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4-Hydroxyestrone (4-OHE1), a typical cancer-inducing metabolite, originating from 17β-estradiol (17β-E2), was chosen as a model for the studies. The aim was to get a deeper insight in the mechanisms of its ability to initiate cancer. It was found, that 4-OHE1 can eject electrons , when excited in the singlet state by monochromatic UV-light (γ = 254 nm) in polar media (water:ethanol = 40:60 vol.%). The quantum yield , determined for various 4-OHE1 concentrations, is found to be as high as that previously observed for 17β-E2. It decreases with increasing substrate concentration, but it is enhanced at higher temperature. The ability of 4-OHE1 to eject as well as to consume and to transfer electrons to other biological systems, classifies it as an electron mediator, similar to 17β-E2.
The 4-OHE1 transients resulting of the electron emission process are leading to the formation of secondary metabolites. Surprisingly, it was established that the secondary metabolites possess likewise the ability to eject as well as to consume electrons. Hence, they behave similar like 17β-E2. However, the structure of the secondary formed metabolites, which determinates their biological properties and carcinogenity, depends on the nature of the available reaction partners involved in their formation. A probable reaction mechanism explaining the subject matter is discussed.
Based on experimental evidence, 17β-estradiol (17β-E2) is blamed for the initiation of breast cancer [1-8]. Almost all of its A-ring metabolites exhibit two different biological actions on cell proliferation: at low concentrations (10−8–10−6 mol/L) 17β-E2 stimulates, but at higher concentrations (>10−5 mol/L) it inhibits the proliferation of cultured cells . This effect was not observed for D-ring metabolites with exception for 16α-hydroxyestrone. At higher 17β-E2 concentrations, the metabolites: 2-hydroxyestrone, 2-hydroxyestradiol, 2-hydroxyestriol, 4-hydroxyestrone and 4-hydroxyestradiol caused significant inhibition of the cell proliferation. Recent studies confirmed the previous findings that estrogens play a major role in promoting the proliferation of both, normal as well as the neoplastic breast epithelium cells [6,7]. It has been also found that patients with fibrocystic diseases are at elevated risk for developing breast cancer. Both, 16α-hydroxyestrone and 4-hydroxyestrone are reported carcinogens [9,10]. Based on the above briefly reported results, it seems that estrogen metabolites obviously influence estrogen-target tissues and modulate estrogen-associated cancer disease by the mentioned metabolites.
In order to learn more about reaction mechanisms responsible for the specific behaviour of some estrogen metabolites initiating breast cancer, 4-hydroxyestrone (4-OHE1) was chosen as a model for the present studies. In this respect it should be mentioned that 17β-E2 as well as progesterone (PRG)  and testosterone (TES)  can emit as well as consume “solvated electrons” in polar media and transfer them to other compounds. The same biological property has also been established for genistein  and adrenaline . This capability of the hormones to act as electron mediator enables them to communicate with other biological systems . The hormone transients resulting from the electron emission processes, e.g. the progesterone radical cation PRG.+, can be regenerated in the presence of an appropriate electron donor . The other types of hormone transients, such as phenoxyl type of ring A in the case of 17β-E2, are leading to the formation of several metabolites. In the case of 17β-E2, the electron ejection occurs from the π-electron structure of the A-ring resulting in the formation of a phenoxyl type radical . It has several mesomeric structures, which enables the formation of various metabolites. As mentioned above, some of them can promote the proliferation of normal cells and other may initiate cancer, as it is the case of 4-OHE1.
Therefore, the aim of the present investigations was besides studying the electron emission of 4-OHE1, to investigate the fate of the resulting transients, their possible ability to form secondary metabolites and the behaviour of those, as well as the involved reaction mechanisms leading to carcinogenesis.
A low pressure Hg-lamp (Osram HNS 12 W) with incorporated Vycor-filter for removal of the 185 nm line was used. The solutions were irradiated with a monochromatic UV(C)-light of 254 nm (4.85 eV/hm) in a special, double-wall 4π-irradiation apparatus connected with a thermostat .
During the irradiation, the temperature was kept constant at 37 ± 1 °C or at 45 ± 1 °C. The intensity of the monochromatic lamp emission was determined at 254 nm (I0 = 1 × 1018 hν mL−1 min−1) by implementing chloroacetic acid actinometer .
