In Vitro Interactions between 17β-Estradiol and DNA Result in Formation of the Hormone-DNA Complexes

Beyond the role of 17β-estradiol (E2) in reproduction and during the menstrual cycle, it has been shown to modulate numerous physiological processes such as cell proliferation, apoptosis, inflammation and ion transport in many tissues. The pathways in which estrogens affect an organism have been partially described, although many questions still exist regarding estrogens’ interaction with biomacromolecules. Hence, the present study showed the interaction of four oligonucleotides (17, 20, 24 and/or 38-mer) with E2. The strength of these interactions was evaluated using optical methods, showing that the interaction is influenced by three major factors, namely: oligonucleotide length, E2 concentration and interaction time. In addition, the denaturation phenomenon of DNA revealed that the binding of E2 leads to destabilization of hydrogen bonds between the nitrogenous bases of DNA strands resulting in a decrease of their melting temperatures (Tm). To obtain a more detailed insight into these interactions, MALDI-TOF mass spectrometry was employed. This study revealed that E2 with DNA forms non-covalent physical complexes, observed as the mass shifts for app. 270 Da (Mr of E2) to higher molecular masses. Taken together, our results indicate that E2 can affect biomacromolecules, as circulating oligonucleotides, which can trigger mutations, leading to various unwanted effects.

. Model expressing the distinct molecular pathways of ER transcription factors regulatory actions. (A) The classical direct, genomic ligand-dependent pathway includes detachment of transcription factors-Estrogen receptors (ER) from complex with heat shock proteins (HSP) and shift of E 2 -ER complex towards estrogen response element (ERE), localized in nucleus, where transcription is triggered after recruitment of coactivators (CoA) and corepressors (CoR) of transcription process [24]. (B) Non-direct genomic ligand-dependent mechanism-Tethering or cross-talk includes protein-protein interaction with other transcription factors (TF) after ligand activation, and thereby gene regulation is affected by indirect DNA binding [29]. (C) Estrogen receptors can be phosphorylated at specific serine sites by growth factors (GF) and other plasma membrane estrogen receptors, coupled to kinase signaling (ERK-extracellular regulated kinase, Akt-Protein kinase B, PI3K-Phosphoinositide 3-kinase). Phosphorylated (P) ER can trigger the transcription in a ligand-independent manner [26]. (D) The non-genomic effect includes the activation of receptor, associated with the membrane receptors including G-protein-coupled receptor (GPER). Signaling through GPER occurs via transactivation of the epidermal growth factor receptor (EGFR) activated by metalloproteinases (MMP). This phenomenon leads to downstream activation of various signaling molecules, triggering possible alterations in protein synthesis. Furthermore, cyclic adenosine monophosphate (cAMP) and intracellular calcium mobilization (Ca ++ ) are stimulated by E 2 -mediated activation of GPER. This leads to rapid physiological responses without gene regulation. Moreover, GPER also regulates transcriptional activity by other signaling mechanisms as cAMP, ERK or PI3K [27].

Chemicals and pH Measurement
Working solutions as buffers or standard solution of 17β-estradiol were prepared daily by diluting the stock solutions. The interactions of E 2 and DNA fragments were carried out in the environment maintained by phosphate buffered saline (PBS, pH = 7.4), whose osmolarity and ion concentrations match with those of the isotonic environment in human body. PBS was prepared according to the protocol as follows: 8.01 g•L −1 of NaCl, 0.20 g•L −1 of KCl; 1.78 g•L −1 of Na 2 HPO 4 ·H 2 O and 0.27 g•L −1 of KH 2 PO 4 , dissolved MilliQ water, obtained by reverse osmosis using Aqual 25 (Aqual s.r.o., Brno, Czech Republic). The water was purified by apparatus Direct-Q 3 UV Water Purification System equipped with the UV lamp, purchased from Millipore (Billerica, MA, USA) with resistance established to 18 MΩ·cm −1 . 17β-estradiol, oligonucleotides and others were purchased from Sigma Aldrich (St. Louis, MO, USA) in ACS purity, unless noted otherwise. The pH was measured using pH meter WTW inoLab (Weilheim, Germany).

