There is a relationship between DNA binding and the mutagenicity of chemical substances (Dashwood, Combes, & Ashby, 1988). Drug binding causes structural and conformational changes in the DNA such as DNA bending, winding and double/single-strand breaks, resulting in DNA damage, which inhibits DNA transcription and replication and subcequently, related disorders(Shaikh, Ahmed, & Jayaram, 2004). UV-visible spectroscopy is the most common method to study drug-DNA interaction. In the spectrum of DNA hypochromic effects occurred due to changes in the conformation and structure of DNA, after binding of a drug to DNA (Valipour, Yilmaz, & Valipour, 2019). A hyperchromic effect is due to the binding of chemicals to DNA which might result in the unwinding of DNA helix(Tabassum, Chandra Sharma, Arjmand, & Azam, 2010). In this research, initial solution (containing only 60 µM of ctDNA) showed 0.4 absorption value at 260 nm while the last solution containing 60 µM of ctDNA and 45.6 µM of Dexamethasone showed absorption value of 1.02 at the same wavelength. Dexamethasone decreased the absorption peak wavelength of DNA solution from 260 nm to 250 nm. Therefore, the DNA solution showed a hyperchromism and blue shift by titration with Dexamethasone. The results of our research were in line with previous research that titrated a constant concentration of ctDNA with a varying concentration of chemicals and reported hyperchromism and blue shift in ctDNA. For example, Firdhouse and his coworkers (Firdhouse & Lalitha, 2015) recorded the absorbance spectra by keeping the ctDNA concentration constant, while varying some gold nanoparticles and they showed blue shift and hyperchromism in the UV-Vis spectra. Zhang and his coworkers (Zhang, Fu, Wang, & Hu, 2011) reported that the absorption peak of DNA at 260 nm exhibited gradual increase and slight blue shift with the increasing concentration of farrerol. Huang and his coworkers showed that with the gradual addition of an anticancer drug [(h6-p-Cymene)Ru(benzaldehyde-N4-phenylthiosemicarbazone) Cl], the absorbance of ctDNA–drug complex increased dramatically, and the peak position of ctDNA–drug complex blue shifted slightly (Huang et al., 2015). Eshkourfu and et al.(Eshkourfu et al., 2011)reported that absorbtion spectra of ct-DNA in the presence of diferent concentration of dinuclear cobalt(III) complex showed blue shift and hyperchromism [31]. The free binding energy (-5.35 kcal/mol) obtained by UV-Vis spectrophotometry matches the free energy (-5.39 kcal/mol) calculated by docking study for one of the ligand conformers.
In this research, it was computationally indicated Dexamethasone was located at the minor groove of DNA. Minor-groove binding usually involves greater binding affinity and higher sequence specificity(Paul & Bhattacharya, 2012). Before hydrogen bonds had been categorizing with a donor-acceptor distance in the ranges of 2.2–2.5 A˚, 2.5–3.2 A˚ and 3.2–4.0 A˚ as strong, moderate, and weak, respectively(Yu, Chen, Wu, & Chen, 2014). In this research, all of five hydrogen bonds shown in Dexamethasone–DNA docked model had a donor-acceptor distance ranging from 2.62 to 3.07 A˚ and one of them had 3.27 A˚. DNA in a human cell undergoes several thousand to a million damaging events per day, generated by both external (exogenous) and internal metabolic (endogenous) processes(Valipour et al., 2019). In order to explore DNA damage caused by chemicals, one of the inexpensive, rapid, and valuable method is incubation of a supercoiled plasmid DNA with the selected chemical followed by agarose gel electrophoresis(De Mattos, Dantas, Caldeira-de‐Araújo, & Moraes, 2004). The native conformation of pUC19 plasmid DNA is supercoil. Any breakage in one of the double strands converts the supercoiled form to open circular (relaxed) form while breakage in the same position on both strands makes linear plasmid DNA. Migration of the linear form through agarose gel is slower than the supercoiled form and faster than the open circle form(Moreno et al., 2007). The range of Dexamethasone concentrations in human plasma estimated by Klintip et al. (Klintip, Boonprasert, & Kolladarungkri, 2016) and by Song et al. (Song, Park, Kim, & Kim, 2004) was