Photo-induced regeneration of hormones by electron transfer processes: Potential biological and medical consequences.

Based on the previous results concerning electron transfer processes in biological substances, it was of interest to investigate if hormone transients resulting by e.g. electron emission can be regenerated.The presented results prove for the first time that the hormone transients originating by the electron emission process can be successfully regenerated by the transfer of electrons from a potent electron donor, such as vitamin C (VitC). Investigations were performed using progesterone (PRG), testosterone (TES) and estrone (E1) as representatives of hormones. By irradiation with monochromatic UV light (λ=254 nm) in a media of 40% water and 60% ethanol, the degradation as well as the regeneration of the hormones was studied with each hormone individually and in the mixture with VitC as a function of the absorbed UV dose, using HPLC. Calculated from the obtained initial yields, the determined regeneration of PRG amounted to 52.7%, for TES to 58.6% and for E1 to 90.9%. The consumption of VitC was determined in the same way.The reported results concerning the regeneration of hormones by the transfer of electrons from an electron donor offer a new, promising method for the therapy with hormones. As a consequence of the regeneration of hormones, a decreased formation of carcinogenic metabolites is expected.

Introduction

It has been established on a number of steroid hormones (Gerschpacher et al., 2010; Getoff et al., 2008, 2009a, 2010a, 2010b, 2010c, 2010d, 2010e) and on phyto-hormone genistein (Getoff et al., 2009b) that hormones in a polar media containing water are able to emit electrons when excited in their singlet state. Above all, the electron yield depends not only on the molecular structure and concentration of the hormone, but also on the polarity and the pH of the media, temperature, energy input, etc. Thereby, the resulting hormone transients induce the formation of metabolites with different biological properties, depending on the substances present in the media, or the transients can undergo decomposition. Some of the hormone degradation products can also emit e−aq, but with a much lower yield. However, hormones can also consume electrons with reaction rate constants (k) from 108 up to more than 1010 L mol−1 s−1, or they can transfer them to other biological systems. Hence, hormones can be classified as “electron mediators” (Getoff et al., 2010b). This fact enables hormones to communicate with themselves and other systems in the organism (Getoff, 2009).

Based on the previous results concerning electron transfer between compounds of biological interest (Getoff, 2001, 2007; Steenken, 1992) as well as on the observed intramolecular electron transfer (Getoff, 2005), it was conceivable that hormone transients resulting from electron emission could be regenerated by potent electron donors. Therefore, the objectives of the present studies focused on a possible electron transfer from ascorbate (vitamin C), acting as an efficient electron donor, to hormone transients in “status nascendi”. The hormones progesterone (PRG), testosterone (TES) and estrone (E1) were chosen as representative compounds because of their molecular structures. The biological processes of the hormones were simulated by irradiation with monochromatic UV light (λ=254 nm).

Materials and methods

All used chemicals were of highest purity available (<99%; Sigma-Aldrich, Vienna, Austria) and were applied as delivered. Due to their insolubility in water the hormones, progesterone (PRG), testosterone (TES) and estrone (E1), were dissolved in a mixture of triply distilled water (40 vol%) and p.a. ethanol (60%). However, in this mixture the hormones form “associates” (unstable complexes) at concentrations above 5×10−7 mol/L hormone (pH ∼7.4), which was detected by spectroscopy.

The excitation of the substrates in their singlet state was achieved by irradiation with monochromatic UV light (λ=254 nm, 4.85 eV/hν) using a low-pressure Hg–UV lamp (HNS 12, OSRAM, 12 W) with incorporated VYCOR-filter for removal of the 185 nm line. The lamp was mounted in a specially designed 4π-geometry irradiation double-wall vessel and connected to a thermostat to maintain a certain temperature of the solution during the experiment (Getoff and Schenck, 1968). The intensity of the UV lamp, I0=1×1018hν mL−1 min−1, was determined by means of monochloracetic acid actinometer (Neumann-Spallart and Getoff, 1975).

The photo-induced degradation of hormones individually as well as their regeneration by electron transfer in a mixture with vitamin C (VitC) was performed by the HPLC method. Samples were analyzed using a Hewlett-Packard/Agilent 1100 HPLC series with a series 1050 diode array detector (DAD). The substances were separated on a Zorbax Eclipse XDB-C18 column (150×4.6 mm2 I.D., 5 μm particle size, Agilent) at a temperature of 30 °C. The total run time was 35 min, followed by 5 min of post-run. Detection was performed at 244  nm for PRG and TES, 280 nm for E1 and at 250 nm for VitC. 25 μL sample was injected, and elution was achieved by a linear gradient between the mobile phases A (2.5×10−4 mol/L ammonium acetate in water) and B (acetonitrile). The gradient started with 80% (A), decreased linearly to 65% (A) in 0.6 min, followed by a linear decrease to 10% (A) in 23.4 min, held at 10% (A) for 3 min, then increased linearly to 80% (A) in 3 min and finally held at 80% (A) for 5 min with a flow rate of 0.25 mL/min.

Results

The results of hormone degradation as well as their regeneration in the presence of VitC by transfer of electrons (given in %) were studied as a function of the absorbed UV-quanta per liter (hν/L) at a pH of ∼7.4 and are presented separately.