Oligonucleotides
Sequences used for experiments were designed as it is shown in Table 1. Table 1. Oligonucleotide sequences employed for interaction experiments.

The Hybridization Process of Single-Stranded DNA Fragments
The double-stranded DNA fragments were acquired by hybridization of two complementary single-stranded DNA oligonucleotides in ratio 1:1, with volume of 30 µL. Subsequently, 30 µL of immobilization solution (composed of 100 mM Na 2 HPO 4 + 100 mM NaH 2 PO 4 ), 0.5 M NaCl, 0.6 M guanidinium thiocyanate, 0.15 M Trizma base adjusted by HCl on pH of 7.5 was added to the solution containing oligonucleotides. The resulting solution was stirred for 40 min to obtain the final dsDNA fragments.
Carousel was heated to 37 °C by a flow thermostat Julabo F25 (Julabo, Seelbach, Germany) and the absorption characteristics of DNA representing EREs (A λmax = 260 nm) were determined during 0-120 min lasting of interaction. For the subsequent denaturation study, the same final concentrations of E 2 (0-40 nM) and DNA (200 nM) were employed. The denaturation study was carried out within the temperature ranges from 25 to 80 °C per 1.5 °C every 90 s using UV-vis spectrophotometer SPECORD S600 (Analytik Jena, Jena, Germany) by measuring of absorption maxima of DNA as A λmax = 260 nm. Denaturation analyses were carried out with E 2 /DNA mixture after 120 min of lasting interaction.

MALDI-TOF MS Analysis of E 2 /DNA Fragments Interaction
Interaction of each ERE oligonucleotide in final concentration of 50 nM with E 2 in final concentration of 20 nM was analyzed using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer Bruker Ultraflextreme (Bruker Daltonik GmbH, Germany) equipped with a laser at the wavelength of 355 nm with an accelerating voltage of 25 kV, cooled with nitrogen. Prior each measurement, the resulting complex was filtered through Amicon 3k (Millipore, Billerica, MA, USA). The matrix used for the analyses was 3-hydroxypicolinic acid (3-HPA) with addition of ammonium citrate (0.8 mg in 100 µL of water). The solutions for analyses were mixed in ratio of 1:1 (matrix/substance). After obtaining a homogeneous solution, 1 µL was applied on the target and dried under atmospheric pressure and ambient temperature (25 °C). The spectra were typically acquired from the average of 20 sub spectra from a total of 500 shots of the laser.

Descriptive Statistics
Mathematical analysis of the data and their graphical interpretation were realized by Microsoft Excel ® , Microsoft Word ® and Microsoft PowerPoint ® (Microsoft, Redmond, WA, USA). Results are expressed as mean ± standard deviation (S.D.) unless noted otherwise.

Results and Discussion
On the basis of cellular functions, DNA is the key macromolecule, which is highly protected against damages coming from various substances including those called carcinogens [30,31]. Metabolism of estrogens via catechol estrogen pathway was previously characterized as a balanced set of activating and protective enzymes. The disruption of this homeostasis with excessive production of catechol estrogen quinones leads to interactions with DNA molecules resulting in inducement of mutagenesis and subsequent initiation of breast cancer [32]. Due to the fact that the metabolic conversion of E 2 plays a crucial role in its adverse effects on organisms, there may exist other ways how the estrogen hormones (among which E 2 exhibits the largest potency [10]) affect macromolecules as the nucleic acids. Therefore, for a better understanding of the fate of DNA upon contact with E 2 , we decided to perform an in vitro interaction study using spectrophotometry and mass spectrometry.