The “solvated electrons” , ejected from 4-OHE1 were scavenged by chloroethanol (1 × 10−2 mol/L), where L mol−1 s− . The absorption of the UV-light by chloroethanol under the given conditions is negligible, however, it was taken into account. The resulting yield of Cl− was determined by mercury (II) thiocyanate method , where .
The substrate, 4-OHE1, as well as all chemicals used, were of highest purity available (Fluka–Aldrich). Since 4-OHE1 is not soluble in water, a mixture of 40 vol.% water and of 60 vol.% ethanol was used as a solvent. It might be mentioned that the polarity of the solvent plays an essential role in the electron ejection process . Therefore, the water content in the solvent mixture is of crucial importance because its polarity is much stronger (dielectric constant, DC = 80) than that of ethanol (DC = 24.3).
In the frame of the previous studies [11-15] it was stated that the above mentioned hormones (17β-E2, PRG, TES, etc.) can eject electrons from their excited singlet states. On the other hand, their molecules in the ground state react with (k = 109 up to 1010 L mol−1 s−1) and consume a part of them. The probability of this reaction depends on the substrate concentration, on the reaction rate constant as well as on the nature of the solvent.
In order to enclose the multifunctional behaviour in the present studies, various substrate concentrations (1 × 10−5 up to 7.5 × 10−5 mol/L 4-OHE1) were used.
As known, the electron emission process increases with rising the temperature at the expense of the fluorescence yield . Hence, in order to verify this behaviour, the electron emission of 4-OHE1 was investigated at two temperatures, 37 and 45 °C. The initial quantum yield of the ejected , , were calculated from the tangent of each curve (Fig. 1--4)4) in respect to the absorbed UV-quanta (hν/mL) (formula (1)):
where = number of ejected electrons (mol/L); N(A) = Avogadro constant (6.0221 × 1023 mol−1); Iab = absorbed UV-quanta/L (Iab × 103) and L = litre (1000 mL).
In Fig. 1, the yield of the ejected electrons (, mol/L) from 10−5 mol/L 4-OHE1 at 37 °C is presented as a function of the absorbed monochromatic UV-quanta. The presented data are mean values of five individual series of experiments with a standard deviation of about 10%. Unexpectedly, the electron emission appeared as two sharp peaks. This indicates that two consecutive electron emitting processes were involved. The calculated initial quantum yields of both peaks are: (A) = 2.1 × 10−2 and (B) = 0.3 × 10−2 given as an insert in Fig. 1. The -yield of peak (A) is very similar to the previously reported of 17β-E2, = 1.76 × 10−2 .
The electron emission expressed by two peaks in respect to the absorbed UV-quanta at 37 °C was also observed at higher substrate concentrations. Fig. 2 shows this effect for 5 × 10−5 mol/L 4-OHE1 The corresponding electron yields are somewhat lower: for peak (A) = 1.20 × 10−2 and (B) = 0.1 × 10−2 as illustrated in Fig. 2 in insert.
Using 7.5 × 10−5 mol/L 4-OHE1 in addition to peaks (A) and (B), a third peak appears at higher absorbed UV-doses (~12 × 1018 hν/mL).
The observed peaks indicate the formation of secondary metabolites. It is surprising that they are also able to emit electrons. From our previous studies  is known that in such case the π-electron structure of ring A of the molecule is the main source of electron ejection. Hence, ring A in the secondary metabolite obviously remains preserved. This effect is of biological importance, because in the formation of secondary metabolites exists the possibility of incorporation of carcinogenous components.
Preliminary HPLC-analyses of the UV-irradiated samples at various UV-doses and of various substrate concentrations showed that several products are formed, however, with very small yields. This indicates that the ring skeleton of the substrate-molecule remains more or less unchanged. The reaction occurs mainly on the periphery of the molecule. The transients resulting from 4-OHE1 as a sequence of the electron emission may scavenge free radicals available in the present medium, leading to the formation of new types of secondary metabolites.
For illustration of the subject matter, the following reaction mechanism is suggested: reaction (2) demonstrates the electron ejection from the excited 4-OHE1 by UV-light. In organism, this process is realized by energy transfer through biological processes. The scavenge of by chloroethanol under Cl− formation is shown by reaction (3).
