Radiation induced DNA damage and its protection by a gadolinium(III) complex: Spectroscopic, molecular docking and gel electrophoretic studies

International Journal of Biological Macromolecules 127 (2019) 520–528

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Radiation induced DNA damage and its protection by a gadolinium(III) complex: Spectroscopic, molecular docking and gel electrophoretic studies Subharthi Banerjee a, Md. Selim d, Abhijit Saha b, Kalyan K. Mukherjea a,c,⁎ a

Department of Chemistry, Jadavpur University, Kolkata 700032, India UGC-DAE-CSR, Kolkata Centre, Bidhannagar, Kolkata 700098, India c Department of Chemistry, Aliah University, Newtown, Kolkata 700160, India d Department of Chemistry, Vivekananda College, Thakurpukur, Kolkata 700063, India b

a r t i c l e

i n f o

Article history: Received 8 October 2018 Received in revised form 21 December 2018 Accepted 7 January 2019 Available online 8 January 2019 Keywords: DNA radioprotection Gadolinium(III) complex Spectroscopy Molecular docking Gel electrophoresis

a b s t r a c t The current work describes the efficacy of an artificially synthesized Gd(III) complex as a potential radioprotecting molecule. The work involves utilization of spectroscopic and electrophoretic techniques to investigate the radioprotecting behavior of the Gd(III) complex. Spectroscopic studies revealed that the complex interacted strongly with DNA while molecular docking studies suggested groove binding through H-bond formation and other non-covalent interactions. The Gd(III) complex was found to impart 94% and 91% protection to irradiatively damaged DNA at radiation doses of 20 and 25 Gy respectively. The protection is believed to occur via radical scavenging mechanism and the antioxidant behavior of the complex suggested a strong radical scavenging property. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Genetic mutations arising from endogenous or exogenous causes are often the roots for the development of cancer. This unnatural and abnormal division of cells in the affected area often spreads to other parts of the body. Cancer, itself, is one of the biggest threats of the current generation, with a predicted 18.1 million new cases and 9.6 million deaths globally in 2018 [1]. Non-malignant neoplasm is treated locally by surgery but malignancy is generally dealt by means of chemotherapy (CT) and/or radiotherapy (RT), often coupled with surgery [2]. Radiotherapeutic methods are excellent in impeding the spread of malignancy in a plethora of cases, but its associated side effects and toxicities are not trivial [3–5]. In case of radiation therapy, an optimum balance must be maintained between the total dose of irradiation and the tolerance level of the surrounding normal tissues so that they are not damaged beyond repair [5]. In spite of technologically driven improvements in cancer radiotherapy, radiation related toxicities to healthy tissues in the vicinity of the area of treatment are omnipresent. The challenge is to protect the normal tissues from radiation induced injury. As a result, the role of radioprotective compounds is very ⁎ Corresponding author at: Department of Chemistry, Jadavpur University, Kolkata 700032, India. E-mail addresses: [email protected], [email protected] (K.K. Mukherjea).

https://doi.org/10.1016/j.ijbiomac.2019.01.031 0141-8130/© 2019 Elsevier B.V. All rights reserved.

important in radiotherapeutic practices. Protection of normal cells from irradiation is an emerging strategy to combat radiation induced damage. Ionizing radiation has a lethal effect on both cancerous and normal tissue cells and DNA, since it can lead to the generation of reactive oxygen species (ROS) through the ionization of cellular cytoplasmic medium. ROS can lead to several complications and DNA damage via multiple pathways, including, but not limited to, single strand breaks (SSBs), double strand breaks (DSBs), apurinic/apyrimidinic sites (AP), etc., the end results of which are often mutagenesis and oncogenesis [6–9]. Therefore, compounds with radical scavenging capabilities have attracted considerable attention for protection of DNA against environmental genotoxins [10–12]. Therapeutic radiation induced toxicities can be reduced to a great extent with technological improvements in radiation delivery and also by the improvements in chemical radioprotectors [13]. The use of radiation protectors prior to or immediately after irradiation is an effective way to combat radiation induced damage to normal tissues. These radioprotective agents are typically natural products or some synthetic compounds that are administrated before irradiation to reduce injuries caused by ionizing radiation. The development of metal based radioprotectors could be of considerable significance, but the reports of metal based radioprotectors in literature are quite rare [14]. Gd(III), a highly paramagnetic entity with seven unpaired electrons, has a very strong hydrogen-proton spin-lattice relaxation effect. Hence, the compounds of it are routinely used in a number of clinical