Progesterone

First, it should be mentioned that in the mixture of 1×10−4 mol/L PRG and 0.92×10−4 mol/L VitC, the effect of degradation is based on the corresponding molar extinction coefficients (ε254, L mol−1 cm−1), considering the fact that PRG absorbs 60% and VitC 40% of the absorbed UV-quanta. This fact has been considered in the calculation of PRG regeneration and VitC consumption.

Fig. 1 illustrates the course of degradation of PRG, VitC and their mixtures. The used concentrations are given in the legend of the figure. Curve A shows the degradation of PRG, while (B) depicts the specific degradation of PRG in a mixture with VitC as a function of the absorbed dose (hν/L). Curve (C) corresponds to the degradation of UV-irradiated VitC alone and curve (D) shows only the degradation of VitC mixed with PRG in dependence of the dose. It is remarkable that the predominant part of the substrate degradation occurs at low UV doses. Similar observations were shown previously when studying the electron emission process of various hormones.

Fig. 1

HPLC analyses: Determination of 1×10−4 mol/L PRG individually (A) and in a mixture with 0.92×10−4 mol/L VitC (B) as well as 0.92×10−4 mol/L VitC individually (C) and in a mixture with 1×10−4 PRG (D) dissolved in an airfree solution of 40% water and 60% ethanol (pH∼7.4) as a function of the absorbed UV-quanta (hν/L), λ=254 nm.

The regeneration of PRG resulting in an electron transfer from VitC as well as in VitC consumption is calculated in percentage and given as an inset of Fig. 1. Thereby, the computation of the data is based on the initial quantum yields (Q) of the corresponding remainder of the substrate resulting in the irradiation of PRG and VitC. (curves (A) and (C), Fig. 1). Curves (B) and (D) represent the individual remainders of PRG and VitC in the corresponding mixtures resulting as a consequence of UV irradiation. The individual UV absorption of PRG and VitC in the mixture was considered as the basis of the corresponding molar extinction coefficients at 254 nm (ε254).

It was further of interest to examine the effect of the concentration of VitC on the regeneration of PRG. The mean values of several experiments concerning the hormone regeneration, using 1×10−4 mol/L PRG and 2.5×10−4 mol/L VitC of the HPLC analysis, are shown in Fig. 2. A high regeneration of PRG of about 80.9% and a consumption of VitC of 31.1% was observed. It is also worth mentioning that an increased concentration of about 1×10−3 mol/L VitC leads to nearly 100% regeneration of PRG.

Fig. 2

HPLC analyses: Determination of 1×10−4 mol/L PRG individually (A) and in a mixture with 2.5×10−4 mol/L VitC (B) as well as 2.5×10−4 mol/L VitC individually (C) and in a mixture with 1×10−4 mol/L PRG (D) dissolved in an airfree solution of 40% water and 60% ethanol (pH∼7.4) as a function of the absorbed UV-quanta (hν/L), λ=254 nm.

Testosterone

Due to its A ring, the molecular structure of TES is similar to that of PRG. However, at position 17 of TES, there is an electron donating OH group, while in the case of PRG there is an electron consuming carbonyl group at position 20 (Figs. 1 and 3). This difference in the molecular structures of TES and PRG and the resulting corresponding reaction rate constants of the consumption of e−aq (k(e−aq+ascorbate)=3.5×108 L mol−1 s−1 and k (e−aq+PRG) ∼4×109 L mol−1 s−1), which are similar to that of TES, is a determining factor for the biological diversity of both hormones. Hence, it was of interest to investigate the possible regeneration of TES based on the transfer of electrons of VitC. The results of the corresponding HPLC analysis are presented in Fig. 3, whereby the evaluation of the experimental data followed the same procedure as mentioned above.

Fig. 3

HPLC analyses: Determination of 0.95×10−4 mol/L TES individually (A) and in a mixture with 1×10−4 mol/L VitC (B) as well as 1×10−4 mol/L VitC individually (C) and in a mixture with 1×10−4 TES (D) dissolved in an airfree solution of 40% water and 60% ethanol (pH∼7.4) as a function of the absorbed UV-quanta (hν/L), λ=254 nm.

Estrone

E1 is labeled through another type of molecular structure. Its A ring is a phenol ring acting as a potent electron emitter (compare the structure formulas shown in Figs. 1, 3 and 4). At position 17 of the E1 molecule, however, there is a strongly electron consuming oxygen atom located. Examinations using 1×10−4 mol/L E1 and 1×10−4 mol/L VitC individually and in mixture were carried out. The resulting mean values are presented in Fig. 4. The course of the curves of the substrate degradation is similar to that observed in the previous systems. The evaluation of curves (A)–(D) yielded up to 90% regeneration of E1, but the transfer of electrons consumes 16% of VitC. Compared to PRG and TES, the system containing E1 has the most efficient regeneration of transfer of electrons from VitC.