An Optical Behavior of DNA Affected by 17β-Estradiol
Firstly, UV-vis spectrophotometry was employed to study 17β-estradiol interactions with DNA on the level of basic in vitro characteristics. To determine the interaction strength, the changes of DNA absorbance (final concentration 200 nM) at A λmax = 260 nm after interactions with E 2 (final concentrations in range 0-40 nM) were evaluated. As it is obvious from curves in Figure 2Aa-Da, shorter oligonucleotides were determined to exhibit weaker interaction in time, but 120 min were shown to be sufficient to change the optical attributes of all tested sequences of DNA. Subsequently, to better understand the obtained results, the DNA absorbance was converted into bar graphs. In these figures, the largest applied concentrations of E 2 (40 nM) were presented for whole interaction interval. The shortest chain (17-mer) was shown to interact weakly. The most significant change in optical behavior was identified after 120 min (0.37 AU compared with absorbance of control-0 nM  It can be concluded, based on the obtained results, that within the time interval of 0-120 min, the interactions are fundamentally dependent on the three factors (I) oligonucleotide length; (II) time of interaction and (III) concentration of E 2 . Particularly, the longer DNA sequences provide more accessible binding sites for hormone interaction, which then results in formation of the complex under lower threshold concentrations and moreover, a shorter time is required for its establishment. Because nucleic acids absorb strongly in the UV region (260 nm) [33], the hypochromic effect under the influence of E 2 is caused by changing of optical behavior of the complex, but not by disintegration of double-stranded structure that commonly leads to the increase of absorbance by unstacking of nitrogenous bases elevating absorbance of single-stranded DNA [34].

An Effect of 17β-Estradiol on Denaturation of Double-Stranded DNA
Differences in energy between stacked G· C and A· T Watson-Crick base pairs in DNA duplex lead to complex melting profiles, observable with optical methods, providing a high degree of precision for the evaluation of thermodynamic quantities associated with the DNA stability [35]. Therefore, we decided to determine this DNA denaturation phenomenon using UV-vis spectrophotometry. In accordance with previous spectrophotometric results, we confirmed that the length of nucleic acid is closely related to willingness of the complexes formation. As it is shown in Figure 3A, melting temperature (T m ) of the shortest DNA sequence (ERE 1 ) exhibited T m disparity at about 3.5 °C (subtraction of DNA +40 nM E 2 from DNA with no E 2 application after 120 min of lasting interaction). Despite the fact that in preliminary experiments, it was determined that 120 min of interaction are sufficient to form complex, and it was shown that the formation of the complex effect on the structure of ERE 1 with 17-mer is not seen. This phenomenon points to a higher stability of shorter sequences against E 2 and is closely related to sequence, where A· T pair is held by only two hydrogen bonds, while G· C has three, as it was described previously [36]. With increasing chain length, larger disparities between control and the highest applied E 2 concentration (40 nM) were observed. As it can be seen in Figure 3B, in 20-mer ERE 2 was observed 9 °C difference in T m . In case of 24-mer ERE 3 the difference in T m reached 15.3 °C ( Figure 3C) and finally in 38-mer ERE 4 22.3 °C difference was determined ( Figure 3D). Such a big melting abnormality is caused by a physical complexation of hormone with DNA, which leads to destabilization of the hydrogen bonds between the nitrogenous bases of DNA strands. This phenomenon further causes decrease of T m , in which the differential stability of A· T and G· C base pairs can be altered with a minimum of disturbance of the other properties of the native DNA helix [37].