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applications such as in Magnetic Resonance Imaging (MRI) as a contrast agents (CAs) [15] and in NMR studies as a paramagnetic proton relaxation probes [16,17]. The paramagnetic Gd(III) ion, chelated with diethylenetriamine pentaacetic acid (DTPA), forms a strong paramagnetic stable complex, that has been reported to reduce proton relaxation times even in very low concentrations [18]. Gd(III) based contrast agents (GBCAs) are in vogue in biomedicine. The utility of Gd (III) in several biotechnological and biochemical work has prompted the present study of developing a Gd (III) complex and explore its potentiality as a lanthanide based artificial radioprotector molecule. 2. Materials and methods 2.1. Materials Gadolinium nitrate hexahydrate, Gd(NO3)3·6H2O (extra-pure), used as the starting material for the synthesis, was obtained from Aldrich. 1,10-Phenanthroline and ammonium thiocyanate were obtained from Merck, India and Sisco Research Laboratory (SRL), India respectively and used without further purification. All other chemicals were obtained from Merck (India). The analytical grade solvents used for physico-chemical studies were further purified by literature method before use [19], wherever necessary. CTDNA and supercoiled plasmid pUC19 DNA were obtained from Sigma Chemical Company, USA, and Genei Bangalore, India, respectively. Triple distilled water was used for the preparation of buffer solutions as and when necessary and for all spectrochemical investigations involving DNA.

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other procedures for titrations were similar to that outlined above for spectrophotometric titrations. Fluorescence lifetime measurements were recorded on a Horiba-Jobin-Yvon FluoroCube emission lifetime system using time-correlated single photon counting (TCSPC). Nano LED (IBH, UK) was used as the excitation source at 280 nm and TBX photon detection module as the detector. The decays were analyzed with IBH DAS-6 decay analysis software. 2.5. Competitive dye displacement assay The nature of the binding mode of the Gd(III) complex with DNA was determined through a competitive dye displacement assay using ethidium bromide (EB) as the fluorescent probe. Emission intensities of EB (2 × 10−5 M) in DNA (10−5 M) were monitored at 600 nm, after excitation at 515 nm, with the gradual addition of incremental amounts of the Gd(III) complex solution (0.1–1.0 × 10−5 M). 2.6. Viscometric studies The viscosity of sonicated DNA and DNA-Gd(III) complex were measured by a fabricated micro-viscometer maintained at 28 °C in a thermostatic water bath. A graph of (η/ηo)1/3 vs. [Gd complex]/[DNA] concentration ratio was plotted (η and ηo = viscosities of the DNA solutions in the presence and absence of complex respectively). The viscosity value (η = t − t0) was calculated from the observed flow time of DNA solutions (t) and was corrected for the buffer solution (t0). 2.7. Circular dichroism (CD) spectroscopic study

2.2. Preparation of the complex The synthesis, characterization and X-ray crystallographic structural details of the synthesized Gd(III) complex, (PhenH) [Gd(Phen)2(SCN) 4], has been reported by us earlier [20].

The CD measurements were performed in a JASCO J-815 CD spectrometer with a path length of 1 cm in each case. A 10−5 M DNA solution was titrated against increasing amounts of the Gd(III) complex (0.2–0.8 × 10−5 M), keeping the total volume same in all of the test solutions. The spectra were recorded in the region of 225–300 nm.

2.3. UV–vis spectral study 2.8. Molecular docking study Perkin Elmer Lambda UV–vis spectrophotometer was used to monitor the spectrophotometric interaction of the synthesized Gd(III) complex with DNA. The absorbances at λmax of a 10−5 M solution of the complex (264 nm) was followed in the presence of increasing amounts of DNA (4.0–20.0 × 10−5 M) and was used to calculate the interaction binding constant. For these measurements, a stock solution of DNA of identical concentration as in the Gd complex-DNA mixture, was taken as the reference solution to eliminate the intrinsic absorption of DNA itself. The following equation was used for the quantitative evaluation of the binding constant for the interaction (Eq. (1)) [21]:

here, [DNA] is the concentration of DNA in base pairs. εa, εf and εb correspond to Aobsd / [complex], the extinction coefficient for the free complex and the extinction coefficient for the complex in the fully bound form respectively. Plots of [DNA] / (εa − εf) vs. [DNA] gave a slope of 1 / (εb − εf) with a Y-intercept of 1 / [Kb (εb − εf)]. The intrinsic binding constant Kb was obtained from the ratio of slope to the intercept [22].