Fig. 4

HPLC analyses: Determination of 1×10−4 mol/L E1 individually (A) and in a mixture with 1.04×10−4 mol/L VitC (B) as well as 1.04×10−4 mol/L VitC individually (C) and in a mixture with 1×10−4 E1 (D) dissolved in an airfree solution of 40% water and 60% ethanol (pH∼7.4) as a function of the absorbed UV-quanta (hν/L), λ=254 nm.

Discussion

The presented results clearly demonstrate that the regeneration of the hormones PRG, TES and E1 by the transfer of electrons from the potent electron donor VitC depends on the molecular structure of the steroid hormone. The same dependence was observed previously in the electron emission process of various hormones (e.g. Gerschpacher et al., 2010; Getoff et al., 2009a, 2010a, 2010b). Obviously, the functional groups and their position in the molecule play an important role in these processes.

For a better understanding of the subject matter, some reaction mechanisms are presented, taking PRG as an example first. The emission of electrons from PRG and its regeneration by VitC are illustrated by Eqs. (1) and (2), respectively.

In addition to this process, the PRG•+ species have a strong oxidizing property; hence, they can react with water and can be regenerated:

The resulting OH radicals could react principally with PRG (reaction rate constant, k ∼6×109 L mol−1 s−1) forming e.g. OH adducts:

However, the OH radicals are completely scavenged by ethanol, hence under the given conditions none PRG OH-adduct is formed. Transients like PRG•+ and R2 can lead to the formation of metabolites or/and the degradation of the hormone. At the same time, as already mentioned, the PRG molecules in the ground state can also react with a part of the emitted e−aq (k(PRG+e−aq) ∼4×109 L mol−1 s−1) (Getoff et al., 2009a), producing PRG radical anions, e.g. R3, R4 etc.:

The produced radical cation of testosterone (TES•+), resulting in electron emission, can be involved in similar reactions (Eqs. (3) and (5)) due to the identical structure of the A ring of TES and PRG. The OH group on position 17 of the TES molecule, acting as an electron emitting site, takes part also in the regeneration process, e.g.

Certainly, TES and its primary transients are involved in the same processes as mentioned for PRG.

Based on the phenolic character of ring A of E1 other types of reactions occur as a consequence of the electron emission (Eq. (8)) as well as in the regeneration process of E1 (Eq. (10a) and (10b)). The emission of e−aq from ring A of the molecule results in the phenoxyl-like hormone transient, R6, which exists in 4 mesomeric structures (Eq. (9)). Each one of them leads to the formation of metabolites, which are not further discussed. Thereby, the following reaction steps (Eq. (8)) play an essential role, e.g. in the initiation of cancer as previously reported (Mueck et al., 2002; Pasqualini and Chetrite, 2008; Seeger et al., 2006):

Radical R6, as mentioned above, appears in several mesomere forms, represented by ring A (Eq. (9)):

The consumption of e−aq donated by VitC is involved in two processes: regeneration of R6-mesomere forms on ring A as well as formation of E1•− radical anions (R7) given in Eq. (10b). E1•− species can lead to the formation of metabolites and/or to the degradation of hormones. Similar reactions can occur with other hormones likewise.

The involved reaction mechanisms are rather complicated, because the hormones in ground state react with e−aq (reaction rate constants, k∼108–1010 L mol−1 s−1), depending on their molecular structure. On the other side, the hormone transients resulting by emission of e−aq can be at least partly regenerated to the original compounds by electron transfer processes (e.g. e−aq donated by VitC).

Considering the rather high reaction rate constants of the hormone involving processes, it is conceivable that the molecules of the electron donor (VitC) and those of the primary hormone transients (in “status nascendi”) should be very close to each other. Based on the results it is assumed that the hormone and VitC molecules are probably pre-forming a kind of “associate”, preventing the formation of undesired metabolites initiating cancer (Mueck et al., 2002; Pasqualini and Chetrite, 2008; Seeger et al., 2006).

The regeneration of hormones by electron transfer process using a potent electron donor, such as VitC, might offer a new pathway for an efficient reduction in the formation of metabolites, also such initiating cancer among others. The reported results concerning the ability of VitC to act as electron donor in the regeneration of hormone transients might also be of benefit in the clinical application of hormones (e.g. contraceptive, HRT).

Conclusion

The highlights of the present investigation can be summarized in the following points:

The hormone transients originating e.g. by electron emission processes, being in “status nascendi”, can be regenerated to their original structure by the transfer of electrons from an efficient donor like VitC.

In order to overcome the negative effect of competitive reactions for e−aq consumption, the concentrations of the donor have to be adjusted accordantly.

The hormone regeneration process depends on the molecular structure of the hormone and the reaction rate constants (in L mol−1 s−1) of competitive processes using up e−aq.

The reported results concerning the regeneration of hormones by the transfer of electrons from a potent electron donor like VitC offer a new, promising method for the therapy with hormones. As a consequence of the regeneration of hormones, a decreased formation of carcinogenic metabolites is expected.

Role of the funding source

The authors thank the FWF Austrian Science Fund for the financial support, which made it possible to perform the project: Free Radical Action on Sexual Hormones in Respect to Cancer (Contract no. P21138-B11).

Disclosure statement

The authors declare that there is no conflict of interest.