Under the given conditions, the 4-OHE1 radical and the ethanol radical can react and result in a new metabolite (M1). Naturally, the biological properties of M1 will be entirely different from those of the starting one (4-OHE1).
Reaction (5) demonstrates a pathway for the formation of secondary metabolites under the present experimental conditions. However, following this pathway, other free radicals available in the media can lead to the formation of metabolites with characteristic biological properties. If the radical originates from a heterocyclic compound (e.g. pyrene, etc.) carcinogenic metabolites might be formed.
The biological attributes of the secondary metabolite, M1, are very likely contingent on environmental conditions, life habits of the person, such as nutrition and may have distinct structures favouring carcinogenesis. The M1-metabolite is on the strength of its molecular structure able to eject likewise electrons, similarly to other biological substances [12,21,22]. The observed electron emission characterized by peak (B) in Figs. Figs.11 and and22 is attributed to a process originating very likely from metabolite M1 reaction (5), but also of radicals originating of 4-OHE1 photolysis (formation of dimers). Obviously, this depends on the substrate concentration and on the specific reaction rate constant (k) of the involved reaction partners.
It was also stated that with increasing 4-OHE1 concentration, the -yield decreases, as shown in Figs. Figs.11 and and22 for both peaks (A) and (B). This effect was also previously observed for other investigated hormones [11-15]. It was attributed to several factors: (i) formation of hormone associates (unstable complexes) in the solvent mixture, where the substrate molecules in ground state consume a part of the ejected . Hence, only a fraction can diffuse away and is scavenged by chloroethanol (reaction (3)) (ii) another part of the emitted reacts with the well dissolved substrate molecules. The reaction rate constant of the above mentioned hormones are in the range of k = 109–1010 L mol−1 s−1/11–14/ (iii) the =CO group on the 17-position of the 4-OHE1 molecule acts as an efficient electron scavenger k ~ 1.6 × 109 L mol−1 s−1 . Therefore, a further part of the emitted electrons can be consumed by reacting with this group. The importance of this process is proportional to the substrate concentration and can initiate the formation of further types of metabolites possibly by reactions (6a)–(6c):
As a sequence of the attack, the resulting transient with the very reactive =.CO− group can subsequently scavenge H+, another radical present in medium or eventually undergo hydrolysis, as shown by reaction (6c).
Naturally, the electron ejection process is resulting into a transient (degradation species; see reaction (2), which subsequently is leading to a metabolite (final product; reactions (5) and (6)) of the process.
Preliminary HPLC-analysis of UV-irradiated 4-OHE1 samples showed that at a low UV-dose (~0.3 × 1018 hν/mL) the formation of just one metabolite is observed. At higher UV-doses, however, in the addition to the formation of the second peak, also the substrate and the metabolite are successively decomposed, resulting into simpler molecules.
Since the skeleton of the 4-OHE1 molecule remains more or less unchanged under relative low UV-doses and the reaction takes place on the “periphery” of the molecule (reactions: (2)-(4) and (6)), several metabolites can be formed. The peak (C) in Fig. 3 represents the electron emission of metabolites resulting probably from the transients shown by reaction (5). The observed -yield was lower as expected.
As previously stated, the electron emission process, fluorescence, etc., depends on the temperature . This effect was also observed in the case of other hormones [13,14]. Hence, for the sake of completeness, the electron ejection of 4-OHE1 was verified at 45 °C. The results are presented in Fig. 4. The obtained quantum yields of for peak A and B are higher compared to those in Fig. 1 for the same substrate concentration, but at 37 °C.
It is worth mentioning that the pH of the media decreases (formation of H+) simultaneously with the increase of the -yield (reaction (1)). This gives a hint that the OH-groups are involved in the electron ejection process, as stated previously . The effect is shown for 37 and 45 °C in Fig. 5. The pH-change as a consequence of the absorbed quanta (hν/mL) does not show any peaks, but is linear, which indicates that the electron ejection proceeds continuously during the UV-irradiation.
4-Hydroxyestrone (4-OHE1) is a carcinogenic metabolite originating from 17β-E2 hormone. In order to get a deeper insight in the reaction mechanisms of its biological properties, especially in respect to cancer, adequate investigations were performed. The obtained results are summarized as follows:
The authors thank the FWF Austrian Science Fund for the financial support, which made possible the performance of the Project: “Free radical action on sexual hormones in respect to cancer”, Contract No. P21138-B11.