Rigid molecular docking was performed with HEX 8.0.0 Protein Docking Software (http://hexserver.loria.fr). HEX can display the possible binding modes between a ligand and a receptor molecule. The crystal structure of the B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) was downloaded from the Protein Data Bank (http://www.rcsb.org./pdb) and was further processed with UCSF Chimera (http://www.rbvi.ucsf.edu/chimera) to remove the water molecules which might interfere with the docking calculations. The coordinates of the Gd(III) complex were taken from its crystal structure (.cif file) and was converted to the PDB format (.pdb) using Mercury software (http://www.ccdc.cam.ac.uk/). Docking was performed with the default three dimensional parameters of the program. PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC (http://pymol. sourceforget.net/) software and Biovia Discovery Studio (Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systèmes, 2016) were used for visualization of the docked pose. All the calculations were run on an AMD FX 6300 6 core CPU system powered with an Nvidia GTX 1050Ti 4 Gb GPU running Windows 10 Pro 64-bit version [23].

2.4. Fluorescence emission study

2.9. Emission spectroscopic assessment of DNA damage and its protection

A solution of the Gd(III) complex, gave emission maxima at 361 nm, when excited at 280 nm. Emission intensity measurements were carried out using a PTI Model QM-40 spectrofluorometer. Fluorescence titration experiments were performed with a fixed concentration (10−5 M) of the complex against increasing concentrations of DNA (0.2–8.0 × 10−5 M). For all measurements, the samples were excited at 280 nm and emission data were recorded from 320 to 450 nm. All

For this study, the DNA solutions were irradiated with gamma radiation from a GC-900 Gamma Chamber fitted with a Co60 radiation source. Sample solutions of DNA pretreated with either the Gd(III) complex or the ligands (Phen and NH4SCN) were incubated at 37 °C for 30 min and were subjected to gamma irradiation (Co60 γ-chamber, dose rate: 42.01 Gy min−1) for a total dose of 3.024 kGy. Irradiation dose rates of 20 and 25 Gy were used for plasmid DNA samples. The

½DNA=ðεa −ε f Þ ¼ ½DNA=ðεb −ε f Þ þ 1=½Kb ðεb −ε f Þ

ð1Þ

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emission intensities were measured with a Perkin Elmer LS-55 spectrofluorometer. Radiation induced DNA damage was monitored spectrofluorometrically using ethidium bromide (EB) as a probe in PBS buffer (pH 7.2). Emission intensity at 600 nm was monitored (λexcitation = 515 nm), after allowing the irradiated DNA solutions (DNA along with either Gd(III) complex or a ligand; 60Co-γ source dose rate: 42.01 Gy min−1 for 72 min) to interact with EB. The data obtained experimentally were plotted as (I − Ia) / (I0 − Ia) versus dose (Ia = fluorescence intensity of EB, I0 = fluorescence intensity of non-irradiated EB-DNA control, I = fluorescence intensity of irradiated EB-DNA sample). From the graph the corresponding dose-response relationship was obtained. 2.10. Gel electrophoretic assessment of DNA damage and its protection Agarose gel electrophoresis was used to assess the ability of the synthesized Gd(III) complex and its component ligands to protect DNA from radiation induced damage. This was done by carrying out gel electrophoresis of the samples (total volume: 15 μl, composition: pUC19 DNA (0.5 μg), the Gd(III) complex or ligands (0–2 mM), TrisHCl buffer) after incubation for 30 min followed by exposure to radiation doses of 20 and 25 Gy. Electrophoresis was carried out on a 0.9% agarose gel at 80 V for 3 h in TAE buffer (Tris base, acetic acid, and 1 mM EDTA, pH 7.2). 1.0 μg ml−1 ethidium bromide solution was used for staining the gel followed by visualization in UV light with the UVP BIO-DOC-IT Gel Documentation System. UVP-BIO-DOC-IT LS Software was used for quantitative assessments of damage induced and the corresponding protection imparted by analyzing the intensities of bands in the electrophoregram. 2.11. Radical scavenging by the Gd(III) complex DPPH or 2,2-diphenyl-1-picrylhydrazyl is a stable nitrogen centered free radical with applications in chemistry and biochemistry, mostly as a tool for assessing the antioxidant properties of a potential radical scavenger [24]. DPPH shows characteristic strong absorption maxima at 517 nm. The molecule is relatively more stable than other free radicals due to the delocalization of the electron over the entire system. The absorption of DPPH decreases if this odd electron is coupled in presence of a radical scavenger and consequently DPPH is reduced to DPPHH. For assessing the scavenging potential of the Gd(III) complex, different concentrations of it were added to a sample of freshly prepared DPPH in methanol, followed by incubation of the solutions at 37 °C for 30 min in the dark and the corresponding absorption spectra were recorded in a Shimadzu UV 1700 spectrophotometer. The scavenging percentage was calculated using Eq. (2) [10]. Scavenging Activity ð%Þ ¼ ðA0 −Ai =A0 Þ  100%

