (Table II, r4) and undergoing subsequent dimerization. In the presence of air the OH-adduct of phenol as well as of 17βE2 can combine with O2 and the obtained peroxyl radicals decompose to various products, e.g. (Table II, r5a, r5b). The OH-adducts at the other positions of the A-ring likewise lead to the formation of a variety of metabolites. The transients of the R1 type of Table II, r1, can naturally result in several 17βE2 metabolites, depending on the available components in the media.
It may be said that the effect on 17βE2 of the oxidizing primary species generated in the organism (OH, O2•− etc.) leads to the formation of a broad spectrum of metabolites having diverse biological properties.
Entirely opposite results were obtained in air-free media. The reducing species (44% e−aq, 10% H) are predominant, whereby 46% OH radicals are also involved in the reaction mechanisms. The survival curves observed under these conditions are shown in Figure 4.
For HBC a ΔD37 value of −100 was observed and for 17βE2+HBC a value of ΔD37 = −60. These negative values indicate that the reactions of the reducing species with the substrate resulted in intermediates with cytostatic abilities. The reaction mechanism is rather complicated. However, based on the molecular structure of 17βE2, it is expected that e−aq and H predominantly attack the A-ring and to a smaller extent other positions of the molecule. Taking again phenol as a model for the A-ring of 17βE2 the reactions shown in Table II (r6, r7) are possible.
The attack of e−aq and H can proceed at various positions on the A-ring as discussed above for the OH reactivity with 17βE2. Thereby, a reaction rate constant of k (e−aq + 17βE2) = 2.7×1010 lmol−1s−1 has been reported (17). Naturally, the radical type R2 can be involved in various processes, finally leading to the formation of metabolites with different biological properties.
Summing up, it can be stated that both types, oxidizing as well as reducing free radicals, which are normally also generated in the human organism, react very efficiently with 17βE2 hormone. Thereby a number of metabolites can be formed, having different biological properties.
Using water-soluble substrates 17βE2+HBC and HBC in neutral media (pH ~7.5) and E. coli bacteria (AB1157) as a model for experiments in vitro, the reaction of oxidizing (OH, O2•−) as well as reducing free radicals (e−aq, H) was investigated.
The toxicity (N/N0 ratio) of HBC as well as of 17βE2+HBC in dependence on substrate concentration (1×10−7 to 4×10−4 mol/l) shows an unusual course. This indicates a complex bifunctional behaviour of the substrates.
Based on the survival curves of the bacteria observed in aerated media (46% OH, 54% O2•−) as a function of the free radical concentration (corresponding to the absorbed radiation dose, Gy), it can be stated that: (i) Both substrates act as very efficient radical scavengers. (ii) The calculated ΔD37 value from the corresponding survival curves are: +110 for HBC and +40 for 17βE2 + HBC, respectively.
In media saturated with N2O (90% OH, 10% H) is this effect even stronger expressed, namely for HBC ΔD37 = +140 and for 17βE2+HBC ΔD37 = +250. In both aerated and N2O-containing media, where only oxidizing primary radicals are operating, the main reaction takes place mainly on the A-ring of 17βE2 hormone. The number of metabolites resulting in such cases depends on the reaction partners available in the media.
In air-free media, however, where the reducing radicals predominate (44% e−aq, 10% H) and fewer oxidizing species (46% OH) are acting, the observed ΔD37 values are negative: −100 for HBC and −60 for 17βE2+HBC, respectively. These data indicate that the intermediates resulting from the attack of e−aq and H atoms on both substrates have a strong cytostatic property.
Putative reaction mechanisms pertaining to oxidizing as well reducing free radical effects on 17βE2 are presented.
The authors are deeply indebted to FWF Austrian Science Fund for the financial support, which it made possible to perform the project: Free radical action on sexual hormones in respect to cancer, Contract No. P21138-B11.