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Keywords:

  • DNA binding;
  • docking;
  • intercalation;
  • naproxen;
  • ROS

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Naproxen is an important non-steroidal anti-inflammatory drug with many pharmacological and biological properties. In this study, we have attempted to ascertain the mode of action and the mechanism of binding of naproxen to DNA. We have also demonstrated that, upon irradiation with white light, naproxen generates reactive oxygen species, causing DNA cleavage. Generation of reactive oxygen species from photo-irradiated naproxen as determined spectrophotometrically was found to lead to nicking of plasmid DNA as analyzed by agarose gel electrophoresis. Without photo-irradiation, naproxen binds to DNA and forms drug–DNA complexes as revealed by spectroscopic techniques. Several experiments such as determination of the effect of urea, iodide-induced quenching and a competitive binding assay with ethidium bromide showed that naproxen binds to DNA primarily in an intercalative manner. These observations were further supported by CD analysis, viscosity measurements and molecular docking. Using DNA as a template, fluorescence resonance energy transfer between naproxen and ethidium bromide was also observed, further strengthening the evidence for intercalative binding of naproxen with DNA.


Abbreviations
ctDNA

calf thymus DNA

EtBr

ethidium bromide

FRET

Förster resonance energy transfer

NBT

nitroblue tetrazolium

NSAIDs

non-steroidal anti-inflammatory drug

ROS

reactive oxygen species

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of rheumatic and arthritic diseases [1]. Naproxen [2-(6-methoxynaphthalen-2-yl) propanoic acid, Fig. 1A] belongs to this class, and its pharmacological activity primarily comprises inhibition of cyclo-oxygenase (COX). A general hallmark of most NSAIDs is the generation of reactive oxygen species (ROS) in the presence of light, and naproxen is a well-known photosensitizer that is able to generate ROS upon illumination [2].

image

Figure 1. (A) Structure of the naproxen (+ isomer) sodium. (B) Agarose gel electrophoresis of EtBr-stained pBR322 DNA after treatment with naproxen. Lane A is the ‘control’, which contains only plasmid DNA. The concentrations of naproxen in lanes B–F were 100, 150, 200, 250 and 300 μm, respectively. The arrows labelled ‘OC’, ‘L’ and ‘SC’ indicate the relaxed, linear and supercoiled forms of plasmid DNA.

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Among the NSAIDs, compounds containing arylpropionic acid (naproxen) in their chemical structure behave as photosensitizers and produce biomolecule modifications that are thought to be responsible for the occurrence of photo-induced effects [3, 4]. Drug–DNA photoreaction results in undesired effects, and DNA molecules are susceptible to damage caused by the absorption of light by photosensitizing molecules [5, 6].

It is well-established that ROS react with nuclear DNA, resulting in breakage of the DNA strand. The DNA strand breakage was found to be facilitated by a non-covalent drug–DNA interaction, induced by both electron and energy transfer to DNA, as recently suggested for some NSAIDs [7, 8]. In this context, the primary event is absorption of photons of the appropriate wavelength, which allows the chromophore to reach an excited state. The excitation energy is then transferred to oxygen molecules, generating ROS such as superoxide and hydroxyl radicals [9, 10]. These generated ROS result in local oxidative stress, causing breakage in genomic DNA and oxidative modifications in proteins and lipids within cell membranes [11]. In order to avoid the undesirable effects of photoreactive compounds, efforts have been made to establish a model system for estimation of photosensitive potential through analytical and biochemical methods [12].

Interaction studies of drugs with DNA are useful for understanding of the reaction mechanism as well as to provide guidance for the application and design of new drugs. Binding studies of drugs with DNA have been performed in order to develop design principles for targeting of specific DNA sequences in order to control gene expression [13]. Important modes of drug–DNA interactions included (a) electrostatic interactions between the negatively charged phosphate groups of DNA and ionic portion of the molecules, (b) binding of molecules to the major or minor grooves, involving van der Waals interactions, and (c) intercalation of the molecules with the base pairs of nucleic acid [14, 15]. Intercalation describes the formation of a ‘molecular sandwich’ between an incoming planar or flat molecule and two layers of stacked nitrogenous bases. This is the most effective mode of interaction for drugs targeting DNA. There are a large number of molecules that interact with DNA, and the factors responsible for such interactions are quite complex [16].