Fig. 1. The crystal structure of the anionic part of the complex (H atoms omitted for clarity).

us to understand the mechanism of action of the complex and its typical binding nature [25–29]. The absorption spectra of the Gd(III) complex in the presence and absence of DNA are shown in Fig. 2. Binding of the complex with DNA is observed as a modification of its UV absorption band in the presence of incremental amounts of DNA. In the UV region the Gd (III) complex exhibited a band at 264 nm, which can be attributed to the π → π* transition of the coordinated phenanthroline ligand. Addition of incremental amounts of DNA (4.0–20.0 × 10−5 M) to the complex solution (10−5 M) caused this ligand-based charge transfer band at 264 nm to exhibit hyperchromism (with no red shift). Electrostatic binding of a complex with DNA often results in the hyperchromism of the observed spectra [30]. Hyperchromism is generally ascribed to an intimate association of the complex with DNA, where the complex binds non-covalently to the DNA duplex [31–34]. These

ð2Þ

here, A0 = Absorbance in absence of test compound and Ai = Absorbance in presence of test compound. 3. Results and discussion 3.1. Synthesis and structural characterization The synthesis, characterization and crystal structure of the compound (PhenH) [Gd(Phen)2(SCN)4] (monoclinic P21/n space group) have been previously reported by us (CCDC 923442) [20]. The crystal structure of the anionic part of the complex is presented in Fig. 1. 3.2. DNA binding study by absorption spectrophotometry The binding modes of a metal complex with DNA is generally ascertained with the help of electronic absorption spectroscopy and the relative binding modes reveal valuable information which enables

Fig. 2. Absorption spectra of the Gd complex (10−5 M) in the presence of incremental amounts of DNA (4.0–20.0 × 10−5 M). Inset: Plot of [DNA] / ((εa − εf) × 1010 M/M−1 cm−1) vs. [DNA] × 105 M.

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interactions predominantly occur via electrostatic and van der Waals attractions and hydrogen bonds. The DNA double helix possesses many hydrogen bonding sites which are accessible both through the minor and the major grooves. Groove binding is often associated with such typical non-covalent interactions, and unlike intercalation, there is no stacking interaction between the complex and the DNA base pairs, and the changes in the double helical structure of DNA are relatively minor. Grove binding interactions are intermolecular and reversible. In order to quantitatively evaluate the binding strength of the complexes with DNA, the intrinsic binding constant (Kb) value was calculated following Eq. (1) and was found to be 2.4 × 103 M−1. The value of the binding constant indicated a strong interaction between the Gd(III) complex and DNA, although the order of binding was not as high as generally reported for typical intercalating molecules [35]. Thus, it could be envisaged that the Gd(III) complex possibly interacted with DNA via hydrogen bonds and other non-covalent interactions and behaved as a typical groove binder, which may have contributed to the observed hyperchromism in its absorption spectra and relatively lower order value of the binding constant as compared to classical intercalators. The Kb value of the DNA-complex system confirmed the nature of binding as suggested qualitatively from the change of intensity of the UV absorption bands. To further verify the nature of binding, the interaction of the Gd(III) complex with DNA, was also studied by monitoring the spectral changes of DNA (10−5 M) itself with increasing amounts of the complex (0.2–1.0 × 10−5 M) and is represented in Fig. S1 (ESI). The changes in the spectral characteristics of DNA on interaction with a small molecule provide a suitable platform for deducing the mode of interaction [36]. The absorption maximum of DNA, around 259 nm, is due to the electronic transitions in the chromophoric groups in purine and pyrimidine bases [37]. The binding of a small molecule with DNA through intercalation results in a hypochromic effect on the DNA absorption spectrum with a concomitant red shift [38]. Groove binding molecules generally cause a hyperchromic effect on the DNA spectral band with negligible or no red shift [39]. Addition of increasing amounts of the Gd(III) complex to DNA resulted in a hyperchromic effect on the DNA absorption band at 259 nm with negligible red shift (b2 nm). This clearly suggested that the Gd(III) complex interacted with DNA and the possible mode of binding was via the groove. 3.3. Fluorescence emission study for DNA interaction A quenching in the emission intensity of the Gd(III) complex without any shift in wavelength was observed on addition of incremental amounts of DNA (Fig. 3), thereby attaining a saturation at high DNA concentration. The data were analyzed using the Stern-Volmer Eq. (3) [40] after necessary corrections for inner filter effect: F0 =F ¼ 1 þ KSV ½Q 