as 5.12–21.03 nmol/L and 2.5-509.6 nmol/L, respectively. In this study, Dexamethasone, ascorbic acid, H2O2, iron and copper concentrations were selected by considering their concentration in human plasma, which had been reported previously. In this research, even in the presence of high concentrations of an oxidative agent (H2O2, 6mM), a reducing agent (ascorbic acid, 60 µM), iron and copper, the DNA was not broken by Dexamethasone concentrations higher than those in human plasma and higher than those used by the previous investigators. In addition, the Dexamethasone could not protect the DNA against damaging hydroxyl radicals generated by fenton reactions. GPx plays a key role in metabolizing H2O2, hydroperoxides by reducing glutathione (GSH). GPX4 reduces membrane-bound phospholipid hydroperoxides and protects cells against membrane damage(Li, Zheng, Zhang, Xu, & Gao, 2018). Dexamethasone docked to the catalytic site of the GPX4 by docking score (-49.067 kcal/mol) almost equal to the docking score of its reference inhibitor, tiopronin, and showed interactions with Asp137 and Trp136 that are important residue in catalytic tetrad of the GPX4(Flohé & Brigelius-Flohé, 2011). Also, the drug showed a hydrogen bond with Lys135 interacting with GSH. In docking simulation, the estimated inhibition constant for dexamethasone was 20 times lower than estimated inhibition constant for tiopronin (154.83 µM). During oxidative stress, GSH was oxidized to glutathione disulfide (GSSG) which converted to GSH by glutathione reductase (GR) enzyme(Matés & Sánchez-Jiménez, 1999). GR is made from two subunits and each subunit forms four domains i.e. i) central catalytic site ii) FAD-binding, iii) NADPH-binding, iv) central interface domainsn(Ithayaraja, 2011). Dexamethasone binds to GR by interacting with Ala155, Asp331, Glu50, Gly157, Gly27, Gly29, Gly31, Gly 56, His52, Ser51, Thr156 and Thr57, which are involved in the interaction between the enzyme GR and FAD(Karplus & Schulz, 1989). Hydrogen bond score of dexametason is -6.455 and is 5 time more than the score related to the GR inhibitor. SOD catalyses the conversion of superoxide anion (O2.−) into hydrogen peroxide (H2O2), and catalase degrades H2O2 into water. SOD1 is a homodimeric enzyme. The active site of each subunit contains both a zinc and a copper ion, the latter being the site of the reaction(Banci, Bertini, Cramaro, Del Conte, & Viezzoli, 2002). Dexamethasone bond to His120, His48, His63, Thr137 of SOD1 that were residues interaction with cupper(Wright, Antonyuk, Kershaw, Strange, & Samar Hasnain, 2013). The docking score of Dexamethasone (-55.403 kcal/mol) was not very lower than the docking score related to isoproterenol (inhibitor of SOD1).
Human erythrocyte catalase (HEC) is a tetrameric protein comprising four identical subunits plus four heme groups and four NADPH molecules(Ko et al., 2000). Although the exact mechanism of NADPH action is unclear, low catalase activity in xeroderma pigmentosum fibroblasts and SV40-transformed human cell lines are linked to low intracellular NADPH(Putnam, Arvai, Bourne, & Tainer, 2000). In this research, Dexamethasone bond to the catalase enzyme by interacting with the same residues including His305, Leu451, Phe198, Phe446, Pro151, Pro304, Ser201, Trp303, Tyr2015, Val302 and Val450. The residues are NADPH binding residues (Putnam et al., 2000). In this case, the docking score related to Dexamethasone was obtained roughly equal to the docking score related to the inhibitor of the catalase enzyme. In accordance with our findings, before Dexamethasone(Mutsaers & Tofighi, 2012) and glucocorticoids(Feng & Tang, 2014) had been reported to induce oxidative stress. In view of the present findings, Dexamethasone did not directly break double helix DNA even in the presence of H2O2, ascorbic acid, iron and copper. According to the docking analysis, dexamethasone was strongly bond to DNA, the catalytic site of the GPX4, FAD-binding site of the GR, active site of the SOD1 and NADPH binding residues of the CAT enzyme. Therefore, although the drug did not directly break DNA, it may make DNA damages by inhibiting antioxidant defens enzymes and caused to induce oxidative stress in cells.