The availability of the genome sequence, the well-studied three-dimensional structure of DNA, and the predictability of accessible chemical and functional groups make DNA as attractive drug target. However, the number of known drugs that target DNA is still very limited compared to the drugs that target proteins [17]. We wished to investigate the interaction of naproxen with DNA in detail. The ultimate objective was to determine the mode of action and mechanism of binding of naproxen with DNA in the presence and absence of light. Moreover, the photosensitizing action of naproxen in formation of strand breaks in the double-stranded supercoiled pBR322 plasmid was also investigated. Our results clearly demonstrate that naproxen binds to DNA primarily in an intercalative manner. Such interaction is also supported by the results of biophysical experiments such as determination of the effect of urea, iodide-induced quenching and displacement of ethidium bromide (EtBr). Our results also suggest that Förster resonance energy transfer (FRET) takes place between naproxen and EtBr in the presence of DNA, providing an opportunity to use such systems in optoelectronic devices and for detection of DNA.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Interaction of photo-irradiated naproxen with DNA: ROS generation assay

In order to demonstrate the photocleavage activity of naproxen, a reaction mixture containing naproxen and plasmid DNA pBR322 was irradiated for 2 h at 37 °C. Significant DNA damage was caused by naproxen after irradiation with white light. Exposure of plasmid pBR322 to increasing concentrations of naproxen in the presence of light caused conformational changes from a supercoiled to an open circular form. As shown in Fig. 1B, the increase in the intensity of the band representing the open circular form and appearance of a DNA band corresponding to linear DNA indicates plasmid DNA strand break activity. Naproxen did not promote DNA strand breaks in the absence of irradiation [6]. It is well documented that ROS such as singlet oxygen and superoxide act as major toxic mediators in drug-induced phototoxic cascades [18].

There are at least three direct mechanisms by which photo-excited molecules can damage DNA. First, photo induced molecules cause formation of photo-adducts by damaging DNA directly through covalent binding. Second, an excited molecule may transfer the excitation energy to DNA, leading to production of a pyrimidine dimer. Last, DNA damage in the form of oxidative modification of guanine may occur due to abstraction of an electron or a hydrogen atom by a photo-excited chromophore. In addition to these direct mechanisms, there are at least two indirect mechanisms of DNA damage by excited photoreactive chemicals, which include (a) reactive oxygen-mediated DNA damage, and (b) production of reactive decomposition products [19]. In a nitroblue tetrazolium (NBT) assay, naproxen produced superoxide anions upon induction with white light; these superoxide anions reduce NBT via a one-electron transfer reaction, producing partially reduced monoformazan (NBT+) as a stable intermediate. Formation of monoformazan was recorded spectrophotometrically at 560 nm (Fig. 2A). In addition, increasing concentrations of naproxen in the presence of white light lead to a progressive increase in the formation of hydroxyl radicals (Fig. 2B). It has been reported that, among oxygen radicals, hydroxyl radicals are the main activated oxygen species involved in DNA cleavage [6]. Therefore, activated species such as superoxide and hydroxyl radicals formed during the photolysis of naproxen result in DNA strand breaks, leading to structural changes.

image

Figure 2. (A) Concentration-dependent photo-generation of superoxide anions by naproxen, as measured by the reduction of NBT. Naproxen was dissolved in 50 mm sodium phosphate buffer (pH 7.8) at the indicated concentrations, and then exposed to white light for 2 h, and the absorbance was measured at 560 nm. (B) Concentration-dependent generation of hydroxyl radicals by photo-irradiated naproxen. Naproxen was dissolved in 10 mm Tris/HCl (pH 7.2) at the indicated concentrations, and then exposed to white light for 1 h, and the absorbance was measured at 532 nm. The increase in absorbance at 532 nm is due to the formation of a coloured complex between malondialdehyde and thiobarbituric acid. Values are means ± SD of three experiments. Asterisks indicate statistically significant differences compared with the control (*P < 0.05).