ð3Þ

where, F0 is the emission intensity of the Gd complex alone, F is the emission intensity of the Gd complex-DNA adduct at different concentrations [Q] of DNA and KSV is the Stern-Volmer quenching constant. The results were plotted as F0/F vs. [Q] at a pre-fixed intercept of 1.0, and from the slope of the corresponding line, the value of KSV was evaluated. The Stern-Volmer plot of the interaction of the Gd(III) complex with DNA was linear with a fluorescence quenching constant (KSV) value of 4.4 × 103 M−1. Fluorescence emission of a complex bound to DNA provides valuable information on the respective mode of binding. Classical intercalators are stacked in-between the nitrogenous bases of DNA and are in a relatively hydrophobic environment. Compounds which associate with DNA through non-covalent interactions in the groove are more exposed to the surrounding hydrophilic medium and are nearer to the surface of the DNA double helix than intercalators. Consequently, there is a decrease in the intensity of the emission spectrum of the DNA bound complex due to non-radiative loss of emissive

523

Fig. 3. Emission spectra of (a) Gd complex only (10−5 M), (b)–(z) Gd complex (10−5 M) + DNA (0.2–8.0) × 10−5 M. Inset: Stern-Volmer plot for the interaction of Gd complex (10−5 M) with incremental amounts of DNA (0.2–8.0 × 10−5 M).

energy on account of the surrounding solvent medium. A variety of molecular interactions can lead to a reduction of emission intensity of a fluorophore, in this case, the Gd(III) complex, such as excited state reactions, molecular rearrangements and energy transfer and groundstate complex formation [41]. From the linear nature of the SternVolmer plot it could be suggested that static quenching was the only mechanism of quenching under operation in this case due to the formation of a Gd complex-DNA adduct in the ground state [42]. To further verify the static nature of quenching, lifetime measurements were carried out and the decay pattern of the Gd(III) complex under the addition of incremental amounts of DNA have been represented in Fig. S2 in ESI. It was found that the decay was mono exponential and the order of the lifetimes of the Gd(III) complex fluorophore solutions remained unchanged (Table T1 in ESI) in presence of increasing DNA concentrations (of the same order as used in the quenching experiment), which was indicative of purely the static mechanism of quenching as discussed from the emission titrations. The binding constant of the interaction between the Gd complex and DNA was also evaluated from the quenching experiment using Modified Stern-Volmer Eq. (4) [43] and is represented in Fig. S3 in ESI. log ½ð F0 − FÞ=F ¼ log K þ n log ½Q 

ð4Þ

where, n is the number of binding sites and K is the binding constant. The binding constant K for the interaction was obtained from the intercept of the graph log [(F0 − F) / F] vs. log [Q], and was found to be 4.5 × 103 M−1. The order of the binding constant obtained from emission spectroscopic data was in good agreement with that obtained from absorption spectroscopic studies. 3.4. Competitive EB binding experiments Dye displacement assays or competitive binding studies can corroborate whether a small molecule is capable of competing with a classical intercalator molecule to introduce itself within the DNA base pairs [44]. Competitive binding experiment, with EB as a probe, was used to study the nature of the interaction between the Gd(III) complex and DNA and is represented in Fig. S4 in ESI. If a molecule is capable of intercalation, it will compete with EB for the same and in the process, some EB, already intercalated between DNA base pairs, will be displaced to the surrounding hydrophilic zone, resulting in the loss of its emission intensity. It was found that increase in concentration of the Gd complex in a EB-DNA mixture, resulted in no change in the fluorescence intensity of EB. In other words, the Gd(III) complex, itself, was unable to displace any

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Fig. 4. Structural surface of B-DNA dodecamer and the docked pose of the Gd(III) complex.