MALDI-TOF Mass Spectrometry for Evaluation of E 2 -DNA Interaction
To confirm the DNA/hormone complex formation, the MALDI-TOF analyses were carried out ( Figure 4A-D). 3-hydroxypicolinic acid (3-HPA) which is commonly used as a DNA specific matrix was employed in this experiment [38][39][40]. Due to the presence of the negatively charged phosphate backbones, the adduct formation with alkali metal ions (Na + or K + ) is often observed in spectra of DNA. Hence a matrix additive represented by ammonium citrate was used to suppress undesirable cationization, whereas ammonium ions exchange with the Na + and K + ions, which are complexed with citrate [41]. The results of control analyses point to denaturation of double-stranded DNA. This phenomenon is caused by 3-HPA matrix as it was described previously, and results from protonation of bases that destabilize hydrogen bonds between two strands that partially denature a duplex upon mixing with 3-HPA [42]. Moreover, in the case of ERE 2 and ERE 3 control measurements, depurinating products, occurred as a result of heating (ionization with laser) and formed typical secondary peaks having 151 mass units (in the case of dG) less than major peak [43], which it is shown in Figure 4B,C.
It was also shown that alkali metal adducts are completely absent in DNA spectra; thus, the ammonium citrate co-matrix performs well to eliminate them. Subsequently, the interactions of DNA with 20 nM E 2 were carried out and lasted 120 min. As it is shown in lower spectra in Figure 4A-D, all oligonucleotides formed complexes shifting by app. 270 Da (Mr of E 2 ) to higher molecular masses. Moreover, the reduction of signal intensity and mass resolution occurred while increased the spectra complexity. This phenomenon is typical for oligonucleotides when forming the complexes [44]. Although both the purine and pyrimidine bases make up the structure of DNA contain nucleophilic sites suitable for reaction with carcinogens, the purines (adenine and guanine) are usually the most reactive sites for reaction [45]. Nevertheless, the metabolic conversion to reactive catechol estrogen quinones is fundamental for covalent interaction by Michael addition that results in depurinating adducts [18,46].
In organisms, the loss of these depurinating adducts generates apurinic sites and results in mutations in the H-ras oncogene by error-prone repair and subsequent mutations in H-ras protein that directs cells to grow and divide without control [47]. Moreover, the formation of covalent adducts with genotoxic carcinogens does not happen with DNA only, but also with all cellular nucleophilic species, e.g., protein, glutathione and/or water [48], but with not so profound genetic consequences as that on DNA.
Our in vitro study shows that excessive amount of E 2 exhibits a potential to form the complexes with nucleic acids, and thus affect their attributes, which result in various pathological phenomena, with no need of conversion via cytochrome P450 pathway to more reactive products. Due to the fact that E 2 is not chemically reactive, the strong complexes arise as a consequence of non-covalent physical reaction as it has been previously demonstrated by Saeed and coworkers with synthetic estrogen diethylstilbestrol (DES) [49]. Hendry and colleagues employed computational approach and they demonstrated that in partially unwound DNA there can be accommodated a variety of small molecules [50]. Estradiol was shown to be hypothetically inserted between base pair in DNA, using its hydroxyl groups at 3-and 17β-positions to form hydrogen bonds to phosphate oxygens on adjacent strands of DNA, because E 2 contains heteroatoms separated by internuclear distances similar to that of phosphate oxygens on strands of dsDNA [51]. Based on our results, it can be stated that estradiol fits well within the topography of the double-helix molecule of DNA and physical complexes that can potentially affect the physiological behavior of the DNA and may form essential precursors for covalent bonds being formed between DNA and reactive estrogen metabolites.
Because of steroidal hormone properties, estrogens may pass through the phospholipid membranes of the cell to accomplish the interactions with circulating nucleotides as miRNA and/or siRNA to disturb their physiological functions. The RNA is not complexed with histones or embedded in chromatin structure, hence nucleotides are particularly sensitive to the binding of genotoxic agents of environmental origin [52]. It is conceivable that environmental carcinogens may modify the structure of miRNAs, thus blocking their access to the catalytic pockets of Dicers and arresting the miRNA maturation process. However, in the case of E 2 complexes with these molecules, the real biological significance has to be elucidated through further, comprehensive in vivo experiments.

Conclusions
In our study, we showed that increased levels of hormone 17β-estradiol (E 2 ) exhibit the potential to form complexes directly with double-stranded DNA. Using spectrophotometry and mass spectrometry it was shown that three major factors crucially influence the interaction: (I) the complexes were observed earlier if higher E 2 concentration was used; (II) on the other hand increased time of interaction was shown to require lower concentrations of hormone to form complexes; and (III) it was revealed that longer oligonucleotide sequences form complex more readily probably due to higher content of E 2 binding sites in their sequences. The stability of longer oligonucleotides was shown to be more influenced by hormone, probably due to a higher amount of binding sites causing destabilization of hydrogen bonds in DNA strands. Finally, the formation of the complexes was revealed by mass spectrometry MALDI-TOF, where shifts by about 272.38 Da were observed, pointing to the ability of E 2 to bind the DNA and form the strong complexes as a result of non-covalent physical interaction, changing the optical and spectrometrical attributes of DNA.