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Interaction of naproxen with DNA without irradiation: UV/vis spectroscopy

UV/vis absorption spectroscopy is used to explore the structural changes and formation of biomacromolecules upon addition of small ligands. The effect of naproxen on the UV absorption spectra is shown in Fig. 3. The absorption spectra showed large hyperchromic shifts (increase in band intensity) upon increasing the concentration of calf thymus DNA (ctDNA), with a band centered at 230 nm, indicating formation of adducts between ctDNA and naproxen. However, no red shift was observed in the spectra of the naproxen–DNA complex. As seen in Fig. 3, there is no clear isosbestic point, which indicates that there is more than one type of binding and/or that 1 : 1 drug:DNA stoichiometry is not maintained during the process [20]. Therefore, we conclude that formation of naproxen–DNA complexes does occur; however, these experiments do not provide mechanistic details.

image

Figure 3. UV/vis absorption spectra for naproxen (20 μm) in the presence of various concentrations of ctDNA in Tris/HCl buffer (pH 7.2). The arrow shows that the absorbance increases with increasing amounts of ctDNA.

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Steady-state fluorescence

In order to study the interaction between naproxen and DNA, the steady-state fluorescence technique was employed. The fluorescence emission spectra showed the effect of ctDNA on the emission spectrum of naproxen. Upon addition of ctDNA to naproxen solution, we observed significant quenching of the fluorescence intensity of naproxen (Fig. 4). The change in the emission spectra of naproxen due to the presence of ctDNA indicated an interaction between naproxen and ctDNA, causing fluorescence quenching of naproxen. The quenching pattern observed is potentially due to formation of a non-fluorescent complex of naproxen with DNA. To rationalize the results of the fluorescence experiments, the ratio of the peak fluorescence intensity in the presence and absence of ctDNA (F/F0) was plotted as a function of DNA concentration for naproxen. The inset in Fig. 4 shows that the fluorescence intensity decreases with an increasing concentration of DNA, i.e. the binding interaction increases gradually with subsequent addition of DNA.

image

Figure 4. Fluorescence emission spectra for naproxen (20 μm) in the presence of various concentrations of ctDNA. The excitation wavelength was 230 nm and the emission wavelengths were 300–500 nm. The inset shows the variation in the respective fluorescence intensities with DNA concentrations. The arrow shows that the intensity decreases upon increasing the concentration of ctDNA. Values are means ± SD of three experiments.

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Determination of the binding mechanism of naproxen and DNA

Iodide quenching

The fluorescence intensity of naproxen in the presence or absence of DNA was analyzed using potassium iodide (KI) as a quencher, as described previously [21], and the correlation between the degree of accessibility of each molecule to the quencher and steric bulk was observed. Interaction of KI with the negatively charged phosphate backbone of DNA was assumed to be repulsive in nature. As a consequence, an intercalatively bound small molecule experiences little protection compared with groove/surface binders in the presence of an anionic quencher. Quenching of the fluorescence intensity of naproxen by KI in the presence and absence of DNA was calculated using the Stern–Volmer equation:

  • display math(1)

where F0 and F are the fluorescence intensities in the absence and presence of the quencher KI (Q), and KSV is the Stern–Volmer quenching constant. KSV indicates the accessibility of the bulky quencher (iodide) to the fluorophore. The slopes of the plots of (F0/– 1) versus [KI] yield the values of KSV (Fig. 5). The KSV values for of naproxen in the presence of KI and the absence and presence of DNA were 30.06 and 7.52 m, respectively. Table 1 summarizes the calculated KSV from Stern–Volmer plots. The value of KSV for naproxen is low in the presence of DNA compared to its absence, clearly indicating intercalative binding.

image

Figure 5. Stern–Volmer plot for fluorescence quenching of naproxen (50 μm) by KI in the absence and presence of ctDNA (13.5 μm) in Tris/HCl buffer (pH 7.2). The concentration of KI was varied from 0 to 72 mm. Values are means ± SD of three experiments.