intercalated EB from DNA, thus establishing that it did not interact with DNA in an intercalative manner. 3.5. Viscometric studies for assessing the DNA binding pattern Viscometric study is probably the least ambiguous and simplest of tests that indicate the nature of binding of a complex with DNA with acceptable accuracy. If a ligand binds to DNA through intercalation, it results in the lengthening of the DNA helix, which in turn increases the viscosity of DNA [42]. The values of relative specific viscosities of DNA in the presence and absence of the Gd(III) complex were plotted against [complex]/[DNA] concentration ratios. The results of the viscometric studies are presented in Fig. S5 in ESI. It was observed that the addition of the complex to the DNA solution could not induce any significant change in the intrinsic viscosity of DNA, clearly demonstrating the non-intercalative nature of binding by the present complex, which, in turn, indirectly supports the proposition of groove binding.

3.7. Molecular docking study for DNA interaction The structure of B-DNA dodecamer d(CGCGAATTCGCG)2 and the docked pose of the Gd complex with DNA are represented in Fig. 4 (and Fig. S7 in ESI). Molecular docking is an attractive technique to understand the drug-DNA interactions and to establish the mechanism of action of the reactants with DNA. Different structural properties lead to different binding modes; in fact, one of the most important factors governing the binding mode is the molecular shape [47]. The docked pose of the Gd(III) complex reveals that it is a predominant groove binder, interacting in the GC rich region of the B-DNA dodecamer and

3.6. CD spectral study The typical CD spectrum of DNA is perturbed on interaction with other molecules, mainly from the deformations in its secondary structure, and from the changes in the spectrum, the nature of binding can be estimated to a considerable degree [45]. Molecules intercalating within the DNA base pairs can significantly alter the CD spectrum of native DNA, while molecules interacting mainly as grove binders, through non-covalent and other electrostatic interactions, have a negligible effect on the CD spectrum. The CD spectrum of native DNA, represented in Fig. S6 in ESI, showed distinctive characteristics of its B form, with a positive peak at 275 nm, arising due to base stacking, and a negative peak at 244 nm, attributed to its classical right handed helicity [46]. With the increase in concentration of the Gd(III) complex, the CD spectrum of DNA showed no appreciable change in its positive band and a negligible decrease was observed in its negative peak. This clearly ruled out the possibility of intercalation of the Gd complex between the DNA base pairs, and is suggestive of the inferences of absorption spectroscopic studies, indicating that the complex is possibly interacting with DNA at its grove.

Fig. 5. Fluorescence emission spectra of EB-DNA in presence of increasing amounts of Gd (III) complex after 72 min of gamma irradiation; (a) Emission spectrum of only EB at 20.0 μM; (b) Emission spectrum of EB (20.0 μM) in presence of DNA (10.0 μM) (unirradiated); (c–m) Emission spectra of EB in presence of irradiated DNA pretreated with increasing Gd (III) complex concentrations [Gd complex]/[DNA]: 0–1 (irradiated).

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Fig. 8. Protection of plasmid pUC19 DNA at 20 Gy with different doses of the Gd(III) complex [0–2 mM] and ligands [0–2 mM] on gamma-radiation induced strand breaks.

3.8. Estimation of the radiation induced DNA damage and its protection

Fig. 6. Fluorescence emission spectra of EB-DNA in presence of increasing amounts of Phen after 72 min of gamma irradiation; (a) Emission spectrum of only EB at 20.0 μM; (b) Emission spectrum of EB (20.0 μM) in presence of DNA (10.0 μM) (unirradiated); (c–m) Emission spectra of EB in presence of irradiated DNA pretreated with increasing Phen concentrations [Phen]/[DNA]: 0–1 (irradiated).

the best pose was selected with the lowest Etotal value of −321. The ligand phenanthroline rings were projected outside and the system was stabilized by 2 H bonded interactions between 2 \\SCN nitrogens and a free amine H from a guanine residue (G4), which was not involved in the GC H-bonded linkage, and by 1 H bond between a\\SCN nitrogen atom and a H from the arene residue of the same guanine moiety (Fig. S7 in ESI). The docked pose of the Gd(III) complex with DNA also involved 4 other non-covalent π interactions, involving the sulphur atoms of the coordinated\\SCN groups and the corresponding nitrogen containing heterocycles of the base residues G4, A5 and G22. The energetically favorable docked pose confirmed the results from absorption spectroscopy and the negative energy value of the pose indicated the stability of the interaction. The Gd(III) complex interacted with DNA at the groove and was mainly stabilized by non-covalent electrostatic interactions like H-bonding.

