Journal of Photochemistry and Photobiology B: Biology 92 (2008) 38–41
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Photo-induced electron emission from 17b-estradiol and progesterone and possible biological consequences Nikola Getoff a,*, Johannes Hartmann a, Johannes C. Huber b, Ruth Maria Quint a a b
Department of Nutritional Sciences, Section Radiation Biology, The University of Vienna, A-1090 Vienna, Althanstrasse 14, UZAII, Austria Department of Gynecology, Medical University, A-1090 Vienna, Währinger Gürtel 18-20, Austria
a r t i c l e
i n f o
Article history: Received 30 January 2008 Received in revised form 31 March 2008 Accepted 7 April 2008 Available online 15 April 2008 Keywords: 17b-Estradiol Progesterone Solvated electrons Excited state
a b s t r a c t It was established for the first time, that the sexual hormones 17b-estradiol (17bE2) and progesterone (PRG) are able to emit electrons from their excited single state in water–ethanol mixtures. The yield of the ‘‘solvated electrons” (e s ) depends on the substrate concentration, the ratio of water–alcohol-mixtures and the temperature. The e s yield obtained from 17bE2 is by two orders of magnitude higher than this of PRG. The possible relationship of the resulting hormone transients from 17bE2 leading via specific metabolites to breast cancer is discussed. Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Recently it has been frequently reported that estrogens are considered to play a double role: promoting the proliferation of normal as well as of neoplastic breast epithelium cells. Various mechanisms for the initiation of carcinogenic effects, particularly of breast cancer, based on several metabolites originating from estradiol were postulated [1–4]. According to our previous experiences, e.g. [5,6] and current studies on cell communication and cell membrane protection [7], it is expected, that the hormones would be capable to transfer or/and emit electrons to an appropriate acceptor. In the present work, we now report for the first time the emission of electrons from the excited single state of 17b-estradiol (17bE2) and progesterone (PRG) in polar media. A possible implication of the resulting transients as precursors of carcinogenic metabolites is briefly discussed under consideration of the hormone molecular structure. The electron ejection in aqueous media has been earlier observed in a number of flavines [5] as well as of various organic compounds, having functional groups as –OH, –OCH3, –NH2, OPO3H2, etc. [6]. This process competes with the fluorescence and is temperature dependent.
Both hormones, 17b-estradiol (17bE2; 1,3,5-estratriene-3,17bdiol) and progesterone (PRG; pregn-4-ene-3,20-dione) used at highest purity available (>99%; Fluka–Aldrich) were dissolved in mixtures of p.a. ethanol and triple distilled water, because of their insolubility in pure water. In order to get a deeper insight in the electron emission process the hormone concentration was varied from about 5 106 up to 103 mol/L. The experiments were performed at 30 °C, but some of them for comparison were carried out at 25 °C and 37 °C. As a scavenger for the ejected electrons 10 2 mol/L chloroethanol was applied, whereby the resulting quantum yield, Q(Cl) = Q(e sol ), was determined spectrophotometrically as a rhodamin-complex at 460 nm [8]. The required excitation energy for the substrates was provided by a low-pressure Hg-lamp (Osram HNS 12 W) with incorporated Vycor-filter for elimination of UV-C line 185 nm. Hence, the solutions were irradiated with a monochromatic UV-C-light of 254 nm (4.8 eV/hm) in a 4p-irradiation apparatus, connected with a thermostat in order to keep the desired temperature constant during the experiment [9]. The temperature can effect the yield of ‘‘solvated electrons” (e s ), since their generation compete with that of the fluorescence, which is temperature dependent [6]. The intensity of the lamp emission at UV-C 254 nm was determined (I0 = 1 1018 hm ml1 min1), implementing chloroacetic acid actinometer, where at 30 °C the quantum yield Q(Cl) = 0.32 and 0.41 at 37 °C, respectively [10]. Under these conditions the absorption of 102 mol/L chloroethanol was negligible, but even the resulting very small Cl-yield was taken into account. From biological point of view the role of water in the used
* Corresponding author. Tel.: +43 (1) 4277 54966; fax: +43 (1) 4277 54965. E-mail address:
[email protected] (N. Getoff). 1011-1344/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2008.04.002
39
N. Getoff et al. / Journal of Photochemistry and Photobiology B: Biology 92 (2008) 38–41
ethanol–water mixtures is of essential interest, since the polarity of water is stronger (dielectric constant, DC = 80), then that of ethanol (DC = 24.3). Therefore, to elucidate the role of water content in the process, two solvent mixtures were implemented throughout the work: (A) 60 vol% ethanol 40 vol% water and (B) 85 vol% ethanol and 15 vol% water. 3. Results and discussion For convenience the structure formulas of 17b-estradiol (17bE2) and progesterone (PRG) are presented in Fig. 1.