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Table 1. Variation of KSV values for naproxen in the absence and presence of ctDNA environments. Values are the means ± SD of four experiments.
DrugKSV (m) R a SDb
  1. a

    R is the correlation coefficient.

  2. b

    SD is standard deviation of naproxen and KI, naproxen, DNA and KI.

Buffer30.06 ± 0.240.998740.7238
Drug–DNA complex7.52 ± 0.200.999210.7830
Effect of urea

Chemical agents such as urea, guanidinium chloride etc. are often used to denature biomacromolecules such as DNA and protein, resulting in release of interacting drug molecules by destabilizing the interaction between the DNA and the drug [22, 23], leading to a change in the fluorescence behaviour of the drug molecule. Urea is used extensively as denaturing agent in biochemistry, destabilizing the double-stranded DNA double helix [24]. As evident from Fig. 6, there is a progressive increase in the fluorescence intensity of naproxen by addition of urea to naproxen-bound ctDNA. The extent of intercalation of naproxen as a function of urea concentration, given by the ratio of its fluorescence intensities in the presence and absence of urea (F/F0), is shown in Fig. 6. This confirmed that urea is able to release naproxen from the DNA strands. This also provides further evidence that the binding mode of naproxen with ctDNA is intercalative.

image

Figure 6. Fluorescence spectra of naproxen in the presence of urea. The fluorescence intensity increases with addition of urea. The inset shows the variation of the fluorescence intensity of ctDNA-bound naproxen as a function of urea concentration. Values are means ± SD of three experiments. Asterisks indicate statistically significant differences compared with the control (*P < 0.05).

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EtBr displacement assay

It is well known that enhancement of fluorescence emission of EtBr occurs as a result of intercalation between DNA base pairs [25, 26]. In order to examine the ability of naproxen to displace EtBr from intercalated complexes between EtBr and DNA, an EtBr displacement assay was performed. As shown in Fig. 7, emission of intense fluorescence from EtBr was observed in the presence of DNA due to its strong intercalation with DNA base pairs. However, addition of naproxen induced a progressive decrease in the DNA-induced EtBr emission, possibly due to displacement of EtBr by naproxen. The observed quenching of the EtBr fluorescence by addition of naproxen suggests that naproxen probably binds to ctDNA in an intercalative manner.

image

Figure 7. Fluorescence titration of the ctDNA–EtBr complex with naproxen. The fluorescence intensity decreases with addition of naproxen. The inset shows the intensity observed at 590 nm versus the concentration of naproxen. The excitation wavelength was 475 nm.

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Viscosity measurement

Viscosity measurements of drug–DNA complexes provide reliable evidence for the intercalative mode of binding because the length of DNA changes upon intercalation with the drug, affecting the viscosity [27, 28]. In the case of classical intercalation binding, a ligand lengthens the DNA helix due to separation of base pairs, resulting in increased DNA viscosity [27, 28]. On the other hand, if the drug is a groove binder or interacts electrostatistically with DNA, the viscosity of solution does not change significantly [29-31]. In Fig. 8, a plot of (η/ηo)1/3 versus the ratio of the naproxen concentration to the DNA concentration gives a measure of the viscosity changes. As seen in Fig. 8, with continuing addition of naproxen to DNA solution, the viscosity of the solution increases gradually. This is explained by the fact that naproxen intercalates between the DNA base pairs. This observation confirms the above results suggesting that naproxen intercalates with DNA.

image

Figure 8. Effect of increasing the concentration of naproxen on the viscosity of ctDNA. The concentration of DNA was kept constant (0.1 mm·L−1) and concentration of the complex was varied. Values are means ± SD of three experiments. Asterisks indicate statistically significant differences compared with the control (*P < 0.05).