Fig. 7. Fluorescence emission spectra of EB-DNA in presence of increasing amounts of NH4SCN after 72 min of gamma irradiation; (a) Emission spectrum of only EB at 20.0 μM; (b) Emission spectrum of EB (20.0 μM) in presence of DNA (10.0 μM) (unirradiated); (c–m) Emission spectra of EB in presence of irradiated DNA pretreated with increasing NH4SCN concentrations [NH4SCN]/[DNA]: 0–1 (irradiated).

An assessment of the protecting ability of the synthesized Gd(III) complex and its constituent ligands was carried out spectrofluorometrically using ethidium bromide (EB) as a probe. EB has a feeble emission in aqueous medium which on intercalation in between the flat adjacent DNA base pairs produces abrupt increase in the emission intensity of the molecule as it moves into a relatively hydrophobic environment [48]. If this EB-treated DNA is subjected to the exposure of gamma radiation, the subsequent strand breaks forces some of the EB to move from the hydrophobic intercalation zone to an aqueous zone on account of the collapse of the DNA double helix in the vicinity. This results in a decrease in emission intensity of EB. This quenching increases gradually with the increase in radiation dose suggesting that the DNA damage varies proportionally to the dose of radiation. DNA solutions in appropriate buffer medium, pretreated with different concentrations of either the Gd(III) complex or the ligands individually, were subjected to gamma radiation, followed by treatment with identical EB concentrations in all cases and subsequent measurement of emission intensities. The fluorescence intensity of EB gradually increased on addition of increasing amounts of the Gd(III) complex (0.5 μM to 10 μM) or ligands (0.5 μM to 10 μM) to a EB-DNA solution before irradiation (Figs. 5–7). This, along with a plot of (I − Ia) / (I0 − Ia) versus [radioprotector] / [CT-DNA] (Fig. S8 in ESI) indicated the extent of radioprotection of DNA imparted by the Gd(III) complex (81%), phen (73%) and NH4SCN (45%). The protection from radiation induced DNA damage was pronounced in the case of the Gd(III) complex compared to the ligands Phen and NH4SCN. Free radicals are one of the most important causes for the damage of radiated biological macromolecules and eliminating them can be a good method to control the effect of radiation. Radical scavengers can thus prove to be very effective in protecting biological materials from radiation induced damage. The Gd (III) complex is a better radio protector than any of the ligands because it has a greater oxidizing potential and higher ability to donate hydrogen atoms or electrons and eliminate free radicals by reacting with them or terminating their chain reactions [49].

Fig. 9. Protection of plasmid pUC19 DNA at 25 Gy with different doses of the Gd(III) complex [0–2 mM] and ligands [0–2 mM] on gamma-radiation induced strand breaks.

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Table 1 Extent of DNA SC pUC19 protection at 20 Gy. Lane no.

Reaction condition

Form I (% SC)

Form II (% NC)

1 2 3 4 5 6 7 8

DNA control (no radiation) DNA irradiated DNA + 1 mM Gd(III) complex DNA + 2 mM Gd(III) complex DNA + 1 mM Phen DNA + 2 mM Phen DNA + 1 mM NH4SCN DNA + 2 mM NH4SCN

98 60 88 92 70 72 64 65

2 40 12 8 30 28 36 35

3.9. Estimation of the protection from DNA damage by gel electrophoresis technique

concomitant decrease in the absorption. The radical scavenging was monitored at 517 nm with different concentrations of the Gd(III) complex and is represented in Fig. S9 in ESI. The inhibitory concentration (IC50) for the Gd(III) complex was obtained from the scatter plot of percentage inhibition vs. complex concentration, followed by calculating the value of x at y = 50% from the trend line. The ability of the Gd(III) complex to scavenge the free radical DPPH was found to be slightly higher than the corresponding standard antioxidant. The IC50 value for the standard antioxidant vitamin C is about 0.9 × 10−3 M, while the IC50 values for the corresponding Gd(III) complex was found to be 1.4 × 10−4 M, which clearly indicated that the Gd(III) complex was a potent radical scavenger and could act as a radioprotecting molecule by eliminating radicals generated by radiolysis. 4. Conclusion