Table 1 Q(e s )-values determined at various 17b-estradiol concentrations at 30 °C in: (A) 60 vol% ethanol and 40 vol% H2O and (B) 85 vol% ethanol and 15 vol% H2O in airfree media Q(e s ) in solvent
17bE2 (mol/L)
A
B
1 105 5 105 1 104 5 104 1 103
1.76 102 1.20 102 0.80 102 0.38 102 0.082 102
0.73 102 0.56 102 0.46 102 0.33 102 0.15 102
1 104a
0.91 102
–
a
4 Q(e mol/L s )-value determined at 37 °C in solvent mixture (A) using 1 10 17bE2.
The electron ejection process of 17bE2 is discussed first. It should be noted, that the absorption of the excitation UV-C-light (k = 254 nm) by 17bE2 is situated in the valley between its 1st and 2nd singlet states. Hence, the absorbed quanta are for 105 mol/L 17bE2 in solvent (A) 3.75% and in solvent (B) 3.4%, respectively. This fact is taken into consideration in the Q(e s )-value calculation for each substrate concentration used. It should be mentioned that the hormone concentration in human organism is in nanomolecular range. However, from experimental point of view it is necessary to take much higher concentration to excite an appropriate amount of hormone molecules in order to obtain well founded results. 5 The Q(e –103 mol/L 17bE2 observed in both s )-data for 10 ethanol–water mixtures given in Table 1 represent mean values of at least five series of experiments, having a standard deviation of less than 10%. Obviously the quantum yield of the solvated electrons, Q(e s )-values, in solvent mixture (A) are essentially higher than in (B) solvent. This can be explained by the predominant content of water and its higher polarity, which favours the electron ejection process. In order to visualize the effect of water, experiments representing the course of the electron emission from 105 mol/L 17bE2 as a function of absorbed quanta (hm/ml) observed in solvent mixture (A) and (B) are shown in Fig. 2. On the other hand it is interesting to state, that with enhancing the substrate concentration from 105 to 103 mol/L 17bE2, the Q(e s )-yields in both solvent mixtures are rapidly decreasing (Table 1). A plausible explanation for this effect is the formation of ‘‘associates ‘‘(unstable molecular complexes) of 17bE2 in the solvent mixtures. This tendency appears commonly with large organic molecules having functional groups. In addition to this, estrogen is reacting with e s , with a rate constant, kðeaq þ estradiolÞ = 10 1 1 2.7 10 L mol s determined by competition reaction in alkaline aqueous solution (pH 13) [11]. Presently experiments are in progress to determine the rate constants of e s with 17bE2 and PRG in ethanol–water mixtures (A) and (B). Consequently, the excited 17bE2-molecules within the substrate-complex are ejecting electrons, but 17bE2-molecules in ground state are consuming
O CH3 OH
CH3 C
CH3
H 3C HO
17β-estradiol (17βE2)
Progesteron (PRG)
Fig. 1. Molecular structures of 17b-estradiol and progesterone.
17
10 x 4.0 3.5 3.0
A: Q (e s ) = 1.76 x 10
-
-2
-
B: Q (e s ) = 0.73 x 10
-2
B
2.5 N (h ν / ml)
3.1. 17b-Estradiol
2.0
A
1.5 1.0 0.5 0.0 0
1
2
3 4 e s (mol/L) -
5
-6
6 x 10
5 Fig. 2. The course of the solvated electron (e mol/L 17bE2 in s ) formation from 10 airfree solutions: (A) 60 vol% ethanol and 40 vol% water, (B) 85 vol% ethanol and 15 vol% water as a function of absorbed quanta (N, hm/ml) of monochromatic UV-C– light (k = 254 nm) at 30 °C. The obtained quantum yields, Q(e s ), in (A) and (B) solvent mixtures are given as insert.
electrons and only a fraction of them is diffusing away and can be scavenged by chloroethanol. The formation of similar ‘‘associates” has been previously observed, e.g. with hematoporphyrine [12]. By application of 5 106 mol/L 17bE2 the obtained Q(e s )-data were not well reproducible, because of the low absorption of UV-Clight (254 nm) of