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Circular dichroism (CD) studies

CD spectroscopy is a very sensitive technique that has been used extensively for analysis of changes that occur in the secondary structure of polypeptides, proteins and DNA under the influence of interacting ligands [32, 33]. Non-covalent drug–DNA interactions affect the structure of DNA, and hence alter the CD spectral behaviour. CD spectroscopy was used to obtain further information on the binding of naproxen to DNA [34, 35]. The CD spectrum of DNA in the absence and presence of naproxen is shown in Fig. 9. In the far-UV wavelength range (200–300 nm), the intrinsic CD profile of DNA is characterized by a positive peak at ~ 276 nm and a negative peak at ~ 247 nm, which represent base pair stacking and the right-handed B-form of DNA, respectively [36, 37]. As shown in Fig. 9, there is a decrease in the ellipticity of DNA with increasing concentration of naproxen. As reported previously [14], the decrease in the peak ellipticity at ~ 276 nm may be explained on the basis of disruption of the stacked nitrogenous bases due to intercalation of naproxen with DNA in order to optimize the binding interaction. Accommodation of the intercalated naproxen within a particular base pair results in adjustment of the relative orientation of bases. The change in the peak position at 247 nm occurs due to the change in the hydration layer of DNA, which in turn affects the helicity of DNA. Minor groove binders do not perturb the CD profile of DNA significantly. Hence, the CD analysis confirms our finding that naproxen binds to DNA in an intercalative manner.

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Figure 9. CD spectra of ctDNA (30 μm) in 10 mm Tris/HCl (pH 7.2) with varying concentrations of naproxen. The numbers 1–3 indicate the spectra at 0, 30 and 60 μm of naproxen, respectively. Each spectrum was obtained at 25 °C with a 10 mm path length cell.

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Förster resonance energy transfer (FRET)

FRET is a phenomenon that is observed when the emission spectrum of the donor molecules (D) overlaps with the excitation spectrum of the acceptor molecules (A). For FRET to be operational, the donor and acceptor molecules must be in close proximity to one another (1–10 nm), the donor and acceptor transition dipole moment must be correctly oriented, and donor must have a high quantum yield [38]. The emission spectrum of DNA is not very pronounced, although it can increase the fluorescence intensity of EtBr significantly. In order to magnify the FRET, we used the naproxen–DNA complex as an energy donor and EtBr as an energy acceptor. As shown in Fig. 10A, the emission spectrum of the donor drug–DNA complex overlaps with the absorption spectrum of the acceptor EtBr. The emission spectra of naproxen in the presence of increasing concentrations of EtBr do not show any appreciable change, indicating limited interaction between EtBr and naproxen (Fig. 10B), but the fluorescence emission of EtBr increases in the presence of DNA (Fig. 10C). These factors make EtBr a suitable candidate to study FRET between naproxen and DNA. In order to understand the relative interactions of naproxen and EtBr with DNA, we monitored the emission intensity upon addition of EtBr, and observed a significant decrease in the excimer emission intensity. However, we also observed the appearance of a new peak at 600 nm (Fig. 10C) with addition of EtBr. The appearance of emission from EtBr at 600 nm (while exciting the solution at 230 nm, at which EtBr has minimum absorption) may be expected due to FRET between the excimer of naproxen and EtBr in the presence of DNA.

image

Figure 10. (A) Spectral overlap between the normalized absorption spectrum of EtBr and the emission spectrum of naproxen in the presence of DNA. (B) Emission spectrum of naproxen in the presence of EtBr. (C) Changes in the emission spectrum of naproxen (20 μm) in the presence of DNA (20 μm) with addition of EtBr. The concentration of EtBr was 0–7 μm.