Exposure of the plasmid DNA to gamma-radiation at different doses can cause single strand breaks resulting in relaxation of plasmid DNA from a supercoiled (SC) form to a nicked coil (NC) form [50]. When pUC19 DNA was subjected to gamma-radiation of different doses (20 Gy and 25 Gy), 40% and 50% of the SC form of plasmid DNA got converted into open circular form respectively (Figs. 8 and 9, Tables 1 and 2). The DNA damage was significantly lowered on pretreatment of it with the Gd(III) complex or the ligands in appropriate concentrations. In such cases, a higher percent of SC DNA (Form I) was observed compared to the NC form (Form II). At a concentration of 2 mM, the Gd(III) complex and the ligands (Phen and NH4SCN) preserved the native SC DNA form to the extent of 92%, 72% and 65% respectively at a dose of 20 Gy and 89%, 70% and 62% respectively at a dose of 25 Gy. The corresponding radiation protection imparted by the Gd(III) complex was superior with respect to the ligands and was measured to show around 94% and 91% protection to plasmid DNA at 20 and 25 Gy respectively. Under the same conditions, Phen imparted 73% and 71% protection, while NH4SCN showed still lesser protection at 66% and 63%. Since radicals are a major cause of DNA damage, it may be suggested that the synthesized complex and ligands have radical scavenging capabilities, thereby possessing the ability to protect DNA from radiation induced strand breaks. 3.10. Assessment of radical scavenging activity using absorption spectroscopy Molecules which are generally employed as potential radioprotectors show significant antioxidant properties by scavenging free radicals in the system generated from ionizing radiation. Amifostine, the FDA approved cytoprotector, also works on that principle, by producing a radical scavenging free thiol unit in the cellular medium [51]. The free radical scavenging property of the Gd(III) complex was investigated by electronic absorbance spectroscopy using DPPH as a stable free radical. Radical scavenging assay using DPPH is a potent and widely accepted technique to ascertain the antioxidant behavior of a new molecule or drug [52]. The gradual removal of DPPH free radical from a system can be correlated with the change of color of the DPPH medium from purple to pale yellow. As more and more radicals are removed from the solution, only a pale yellow color persists due to the picryl group in the system with a

The phenanthroline and thiocyanate derived gadolinium (III) complex, (PhenH) [Gd(Phen)2(SCN)4], had been previously designed, synthesized and structurally characterized by us using single crystal X-ray diffraction. The current communication shows that the complex is an efficient DNA grove binder through external non-covalent interactions. DNA binding studies using absorption spectroscopy showed that the intrinsic binding constants for the Gd(III) complex was 2.4 × 103 M−1. Furthermore, the Stern-Volmer quenching constant, KSV, was evaluated to be 4.4 × 103 M−1. Viscometric study conclusively proved that the complex did not behave like a classical intercalator. Molecular docking studies performed on the complex verified the inferences obtained from other techniques and showed that the Gd(III) complex interacted with DNA predominantly at its groove and is stabilized by non-covalent interactions. The Gd(III) complex and the ligands, Phen and NH4SCN, exhibited effective protecting ability against radiation induced DNA damage. Fluorometric assessment revealed that 81% of the damaged DNA was revived when the concentration of the Gd(III) complex was about 10 μM, whereas the ligands Phen and NH4SCN imparted lesser protection to damaged DNA than the complex. Gel electrophoresis study with plasmid DNA indicated that the Gd(III) complex showed a superior protection in comparison to the individual ligands and the protective ability was estimated to be 94% and 91% respectively at 20 and 25 Gy. The DPPH radical scavenging study showed that the Gd(III) complex was a potent radical scavenger and probably eliminated the radicals generated during radiolysis by adduct formation. Thus, the present study achieves the development of a lanthanide based complex as a potential radioprotecting agent. Acknowledgements Authors are thankful to the UGC-DAE-CSR-KC for funding in the form of a Major Research Project to KKM [UGC-DAE-CSR-KC/CRS/13/ RC05/0888] where SB is a project fellow. The authors extend their thanks to Dr. A. Dutta of UGC-DAE-CSR-KC for assisting in running the GC-900 gamma radiation equipment. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.01.031.

Table 2 Extent of DNA SC pUC19 protection at 25 Gy. Lane no.

Reaction condition

Form I (% SC)

Form II (% NC)

1 2 3 4 5 6 7 8

DNA control (no radiation) DNA irradiated DNA + 1 mM Gd(III) complex DNA + 2 mM Gd(III) complex DNA + 1 mM Phen DNA + 2 mM Phen DNA + 1 mM NH4SCN DNA + 2 mM NH4SCN

98 50 84 89 66 70 60 62

2 50 16 11 34 30 40 38

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