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According to Förster's theory [39], the efficiency of FRET depends on the inverse sixth power of the distance between the two components:

  • display math(2)

The term Ro is known as the Förster distance, and represents the distance at which the efficiency of transfer is 50%. The Förster distance [39] may be calculated as [39]

  • display math(3)

where κ2 is the orientation factor (value 2/3; the value is valid when both components are freely rotating and may be considered to be isotropically oriented during the lifetime of the excited state), ϕD is the fluorescence quantum yield of the donor in the absence of FRET, N is the refractive index of the medium (assumed to be equal to 1.3) [40], and J represents the spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, and is given as

  • display math(4)

where F(λ) is the fluorescence intensity of the donor in the wavelength range λ to λ + dλ and is dimensionless, and ε(λ) is the extinction coefficient (in m−1·cm−1) of the acceptor at λ. EFRET may also be estimated using the fluorescence emission intensity both in the absence and presence of acceptor by using the following equation:

  • display math(5)

On solving the above equations (in the presence of DNA), we calculated the spectral overlap, the Förster distance, the distance between the energy donor and acceptor and the FRET efficiency, and these were found to be 3.2 × 10−15, 1.92 nm, 2.11 nm and 35%, respectively. Hence, the value of RDA is close to that of Ro, i.e. the Förster distance or critical distance at which the energy transfer is 50%, indicating that the energy transfer from naproxen–DNA to EtBr occurs with high probability. As the UV energy absorbed by naproxen may be efficiently transferred to the intercalated EtBr in the presence of DNA, this strongly suggests that naproxen binds to DNA in intercalating mode. In the case of groove binders, no FRET is observed because of the greater distance between donor and acceptor molecules and the fact that orientation of the dipoles is inadequate for the energy transfer.

Molecular docking

Molecular docking is a useful tool for obtaining information about the structural features of ligand–receptor complexes and the binding affinity of a ligand with its receptor [41-43]. Docking studies were therefore performed in an attempt to ascertain the type and amount of interaction between a double-stranded DNA dodecamer [d(CGCGAATTCGCG)2, PDB ID 1BNA] and naproxen. The drug was made flexible to attain different conformations in order to predict the best-fit orientation, and the best energy-docked structure was analyzed. As evident from Fig. 11, naproxen binds to DNA by intercalating within the nucleotide base pairs of DNA. Furthermore, there is possibility of hydrogen bonding as the oxygen-bearing groups of naproxen are in proximity to the deoxyadenosines (DA4 and DA6 of strand A) of the dodecamer (Fig. 11C). The resulting relative binding energy of the docked DNA–naproxen complex was found to be ~ −170.11 kJ·m. Despite the electrostatic repulsion between naproxen and DNA (which bear the same charge), the large negative value of binding energy indicates a high binding potential of the naproxen with DNA. Hence, there is agreement between the results of spectral techniques and molecular modeling, providing important information about the mode and mechanism of interaction of naproxen with DNA.

image

Figure 11. (A, B) Molecular docked structure of naproxen complexed with DNA. (C) The possibility of hydrogen bonding to adjacent deoxyadenosines (DA4 and DA6 of strand A) of the dodecamer duplex of sequence (CGCGAATTCGCG)2 (PDB ID 1BNA).

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In conclusion, the present study clearly demonstrates the action of naproxen in the presence and absence of light based on studies on ROS generation as well as DNA binding by naproxen. These results may be very useful from the medical point of view as they show that naproxen has photochemical and/or photobiological properties in vitro. Our study also established the binding mode of naproxen with DNA. The results of the detailed studies performed here are in total agreement with interaction of naproxen with DNA by an intercalative mode of binding. The observation of FRET between naproxen and EtBr in the presence of DNA suggests the possibility of using such a system for efficient DNA drug design.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Materials

Naproxen and calf thymus DNA were purchased from Sigma-Aldrich (St Louis, MO). Ethidium bromide was purchased from Himedia (Mumbai, India). Plasmid pBR322 DNA was purified as described previously [44]. All other chemicals and solvents were of reagent grade and used without purification.

Calf thymus DNA preparation

ctDNA was dissolved in 0.1 m Tris/HCl buffer (pH 7.2) at room temperature with occasional stirring to ensure formation of a homogeneous solution. The purity of the DNA solution was determined by measuring the absorbance ratio A260 nm/A280 nm. As this ratio was in the range 1.8–1.9, no further purification was necessary. The concentrations of DNA solutions were determined by using a mean extinction coefficient value of 6600 m−1·cm−1 for a single nucleotide at 260 nm [45].

Plasmid nicking assay

To examine the generation of nicks in double-stranded DNA by ROS generated by naproxen, a plasmid nicking assay was performed. The reaction mixture contained 0.5 μg pBR322 plasmid DNA, the desired concentration of naproxen, and 10 mm Tris/HCl (pH 7.5) to a final volume of 25 μL in all tubes. Irradiation was performed with white light for 2 h at 37 °C. After incubation, 5 μL of 6X tracking dye solution containing 40 mm EDTA, 0.05% bromophenol blue and 50% glycerol was added, and the reaction mixture was subjected to electrophoresis using a 1% w/v agarose gel. The gel was stained with EtBr and viewed and photographed on a UV transilluminator (Wealtec Corporation, Model MD-25, Sparks, Nevada, USA).

Irradiation procedure

Irradiations were performed using a fluorescent lamp at a distance of 10 cm. The irradiation test was performed at 37 °C using an irradiance of 38.6 W/m2 as measured using a Lasermate coherent power meter (Coherent Inc., Santa Clara, CA, USA), and there was no measurable change in the temperature of the solution after irradiation for 2 h.

Superoxide generation assay

In order to study the superoxide generation by naproxen, the NBT reduction assay was performed essentially as described previously [46]. The assay mixture contained 50 mm sodium phosphate buffer (pH 7.8), 0.3 mm NBT, 0.1 mm EDTA and 0.06% Triton X-100 in a total volume of 3.0 mL. The reaction was started by addition of increasing concentrations of naproxen (0–200 μm). After mixing, the absorbance was recorded at 560 nm against a blank that did not contain naproxen.

Hydroxyl radical assay

The generation of hydroxyl radicals was measured as described previously [47]. ctDNA (300 μg) dissolved in 10 mm Tris/HCl (pH 7.2) was used as a substrate with increasing concentrations of naproxen (0–300 μm), and volume of the reaction mixture was adjusted to 3 mL by adding 10 mm Tris/HCl (pH 7.2). Incubation was performed for 60 min at 37 °C in the presence of white light. Formation of hydroxyl radicals generates malondialdehyde from deoxyribose, which then reacts with thiobarbituric acid to give a coloured complex that was assayed by recording the absorbance at 532 nm.

UV spectroscopic method

The UV spectrum was recorded using a Beckman DU40 spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA) with 1 × 1 cm quartz cuvettes. The UV spectra of naproxen and the DNA–naproxen complex were recorded in the wavelength range 200–250 nm. The experiment was performed in the presence of a fixed concentration of naproxen (20 μm) in a total volume of 3 mL, and titrated using varying concentrations of DNA (0–12 μm). DNA solutions of the same concentrations without naproxen were used as blanks to observe the UV spectra specific to the naproxen–DNA complex.

Fluorescence measurements

All fluorescence emission spectra were recorded on a Shimadzu 5000 spectrofluorometer (Kyoto, Japan) equipped with a xenon flash lamp using 1.0 cm quartz cells. Fluorescence emission spectra of naproxen–DNA, naproxen–DNA with KI and naproxen–DNA with urea were recorded at 300–500 nm upon excitation at 230 nm, with the widths of both the excitation and the emission slits set to 10.0 nm. All reaction mixtures were prepared in 10 mm Tris/HCl (pH 7.2).

Naproxen–DNA interaction

The fluorescence emission spectra were recorded by keeping the concentration of naproxen constant (20 μm) and varying the DNA concentration from 0–5.75 μm in a total volume of 3 mL containing 10 mm Tris/HCl (pH 7.2).

Potassium iodide (KI) quenching method

Potassium iodide quenching was performed in the presence and absence of DNA. Naproxen (50 μm) was dissolved into a 3 mL reaction mixture containing 10 mm Tris/HCl (pH 7.2), and emission spectra were recorded for varying concentrations of KI (0–72 mm). In another experiment, fixed concentrations of naproxen (50 μm) and ctDNA (13.5 μm) were used, and KI was added subsequently from 0–72 mm. The volume of the reaction mixture was made up to 3 mL by adding 10 mm Tris/HCl (pH 7.2).

Effect of urea

This was performed by using fixed concentrations of naproxen (50 μm) and ctDNA (13.5 μm) in a 3 mL reaction mixture containing 10 mm Tris/HCl (pH 7.2). Emission spectra were recorded for varying concentrations of urea (0–3.6 m).

Ethidium bromide displacement assay

The EtBr displacement assay was performed as described previously [48]. ctDNA (2.4 × 10−4 m) was dissolved in 10 mm Tris/HCl (pH 7.2), and EtBr was added to a final concentration of 2 μm. The concentration of naproxen was varied from 0–264 μm. The solution was excited at a wavelength of 475 nm, and emission was recorded at a wavelength of 500–700 nm.

Viscosity measurement

To further elucidate the binding mode of naproxen, viscosity measurements were performed by keeping the DNA concentration constant (0.1 mm·L−1) [49] and varying the concentration of the complexes. Viscosity measurements were performed using an Ubbelohde viscometer (Canon, Model-9721-K56, Coleparmer, USA) suspended vertically in a thermostat at 25 °C (accuracy ± 0.1 °C). The flow time was measured using a digital stopwatch, and each sample was tested three times to obtain a mean calculated time. The data are presented as η/η0 versus the ratio of the DNA concentration to the naproxen concentration, where η and η0 are the viscosity of naproxen in the presence and absence of DNA, respectively [50].

CD studies

In order to study the structural changes in DNA due to the presence of naproxen, CD spectra were analyzed. CD spectra were recorded in the range of 220–320 nm for ctDNA dissolved in 10 mm Tris/HCl (pH 7.2) in the absence and presence of various concentrations of naproxen (0–60 μm). Spectra were recorded on a CIRASCAN spectrophotometer (Applied Photophysics, Leatherhead, UK) equipped with a Peltier temperature controller. All CD spectra were collected with a scan speed of 200 nm/min and a spectral band width of 10 nm. Each spectrum was the average of four scans.

Molecular docking

An interactive molecular graphics program, Hex 6.3 [51] was used to understand the naproxen–DNA interactions. The structure of the B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID 1BNA) was downloaded from the Protein Data Bank (http://www.rcsb.org./pdb). The strucuture-data file (SDF) of naproxen was obtained from http://www.drugbank.ca/drugs/DB00788 and converted into PDB format using Avagadro's 1.01 [52]. Hex 6.3 performs docking using spherical polar Fourier correlations, and requires the ligand and the receptor to be input in PDB format. The parameters used for docking include: correlation type, shape only; FFT mode, 3D; grid dimension, 0.6; receptor range, 180; ligand range, 180; twist range, 360; distance range, 40. Visualization of the docked pose was performed using PyMol (DeLano Scientific, San Carlos, CA, USA).

Statistical analysis

The results are expressed as means ± SE of at least three independent observations. Student's t test was used to examine statistically significant differences. Analysis of variance was performed by ANOVA. P values < 0.05 were considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

The authors are thankful to University Grants Commission (UGC) (New Delhi, India) for the award of a University Grants Comission-Maulana Azad National Fellowship and A.M. University (Aligarh, India) for providing the necessary facilities. We also thank the Advanced Instrumentation Research Facility, Jawaharlal Nehru University (New Delhi, India) for performing CD experiments.

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  3. Introduction
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  5. Experimental procedures
  6. Acknowledgements
  7. References
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